專利名稱：用于鹵化反應(yīng)的組合物和方法
背景技術(shù)：
本發(fā)明主要涉及用于鹵化天然產(chǎn)物的生物合成的方法、轉(zhuǎn)基因植物、和轉(zhuǎn)基因微生物，其中鹵化具有底物和區(qū)域?qū)Ｒ恍?。一方面，本發(fā)明涉及通過本發(fā)明方法生成的鹵化代謝物用于保護(hù)宿主生物體免于病原體的危害(更具體的說是保護(hù)植物免于植物病原體的危害)的用途。在這方面，本發(fā)明提供了對植物病原體的抵抗力增強(qiáng)的轉(zhuǎn)基因植物，和生物防治特性增強(qiáng)的生物防治生物體。
長期以來認(rèn)為超過2000種已知的天然發(fā)生的鹵化代謝物的生物合成是兩類酶的功能鹵素過氧化物酶和非血紅素過氧化物酶(GribbleGW，“The natural production of chlorinated compounds”(氯化化合物的天然生成)，Environ Sci Technol，28310-319，1994；van Pee K-H，“Biosynthesis of halogenated metabolites bybacteria”(由細(xì)菌進(jìn)行的鹵化代謝物的生物合成)，Annu RevMicrobiol，50375-399，1996)。在第一組中，溴過氧化物酶和氯過氧化物酶都擁有原卟啉IX作為含血紅素的輔基。這組酶發(fā)揮催化作用，即與過氧化氫發(fā)生反應(yīng)而形成酶的氫過氧化物(化合物1)，然后與鹵化物(X；X＝Br-、Cl-、或I-)發(fā)生反應(yīng)而形成酶(E)結(jié)合中間物EOX。尚不知道EOX是否是鹵化劑，或者EOX的分解是否產(chǎn)生活化的、半壽期較短的鹵化劑X+或其衍生物(如HOX、X2、或X3-)。但是，由這類鹵化酶展示的底物專一性的缺乏和區(qū)域?qū)Ｒ恍缘娜狈τ辛Φ恼f明，鹵化發(fā)生于活性位點以外而且是由EOX的分解產(chǎn)物之一催化的(FranssenMCR，“Halogenation and oxidation reactions withhaloperoxi dases”(鹵素過氧化物酶的鹵化和氧化反應(yīng))，Biocatalysis，1087-111，1994)。
非血紅素鹵素過氧化物酶有兩類，一類擁有釩，另一類擁有絲氨酸蛋白酶的特征性ser/Asp/His催化三聯(lián)體。前一組催化依賴釩和過氧化氫的HOX形成，繼而導(dǎo)致活性位點以外的鹵化和底物專一性的顯著缺乏(Franssen MCR，“Halogenation and oxidation reactions withhaloperoxidases”(鹵素過氧化物酶的鹵化和氧化反應(yīng))，Biocatalysis，1087-111，1994)。不含釩的非血紅素鹵素過氧化物酶被假定在活潑Ser殘基位點處形成醋酸酯，然后在存在過氧化氫時轉(zhuǎn)變成過醋酸；過醋酸將鹵化物離子氧化成活化的鹵化種類(PelletierI、AltenbucherJ、MattesR，“Acatalytic triad isrequ ired by the non-heme haloperoxidase to performhalogenation”(非血紅素鹵素過氧化物酶進(jìn)行鹵化需要催化性三聯(lián)體)，Biochim Biophys Acta，1250149-157，1995)。同樣，結(jié)果是發(fā)生不具有底物專一性或區(qū)域?qū)Ｒ恍缘姆磻?yīng)(van Pee K-H，“Biosynthesis of halogenated metabolites by bacteria”(細(xì)菌中鹵化代謝物的生物合成)，Annu Rev Microbiol，50375-399，1996)。
最近描述了另外一類鹵化酶基因，其產(chǎn)物展示對廣泛天然產(chǎn)物進(jìn)行區(qū)域?qū)Ｒ恍喳u化的能力(HammerPE、Hill DS、Lam ST、van PeeK-H，Ligon JM，“Four genes from Pseudomonas fluorescens that encodethe biosynthesis of pyrrolinitrin”(來自熒光假單胞菌的編碼硝吡咯菌素生物合成的四種基因)，Appl Env iron Microbiol，632147-2154，1997)。
發(fā)明概述本發(fā)明描述了以區(qū)域?qū)Ｒ坏姆绞綄Ⅺu素轉(zhuǎn)移至底物的方法，包括在存在氧化劑、鹵素供體、電子轉(zhuǎn)移酶、和還原劑時使底物接觸區(qū)域?qū)Ｒ恍喳u化酶，其中若轉(zhuǎn)移發(fā)生于體內(nèi)，則電子轉(zhuǎn)移酶是由異源核酸分子編碼的。
具體而言，描述了這樣的方法●其中依照本發(fā)明的方法還包含F(xiàn)AD或FMN成份，特別是FAD
●其中電子轉(zhuǎn)移酶是能夠催化電子由NADH或NADPH或鐵氧還蛋白轉(zhuǎn)移至FAD的酶●其中電子轉(zhuǎn)移酶是能夠催化電子由NADH或NADPH或鐵氧還蛋白轉(zhuǎn)移至區(qū)域?qū)Ｒ恍喳u化酶的酶●其中電子轉(zhuǎn)移酶是黃素還原酶、鐵氧還蛋白NADP還原酶、鐵氧還蛋白、心肌黃酶巰基還原酶或NADH-cyt-B5還原酶、NADPH-FMN還原酶、NADPH-cyt-p450還原酶、或硝酸還原酶●其中電子轉(zhuǎn)移酶包含與依照SEQ ID NO19、21、23、25、27、29、或31的任一氨基酸序列具有至少30％同一性的氨基酸序列●其中電子轉(zhuǎn)移酶包含SEQ ID NO19、21、23、25、29、或31任一的氨基酸序列●其中區(qū)域?qū)Ｒ恍喳u化酶是prnA、prnC、藤黃綠膿菌素鹵化酶pltA、pltD、和pltM、四環(huán)素鹵化酶cts4、水解酶a、或balhimycin鹵化酶bhaA●其中區(qū)域?qū)Ｒ恍喳u化酶包含SEQ ID NO1●其中區(qū)域?qū)Ｒ恍喳u化酶是包含依照SEQ ID NO3、5、7、9、11、13、15、或17任一的氨基酸結(jié)構(gòu)域的多肽。
本發(fā)明還提供了表達(dá)與SEQ ID NO18、10、22、24、26、28、或30任一基本相似的一種異源核酸和與SEQ ID NO2、4、6、8、10、12、14、或16任一基本相似的至少一種異源核酸的宿主細(xì)胞，具體而言，其中●宿主細(xì)胞是細(xì)菌、真菌、或植物細(xì)胞●宿主細(xì)胞是微生物細(xì)胞●宿主細(xì)胞還表達(dá)編碼prnB和prnD的核酸序列。
本發(fā)明還提供了●生成硝吡咯菌素的方法，包括培養(yǎng)上述宿主細(xì)胞●保護(hù)植物免于病原體的危害的方法，包括用上述宿主細(xì)胞處理植物，由此宿主以抑制病原體的量生成硝吡咯菌素●上述方法，還包括由宿主收集硝吡咯菌素。
本發(fā)明還提供了
●包含依照本發(fā)明的宿主細(xì)胞的植物●保護(hù)植物免于病原體的危害的方法，包括培養(yǎng)上述植物，由此在植物中以抑制病原體的量生成硝吡咯菌素●上述植物的種子●預(yù)防真菌在農(nóng)作物上生長的方法，包括培養(yǎng)依照本發(fā)明的植物，其中植物是農(nóng)作物植物●用于改進(jìn)宿主的鹵化底物生成的方法，包括在宿主中表達(dá)編碼電子轉(zhuǎn)移酶的異源核酸分子，其中宿主表達(dá)具有區(qū)域?qū)Ｒ恍喳u化酶活性的至少一種內(nèi)源多肽。
在本發(fā)明中令人驚訝的發(fā)現(xiàn)，區(qū)域?qū)Ｒ恍喳u化酶能夠在體外將鹵素轉(zhuǎn)移至底物，但是，為了這樣做，它們需要額外的蛋白質(zhì)因子即電子轉(zhuǎn)移酶。這些酶在體外實現(xiàn)鹵化需要額外蛋白質(zhì)因子的現(xiàn)象是在PrnA的純化中發(fā)現(xiàn)的，PrnA是一種D-色氨酸鹵化酶，它在硝吡咯菌素的生物合成中發(fā)揮功能，而硝吡咯菌素是由熒光假單胞菌(Pseudomonas fluorescens)生成的二氯化硝基苯基吡咯抗生素。這種依賴NADH和黃素腺嘌呤二核苷酸(以下稱為“FAD”)的鹵化酶的純化伴隨著鹵化活性的逐漸降低。在來自過度表達(dá)PrnA的熒光假單胞菌的提取物的離子交換層析過程中，可以通過加入一些缺乏PrnA的層析級分而重新激活部分純化且無活性的PrnA。根據(jù)對加熱和蛋白酶的敏感性，本文稱為熒光假單胞菌P2的、負(fù)責(zé)重激活的因子后來顯示是蛋白質(zhì)。將PrnA純化至均質(zhì)，導(dǎo)致活性完全喪失，但是可以通過加入本發(fā)明的電子轉(zhuǎn)移酶而恢復(fù)。
硝吡咯菌素途徑中的第二種鹵化酶PrnC展示與PrnA的序列的相似性，雖然與PrnA的序列相似性小于下列已知涉及鹵化天然產(chǎn)物的生物合成的區(qū)域?qū)Ｒ恍喳u化酶藤黃綠膿菌素(參閱Nowak-Thompson B、Chaney N、Wing JS、Gould SJ、Loper JE，“Characterization ofthe pyoluteorin biosynthetic gene cluster of Pseudomonasfluorescens Pf-5”(熒光假單胞菌Pf-5的藤黃綠膿菌素生物合成基因簇的表征)，J Bacteriol，1812166-2174，1999)；chloroeremomycin(參閱van Wageningen AM、Kirkpatrick PN、Williams DH、Harris BR、Kershaw JK、Lennard NJ、Jones M、JonesSJ、Solenberg PJ，“Sequencing and analysis of genes involvedin the biosynthesis of a vancomycin group antibiotic”(涉及一種萬古霉素組抗生素的生物合成的基因的測序和分析)，Chem Biol，5155-162，1998)；balhimycin(參閱Pelzer S、Sussmuth R、Heckmann D、Recktenwald J、Huber P、Jung G、Wohlleben W，“Identification and analysis of the balhimycin biosyntheticgene cluster and its use for manipulating glycopeptidebiosynthesis in Amycolatopsis mediterranei DSM5908”(balhimycin生物合成基因簇的鑒定和分析及其在地中海擬無枝酸菌DSM5908中操作糖肽生物合成的用途)，Antimicrob AgentsChemother，431565-1573，1999和Pelzer S、Reichert W、HuppertM、HeckmannD、WohllebenW，“Cloning and analysis of a peptidesynthetase gene of the balhimycin producer Amycolatopsismedi terranei DSM5908 and development of a genedisruption/replacement system”(balhimycin生產(chǎn)者地中海擬無枝酸菌DSM5908的肽合成酶基因的克隆和分析及基因破壞/取代系統(tǒng)的開發(fā))，J Biotechnol，56115-128，1997)；和氯四環(huán)素(參閱Dairi T、Nakano T、Mizukami T、Aisaka K、Hasegawa M、KatsumataR，“Conserved organization of genes for biosynthesis ofchlorotetracycline in Streptomyces strains”(鏈霉菌菌株中氯四環(huán)素生物合成基因的保守組織)，Biosci Biotechnol Biochem，591360-1361，1995和DairiT、NakanoT、AisakaK、KatsumataR、HasegawaM，“Cloning and nucleotide sequence of the generesponsible for chlorination of tetracycline”(負(fù)責(zé)四環(huán)素氯化的基因的克隆和核苷酸序列)，Biosci Biotechnol Biochem，591099-1106，1995)。與PrnA相似，PrnC的純化也伴隨著鹵化活性的喪失，但是可以通過加入本發(fā)明的電子轉(zhuǎn)移酶而恢復(fù)。
硝吡咯菌素途徑先前顯示在大腸桿菌中發(fā)揮功能，此時表達(dá)編碼PrnA、PrnB、PrnC、和PrnD的硝吡咯菌素操縱子(硝吡咯菌素操縱子的核苷酸序列請參閱美國專利號5,723,759中敘述的5.8X/N，完整收入本文作為參考)。PrnA和PrnC發(fā)揮鹵化酶的功能；PrnB催化色氨酸的吲哚基重排至氨基苯基吡咯；而PrnD將氨基苯基氧化成硝基苯基取代基。在本發(fā)明中令人驚訝的發(fā)現(xiàn)，當(dāng)本發(fā)明的電子轉(zhuǎn)移酶大腸桿菌黃素還原酶(以下稱為“Fre”)在這種情況下過度表達(dá)時，硝吡咯菌素的體內(nèi)生成顯著增強(qiáng)。
通過向純化且無活性的PrnA中加入大腸桿菌提取物，在大腸桿菌中證實了“P2樣活性”的存在。然后通過離子交換、羥基磷灰石、和凝膠滲透柱層析，部分純化了大腸桿菌P2樣活性。用胰蛋白酶處理含該活性的柱級分和無活性的側(cè)翼級分，隨后通過質(zhì)譜進(jìn)行分離；由在有活性的、含大腸桿菌P2樣活性的級分中存在的肽扣除在無活性的級分中鑒定的肽，剩余的肽稱為大腸桿菌基因組數(shù)據(jù)庫。由此，唯一鑒定了一種核酸序列，即依賴NADH的黃素還原酶(以下稱為“fre”，Genbank編號23486)。
正如下文詳述將明確描述的，然后克隆并過度表達(dá)大腸桿菌fre，過度表達(dá)細(xì)胞顯示大腸桿菌P2樣活性增加，而且與黃素還原酶活性的增加成正比例。將fre與硝吡咯菌素操縱子在分開的質(zhì)粒上共轉(zhuǎn)染到大腸桿菌中。包含這兩種質(zhì)粒的細(xì)胞生成的硝吡咯菌素或硝吡咯菌素代謝物顯著高于只包含硝吡咯菌素操縱子的細(xì)胞，確認(rèn)了Fre作為PrnA和PrnC的輔助因子的身份，并指示在大腸桿菌中黃素還原酶活性是限制硝吡咯菌素生成的主要因子。
在本發(fā)明的一個實施方案中，提供了以區(qū)域?qū)Ｒ坏姆绞綄Ⅺu素轉(zhuǎn)移至底物的方法，包括在存在氧化劑、鹵素供體、電子轉(zhuǎn)移酶、和還原劑時使底物接觸區(qū)域?qū)Ｒ恍喳u化酶，其中若轉(zhuǎn)移發(fā)生于體內(nèi)，則電子轉(zhuǎn)移酶對宿主而言是異源的。
在本發(fā)明的另一個實施方案中，提供了以區(qū)域?qū)Ｒ坏姆绞綄Ⅺu素轉(zhuǎn)移至底物的方法，包括在存在氧化劑、鹵素供體、電子轉(zhuǎn)移酶、還原劑、和FAD或FMN時使底物接觸區(qū)域?qū)Ｒ恍喳u化酶，其中若轉(zhuǎn)移發(fā)生于體內(nèi)，則電子轉(zhuǎn)移酶對宿主而言是異源的。在特別優(yōu)選的實施方案中，反應(yīng)導(dǎo)致硝吡咯菌素的生成。
在一個優(yōu)選的實施方案中，電子轉(zhuǎn)移酶是能夠催化電子由NADH或NADPH或鐵氧還蛋白轉(zhuǎn)移至FAD的酶，或者電子轉(zhuǎn)移酶是能夠催化電子由NADH或NADPH或鐵氧還蛋白轉(zhuǎn)移至區(qū)域?qū)Ｒ恍喳u化酶的酶。
在一個優(yōu)選的實施方案中，電子轉(zhuǎn)移酶的氨基酸序列與NADPH-FMN還原酶、大鼠肝NADPH-cyt-P450還原酶、菠菜鐵氧還蛋白NADP還原酶、細(xì)胞色素b5還原酶、或亞硝酸還原酶至少30％同一、優(yōu)選40％同一、更優(yōu)選50％同一、更優(yōu)選60％同一、更優(yōu)選70％同一、更優(yōu)選80％同一、或更優(yōu)選90％同一。
在一個優(yōu)選的實施方案中，區(qū)域?qū)Ｒ恍喳u化酶的氨基酸序列與來自熒光假單胞菌(Pseudomonas fluorescens)的PrnA、PrnC、藤黃綠膿菌素鹵化酶PltA、PltD、和PltM、來自金霉素鏈霉菌(Streptomyces aurofaciens)的四環(huán)素鹵化酶cts4、來自東方擬無枝酸菌(Amycolatopsis orientalis)的水解酶、或來自地中海擬無枝酸菌(Amycolatopsis mediterranei)的balhimycin鹵化酶bhaA至少30％同一、優(yōu)選40％同一、更優(yōu)選50％同一、更優(yōu)選60％同一、更優(yōu)選70％同一、更優(yōu)選80％同一、或更優(yōu)選90％同一。
在一個優(yōu)選的實施方案中，提供了表達(dá)與本發(fā)明電子轉(zhuǎn)移酶的核酸基本相似的異源核酸并表達(dá)編碼本發(fā)明區(qū)域?qū)Ｒ恍喳u化酶的異源核酸的宿主細(xì)胞。在一個優(yōu)選的實施方案中，宿主細(xì)胞是細(xì)菌、真菌、或植物細(xì)胞。
在一個優(yōu)選的實施方案中，提供了表達(dá)編碼prnA、prnB、prnC、prnD、和fre的異源核酸分子的宿主細(xì)胞。
在一個優(yōu)選的實施方案中，提供了通過培養(yǎng)宿主細(xì)胞來生成硝吡咯菌素的方法，可包括表達(dá)編碼prnA、prnB、prnC、prnD、和fre的異源核酸分子的植物細(xì)胞。
在一個優(yōu)選的實施方案中，提供了包含本發(fā)明宿主細(xì)胞的植物，所述宿主細(xì)胞表達(dá)與編碼本發(fā)明電子轉(zhuǎn)移酶的核酸基本相似的異源核酸并表達(dá)編碼本發(fā)明區(qū)域?qū)Ｒ恍喳u化酶的異源核酸。
在一個優(yōu)選的實施方案中，提供了表達(dá)編碼本發(fā)明的prnA、prnB、prnC、prnD、和電子轉(zhuǎn)移酶的異源核酸分子的植物。
序列簡述SEQ ID NO1是本發(fā)明的區(qū)域?qū)Ｒ恍喳u化酶中存在的保守氨基酸基元。SEQ ID NO2是編碼來自熒光假單胞菌的PrnA的核酸序列。SEQ ID NO3是來自熒光假單胞菌的PrnA的氨基酸序列。SEQ ID NO4是編碼來自熒光假單胞菌的PrnC的核酸序列。SEQ ID NO5是來自熒光假單胞菌的PrnC的氨基酸序列。SEQ ID NO6是編碼來自熒光假單胞菌的PltA的核酸序列。SEQ ID NO7是來自熒光假單胞菌的PltA的氨基酸序列。SEQ ID NO8是編碼來自熒光假單胞菌的PltD的核酸序列。SEQ ID NO9是來自熒光假單胞菌的PltD的氨基酸序列。SEQ ID NO10是編碼來自熒光假單胞菌的PltM的核酸序列。SEQ ID NO11是來自熒光假單胞菌的PltM的氨基酸序列。SEQ ID NO12是編碼來自東方擬無枝酸菌的水解酶A的核酸序列。SEQ ID NO13是來自東方擬無枝酸菌的水解酶A的氨基酸序列。SEQ ID NO14是編碼來自金霉素鏈霉菌的cts4的核酸序列。SEQ ID NO15是來自金霉素鏈霉菌的cts4的氨基酸序列。SEQ ID NO16是編碼來自地中海擬無枝酸菌的bhaA的核酸序列。SEQ ID NO17是來自地中海擬無枝酸菌的bhaA的氨基酸序列。SEQ ID NO18是編碼來自大腸桿菌的Fre的核酸序列。SEQ ID NO19是來自大腸桿菌的Fre的氨基酸序列。SEQ ID NO20是編碼來自大鼠的NADH細(xì)胞色素b5還原酶的核酸序列。SEQ ID NO21是來自大鼠的NADH細(xì)胞色素b5還原酶的氨基酸序列。SEQ ID NO22是編碼來自兔的NADPH-cyt-p450還原酶的核酸序列。SEQ ID NO23是來自兔的NADPH-cyt-p450還原酶的氨基酸序列。SEQID NO24是編碼來自蔬菜沙門氏菌(S.oleracea)的鐵氧還蛋白的核酸序列。SEQ ID NO25是來自蔬菜沙門氏菌的鐵氧還蛋白的氨基酸序列。SEQ ID NO26是編碼來自費氏弧菌(Vibriofischeri)的NADPH-FMN還原酶的核酸序列。SEQ ID NO27是來自費氏弧菌的NADPH-FMN還原酶的氨基酸序列。SEQ ID NO28是編碼來自蔬菜沙門氏菌的鐵氧還蛋白-NADP還原酶的核酸序列。SEQ ID NO29是來自蔬菜沙門氏菌的鐵氧還蛋白-NADP還原酶的氨基酸序列。SEQ ID NO30是編碼來自寄生曲霉(Aspergullus parasiticus)的硝酸還原酶的核酸序列。SEQ ID NO31是來自寄生曲霉的硝酸還原酶的氨基酸序列。SEQ ID NO32是用于大腸桿菌黃素還原酶的引物。SEQ ID NO33是用于大腸桿菌黃素還原酶的引物。SEQ ID NO34是質(zhì)粒pNOV523。SEQ ID NO35是pNOV524。
發(fā)明詳述體外鹵化天然產(chǎn)物的生成依照本發(fā)明，可以通過在存在鹵素供體、氧化劑、還原劑、和本發(fā)明的電子轉(zhuǎn)移酶時使區(qū)域?qū)Ｒ恍喳u化酶與底物發(fā)生反應(yīng)而在體外生成鹵化天然產(chǎn)物。
本發(fā)明的區(qū)域?qū)Ｒ恍喳u化酶是能夠與鹵化物、氧化劑、和還原系統(tǒng)相互作用而在生物學(xué)鹵化反應(yīng)過程中催化一個或多個碳-鹵素鍵取代一個或多個碳-氫鍵，且具有底物和/或區(qū)域?qū)Ｒ恍缘柠u化酶。區(qū)域?qū)Ｒ恢柑?鹵素鍵只于底物的特定位置形成。
本發(fā)明優(yōu)選的區(qū)域?qū)Ｒ恍喳u化酶包括包含如下保守基元并在特定位置催化至少一個碳-鹵素鍵取代碳-氫鍵的鹵化酶X1-W-X2-W-X3-I-P-X4(SEQ ID N01)，其中X1是G或T；X2是V、L、T、F、或M；X3是任意氨基酸殘基；X4是I、F、M、或L。
在一個優(yōu)選實施方案中，本發(fā)明的鹵化酶包括色氨酸鹵化酶。本發(fā)明的色氨酸鹵化酶包括PrnA(SEQ ID NO3)(參閱蛋白質(zhì)編號#AAB97504；Hammer PE、Burd W、Hill DS、Ligon JM、van Pee K，“Conservation of the pyrrolnitrin biosynthetic gene clusteramong six pyrrolnitrin-producing strains”(六種硝吡咯菌素生成菌株中硝吡咯菌素生物合成基因簇的保守性)，F(xiàn)EMS MicrobiolLett，180(1)39-44，1999年11月1日)和與SEQ ID NO3優(yōu)選具有90％同一性、80％同一性、70％同一性、60％同一性、50％同一性、或40％同一性的區(qū)域?qū)Ｒ恍喳u化酶。在本中請書全文中所用的氨基酸序列之間的同一性百分比是通過BASTP 2.09程序(可以由http//www.ncbi.nlm.nih.gov/gorf/bl2.html獲得)測定的，參數(shù)設(shè)置是評分矩陣＝blosum62，缺口開口罰分＝7，缺口延伸罰分＝2，x_dropoff＝50，預(yù)期(expect)＝10.00，字長(wordsize)＝3。
在另一個優(yōu)選實施方案中，本發(fā)明的區(qū)域?qū)Ｒ恍喳u化酶包括單氯氨基硝吡咯菌素(monochchloroaminopyrrolnitrin)鹵化酶。單氯氨基硝吡咯菌素鹵化酶包括PrnC(SEQ ID NO5)(參閱蛋白質(zhì)編號#AAB97506)和與其優(yōu)選具有90％同一性、80％同一性、70％同一性、60％同一性、50％同一性、或40％同一性的區(qū)域?qū)Ｒ恍喳u化酶。
在本發(fā)明的一個特別優(yōu)選的實施方案中，本發(fā)明的區(qū)域?qū)Ｒ恍喳u化酶包括與來自熒光假單胞菌的prnA(SEQ ID NO3)、prnC(SEQ IDNO5)、藤黃綠膿菌素鹵化酶pltA(SEQ ID NO7)、pltD(SEQ IDNO9)、和pltM(SEQ ID NO11)、來自金霉素鏈霉菌的四環(huán)素鹵化酶cts4(SEQ ID NO15)、來自東方擬無枝酸菌的水解酶a(SEQ IDNO13)、來自地中海擬無枝酸菌的balhimycin鹵化酶bhaA(SEQ ID NO17)中的任一種30％同一、優(yōu)選40％同一、更優(yōu)選50％同一、更優(yōu)選60％同一、更優(yōu)選70％同一、更優(yōu)選80％同一、更優(yōu)選90％同一、更優(yōu)選95％同一、或更優(yōu)選99％同一的任何鹵化酶，包括下表中鑒定的鹵化酶
本發(fā)明的電子轉(zhuǎn)移酶可包括通過依賴NAD(P)H的氧化還原酶或氧化還原酶及其它電子供體(諸如葉綠體光合系統(tǒng)、乳酸鹽、黃嘌呤等)，能夠?qū)㈦娮佑蒒ADH或NADPH或鐵氧還蛋白或其它還原劑轉(zhuǎn)移至FAD或FMN的電子轉(zhuǎn)移酶，或者能夠?qū)㈦娮佑蒒ADH或NADPH或鐵氧還蛋白或其它還原劑轉(zhuǎn)移至鹵化酶的電子轉(zhuǎn)移酶。
可以通過選擇這樣的電子轉(zhuǎn)移酶來確定本發(fā)明的電子轉(zhuǎn)移酶，其中可以通過監(jiān)控NADH或NADPH或鐵氧還蛋白的氧化(通過與還原劑的氧化有關(guān)的吸光度的特征性變化)來檢測電子轉(zhuǎn)移。這種變化(或變化速率的增加)依賴FAD或FMN的存在?？梢酝ㄟ^監(jiān)控340nm的吸光度來檢測NADH和NADPH的氧化；氧化導(dǎo)致吸光度降低?？梢酝ㄟ^監(jiān)控420nm的吸光度來檢測鐵氧還蛋白的氧化；氧化導(dǎo)致吸光度升高。還可以通過監(jiān)控NADH或NADPH的氧化(通過340nm處的激發(fā)、＞380nm處的發(fā)射的熒光特征性降低)來檢測電子轉(zhuǎn)移。這種熒光降低依賴FAD或FMN的存在。
還可以通過選擇這樣的電子轉(zhuǎn)移酶來確定本發(fā)明的電子轉(zhuǎn)移酶，其中可以通過混合電子轉(zhuǎn)移酶與50μmolar NADH或50μmolar NADPH、包含或不含50μmolar鹵化酶(鹵化酶必須是全酶的狀態(tài)，即早已結(jié)合了所有必需的輔因子，諸如FAD)并觀察依賴鹵化酶的NADH或NADPH氧化速率的增加來鑒定由NADH或NADPH向本發(fā)明區(qū)域?qū)Ｒ恍喳u化酶的電子轉(zhuǎn)移；其中通過340nm處吸光度的降低或上述熒光的降低來測量氧化。
可以通過選擇這樣的電子轉(zhuǎn)移酶來確定本發(fā)明的電子轉(zhuǎn)移酶，其中可以通過混合電子轉(zhuǎn)移酶與50mol還原型鐵氧還蛋白、包含或不含50μmol鹵化酶(鹵化酶必須是全酶的狀態(tài)，即早已結(jié)合了所有必需的輔因子，諸如FAD)并觀察依賴鹵化酶的氧化速率的增加來鑒定由鐵氧還蛋白向鹵化酶的電子轉(zhuǎn)移；通過340nm吸光度的增加來測量鐵氧還蛋白的氧化。
在本發(fā)明的優(yōu)選實施方案中，電子轉(zhuǎn)移酶與下列任一至少30％同一、優(yōu)選40％同一、更優(yōu)選50％同一、更優(yōu)選60％同一、更優(yōu)選70％同一、更優(yōu)選80％同一、更優(yōu)選90％同一、或同一包含SEQ ID NO19的氨基酸序列的大腸桿菌黃素還原酶(描述于FieschiF、NiviereV、Frier C、Decout JL、Fontecave M，“The mechanism and substratespecificity of the NADPHflaVin oxidoreductase fromEscherichia coli”(來自大腸桿菌的NADPH黃素氧化還原酶的機(jī)制和底物專一性)，J Biol Chem，270(51)30392-30400，1995年12月22日)；依照Richarme G，“Purification of a new dihydrolipoamidedehydrogenase from Escherichia coli”(來自大腸桿菌的一種新的二氫硫辛酰胺脫氫酶的純化)，J Bacteriol，171(12)680-685，1989年12月純化的心肌黃酶巰基還原酶；NADH細(xì)胞色素b5還原酶(SEQID NO21)(描述于Barber MJ、Quinn GB，“High-level expressionin Escherichial coli of soluble，catalytic domain of rat hepaticcytochrome b5 reductase”(大鼠肝細(xì)胞色素b5還原酶的可溶性催化結(jié)構(gòu)域在大腸桿菌中的高水平表達(dá))，Protein Expr Purif，8(1)41-47，1996年8月；來自大鼠的NADPH-cyt-P450還原酶(SEQ ID NO23)、來自蔬菜沙門氏菌的鐵氧還蛋白-NADP還原酶(SEQ ID NO29)、來自蔬菜沙門氏菌的鐵氧還蛋白(SEQ ID NO25)、來自寄生曲霉的硝酸還原酶(SEQ ID NO31)、和來自費氏弧菌的NAD(P)H-FMN還原酶(SEQ ID NO27)(描述于Zenno S、SaigoK，“Identificationof the genes encoding NAD(P)H-flavin oxidoreductases that aresimi lar in sequence to Escherichia coli Fre in four species ofluminous bacteriaPhotorhabdus luminescens，Vibrio fischeri，Vibrio harveyi，and Vibrio orientails”(四種發(fā)光細(xì)菌(發(fā)光光桿狀菌、費氏弧菌、哈氏弧菌、和東方弧菌)中序列與大腸桿菌Fre相似的NAD(P)H-黃素氧化還原酶的編碼基因的鑒定)，J Bacteriol)，176(12)3544-3551，1994年6月)?？梢砸蕴崛∥锘蚣兓问绞褂帽景l(fā)明的電子轉(zhuǎn)移酶。
在特別優(yōu)選的實施方案中，本發(fā)明的電子轉(zhuǎn)移酶與SEQ ID NO21、23、25、27、29、或31任一至少30％同一、優(yōu)選40％同一、更優(yōu)選50％同一、更優(yōu)選60％同一、更優(yōu)選70％同一、更優(yōu)選80％同一、更優(yōu)選90％同一，而且在任一上述測試中電子轉(zhuǎn)移測試為陽性。
還原劑的選擇，諸如吡啶核苷酸，如還原型煙酰胺腺嘌呤二核苷酸或還原型煙酰胺腺嘌呤二核苷酸磷酸或還原型鐵氧還蛋白，取決于本發(fā)明電子轉(zhuǎn)移酶的選擇。一般而言，本發(fā)明的所有電子轉(zhuǎn)移酶對一種吡啶核苷酸具有高度催化活性，但通常對另一種吡啶核苷酸具有一些活性。因而，如果出于其它考慮而希望這樣，那么可以在特定電子轉(zhuǎn)移酶的鹵化反應(yīng)中使用非優(yōu)選的吡啶核苷酸。每種電子轉(zhuǎn)移酶的優(yōu)選吡啶核苷酸如下NADPH是NADPH-cyt-P450還原酶和鐵氧還蛋白NADP還原酶的優(yōu)選吡啶核苷酸；NADH是大腸桿菌黃素還原酶、NADH-cyt-b5還原酶、硝酸還原酶、和心肌黃酶巰基還原酶的優(yōu)選吡啶核苷酸。
鐵氧還蛋白NADP還原酶還可使用還原型鐵氧還蛋白，它可以通過植物、分離葉綠體、或含葉綠體片段的光合系統(tǒng)I的光照而生成。鐵氧還蛋白還可以由依賴鐵氧還蛋白的脫氫酶還原，諸如丙酮酸鐵氧還蛋白氧化還原酶(Horner DS、Hirt RP、Embley TM，“A single eubacterialorigin of eukaryotic pyruvateferredoxin oxidoreductase genesimplications for the evolution of anaerobic eukaryotes”(真核生物丙酮酸鐵氧還蛋白氧化還原酶基因的單一真細(xì)菌起源厭氧真核生物進(jìn)化的暗示)，Mol Biol Evol，16(9)1280-1291，1999年9月)。
在優(yōu)選的實施方案中，可以在體外反應(yīng)中包含F(xiàn)AD以增加反應(yīng)效率。在特別優(yōu)選的實施方案中，反應(yīng)包含F(xiàn)AD，而且選擇的區(qū)域?qū)Ｒ恍喳u化酶是PrnA。
在候選實施方案中，本發(fā)明包括聯(lián)合鹵化酶(其中鹵化酶是純化的本發(fā)明區(qū)域?qū)Ｒ恍喳u化酶)與底物鹵素離子諸如Cl-，和活性氧供體諸如H2O2、KIO4、亞碘酰苯、亞碘酰基苯甲酸(鹽)、叔丁基過氧化氫、過氧化苯甲酰、氫過氧化枯烯(cumene hydroperoxide)、過氧化二異丙苯(dicumyl peroxide)、過氧乙酸、或近似化合物。活性氧供體省卻了供給O2和還原劑的需要。
本發(fā)明的底物將取決于選擇的本發(fā)明區(qū)域?qū)Ｒ恍喳u化酶。本發(fā)明的底物可包括色氨酸、吲哚、氨基苯基吡咯、及其衍生物、和四環(huán)素，bhaA的底物包括balhimycin底物種類B1-1、B1-2、B2-1、B2-2、和B3的所有化合物(描述于PelzerS、SussmuthR、HeckmannD、RecktenwaldJ、HuberP、JungG、WohllebenW，“Identification and analysisof the balhimycin biosynthetic gene cluster and its use formanipulating glycopeptide biosynthesis in Amycolatopsismediterranei DSM5908”(balhimycin生物合成基因簇的鑒定和分析及其在地中海擬無枝酸菌DSM5908中操作糖肽生物合成的用途)，Antimicrob Agents Chemother，431565-1573，1999)。
可以以無機(jī)或有機(jī)陽離子的鹽或其相應(yīng)的酸的形式向反應(yīng)供給可用于本發(fā)明的鹵素供體。本發(fā)明的鹵素供體可提供F-、Cl-、Br-、或I離子。
可以在pH4-10、溫度0-65℃的緩沖液中進(jìn)行本發(fā)明的反應(yīng)?？梢砸喳}的形式加入鹵素供體，如氯化物鹽類可包括LiCl、NaCl、KCl、CsCl、MgCl2、CaCl2、和NH4Cl。反應(yīng)時間可以在1分鐘-48小時之間變化。最佳條件是pH7.5、溫度30℃、反應(yīng)時間12小時。
可以通過共價偶聯(lián)電子轉(zhuǎn)移酶與鹵化酶，使得由還原劑向鹵化酶的電子轉(zhuǎn)移成為一級過程而非二級過程(就鹵化酶濃度而言)，從而提高體外鹵化的催化效率?？梢酝ㄟ^遺傳工程構(gòu)建包含本發(fā)明的電子轉(zhuǎn)移酶和區(qū)域?qū)Ｒ恍喳u化酶的融合蛋白(通過在同一讀碼框中融合它們的編碼區(qū))來獲得相同結(jié)果?？梢允褂没虿挥镁幋a將電子轉(zhuǎn)移酶和鹵化酶蛋白質(zhì)結(jié)構(gòu)域分隔開來的短肽序列的間插序列來進(jìn)行融合?？梢砸詢煞N取向構(gòu)建融合蛋白(1)N端-電子轉(zhuǎn)移酶-(任選的接頭)-鹵化酶-C端；(2)N端-鹵化酶-(任選的接頭)-電子轉(zhuǎn)移酶-C端。
在本發(fā)明的另一個實施方案中，可以將包含區(qū)域?qū)Ｒ恍喳u化酶和電子轉(zhuǎn)移酶的系統(tǒng)的蛋白質(zhì)成份進(jìn)行固定，正如下文進(jìn)一步所述，使之與底物反應(yīng)而生成產(chǎn)物。鹵化酶和電子轉(zhuǎn)移酶可以作為共固定的單獨酶使用，或者作為具有電子轉(zhuǎn)移酶和鹵化酶活性的融合蛋白(其中兩種成份的編碼序列融合在一起而生成單一蛋白質(zhì))使用。系統(tǒng)中可以包含額外酶和適當(dāng)?shù)牡诙€原劑以再生NADH或NADPH。這種酶和第二還原劑配對的范例包括乙醇脫氫酶與乙醇、葡萄糖-6-磷酸脫氫酶與葡萄糖-6-磷酸、醛脫氫酶與乙醛、硫辛酰胺脫氫酶與還原型硫醇(諸如硫辛酰胺、二硫蘇糖醇、或巰基磺酸)。
在這個實施方案中，可以通過幾種方法將酶(將包括NADH或NADPH再生系統(tǒng)的酶，如果使用這種系統(tǒng)的話)進(jìn)行固定。范例包括(1)將酶置于具有半透膜(透析膜)的容器內(nèi)，所述半透膜允許底物和核苷酸通過，但是酶不能通過；(2)將酶共價附著于不溶性基質(zhì)；(3)經(jīng)針對酶的抗體或針對酶所融合的抗原的抗體使酶結(jié)合基質(zhì)；(4)經(jīng)生物素和生物素結(jié)合結(jié)構(gòu)域(諸如親和素)使酶結(jié)合基質(zhì)；(5)使酶周圍的基質(zhì)聚合(諸如異丁烯酸酯聚合物)。
然后可以將固定的酶暴露于含還原劑、第二還原劑(如果使用NAD(P)H再生系統(tǒng)的話)、底物、和鹵化物鹽的緩沖液?？梢园袡C(jī)溶劑以促進(jìn)底物的溶解。典型條件包括pH4-10、0-65℃。生成足夠的鹵化產(chǎn)物后，由反應(yīng)混合液提取鹵化天然產(chǎn)物。異源宿主中鹵化天然產(chǎn)物的生成可以在細(xì)菌或真菌宿主中表達(dá)編碼本發(fā)明電子轉(zhuǎn)移酶的異源核酸分子，從而能夠以比天然宿主的可能更高的效率進(jìn)行天然產(chǎn)物的鹵化生產(chǎn)。例如，為了增強(qiáng)天然產(chǎn)物生產(chǎn)，可以在硝吡咯菌素生產(chǎn)者諸如熒光假單胞菌、吡咯伯克霍爾德氏菌、橙色粘球菌、洋蔥伯克霍爾德氏菌、致金色假單胞菌，藤黃綠膿菌素生產(chǎn)者諸如熒光假單胞菌，萬古霉素類抗生素生產(chǎn)生物體諸如各種擬無枝酸菌物種諸如東方擬無枝酸菌和地中海擬無枝酸菌，和氯四環(huán)素生產(chǎn)者金霉素鏈霉菌，或其它抗生素生產(chǎn)鏈霉菌物種中表達(dá)編碼本發(fā)明電子轉(zhuǎn)移酶的異源核酸分子。
另外，可以在細(xì)菌或真菌宿主中共表達(dá)編碼區(qū)域?qū)Ｒ恍喳u化酶和電子轉(zhuǎn)移酶的異源核酸分子，從而能夠進(jìn)行或增加鹵化天然產(chǎn)物的生產(chǎn)。在有些情況下，本發(fā)明鹵化天然產(chǎn)物的合成將只需要一個生物合成步驟，即鹵化步驟，因而將要表達(dá)的唯一異源核酸分子將是包含本發(fā)明鹵化酶和電子轉(zhuǎn)移酶的編碼序列的異源核酸分子。在其它情況下，生成鹵化天然產(chǎn)物的生物合成途徑將包含一個或多個鹵化步驟作為其中一部分。在這種情況下，將表達(dá)多種異源核酸分子。
術(shù)語“異源核酸分子”在用于本申請書全文時指與它所導(dǎo)入的宿主細(xì)胞天然無關(guān)的核酸分子，包括遺傳構(gòu)建物、天然發(fā)生核酸分子的非天然發(fā)生多拷貝；和可操作連接非天然核酸分子的原本同源的核酸分子。
在廣義上，術(shù)語“基本相似”在用于本申請書全文中的核酸分子方面時指對應(yīng)于參考核苷酸序列的核酸分子，其中相應(yīng)的核酸分子編碼的多肽與參考核苷酸序列編碼的多肽具有基本相同的結(jié)構(gòu)和功能，如其中只發(fā)生不影響多肽功能的氨基酸變化。理想的是，基本相似的核酸分子編碼由參考核苷酸序列編碼的多肽。術(shù)語“基本相似”明確的意欲包括其序列經(jīng)修飾而使得在特定細(xì)胞中的表達(dá)最優(yōu)化的核酸分子。基本相似的核酸分子與參考核苷酸序列之間的同一性百分比理想的是至少30％、優(yōu)選至少45％、更希望是至少65％、更希望是至少75％、優(yōu)選至少85％、更優(yōu)選至少90％、仍更優(yōu)選至少95％、仍然更優(yōu)選至少99％同一。序列比較是使用Smith-Waterman序列比對算法(參閱如Waterman MS，《Introduction to Computational BiologyMaps，sequences and genomes》(計算生物學(xué)入門圖譜、序列、和基因組)，Chapman & Hall，倫敦，1995，ISBN 0-412-99391-0；或http//www-hto.usc.edu/software/seqaln/index.html)進(jìn)行的。使用局部S程序1.16版，參數(shù)如下匹配＝1，錯配罰分＝0.33，缺口開放罰分(open-gap penalty)＝2，缺口延伸罰分(extended-gappenalty)＝2。
與參考核苷酸序列“基本相似”的核酸分子與參考核苷酸序列在下列條件下發(fā)生雜交在7％十二烷基磺酸鈉(SDS)、0.5M NaPO4、1mMEDTA中于50℃雜交，在2xSSC、0.1％SDS中于50℃清洗；更理想的是在7％十二烷基磺酸鈉(SDS)、0.5M NaPO4、1mM EDTA中于50℃雜交，在1x SSC、0.1％SDS中于50℃清洗；更理想的是仍在7％十二烷基磺酸鈉(SDS)、0.5M NaPO4、1mM EDTA中于50℃雜交，在0.5x SSC、0.1％SDS中于50℃清洗；優(yōu)選在7％十二烷基磺酸鈉(SDS)、0.5M NaPO4、1mM EDTA中于50℃雜交，在0.1x SSC、0.1％SDS中于50℃清洗；更優(yōu)選在7％十二烷基磺酸鈉(SDS)、0.5M NaPO4、1mM EDTA中于50℃雜交，在0.1x SSC、0.1％SDS中于65℃清洗。在上述條件下發(fā)生雜交的本發(fā)明多核苷酸優(yōu)選包含至少80堿基對、更優(yōu)選至少50堿基對、特別是至少21堿基對、和更特別是18堿基對。
這些遺傳操作的技術(shù)對于不同的可利用宿主是特異的，在本領(lǐng)域是知道的。例如，表達(dá)載體pKK223可用于在大腸桿菌中以轉(zhuǎn)錄或翻譯融合的方式在tac啟動子之后表達(dá)異源基因。對于編碼多個開放讀碼框(以下稱為“ORF”)的操縱子的表達(dá)，最簡單的流程是以轉(zhuǎn)錄融合的方式將操縱子插入載體諸如pKK233，從而使得使用異源基因的同源核糖體結(jié)合位點。在革蘭氏陽性物種諸如芽孢桿菌屬(Bacillus)中過度表達(dá)的技術(shù)在本領(lǐng)域是知道的，而且可用于本發(fā)明的內(nèi)容(Quax等人，在Baltz等人編的《Industrial MicroorganismsBasic andApplied Molecular Genetics》(工業(yè)微生物分子遺傳學(xué)基礎(chǔ)和應(yīng)用)一書中，Ameri can Society for Microbiology(美國微生物學(xué)學(xué)會)，華盛頓，1993)。過度表達(dá)的候選系統(tǒng)依賴酵母載體，包括畢赤酵母屬(Pichia)、酵母屬(Saccharomyces)、和克魯維酵母屬(Kluyveromyces)的使用(Sreekrishna，在Baltz、Hegeman、和Skatrud編的《Industrial MicroorganismsBasic and AppliedMolecular Genetics》(工業(yè)微生物分子遺傳學(xué)基礎(chǔ)和應(yīng)用)一書中，American Society for Microbiology(美國微生物學(xué)學(xué)會)，華盛頓，1993；Dequin和Barre，Biotechnology，12173-177，1994；van den Berg等人，Biotechnology，8135-139，1990)。
這些鹵化天然產(chǎn)物中的有些在抑制微生物生長中可能是有效的，特別是致植物病微生物?？梢杂蛇^度表達(dá)鹵化酶和/或電子轉(zhuǎn)移酶的生物體生產(chǎn)鹵化天然產(chǎn)物，適用于該目的的生物體包括革蘭氏陰性和革蘭氏陽性的細(xì)菌和酵母，以及下文將更詳細(xì)描述的植物。為了鹵化天然產(chǎn)物的生產(chǎn)目的，選擇宿主生物體的重要標(biāo)準(zhǔn)是易于操作、快速生長(即在微生物的情況下是發(fā)酵)、和缺乏對過度生成的鹵化天然產(chǎn)物的敏感性。鹵化天然產(chǎn)物的這些生產(chǎn)方法相比于鹵化天然產(chǎn)物制備中常用的化學(xué)合成技術(shù)具有顯著優(yōu)勢。本文所述方法的應(yīng)用將可提高通過發(fā)酵生產(chǎn)鹵化天然產(chǎn)物的效率和產(chǎn)量，而且將可用于在天然產(chǎn)物中先前不存在的且合成將難以實現(xiàn)的位置導(dǎo)入新的鹵素原子。
相比于化學(xué)合成的一些優(yōu)勢是生產(chǎn)成本更便宜，和合成區(qū)域?qū)Ｒ恍喳u化的優(yōu)選化合物的能力。摻入電子轉(zhuǎn)移酶可提高鹵化產(chǎn)物的效率和產(chǎn)量。另外，使用具有期望的底物和區(qū)域?qū)Ｒ恍缘奶烊话l(fā)生鹵化酶，或者使用具有新的底物和區(qū)域?qū)Ｒ恍缘母脑旌篼u化酶，可以通過向已知天然產(chǎn)物中添加鹵素來生成新的鹵化產(chǎn)物。使用化學(xué)手段將很難鹵化具有區(qū)域?qū)Ｒ恍院蛯τ丑w專一性的許多天然產(chǎn)物，例如大環(huán)內(nèi)酯、聚酮、和非核糖體肽。芳基或烷基的鹵化所需要的條件將通常引起天然產(chǎn)物的其它結(jié)構(gòu)變化。
鹵化酶還可生成對映異構(gòu)方面純的產(chǎn)物(在前手性碳鹵化的情況下)，這與有機(jī)合成通常產(chǎn)生的外消旋混合物相反。生成立體化學(xué)適當(dāng)?shù)幕衔锏哪芰τ诰哂性S多手性碳原子的分子是特別重要的。由異源宿主生成的鹵化天然產(chǎn)物可用于許多目的，包括醫(yī)學(xué)(即控制病原體和/或傳染病)和農(nóng)業(yè)應(yīng)用。
當(dāng)鹵化產(chǎn)物的生成需要超過一種酶時，可以在單一生物體中表達(dá)編碼目的鹵化產(chǎn)物生物合成的酶的核酸分子。在一個優(yōu)選的實施方案中，將所有編碼生產(chǎn)天然產(chǎn)物所需要的酶的核酸序列整合到生物體的染色體中，作為單一操縱子，并受到合適調(diào)控元件的控制。在另一優(yōu)選實施方案中，可以在具有選擇標(biāo)記的質(zhì)粒上攜帶核酸序列。另一個候選的優(yōu)選實施方案包括在兩種或更多相容質(zhì)粒上表達(dá)所需核酸序列，或者可以將所需核酸序列分配到染色體和一種或更多相容質(zhì)粒中?？梢酝ㄟ^天然產(chǎn)物生物合成核酸編碼序列的天然調(diào)控元件或者通過為了更精確的控制該途徑核酸序列的表達(dá)而選擇的啟動子來控制核酸分子的表達(dá)。任選的是，操縱子中將包含電子轉(zhuǎn)移酶核酸序列，以及編碼本發(fā)明的區(qū)域?qū)Ｒ恍喳u化酶(或鹵化酶)的核酸序列?；蛘?，可以分開表達(dá)電子轉(zhuǎn)移酶序列。
本發(fā)明用于生產(chǎn)鹵化產(chǎn)物的另一種方法包括在兩種或更多的分開生物體中分配生物合成途徑的核酸分子?？梢苑珠_培養(yǎng)生物體，將一種培養(yǎng)物生成的生物合成中間物轉(zhuǎn)移至表達(dá)生物合成途徑隨后步驟的另一種培養(yǎng)物中?；蛘撸梢怨才囵B(yǎng)生物體，根據(jù)需要，中間物由一種生物體傳遞至另一種生物體。在任何這些應(yīng)用中，每種鹵化酶都需要在相同的生物體中、在相同的亞細(xì)胞位置共表達(dá)合適的電子轉(zhuǎn)移酶。
可以通過將鹵化酶導(dǎo)入早已表達(dá)生成目的未鹵化結(jié)構(gòu)所需要的基因的生物體來生成新的鹵化產(chǎn)物。可以改造鹵化酶以具有對完成結(jié)構(gòu)中特定位點的專一性，或者它可具有對在天然生物體中隨后摻入最終結(jié)構(gòu)的結(jié)構(gòu)成份的專一性。例如，可以改造鹵化酶以專一性鹵化隨后將摻入含肽抗生素的氨基酸。于是，得到的產(chǎn)物可以在天然產(chǎn)物中未發(fā)現(xiàn)的位置擁有新的鹵素修飾。
在任何上述系統(tǒng)中，可以通過融合編碼電子轉(zhuǎn)移酶和區(qū)域?qū)Ｒ恍喳u化酶的核酸序列從而生成具有兩種功能性的融合蛋白來實現(xiàn)鹵化效率的顯著優(yōu)勢；這種融合可導(dǎo)致電子以更高效率由還原劑轉(zhuǎn)移至鹵化酶。電子轉(zhuǎn)移酶核酸序列可以融合在鹵化酶的5’或3’端。可以在融合中摻入連接短肽(接頭)的編碼序列，將電子轉(zhuǎn)移酶和鹵化酶蛋白質(zhì)結(jié)構(gòu)域的編碼序列分隔開來；接頭的長度可以在1-30個氨基酸殘基之間變化。
還可以在異源細(xì)菌和真菌宿主中表達(dá)本發(fā)明的鹵化酶和/或電子轉(zhuǎn)移酶以生成鹵化天然產(chǎn)物，目的是提高這些細(xì)菌和真菌宿主的生物防治菌株的功效。適合于抗病原體鹵化天然產(chǎn)物的異源過度表達(dá)的微生物是能夠在植物或根際定居的所有微生物。由此它們將接觸致植物病的真菌、細(xì)菌、和線蟲，引起對病原體生長的抑制。這些病原體包括革蘭氏陰性微生物諸如假單胞菌屬、腸桿菌屬(Enterobacter)、和沙雷氏菌屬(Serratia)，革蘭氏陽性微生物芽孢桿菌屬，和真菌木霉屬(Trichoderma)和粘帚霉屬(Gliocladium)。特別優(yōu)選的異源宿主是熒光假單胞菌、惡臭假單胞菌(Pseudomonas putida)、洋蔥假單胞菌、致金色假單胞菌、桔橙假單胞菌(Pseudomonasaurantiaca)、陰溝腸桿菌(Enterobacter cloacae 、粘質(zhì)沙雷氏菌(Serratia marcescens)、枯草芽孢桿菌(Bacillus subtilis)、蠟狀芽孢桿菌(Bacillus cereus)、綠色木霉(Trichoderma viride)、Trichoderma harzianum、和綠粘帚霉(Gliocladium virens)。
異源生物防治菌株中的表達(dá)需要選擇適合于在選定宿主中復(fù)制的載體和選擇合適的啟動子。用于在革蘭氏陰性和革蘭氏陽性細(xì)菌和真菌中表達(dá)的技術(shù)在本領(lǐng)域是眾所周知的，而且在本申請書的其它章節(jié)有所描述。轉(zhuǎn)基因植物中鹵化產(chǎn)物的生成在轉(zhuǎn)基因植物中表達(dá)本發(fā)明的鹵化酶和/或電子轉(zhuǎn)移酶，由此引起選定鹵化天然產(chǎn)物在轉(zhuǎn)基因植物中的生物合成。在有些情況下，本發(fā)明的鹵化天然產(chǎn)物將只需要一個生物合成步驟，即鹵化步驟，因而將要表達(dá)的唯一異源核酸分子將是包含本發(fā)明的區(qū)域?qū)Ｒ恍喳u化酶和電子轉(zhuǎn)移酶的編碼序列的異源核酸分子。在其它情況下，生成鹵化天然產(chǎn)物的生物合成途徑將包含一個或多個鹵化步驟作為其中一部分。在這種情況下，將表達(dá)多種異源核酸分子。
在用于本申請書時，“植物”指處于任何發(fā)育階段的任何植物或植物部分。還包括插條、細(xì)胞或組織培養(yǎng)物、和種子。在與本發(fā)明結(jié)合使用時，術(shù)語“植物組織”包括但不限于完整植株、植物細(xì)胞、植物器官、植物種子、原生質(zhì)體、愈傷組織、細(xì)胞培養(yǎng)物、和組織成結(jié)構(gòu)和/或功能單位的任何植物細(xì)胞群。當(dāng)鹵化天然產(chǎn)物具有抗病原體特性時，則生成了對致植物病的真菌和細(xì)胞的抵抗力增強(qiáng)的轉(zhuǎn)基因植物。為了它們在轉(zhuǎn)基因植物中的表達(dá)，編碼本發(fā)明鹵化酶和/或電子轉(zhuǎn)移酶的核酸分子和鄰近序列可能需要修飾和優(yōu)化。
雖然在許多情況下可以在植物中高水平表達(dá)來自其它生物體的核酸分子而無需修飾，但是轉(zhuǎn)基因植物中的低表達(dá)可能是由于核酸分子具有植物不優(yōu)選的密碼子。本領(lǐng)域知道，所有生物體對密碼子使用具有特異的偏愛，而且可以改變來自其它生物體的密碼子以符合植物的偏愛，同時維持所編碼的氨基酸。此外，植物中的高表達(dá)由具有至少35％、更優(yōu)選超過45％GC含量的編碼序列得到最好的實現(xiàn)。由于存在可使信息不穩(wěn)定的ATTTA基序和可引起不適當(dāng)聚腺苷酸化的AATAAA基序，具有低GC含量的微生物基因在植物中的表達(dá)可能較差。另外，可以對編碼本發(fā)明鹵化酶或電子轉(zhuǎn)移酶的核酸分子篩選可引起mRNA截短的不正常剪接位點的存在情況。可以使用眾所周知的定點誘變、PCR、和合成基因構(gòu)建技術(shù)，使用已發(fā)表的專利申請書EP 0 385 962、EP 0 359472、和WO 93/07278中所述方法，進(jìn)行需要在編碼序列中產(chǎn)生的所有改變(諸如上文所述)。本發(fā)明的優(yōu)選核酸分子可以是未修飾的，只要它們在目標(biāo)轉(zhuǎn)基因植物物種中高水平表達(dá)即可；或者可以是經(jīng)修飾的核酸分子，其中通過除去不穩(wěn)定和不適當(dāng)聚腺苷酸化基序以及不正常剪接位點而進(jìn)行了修飾，通過摻入植物優(yōu)選密碼子而得到進(jìn)一步修飾，而且具有在植物中表達(dá)的優(yōu)選GC含量。雖然優(yōu)選核酸序列可以在單子葉植物和雙子葉植物物種中充分表達(dá)，但是可以修飾序列以解決單子葉植物或雙子葉植物特異的密碼子偏愛和GC含量偏愛，因為這些偏愛顯示是不同的(Murray等人，Nucl Acid Res，17477-498，1989)。
為了有效起始翻譯，可能需要修飾起始甲硫氨酸的鄰近序列。與選定核酸分子同源的序列可以在植物中有效起始翻譯，或者可能是低效的。在它們低效起始的情況下，可以通過包含已知在植物中有效的序列來修飾它們。Joshi建議了用于植物的適當(dāng)共有翻譯起始子(NAR，156643-6653，1987；SEQ ID NO15)，而Clontech建議了另一種共有翻譯起始子(1993/1994產(chǎn)品目錄，第210頁；SEQ ID NO16)。這些共有序列適用于本發(fā)明的核酸分子。將序列摻入核酸分子構(gòu)建物，可直至并包含ATG(同時保持選定核酸分子的第二個氨基酸未修飾)，或者直至并包含ATG后面的GTC(有可能修飾轉(zhuǎn)基因的第二個氨基酸)。
編碼本發(fā)明鹵化酶或電子轉(zhuǎn)移酶的核酸分子在轉(zhuǎn)基因植物中的表達(dá)位于顯示在植物中有功能的啟動子之后。啟動子的選擇將根據(jù)表達(dá)的時空要求和目標(biāo)物種而變化。當(dāng)鹵化天然產(chǎn)物是抗病原體的而且希望保護(hù)植物免于葉病原體的危害時，優(yōu)選在葉中的表達(dá)；為了保護(hù)植物免于穗病原體的危害，優(yōu)選在花序(如穗狀花序、圓錐花序、穗軸等)中的表達(dá)；為了保護(hù)植物免于根病原體的危害，優(yōu)選在根中的表達(dá)；為了保護(hù)幼苗免于土傳病原體的危害，優(yōu)選在根和/或幼苗中的表達(dá)。然而，在許多情況下，將尋找針對超過一種類型的植物病原體的表達(dá)，因而將希望在多種組織中的表達(dá)。雖然來自雙子葉植物的許多啟動子顯示在單子葉植物中可運作且反之亦然，但是理想的是選擇雙子葉啟動子用于雙子葉植物中的表達(dá)，單子葉啟動子用于單子葉植物中的表達(dá)。但是，對于選定啟動子的出處沒有限制，只要它們在驅(qū)動本發(fā)明核酸分子的表達(dá)中可運作就足夠了。組成性表達(dá)的優(yōu)選啟動子包括CaMV 35S和19S啟動子，和來自編碼肌動蛋白或泛素的基因的啟動子。
還可以在化學(xué)調(diào)控的啟動子的調(diào)控下表達(dá)本發(fā)明的核酸分子。這使得能夠只在用誘導(dǎo)性化學(xué)藥品處理農(nóng)作物植物時合成鹵化天然產(chǎn)物，而且鹵化天然產(chǎn)物生物合成隨后下降。用于化學(xué)誘導(dǎo)基因表達(dá)的優(yōu)選技術(shù)詳述于已發(fā)表的申請EP 0 332 104和美國專利5,614,395(收入本文作為參考)。用于化學(xué)誘導(dǎo)的優(yōu)選啟動子是煙草PR-1a啟動子。
啟動子的優(yōu)選種類是創(chuàng)傷誘導(dǎo)型。已經(jīng)描述了在創(chuàng)傷位點以及致植物病原體感染位點表達(dá)的許多啟動子。理想的是，這種啟動子應(yīng)當(dāng)只在感染位點局部有活性，這樣，抗病原體的鹵化天然產(chǎn)物只在需要合成它以抑制侵入病原體生長的細(xì)胞中積累。這類優(yōu)選啟動子包括由Stanford等人，Mol Gen Genet，215200-208，1989；Xu等人，PlantMolec Biol，22573-588，1993；Logemann等人，Plant Cell，1151-158，1989；Rohrmeier和Lehle，Plant Molec Biol，22783-792，1993；Firek等人，Plant Molec Biol，22129-142，1993；和Warner等人，Plant J，3191-201，1993描述的啟動子。
優(yōu)選的組織特異性表達(dá)模式包括綠色組織特異性、根特異性、莖特異性、和花特異性。適用于在綠色組織中表達(dá)的啟動子包括調(diào)控涉及光合作用的基因的許多啟動子，而且這些啟動子中的許多已經(jīng)由單子葉植物和雙子葉植物二者進(jìn)行了克隆。一種優(yōu)選啟動子是來自磷酸烯醇丙酮酸羧化酶基因的玉米PEPC啟動子(Hudspeth和Crula，PlantMolec Biol，125779-589，1989)。de Framond描述了用于根特異性表達(dá)的一種優(yōu)選啟動子(FEBS，290103-106，1991；EP 0 452269，1479)，而另一種優(yōu)選的根特異性啟動子是由本發(fā)明提供的來自T-1基因的啟動子。一種優(yōu)選的莖特異性啟動子描述于專利申請WO93/07278，它驅(qū)動玉米trpA基因的表達(dá)。
本發(fā)明的一個優(yōu)選實施方案是以根特異性方式生成鹵化天然產(chǎn)物硝吡咯菌素的轉(zhuǎn)基因植物。在本發(fā)明的一個尤其優(yōu)選的實施方案中，在根特異性啟動子之后表達(dá)硝吡咯菌素的生物合成基因，以保護(hù)轉(zhuǎn)基因植物免于植物病原體絲核菌(Rhizoctonia)的危害。其它優(yōu)選實施方案是以創(chuàng)傷可誘導(dǎo)方式或病原體感染可誘導(dǎo)方式生成抗病原體的鹵化天然產(chǎn)物的轉(zhuǎn)基因植物。
除了合適啟動子的選擇，用于在植物中生成鹵化天然產(chǎn)物的構(gòu)建物需要適當(dāng)?shù)霓D(zhuǎn)錄終止子附著于異源鹵化酶和/或電子轉(zhuǎn)移酶核酸分子的下游?？梢垣@得這樣的幾種終止子，而且在本領(lǐng)域是知道的(如來自CaMV的tml、來自rbcS的E9)。已知在植物中發(fā)揮功能的任何可利用終止子都可用于本發(fā)明的內(nèi)容。
可以將許多其它序列摻入鹵化酶和/或電子轉(zhuǎn)移酶核酸分子的表達(dá)盒。這些序列包括顯示增強(qiáng)表達(dá)的序列，諸如內(nèi)含子序列(如來自Adh1和bronzel)和病毒前導(dǎo)序列(如來自TMV、MCMV、和AMV)。
鹵化天然產(chǎn)物在植物中的生成要求編碼該途徑第一步的鹵化天然產(chǎn)物生物合成核酸分子能接觸該途徑底物。對于所涉及的每種鹵化天然產(chǎn)物和途徑，這種底物將有可能是不同的，而且它在植物中的細(xì)胞定位也可能是不同的。在許多情況下，底物可位于細(xì)胞溶膠中；而在其它情況下，它可位于一些亞細(xì)胞細(xì)胞器中。由于植物中的許多生物合成活性發(fā)生于葉綠體中，因此底物常常可能位于葉綠體中，因而本發(fā)明的鹵化酶和電子轉(zhuǎn)移酶最好靶向適當(dāng)?shù)募?xì)胞器(如葉綠體)?？梢允褂帽绢I(lǐng)域眾所周知的技術(shù)來保證編碼酶的轉(zhuǎn)基因的亞細(xì)胞定位。通常，操作來自已知細(xì)胞器靶向基因產(chǎn)物的靶向肽的編碼DNA，并融合在所需鹵化酶和電子轉(zhuǎn)移酶核酸分子的上游。已知許多可用于葉綠體的這種靶向序列，而且已顯示了它們在異源構(gòu)建物中發(fā)揮功能。在本發(fā)明的一個優(yōu)選實施方案中，將硝吡咯菌素生物合成所需要的核酸分子靶向葉綠體，因為該途徑底物色氨酸是在葉綠體中合成的。
在有些情形中，生成鹵化天然產(chǎn)物所需要的核酸的過度表達(dá)可耗盡特定途徑底物的細(xì)胞利用度，而這對細(xì)胞可能具有有害影響。在諸如這種情形中，希望通過編碼底物生物合成的酶的核酸分子的過度表達(dá)來增加可利用的底物量。在色氨酸(硝吡咯菌素生物合成的底物)的情況下，這可以通過trpA和trpB編碼核酸分子的過度表達(dá)來實現(xiàn)。生成更多可利用底物的另一種方法是關(guān)閉利用特定底物的已知途徑(條件是可以這樣做而沒有有害副作用)。這樣，合成的底物被引導(dǎo)至鹵化天然產(chǎn)物的生物合成而不通向其它化合物。
適用于植物轉(zhuǎn)化的載體描述于本說明書的其它地方。對于由農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化，二元載體或攜帶至少一個T-DNA邊界序列的載體是合適的；對于直接轉(zhuǎn)移，任何載體都是合適的，而只包含目的構(gòu)建物的線性DNA可能是優(yōu)選的。在直接轉(zhuǎn)移的情況下，可以使用單一DNA種類的轉(zhuǎn)化或共轉(zhuǎn)化(Schocher等人，Biotechnology，41093-1096，1986)。對于直接轉(zhuǎn)移和由農(nóng)桿菌介導(dǎo)的轉(zhuǎn)移二者，通常(但非必需)通過可提供抗生素(卡那霉素、潮霉素、或氨甲蝶呤)或除草劑(草銨膦)抗性的選擇標(biāo)記來進(jìn)行轉(zhuǎn)化。然而，選擇標(biāo)記的選擇對于本發(fā)明而言不是決定性的。
轉(zhuǎn)基因植物中鹵化天然產(chǎn)物的合成將頻繁需要編碼鹵化天然產(chǎn)物生物合成酶的多種核酸分子的同時過度表達(dá)。這可以如下實現(xiàn)，將單個鹵化天然產(chǎn)物生物合成核酸分子各個分別轉(zhuǎn)化到不同植物品系中，然后將得到的品系雜交。若各種轉(zhuǎn)化構(gòu)建物利用不同的選擇標(biāo)記，則將有益于攜帶多種核酸序列的品系的選擇和維持。其中需要的所有鹵化天然產(chǎn)物生物合成核酸分子漸增的品系將合成鹵化天然產(chǎn)物，而其它品系則不能。這種方法可能適用于雜交農(nóng)作物(諸如玉米)，其中最終雜種必須是兩種親本間的雜交。具有不同異源核酸分子的不同近交品系的維持在其中特定鹵化天然產(chǎn)物途徑可產(chǎn)生多種鹵化天然產(chǎn)物且每一種都具有效用的情形中也可能是有利的。通過將攜帶途徑中較后步驟的不同候選核酸序列的不同品系用于與攜帶所有剩余所需核酸分子的品系生成雜種，有可能生成攜帶可能具有不同效用的不同選定鹵化天然產(chǎn)物的不同雜種。
生成攜帶多種核酸序列的植物品系的候選方法包括用鹵化天然產(chǎn)物生物合成核酸分子再次轉(zhuǎn)化早已轉(zhuǎn)化的現(xiàn)有品系(并用不同標(biāo)記進(jìn)行選擇)，以及攜帶多種生物合成核酸分子(每一種都處于適當(dāng)調(diào)控控制下，即啟動子、終止子等)的單一轉(zhuǎn)化載體的使用?？紤]到DNA構(gòu)建的容易，操作克隆載體以攜帶多種生物合成核酸分子是優(yōu)選方法。
另一種優(yōu)選方法是構(gòu)建本發(fā)明鹵化酶與本發(fā)明電子轉(zhuǎn)移酶的上述融合蛋白，并在本發(fā)明的轉(zhuǎn)基因植物中表達(dá)編碼這種融合蛋白的核酸。編碼電子轉(zhuǎn)移酶的核酸分子可以融合在鹵化酶編碼核酸分子的5’或3’端。任選的是，可以將接頭摻入融合體，將電子轉(zhuǎn)移酶和鹵化酶蛋白質(zhì)結(jié)構(gòu)域分隔開來。在一個優(yōu)選的實施方案中，融合蛋白包含由(G1y)6組成的接頭。然而，本領(lǐng)域技術(shù)人員將認(rèn)識到，也可以選擇其它合適長度和/或組成的接頭。
在另一個優(yōu)選實施方案中，可以通過直接質(zhì)體轉(zhuǎn)化來實現(xiàn)鹵化天然產(chǎn)物在植物中的生成。質(zhì)體表達(dá)(其中通過同源重組將基因插入每個植物細(xì)胞中存在的幾千個拷貝的環(huán)狀質(zhì)體基因組中)利用了龐大拷貝數(shù)相對于核表達(dá)基因的優(yōu)勢，從而使表達(dá)水平可容易的超過總可溶性植物蛋白的10％。在一個優(yōu)選的實施方案中，將核苷酸序列插入質(zhì)體靶向載體，并轉(zhuǎn)化到期望植物宿主的質(zhì)體基因組中。獲取對含核苷酸序列的質(zhì)體基因組而言同質(zhì)且優(yōu)選能夠高度表達(dá)核苷酸序列的植物。
質(zhì)體轉(zhuǎn)化技術(shù)廣泛的描述于例如美國專利號5,451,513、5,545,817、5,545,818、和5,877,462、PCT發(fā)表號WO95/16783和WO97/32977、和McBride等人，Proc Natl Acad Sci USA，917301-7305，1994(都完整收入本文作為參考)。質(zhì)體轉(zhuǎn)化的基本技術(shù)包括將所克隆質(zhì)體DNA中選擇標(biāo)記側(cè)翼的區(qū)域與核苷酸序列一起導(dǎo)入合適的靶組織，如使用biolistics或原生質(zhì)體轉(zhuǎn)化(如由氯化鈣或PEG介導(dǎo)的轉(zhuǎn)化)。1-1.5kb側(cè)翼區(qū)(稱為打靶序列)有助于與質(zhì)體基因組的同源重組，由此允許質(zhì)體基因組中特定區(qū)域的取代或修飾。首先，利用賦予壯觀霉素和/或鏈霉素的葉綠體16S rRNA和rps12基因中的點突變作為轉(zhuǎn)化的選擇標(biāo)記(SvabZ、Hajdukiewicz P、和Maliga P，ProcNatl Acad Sci USA，878526-8530，1990；Staub JM和Maliga P，Plant Cell，439-45，1992)。這些標(biāo)記間克隆位點的存在允許生成用于導(dǎo)入外源基因的質(zhì)體打靶載體(Staub JM和Maliga P，EMBO J，12601-606，1993)。通過用顯性選擇標(biāo)記編碼壯觀霉素解毒酶氨基糖苷-3’-腺苷?；D(zhuǎn)移酶的細(xì)菌aadA基因取代隱性rRNA或r-蛋白質(zhì)抗生素抗性基因而獲得了轉(zhuǎn)化頻率的顯著升高(Staub JM和Maliga P，Proc Natl Acad Sci USA，90913-917，1993)?？捎糜谫|(zhì)體轉(zhuǎn)化的其它選擇標(biāo)記在本領(lǐng)域是知道的，而且屬于本發(fā)明的范圍之內(nèi)。
在本發(fā)明的一個特別優(yōu)選的實施方案中，硝吡咯菌素的可誘導(dǎo)質(zhì)體生成是通過fre、prnA、prnB、prnC、和prnD作為噬菌體T7啟動子控制下的操縱子的直接葉綠體轉(zhuǎn)化而實現(xiàn)的?？烧T導(dǎo)表達(dá)是通過與擁有編碼T7RNA聚合酶、改造后擁有葉綠體運輸肽、且在PR1啟動子控制下允許BTH可誘導(dǎo)表達(dá)的核構(gòu)建物的植物雜交而實現(xiàn)的。
通過本發(fā)明方法生產(chǎn)鹵化天然產(chǎn)物，可于廣泛的植物細(xì)胞中進(jìn)行，包括裸子植物、單子葉植物、和雙子葉植物的細(xì)胞。雖然可以將基因插入屬于這些廣泛種類的任何植物細(xì)胞，但是在(但不限于)下列農(nóng)作物植物細(xì)胞中特別有用諸如稻、小麥、大麥、黑麥、玉米、馬鈴薯、胡蘿卜、甘薯、甜菜、菜豆、豌豆、菊苣、萵苣、甘藍(lán)、花椰菜、嫩莖花椰菜、蕪菁、蘿卜、菠菜、蘆筍、洋蔥、大蒜、茄子、胡椒、芹菜、胡蘿卜、南瓜、西葫蘆、夏南瓜、黃瓜、蘋果、梨、榅桲、甜瓜、李、櫻桃、桃、油桃、杏、草莓、葡萄、懸鉤子、黑莓、鳳梨、鱷梨、番木瓜、芒果、香蕉、大豆、煙草、番茄、高粱、和甘蔗。
在通過在農(nóng)作物植物或可再生農(nóng)作物植物的植物細(xì)胞培養(yǎng)物中進(jìn)行直接選擇獲得了本發(fā)明的區(qū)域?qū)Ｒ坏柠u化酶和/或電子轉(zhuǎn)移酶的等位基因之后，使用傳統(tǒng)育種技術(shù)將它轉(zhuǎn)移到商品化品種中，而無需遺傳工程改造等位基因并將其轉(zhuǎn)化到植物中。
實施例下列實施例作為本發(fā)明的進(jìn)一步描述和用于實踐本發(fā)明的方法。它們并非意欲限制而是提供如何實踐本發(fā)明的方針。實施例1PrnA的體外鹵化反應(yīng)A.大腸桿菌黃素還原酶、P2、曲霉硝酸還原酶、和細(xì)胞色素b5還原酶對PrnA的激活通過離子交換層析由包含質(zhì)粒pPEH14(prnA)(Kirner S等人，JBacteriol，180(7)1939-1943，1998年4月)的熒光假單胞菌BL915ΔORF1-4純化PrnA。在沒有加入如上文發(fā)明背景中所述制備的P2時，純化的酶具有的活性可忽略。蛋白質(zhì)濃度或制劑是0.36mmg/ml。
配制測定混合液，包含HEPES緩沖液pH7.5(50mM)、葡萄糖-6-磷酸(14.3mM)、D-Trp(7mM)、NaCl(7mM)。黑曲霉(Aspergillusniger)過氧化氫酶購自Sigma Chemical公司(13U/ml)，牛紅血球超氧化物歧化酶(以下稱為“SOD”)購自Sigma Chemical公司(5U/ml)，腸膜明串珠菌(Leuconostoc mesenteroides)葡萄糖-6-磷酸脫氫酶購自Sigma(5U/ml)，F(xiàn)AD(7μmolar)。如下文所示使用依賴NADH的混合液或NADPH混合液。依賴NADH的測定混合液是通過向4.5ml上述測定混合液中加入12mg NADH而配制的。依賴NADPH的測定混合液是通過向1ml上述測定混合液中加入3mg NADPH而配制的。
在聚丙烯管中建立下文所述反應(yīng)1-7，平行3份。將PrnA、所示測定混合液、與電子轉(zhuǎn)移酶混勻后，將樣品旋渦震動，然后通過室溫倒置而混勻。反應(yīng)開始20.5小時后通過煮沸2分鐘終止反應(yīng)，然后通過Microcon 10濾膜的超濾(14000xg，30分鐘)來制備用于HPLC分析的樣品。HPLC分析使用Method Set PrnAl(下文所述)，注射體積是50μl，收集前6分鐘的數(shù)據(jù)。
通過混合5或10μl7-Cl-Trp(1mM)與足夠的50mM HEPES pH7.5至終體積200μl來配制標(biāo)準(zhǔn)物。正如真正的D-Trp和7-Cl-Trp的洗脫所示，D-Trp洗脫于約2分鐘，7-Cl-Trp洗脫于4.3分鐘。通過與標(biāo)準(zhǔn)曲線的比較來測定7-Cl-Trp的量。報告活性是加入電子轉(zhuǎn)移酶之后的7-Cl-Trp凈增長。B.PrnA的HPLC分析法7-Cl-Trp的測定使用裝備光電二極管陣列檢測器的Waters Alliance HPLC系統(tǒng)。Waters Alliance HPLC配備填充了粒度3微米的C18硅膠的4.6×50mm柱。使用本文稱為PrnAl的梯度洗脫方法。流速是1ml/min，收集210-400nm的吸光度數(shù)據(jù)，分辨率為1.2nm，取樣速率1/s。將系統(tǒng)用85∶15的水∶甲醇混合液預(yù)先平衡。注入樣品后，用6分鐘的梯度(由起始條件至40∶60的水∶甲醇混合液)展開柱。然后在6.0-7.0分鐘以線性梯度將甲醇濃度提高至100％。將柱用100％甲醇清洗1分鐘，然后再次平衡。正如真正的D-Trp和7-Cl-Trp的洗脫所示，D-Trp洗脫于約2分鐘，7-Cl-Trp洗脫于4.3分鐘。
1.大腸桿菌黃素還原酶對PrnA的激活以基于Fieschi等人，J Biol Chem，270303921-30400，1995(完整收入本文作為參考)的方案的方法，通過硫酸銨沉淀隨后通過疏水層析，純化大腸桿菌黃素還原酶(以下縮寫為Fre)。黃素還原酶純化在細(xì)菌勻漿和硫酸銨分級分離過程中遵循Fieschi的流程。此時沉淀黃素還原酶活性。通過離心收集沉淀，重懸于25mM Tris/Cl pH7.5、0.5M KCl、10％甘油。然后遵循Fontcave等人，J Biol Chem，262(25)12325-12331，1987年9月5日(完整收入本文作為參考)的方法直至完成。收集的純化Fre樣品的蛋白質(zhì)濃度是21μg/ml。每個反應(yīng)含20μl PrnA、160μl上述NADH混合液、和20μl Fre。得到的凈產(chǎn)物形成是21.46±1.02nmol7-Cl-Trp。
2.P2對PrnA的激活
P2是來自熒光假單胞菌的電子轉(zhuǎn)移酶蛋白質(zhì)制劑，通過離子交換層析純化，且描述于上文發(fā)明背景。它不具有PrnA活性。P2樣品的蛋白質(zhì)濃度是4.8mg/ml。每個反應(yīng)含20μl PrnA、160μl NADH混合液、和20μl P2。得到的凈產(chǎn)物形成是12.50±2.02nmol 7-Cl-Trp。
3.菠菜硝酸還原酶對PrnA的激活菠菜硝酸還原酶(以下稱為“SNIR”)的重組FAD結(jié)構(gòu)域(18.6μmolar)。每個反應(yīng)含20μl PrnA、160μl NADH混合液、和20μl SNIR。得到的凈產(chǎn)物形成是0.048±0.73nmol 7-Cl-Trp。
4.曲霉硝酸還原酶對PrnA的激活來自曲霉的硝酸還原酶(10U/ml)購自ICN。每個反應(yīng)含20μlPrnA、160μl NADH混合液、和20μl硝酸還原酶。得到的凈產(chǎn)物形成是1.49±0.18nmol 7-Cl-Trp。
5.大鼠NADH-細(xì)胞色素-b5還原酶對PrnA的激活獲得了大鼠肝細(xì)胞色素b5還原酶的重組可溶性結(jié)構(gòu)域(11.7μmolar)。每個反應(yīng)含20μl PrnA、160μl NADH混合液、和20μl細(xì)胞色素b5還原酶。凈產(chǎn)物形成是0.31±0.11nmol 7-Cl-Trp。
6.心肌黃酶巰基還原酶對PrnA的激活心肌黃酶巰基還原酶(200U/ml)購自United StatesBiochemicals。每個反應(yīng)含20μl PrnA、160μl NADH混合液、和20μl心肌黃酶。凈產(chǎn)物形成是2.24±0.04nmol 7-Cl-Trp。
7.兔NADPH-cyt-P450還原酶對PrnA的激活兔肝NADPH-cyt-P450還原酶(0.069mg/ml)購自Sigma Chemical公司。每個反應(yīng)含20μl PrnA、160μl NADPH混合液、和20μl細(xì)胞色素P450還原酶。得到的凈產(chǎn)物形成是3.35±0.23nmol 7-Cl-Trp。實施例2大腸桿菌黃素還原酶；菠菜鐵氧還蛋白NADP還原酶、菠菜鐵氧還蛋白還原酶+菠菜鐵氧還蛋白；和費氏發(fā)光桿菌NAD(P)HFMN還原酶對PrnA的激活下文實例1-4中使用了下列成份PrnA(如上文實施例1所述)(0.36mg/ml)，包含HEPES(100mM)、葡萄糖-6-磷酸、二鈉鹽(50mM)、D-Trp(5mM)、NaCl(5mM)的測定混合液，黑曲霉過氧化氫酶(39U/ml)、牛紅血球超氧化物歧化酶(15U/ml)、腸膜明串珠菌葡萄糖-6-磷酸脫氫酶(10U/ml)、NADH(3mg/ml)、NADPH(3mg/ml)。
每次測定包含測定混合液、NADH(用于包含F(xiàn)re和NAD(P)HFMN還原酶的樣品)或NADPH(用于包含F(xiàn)NR或FNR和Fd的樣品)、PrnA、和指定電子轉(zhuǎn)移酶。將陰性對照樣品平行保溫；它們?nèi)〈鶳rnA的緩沖液。通過在100μl測定混合液、50μl NADH、20μl PrnA、和50μl緩沖液中稀釋0、1、2、或5μl 7-Cl-Trp標(biāo)準(zhǔn)品(1mM)配制定量標(biāo)準(zhǔn)物；在添加PrnA之前將管加熱至100℃，然后繼續(xù)加熱2分鐘。其它處理與酶促反應(yīng)平行。將所有樣品于室溫混合2小時。終止反應(yīng)并如上文實施例1中所述處理樣品，包括使用實施例1中所述的HPLC分析方法PrnAl。1.與Fre一起時的PrnA活性如上所述混合100μl測定混合液、50μlNADH、20μl PrnA、和50μl Fre(0.84μg/ml)。生成的凈7-Cl-Trp是8.44nmol。
2.與鐵氧還蛋白NADP還原酶一起時的PrnA活性如上所述混合100μl測定混合液、50μl NADH、20μl PrnA、和50μl FNR(4.1μmol)。生成的凈7-Cl-Trp是4.22nmol。
3.與鐵氧還蛋白NADP還原酶和鐵氧還蛋白一起時的PrnA活性如上所述混合100μl測定混合液、50μl NADH、20μl PrnA、和50μl FNR(4.1μmol)和Fd(7μmol)。生成的凈7-Cl-Trp是9.15nmol。
4.與費氏發(fā)光桿菌NAD(P)HFMN還原酶一起時的PrnA活性如上所述混合100μl測定混合液、50μl NADH、20μl PrnA、和50μl購自Roche的NAD(P)HFMN還原酶(4U/ml)。生成的凈7-Cl-Trp是0.11nmol。實施例3PrnC的體外鹵化反應(yīng)對Fre、即鐵氧還蛋白NADP還原酶、鐵氧還蛋白、和NADPHFMN還原酶測試了激活熒光假單胞菌PrnC的能力，所述PrnC如下所述耗盡了內(nèi)源電子轉(zhuǎn)移酶(P2)。PrnC催化單脫氯氨基硝吡咯菌素(MDA)氯化而生成氨基硝吡咯菌素(APRN)。
制備下列材料用于下文所述測定法。緩沖液100mM Tris/Cl、1mMEDTA pH7.5。由如Kirner等人，1998(完整收入本文作為參考)所述表達(dá)PrnA和PrnB的熒光假單胞菌培養(yǎng)物來制備74.2mM單脫氯氨基硝吡咯菌素(MDA)。測定混合液在緩沖液中包含F(xiàn)AD(5μM)和MDA(742μM)。將NADH以6mg/ml的濃度溶于緩沖液，或者將NADPH以6mg/ml的濃度溶于緩沖液。提取物#1是在緩沖液中包含PrnC和上文實施例1中所述內(nèi)源電子轉(zhuǎn)移酶P2的粗制提取物。PrnC表達(dá)于刪除染色體prn操縱子但包含在質(zhì)粒pPEH-PrnC(Kirner等人，1998)上在tac啟動子之后編碼PrnC的核酸序列(SEQ ID NO4)的熒光假單胞菌(pPEH/prnC/134Δprn)中。在該系統(tǒng)中，tac啟動子引起PrnC的組成性表達(dá)。提取物#2，通過混合提取物#1與陰離子交換樹脂然后通過離心除去樹脂來純化提取物#1中的PrnC。為了耗盡PrnC的熒光假單胞菌P2活性，使用100mM Tris/Cl緩沖液。
如下進(jìn)行下文所述測定法將提取物#2與指定的電子轉(zhuǎn)移酶、測定混合液、和指定的NADH或NADPH混合。通過平行樣品(其中將提取物#1與測定混合液和NADH混合)測定在除去P2活性之前PrnC的天然活性。通過倒置過夜混合所有樣品，然后通過加入10μl KOH(6M)終止反應(yīng)，隨后用乙酸乙酯(1ml)萃取。將0.6ml有機(jī)可溶層轉(zhuǎn)移到另一個管中，并通過真空離心除去溶劑。將殘余物重新溶于200μl 60∶40的H2O∶CH3CN+100μl CH3CN。將樣品濾過0.2μm尼龍濾器以除去微粒物。通過下文所述PrnC_Iso方法分析樣品。分析并整合290nm吸光度層析圖。PrnC活性表述成APRN峰面積占APRN和MDA峰面積總和的比率的100倍。假定220nm的消光系數(shù)相等，那么計算得到的比率等同于MDA通過鹵化轉(zhuǎn)變成APRN的凈百分比。HPLC分析方法PrnC Iso所用HPLC設(shè)備是裝備光電二極管陣列檢測器的Waters AllianceHPLC系統(tǒng)，并配備填充了粒度3微米的C18硅石的4.6×50mm柱。HPLC方法是等度洗脫法，流速1.5ml/min，溶劑是58∶42的水∶乙腈。收集210-400nm的吸光度數(shù)據(jù)，分辨率為2.4nm，取樣速率5/s。注射前將系統(tǒng)預(yù)先平衡最少6分鐘。注射體積是50μl，數(shù)據(jù)收集時間是6分鐘，隨后是注入下一個樣品前的又一個6分鐘等度洗脫。在這種方法中，MDA洗脫于2.16分鐘，氨基硝吡咯菌素(APRN)洗脫于3.05分鐘。蛋白質(zhì)濃度蛋白質(zhì)濃度是通過BCA方法使用賣主(Pierce)描述的標(biāo)準(zhǔn)流程測定的。
1.與大腸桿菌Fre一起時的PrnC活性將50μl提取物#2與20μl大腸桿菌黃素還原酶(21μg/ml)、100μl測定混合液、和50μl NADH混合；繼續(xù)混合過夜，隨后如上所述分析樣品。觀察到的活性是51.8％的MDA轉(zhuǎn)變成APRN。
2.與菠菜鐵氧還蛋白NADP還原酶一起時的PrnC活性將50μl提取物#2與20μl菠菜鐵氧還蛋白NADP還原酶(20.7μmol)、100μl測定混合液、和50μl NADH混合；繼續(xù)混合過夜，隨后如上所述分析樣品。觀察到的活性是1.8％的MDA轉(zhuǎn)變成APRN。
3.與菠菜鐵氧還蛋白NADP還原酶和菠菜鐵氧還蛋白一起時的PrnC活性將50μl提取物#2與20μl菠菜鐵氧還蛋白NADP還原酶(20.7μmol)和菠菜鐵氧還蛋白(Fd)(35μmol)、100μl測定混合液、和50μl NADH混合；繼續(xù)混合過夜，隨后如上所述分析樣品。觀察到的活性是2.5％的MDA轉(zhuǎn)變成APRN。
4.與NADPHFMN還原酶一起時的PrnC活性將50μl提取物#2與20μl來自費氏發(fā)光桿菌的NAD(P)HFMN還原酶(10U/ml)、100μl測定混合液、和50μl NADH混合；繼續(xù)混合過夜，隨后如上所述分析樣品。觀察到的活性是4.0％的MDA轉(zhuǎn)變成APRN。
5.通過平行樣品測定除去P2活性前的PrnC天然活性將50μl提取物#1與100μl測定混合液和50μl NADH混合；繼續(xù)混合過夜，隨后如上所述分析樣品。觀察到的活性是7.8％的MDA轉(zhuǎn)變成APRN。實施例4大腸桿菌中的鹵化A.編碼大腸桿菌黃素還原酶的核酸的克隆使用引物5’GCGCGAATTCATGACAACCTTAAGCTGTAAAGTGACC(SEQ IDNO32)和3’GCGCCTGCAGTCAGATAAATGCAAACGCATCGCC(SEO ID NO33)，由大腸桿菌菌株XL-1 Blue(Stratagen)PCR擴(kuò)增編碼大腸桿菌黃素還原酶的核酸序列(以下稱為“fre”)。然后將核酸分子Topo克隆(Invitrogen)，轉(zhuǎn)化到大腸桿菌XL-1 Blue(Stratagen)中，并通過在添加氨芐青霉素的Luria肉湯(LB)固體培養(yǎng)基上涂板來選擇轉(zhuǎn)化體。選擇幾個菌落，并通過DNA測序進(jìn)行分析，以確認(rèn)它們的身份。在這些菌落中，發(fā)現(xiàn)有一個擁有的核酸分子包含與報導(dǎo)的fre(Genbank編號23486)相同的序列。另一個擁有的核酸序列在第247位核苷酸處包含突變，導(dǎo)致一個帶電荷氨基酸替代Lys83→Glu83(該突變體以下稱為freE83)。B.fre和freE83突變體的可誘導(dǎo)過度表達(dá)fre和frE83突變體的可誘導(dǎo)過度表達(dá)是如下實現(xiàn)的，將野生型fre和替代突變體freE83克隆到pKK223-3(Pharmacia)的EcoRl/Pst1位點中，置于tac啟動子的控制之下。轉(zhuǎn)化后，將包含fre-pKK223-3、freE83-pKK223-3、和空載體pKK223-3的細(xì)胞在6ml LB+amp中于37℃培養(yǎng)過夜，然后在30ml LB+amp 5mM IPTG(Fisher)中稀釋達(dá)5小時，并通過離心進(jìn)行收獲。將細(xì)菌沉淀懸浮于4.5ml 50mM HEPES pH7.5、1mM EDTA+0.5ml 5mg/ml溶菌酶于25℃達(dá)15分鐘，進(jìn)行兩輪凍融。在冰上超聲處理1分鐘后，將勻漿物以16000xg離心20分鐘。然后將上清液用50mM HEPES pH7.5、1mM EDTA系列稀釋，產(chǎn)生相對濃度范圍1-1/10000的8份樣品。
對每份細(xì)菌提取物和稀釋后的細(xì)菌提取物如下測定PrnA活性的互補(bǔ)向180μl由7.2μg PrnA(0.36μg/μl)、3.3μmol FAD、3.3mMNaCl、1.67mM D-Trp、0.67mg/ml NADH、和50mM HEPES pH7.5組成的溶液中加入20μl提取物。將反應(yīng)液于30℃保溫2小時。通過加熱至100℃達(dá)2分鐘來終止反應(yīng)，隨后以21000xg離心5分鐘。然后將上清液濾過10kDa截留離心超濾膜。然后使用上文實施例1關(guān)于PrnA1所述的分析方法，通過反相HPLC測定濾出液，將D-Trp向D-7-氯色氨酸的轉(zhuǎn)變量化。加入來自包含空載體pKK223-3的大腸桿菌的提取物，產(chǎn)生0.34pmol7-Cl-Trp每分鐘每微克所加入提取物中的蛋白質(zhì)。加入來自包含freE83-pKK223-3的大腸桿菌的提取物，產(chǎn)生1.14pmol 7-Cl-Trp每分鐘每微克所加入提取物中的蛋白質(zhì)。加入來自包含fre-pKK223-3的大腸桿菌的提取物，產(chǎn)生301pmol 7-Cl-Trp每分鐘每微克所加入提取物中的蛋白質(zhì)。
如下進(jìn)行黃素還原酶測定法向990μl含0.1mg/ml NADPH和9.5μmol核黃素的50mM HEPES pH7.5中加入10μl細(xì)菌提取物。若活性過高而不能觀察反應(yīng)的前20％，則將細(xì)菌提取物在50mM HEPES緩沖液中1/10稀釋，然后如上測定。然后通過分光光度法于340nm監(jiān)測NADH向NADP的轉(zhuǎn)變。加入來自包含空載體pKK223-3的大腸桿菌的提取物，具有0.055nmol黃素還原酶活性每分鐘每微克所加入提取物中的蛋白質(zhì)。加入來自包含freE83-pKK223-3的大腸桿菌的提取物，具有0.157nmol黃素還原酶活性每分鐘每微克所加入提取物中的蛋白質(zhì)。加入來自包含fre-pKK223-3的大腸桿菌的提取物，具有25.4nmol黃素還原酶活性每分鐘每微克所加入提取物中的蛋白質(zhì)。這證明黃素還原酶活性的變化與鹵化活性成比例。C.fre和Drn操縱子在大腸桿菌中的共表達(dá)將pKK223-3(Pharmacia)中的完整熒光假單胞菌硝吡咯菌素操縱子(美國專利號5,723,759中敘述的5.8X/N，上文將其收入本文作為參考)轉(zhuǎn)化到大腸桿菌中。將fre序列(包括Taq啟動子由pKK223-3轉(zhuǎn)移到包含相容復(fù)制起點p15A的pACYC184(NEB)的四環(huán)素標(biāo)記中。然后將該質(zhì)粒與5.8X/N進(jìn)行共轉(zhuǎn)化，并通過氨芐青霉素和氯霉素選擇這兩種載體的存在。還生成只包含fre的宿主菌株作為陰性對照。將每種株系在60ml培養(yǎng)基中于37℃培養(yǎng)、200rpm振搖培養(yǎng)48小時。由每種培養(yǎng)物取5ml用于質(zhì)粒分析，確認(rèn)一種或兩種質(zhì)粒的存在。取15ml培養(yǎng)物用于蛋白質(zhì)和活性分析。將剩余的40ml培養(yǎng)物用2倍體積的乙酸乙酯萃取2次。將乙酸乙酯級分在真空中濃縮至干燥，然后轉(zhuǎn)移至50μl 6∶4的H2O/CH3CN和60μl MeOH中。然后取20μl得到的溶液，通過下文所述HPLC方法Prn_BCD分析氨基硝吡咯菌素和硝吡咯菌素。D.HPLC分析方法Prn BCD MDA、APRN、和PRN的測定HPLC設(shè)備是裝備光電二極管陣列檢測器的Waters Alliance HPLC系統(tǒng)，并配備填充了粒度3微米的C18硅石的4.6×50mm柱。HPLC方法是梯度洗脫法，流速1.2ml/min。收集210-400nm的吸光度數(shù)據(jù)，分辨率為2.4nm，取樣速率5/s。將系統(tǒng)用65∶35的水∶乙腈預(yù)先平衡。注入樣品后，由起始條件至40∶60的水∶乙腈的線性梯度展開柱。氨基硝吡咯菌素洗脫于5.0分鐘，硝吡咯菌素洗脫于6.6分鐘。通過整合于診斷波長測量的層析圖中的峰面積來測量氨基硝吡咯菌素和硝吡咯菌素二者。對于氨基硝吡咯菌素，使用300nm吸光度。對于硝吡咯菌素，使用250nm吸光度。
結(jié)果顯示，與只表達(dá)硝吡咯菌素操縱子的細(xì)胞相比，在共表達(dá)包含fre和硝吡咯菌素操縱子的質(zhì)粒的大腸桿菌細(xì)胞中，氨基硝吡咯菌素積累的增加超過10倍，硝吡咯菌素積累的增加超過4倍。實施例5在轉(zhuǎn)基因植物中表達(dá)然后在體外純化并測定的PrnA的鹵化用硝吡咯菌素操縱子的4種核酸分子(編碼PrnA、PrnB、PrnC、和PrnD，每一種都處于下文實施例6中所述泛素啟動子之后)轉(zhuǎn)化(通過由農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化方法)擬南芥(Arabidopsis thaliana)哥倫比亞(Columbia)生態(tài)型。
由包含來自熒光假單胞菌BL915的粘?？寺〉膒CIB169(美國專利號5,723,759)(Genbank編號U74493)PCR擴(kuò)增各種硝吡咯菌素核酸分子(包括適當(dāng)?shù)南拗菩晕稽c)。將核酸分子亞克隆并測序。由擬南芥基因組PCR擴(kuò)增泛素3啟動子和第一個內(nèi)含子(Caiis J等人，Journalof Biological Chemistry，26512486-12493，1990和Norris SR等人，Plant Molecular Biology，21895-906，1993)以包含5’KpnI和3’BamHI位點。將泛素啟動子、nos終止子(D epicker等人，Journal of Molecular and Applied Genetics，1561-573，1982)、和每一種硝吡咯菌素核酸分子(參閱美國專利號5,723,759和5,955,348，都完整收入本文作為參考)克隆到修飾后的pSort1載體中。在PrnA、B、和D的每一種中緊挨著起始ATG的5’加入Kozak共有-3ACC核苷酸三聯(lián)體。PrnC核酸分子不被修飾。將PrnB中的起始GTG密碼子變成ATG密碼子。這些修飾產(chǎn)生一組載體pPEH7826、27、28、和29(分別是PrnA、B、C、和D)。所有其它序列與野生型序列是共有的。通過將來自pCIB7826(PrnA)的KpnI片段插入pCIB7828(PrnC)的KpnI位點生成pCIB7830而構(gòu)建了PrnAC雙聯(lián)體。通過將來自pCIB7827(PrnB)的KpnI片段插入pCIB7829(PrnD)的KpnI位點生成pCIB7831而構(gòu)建了PrnBD雙聯(lián)體。通過將來自pCIB7830的NotI片段插入pCIB7831的NotI位點生成pCIB7832而構(gòu)建了4種核酸分子操縱子。將來自pCIB7832的XbaI片段插入二元載體pCIB200生成轉(zhuǎn)化載體pCIB7819。將最終載體電穿孔到農(nóng)桿菌中并用于擬南芥轉(zhuǎn)化。
通過Bechtold N等人的方法轉(zhuǎn)化擬南芥(Bechtold N等人，C.R.Acad.Sci.Paris，Life Science，3161194-1199，1993)。
培養(yǎng)2種轉(zhuǎn)化品系(3和12)和非轉(zhuǎn)化對照品系并收獲葉片(1g)。將葉片在液氮中凍結(jié)，在研缽中磨成粉，并用6ml Ls緩沖液(50mM HEPESpH7.5、5mM NaCl)提取。以5000xg離心15分鐘以沉淀碎片后，將上清液濾過玻璃絨以除去殘余微粒。
通過混合提取物(3ml)與親和基質(zhì)來如下免疫純化PrnA。通過于室溫混合100μl兔抗山羊IgG-瓊脂糖(購自Sigma)與50μl山羊抗PrnA血清來制備親和基質(zhì)。然后將瓊脂糖珠用1ml LS緩沖液清洗3次。將3ml樣品與親和基質(zhì)混勻后，通過LS緩沖液清洗由珠除去未吸收的物質(zhì)。通過混合5μl如實施例1中所述由熒光假單胞菌純化的PrnA(0.36μg/μl)與3ml LS、然后與植物提取物樣品平行處理來制備陽性對照樣品。
向包含免疫純化的PrnA的瓊脂糖珠中加入200μl測定緩沖液(50mM HEPES pH7.5、5mM D-Trp、5mM NaCl、5μM FAD、5mM葡萄糖-6-磷酸+2mg/ml NADH+6.25U/ml葡萄糖-6-磷酸脫氫酶+44U/ml過氧化氫酶+30U/ml SOD)和20μl如實施例1中所述由大腸桿菌純化的Fre(21μg/ml)，除了品系3和12各有一個樣品不加。然后通過倒置將樣品混合過夜，濾過Microcon-10濾器，并通過HPLC方法PrnAl(描述于上文實施例1)分析產(chǎn)物。樣品的注射體積是50μl。發(fā)現(xiàn)了下列7-Cl-Trp水平陽性對照(向非轉(zhuǎn)化植物提取物中加入外源PrnA)185pmol，品系3加Fre(兩份單獨的樣品)83pmol和113pmol，品系3不加Fre 0pmol，品系12加Fre(兩份單獨的樣品)120pmol和64pmol，品系12不加Fre 0pmol，非轉(zhuǎn)化對照0pmol。
這些數(shù)據(jù)證明轉(zhuǎn)化植物表達(dá)活性形式的PrnA，其活性依賴Fre的加入。實施例6轉(zhuǎn)基因植物中的鹵化A.通過將編碼大腸桿菌黃素還原酶的核酸轉(zhuǎn)化到包含編碼PrnA、PrnB、PrnC、和PrnD的核酸的植物中而在轉(zhuǎn)基因植物中進(jìn)行鹵化化合物的胞質(zhì)生產(chǎn)將SEQ ID NO6的核酸序列(編碼來自大腸桿菌的黃素還原酶)克隆到載體pNOV019中，從而將核酸分子置于擬南芥泛素10(UB1O)啟動子(Callis J等人，Journal of Biological Chemistry，26512486-12493，1990和Norris SR等人，Plant Molecular Biology，21895-906，1993)的控制之下，并用來自農(nóng)桿菌的胭脂堿合酶終止子(Depicker等人，Journal of Molecular and Applied Genetics，1561-573，1982)終止。
嘗試了由pNOV507(KanR)、508(ChlorR)、和509(AmpR)組成的二元載體系統(tǒng)。與fre核酸分子和除草劑抗性選擇標(biāo)記一起用于構(gòu)建硝吡咯菌素操縱子的三種載體如下。pNov507(KanR)是用在啟動子、終止子、硝吡咯菌素、fre、或選擇標(biāo)記核酸分子任一中未發(fā)現(xiàn)的唯一限制性位點選擇取代左邊界與右邊界之間的多接頭后的二元載體。其它兩種載體pNOV508(ChlorR)和pNOV509(AmpR)是包含一部分pNOV507多接頭以及添加用于克隆硝吡咯菌素操縱子的分開核酸分子盒的額外限制性位點的載體。這兩種載體是構(gòu)建或裝配載體。將fre盒與來自pNOV111的UB3選擇標(biāo)記盒一起在pNOV509中連接。然后將這種雙重盒轉(zhuǎn)移到二元載體pNOV507中，產(chǎn)生最終載體pNOV510。將該載體電穿孔到農(nóng)桿菌中。通過Bechtold N等人的方法(Bechtold N等人，C.R.Acad.Sci.Paris，Life Sciences，3161194-1199，1993)轉(zhuǎn)化如實施例5中所述用PrnA、PrnB、PrnC、和PrnD核酸分子轉(zhuǎn)化的擬南芥品系。
植物和各種構(gòu)建物中的所有硝吡咯菌素途徑核酸分子都是由擬南芥泛素3(UB3)啟動子(Callis J等人，Journal of Biology Chemistry，26512486-12493，1990和Norris sR等人，Plant Molecular Biology，21895-906，1993)驅(qū)動的，并用來自農(nóng)桿菌的nos終止子終止。通過上文所述Bechtold等人的方法的農(nóng)桿菌滲透，用pNOV510轉(zhuǎn)化包含prnA、prnB、prnC、和prnD的純合擬南芥品系和野生型哥倫比亞品系。收集種子，干燥，并種到土里。通過對幼苗噴灑0.025％選擇劑3次達(dá)8天來鑒定轉(zhuǎn)化植株。然后通過HPLC或氣相層析-質(zhì)譜法對植物確認(rèn)硝吡咯菌素的存在情況和水平。同樣/或者，可如上所述對植物提取物確認(rèn)prnA和/或prnC活性。B.通過大腸桿菌黃素還原酶和硝吡咯菌素操縱子的其轉(zhuǎn)化而在轉(zhuǎn)基因植物中進(jìn)行鹵化化合物的胞質(zhì)生產(chǎn)將列于美國專利號5,723,759(上文將其收入本文作為參考)的編碼硝吡咯菌素途徑PrnA、prnB、prnC、和prnD的核酸序列，和編碼大腸桿菌黃素還原酶的SEQ ID NO7在單個t-DNA構(gòu)建物中導(dǎo)入植物。每種硝吡咯菌素生物合成核酸分子的表達(dá)都是由UB3啟動子驅(qū)動的，而fre SEQ ID NO7是由UB10驅(qū)動的。所有5種核酸分子符合或改變后精確符合Kozak翻譯起始序列，即在第-3位擁有A。所有核酸分子都是由nos終止子終止的。在一個優(yōu)選的實施方案中，最終載體是通過在包含右邊界-UB3-prnA-nos-UB3-prnC-nos-UB3-prnB-nos-UB3-prnD-nos-UB10-fre-nos-UB3-選擇標(biāo)記-nos-左邊界的二元載體中裝配UB3啟動子-胞質(zhì)溶膠靶向硝吡咯菌素生物合成基因和UB10-fre盒而構(gòu)建的。該載體稱為pNOV523(SEQ ID NO34)。
在另一個實施方案中，胞質(zhì)溶膠靶向硝吡咯菌素操縱子是通過將來自pCIB7830的NotI A/B雙聯(lián)體片段連接到C/D雙聯(lián)體載體pCIB7831中而生成的。將操縱子作為XbaI盒轉(zhuǎn)移到pNOV507中。將來自pCIB10253的NotI A/B雙聯(lián)體連接到C/D雙聯(lián)體載體pCIB10254中。同樣將該構(gòu)建物作為XbaI盒轉(zhuǎn)移到pNOV507中。
最終載體包含右邊界-UB3-prnA-nos-UB3-prnB-nos-UB3-prnC-nos-UB3-prnD-nos-UB10-fre-nos-UB3-選擇標(biāo)記-nos-左邊界。
然后將該載體電穿孔到農(nóng)桿菌中，并通過農(nóng)桿菌滲透(Bechtold N等人，C.R.Acad.Sci.Paris，Life sciences，3161194-1199，1993)轉(zhuǎn)化擬南芥(哥倫比亞品系)。收集種子，干燥，并種到土里。通過對幼苗噴灑0.025％選擇劑3次達(dá)8天來鑒定轉(zhuǎn)化植株。然后通過HPLC或氣相層析-質(zhì)譜法對植物確認(rèn)硝吡咯菌素的存在情況和水平。C.鹵化化合物在轉(zhuǎn)基因植物質(zhì)體中的生產(chǎn)改造編碼prnA和prnB的核酸構(gòu)建物以表達(dá)葉綠體運輸肽(Wong EY等人，Plant Molecular Bilogy，2081-93，1992，并一起置于允許卡那霉素選擇的載體上。轉(zhuǎn)化方案在先前的實施例中有詳述(Bechtold N等人，C.R.Acad.Sci.Paris，Life Sciences，3161194-1199，1993)。質(zhì)體靶向硝吡咯菌素核酸分子載體的構(gòu)建由pCIB10230、31、32、33(分別是PrnA、B、C、D)PCR擴(kuò)增各種硝吡咯菌素途徑核酸分子，以包含5’NheI和3’BamHI限制性位點。將核酸分子Topo克隆到pCR2.1(Invitrogen，US Office Calsbad，CA 92008，產(chǎn)品目錄編號K2030-01)中，用于序列確認(rèn)。由pFL61中的擬南芥cDNA文庫(Wong等人，Plant Mol Biol，2081-93，1992)PCR復(fù)制RuBPcase小亞基肽運輸序列。將該核酸序列連接到pPEH31、30、29、和28(分別是PrnA、B、C、和D)中每種硝吡咯菌素核酸分子的5’端。這組pPEH載體包含UB3-內(nèi)含子-nos盒。合成額外成熟肽作為互補(bǔ)寡聚物，退火，并連接到運輸肽硝吡咯菌素核酸分子構(gòu)建物的5’部分。這產(chǎn)生了質(zhì)體靶向硝吡咯菌素核酸分子載體pCIB10249、50、51、和52(分別是PrnA、B、C、和D)。通過將來自pCIB10249的含PrnA的KpnI核酸分子盒連接到pCIB10250中而構(gòu)建了PrnAB雙聯(lián)體pCIB10253。通過將來自pCIB10251的含PrnC的xhoI核酸分子盒連接而pCIB10252中而構(gòu)建了PrnCD雙聯(lián)體pCIB10254。將每種雙聯(lián)體作為XbaI盒轉(zhuǎn)移到二元載體pCIB200(KanR)中。用于質(zhì)體靶向載體的選擇標(biāo)記方案是對于fre載體為右邊界-UB10-clp-fre-nos-UB3-選擇標(biāo)記-nos-左邊界；對于PrnA/B載體為右邊界-UB3-prnA-nos-UB3-prnB-nos-UB3-選擇標(biāo)記-nos-左邊界；對于PrnC/D載體為右邊界-UB3-prnC-nos-UB3-prnD-nos-UB3-選擇標(biāo)記-nos-左邊界。
然后將質(zhì)體靶向prnAB-fre載體電穿孔到農(nóng)桿菌中，并通過上文所述Bechtold N等人的方法轉(zhuǎn)化哥倫比亞擬南芥(Arabadopsiscolumbia)。收集種子，干燥，并種到土里。通過對幼苗噴灑選擇劑來鑒定轉(zhuǎn)化植株，并自交至純合。
相似的，如上所述將質(zhì)體靶向prnCD/選擇標(biāo)記載體導(dǎo)入擬南芥，并將得到的轉(zhuǎn)化體自交至純合。
然后將包含質(zhì)體靶向prnAB-fre/選擇標(biāo)記構(gòu)建物的純合轉(zhuǎn)化植株與純合質(zhì)體靶向prnCD/選擇標(biāo)記植株雜交。在另一個實施方案中，將質(zhì)體靶向prnCD盒轉(zhuǎn)移到包含UB10-質(zhì)體靶向fre盒的二元載體中。該載體稱為pNOV524(SEQ ID N035)。然后將載體pNOV524電穿孔到擬南芥中，并通過上文所述Bechtold N等人的方法轉(zhuǎn)化哥倫比亞擬南芥。用pNOV524轉(zhuǎn)化野生型擬南芥和先前用pCIB10253(包含質(zhì)體靶向prnA/B)轉(zhuǎn)化的擬南芥。收集種子，干燥，并種到土里。通過對幼苗噴灑選擇劑來鑒定轉(zhuǎn)化植株，并自交至純合。
對得到的子代施用適當(dāng)?shù)倪x擇劑。抵抗這種選擇劑方案的植物擁有半合子狀態(tài)的fre和prnA、B、C、D。本領(lǐng)域技術(shù)人員將領(lǐng)會這種方法可能的許多變異。在所有情況下，硝吡咯菌素表達(dá)是通過HPLC或氣相層析量化的。實施例7在供給MDA的轉(zhuǎn)基因植物葉中表達(dá)的PrnC的鹵化在草銨膦選擇后，對用pNOV524構(gòu)建物(包含質(zhì)體靶向prnC、prnD、和fre)轉(zhuǎn)化的哥倫比亞品系進(jìn)行Western印跡分析。另外，在草銨膦選擇后，對用pCIB10253(包含質(zhì)體靶向prnA和prnB)轉(zhuǎn)化隨后用pNOV524轉(zhuǎn)化的擬南芥品系進(jìn)行Western印跡分析。將來自每種品系的單一葉片在1x蛋白質(zhì)樣品緩沖液中勻漿，煮沸，并通過10％SDS-PAGE分離。隨后，分別用針對prnC和prnD產(chǎn)生的抗體對膜探查prnC和prnD蛋白質(zhì)的存在情況。鑒定到了prnC和prnD表達(dá)陽性的擬南芥品系。如下對相同蛋白質(zhì)提取物再次檢驗黃素還原酶(fre)蛋白質(zhì)的存在使用10-20％梯度凝膠，隨后用針對fre的抗體對膜進(jìn)行探查。鑒定到了fre表達(dá)陽性的品系。
由質(zhì)體靶向prnC、prnD、和fre表達(dá)陽性的擬南芥品系以及prnC和prnD陰性的擬南芥品系(通過Western印跡鑒定)采集葉片。在浸沒在5mM MES pH5.7、400mM甘露醇緩沖液中時用MDA真空滲透葉片，并于室溫在黑暗中放置過夜。隨后，用乙酸乙酯萃取緩沖液，濃縮至干燥，并在HPLC上進(jìn)行分析(如上文實施例4中所述)。
來自prnC、prnD、和fre陽性植株的葉片將MDA轉(zhuǎn)變成APRN(大約5％)。在3小時的保溫時間里檢測到轉(zhuǎn)變。此外，大約30％的APRN轉(zhuǎn)變成硝吡咯菌素。另外，陰性對照即來自不表達(dá)prnC或prnD的植株的葉片不顯示MDA向APRN或硝吡咯菌素的轉(zhuǎn)變。
將上文引用的參考發(fā)表物都完整收入本文作為參考。
序列表<110>Novartis AG<120>用于鹵化反應(yīng)的組合物和方法<130>Case S-31082A<140><141><150>US 60/228801<151>1999-12-15<150>US 60/219343<151>2000-01-03<160>35<170>PatentIn Ver.2.0<210>1<211>16<212>PRT<213>人工序列<220><223>人工序列的描述氨基酸共有結(jié)構(gòu)域<400>1Xaa Ala Ala Trp Xaa Ala Ala Trp Xaa Ala Ala Ile Pro Xaa Ala Ala1 5 10 15<210>2<211>1617<212>DNA<213>熒光假單胞菌(Pseudomonas fluorescens)<220><221>CDS<222>(1)..(1617)<400>2atg aac aag ccg atc aag aat atc gtc atc gtg ggc ggc ggt act gcg48Met Asn Lys pro Ile Lys Asn Ile Val Ile Val Gly Gly Gly Thr Ala1 5 10 15ggc tgg atg gcc gcc tcg tac ctc gtc cgg gcc ctc caa cag cag gcg96Gly Trp Met Ala Ala Ser Tyr Leu Val Arg Ala Leu Gln Gln Gln Ala20 25 30aac att acg ctc atc gaa tct gcg gcg atc cct cgg atc ggc gtg ggc144Asn Ile Thr Leu Ile Glu Ser Ala Ala Ile Pro Arg Ile Gly Val Gly35 40 45gaa gcg acc atc cca agt ttg cag aag gtg ttc ttc gat ttc ctc ggg192Glu Ala Thr Ile Pro Ser Leu Gln Lys Val Phe Phe Asp Phe Leu Gly50 55 60ata ccg gag cgg gaa tgg atg ccc caa gtg aac ggc gcg ttc aag gcc240Ile Pro Glu Arg Glu Trp Met Pro Gln Val Asn Gly Ala Phe Lys Ala65 70 75 80gcg atc aag ttc gtg aat tgg aga aag tct ccc gac ccc tcg cgc gac288Ala Ile Lys Phe Val Asn Trp Arg Lys Ser Pro Asp Pro Ser Arg Asp85 90 95gat cac ttc tac cat ttg ttc ggc aac gtg ccg aac tgc gac ggc gtg336Asp His Phe Tyr His Leu Phe Gly Asn Val Pro Asn Cys Asp Gly Val100 105110ccg ctt acc cac tac tgg ctg cgc aag cgc gaa cag ggc ttc cag cag384Pro Leu Thr His Tyr Trp Leu Arg Lys Arg Glu Gln Gly Phe Gln Gln115 120 125ccg atg gag tac gcg tgc tac ccg cag ccc ggg gca ctc gac ggc aag432Pro Met Glu Tyr Ala Cys Tyr Pro Gln Pro Gly Ala Leu Asp Gly Lys130 135 140ctg gca ccg tgc ctg tcc gac ggc acc cgc cag atg tcc cac gcg tgg480Leu Ala Pro Cys Leu Ser Asp Gly Thr Arg Gln Met Ser His Ala Trp145 150 155 160cac ttc gac gcg cac ctg gtg gcc gac ttc ttg aag cgc tgg gcc gtc528His Phe Asp Ala His Leu Val Ala Asp Phe Leu Lys Arg Trp Ala Val165 170 175gag cgc ggg gtg aac cgc gtg gtc gat gag gtg gtg gac gtt cgc ctg576Glu Arg Gly Val Asn Arg Val Val Asp Glu Val Val Asp Val Arg Leu180 185 190aac aac cgc ggc tac atc tcc aac ctg ctc acc aag gag ggg cgg acg624Asn Asn Arg Gly Tyr Ile Ser Asn Leu Leu Thr Lys Glu Gly Arg Thr195 200 205ctg gag gcg gac ctg ttc atc gac tgc tcc ggc atg cgg ggg ctc ctg672Leu Glu Ala Asp Leu Phe Ile Asp Cys Ser Gly Met Arg Gly Leu Leu210 215 220atc aat cag gcg ctg aag gaa ccc ttc atc gac atg tcc gac tac ctg720Ile Asn Gln Ala Leu Lys Glu Pro Phe Ile Asp Met Ser Asp Tyr Leu225 230 235 240ctg tgc gac agc gcg gtc gcc agc gcc gtg ccc aac gac gac gcg cgc768Leu Cys Asp Ser Ala Val Ala Ser Ala Val Pro Asn Asp Asp Ala Arg245 250 255gat ggg gtc gag ccg tac acc tcc tcg atc gcc atg aac tcg gga tgg816Asp Gly Val Glu Pro Tyr Thr Ser Ser Ile Ala Met Asn Ser Gly Trp260 265 270acc tgg aag att ccg atg ctg ggc cgg ttc ggc agc ggc tac gtc ttc864Thr Trp Lys Ile Pro Met Leu Gly Arg Phe Gly Ser Gly Tyr Val Phe275 280 285tcg agc cat ttc acc tcg cgc gac cag gcc acc gcc gac ttc ctc aaa912Ser Ser His Phe Thr Ser Arg Asp Gln Ala Thr Ala Asp Phe Leu Lys290 295 300ctc tgg ggc ctc tcg gac aat cag ccg ctc aac cag atc aag ttc cgg960Leu Trp Gly Leu Ser Asp Asn Gln Pro Leu Asn Gln Ile Lys Phe Arg305 310 315 320gtc ggg cgc aac aag cgg gcg tgg gtc aac aac tgc gtc tcg atc ggg1008Val Gly Arg Asn Lys Arg Ala Trp Val Asn Asn Cys Val Ser Ile Gly325 330 335ctg tcg tcg tgc ttt ctg gag ccc ctg gaa tcg acg ggg atc tac ttc1056Leu Ser Ser Cys Phe Leu Glu Pro Leu Glu Ser Thr Gly Ile Tyr Phe340 345 350atc tac gcg gcg ctt tac cag ctc gtg aag cac ttc ccc gac acc tcg1104Ile Tyr Ala Ala Leu Tyr Gln Leu Val Lys His Phe Pro Asp Thr Ser355 360 365ttc gac ccg cgg ctg agc gac gct ttc aac gcc gag atc gtc cac atg1152Phe Asp Pro Arg Leu Ser Asp Ala Phe Asn Ala Glu Ile Val His Met370 375 380ttc gac gac tgc cgg gat ttc gtc caa gcg cac tat ttc acc acg tcg1200Phe Asp Asp Cys Arg Asp Phe Val Gln Ala His Tyr Phe Thr Thr Ser385 390 395 400cgc gat gac acg ccg ttc tgg ctc gcg aac cgg cac gac ctg cgg ctc1248Arg Asp Asp Thr Pro Phe Trp Leu Ala Asn Arg His Asp Leu Arg Leu405 410 415tcg gac gcc atc aaa gag aag gtt cag cgc tac aag gcg ggg ctg ccg1296Ser Asp Ala Ile Lys Glu Lys Val Gln Arg Tyr Lys Ala Gly Leu Pro420 425 430ctg acc acc acg tcg ttc gac gat tcc acg tac tac gag acc ttc gac1344Leu Thr Thr Thr Ser Phe Asp Asp Ser Thr Tyr Tyr Glu Thr Phe Asp435 440 445tac gaa ttc aag aat ttc tgg ttg aac ggc aac tac tac tgc atc ttt1392Tyr Glu phe Lys Asn Phe Trp Leu Asn Gly Asn Tyr Tyr Cys I1e Phe450 455 460gcc ggc ttg ggc atg ctg ccc gac cgg tcg ctg ccg ctg ttg cag cac1440Ala Gly Leu Gly Met Leu Pro Asp Arg Ser Leu Pro Leu Leu Gln His465 470 475 480cga ccg gag tcg atc gag aaa gcc gag gcg atg ttc gcc agc atc cgg1488Arg Pro Glu Ser Ile Glu Lys Ala Glu Ala Met Phe Ala Ser Ile Arg485 490 495cgc gag gcc gag cgt ctg cgc acc agc ctg ccg aca aac tac gac tac1536Arg Glu Ala Glu Arg Leu Arg Thr Ser Leu Pro Thr Asn Tyr Asp Tyr500 505 510ctg cgg tcg ctg cgt gac ggc gac gcg ggg ctg tcg cgc ggc cag cgt1584Leu Arg Ser Leu Arg Asp Gly Asp Ala Gly Leu Ser Arg Gly Gln Arg515 520 525ggg ccg aag ctc gca gcg cag gaa agc ctg tag1617Gly Pro Lys Leu Ala Ala Gln Glu Ser Leu530 535<210>3<211>538<212>PRT<213>熒光假單胞菌<400>3Met Asn Lys Pro Ile Lys Asn Ile Val Ile Val Gly Gly Gly Thr Ala1 5 10 15Gly Trp Met Ala Ala Ser Tyr Leu Val Arg Ala Leu Gln Gln Gln Ala20 25 30Asn Ile Thr Leu Ile Glu Ser Ala Ala Ile Pro Arg Ile Gly Val Gly35 40 45Glu Ala Thr Ile Pro Ser Leu Gln Lys Val Phe Phe Asp Phe Leu Gly50 55 60Ile Pro Glu Arg Glu Trp Met Pro Gln Val Asn Gly Ala Phe Lys Ala65 70 75 80Ala Ile Lys Phe Val Asn Trp Arg Lys Ser Pro Asp Pro Ser Arg Asp85 90 95Asp His Phe Tyr His Leu Phe Gly Asn Val Pro Asn Cys Asp Gly Val100 105 110Pro Leu Thr His Tyr Trp Leu Arg Lys Arg Glu Gln Gly Phe Gln Gln115 120 125Pro Met Glu Tyr Ala Cys Tyr Pro Gln Pro Gly Ala Leu Asp Gly Lys130 135 140Leu Ala Pro Cys Leu Ser Asp Gly Thr Arg Gln Met Ser His Ala Trp145 150 155 160His Phe Asp Ala His Leu Val Ala Asp Phe Leu Lys Arg Trp Als Val165 170 175Glu Arg Gly Val Asn Arg Val Val Asp Glu Val Val Asp Val Arg Leu180 185 190Asn Asn Arg Gly Tyr Ile Ser Asn Leu Leu Thr Lys Glu Gly Arg Thr195 200 205Leu Glu Ala Asp Leu Phe Ile Asp Cys Ser Gly Met Arg Gly Leu Leu210 215 220Ile Asn Gln Ala Leu Lys Glu Pro Phe Ile Asp Met Ser Asp Tyr Leu225 230 235 240Leu Cys Asp Ser Ala Val Ala Ser Ala Val Pro Asn Asp Asp Ala Arg245 250 255Asp Gly Val Glu Pro Tyr Thr Ser Ser Ile Ala Met Asn Ser Gly Trp260 265 270Thr Trp Lys Ile Pro Met Leu Gly Arg Phe Gly Ser Gly Tyr Val Phe275 280 285Ser Ser His Phe Thr Ser Arg Asp Gln Ala Thr Ala Asp Phe Leu Lys290 295 300Leu Trp Gly Leu Ser Asp Asn Gln Pro Leu Asn Gln Ile Lys Phe Arg305 310 315 320Val Gly Arg Asn Lys Arg Ala Trp Val Asn Asn Cys Val Ser Ile Gly325 330 335Leu Ser Ser Cys Phe Leu Glu Pro Leu Glu Ser Thr Gly Ile Tyr Phe340 345 350Ile Tyr Ala Ala Leu Tyr Gln Leu Val Lys His Phe Pro Asp Thr Ser355 360 365Phe Asp Pro Arg Leu Ser Asp Ala Phe Asn Ala Glu Ile Val His Met370 375 380Phe Asp Asp Cys Arg Asp Phe Val Gln Ala His Tyr Phe Thr Thr Ser385 390 395 400Arg Asp Asp Thr Pro Phe Trp Leu Ala Asn Arg His Asp Leu Arg Leu405 410 415Ser Asp Ala Ile Lys Glu Lys Val Gln Arg Tyr Lys Ala Gly Leu Pro420 425 430Leu Thr Thr Thr Ser Phe Asp Asp Ser Thr Tyr Tyr Glu Thr Phe Asp435 440 445Tyr Glu Phe Lys Asn Phe Trp Leu Asn Gly Asn Tyr Tyr Cys Ile Phe450 455 460Ala Gly Leu Gly Met Leu Pro Asp Arg Ser Leu Pro Leu Leu Gln His465 470 475 480Arg Pro Glu Ser Ile Glu Lys Ala Glu Ala Met Phe Ala Ser Ile Arg485 490 495Arg Glu Ala Glu Arg Leu Arg Thr Ser Leu Pro Thr Asn Tyr Asp Tyr500 505 510Leu Arg Ser Leu Arg Asp Gly Asp Ala Gly Leu Ser Arg Gly Gln Arg515 520 525Gly Pro Lys Leu Ala Ala Gln Glu Ser Leu530 535<210>4<21l>1704<212>DNA<213>熒光假單胞菌<220><221>CDS<222>(1)..(1704)<400>4atg act cag aag agc ccc gcg aac gaa cac gat agc aat cac ttc gac48Met Thr Gln Lys Ser Pro Ala Asn Glu His Asp Ser Asn His Phe Asp1 5 10 15gta atc atc ctc ggc tcg ggc atg tcc ggc acc cag atg ggg gcc atc96Val Ile Ile Leu Gly Ser Gly Met Ser Gly Thr Gln Met Gly Ala Ile20 25 30ttg gcc aaa caa cag ttt cgc gtg ctg atc atc gag gag tcg tcg cac144Leu Ala Lys Gln Gln Phe Arg Val Leu Ile Ile Glu Glu Ser Ser His35 40 45ccg cgg ttc acg atc ggc gaa tcg tcg atc ccc gag acg tct ctt atg192Pro Arg Phe Thr Ile Gly Glu Ser Ser Ile Pro Glu Thr Ser Leu Met50 55 60aac cgc atc atc gct gat cgc tac ggc att ccg gag ctc gac cac atc240Asn Arg Ile Ile Ala Asp Arg Tyr Gly Ile Pro Glu Leu Asp His Ile65 70 75 80acg tcg ttt tat tcg acg caa cgt tac gtc gcg tcg agc acg ggc att288Thr Ser Phe Tyr Ser Thr Gln Arg Tyr Val Ala Ser Ser Thr Gly Ile85 90 95aag cgc aac ttc ggc ttc gtg ttc cac aag ccc ggc cag gag cac gac336Lys Arg Asn Phe Gly Phe Val Phe His Lys Pro Gly Gln Glu His Asp100 105 110ccg aag gag ttc acc cag tgc gtc att ccc gag ctg ccg tgg ggg ccg384Pro Lys Glu Phe Thr Gln Cys Val Ile Pro Glu Leu Pro Trp Gly Pro115 120 125gag agc cat tat tac cgg caa gac gtc gac gcc tac ttg ttg caa gcc432Glu Ser His Tyr Tyr Arg Gln Asp Val Asp Ala Tyr Leu Leu Gln Ala130 135 140gcc att aaa tac ggc tgc aag gtc cac cag aaa act acc gtg acc gaa480Ala Ile Lys Tyr Gly Cys Lys Val His Gln Lys Thr Thr Val Thr Glu145 150 155 160tac cac gcc gat aaa gac ggc gtc gcg gtg acc acc gcc cag ggc gaa528Tyr His Ala Asp Lys Asp Gly Val Ala Val Thr Thr Ala Gln Gly Glu165 170 175cgg ttc acc ggc cgg tac atg atc gac tgc gga gga cct cgc gcg ccg576Arg Phe Thr Gly Arg Tyr Met Ile Asp Cys Gly Gly Pro Arg Ala Pro180 185 190ctc gcg acc aag ttc aag ctc cgc gaa gaa ccg tgt cgc ttc aag acg624Leu Ala Thr Lys Phe Lys Leu Arg Glu Glu Pro Cys Arg Phe Lys Thr195 200 205cac tcg cgc agc ctc tac acg cac atg ctc ggg gtc aag ccg ttc gac672His Ser Arg Ser Leu Tyr Thr His Met Leu Gly Val Lys Pro Phe Asp210 215 220gac atc ttc aag gtc aag ggg cag cgc tgg cgc tgg cac gag ggg acc720Asp Ile Phe Lys Val Lys Gly Gln Arg Trp Arg Trp His Glu Gly Thr225 230 235 240ttg cac cac atg ttc gag ggc ggc tgg ctc tgg gtg att ccg ttc aac768Leu His His Met Phe Glu Gly Gly Trp Leu Trp Val Ile Pro Phe Asn245 250 255aac cac ccg cgg tcg acc aac aac ctg gtg agc gtc ggc ctg cag ctc816Asn His Pro Arg Ser Thr Asn Asn Leu Val Ser Val Gly Leu Gln Leu260 265 270gac ccg cgt gtc tac ccg aaa acc gac atc tcc gca cag cag gaa ttc864Asp Pro Arg Val Tyr Pro Lys Thr Asp Ile Ser Ala Gln Gln Glu Phe275 280 285gat gag ttc ctc gcg cgg ttc ccg agc atc ggg gct cag ttc cgg gac912Asp Glu Phe Leu Ala Arg Phe Pro Ser Ile Gly Ala Gln Phe Arg Asp290 295 300gcc gtg ccg gtg cgc gac tgg gtc aag acc gac cgc ctg caa ttc tcg960Ala Val Pro Val Arg Asp Trp Val Lys Thr Asp Arg Leu Gln Phe Ser305 310 315 320tcg aac gcc tgc gtc ggc gac cgc tac tgc ctg atg ctg cac gcg aac1008Ser Asn Ala Cys Val Gly Asp Arg Tyr Cys Leu Met Leu His Ala Asn325 330 335ggc ttc atc gac ccg ctc ttc tcc cgg ggg ctg gaa aac acc gcg gtg1056Gly Phe Ile Asp Pro Leu Phe Ser Arg Gly Leu Glu Asn Thr Ala Val340 345 350acc atc cac gcg ctc gcg gcg cgc ctc atc aag gcg ctg cgc gac gac1104Thr Ile His Ala Leu Ala Ala Arg Leu Ile Lys Ala Leu Arg Asp Asp355 360 365gac ttc tcc ccc gag cgc ttc gag tac atc gag cgc ctg cag caa aag1152Asp Phe Ser Pro Glu Arg Phe Glu Tyr Ile Glu Arg Leu Gln Gln Lys370 375 380ctt ttg gac cac aac gac gac ttc gtc agc tgc tgc tac acg gcg ttc1200Leu Leu Asp His Asn Asp Asp Phe Val Ser Cys Cys Tyr Thr Ala Phe385 390 395 400tcg gac ttc cgc cta tgg gac gcg ttc cac agg ctg tgg gcg gtc ggc1248Ser Asp Phe Arg Leu Trp Asp Ala Phe His Arg Leu Trp Ala Val Gly405 410 415acc atc ctc ggg cag ttc cgg ctc gtg cag gcc cac gcg agg ttc cgc1296Thr Ile Leu Gly Gln Phe Arg Leu Val Gln Ala His Ala Arg Phe Arg420 425 430gcg tcg cgc aac gag ggc gac ctc gat cac ctc gac aac gac cct ccg1344Ala Ser Arg Asn Glu Gly Asp Leu Asp His Leu Asp Asn Asp Pro Pro435 440 445tat ctc gga tac ctg tgc gcg gac atg gag gag tac tac cag ttg ttc1392Tyr Leu Gly Tyr Leu Cys Ala Asp Met Glu Glu Tyr Tyr Gln Leu Phe450 455 460aac gac gcc aaa gcc gag gtc gag gcc gtg agt gcc ggg cgc aag ccg1440Asn Asp Ala Lys Ala Glu Val Glu Ala Val Ser Ala Gly Arg Lys Pro465 470 475 480gcc gat gag gcc gcg gcg cgg att cac gcc ctc att gac gaa cga gac1488Ala Asp Glu Ala Ala Ala Arg Ile His Ala Leu Ile Asp Glu Arg Asp485 490 495ttc gcc aag ccg atg ttc ggc ttc ggg tac tgc atc acc ggg gac aag1536Phe Ala Lys Pro Met Phe Gly Phe Gly Tyr Cys Ile Thr Gly Asp Lys500 505 510ccg cag ctc aac aac tcg aag tac agc ctg ctg ccg gcg atg cgg ctg1584Pro Gln Leu Asn Asn Ser Lys Tyr Ser Leu Leu Pro Ala Met Arg Leu515 520 525atg tac tgg acg caa acc cgc gcg ccg gca gag gtg aaa aag tac ttc1632Met Tyr Trp Thr G1n Thr Arg Ala Pro Ala Glu Val Lys Lys Tyr Phe530 535 540gac tac aac ccg atg ttc gcg ctg ctc aag gcg tac atc acg acc cgc1680Asp Tyr Asn Pro Met Phe Ala Leu Leu Lys Ala Tyr Ile Thr Thr Arg545 550 555 560atc ggc ctg gcg ctg aag aag tag1704Ile Gly Leu Ala Leu Lys Lys565<210>5<211>567<212>PRT<213>熒光假單胞菌<400>5Met Thr Gln Lys Ser Pro Ala Asn Glu His Asp Ser Asn His Phe Asp1 5 10 15Val Ile Ile Leu Gly Ser Gly Met Ser Gly Thr Gln Met Gly Ala Ile20 25 30Leu Ala Lys Gln Gln Phe Arg Val Leu Ile Ile Glu Glu Ser Ser His35 40 45Pro Arg Phe Thr Ile Gly Glu Ser Ser Ile Pro Glu Thr Ser Leu Met50 55 60Asn Arg Ile Ile Ala Asp Arg Tyr Gly Ile Pro Glu Leu Asp His Ile65 70 75 80Thr Ser Phe Tyr Ser Thr Gln Arg Tyr Val Ala Ser Ser Thr Gly Ile85 90 95Lys Arg Asn Phe Gly Phe Val Phe His Lys Pro Gly Gln Glu His Asp100 105 110Pro Lys Glu Phe Thr Gln Cys Val Ile Pro Glu Leu Pro Trp Gly Pro115 120 125Glu Ser His Tyr Tyr Arg Gln Asp Val Asp Ala Tyr Leu Leu Gln Ala130 135 140Ala Ile Lys Tyr Gly Cys Lys Val His Gln Lys Thr Thr Val Thr Glu145 150 155 160Tyr His Ala Asp Lys Asp Gly Val Ala Val Thr Thr Ala Gln Gly Glu165 170 175Arg Phe Thr Gly Arg Tyr Met Ile Asp Cys Gly Gly Pro Arg Ala Pro180 185 190Leu Ala Thr Lys Phe Lys Leu Arg Glu Glu Pro Cys Arg Phe Lys Thr195 200 205His Ser Arg Ser Leu Tyr Thr His Met Leu Gly Val Lys Pro Phe Asp210 215 220Asp Ile Phe Lys Val Lys Gly Gln Arg Trp Arg Trp His Glu Gly Thr225 230 235 240Leu His His Met Phe Glu Gly Gly Trp Leu Trp Val Ile Pro Phe Asn245 250 255Ash His Pro Arg Ser Thr Asn Asn Leu Val Ser Val Gly Leu Gln Leu260 265 270Asp Pro Arg Val Tyr Pro Lys Thr Asp Ile Ser Ala Gln Gln Glu Phe275 280 285Asp Glu Phe Leu Ala Arg Phe Pro Ser Ile Gly Ala Gln Phe Arg Asp290 295 300Ala Val Pro Val Arg Asp Trp Val Lys Thr Asp Arg Leu Gln Phe Ser305 310 315 320Ser Asn Ala Cys Val Gly Asp Arg Tyr Cys Leu Met Leu His Ala Asn325 330 335Gly Phe Ile Asp Pro Leu Phe Ser Arg Gly Leu Glu Asn Thr Ala Val340 345 350Thr Ile His Ala Leu Ala Ala Arg Leu Ile Lys Ala Leu Arg Asp Asp355 360 365Asp Phe Ser Pro Glu Arg Phe Glu Tyr Ile Glu Arg Leu Gln Gln Lys370 375 380Leu Leu Asp His Asn Asp Asp Phe Val Ser Cys Cys Tyr Thr Ala Phe385390 395 400Ser Asp Phe Arg Leu Trp Asp Ala Phe His Arg Leu Trp Ala Val Gly405 410 415Thr Ile Leu Gly Gln Phe Arg Leu Val Gln Ala His Ala Arg Phe Arg420 425 430Ala Ser Arg Asn Glu Gly Asp Leu Asp His Leu Asp Asn Asp Pro Pro435 440 445Tyr Leu Gly Tyr Leu Cys Ala Asp Met Glu Glu Tyr Tyr Gln Leu Phe450 455 460Asn Asp Ala Lys Ala Glu Val Glu Ala Val Ser Ala Gly Arg Lys Pro465 470 475 480Ala Asp Glu Ala Ala Ala Arg Ile His Ala Leu Ile Asp Glu Arg Asp485 490 495Phe Ala Lys Pro Met Phe Gly Phe Gly Tyr Cys Ile Thr Gly Asp Lys500 505 510Pro Gln Leu Asn Asn Ser Lys Tyr Ser Leu Leu Pro Ala Met Arg Leu515 520 525Met Tyr Trp Thr Gln Thr Arg Ala Pro Ala Glu Val Lys Lys Tyr Phe530 535 540Asp Tyr Asn Pro Met Phe Ala Leu Leu Lys Ala Tyr Ile Thr Thr Arg545 550 555 560Ile Gly Leu Ala Leu Lys Lys565<210>6<211>1350<212>DNA<213>熒光假單胞菌<220><221>CDS<222>(1)..(1350)<400>6atg agc gat cat gat tat gat gta gtg att atc ggt ggc ggg ccg gcg48Met Ser Asp His Asp Tyr Asp Val Val Ile Ile Gly Gly Gly Pro Ala1 5 10 15ggt tcg acc atg gcc tcc tac ctg gca aaa gcc ggt gtc aaa tgc gcg96Gly Ser Thr Met Ala Ser Tyr Leu Ala Lys Ala Gly Val Lys Cys Ala20 25 30gtg ttc gaa aaa gaa ctg ttc gag cgc gag cat gtt ggc gag tcg ctg144Val Phe Glu Lys Glu Leu Phe Glu Arg Glu His Val Gly Glu Ser Leu35 40 45gta ccg gcc acc act ccg gtg ctg ctg gaa atc ggg gtg atg gaa aag192Val Pro Ala Thr Thr Pro Val Leu Leu Glu Ile Gly Val Met Glu Lys50 55 60atc gag aaa gcc aac ttc ccg aag aag ttc ggc gct gcc tgg acc tcg240Ile Glu Lys Ala Asn Phe Pro Lys Lys Phe Gly Ala Ala Trp Thr Ser65 70 75 80gca gat tcc ggc ccc gaa gac aag atg ggc ttc cag ggg ctg gac cac288Ala Asp Ser Gly Pro Glu Asp Lys Met Gly Phe Gln Gly Leu Asp His85 90 95gat ttc cgt tcg gcg gaa atc ctc ttc aac gag cgc aag cag gaa ggg336Asp Phe Arg Ser Ala Glu Ile Leu Phe Asn Glu Arg Lys Gln Glu Gly100 105 110gtc gat cgc gac ttc acg ttc cac gtc gac cgc ggc aag ttc gac cgc384Val Asp Arg Asp Phe Thr Phe His Val Asp Arg Gly Lys Phe Asp Arg115 120 125att ctt ctg gag cac gca ggt tcg ctg ggg gcc aag gtc ttc cag ggc432Ile Leu Leu Glu His Ala Gly Ser Leu Gly Ala Lys Val Phe Gln Gly130 135 140gtg gag atc gct gac gtc gag ttt ctc agc ccg ggc aat gtc att gtc480Val Glu Ile Ala Asp Val Glu Phe Leu Ser Pro Gly Asn Val Ile Val145 150 155 160aat gcc aag ctg ggc aag cgc agc gtg gag atc aag gcc aag atg gtg528Ash Ala Lys Leu Gly Lys Arg Ser Val Glu Ile Lys Ala Lys Met Val165 170 175gtg gat gcc agc ggt cgc aac gtg ctg ctg ggc cgc cgg ctg ggc ttg576Val Asp Ala Ser Gly Arg Asn Val Leu Leu Gly Arg Arg Leu Gly Leu180 185 190cga gaa aag gac ccg gtc ttc aac cag ttc gcg att cac tcc tgg ttc624Arg Glu Lys Asp Pro Val Phe Asn Gln Phe Ala Ile His Ser Trp Phe195 200 205gac aac ttc gac cgc aag tcg gcg acg caa agc ccg gac aag gtc gac672Asp Asn Phe Asp Arg Lys Ser Ala Thr Gln Ser Pro Asp Lys Val Asp210 215 220tac atc ttc att cac ttc ctg ccg atg acc aat acc tgg gtc tgg cag720Tyr Ile Phe Ile His Phe Leu Pro Met Thr Asn Thr Trp Val Trp Gln225 230 235 240atc ccg atc acc gaa acc att acc agc gtg ggc gtg gtt acg cag aag768Ile Pro Ile Thr Glu Thr Ile Thr Ser Val Gly Val Val Thr Gln Lys245 250 255cag aac tac acc aac tcc gac ctc acc tat gaa gag ttc ttc tgg gaa816Gln Asn Tyr Thr Asn Ser Asp Leu Thr Tyr Glu Glu Phe Phe Trp Glu260 265 270gcg gtg aag acc cgg gaa aac ctg cat gac gcg ctg aag gca tcg gag864Ala Val Lys Thr Arg Glu Asn Leu His Asp Ala Leu Lys Ala Ser Glu275 280 285cag gtc cgc ccg ttc aag aaa gag gcg gac tac agc tac ggc atg aaa912Gln Val Arg Pro Phe Lys Lys Glu Ala Asp Tyr Ser Tyr Gly Met Lys290 295 300gaa gtc tgt ggc gac agc ttc gtg ctg atc ggc gat gcc gca cgg ttc960Glu Val Cys Gly Asp Ser Phe Val Leu Ile Gly Asp Ala Ala Arg Phe305 310 315 320gtc gac ccg atc ttc tcc agc ggc gtc agc gtt gca ctc aac agt gcg1008Val Asp Pro Ile Phe Ser Ser Gly Val Ser Val Ala Leu Asn Ser Ala325 330 335cgc atc gcc agc ggc gac atc atc gag gcg gtg aag aac aac gac ttt1056Arg Ile Ala Ser Gly Asp Ile Ile Glu Ala Val Lys Asn Asn Asp Phe340 345 350agc aag tcc agt ttc act cac tac gaa ggc atg atc agg aat ggc atc1104Ser Lys Ser Ser Phe Thr His Tyr Glu Gly Met Ile Arg Asn Gly Ile355 360 365aag aac tgg tat gag ttc atc acg ctc tat tac cgc ctg aac atc ctc1152Lys Ash Trp Tyr Glu Phe Ile Thr Leu Tyr Tyr Arg Leu Asn Ile Leu370 375 380ttc acc gcg ttc gtt caa gac cca cgc tac cgc ctg gac atc ctg caa1200Phe Thr Ala Phe Val Gln Asp Pro Arg Tyr Arg Leu Asp Ile Leu Gln385 390 395 400ttg ctg caa ggg gac gtc tac agc ggc aag cgc ctg gaa gtg ctg gac1248Leu Leu Gln Gly Asp Val Tyr Ser Gly Lys Arg Leu Glu Val Leu Asp405 410 415aag atg cgc gaa atc atc gct gcg gtt gaa agc gac ccg gaa cac ctc1296Lys Met Arg Glu Ile Ile Ala Ala Val Glu Ser Asp Pro Glu His Leu420 425 430tgg cac aag tac ctg ggc gac atg cag gtt cct acc gcc aaa ccc gcg1344Trp His Lys Tyr Leu Gly Asp Met Gln Val Pro Thr Ala Lys Pro Ala435 440 445ttc taa1350Phe450<210>7<211>449<212>PRT<213>熒光假單胞菌<400>7Met Ser Asp His Asp Tyr Asp Val Val Ile Ile Gly Gly Gly Pro Ala1 5 10 15Gly Ser Thr Met Ala Ser Tyr Leu Ala Lys Ala Gly Val Lys Cys Ala
20 25 30Val Phe Glu Lys Glu Leu Phe Glu Arg Glu His Val Gly Glu Ser Leu35 40 45Val Pro Ala Thr Thr Pro Val Leu Leu Glu Ile Gly Val Met Glu Lys50 55 60Ile Glu Lys Ala Asn Phe Pro Lys Lys Phe Gly Ala Ala Trp Thr Ser65 70 75 80Ala Asp Ser Gly Pro Glu Asp Lys Met Gly Phe Gln Gly Leu Asp His85 90 95Asp Phe Arg Ser Ala Glu Ile Leu Phe Asn Glu Arg Lys Gln Glu Gly100 105 110Val Asp Arg Asp Phe Thr Phe His Val Asp Arg Gly Lys Phe Asp Arg115 120 125Ile Leu Leu Glu His Ala Gly Ser Leu Gly Ala Lys Val Phe Gln Gly130 135 140Val Glu Ile Ala Asp Val Glu Phe Leu Ser Pro Gly Asn Val Ile Val145 150 155 160Asn Ala Lys Leu Gly Lys Arg Ser Val Glu Ile Lys Ala Lys Met Val165 170 175Val Asp Ala Ser Gly Arg Asn Val Leu Leu Gly Arg Arg Leu Gly Leu
180 185 190Arg Glu Lys Asp Pro Val Phe Asn Gln Phe Ala Ile His Ser Trp Phe195 200 205Asp Asn Phe Asp Arg Lys Ser Ala Thr Gln Ser Pro Asp Lys Val Asp210 215 220Tyr Ile Phe Ile His Phe Leu Pro Met Thr Asn Thr Trp Val Trp Gln225 230 235 240Ile Pro Ile Thr Glu Thr Ile Thr Ser Val Gly Val Val Thr Gln Lys245 250 255Gln Asn Tyr Thr Asn Ser Asp Leu Thr Tyr Glu Glu Phe Phe Trp Glu260 265 270Ala Val Lys Thr Arg Glu Asn Leu His Asp Ala Leu Lys Ala Ser Glu275 280 285Gln Val Arg Pro Phe Lys Lys Glu Ala Asp Tyr Ser Tyr Gly Met Lys290 295 300Glu Val Cys Gly Asp Ser Phe Val Leu Ile Gly Asp Ala Ala Arg Phe305 310 315 320Val Asp Pro Ile Phe Ser Ser Gly Val Ser Val Ala Leu Asn Ser Ala325 330 335Arg Ile Ala Ser Gly Asp Ile Ile Glu Ala Val Lys Asn Asn Asp Phe
340 345 350Ser Lys Ser Ser Phe Thr His Tyr Glu Gly Met Ile Arg Ash Gly Ile355 360 365Lys Asn Trp Tyr Glu Phe Ile Thr Leu Tyr Tyr Arg Leu Asn Ile Leu370 375 380Phe Thr Ala Phe Val Gln Asp Pro Arg Tyr Arg Leu Asp Ile Leu Gln385 390 395 400Leu Leu Gln Gly Asp Val Tyr Ser Gly Lys Arg Leu Glu Val Leu Asp405 410 415Lys Met Arg Glu Ile Ile Ala Ala Val Glu Ser Asp Pro Glu His Leu420 425 430Trp His Lys Tyr Leu Gly Asp Met Gln Val Pro Thr Ala Lys Pro Ala435 440 445Phe<210>8<211>1641<212>DNA<213>熒光假單胞菌<220><221>CDS<222>(1)..(1641)<400>8gtg gtt atg aac gat gtg cag tct ggc aag gcg cca gag cat tac gac48Val Val Met Asn Asp Val Gln Ser Gly Lys Ala Pro Glu His Tyr Asp1 5 10 15att ctc ttg gcg ggc aac agc atc agc gtg atc atg ctc gcc gcc tgc96Ile Leu Leu Ala Gly Asn Ser Ile Ser Val Ile Met Leu Ala Als Cys20 25 30ctg gcc cgg aac aag gtc cgg gtc ggt ttg ttg cgc aac cgg cag atg144Leu Ala Arg Asn Lys Val Arg Val Gly Leu Leu Arg Asn Arg Gln Met35 40 45ccc ccc gac ctt acc ggt gag gcg acg att ccc tat acc tcg atg att192Pro Pro Asp Leu Thr Gly Glu Ala Thr Ile Pro Tyr Thr Ser Met Ile50 55 60ttc gag ctg att gcc gac cgc tat ggc gtg ccg gaa ata aag aat atc240Phe Glu Leu Ile Ala Asp Arg Tyr Gly Val Pro Glu Ile Lys Asn Ile65 70 75 80gcc cgc acc cgg gat atc cag cag aag gtg atg ccg tct tcc ggg gtc288Ala Arg Thr Arg Asp Ile Gln Gln Lys Val Met Pro Ser Ser Gly Val85 90 95aag aag aac ctc ggg ttc atc tat cac cag cgc agc cgg gcg gtg gac336Lys Lys Asn Leu Gly Phe Ile Tyr His Gln Arg Ser Arg Ala Val Asp100 105 110ctg ggc cag gcg ctg caa ttc aac gtg ccc tcc gag cat ggc gag aac384Leu Gly Gln Ala Leu Gln Phe Asn Val Pro Ser Glu His Gly Glu Asn115 120 125cat ctg ttc agg ccc gat atc gat gcc tat ctg ctg gcg gcg gcc atc432His Leu Phe Arg Pro Asp Ile Asp Ala Tyr Leu Leu Ala Ala Ala Ile130 135 140ggt tat ggc gcg cag ctg gtg gag atc gat aac agc cca gag gtg ctg480Gly Tyr Gly Ala Gln Leu Val Glu Ile Asp Asn Ser Pro Glu Val Leu145 150 155 160gtc gag gac agc ggg gtc aag gta gct acg gca ctg ggg cgc tgg gtc528Val Glu Asp Ser Gly Val Lys Val Ala Thr Ala Leu Gly Arg Trp Val165 170 175act gcc gat ttc atg gtt gat ggc agc cag ggc ggc cag gtg ctg gcg576Thr Ala Asp Phe Met Val Asp Gly Ser Gln Gly Gly Gln Val Leu Ala180 185 190cgg cag gct ggc ctg gtc agc cag gct tcg acg cag aag acc cgg acc624Arg Gln Ala Gly Leu Val Ser Gln Ala Ser Thr Gln Lys Thr Arg Thr195 200 205ctg gaa ttc tcc act cat atg ctc ggg gtg gtg ccg ttc gat gag tgc672Leu Glu Phe Ser Thr His Met Leu Gly Val Val Pro Phe Asp Glu Cys210 215 220gtg cag ggc gat ttt ccc ggc cag tgg cat ggc ggc act ctg cat cac720Val Gln Gly Asp Phe Pro Gly Gln Trp His Gly Gly Thr Leu His His225 230 235 240gtg ttc gat ggg ggc tgg gtg ggg gtc atc ccg ttc aac aac cat cag768Val Phe Asp Gly Gly Trp Val Gly Val Ile Pro Phe Asn Asn His Gln245 250 255cac tcg cgc aac cct ttg gtc agc gtg ctg gtt tca ctg cgt gag gac816His Ser Arg Asn Pro Leu Val Ser Val Leu Val Ser Leu Arg Glu Asp260 265 270ctc tgc ccg agc atg gac ggc gac cag gtc ctg gcc ggc ctg atc gag864Leu Cys Pro Ser Met Asp Gly Asp Gln Val Leu Ala Gly Leu Ile Glu275 280 285ctg tac ccc ggc ctg ggg cgg cac ctg tcc ggc gcc cgg cgg gtg cgc912Leu Tyr Pro Gly Leu Gly Arg His Leu Ser Gly Ala Arg Arg Val Arg290295 300gag tgg gtg ctg cgc cag ccg ccc cgg cag gtc tat cgc acg gcg ctc960Glu Trp Val Leu Arg Gln Pro Pro Arg Gln Val Tyr Arg Thr Ala Leu305 310315 320gaa cgc cgc tgc ctg atg ttc gac gag ggc gcc gcg agc aac gat ctg1008Glu Arg Arg Cys Leu Met Phe Asp Glu Gly Ala Ala Ser Asn Asp Leu325 330 335ttg ttc tcg cgc aag ctg tcc aat gct gcg gaa ctg gtt ctg gcc ctg1056Leu Phe Ser Arg Lys Leu Ser Asn Ala Ala Glu Leu Val Leu Ala Leu340 345 350gcg cac cgg ctg atc aag gcg gcg cac agc ggt gac tac cgc agc ccg1104Ala His Arg Leu Ile Lys Ala Ala His Ser Gly Asp Tyr Arg Ser Pro355 360 365gcc ctg aat gat ttt gtc ctg acc cag gac agc atc atc agc ttg agt1152Ala Leu Asn Asp Phe Val Leu Thr Gln Asp Ser Ile Ile Ser Leu Ser370 375 380gac cgg atc gcc tta gcg gct tat gtg tcg ttt cgc gac ccc gag ttg1200Asp Arg Ile Ala Leu Ala Ala Tyr Val Ser Phe Arg Asp Pro Glu Leu385 390 395 400tgg aat gcc ttc gcc cgt gtc tgg ctg ctg cag tcg att gcc gcc acc1248Trp Asn Ala Phe Ala Arg Val Trp Leu Leu Gln Ser Ile Ala Ala Thr405 410 415atc acc gcg cgc aag atc aac gat gcc ttt gcc aag gac ctg gac ccg1296Ile Thr Ala Arg Lys Ile Asn Asp Ala Phe Ala Lys Asp Leu Asp Pro420 425 430cga gtg ttc gat gaa atc gac cag ctc gca gag gac ggt ttc tgg atg1344Arg Val Phe Asp Glu Ile Asp Gln Leu Ala Glu Asp Gly Phe Trp Met435 440 445cct ctg tat cgg ggg tac aag gat att ctc aac act acg ctg ggc ctt1392Pro Leu Tyr Arg Gly Tyr Lys Asp Ile Leu Asn Thr Thr Leu Gly Leu450 455 460tgt gat gac gtc aaa agc gcc aag gtc tct gct gcg cac gcg gcg agc1440Cys Asp Asp Val Lys Ser Ala Lys Val Ser Ala Ala His Ala Ala Ser465 470 475 480agc atc ttt gcg gag ctt gcc aac gcc agt ttt gtt ccg cct att ttt1488Ser Ile Phe Ala Glu Leu Ala Asn Ala Ser Phe Val Pro Pro Ile Phe485 490 495gat ttt gct aat cct cac gct cgt gtc tat caa ctg acc acc ttg aga1536Asp Phe Ala Asn Pro His Ala Arg Val Tyr Gln Leu Thr Thr Leu Arg500 505 510aag ctc aag gcg ctc tgg tgg ggc ctg atg caa gtg ccc tca gag gtc1584Lys Leu Lys Ala Leu Trp Trp Gly Leu Met Gln Val Pro Ser Glu Val515 520 525gga cgg ctg att ttc tat cga tcc ttc aga aaa cct tcc ctg cgc aag1632Gly Arg Leu Ile Phe Tyr Arg Ser Phe Arg Lys Pro Ser Leu Arg Lys530 535 540gag agt tga 1641Glu Ser545<210>9<211>546<212>PRT<213>熒光假單胞菌<400>9Val Val Met Asn Asp Val Gln Ser Gly Lys Ala Pro Glu His Tyr Asp1 5 10 15Ile Leu Leu Ala Gly Asn Ser Ile Ser Val Ile Met Leu Ala Ala Cys20 25 30Leu Ala Arg Asn Lys Val Arg Val Gly Leu Leu Arg Asn Arg Gln Met35 40 45Pro Pro Asp Leu Thr Gly Glu Ala Thr Ile Pro Tyr Thr Ser Met Ile50 55 60Phe Glu Leu Ile Ala Asp Arg Tyr Gly Val Pro Glu Ile Lys Asn Ile65 70 75 80Ala Arg Thr Arg Asp Ile Gln Gln Lys Val Met Pro Ser Ser Gly Val85 90 95Lys Lys Asn Leu Gly Phe Ile Tyr His Gln Arg Ser Arg Ala Val Asp100 105 110Leu Gly Gln Ala Leu Gln Phe Asn Val Pro Ser Glu His Gly Glu Asn115 120 125His Leu Phe Arg Pro Asp Ile Asp Ala Tyr Leu Leu Ala Ala Ala Ile130 135 140Gly Tyr Gly Ala Gln Leu Val Glu Ile Asp Asn Ser Pro Glu Val Leu145 150 155 160Val Glu Asp Ser Gly Val Lys Val Ala Thr Ala Leu Gly Arg Trp Val165 170 175Thr Ala Asp Phe Met Val Asp Gly Ser Gln Gly Gly Gln Val Leu Ala180 185 190Arg Gln Ala Gly Leu Val Ser Gln Ala Ser Thr Gln Lys Thr Arg Thr195 200 205Leu Glu Phe Ser Thr His Met Leu Gly Val Val Pro Phe Asp Glu Cys210 215 220Val Gln Gly Asp Phe Pro Gly Gln Trp His Gly Gly Thr Leu His His225 230 235 240Val Phe Asp Gly Gly Trp Val Gly Val Ile Pro Phe Asn Asn His Gln245 250 255His Ser Arg Asn Pro Leu Val Ser Val Leu Val Ser Leu Arg Glu Asp260 265 270Leu Cys Pro Ser Met Asp Gly Asp Gln Val Leu Ala Gly Leu Ile Glu275 280 285Leu Tyr Pro Gly Leu Gly Arg His Leu Ser Gly Ala Arg Arg Val Arg290 295 300Glu Trp Val Leu Arg Gln Pro Pro Arg Gln Val Tyr Arg Thr Ala Leu305 310 315 320Glu Arg Arg Cys Leu Met Phe Asp Glu Gly Ala Ala Ser Asn Asp Leu325 330 335Leu Phe Ser Arg Lys Leu Ser Asn Ala Ala Glu Leu Val Leu Ala Leu340 345 350Ala His Arg Leu Ile Lys Ala Ala His Ser Gly Asp Tyr Arg Ser Pro355 360 365Ala Leu Asn Asp Phe Val Leu Thr Gln Asp Ser Ile Ile Ser Leu Ser370 375 380Asp Arg Ile Ala Leu Ala Ala Tyr Val Ser Phe Arg Asp Pro Glu Leu385 390 395 400Trp Asn Ala Phe Ala Arg Val Trp Leu Leu Gln Ser Ile Ala Ala Thr405 410 415Ile Thr Ala Arg Lys Ile Asn Asp Ala Phe Ala Lys Asp Leu Asp Pro420 425 430Arg Val Phe Asp Glu Ile Asp Gln Leu Ala Glu Asp Gly Phe Trp Met435 440 445Pro Leu Tyr Arg Gly Tyr Lys Asp Ile Leu Asn Thr Thr Leu Gly Leu450 455 460Cys Asp Asp Val Lys Ser Ala Lys Val Ser Ala Ala His Ala Ala Ser465 470 475 480Ser Ile Phe Ala Glu Leu Ala Ash Ala Ser Phe Val Pro Pro Ile Phe485 490 495Asp Phe Ala Asn Pro His Ala Arg Val Tyr Gln Leu Thr Thr Leu Arg500 505 510Lys Leu Lys Ala Leu Trp Trp Gly Leu Met Gln Val Pro Ser Glu Val515 520 525Gly Arg Leu Ile Phe Tyr Arg Ser Phe Arg Lys Pro Ser Leu Arg Lys530535540Glu Ser545<210>10<211>1510<212>DNA<213>熒光假單胞菌<220><221>CDS<222>(1)..(1509)<400>10atg aat cag tac gac gtc att atc atc ggt agt ggt atc gcc ggc gcg48Met Asn Gln Tyr Asp Val Ile Ile Ile Gly Ser Gly Ile Ala Gly Ala1 5 10 15ctg acc ggc gcc gtc ctc gcg aag tcc ggg ctg aac gtt ctg atc ctc96Leu Thr Gly Ala Val Leu Ala Lys Ser Gly Leu Asn Val Leu Ile Leu20 25 30gac tcg gcc cag cac cca cga ttc tcc gtc ggc gaa gcg gcg aca ccg144Asp Ser Ala Gln His Pro Arg Phe Ser Val Gly Glu Ala Ala Thr Pro35 40 45gaa agc ggt ttt ctg ctg cgt ttg ctc tca aag cgc ttc gac atc cct192Glu Ser Gly Phe Leu Leu Arg Leu Leu Ser Lys Arg Phe Asp Ile Pro50 55 60gaa atc gcc tac ctc tcg cac ccc gac aag atc atc cag cac gtc ggt240Glu Ile Ala Tyr Leu Ser His Pro Asp Lys Ile Ile Gln His Val Gly65 70 75 80tcg agc gcc tgc ggg atc aag ctg ggc ttc agt ttt gcc tgg cat caa288Ser Ser Ala Cys Gly Ile Lys Leu Gly Phe Ser Phe Ala Trp His Gln85 90 95gag aac gcg ccg tcg tcc ccc gac cac ctt gtg gcc ccg ccg ctg aag336Glu Asn Ala Pro Ser Ser Pro Asp His Leu Val Ala Pro Pro Leu Lys100 105 110gtg ccg gaa gcc cat ctt ttc cgg cag gac atc gac tat ttc gcc ctg384Val Pro Glu Ala His Leu Phe Arg Gln Asp Ile Asp Tyr Phe Ala Leu115 120 125atg att gcc ctg aaa cac ggc gcc gaa tcc aga cag aac atc aag atc432Met Ile Ala Leu Lys His Gly Ala Glu Ser Arg Gln Asn Ile Lys Ile
130 135 140gag tcg atc agc ctc aac gac gac ggg gtc gag gtg gca ttg tcc aac480Glu Ser Ile Ser Leu Asn Asp Asp Gly Val Glu Val Ala Leu Ser Asn145 150 155 160gcc gcc ccc gtc aag gcc gcg ttc atc att gac gct gct gcc cag ggc528Ala Ala Pro Val Lys Ala Ala Phe Ile Ile Asp Ala Ala Ala Gln Gly165 170 175tct ccg ctt tcc cgc caa ctg ggc ttg cgc acc acc gaa ggg ctg gcg576Ser Pro Leu Ser Arg Gln Leu Gly Leu Arg Thr Thr Glu Gly Leu Ala180 185 190acc gac acc tgc tca ttc ttc acc cac atg ctc aat gtg aag agc tac624Thr Asp Thr Cys Ser Phe Phe Thr His Met Leu Asn Val Lys Ser Tyr195 200 205gaa gat gcc ctg gct ccg ttg tcc cgc act cgt tcc ccc atc gaa ctg672Glu Asp Ala Leu Ala Pro Leu Ser Arg Thr Arg Ser Pro Ile Glu Leu210 215 220ttc aag agc acc ttg cac cac atc ttc gaa gag ggc tgg ttg tgg gtc720Phe Lys Ser Thr Leu His His Ile Phe Glu Glu Gly Trp Leu Trp Val225 230 235 240atc ccc ttc aac aac cac ccg cag ggc acc aat cag ttg tgc agc atc768Ile Pro Phe Asn Asn His Pro Gln Gly Thr Asn Gln Leu Cys Set Ile245 250 255ggc ttc cag ttc aac aac gcc aag tac cgt ccc acc gag gcg ccg gag816Gly Phe Gln Phe Asn ASn Ala Lys Tyr Arg Pro Thr Glu Ala Pro Glu260 265 270atc gag ttt cgc aaa ctg ctg aaa aag tac ccg gcc atc ggc gaa cac864Ile Glu Phe Arg Lys Leu Leu Lys Lys Tyr Pro Ala Ile Gly Glu His275 280 285ttc aag gat gcg gtc aat gcc cgg gag tgg atc tac gcg ccg cgc atc912Phe Lys Asp Ala Val Asn Ala Arg Glu Trp Ile Tyr Ala Pro Arg Ile290 295 300aac tac cgc agc gtg caa aat gtc ggg gat cgc ttc tgc ctg ctg ccg960Asn Tyr Arg Ser Val Gln Asn Val Gly Asp Arg Phe Cys Leu Leu Pro305 310 315 320caa gcc aca ggg ttt atc gac ccg ctg ttc tcc agg ggg ttg atc acc1008Gln Ala Thr Gly Phe Ile Asp Pro Leu Phe Ser Arg Gly Leu Ile Thr325 330 335acc ttc gag tcc atc ctc agg ctg gcc ccc aag gtg ctg gac gcc gcc1056Thr Phe Glu Set Ile Leu Arg Leu Ala Pro Lys Val Leu Asp Ala Ala340 345 350cgc agc aac cgc tgg caa cgg gaa cag ttc atc gaa gtc gag cgc cat1104Arg Ser Asn Arg Trp Gln Arg Glu Gln Phe Ile Glu Val Glu Arg His355 360 365tgc ctg aac gcg gtg gcg acc aat gac cag ttg gtc tcc tgc tcc tat1152Cys Leu Asn Ala Val Ala Thr Asn Asp Gln Leu Val Ser Cys Ser Tyr
370 375 380gaa gcc ttc agc gac ttt cac ctg tgg aac gtg tgg cat cgg gtc tgg1200Glu Ala Phe Ser Asp Phe His Leu Trp Asn Val Trp His Arg Val Trp385 390 395 400ctc agc ggc tcc aac ctg ggc agt gcc ttt ctg caa aag ctg ctg cac1248Leu Ser Gly Ser Asn Leu Gly Ser Ala Phe Leu Gln Lys Leu Leu His405 410 415gac ctg gaa cac agt ggc gac gcc cgc cag ttc gat gca gcg ctt gag1296Asp Leu Glu His Ser Gly Asp Ala Arg Gln Phe Asp Ala Ala Leu Glu420 425 430gcg gtg cgc ttc cct ggc tgc ctg tcc ctg gac tcg ccc gcc tac gaa1344Ala Val Arg Phe Pro Gly Cys Leu Ser Leu Asp Ser Pro Ala Tyr Glu435 440 445agc ctg ttc agg cag tcg tgc cag gtc atg caa cag gcc agg gag caa1392Ser Leu Phe Arg Gln Ser Cys Gln Val Met Gln Gln Ala Arg Glu Gln450 455 460gcc agg ccg gtg gcc gaa acc gcc aac gcg ctg cat gag ctg atc aag1440Ala Arg Pro Val Ala Glu Thr Ala Asn Ala Leu His Glu Leu Ile Lys465 470 475 480gag cac gaa gcc gag ttg ttg ccc ctg ggc tat tca cgg ata tcc aat1488Glu His Glu Ala Glu Leu Leu Pro Leu Gly Tyr Ser Arg Ile Ser Asn485 490 495cgt ttc atc ctc aaa gtc tga a1510Arg Phe Ile Leu Lys Val500<210>11<211>502<212>PRT<213>熒光假單胞菌<400>11Met Asn Gln Tyr Asp Val Ile Ile Ile Gly Ser Gly Ile Ala Gly Ala1 5 10 15Leu Thr Gly Ala Val Leu Ala Lys Ser Gly Leu Asn Val Leu Ile Leu20 25 30Asp Ser Ala Gln His Pro Arg Phe Ser Val Gly Glu Ala Ala Thr Pro35 40 45Glu Ser Gly Phe Leu Leu Arg Leu Leu Ser Lys Arg Phe Asp Ile Pro50 55 60Glu Ile Ala Tyr Leu Ser His Pro Asp Lys Ile Ile Gln His Val Gly65 70 75 80Ser Ser Ala Cys Gly Ile Lys Leu Gly Phe Ser Phe Ala Trp His Gln85 90 95Glu Asn Ala Pro Ser Ser Pro Asp His Leu Val Ala Pro Pro Leu Lys
100 105 110Val Pro Glu Ala His Leu Phe Arg Gln Asp Ile Asp Tyr Phe Ala Leu115 120 125Met Ile Ala Leu Lys His Gly Ala Glu Ser Arg Gln Asn Ile Lys Ile130 135 140Glu Ser Ile Ser Leu Asn Asp Asp Gly Val Glu Val Ala Leu Ser Asn145 150 155 160Ala Ala Pro Val Lys Ala Ala Phe Ile Ile Asp Ala Ala Ala Gln Gly165 170 175Ser Pro Leu Ser Arg Gln Leu Gly Leu Arg Thr Thr Glu Gly Leu Ala180 185 190Thr Asp Thr Cys Ser Phe Phe Thr His Met Leu Asn Val Lys Ser Tyr195 200 205Glu Asp Ala Leu Ala Pro Leu Ser Arg Thr Arg Ser Pro Ile Glu Leu210 215 220Phe Lys Ser Thr Leu His His Ile Phe Glu Glu Gly Trp Leu Trp Val225 230 235 240Ile Pro Phe Asn Asn His Pro Gln Gly Thr Asn Gln Leu Cys Ser Ile245 250 255Gly Phe Gln Phe Asn Asn Ala Lys Tyr Arg Pro Thr Glu Ala Pro Glu
260 265 270Ile Glu Phe Arg Lys Leu Leu Lys Lys Tyr Pro Ala Ile Gly Glu His275 280 285Phe Lys Asp Ala Val Asn Ala Arg Glu Trp Ile Tyr Ala Pro Arg Ile290 295 300Asn Tyr Arg Ser Val Gln Asn Val Gly Asp Arg Phe Cys Leu Leu Pro305 310 315 320Gln Ala Thr Gly Phe Ile Asp Pro Leu Phe Ser Arg Gly Leu Ile Thr325 330 335Thr Phe Glu Ser Ile Leu Arg Leu Ala Pro Lys Val Leu Asp Ala Ala340 345 350Arg Ser Asn Arg Trp Gln Arg Glu Gln Phe Ile Glu Val Glu Arg His355 360 365Cys Leu Asn Ala Val Ala Thr Asn Asp Gln Leu Val Ser Cys Ser Tyr370 375 380Glu Ala Phe Ser Asp Phe His Leu Trp Asn Val Trp His Arg Val Trp385 390 395 400Leu Ser Gly Ser Asn Leu Gly Ser Ala Phe Leu Gln Lys Leu Leu His405 410 415Asp Leu Glu His Ser Gly Asp Ala Arg Gln Phe Asp Ala Ala Leu Glu
420 425 430Ala Val Arg Phe Pro Gly Cys Leu Ser Leu Asp Ser Pro Ala Tyr Glu435 440 445Ser Leu Phe Arg Gln Ser Cys Gln Val Met Gln Gln Ala Arg Glu Gln450 455 460Ala Arg Pro Val Ala Glu Thr Ala Asn Ala Leu His Glu Leu Ile Lys465 470 475 480Glu His Glu Ala Glu Leu Leu Pro Leu Gly Tyr Ser Arg Ile Ser Asn485 490 495Arg Phe Ile Leu Lys Val500<210>12<211>1476<212>DNA<213>東方擬無枝酸菌(Amycolatopsis orientalis)<220><221>CDS<222>(1)..(1476)<400>12atg tcg gtc gaa gat ttc gat gtt gtg gtg gcg ggc ggc ggg ccg ggt48Met Ser Val Glu Asp Phe Asp Val Val Val Ala Gly Gly Gly Pro Gly1 5 10 15ggt tcg acg gtg gcc acc ctg gtg gcg atg cag gga cac cgg gtc ctg96Gly Ser Thr Val Ala Thr Leu Val Ala Met Gln Gly His Arg Val Leu20 25 30ctg ctg gag aaa gag gtc ttc ccc cgg tac cag atc ggt gag tcg ctg144Leu Leu Glu Lys Glu Val Phe Pro Arg Tyr Gln Ile Gly Glu Ser Leu35 40 45ctg ccc gcc acg gtg cac ggg gtc tgc cgg atg ctc ggc gtc gcg gac192Leu Pro Ala Thr Val His Gly Val Cys Arg Met Leu Gly Val Ala Asp50 55 60gag ctg gcg aat tcc ggg ttc cog ate aaa cgc ggc ggc acg ttc cgc240Glu Leu Ala Asn Ser Gly Phe Pro Ile Lys Arg Gly Gly Thr Phe Arg65 70 75 80tgg ggc gcc cgt ccg gag ccg tgg acg ttc cac ttc ggg atc tcg gcc288Trp Gly Ala Arg Pro Glu Pro Trp Thr Phe His Phe Gly Ile Ser Ala85 90 95aag atg gcg ggc tcg acg tcg cac gcc tat cag gtc gag cgg gcg aag336Lys Met Ala Gly Ser Thr Ser His Ala Tyr Gln Val Glu Arg Ala Lys100 105 110ttc gac gac atc ctg ctg aag aac gcc aag agc aag ggc gtc gtc gtg384Phe Asp Asp Ile Leu Leu Lys Asn Ala Lys Ser Lys Gly Val Val Val115 120 125cgg gaa ggc tgc tcg gtc aac gac gtc gtg gag gac ggc gag cgg gtc432Arg Glu Gly Cys Ser Val Asn Asp Val Val Glu Asp Gly Glu Arg Val130 135 140acc ggc gcg cgc tac acc gac gcg gac ggc aac gcg cac gaa gtc tog480Thr Gly Ala Arg Tyr Thr Asp Ala Asp Gly Asn Ala His Glu Val Ser145 150 155 160gcc cgg ttc gtg atc gac gcg tcg ggc aac aag agc cgg ctc tac acg528Ala Arg Phe Val Ile Asp Ala Ser Gly Asn Lys Ser Arg Leu Tyr Thr165 170 175aag gtc aac ggt tcg cgg aac tac tcg gag ttc ttc cgc agc ctc gcg576Lys Val Asn Gly Ser Arg Asn Tyr Ser Glu Phe Phe Arg Ser Leu Ala180 185 190ctg ttc ggc tat ttc gag ggt ggc aaa cgg ctg ccc gag ccg gtg tcg624Leu Phe Gly Tyr Phe Glu Gly Gly Lys Arg Leu Pro Glu Pro Val Ser195 200 205ggc aac atc ctg agc gtc gcc ttc gac agc ggc tgg ttc tgg tac atc672Gly Asn Ile Leu Ser Val Ala Phe Asp Ser Gly Trp Phe Trp Tyr Ile210 215 220ccc ctg agc gac acg ctg acc agc gtc ggc gcg gtc gtg cgc cgg gag720Pro Leu Ser Asp Thr Leu Thr Ser Val Gly Ala Val Val Arg Arg Glu225 230 235 240gac gcc gac aag atc cag ggc gac cgc gag aag gcc ctc aac acc ttg768Asp Ala Asp Lys Ile Gln Gly Asp Arg Glu Lys Ala Leu Asn Thr Leu
245 250 255atc gcc gaa tgc ccg ctg atc tcg gag tac ctc tcg aac gcg acc agg816Ile Ala Glu Cys Pro Leu Ile Ser Glu Tyr Leu Ser Asn Ala Thr Arg260 265 270gtg acc acc ggc agg tac ggc gaa ctg cgg gtg cgc aag gac tac tcg864Val Thr Thr Gly Arg Tyr Gly Glu Leu Arg Val Arg Lys Asp Tyr Ser275 280 285tac cag cag gac agc tac tgg cgg ccc ggg atg gtc ctg gtc ggc gac912Tyr Gln Gln Asp Ser Tyr Trp Arg Pro Gly Met Val Leu Val Gly Asp290 295 300gcc gcg tgc ttc gtg gac ccg gtg ttc tcc tcc ggg gtg cac ctg gcg960Ala Ala Cys Phe Val Asp Pro Val Phe Ser Ser Gly Val His Leu Ala305 310 315 320acc tac agc gcg ctg ctc gcg gcc cgg tcg atc aac agc gtc ctc gcg1008Thr Tyr Ser Ala Leu Leu Ala Ala Arg Ser Ile Asn Ser Val Leu Ala325 330 335ggc gac ctc gac gag aag acc gcg ctg aac gag ttc gag gcg cgc tat1056Gly Asp Leu Asp Glu Lys Thr Ala Leu Asn Glu Phe Glu Ala Arg Tyr340 345 350cgc cgc gag tac ggc gtc ttc tac gag ttc ctc gtc tcc ttc tat cag1104Arg Arg Glu Tyr Gly Val Phe Tyr Glu Phe Leu Val Ser Phe Tyr Gln355 360 365atg aac gtc aac gag gaa tcg tat ttc tgg cag gcc aag aag gtc acg1152Met Asn Val Asn Glu Glu Ser Tyr Phe Trp Gln Ala Lys Lys Val Thr370 375 380cag aac cag agc acc gac atc gag tcg ttc gtc gag ctg atc ggc ggg1200Gln Asn Gln Ser Thr Asp Ile Glu Ser Phe Val Glu Leu Ile Gly Gly385 390 395 400gtg tcg tcc ggc gag acc gcg ctg acg gcc gcc gac cgg atc gcc gcg1248Val Ser Ser Gly Glu Thr Ala Leu Thr Ala Ala Asp Arg Ile Ala Ala405 410 415aac agt gcc gaa ttc gcc gcc gcc gtc gac aag atg gcg acg ggc gac1296Asn Ser Ala Glu Phe Ala Ala Ala Val Asp Lys Met Ala Thr Gly Asp420 425 430ggc gac gac atg gtg ccg atg ttc aag tcg acc gtg gtc aag cag gcg1344Gly Asp Asp Met Val Pro Met Phe Lys Ser Thr Val Val Lys Gln Ala435 440 445atg cag gag gcg ggc cag gtc cag atg aag gcg ctg ctc ggc gag gac1392Met Gln Glu Ala Gly Gln Val Gln Met Lys Ala Leu Leu Gly Glu Asp450 455 460gcc gaa ccc gag ctg ccg ctg ttc ccc ggc ggc ctg gtg act tcg ccc1440Ala Glu Pro Glu Leu Pro Leu Phe Pro Gly Gly Leu Val Thr Ser Pro465 470 475 480gac ggg atg aag tgg ctg ccg cac cac ccg gcc tga1476Asp Gly Met Lys Trp Leu Pro His His Pro Ala
485 490<210>13<211>491<212>PRT<213>東方擬無枝酸菌<400>13Met Ser Val Glu Asp Phe Asp Val Val Val Ala Gly Gly Gly Pro Gly1 5 10 15Gly Ser Thr Val Ala Thr Leu Val Ala Met Gln Gly His Arg Val Leu20 25 30Leu Leu Glu Lys Glu Val Phe Pro Arg Tyr Gln Ile Gly Glu Ser Leu35 40 45Leu Pro Ala Thr Val His Gly Val Cys Arg Met Leu Gly Val Ala Asp50 55 60Glu Leu Ala Asn Ser Gly Phe Pro Ile Lys Arg Gly Gly Thr Phe Arg65 70 75 80Trp Gly Ala Arg Pro Glu Pro Trp Thr Phe His Phe Gly Ile Ser Ala85 90 95Lys Met Ala Gly Ser Thr Ser His Ala Tyr Gln Val Glu Arg Ala Lys100 105 110Phe Asp Asp Ile Leu Leu Lys Asn Ala Lys Ser Lys Gly Val Val Val115 120 125Arg Glu Gly Cys Ser Val Asn Asp Val Val Glu Asp Gly Glu Arg Val130 135 140Thr Gly Ala Arg Tyr Thr Asp Ala Asp Gly Asn Ala His Glu Val Ser145 150 155 160Ala Arg Phe Val Ile Asp Ala Ser Gly Asn Lys Ser Arg Leu Tyr Thr165 170 175Lys Val Asn Gly Ser Arg Asn Tyr Ser Glu Phe Phe Arg Ser Leu Ala180 185 190Leu Phe Gly Tyr Phe Glu Gly Gly Lys Arg Leu Pro Glu Pro Val Ser195 200 205Gly Asn Ile Leu Ser Val Ala Phe Asp Ser Gly Trp Phe Trp Tyr Ile210 215 220Pro Leu Ser Asp Thr Leu Thr Ser Val Gly Ala Val Val Arg Arg Glu225 230 235 240Asp Ala Asp Lys Ile Gln Gly Asp Arg Glu Lys Ala Leu Asn Thr Leu245 250 255Ile Ala Glu Cys Pro Leu Ile Ser Glu Tyr Leu Ser Asn Ala Thr Arg260 265 270Val Thr Thr Gly Arg Tyr Gly Glu Leu Arg Val Arg Lys Asp Tyr Ser275 280 285Tyr Gln Gln Asp Ser Tyr Trp Arg Pro Gly Met Val Leu Val Gly Asp290 295 300Ala Ala Cys Phe Val Asp Pro Val Phe Ser Ser Gly Val His Leu Ala305 310 315 320Thr Tyr Ser Ala Leu Leu Ala Ala Arg Ser Ile Asn Ser Val Leu Ala325 330 335Gly Asp Leu Asp Glu Lys Thr Ala Leu Asn Glu Phe Glu Ala Arg Tyr340 345 350Arg Arg Glu Tyr Gly Val Phe Tyr Glu Phe Leu Val Ser Phe Tyr Gln355 360 365Met Asn Val Asn Glu Glu Ser Tyr Phe Trp Gln Ala Lys Lys Val Thr370 375 380Gln Asn Gln Ser Thr Asp Ile Glu Ser Phe Val Glu Leu Ile Gly Gly385 390 395 400Val Ser Ser Gly Glu Thr Ala Leu Thr Ala Ala Asp Arg Ile Ala Ala405 410 415Asn Ser Ala Glu Phe Ala Ala Ala Val Asp Lys Met Ala Thr Gly Asp420 425 430Gly Asp Asp Met Val Pro Met Phe Lys Ser Thr Val Val Lys Gln Ala435 440 445Met Gln Glu Ala Gly Gln Val Gln Met Lys Ala Leu Leu Gly Glu Asp450 455 460Ala Glu Pro Glu Leu Pro Leu Phe Pro Gly Gly Leu Val Thr Ser Pro465 470 475 480Asp Gly Met Lys Trp Leu Pro His His Pro Ala485 490<210>14<211>1359<212>DNA<213>金霉素鏈霉菌(Streptomyces aureofaciens)<220><221>CDS<222>(1)..(1359)<400>14atg ttc cac cgg gac ggc gag gag ccg gac ccg aac gag acc agc cag48Met Phe His Arg Asp Gly Glu Glu Pro Asp Pro Asn Glu Thr Ser Gln1 5 10 15ttc cgc atc ccc tcg atc gtc ggc aac gcg gcc cac ttc ttc cgc cag96Phe Arg Ile Pro Ser Ile Val Gly Asn Ala Ala His Phe Phe Arg Gln20 25 30gac acc gac tcc tac atg ttc cac gcc gcg gtg cgc tac ggc tgc gac144Asp Thr Asp Ser Tyr Met Phe His Ala Ala Val Arg Tyr Gly Cys Asp35 40 45gcc cgg cag tac tac cgg gtg gag aac atc gag ttc gac gac ggc ggg192Ala Arg Gln Tyr Tyr Arg Val Glu Asn Ile Glu Phe Asp Asp Gly Gly50 55 60gtg acc gtc tcc ggc gcg gac ggc agc acc gtc cgg gcc cgc tac ctg240Val Thr Val Ser Gly Ala Asp Gly Ser Thr Val Arg Ala Arg Tyr Leu65 70 75 80gtc gac gcc agc ggc ttc cgc tcg ccg ctg gca cgg cag ttg ggg ttg288Val Asp Ala Ser Gly Phe Arg Ser Pro Leu Ala Arg Gln Leu Gly Leu85 90 95cgg gag gag ccg agc cgg ctc aag cac cac gcc cgc tcg atc ttc acc336Arg Glu Glu Pro Ser Arg Leu Lys His His Ala Arg Ser Ile Phe Thr100 105 110cac atg gtc gga gtg gac gcg atc gac gac cac gtg gac atg ccg gcc384His Met Val Gly Val Asp Ala Ile Asp Asp His Val Asp Met Pro Ala115 120 125gag ctt cgc ccg ccg gtg ccg tgg aac gac ggg acg atg cac cac atc432Glu Leu Arg Pro Pro Val Pro Trp Asn Asp Gly Thr Met His His Ile130 135 140ttc gag cgc ggc tgg atg tgg atc atc ccg ttc aac aac cac ccc ggg480Phe Glu Arg Gly Trp Met Trp Ile Ile Pro Phe Asn Asn His Pro Gly145 150 155 160gcc acc aac ccg ctg tgc agc gtc ggc atc cag ctc gac gag cgc cgc528Ala Thr Asn Pro Leu Cys Ser Val Gly Ile Gln Leu Asp Glu Arg Arg165 170 175tac ccc gcc cgg ccg gac ctg acg ccc gag gag gag ttc cgg tcc cac576Tyr Pro Ala Arg Pro Asp Leu Thr Pro Glu Glu Glu Phe Arg Ser His180 185 190gtg gac cgc ttc ccg gcg gtg cag cgg cag ttg aag ggc gcc cgc agc624Val Asp Arg Phe Pro Ala Val Gln Arg Gln Leu Lys Gly Ala Arg Ser195 200 205gtg cgc gag tgg gtg cga acg gac cgc atg cag tac tcc tcg agc cgg672Val Arg Glu Trp Val Arg Thr Asp Arg Met Gln Tyr Ser Ser Ser Arg210 215 220acg gtc ggc gag cgc tgg tgc ctg atg tcg cac gcg gcc ggc ttc atc720Thr Val Gly Glu Arg Trp Cys Leu Met Ser His Ala Ala Gly Phe Ile225 230 235 240gac ccg ctc ttc ctt cgc ggc ctg tcc aac acc tgc gag atc atc aac768Asp Pro Leu Phe Leu Arg Gly Leu Ser Asn Thr Cys Glu Ile Ile Asn245 250 255gcg ctg tcc tgg cgg ctg atg gcc gcg ctg cgc gag gac gac ttc gcg816Ala Leu Ser Trp Arg Leu Met Ala Ala Leu Arg Glu Asp Asp Phe Ala260 265 270gtc gag cgc ttc gcc tac gtg gag gaa ctg gag cag ggc ctg ctg gac864Val Glu Arg Phe Ala Tyr Val Glu Glu Leu Glu Gln Gly Leu Leu Asp275 280 285tgg aac gac aag ctg gtc aac aac tcc ttc atc tcc ttc tcg cac tac912Trp Asn Asp Lys Leu Val Asn Asn Ser Phe Ile Ser Phe Ser His Tyr290 295 300ccg ctg tgg aac tcg gcg ttc cgg atc tgg gcc tcg gcc agc gtg atc960Pro Leu Trp Asn Ser Ala Phe Arg Ile Trp Ala Ser Ala Ser Val Ile305 310 315 320ggc ggc aag cgc atc ctc aac gca ctg acc agg acc aag gag acc ggc1008Gly Gly Lys Arg Ile Leu Asn Ala Leu Thr Arg Thr Lys Glu Thr Gly325 330 335gac gac agc cac tgc cag gcg ctg gac gac aac ccg tac ccg ggc ctg1056Asp Asp Ser His Cys Gln Ala Leu Asp Asp Asn Pro Tyr Pro Gly Leu340 345 350tgg tgt ccg ctg gac ttc tac aag gag gcc ttc gac gag ctc acc gag1104Trp Cys Pro Leu Asp Phe Tyr Lys Glu Ala Phe Asp Glu Leu Thr Glu355 360 365ctg tgc gag gcc gtg gac gcc ggg cac acc acg gcc gag gag gcc gcg1152Leu Cys Glu Ala Val Asp Ala Gly His Thr Thr Ala Glu Glu Ala Ala370 375 380cgg ctg ctg gag cag cgg gtc cgc gag tcg gac tgg atg ctg ccg gcc1200Arg Leu Leu Glu Gln Arg Val Arg Glu Ser Asp Trp Met Leu Pro Ala385 390 395 400ctg ggc ttc aac gac ccc gac acc cac cac atc aac ccg acg gcg gac1248Leu Gly Phe Asn Asp Pro Asp Thr His His Ile Asn Pro Thr Ala Asp405 410 415aag atg atc cgg atc gcg gag tgg gcc acc ggt cac cac cgc ccg gag1296Lys Met Ile Arg Ile Ala Glu Trp Ala Thr Gly His His Arg Pro Glu420 425 430atc cgt gag ctg ctg gcc gcc agc gcc gag gag gtc agg gcg gcg atg1344Ile Arg Glu Leu Leu Ala Ala Ser Ala Glu Glu Val Arg Ala Ala Met435 440 445cgg gtc aag ccg taa1359Arg Val Lys Pro450<210>15<211>452<212>PRT<213>金霉素鏈霉菌<400>15Met Phe His Arg Asp Gly Glu Glu Pro Asp Pro Asn Glu Thr Ser Gln1 5 10 15Phe Arg Ile Pro Ser Ile Val Gly Asn Ala Ala His Phe Phe Arg Gln
20 25 30Asp Thr Asp Ser Tyr Met Phe His Ala Ala Val Arg Tyr Gly Cys Asp35 40 45Ala Arg Gln Tyr Tyr Arg Val Glu Asn Ile Glu Phe Asp Asp Gly Gly50 55 60Val Thr Val Ser Gly Ala Asp Gly Ser Thr Val Arg Ala Arg Tyr Leu65 70 75 80Val Asp Ala Ser Gly Phe Arg Ser Pro Leu Ala Arg Gln Leu Gly Leu85 90 95Arg Glu Glu Pro Ser Arg Leu Lys His His AIa Arg Ser Ile Phe Thr100 105 110His Met Val Gly Val Asp Ala Ile Asp Asp His Val Asp Met Pro Ala115 120 125Glu Leu Arg Pro Pro Val Pro Trp Asn Asp Gly Thr Met His His Ile130 135 140Phe Glu Arg Gly Trp Met Trp Ile Ile Pro Phe Asn Asn His Pro Gly145 150 155 160Ala Thr Asn Pro Leu Cys Ser Val Gly Ile Gln Leu Asp Glu Arg Arg165 170 175Tyr Pro Ala Arg Pro Asp Leu Thr Pro Glu Glu Glu Phe Arg Ser His
180 185 190Val Asp Arg Phe Pro Ala Val Gln Arg Gln Leu Lys Gly Ala Arg Ser195 200 205Val Arg Glu Trp Val Arg Thr Asp Arg Met Gln Tyr Ser Ser Ser Arg210 215 220Thr Val Gly Glu Arg Trp Cys Leu Met Ser His Ala Ala Gly Phe Ile225 230 235 240Asp Pro Leu Phe Leu Arg Gly Leu Ser Asn Thr Cys Glu Ile Ile Asn245 250 255Ala Leu Ser Trp Arg Leu Met Ala Ala Leu Arg Glu Asp Asp Phe Ala260 265 270Val Glu Arg Phe Ala Tyr Val Glu Glu Leu Glu Gln Gly Leu Leu Asp275 280 235Trp Asn Asp Lys Leu Val Asn Asn Ser Phe Ile Ser Phe Ser His Tyr290 295 300Pro Leu Trp Asn Ser Ala Phe Arg Ile Trp Ala Ser Ala Ser Val Ile305 310 315 320Gly Gly Lys Arg Ile Leu Asn Ala Leu Thr Arg Thr Lys Glu Thr Gly325 330 335Asp Asp Ser His Cys Gln Ala Leu Asp Asp Asn Pro Tyr Pro Gly Leu
340 345 350Trp Cys Pro Leu Asp Phe Tyr Lys Glu Ala Phe Asp Glu Leu Thr Glu355 360 365Leu Cys Glu Ala Val Asp Ala Gly His Thr Thr Ala Glu Glu Ala Ala370 375 380Arg Leu Leu Glu Gln Arg Val Arg Glu Ser Asp Trp Met Leu Prc Ala385 390 395 400Leu Gly Phe Asn Asp Pro Asp Thr His His Ile Asn Pro Thr Ala Asp405 410 415Lys Met Ile Arg Ile Ala Glu Trp Ala Thr Gly His His Arg Pro Glu420 425 430Ile Arg Glu Leu Leu Ala Ala Ser Ala Glu Glu Val Arg Ala Ala Met435 440 445Arg Val Lys Pro450<210>16<211>1476<212>DNA<213>地中海擬無枝酸菌(Amycolatopsis mediterranei)<220><221>CDS<222>(1)..(1476)<400>16atg tcg gtc gaa gac ttc gac gtg gtg gtg gcg ggc ggc ggg ccg ggt48Met Ser Val Glu Asp Phe Asp Val Val Val Ala Gly Gly Gly Pro Gly1 5 10 15ggt tcg acg gtg gcc acg ctg gtg gcc atg cag gga cac cgg gtg ctg96Gly Ser Thr Val Ala Thr Leu Val Ala Met Gln Gly His Arg Val Leu20 25 30ctg ctg gag aaa gag gtt ttc ccg cgg tat cag atc ggt gag tcg ctg144Leu Leu Glu Lys Glu Val Phe Pro Arg Tyr Gln Ile Gly Glu Ser Leu35 40 45ctg ccc gcc acg gtg cac ggc gtg tgc cgg atg ctc ggc atc tcc gac192Leu Pro Ala Thr Val His Gly Val Cys Arg Met Leu Gly Ile Ser Asp50 55 60gag ctg gcc aat gcc ggg ttc ccg atc aag cgc ggc ggc acg ttc cgc240Glu Leu Ala Asn Ala Gly Phe Pro Ile Lys Arg Gly Gly Thr Phe Arg65 70 75 80tgg ggc gcc cgg ccg gag ccg tgg acg ttc cac ttc ggc atc tcg gcc288Trp Gly Ala Arg Pro Glu Pro Trp Thr Phe His Phe Gly Ile Ser Ala85 90 95aag atg gcc ggc tcg acg tcg cac gcc tac cag gtc gag cgg gcg cgg336Lys Met Ala Gly Ser Thr Ser His Ala Tyr Gln Val Glu Arg Ala Arg
100 105 110ttc gac gag atg ctg ctg aac aac gcc aag cgc aag ggc gtg gtc gtg384Phe Asp Glu Met Leu Leu Asn Asn Ala Lys Arg Lys Gly Val Val Val115 120 125cgg gag ggg tgc gcg gtc acc gat gtg gtg gaa gac ggc gag cgg gtc432Arg Glu Gly Cys Ala Val Thr Asp Val Val Glu Asp Gly Glu Arg Val130 135 140acc ggt gcg cgg tac acc gat ccc gac ggc acc gag cgg gaa gtg tcg480Thr Gly Ala Arg Tyr Thr Asp Pro Asp Gly Thr Glu Arg Glu Val Ser145 150 155 160gcg cgg ttc gtg atc gac gcg tcg ggc aac aag agc cgg ctc tac acc528Ala Arg Phe Val Ile Asp Ala Ser Gly Asn Lys Ser Arg Leu Tyr Thr165 170 175aag gtc ggc ggt tcg cgg aac tat tcg gag ttc ttc cgc agc ctc gcg576Lys Val Gly Gly Ser Arg Asn Tyr Ser Glu Phe Phe Arg Ser Leu Ala180 185 190ctg ttc ggt tac ttc gag ggt ggc aag cgg ctg ccc gag ccg gtc tcc624Leu Phe Gly Tyr Phe Glu Gly Gly Lys Arg Leu Pro Glu Pro Val Ser195 200 205ggg aac atc ctg agt gtg gcc ttc gac agc ggc tgg ttc tgg tac atc672Gly Asn Ile Leu Ser Val Ala Phe Asp Ser Gly Trp Phe Trp Tyr Ile210 215 220ccg ctg agc gac acg ctg acc agc gtc ggc gcg gtg gtg cgc cgg gag720Pro Leu Ser Asp Thr Leu Thr Ser Val Gly Ala Val Val Arg Arg Glu225 230 235 240gac gcc gag aag atc cag ggt gac cgg gag aag gcc ctc aac acg ctg768Asp Ala Glu Lys Ile Gln Gly Asp Arg Glu Lys Ala Leu Asn Thr Leu245 250 255atc gcc gag tgc ccg ctg atc tcg gaa tac ctc gcg gac gcg acc cgg816Ile Ala Glu Cys Pro Leu Ile Ser Glu Tyr Leu Ala Asp Ala Thr Arg260 265 270gtg acg acc ggc cgg tac ggg gaa ctg cgc gtc cgc aag gac tac tcc864Val Thr Thr Gly Arg Tyr Gly Glu Leu Arg Val Arg Lys Asp Tyr Ser275 280 285tac cag cag gag acc tac tgg cgg ccg ggc atg atc ctg gtc ggc gac912Tyr Gln Gln Glu Thr Tyr Trp Arg Pro Gly Met Ile Leu Val Gly Asp290 295 300gcc gcg tgt ttc gtg gac ccg gtg ttc tcc tcc ggt gtg cac ctg gcg960Ala Ala Cys Phe Val Asp Pro Val Phe Ser Ser Gly Val His Leu Ala305 310 315 320acc tac agc gcg ctg ctc gcg gcc cgg tcg atc aac agc gtc ctc gcc1008Thr Tyr Ser Ala Leu Leu Ala Ala Arg Ser Ile Asn Ser Val Leu Ala325 330 335ggc gac ctg gac gag aag acc gcg ctg aac gag ttc gag ctg cgg tat1056Gly Asp Leu Asp Glu Lys Thr Ala Leu Asn Glu Phe Glu Leu Arg Tyr
340 345 350cgc cgt gag tac ggc gtg ttc tac gag ttc ctc gtg tcc ttc tac cag1104Arg Arg Glu Tyr Gly Val Phe Tyr Glu Phe Leu Val Ser Phe Tyr Gln355 360 365atg aac gtg aac gag gag tcg tac ttc tgg cag gcc aag aag gtc acg1152Met Asn Val Asn Glu Glu Ser Tyr Phe Trp Gln Ala Lys Lys Val Thr370 375 380cag aac cag agc acc gac gtc gag tcg ttc gtc gag ctg atc ggc gga1200Gln Asn Gln Ser Thr Asp Val Glu Ser Phe Val Glu Leu Ile Gly Gly385 390 395 400gtg tcg tcc ggg gag acc gcg ctg acg gcc gcc gac cgc atc gcc gcg1248Val Ser Ser Gly Glu Thr Ala Leu Thr Ala Ala Asp Arg Ile Ala Ala405 410 415cgc agt gcc gag ttc gcc gcg gcg gtg gac gag atg gcg ggc ggg gac1296Arg Ser Ala Glu Phe Ala Ala Ala Val Asp Glu Met Ala Gly Gly Asp420 425 430ggc gac aac atg gtg ccg atg ttc aag tcg acg gtg gtc cag cag gcg1344Gly Asp Asn Met Val Pro Met Phe Lys Ser Thr Val Val Gln Gln Ala435 440 445atg cag gaa gcg ggc cag gtg cag atg aag gcg ctg ctc ggc gag gac1392Met Gln Glu Ala Gly Gln Val Gln Met Lys Ala Leu Leu Gly Glu Asp450 455 460gcc gaa ccc gag ctg ccc ctg ttc ccc ggt ggc ctg gtg acc tcg ccc1440Ala Glu Pro Glu Leu Pro Leu Phe Pro Gly Gly Leu Val Thr Ser Pro465 470 475 480gaa cgg atg aag tgg ctg cct cac cac cct gcg tga1476Glu Arg Met Lys Trp Leu Pro His His Pro Ala485 490<210>17<211>491<212>PRT<213>地中海擬無枝酸菌<400>17Met Ser Val Glu Asp Phe Asp Val Val Val Ala Gly Gly Gly Pro Gly1 5 10 15Gly Ser Thr Val Ala Thr Leu Val Ala Met Gln Gly His Arg Val Leu20 25 30Leu Leu Glu Lys Glu Val Phe Pro Arg Tyr Gln Ile Gly Glu Ser Leu35 40 45Leu Pro Ala Thr Val His Gly Val Cys Arg Met Leu Gly Ile Ser Asp50 55 60Glu Leu Ala Asn Ala Gly Phe Pro Ile Lys Arg Gly Gly Thr Phe Arg65 70 75 80Trp Gly Ala Arg Pro Glu Pro Trp Thr Phe His Phe Gly Ile Ser Ala85 90 95Lys Met Ala Gly Ser Thr Ser His Ala Tyr Gln Val Glu Arg Ala Arg100 105 110Phe Asp Glu Met Leu Leu Asn Asn Ala Lys Arg Lys Gly Val Val Val115 120 125Arg Glu Gly Cys Ala Val Thr Asp Val Val Glu Asp Gly Glu Arg Val130 135 140Thr Gly Ala Arg Tyr Thr Asp Pro Asp Gly Thr Glu Arg Glu Val Ser145 150 155 160Ala Arg Phe Val Ile Asp Ala Ser Gly Asn Lys Ser Arg Leu Tyr Thr165 170 175Lys Val Gly Gly Ser Arg Asn Tyr Ser Glu Phe Phe Arg Ser Leu Ala180 185 190Leu Phe Gly Tyr Phe Glu Gly Gly Lys Arg Leu Pro Glu Pro Val Ser195 200 205Gly Asn Ile Leu Ser Val Ala Phe Asp Ser Gly Trp Phe Trp Tyr Ile210 215 220Pro Leu Ser Asp Thr Leu Thr Ser Val Gly Ala Val Val Arg Arg Glu225 230 235 240Asp Ala Glu Lys Ile Gln Gly Asp Arg Glu Lys Ala Leu Asn Thr Leu245 250 255Ile Ala Glu Cys Pro Leu Ile Ser Glu Tyr Leu Ala Asp Ala Thr Arg260 265 270Val Thr Thr Gly Arg Tyr Gly Glu Leu Arg Val Arg Lys Asp Tyr Ser275 280 285Tyr Gln Gln Glu Thr Tyr Trp Arg Pro Gly Met Ile Leu Val Gly Asp290 295 300Ala Ala Cys Phe Val Asp Pro Val Phe Ser Ser Gly Val His Leu Ala305 310 315 320Thr Tyr Ser Ala Leu Leu Ala Ala Arg Ser Ile Asn Ser Val Leu Ala325 330 335Gly Asp Leu Asp Glu Lys Thr Ala Leu Asn Glu Phe Glu Leu Arg Tyr340 345 350Arg Arg Glu Tyr Gly Val Phe Tyr Glu Phe Leu Val Ser Phe Tyr Gln355 360 365Met Asn Val Asn Glu Glu Ser Tyr Phe Trp Gln Ala Lys Lys Val Thr370 375 380Gln Asn Gln Ser Thr Asp Val Glu Ser Phe Val Glu Leu Ile Gly Gly385 390 395 400Val Ser Ser Gly Glu Thr Ala Leu Thr Ala Ala Asp Arg Ile Ala Ala405 410 415Arg Ser Ala Glu Phe Ala Ala Ala Val Asp Glu Met Ala Gly Gly Asp420 425 430Gly Asp Asn Met Val Pro Met Phe Lys Ser Thr Val Val Gln Gln Ala435 440 445Met Gln Glu Ala Gly Gln Val Gln Met Lys Ala Leu Leu Gly Glu Asp450 455 460Ala Glu Pro Glu Leu Pro Leu Phe Pro Gly Gly Leu Val Thr Ser Pro465 470 475 480Glu Arg Met Lys Trp Leu Pro His His Pro Ala485 490<210>18<211>702<212>DNA<213>大腸桿菌(Escherichia coli)<220><221>CDS<222>(1)..(702)<400>18atg aca acc tta agc tgt aaa gtg acc tcg gta gaa gct atc acg gat48Met Thr Thr Leu Ser Cys Lys Val Thr Ser Val Glu Ala Ile Thr Asp1 5 10 15acc gta tat cgt gtc cgc atc gtg cca gac gcg gcc ttt tct ttt cgt96Thr Val Tyr Arg Val Arg Ile Val Pro Asp Ala Ala Phe Ser Phc Arg20 25 30gct ggt cag tat ttg atg gta gtg atg gat gag cgc gac aaa cgt ccg144Ala Gly Gln Tyr Leu Met Val Val Met Asp Glu Arg Asp Lys Arg Pro35 40 45ttc tca atg gct tcg acg ccg gat gaa aaa ggg ttt atc gag ctg cat192Phe Ser Met Ala Ser Thr Pro Asp Glu Lys Gly Phe Ile Glu Leu His50 55 60att ggc gct tct gaa atc aac ctt tac gcg aaa gca gtc atg gac cgc240Ile Gly Ala Ser Glu Ile Asn Leu Tyr Ala Lys Ala Val Met Asp Arg65 70 75 80atc ctc aaa gat cat caa atc gtg gtc gac att ccc cac gga gaa gcg288Ile Leu Lys Asp His Gln Ile Val Val Asp Ile Pro His Gly Glu Ala85 90 95tgg ctg cgc gat gat gaa gag cgt ccg atg att ttg att gcg ggc ggc336Trp Leu Arg Asp Asp Glu Glu Arg Pro Met Ile Leu Ile Ala Gly Gly100 105 110acc ggg ttc tct tat gcc cgc tcg att ttg ctg aca gcg ttg gcg cgt384Thr Gly Phe Ser Tyr Ala Arg Ser Ile Leu Leu Thr Ala Leu Ala Arg115 120 125aac cca aac cgt gat atc acc att tac tgg ggc ggg cgt gaa gag cag432Asn Pro Asn Arg Asp Ile Thr Ile Tyr Trp Gly Gly Arg Glu Glu Gln130 135 140cat ctg tat gat ctc tgc gag ctt gag gcg ctt tcg ttg aag cat cct480His Leu Tyr Asp Leu Cys Glu Leu Glu Ala Leu Ser Leu Lys His Pro145 150 155 160ggt ctg caa gtg gtg ccg gtg gtt gaa caa ccg gaa gcg ggc tgg cgt528Gly Leu Gln Val Val Pro Val Val Glu Gln Pro Glu Ala Gly Trp Arg165 170 175ggg cgt act ggc acc gtg tta acg gcg gta ttg cag gat cac ggt acg576Gly Arg Thr Gly Thr Val Leu Thr Ala Val Leu Gln Asp His Gly Thr180 185 190ctg gca gag cat gat atc tat att gcc gga cgt ttt gag atg gcg aaa624Leu Ala Glu His Asp Ile Tyr Ile Ala Gly Arg Phe Glu Met Ala Lys195 200 205att gcc cgc gat ctg ttt tgc agt gag cgt aat gcg cgg gaa gat cgc672Ile Ala Arg Asp Leu Phe Cys Ser Glu Arg Asn Ala Arg Glu Asp Arg210 215 220ctg ttt ggc gat gcg ttt gca ttt atc tga702Leu Phe Gly Asp Ala Phe Ala Phe Ile225 230<210>19<211>233<212>PRT<213>大腸桿菌<400>19Met Thr Thr Leu Ser Cys Lys Val Thr Ser Val Glu Ala Ile Thr Asp1 5 10 15Thr Val Tyr Arg Val Arg Ile Val Pro Asp Ala Ala Phe Ser Phe Arg20 25 30Ala Gly Gln Tyr Leu Met Val Val Met Asp Glu Arg Asp Lys Arg Pro35 40 45Phe Ser Met Ala Ser Thr Pro Asp Glu Lys Gly Phe Ile Glu Leu His50 55 60Ile Gly Ala Ser Glu Ile Asn Leu Tyr Ala Lys Ala Val Met Asp Arg65 70 75 80Ile Leu Lys Asp His Gln Ile Val Val Asp Ile Pro His Gly Glu Ala85 90 95Trp Leu Arg Asp Asp Glu Glu Arg Pro Met Ile Leu Ile Ala Gly Gly100 105 110Thr Gly Phe Ser Tyr Ala Arg Ser Ile Leu Leu Thr Ala Leu Ala Arg115 120 125Asn Pro Asn Arg Asp Ile Thr Ile Tyr Trp Gly Gly Arg Glu Glu Gln130 135 140His Leu Tyr Asp Leu Cys Glu Leu Glu Ala Leu Ser Leu Lys His Pro145 150 155 160Gly Leu Gln Val Val Pro Val Val Glu Gln Pro Glu Ala Gly Trp Arg165 170 175Gly Arg Thr Gly Thr Val Leu Thr Ala Val Leu Gln Asp His Gly Thr180 185 190Leu Ala Glu His Asp Ile Tyr Ile Ala Gly Arg Phe Glu Met Ala Lys195 200 205Ile Ala Arg Asp Leu Phe Cys Ser Glu Arg Asn Ala Arg Glu Asp Arg210 215 220Leu Phe Gly Asp Ala Phe Ala Phe Ile225 230<210>20<211>906<212>DNA<213>大鼠<220><221>CDS<222>(1)..(906)<400>20atg ggg gcc cag ctg agc acg ttg agc cga gtg gta ctc tcc ccg gtc48Met Gly Ala Gln Leu Ser Thr Leu Ser Arg Val Val Leu Ser Pro Val1 5 10 15tgg ttc gtc tac agc ctc ttc atg aag ctg ttt cag cgc tcc tca ccg96Trp Phe Val Tyr Ser Leu Phe Met Lys Leu Phe Gln Arg Ser Ser Pro20 25 30gcc atc acc ctc gag aac ccc gac atc aag tac cct ctg cgg ctc atc144Ala Ile Thr Leu Glu Asn Pro Asp Ile Lys Tyr Pro Leu Arg Leu Ile35 40 45gac aag gag att atc agc cat gac act cgg cgc ttc cga ttt gca ctc192Asp Lys Glu Ile Ile Ser His Asp Thr Arg Arg Phe Arg Phe Ala Leu50 55 60cct tcg ccc cag cac atc ctg ggc ctt cct atc ggc cag cac atc tac240Pro Ser Pro Gln His Ile Leu Gly Leu Pro Ile Gly Gln His Ile Tyr65 70 75 80ctc tcc acc agg atc gat ggc aac ttg gtc att cgt ccc tac acc cct288Leu Ser Thr Arg Ile Asp Gly Asn Leu Val Ile Arg Pro Tyr Thr Pro85 90 95gtg tct agt gat gat gac aag ggc ctt gtg gac ttg gtg gtc aag gtt336Val Ser Ser Asp Asp Asp Lys Gly Leu Val Asp Leu Val Val Lys Val100 105 110tac ttc aag gac acg cat ccc aag ttt cca gct gga ggg aaa atg tct384Tyr Phe Lys Asp Thr His Pro Lys Phe Pro Ala Gly Gly Lys Met Ser115 120 125cag tac ctg gaa aac atg aat att gga gac acc att gaa ttc cgg ggc432Gln Tyr Leu Glu Asn Met Asn Ile Gly Asp Thr Ile Glu Phe Arg Gly130 135 140ccc aat ggg cta ctg gtc tac cag ggc aaa ggg aag ttc gcc atc cgt480Pro Asn Gly Leu Leu Val Tyr Gln Gly Lys Gly Lys Phe Ala Ile Arg145 150 155 160gca gac aag aag tcc aac cct gtt gtc agg acg gtg aag tct gta ggc528Ala Asp Lys Lys Ser Asn Pro Val Val Arg Thr Val Lys Ser Val Gly165 170 175atg att gca gga ggg aca ggc atc acc cca atg ctg cag gtg atc cga576Met Ile Ala Gly Gly Thr Gly Ile Thr Pro Met Leu Gln Val Ile Arg180 185 190gcc gtc ttg aag gac ccg aac gac cac act gtg tgc tat ctg ctc ttc624Ala Val Leu Lys Asp Pro Asn Asp His Thr Val Cys Tyr Leu Leu Phe195 200 205gcc aac cag tcc gag aaa gac atc ctg ctg cgg cct gag ctg gag gaa672Ala Asn Gln Ser Glu Lys Asp Ile Leu Leu Arg Pro Glu Leu Glu Glu210 215 220ctg agg aac gaa cat tct tct cgc ttc aag ctc tgg tac aca gtg gac720Leu Arg Asn Glu His Ser Ser Arg Phe Lys Leu Trp Tyr Thr Val Asp225 230235 240aaa gcc ccc gat gcc tgg gac tat agc caa ggc ttc gtg aat gag gag768Lys Ala Pro Asp Ala Trp Asp Tyr Ser Gln Gly Phe Val Asn Glu Glu245 250 255atg atc agg gac cat ctt cca cct cct ggg gag gag aca ctg ata ctg816Met Ile Arg Asp His Leu Pro Pro Pro Gly Glu Glu Thr Leu Ile Leu260 265 270atg tgt gga ccc cca ccg atg atc cag ttt gcc tgt ttg cca aac ctg864Met Cys Gly Pro Pro Pro Met Ile Gln Phe Ala Cys Leu Pro Asn Leu275 280 285gag cgt gtg ggc cat ccc aag gag cga tgc ttc acc ttc tga906Glu Arg Val Gly His Pro Lys Glu Arg Cys Phe Thr Phe290 295 300<210>21<211>301<212>PRT<213>大鼠<400>21Met Gly Ala Gln Leu Ser Thr Leu Ser Arg Val Val Leu Ser Pro Val1 5 10 15Trp Phe Val Tyr Ser Leu Phe Met Lys Leu Phe Gln Arg Ser Ser Pro20 25 30Ala Ile Thr Leu Glu Asn Pro Asp Ile Lys Tyr Pro Leu Arg Leu Ile35 40 45Asp Lys Glu Ile Ile Ser His Asp Thr Arg Arg Phe Arg Phe Ala Leu50 55 60Pro Ser Pro Gln His Ile Leu Gly Leu Pro Ile Gly Gln His Ile Tyr65 70 75 80Leu Ser Thr Arg Ile Asp Gly Asn Leu Val Ile Arg Pro Tyr Thr Pro85 90 95Val Ser Ser Asp Asp Asp Lys Gly Leu Val Asp Leu Val Val Lys Val100 105 110Tyr Phe Lys Asp Thr His Pro Lys Phe Pro Ala Gly Gly Lys Met Ser115 120 125Gln Tyr Leu Glu Asn Met Asn Ile Gly Asp Thr Ile Glu Phe Arg Gly130 135 140Pro Asn Gly Leu Leu Val Tyr Gln Gly Lys Gly Lys Phe Ala Ile Arg145 150 155 160Ala Asp Lys Lys Ser Asn Pro Val Val Arg Thr Val Lys Ser Val Gly165 170 175Met Ile Ala Gly Gly Thr Gly Ile Thr Pro Met Leu Gln Val Ile Arg180 185 190Ala Val Leu Lys Asp Pro Asn Asp His Thr Val Cys Tyr Leu Leu Phe195 200 205Ala Asn Gln Ser Glu Lys Asp Ile Leu Leu Arg Pro Glu Leu Glu Glu210 215 220Leu Arg Asn Glu His Ser Ser Arg Phe Lys Leu Trp Tyr Thr Val Asp225 230 235 240Lys Ala Pro Asp Ala Trp Asp Tyr Ser Gln Gly Phe Val Asn Glu Glu245 250 255Met Ile Arg Asp His Leu Pro Pro Pro Gly Glu Glu Thr Leu Ile Leu260 265 270Met Cys Gly Pro Pro Pro Met Ile Gln Phe Ala Cys Leu Pro Asn Leu275 280 285Glu Arg Val Gly His Pro Lys Glu Arg Cys Phe Thr Phe290 295 300<210>22<211>2049<212>DNA<213>兔<220><221>CDS<222>(1)..(2049)<400>22ctg atc aac atg gcg gac tcc cac ggg gac acc ggc gcc acc atg cct48Leu Ile Asn Met Ala Asp Ser His Gly Asp Thr Gly Ala Thr Met Pro1 5 10 15gaa gcg gcg gcc cag gag gcg tcg gtc ttc agc atg acg gac gtg gtt96Glu Ala Ala Ala Gln Glu Ala Ser Val Phe Ser Met Thr Asp Val Val20 25 30ctg ttc tcg ctc atc gtg ggg ctg atc acc tac tgg ttc ctc ttc aga144Leu Phe Ser Leu Ile Val Gly Leu Ile Thr Tyr Trp Phe Leu Phe Arg35 40 45aag aaa aag gag gaa gtg ccc gag ttc acc aag atc cag gcc ccg acg192Lys Lys Lys Glu Glu Val Pro Glu Phe Thr Lys Ile Gln Ala Pro Thr50 55 60tcg tcg tca gtg aag gag agc agc ttc gtg gag aag atg aag aag acg240Ser Ser Ser Val Lys Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr65 70 75 80ggc cgg aac atc gtg gtc ttc tac ggc tcc cag acg ggc acc gcc gag288Gly Arg Asn Ile Val Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu85 90 95gag ttt gcc aac cgc ctg tcc aag gat gcc cac cgc tac ggg atg cgg336Glu Phe Ala Asn Arg Leu Ser Lys Asp Ala His Arg Tyr Gly Met Arg100 105 110ggc atg gcc gcc gac ccc gag gag tac gac ctg gcc gac ctg agc agc384Gly Met Ala Ala Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser115 120 125ctg ccc gag atc aac aac gcc ctg gcc gtc ttc tgc atg gcc acc tac432Leu Pro Glu Ile Asn Asn Ala Leu Ala Val Phe Cys Met Ala Thr Tyr130 135 140ggt gag ggg gac ccc acc gac aac gcc cag gac ttc tac gac tgg ctg480Gly Glu Gly Asp Pro Thr Asp Asn Ala Gln Asp Phe Tyr Asp Trp Leu145 150 155 160cag gag acc gac gtg gac ctc tcg ggg gtc aag tac gcg gtg ttt ggc528Gln Glu Thr Asp Val Asp Leu Ser Gly Val Lys Tyr Ala Val Phe Gly165 170 175ctc ggg aac aag acc tac gag cac ttc aac gcc atg ggc aag tac gtg576Leu Gly ASn Lys Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val180 185 190gac cag cgg ctg gag cag ctt ggc gcc cag cgc atc ttc gag ctg ggc624Asp Gln Arg Leu Glu Gln Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly195 200 205atg ggc gac gat gat gca aac ctg gag gag gac ttc atc acg tgg cgg672Met Gly Asp Asp Asp Ala Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg210 215 220gag cag ttc tgg ccg gcg gtg tgc gag cac ttc ggt gtg gag gcc aca720Glu Gln Phe Trp Pro Ala Val Cys Glu His Phe Gly Val Glu Ala Thr225 230 235 240gga gag gag tcc agc att cgg cag tac gag ctc gtg ttg cac aca gac768Gly Glu Glu Ser Ser Ile Arg Gln Tyr Glu Leu Val Leu His Thr Asp245 250 255atc gac gtg gcc aag gtg tac cag ggc gag atg ggc cgc ctc aag agc816Ile Asp Val Ala Lys Val Tyr Gln Gly Glu Met Gly Arg Leu Lys Ser260 265 270tac gag aac cag aaa ccc ccc ttc gat gcc aag aat ccc ttc ctg gcc864Tyr Glu Asn Gln Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala275 280 285acg gtc acc acc aac cgg aag ctg aac cag ggc acc gag cgc cac ctc912Thr Val Thr Thr Asn Arg Lys Leu Asn Gln Gly Thr Glu Arg His Leu290 295 300atg cac ctg gag ctg gac atc tcg gac tcc aag atc agg tat gag tct960Met His Leu Glu Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser305 310 315 320ggg gac cac gtg gct gtg tat ccg gcc aac gac tct gcc ctc gtc aac1008Gly Asp His Val Ala Val Tyr Pro Ala Asn Asp Ser Ala Leu Val Asn325 330 335cag ctg ggg gag atc ctg ggt gcc gac ctg gac gtc gtc atg tcc ctg1056Gln Leu Gly Glu Ile Leu Gly Ala Asp Leu Asp Val Val Met Ser Leu340 345 350aac aac ctc gat gag gag tcc aac aag aag cac cca ttc ccc tgc ccc1104Asn Asn Leu Asp Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro355 360 365act tcc tac cgc acg gcc ctc acc tac tac ctg gac atc acc aac ccg1152Thr Ser Tyr Arg Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro370 375 380ccg cgc acc aac gtg ctc tac gag ctg gcc cag tac gcc gcc gac ccc1200Pro Arg Thr Asn Val Leu Tyr Glu Leu Ala Gln Tyr Ala Ala Asp Pro385 390 395 400gct gag cag gag cag ctg cgc aag atg gcc tca tcc tcg ggc gag ggc1248Ala Glu Gln Glu Gln Leu Arg Lys Met Ala Ser Ser Ser Gly Glu Gly405 410 415aag gag ctg tac ctg agc tgg gtg gta gag gcg cgg agg cac atc ctg1296Lys Glu Leu Tyr Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu420 425 430gcc atc ctc caa gac tac ccg tcc ctg cgg ccg ccc atc gac cac ctg1344Ala Ile Leu Gln Asp Tyr Pro Ser Leu Arg Pro Pro Ile Asp His Leu435 440 445tgt gag ctg ctg ccc cgg ctg cag gcg cgc tac tac tcc atc gcc tcc1392Cys Glu Leu Leu Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala Ser450 455 460tcc tcc aag gtc cac ccc aac tcc gtg cac atc tgc gcc gtg gcc gtg1440Ser Ser Lys Val His Pro Asn Ser Val His Ile Cys Ala Val Ala Val465 470 475 480gag tac gag acc aag gcc ggc cgc ctc aac aaa ggc gtg gcc acc agc1488Glu Tyr Glu Thr Lys Ala Gly Arg Leu Asn Lys Gly Val Ala Thr Ser485 490 495tgg ctg cgg gcc aag gag ccg gcc ggg gag aat ggc ggc cgt gcc ctg1536Trp Leu Arg Ala Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala Leu500 505 510gtg ccc atg ttc gtg cgc aag tcc cag ttc cgc ctg ccc ttc aag gcc1584Val Pro Met Phe Val Arg Lys Ser Gln Phe Arg Leu Pro Phe Lys Ala515 520 525acc acg ccg gtc atc atg gtg ggc ccc ggc acc ggc gtg gcc ccc ttc1632Thr Thr Pro Val Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe530 535 540atc ggc ttt atc cag gag cgg gcc tgg ctg cgg cag cag ggc aag gaa1680Ile Gly Phe Ile Gln Glu Arg Ala Trp Leu Arg Gln Gln Gly Lys Glu545 550 555 560gtg ggc gag acg ctg ctg tac tac ggc tgc cgg cgc gcg gcc gag gac1728Val Gly Glu Thr Leu Leu Tyr Tyr Gly Cys Arg Arg Ala Ala Glu Asp565 570 575tac ctg tac cgc gag gag ctc gcc ggc ttc caa aag gac ggc acg ctc1776Tyr Leu Tyr Arg Glu Glu Leu Ala Gly Phe Gln Lys Asp Gly Thr Leu580 585 590agc cag ctc aac gtg gcc ttc tcc cgc gag cag gcc cag aag gtc tac1824Ser Gln Leu Asn Val Ala Phe Ser Arg Glu Gln Ala Gln Lys Va1 Tyr595 600 605gtg cag cac ttg ctg agg agg gac aag gag cac ctg tgg cgg ctc atc1872Val Gln His Leu Leu Arg Arg Asp Lys Glu His Leu Trp Arg Leu Ile610 615 620cac gag ggg ggc gcc cac atc tac gtg tgc ggg gac gct cgg aac atg1920His Glu Gly Gly Ala His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met625 630 635 640gcc agg gac gtg cag aac acc ttc tac gac atc gtg gcc gag ctg ggg1968Ala Arg Asp Val Gln Asn Thr Phe Tyr Asp Ile Val Ala Glu Leu Gly645 650 655gcc atg gag cac gcg cag gcc gtg gac tac gtg aag aag ctc atg acc2016Ala Met Glu His Ala Gln Ala Val Asp Tyr Val Lys Lys Leu Met Thr660 665 670aag ggc cgc tac tcc ctg gac gtg tgg agc tag2049Lys Gly Arg Tyr Ser Leu Asp Val Trp Ser675 680<210>23<211>682<212>PRT<213>兔<400>23Leu Ile Asn Met Ala Asp Ser His Gly Asp Thr Gly Ala Thr Met Pro1 5 10 15Glu Ala Ala Ala Gln Glu Ala Ser Val Phe Ser Met Thr Asp Val Val20 25 30Leu Phe Ser Leu Ile Val Gly Leu Ile Thr Tyr Trp Phe Leu Phe Arg35 40 45Lys Lys Lys Glu Glu Val Pro Glu Phe Thr Lys Ile Gln Ala Pre Thr50 55 60Ser Ser Ser Val Lys Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr65 70 75 80Gly Arg Asn Ile Val Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu85 90 95Glu Phe Ala Asn Arg Leu Ser Lys Asp Ala His Arg Tyr Gly Met Arg100 105 110Gly Met Ala Ala Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser115 120 125Leu Pro Glu Ile Asn Asn Ala Leu Ala Val Phe Cys Met Ala Thr Tyr130 135 140Gly Glu Gly Asp Pro Thr Asp Asn Ala Gln Asp Phe Tyr Asp Trp Leu145 150 155 160Gln Glu Thr Asp Val Asp Leu Ser Gly Val Lys Tyr Ala Val Phe Gly165 170 175Leu Gly Asn Lys Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val180 185 190Asp Gln Arg Leu Glu Gln Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly195 200 205Met Gly Asp Asp Asp Ala Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg210 215 220Glu Gln Phe Trp Pro Ala Val Cys Glu His Phe Gly Val Glu Ala Thr225 230 235 240Gly Glu Glu Ser Ser Ile Arg Gln Tyr Glu Leu Val Leu His Thr Asp245 250 255Ile Asp Val Ala Lys Val Tyr Gln Gly Glu Met Gly Arg Leu Lys Ser260 265 270Tyr Glu Asn Gln Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala275 280 285Thr Val Thr Thr Asn Arg Lys Leu Asn Gln Gly Thr Glu Arg His Leu290 295 300Met His Leu Glu Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser305 310 315 320Gly Asp His Val Ala Val Tyr Pro Ala Asn Asp Ser Ala Leu Val Asn325 330 335Gln Leu Gly Glu Ile Leu Gly Ala Asp Leu Asp Val Val Met Ser Leu340 345 350Asn Asn Leu Asp Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro355 360 365Thr Ser Tyr Arg Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro370 375 380Pro Arg Thr Asn Val Leu Tyr Glu Leu Ala Gln Tyr Ala Ala Asp Pro385 390 395 400Ala Glu Gln Glu Gln Leu Arg Lys Met Ala Ser Ser Ser Gly Glu Gly405 410 415Lys Glu Leu Tyr Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu420 425 430Ala Ile Leu Gln Asp Tyr Pro Ser Leu Arg Pro Pro Ile Asp His Leu435 440 445Cys Glu Leu Leu Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala Ser450 455 460Ser Ser Lys Val His Pro Asn Ser Val His Ile Cys Ala Val Ala Val465 470 475 480Glu Tyr Glu Thr Lys Ala Gly Arg Leu Asn Lys Gly Val Ala Thr Ser485 490 495Trp Leu Arg Ala Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala Leu500 505 510Val Pro Met Phe Val Arg Lys Ser Gln Phe Arg Leu Pro Phe Lys Ala515 520 525Thr Thr Pro Val Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe530 535 540Ile Gly Phe Ile Gln Glu Arg Ala Trp Leu Arg Gln Gln Gly Lys Glu545 550 555 560Val Gly Glu Thr Leu Leu Tyr Tyr Gly Cys Arg Arg Ala Ala Glu Asp565 570 575Tyr Leu Tyr Arg Glu Glu Leu Ala Gly Phe Gln Lys Asp Gly Thr Leu580 585 590Ser Gln Leu Asn Val Ala Phe Ser Arg Glu Gln Ala Gln Lys Val Tyr595 600 605Val Gln His Leu Leu Arg Arg Asp Lys Glu His Leu Trp Arg Leu Ile610 615 620His Glu Gly Gly Ala His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met625 630 635 640Ala Arg Asp Val Gln Asn Thr Phe Tyr Asp Ile Val Ala Glu Leu Gly645 650 655Ala Met Glu His Ala Gln Ala Val Asp Tyr Val Lys Lys Leu Met Thr660 665 670Lys Gly Arg Tyr Ser Leu Asp Val Trp Ser675 680<210>24<211>444<212>DNA<213>蔬菜沙門氏菌(Spinacia oleracea)<220><221>CDS<222>(1)..(444)<400>24atg gca gca acc acc aca aca atg atg ggc atg gcc acc acc ttt gtc48Met Ala Ala Thr Thr Thr Thr Met Met Gly Met Ala Thr Thr Phe Val1 5 10 15cca aaa ccc caa gca cca cca atg atg gcg gcg ctt cca tcc aac acc96Pro Lys Pro Gln Ala Pro Pro Met Met Ala Ala Leu Pro Ser Asn Thr20 25 30ggc cgc tct ttg ttc gga ctc aag acc ggt agc cgt ggc gga agg atg144Gly Arg Ser Leu Phe Gly Leu Lys Thr Gly Ser Arg Gly Gly Arg Met35 40 45aca atg gct gcc tac aag gta acc ttg gta aca ccc acc ggt aac gta192Thr Met Ala Ala Tyr Lys Val Thr Leu Val Thr Pro Thr Gly Asn Val50 55 60gag ttt caa tgc cca gac gat gtt tac atc ttg gat gct gct gaa gaa240Glu Phe Gln Cys Pro Asp Asp Val Tyr Ile Leu Asp Ala Ala Glu Glu65 70 75 80gaa ggc att gac ttg cct tac tca tgc aga gct ggg tcg tgc tct tca288Glu Gly Ile Asp Leu Pro Tyr Ser Cys Arg Ala Gly Ser Cys Ser Ser85 90 95tgc gcc gga aag ctt aag aca ggt agt ctt aac caa gat gat cag agt336Cys Ala Gly Lys Leu Lys Thr Gly Ser Leu Asn Gln Asp Asp Gln Ser100 105 110ttt ttg gat gac gat cag atc gat gaa gga tgg gtt ctt acc tgt gct384Phe Leu Asp Asp Asp Gln Ile Asp Glu Gly Trp Val Leu Thr Cys Ala115 120 125gct tac cct gtt agt gat gtt act att gag acc cac aag gaa gag gag432Ala Tyr Pro Val Ser Asp Val Thr Ile Glu Thr His Lys Glu Glu Glu130 135 140ctt act gcc taa444Leu Thr Ala145<210>25<211>147<212>PRT<213>蔬菜沙門氏菌<400>25Met Ala Ala Thr Thr Thr Thr Met Met Gly Met Ala Thr Thr Phe Val1 5 10 15Pro Lys Pro Gln Ala Pro Pro Met Met Ala Ala Leu Pro Ser Asn Thr20 25 30Gly Arg Ser Leu Phe Gly Leu Lys Thr Gly Ser Arg Gly Gly Arg Met35 40 45Thr Met Ala Ala Tyr Lys Val Thr Leu Val Thr Pro Thr Gly Asn Val50 55 60Glu Phe Gln Cys Pro Asp Asp Val Tyr Ile Leu Asp Ala Ala Glu Glu65 70 75 80Glu Gly Ile Asp Leu Pro Tyr Ser Cys Arg Ala Gly Ser Cys Ser Ser85 90 95Cys Ala Gly Lys Leu Lys Thr Gly Ser Leu Asn Gln Asp Asp Gln Ser100 105 110Phe Leu Asp Asp Asp Gln Ile Asp Glu Gly Trp Val Leu Thr Cys Ala115 120 125Ala Tyr Pro Val Ser Asp Val Thr Ile Glu Thr His Lys Glu Glu Glu130 135 140Leu Thr Ala145<210>26<211>711<212>DNA<213>費氏弧菌(Vibrio fischeri)<220><221>CDS<222>(1)..(711)<400>26atg cca atc aat tgc aaa gta aag tct atc gag cca ttg gct tgt aat48Met Pro Ile Asn Cys Lys Val Lys Ser Ile Glu Pro Leu Ala Cys Asn1 5 10 15act ttt cga att tta ctt cac cca gaa cag cct gtt gct ttt aaa gca96Thr Phe Arg Ile Leu Leu His Pro Glu Gln Pro Val Ala Phe Lys Ala20 25 30ggc caa tac cta acg gtt gtt atg ggt gaa aaa gac aaa cgc cca ttc144Gly Gln Tyr Leu Thr Val Val Met Gly Glu Lys Asp Lys Arg Pro Phe
35 40 45tca atc gca agt agt cct tgt cgc cac gaa ggt gaa att gag tta cat192Ser Ile Ala Ser Ser Pro Cys Arg His Glu Gly Glu Ile Glu Leu His50 55 60att ggt gcc gca gag cac aat gct tat gcc gga gaa gtg gtt gaa tca240Ile Gly Ala Ala Glu His Asn Ala Tyr Ala Gly Glu Val Val Glu Ser65 70 75 80atg aaa tcg gca cta gaa acg ggt ggt gat att tta att gat gcg cct288Met Lys Ser Ala Leu Glu Thr Gly Gly Asp Ile Leu Ile Asp Ala Pro85 90 95cat ggt gaa gcg tgg atc cgt gaa gac agc gat cgt tca atg tta ttg336His Gly Glu Ala Trp Ile Arg Glu Asp Ser Asp Arg Ser Met Leu Leu100 105 110att gct ggc ggt aca ggt ttt agt tac gta cgt tca att ctt gat cac384Ile Ala Gly Gly Thr Gly Phe Ser Tyr Val Arg Ser Ile Leu Asp His115 120 125tgt att agc caa cag att caa aaa cca att tac cta tac tgg ggt ggt432Cys Ile Ser Gln Gln Ile Gln Lys Pro Ile Tyr Leu Tyr Trp Gly Gly130 135 140cgt gat gaa tgc caa ctg tat gca aaa gca gaa tta gag agc att gct480Arg Asp Glu Cys Gln Leu Tyr Ala Lys Ala Glu Leu Glu Ser Ile Ala145 150 155 160caa gcg cat agc cat att acg ttt gtg cca gtg gtt gag aaa agt gaa528Gln Ala His Ser His Ile Thr Phe Val Pro Val Val Glu Lys Ser Glu165 170 175ggc tgg aca ggt aaa acg ggt aat gtg tta gaa gcg gta aaa gcc gat576Gly Trp Thr Gly Lys Thr Gly Asn Val Leu Glu Ala Val Lys Als Asp180 185 190ttt aac tca cta gca gat atg gat att tac atc gca ggt cgc ttt gaa624Phe Asn Ser Leu Ala Asp Met Asp Ile Tyr Ile Ala Gly Arg Phe Glu195 200 205atg gct ggt gca gca cgt gag cag ttc acc act gaa aaa caa gcg aag672Met Ala Gly Ala Ala Arg Glu Gln Phe Thr Thr Glu Lys Gln Ala Lys210 215 220aaa gag cag ctg ttt ggt gat gca ttc gca ttt atc taa711Lys Glu Gln Leu Phe Gly Asp Ala Phe Ala Phe Ile225 230 235<210>27<211>236<212>PRT<213>費氏弧菌<400>27Met Pro Ile Asn Cys Lys Val Lys Ser Ile Glu Pro Leu Ala Cys Asn1 5 10 15Thr Phe Arg Ile Leu Leu His Pro Glu Gln Pro Val Ala Phe Lys Ala20 25 30Gly Gln Tyr Leu Thr Val Val Met Gly Glu Lys Asp Lys Arg Pro Phe35 40 45Ser Ile Ala Ser Ser Pro Cys Arg His Glu Gly Glu Ile Glu Leu His50 55 60Ile Gly Ala Ala Glu His Asn Ala Tyr Ala Gly Glu Val Val Glu Ser65 70 75 80Met Lys Ser Ala Leu Glu Thr Gly Gly Asp Ile Leu Ile Asp Ala Pro85 90 95His Gly Glu Ala Trp Ile Arg Glu Asp Ser Asp Arg Ser Met Leu Leu100 105 110Ile Ala Gly Gly Thr Gly Phe Ser Tyr Val Arg Ser Ile Leu Asp His115 120 125Cys Ile Ser Gln Gln Ile Gln Lys Pro Ile Tyr Leu Tyr Trp Gly Gly130 135 140Arg Asp Glu Cys Gln Leu Tyr Ala Lys Ala Glu Leu Glu Ser Ile Ala145 150 155 160Gln Ala His Ser His Ile Thr Phe Val Pro Val Val Glu Lys Ser Glu165 170 175Gly Trp Thr Gly Lys Thr Gly Asn Val Leu Glu Ala Val Lys Ala Asp180 185 190Phe Asn Ser Leu Ala Asp Met Asp Ile Tyr Ile Ala Gly Arg Phe Glu195 200 205Met Ala Gly Ala Ala Arg Glu Gln Phe Thr Thr Glu Lys Gln Ala Lys210 215 220Lys Glu Gln Leu Phe Gly Asp Ala Phe Ala Phe Ile225 230 235<210>28<211>1110<212>DNA<213>蔬菜沙門氏菌<220><221>CDS<222>(1)..(1110)<400>28atg acc acc gct gtc acc gcc gct gtt tct ttc ccc tct acc aaa acc48Met Thr Thr Ala Val Thr Ala Ala Val Ser Phe Pro Ser Thr Lys Thr1 5 10 15acc tct ctc tcc gcc cga agc tcc tcc gtc att tcc cct gac aaa atc96Thr Ser Leu Ser Ala Arg Ser Ser Ser Val Ile Ser Pro Asp Lys Ile20 25 30agc tac aaa aag gtt cct ttg tac tac agg aat gta tct gca act ggg144Ser Tyr Lys Lys Val Pro Leu Tyr Tyr Arg Asn Val Ser Ala Thr Gly35 40 45aaa atg gga ccc atc agg gcc cag atc gcc tct gat gtg gag gca cct192Lys Met Gly Pro Ile Arg Ala Gln Ile Ala Ser Asp Val Glu Ala Pro50 55 60cca cct gct cct gct aag gta gag aaa cat tca aag aaa atg gag gaa240Pro Pro Ala Pro Ala Lys Val Glu Lys His Ser Lys Lys Met Glu Glu65 70 75 80ggc att aca gtg aac aag ttt aag cct aag acc cct tac gtt gga aga288Gly Ile Thr Val Ash Lys Phe Lys Pro Lys Thr Pro Tyr Val Gly Arg85 90 95tgt ctt ctt aac acc aaa att act ggg gat gat gca ccc gga gag acc336Cys Leu Leu Asn Thr Lys Ile Thr Gly Asp Asp Ala Pro Gly Glu Thr100 105 110tgg cac atg gtt ttt tcc cat gaa gga gag atc cct tac aga gaa ggg384Trp His Met Val Phe Ser His Glu Gly Glu Ile Pro Tyr Arg Glu Gly115 120 125caa tcc gtt ggg gtt att cca gat ggg gaa gac aag aat gga aag ccc432Gln Ser Val Gly Val Ile Pro Asp Gly Glu Asp Lys Asn Gly Lys Pro130 135 140cat aag ttg aga ttg tac tcg atc gcc agc agt gct ctt ggt gat ttt480His Lys Leu Arg Leu Tyr Ser Ile Ala Ser Ser Ala Leu Gly Asp Phe145 150 155 160ggt gat gct aaa tct gtt tcg ttg tgt gta aaa cga ctc atc tac acc528Gly Asp Ala Lys Ser Val Ser Leu Cys Val Lys Arg Leu Ile Tyr Thr165 170 175aat gac gct gga gag acg atc aag gga gtc tgc tcc aac ttc ttg tgt576Asn Asp Ala Gly Glu Thr Ile Lys Gly Val Cys Ser Asn Phe Leu Cys180 185 190gac ttg aaa ccc ggt gct gaa gtg aag tta aca gga cca gtt gga aag624Asp Leu Lys Pro Gly Ala Glu Val Lys Leu Thr Gly Pro Val Gly Lys195 200 205gag atg ctc atg ccc aaa gac cct aac gcg aca att atc atg ctt gga672Glu Met Leu Met Pro Lys Asp Pro Asn Ala Thr Ile Ile Met Leu Gly210 215 220act gga acg ggg att gct cct ttc cgt tca ttc ttg tgg aag atg ttc720Thr Gly Thr Gly Ile Ala Pro Phe Arg Ser Phe Leu Trp Lys Met Phe225 230 235 240ttc gaa aag cat gat gat tac aag ttt aac ggc ttg gct tgg ctt ttc768Phe Glu Lys His Asp Asp Tyr Lys Phe Asn Gly Leu Ala Trp Leu Phe245 250 255ttg ggt gta ccc aca agc agt tot ctt ctc tac aaa gag gaa ttt gag816Leu Gly Val Pro Thr Ser Ser Ser Leu Leu Tyr Lys Glu Glu Phe Glu260 265 270aag atg aag gaa aag gct cca gac aac ttc agg ctg gat ttt gca gtg 864Lys Met Lys Glu Lys Ala Pro Asp Asn Phe Arg Leu Asp Phe Ala Val275 280 285agc aga gag caa act aac gag aaa ggg gag aag atg tac att caa acc 912Ser Arg Glu Gln Thr Asn Glu Lys Gly Glu Lys Met Tyr Ile Gln Thr290 295 300cga atg gca caa tac gca gtt gag cta tgg gaa atg ttg aag aaa gat 960Arg Met Ala Gln Tyr Ala Val Glu Leu Trp Glu Met Leu Lys Lys Asp305 310 315 320aat act tat gtc tac atg tgt ggt ctc aag gga atg gaa aag gga att1008Asn Thr Tyr Val Tyr Met Cys Gly Leu Lys Gly Met Glu Lys Gly Ile325 330 335gac gac att atg gtt tca ttg gct gct gca gaa ggc att gat tgg att1056Asp Asp Ile Met Val Ser Leu Ala Ala Ala Glu Gly Ile Asp Trp Ile340 345 350gaa tac aag agg cag ttg aag aag gca gaa caa tgg aac gtt gaa gtc1104Glu Tyr Lys Arg Gln Leu Lys Lys Ala Glu Gln Trp Asn Val Glu Val355 360 365tac taa1110Tyr370<210>29<211>369<212>PRT<213>蔬菜沙門氏菌<400>29Met Thr Thr Ala Val Thr Ala Ala Val Ser Phe Pro Ser Thr Lys Thr1 5 10 15Thr Ser Leu Ser Ala Arg Ser Ser Ser Val Ile Ser Pro Asp Lys Ile20 25 30Ser Tyr Lys Lys Val Pro Leu Tyr Tyr Arg Asn Val Ser Ala Thr Gly35 40 45Lys Met Gly Pro Ile Arg Ala Gln Ile Ala Ser Asp Val Glu Ala Pro50 55 60Pro Pro Ala Pro Ala Lys Val Glu Lys His Ser Lys Lys Met Glu Glu65 70 75 80Gly Ile Thr Val Asn Lys Phe Lys Pro Lys Thr Pro Tyr Val Gly Arg85 90 95Cys Leu Leu Asn Thr Lys Ile Thr Gly Asp Asp Ala Pro Gly Glu Thr100 105 110Trp His Met Val Phe Ser His Glu Gly Glu Ile Pro Tyr Arg Glu Gly115 120 125Gln Ser Val Gly Val Ile Pro Asp Gly Glu Asp Lys Asn Gly Lys Pro130 135 140His Lys Leu Arg Leu Tyr Ser Ile Ala Ser Ser Ala Leu Gly Asp Phe145 150 155 160Gly Asp Ala Lys Ser Val Ser Leu Cys Val Lys Arg Leu Ile Tyr Thr165 170 175Asn Asp Ala Gly Glu Thr Ile Lys Gly Val Cys Ser Asn Phe Leu Cys180 185 190Asp Leu Lys Pro Gly Ala Glu Val Lys Leu Thr Gly Pro Val Gly Lys195 200 205Glu Met Leu Met Pro Lys Asp Pro Asn Ala Thr Ile Ile Met Leu Gly210 215 220Thr Gly Thr Gly Ile Ala Pro Phe Arg Ser Phe Leu Trp Lys Met Phe225 230 235 240Phe Glu Lys His Asp Asp Tyr Lys Phe Asn Gly Leu Ala Trp Leu Phe245 250 255Leu Gly Val Pro Thr Ser Ser Ser Leu Leu Tyr Lys Glu Glu Phe Glu260 265 270Lys Met Lys Glu Lys Ala Pro Asp Asn Phe Arg Leu Asp Phe Ala Val275 280 285Ser Arg Glu Gln Thr Asn Glu Lys Gly Glu Lys Met Tyr Ile Gln Thr290 295 300Arg Met Ala Gln Tyr Ala Val Glu Leu Trp Glu Met Leu Lys Lys Asp305 310 315 320Asn Thr Tyr Val Tyr Met Cys Gly Leu Lys Gly Met Glu Lys Gly Ile325 330 335Asp Asp Ile Met Val Ser Leu Ala Ala Ala Glu Gly Ile Asp Trp Ile340 345 350Glu Tyr Lys Arg Gln Leu Lys Lys Ala Glu Gln Trp Asn Val Glu Val355 360 365Tyr<210>30<2ll>2580<212>DNA<213>寄生曲霉(Aspergillus parasiticus)<220><221>CDS<222>(1)..(2580)<400>30atg gca acc atc acg gag gtt cgg acg gat gcg ctc gtc cca act gac48Met Ala Thr Ile Thr Glu Val Arg Thr Asp Ala Leu Val Pro Thr Asp1 5 10 15ctc gtc ctt aag aca ggt cag atc aaa att caa agc gaa gag atc tcg96Leu Val Leu Lys Thr Gly Gln Ile Lys Ile Gln Ser Glu Glu Ile Ser20 25 30acg aaa gac ctg tcc gat atc cct ctg cca cca cca tca aaa cgg ccg144Thr Lys Asp Leu Ser Asp Ile Pro Leu Pro Pro Pro Ser Lys Arg Pro35 40 45aca gaa gtg ctg agc gta gat aaa gga acc cca gat agc cat gtt ccg192Thr Glu Val Leu Ser Val Asp Lys Gly Thr Pro Asp Ser His Val Pro50 55 60cgt gat cct cga ctc atc aga tta acg ggt gtt cat ccg ttt aac gtt240Arg Asp Pro Arg Leu Ile Arg Leu Thr Gly Val His Pro Phe Asn Val65 70 75 80gag cca cct ctt aca gat ctg tat aaa gaa ggg ttt tta aca tcg ccg288Glu Pro Pro Leu Thr Asp Leu Tyr Lys Glu Gly Phe Leu Thr Ser Pro85 90 95gag ctc ttc tat gtt cga aat cat ggc cca gtc cct cat gtc aag gat336Glu Leu Phe Tyr Val Arg Asn His Gly Pro Val Pro His Val Lys Asp100 105 110gaa gat atc cct cac tgg gaa att act atc gaa gga ctg gta gag aag384Glu Asp Ile Pro His Trp Glu Ile Thr Ile Glu Gly Leu Val Glu Lys115 120 125cct ttg gta cta aac ttc cga caa gtg ttg cag cag tac gac caa ata432Pro Leu Val Leu Asn Phe Arg Gln Val Leu Gln Gln Tyr Asp Gln Ile130 135 140acg gcg ccc atc acc ctc gta tgt gca ggc aat cga cgc aaa gag caa480Thr Ala Pro Ile Thr Leu Val Cys Ala Gly Asn Arg Arg Lys Glu Gln145 150 155 160aac att gta cgt aaa acg aaa ggt ttt tct tgg gga tcc gcg gga cta528Asn Ile Val Arg Lys Thr Lys Gly Phe Ser Trp Gly Ser Ala Gly Leu165 170 175tcg act gcc ctc ttc act ggc cca ttg ctg gcg gat atc ctc cgc agt576Ser Thr Ala Leu Phe Thr Gly Pro Leu Leu Ala Asp Ile Leu Arg Ser180 185 190ggc aaa ccc ctg cgt caa gcg aaa tac gtc tgt atg gaa gga gcg gat624Gly Lys Pro Leu Arg Gln Ala Lys Tyr Val Cys Met Glu Gly Ala Asp195 200 205aag ctg ccc aat ggt cac tac ggc aca ctc att aaa ttg aac tgg gcc672Lys Leu Pro Asn Gly His Tyr Gly Thr Leu Ile Lys Leu Asn Trp Ala210 215 220cta gac ccc aac agg ggg atc atg ctt gca cat aaa atg aac ggg gag720Leu Asp Pro Asn Arg Gly Ile Met Leu Ala His Lys Met Asn Gly Glu225 230 235 240tct ctt cgc cca gat cat ggt cgt ccg ctg agg gcc gtc gtg ccc ggt768Ser Leu Arg Pro Asp His Gly Arg Pro Leu Arg Ala Val Val Pro Gly
245 250 255caa ata gga gga cga agt gtc aag tgg ctg aag agg ctg atc ttg acc816Gln Ile Gly Gly Arg Ser Val Lys Trp Leu Lys Arg Leu Ile Leu Thr260 265 270gat gca cca agc gat aac tgg tac cat atc aat gac aac cgc gtc tta864Asp Ala Pro Ser Asp Asn Trp Tyr His Ile Asn Asp Asn Arg Val Leu275 280 285cca aca atg gtc tcg ccc gat atg gca tca aat aac cga aat tgg tgg912Pro Thr Met Val Ser Pro Asp Met Ala Ser Asn Asn Arg Asn Trp Trp290 295 300cac gat gag cgg gat gcg att tat gac cta aac acc aac tcc gcc gtt960His Asp Glu Arg Asp Ala Ile Tyr Asp Leu Asn Thr Asn Ser Ala Val305 310 315 320gga tat cct caa aac aat gag gtc tta aat atc ctg gag gcc agg gcc1008Gly Tyr Pro Gln Asn Asn Glu Val Leu Asn Ile Leu Glu Ala Arg Ala325 330 335gtc ata tac tgt cag agg ata gct tac gct ggt ggg ggc cgt agg gtt1056Val Ile Tyr Cys Gln Arg Ile Ala Tyr Ala Gly Gly Gly Arg Arg Val340 345 350acc agg gta gaa ata tcc cta gac aaa ggc aaa tct tgg aga ttg gcg1104Thr Arg Val Glu Ile Ser Leu Asp Lys Gly Lys Ser Trp Arg Leu Ala355 360 365gat atc gaa tat gcc gaa gac aag tat cgt gat ttc gaa ggc gag ctt1152Asp Ile Glu Tyr Ala Glu Asp Lys Tyr Arg Asp Phe Glu Gly Glu Leu370 375 380ttt gga ggc aaa gta gat atg tac tgg cgc gaa act tgc ttc tgc tgg1200Phe Gly Gly Lys Val Asp Met Tyr Trp Arg Glu Thr Cys Phe Cys Trp385 390 395 400tgt ttt tgg tct cta agc atc gcc atc cca gag ctt gag aac agt gat1248Cys Phe Trp Ser Leu Ser Ile Ala Ile Pro Glu Leu Glu Asn Ser Asp405 410 415gcc atc ctt gta aga gcc atg gat gaa gca ttg ggc gtg cag cct cgc1296Ala Ile Leu Val Arg Ala Met Asp Glu Ala Leu Gly Val Gln Pro Arg420 425 430gat atg tac tgg tcc gtt ctc gga atg atg aac aac cct tgg ttc cgg1344Asp Met Tyr Trp Ser Val Leu Gly Met Met Asn Asn Pro Trp Phe Arg435 440 445gtt aca att acg aag gaa aac ggg aac ttg aga ttc gag cac cct acc1392Val Thr Ile Thr Lys Glu Asn Gly Asn Leu Arg Phe Glu His Pro Thr450 455 460cac cct agt atg cct aca gga tgg atg gaa cgc gtc aaa aaa gct ggg1440His Pro Ser Met Pro Thr Gly Trp Met Glu Arg Val Lys Lys Ala Gly465 470 475 480ggt gac ccg acg aat ggt aac tgg gga gaa aga cac gaa gga gag gag1488Gly Asp Pro Thr Asn Gly Asn Trp Gly Glu Arg His Glu Gly Glu Glu
485 490 495ccg acg gag ccg gag ccc gtg caa gac att aat atg aag aaa gac ggg1536Pro Thr Glu Pro Glu Pro Val Gln Asp Ile Asn Met Lys Lys Asp Gly500 505 510cca agc cga acg att agt ttt gaa gaa ttc aag gag aat tcc tgt gat1584Pro Ser Arg Thr Ile Ser Phe Glu Glu Phe Lys Glu Asn Ser Cys Asp515 520 525gag aag cca tgg ttc atc gtg aat gga gaa gtg tat gat ggt caa gca1632Glu Lys Pro Trp Phe Ile Val Asn Gly Glu Val Tyr Asp Gly Gln Ala530 535 540ttt ctt gaa ggc cac cct ggc gga cgg cag agt att atc tcc tct gcc1680Phe Leu Glu Gly His Pro Gly Gly Arg Gln Ser Ile Ile Ser Ser Ala545 550 555 560ggt cag gac gtc tcc gag gaa ttc ctt gct att cat agc gag acg gca1728Gly Gln Asp Val Ser Glu Glu Phe Leu Ala Ile His Ser Glu Thr Ala565 570 575aag gcg atg atg cct gag tac cat att gga acg acg gat ccg gaa ggc1776Lys Ala Met Met Pro Glu Tyr His Ile Gly Thr Thr Asp Pro Glu Gly580 585 590ttg ata gca ctc aag gat gat gca tca tcc tcc acc gat gaa att cgc1824Leu Ile Ala Leu Lys Asp Asp Ala Ser Ser Ser Thr Asp Glu Ile Arg595 600 605cca gtg ttc ctc caa tca cgg tct tgg aca aag gca aca ttg aaa gaa1872Pro Val Phe Leu Gln Ser Arg Ser Trp Thr Lys Ala Thr Leu Lys Glu610 615 620agg aaa gac ata tca tgg gat aca cga ata ttt agt ttc aaa ttg gaa1920Arg Lys Asp Ile Ser Trp Asp Thr Arg Ile Phe Ser Phe Lys Leu Glu625 630 635 640cac gaa gat caa aca ttg ggt tta cca gtc ggc cag cat ctt atg atc1968His Glu Asp Gln Thr Leu Gly Leu Pro Val Gly Gln His Leu Met Ile645 650 655aaa gtc ctc gac aga tca tcc aac aac gaa gcc atc atc cgc tca tac2016Lys Val Leu Asp Arg Ser Ser Ash Asn Glu Ala Ile Ile Arg Ser Tyr660 665 670acc ccg att tct gaa acc agc caa aaa ggg act gtg gac ttg ctg gtt2064Thr Pro Ile Ser Glu Thr Ser Gln Lys Gly Thr Val Asp Leu Leu Val675 680 685aaa gta tac ttt gca aca gcc acc tcg gca ggc ggc aag atg acg atg2112Lys Val Tyr Phe Ala Thr Ala Thr Ser Ala Gly Gly Lys Met Thr Met690 695 700gcc ctg gat agg ctg cca ttg ggc tcc gtg gtc gaa tat ctt gga aat2160Ala Leu Asp Arg Leu Pro Leu Gly Ser Val Val Glu Tyr Leu Gly Asn705 710 715 720gga cga gtt ctc ata agt ggc aag gag cgc cat gtt cgg tcg ttt aag2208Gly Arg Val Leu Ile Ser Gly Lys Glu Arg His Val Arg Ser Phe Lys
725 730 735atg att tgt gga gga acc ggt atc aca ccg atc ttg cag gtc ttg cgc2256Met Ile Cys Gly Gly Thr Gly Ile Thr Pro Ile Leu Gln Val Leu Arg740 745 750gcc gtg gtt cag gac cat caa gat cct acc tct tgt gta gtc ctc aat2304Ala Val Val Gln Asp His Gln Asp Pro Thr Ser Cys Val Val Leu Asn755 760 765gga aac aga cag gag gaa gat atc ctt cgc cgg gct gag ctc gac ggc2352Gly Asn Arg Gln Glu Glu Asp Ile Leu Arg Arg Ala Glu Leu Asp Gly770 775 780ttc atg gcg tcc gac agc aga agg tgt aat ata ata cac act cta tcc2400Phe Met Ala Ser Asp Ser Arg Arg Cys Asn Ile Ile His Thr Leu Ser785 790 795 800aaa gcg ccg gac tca tgg act ggc cgc cga gga cgc ata tcc gaa gag2448Lys Ala Pro Asp Ser Trp Thr Gly Arg Arg Gly Arg Ile Ser Glu Glu805 810 815ctc cta aag gag tac gcg gct cca gaa gat gag agt atg gta ctg att2496Leu Leu Lys Glu Tyr Ala Ala Pro Glu Asp Glu Ser Met Val Leu Ile820 825 830tgt ggt ccg cca gcc atg gaa gaa tcg gct cgg agg ata ctg ttg gcg2544Cys Gly Pro Pro Ala Met Glu Glu Ser Ala Arg Arg Ile Leu Leu Ala835 840 845gaa gga tgg aaa gaa tca gac ctt cac ttt ttc tga 2580Glu Gly Trp Lys Glu Ser Asp Leu His Phe Phe850 855 860<210>31<211>859<212>PRT<213>寄生曲霉<400>31Met Ala Thr Ile Thr Glu Val Arg Thr Asp Ala Leu Val Pro Thr Asp1 5 10 15Leu Val Leu Lys Thr Gly Gln Ile Lys Ile Gln Ser Glu Glu Ile Ser20 25 30Thr Lys Asp Leu Ser Asp Ile Pro Leu Pro Pro Pro Ser Lys Arg Pro35 40 45Thr Glu Val Leu Ser Val Asp Lys Gly Thr Pro Asp Ser His Val Pro50 55 60Arg Asp Pro Arg Leu Ile Arg Leu Thr Gly Val His Pro Phe Asn Val65 70 75 80Glu Pro Pro Leu Thr Asp Leu Tyr Lys Glu Gly Phe Leu Thr Ser Pro85 90 95Glu Leu Phe Tyr Val Arg Asn His Gly Pro Val Pro His Val Lys Asp
100 105 110Glu Asp Ile Pro His Trp Glu Ile Thr Ile Glu Gly Leu Val Glu Lys115 120 125Pro Leu Val Leu Asn Phe Arg Gln Val Leu Gln Gln Tyr Asp Gln Ile130 135 140Thr Ala Pro Ile Thr Leu Val Cys Ala Gly Asn Arg Arg Lys Glu Gln145 150 155 160Asn Ile Val Arg Lys Thr Lys Gly Phe Ser Trp Gly Ser Ala Gly Leu165 170 175Ser Thr Ala Leu Phe Thr Gly Pro Leu Leu Ala Asp Ile Leu Arg Ser180 185 190Gly Lys Pro Leu Arg Gln Ala Lys Tyr Val Cys Met Glu Gly Ala Asp195 200 205Lys Leu Pro Asn Gly His Tyr Gly Thr Leu Ile Lys Leu Asn Trp Ala210 215 220Leu Asp Pro Asn Arg Gly Ile Met Leu Ala His Lys Met Asn Gly Glu225 230 235 240Ser Leu Arg Pro Asp His Gly Arg Pro Leu Arg Ala Val Val Pro Gly245 250 255Gln Ile Gly Gly Arg Ser Val Lys Trp Leu Lys Arg Leu Ile Leu Thr
260 265 270Asp Ala Pro Ser Asp Asn Trp Tyr His Ile Asn Asp Asn Arg Val Leu275 280285Pro Thr Met Val Ser Pro Asp Met Ala Ser Asn Asn Arg Asn Trp Trp290 295 300His Asp Glu Arg Asp Ala Ile Tyr Asp Leu Asn Thr Asn Ser Ala Val305 310 315 320Gly Tyr Pro Gln Asn Asn Glu Val Leu Asn Ile Leu Glu Ala Arg Ala325 330 335Val Ile Tyr Cys Gln Arg Ile Ala Tyr Ala Gly Gly Gly Arg Arg Val340 345 350Thr Arg Val Glu Ile Ser Leu Asp Lys Gly Lys Ser Trp Arg Leu Ala355 360 365Asp Ile Glu Tyr Ala Glu Asp Lys Tyr Arg Asp Phe Glu Gly Glu Leu370 375 380Phe Gly Gly Lys Val Asp Met Tyr Trp Arg Glu Thr Cys Phe Cys Trp385 390 395 400Cys Phe Trp Ser Leu Ser Ile Ala Ile Pro Glu Leu Glu Asn Ser Asp405 410 415Ala Ile Leu Val Arg Ala Met Asp Glu Ala Leu Gly Val Gln Pro Arg
420 425 430Asp Met Tyr Trp Ser Val Leu Gly Met Met Asn Asn Pro Trp Phe Arg435 440 445Val Thr Ile Thr Lys Glu Asn Gly Asn Leu Arg Phe Glu His Pro Thr450 455 460His Pro Ser Met Pro Thr Gly Trp Met Glu Arg Val Lys Lys Ala Gly465 470 475 480Gly Asp Pro Thr Asn Gly Asn Trp Gly Glu Arg His Glu Gly Glu Glu485 490 495Pro Thr Glu Pro Glu Pro Val Gln Asp Ile Asn Met Lys Lys Asp Gly500 505 510Pro Ser Arg Thr Ile Ser Phe Glu Glu Phe Lys Glu Asn Ser Cys Asp515 520 525Glu Lys Pro Trp Phe Ile Val Asn Gly Glu Val Tyr Asp Gly Gln Ala530 535 540Phe Leu Glu Gly His Pro Gly Gly Arg Gln Ser Ile Ile Ser Ser Ala545 550 555 560Gly Gln Asp Val Ser Glu Glu Phe Leu Ala Ile His Ser Glu Thr Ala565 570 575Lys Ala Met Met Pro Glu Tyr His Ile Gly Thr Thr Asp Pro Glu Gly
580 585 590Leu Ile Ala Leu Lys Asp Asp Ala Ser Ser Ser Thr Asp Glu Ile Arg595 600 605Pro Val Phe Leu Gln Ser Arg Ser Trp Thr Lys Ala Thr Leu Lys Glu610 615 620Arg Lys Asp Ile Ser Trp Asp Thr Arg Ile Phe Ser Phe Lys Leu Glu625 630 635 640His Glu Asp Gln Thr Leu Gly Leu Pro Val Gly Gln His Leu Met Ile645 650 655Lys Val Leu Asp Arg Ser Ser Asn Asn Glu Ala Ile Ile Arg Ser Tyr660 665 670Thr Pro Ile Ser Glu Thr Ser Gln Lys Gly Thr Val Asp Leu Leu Val675 680 685Lys Val Tyr Phe Ala Thr Ala Thr Ser Ala Gly Gly Lys Met Thr Met690 695 700Ala Leu Asp Arg Leu Pro Leu Gly Ser Val Val Glu Tyr Leu Gly Asn705 710 715 720Gly Arg Val Leu Ile Ser Gly Lys Glu Arg His Val Arg Ser Phe Lys725 730 735Met Ile Cys Gly Gly Thr Gly Ile Thr Pro Ile Leu Gln Val Leu Arg
740 745 750Ala Val Val Gln Asp His Gln Asp Pro Thr Ser Cys Val Val Leu Asn755 760 765Gly Asn Arg Gln Glu Glu Asp Ile Leu Arg Arg Ala Glu Leu Asp Gly770 775 780Phe Met Ala Ser Asp Ser Arg Arg Cys Asn Ile Ile His Thr Leu Ser785 790 795 800Lys Ala Pro Asp Ser Trp Thr Gly Arg Arg Gly Arg Ile Ser Glu Glu805 810 815Leu Leu Lys Glu Tyr Ala Ala Pro Glu Asp Glu Ser Met Val Leu Ile820 825 830Cys Gly Pro Pro Ala Met Glu Glu Ser Ala Arg Arg Ile Leu Leu Ala835 840 845Glu Gly Trp Lys Glu Ser Asp Leu His Phe Phe850 855<210>32<211>37<212>DNA<213>人工<220><223>引物<220><221>misc feature<222>(1)..(37)<400>32gcgcgaattc atgacaacct taagctgtaa agtgacc 37<210>33<211>34<212>DNA<213>人工<220><223>引物<220><221>misc feature<222>(1)..(34)<400>33gcgcctgcag tcagataaat gcaaacgcat cgcc 34<210>34<211>26019<212>DNA<213>合成<220><221>啟動子<222>(233)..(1582)<220><221>內(nèi)含子<222>(1583)..(1958)<220><221>基因<222>(1969)..(3585)<223>PrnA<220><221>終子<222>(3603)..(3877)<220><221>啟動子<222>(3888)..(5237)<220><221>內(nèi)含子<222>(5238)..(5613)<220><221>基因<222>(5624)..(7327)<223>prnC<220><221>終止子<222>(7338)..(7612)<220><221>啟動子<222>(7636)..(8985)<220><221>內(nèi)含子<222>(8986)..(9361)<220><221>基因<222>(9372)..(10457)<223>PrnB<220><221>終止子<222>(10464)..(10738)<220><221>啟動子<222>(10749)..(12098)<220><221>內(nèi)含子<222>(12099)..(12474)<220><221>基因<222>(12485)..(13576)<223>PrnD<220><221>終止子<222>(13583)..(13857)<220><221>啟動子<222>(13942)..(14942)<220><221>基因<222>(14948)..(15649)<223>fre<220><22l>終止子<222>(15655)..(15936)<220><221>啟動子<222>(15949)..(17285)<220><221>內(nèi)含子<222>(17283)..(17679)<220><22l>基因<222>(17696)..(18238)<223>選擇標(biāo)記<220><221>終子<222>(18245)..(18526)<400>34tggggaaccc tgtggttggc atgcacatac aaatggacga acggataaac cttttcacgc 60ccttttaaat atccgattat tctaataaac gctcttttct cttaggttta cccgccaata 120tatcctgtca aacactgata gtttaaactg aaggcgggaa acgacaatct gatctatcgt 180tctagtcgta cgttttgcga tcggtctcac tagagcggcc gcctcgaggt accggatttg 240gagccaagtc tcataaacgc cattgtggaa gaaagtcttg agttggtggt aatgtaacag 300agtagtaaga acagagaaga gagagagtgt gagatacatg aattgtcggg caacaaaaat 360cctgaacatc ttattttagc aaagagaaag agttccgagt ctgtagcaga agagtgagga 420gaaatttaag ctcttggact tgtgaattgt tccgcctctt gaatacttct tcaatcctca 480tatattcttc ttctatgtta cctgaaaacc ggcatttaat ctcgcgggtt tattccggtt 540caacattttt tttgttttga gttattatct gggcttaata acgcaggcct gaaataaatt 600caaggcccaa ctgttttttt ttttaagaag ttgctgttaa aaaaaaaaaa agggaattaa 660caacaacaac aaaaaaagat aaagaaaata ataacaatta ctttaattgt agactaaaaa 720aacatagatt ttatcatgaa aaaaagagaa aagaaataaa aacttggatc aaaaaaaaaa 780acatacagat cttctaatta ttaacttttc ttaaaaatta ggtccttttt cccaacaatt 840aggtttagag ttttggaatt aaaccaaaaa gattgttcta aaaaatactc aaatttggta 900gataagtttc cttattttaa ttagtcaatg gtagatactt ttttttcttt tctttattag 960agtagattag aatcttttat gccaagtttt gataaattaa atcaagaaga taaactatca 1020taatcaacat gaaattaaaa gaaaaatctc atatatagta ttagtattct ctatatatat 1080tatgattgct tattcttaat gggttgggtt aaccaagaca tagtcttaat ggaaagaatc 1140ttttttgaac tttttcctta ttgattaaat tcttctatag aaaagaaaga aattatttga 1200ggaaaagtat atacaaaaag aaaaatagaa aaatgtcagt gaagcagatg taatggatga 1260cctaatccaa ccaccaccat aggatgtttc tacttgagtc ggtcttttaa aaacgcacgg 1320tggaaaatat gacacgtatc atatgattcc ttcctttagt ttcgtgataa taatcctcaa 1380ctgatatctt cctttttttg ttttggctaa agatatttta ttctcattaa tagaaaagac 1440ggttttgggc ttttggtttg cgatataaag aagaccttcg tgtggaagat aataattcat 1500cctttcgtct ttttctgact cttcaatctc tcccaaagcc taaagcgatc tctgcaaatc 1560tctcgcgact ctctctttca aggtatattt tctgattctt tttgtttttg attcgtatct 1620gatctccaat ttttgttatg tggattattg aatcttttgt ataaattgct tttgacaata 1680ttgttcgttt cgtcaatcca gcttctaaat tttgtcctga ttactaagat atcgattcgt1740agtgtttaca tctgtgtaat ttcttgcttg attgtgaaat taggattttc aaggacgatc1800tattcaattt ttgtgttttc tttgttcgat tctctctgtt ttaggtttct tatgtttaga1860tccgtttctc tttggtgttg ttttgatttc tcttacggct tttgatttgg tatatgttcg1920ctgattggtt tctacttgtt ctattgtttt atttcaggtg gatccaccat gaacaagccg1980atcaagaata tcgtcatcgt gggcggcggt actgcgggct ggatggccgc ctcgtacctc2040gtccgggccc tccaacagca ggcgaacatt acgctcatcg aatctgcggc gatccctcgg2100atcggcgtgg gcgaagcgac catcccaagt ttgcagaagg tgttcttcga tttcctcggg2160ataccggagc gggaatggat gccccaagtg aacggcgcgt tcaaggccgc gatcaagttc2220gtgaattgga gaaagtctcc cgacccctcg cgcgacgatc acttctacca tttgttcggc2280aacgtgccga actgcgacgg cgtgccgctt acccactact ggctgcgcaa gcgcgaacag2340ggcttccagc agccgatgga gtacgcgtgc tacccgcagc ccggggcact cgacggcaag2400ctggcaccgt gcctgtccga cggcacccgc cagatgtccc acgcgtggca cttcgacgcg2460cacctggtgg ccgacttctt gaagcgctgg gccgtcgagc gcggggtgaa ccgcgtggtc2520gatgaggtgg tggacgttcg cctgaacaac cgcggctaca tctccaacct gctcaccaag2580gaggggcgga cgctggaggc ggacctgttc atcgactgct ccggcatgcg ggggctcctg2640atcaatcagg cgctgaagga acccttcatc gacatgtccg actacctgct gtgcgacagc2700gcggtcgcca gcgccgtgcc caacgacgac gcgcgcgatg gggtcgagcc gtacacctcc2760tcgatcgcca tgaactcggg atggacctgg aagattccga tgctgggccg gttcggcagc2820ggctacgtct tctcgagcca tttcacctcg cgcgaccagg ccaccgccga cttcctcaaa2880ctctggggcc tctcggacaa tcagccgctc aaccagatca agttccgggt cgggcgcaac2940aagcgggcgt gggtcaacaa ctgcgtctcg atcgggctgt cgtcgtgctt tctggagccc3000ctggaatcga cggggatcta cttcatctac gcggcgcttt accagctcgt gaagcacttc3060cccgacacct cgttcgaccc gcggctgagc gacgctttca acgccgagat cgtccacatg3120ttcgacgact gccgggattt cgtccaagcg cactatttca ccacgtcgcg cgatgacacg3180ccgttctggc tcgcgaaccg gcacgacctg cggctctcgg acgccatcaa agagaaggtt3240cagcgctaca aggcggggct gccgctgacc accacgtcgt tcgacgattc cacgtactac3300gagaccttcg actacgaatt caagaatttc tggttgaacg gcaactacta ctgcatcttt3360gccggcttgg gcatgctgcc cgaccggtcg ctgccgctgt tgcagcaccg accggagtcg3420atcgagaaag ccgaggcgat gttcgccagc atccggcgcg aggccgagcg tctgcgcacc3480agcctgccga caaactacga ctacctgcgg tcgctgcgtg acggcgacgc ggggctgtcg3540cgcggccagc gtgggccgaa gctcgcagcg caggaaagcc tgtagtggaa cgcaccttgg3600aggatccccc gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga3660atcctgttgc cggtcttgcg atgattatca tctaatttct gttgaattac gttaagcatg3720taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc3780cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat3840tatcgcgcgc ggtgtcatct atgttactag atccgggaat tgggtaccgg atttggagcc3900aagtctcata aacgccattg tggaagaaag tcttgagttg gtggtaatgt aacagagtag3960taagaacaga gaagagagag agtgtgagat acatgaattg tcgggcaaca aaaatcctga4020acatcttatt ttagcaaaga gaaagagttc cgagtctgta gcagaagagt gaggagaaat4080ttaagctctt ggacttgtga attgttccgc ctcttgaata cttcttcaat cctcatatat4140tcttcttcta tgttacctga aaaccggcat ttaatctcgc gggtttattc cggttcaaca4200ttttttttgt tttgagttat tatctgggct taataacgca ggcctgaaat aaattcaagg4260cccaactgtt ttttttttta agaagttgct gttaaaaaaa aaaaaaggga attaacaaca4320acaacaaaaa aagataaaga aaataataac aattacttta attgtagact aaaaaaacat4380agattttatc atgaaaaaaa gagaaaagaa ataaaaactt ggatcaaaaa aaaaaacata4440cagatcttct aattattaac ttttcttaaa aattaggtcc tttttcccaa caattaggtt4500tagagttttg gaattaaacc aaaaagattg ttctaaaaaa tactcaaatt tggtagataa4560gtttccttat tttaattagt caatggtaga tacttttttt tcttttcttt attagagtag4620attagaatct tttatgccaa gttttgataa attaaatcaa gaagataaac tatcataatc4680aacatgaaat taaaagaaaa atctcatata tagtattagt attctctata tatattatga4740ttgcttattc ttaatgggtt gggttaacca agacatagtc ttaatggaaa gaatcttttt4800tgaacttttt ccttattgat taaattcttc tatagaaaag aaagaaatta tttgaggaaa4860agtatataca aaaagaaaaa tagaaaaatg tcagtgaagc agatgtaatg gatgacctaa4920tccaaccacc accataggat gtttctactt gagtcggtct tttaaaaacg cacggtggaa4980aatatgacac gtatcatatg attccttcct ttagtttcgt gataataatc ctcaactgat5040atcttccttt ttttgttttg gctaaagata ttttattctc attaatagaa aagacggttt5100tgggcttttg gtttgcgata taaagaagac cttcgtgtgg aagataataa ttcatccttt5160cgtctttttc tgactcttca atctctccca aagcctaaag cgatctctgc aaatctctcg5220cgactctctc tttcaaggta tattttctga ttctttttgt ttttgattcg tatctgatct5280ccaatttttg ttatgtggat tattgaatct tttgtataaa ttgcttttga caatattgtt5340cgtttcgtca atccagcttc taaattttgt cctgattact aagatatcga ttcgtagtgt5400ttacatctgt gtaatttctt gcttgattgt gaaattagga ttttcaagga cgatctattc5460aatttttgtg ttttctttgt tcgattctct ctgttttagg tttcttatgt ttagatccgt5520ttctctttgg tgttgttttg atttctctta cggcttttga tttggtatat gttcgctgat5580tggtttctac ttgttctatt gttttatttc aggtggatcc accatgactc agaagagccc5640cgcgaacgaa cacgatagca atcacttcga cgtaatcatc ctcggctcgg gcatgtccgg5700cacccagatg ggggccatct tggccaaaca acagtttcgc gtgctgatca tcgaggagtc5760gtcgcacccg cggttcacga tcggcgaatc gtcgatcccc gagacgtctc ttatgaaccg5820catcatcgct gatcgctacg gcattccgga gctcgaccac atcacgtcgt tttattcgac5880gcaacgttac gtcgcgtcga gcacgggcat taagcgcaac ttcggcttcg tgttccacaa5940gcccggccag gagcacgacc cgaaggagtt cacccagtgc gtcattcccg agctgccgtg6000ggggccggag agccattatt accggcaaga cgtcgacgcc tacttgttgc aagccgccat6060taaatacggc tgcaaggtcc accagaaaac taccgtgacc gaataccacg ccgataaaga6120cggcgtcgcg gtgaccaccg cccagggcga acggttcacc ggccggtaca tgatcgactg6180cggaggacct cgcgcgccgc tcgcgaccaa gttcaagctc cgcgaagaac cgtgtcgctt6240caagacgcac tcgcgcagcc tctacacgca catgctcggg gtcaagccgt tcgacgacat6300cttcaaggtc aaggggcagc gctggcgctg gcacgagggg accttgcacc acatgttcga6360gggcggctgg ctctgggtga ttccgttcaa caaccacccg cggtcgacca acaacctggt6420gagcgtcggc ctgcagctcg acccgcgtgt ctacccgaaa accgacatct ccgcacagca6480ggaattcgat gagttcctcg cgcggttccc gagcatcggg gctcagttcc gggacgccgt6540gccggtgcgc gactgggtca agaccgaccg cctgcaattc tcgtcgaacg cctgcgtcgg6600cgaccgctac tgcctgatgc tgcacgcgaa cggcttcatc gacccgctct tctcccgggg6660gctggaaaac accgcggtga ccatccacgc gctcgcggcg cgcctcatca aggcgctgcg6720cgacgacgac ttctcccccg agcgcttcga gtacatcgag cgcctgcagc aaaagctttt6780ggaccacaac gacgacttcg tcagctgctg ctacacggcg ttctcggact tccgcctatg6840ggacgcgttc cacaggctgt gggcggtcgg caccatcctc gggcagttcc ggctcgtgca6900ggcccacgcg aggttccgcg cgtcgcgcaa cgagggcgac ctcgatcacc tcgacaacga6960ccctccgtat ctcggatacc tgtgcgcgga catggaggag tactaccagt tgttcaacga7020cgccaaagcc gaggtcgagg ccgtgagtgc cgggcgcaag ccggccgatg aggccgcggc7080gcggattcac gccctcattg acgaacgaga cttcgccaag ccgatgttcg gcttcgggta7140ctgcatcacc ggggacaagc cgcagctcaa caactcgaag tacagcctgc tgccggcgat7200gcggctgatg tactggacgc aaacccgcgc gccggcagag gtgaaaaagt acttcgacta7260caacccgatg ttcgcgctgc tcaaggcgta catcacgacc cgcatcggcc tggcgctgaa7320gaagtagccg ctcgagggat cccccgaatt tccccgatcg ttcaaacatt tggcaataaa7380gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatctaa tttctgttga7440attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt7500ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg7560caaactagga taaattatcg cgcgcggtgt catctatgtt actagatccg ggaattagcg7620gccgcctcga ggtaccggat ttggagccaa gtctcataaa cgccattgtg gaagaaagtc7680ttgagttggt ggtaatgtaa cagagtagta agaacagaga agagagagag tgtgagatac7740atgaattgtc gggcaacaaa aatcctgaac atcttatttt agcaaagaga aagagttccg7800agtctgtagc agaagagtga ggagaaattt aagctcttgg acttgtgaat tgttccgcct7860cttgaatact tcttcaatcc tcatatattc ttcttctatg ttacctgaaa accggcattt7920aatctcgcgg gtttattccg gttcaacatt ttttttgttt tgagttatta tctgggctta7980ataacgcagg cctgaaataa attcaaggcc caactgtttt tttttttaag aagttgctgt8040taaaaaaaaa aaaagggaat taacaacaac aacaaaaaaa gataaagaaa ataataacaa8100ttactttaat tgtagactaa aaaaacatag attttatcat gaaaaaaaga gaaaagaaat8160aaaaacttgg atcaaaaaaa aaaacataca gatcttctaa ttattaactt ttcttaaaaa8220ttaggtcctt tttcccaaca attaggttta gagttttgga attaaaccaa aaagattgtt8280ctaaaaaata ctcaaatttg gtagataagt ttccttattt taattagtca atggtagata8340cttttttttc ttttctttat tagagtagat tagaatcttt tatgccaagt tttgataaat8400taaatcaaga agataaacta tcataatcaa catgaaatta aaagaaaaat ctcatatata8460gtattagtat tctctatata tattatgatt gcttattctt aatgggttgg gttaaccaag8520acatagtctt aatggaaaga atcttttttg aactttttcc ttattgatta aattcttcta8580tagaaaagaa agaaattatt tgaggaaaag tatatacaaa aagaaaaata gaaaaatgtc8640agtgaagcag atgtaatgga tgacctaatc caaccaccac cataggatgt ttctacttga8700gtcggtcttt taaaaacgca cggtggaaaa tatgacacgt atcatatgat tccttccttt8760agtttcgtga taataatcct caactgatat cttccttttt ttgttttggc taaagatatt8820ttattctcat taatagaaaa gacggttttg ggcttttggt ttgcgatata aagaagacct8880tcgtgtggaa gataataatt catcctttcg tctttttctg actcttcaat ctctcccaaa8940gcctaaagcg atctctgcaa atctctcgcg actctctctt tcaaggtata ttttctgatt9000ctttttgttt ttgattcgta tctgatctcc aatttttgtt atgtggatta ttgaatcttt9060tgtataaatt gcttttgaca atattgttcg tttcgtcaat ccagcttcta aattttgtcc9120tgattactaa gatatcgatt cgtagtgttt acatctgtgt aatttcttgc ttgattgtga9180aattaggatt ttcaaggacg atctattcaa tttttgtgtt ttctttgttc gattctctct9240gttttaggtt tcttatgttt agatccgttt ctctttggtg ttgttttgat ttctcttacg9300gcttttgatt tggtatatgt tcgctgattg gtttctactt gttctattgt tttatttcag9360gtggatccac catggaacgc accttggacc gggtaggcgt attcgcggcc acccacgctg9420ccgtggcggc ctgcgatccg ctgcaggcgc gcgcgctcgt tctgcaactg ccgggcctga9480accgtaacaa ggacgtgccc ggtatcgtcg gcctgctgcg cgagttcctt ccggtgcgcg9540gcctgccctg cggctggggt ttcgtcgaag ccgccgccgc gatgcgggac atcgggttct9600tcctggggtc gctcaagcgc cacggacatg agcccgcgga ggtggtgccc gggcttgagc9660cggtgctgct cgacctggca cgcgcgacca acctgccgcc gcgcgagacg ctcctgcatg9720tgacggtctg gaaccccacg gcggccgacg cgcagcgcag ctacaccggg ctgcccgacg9780aagcgcacct gctcgagagc gtgcgcatct cgatggcggc cctcgaggcg gccatcgcgt9840tgaccgtcga gctgttcgat gtgtccctgc ggtcgcccga gttcgcgcaa aggtgcgacg9900agctggaagc ctatctgcag aaaatggtcg aatcgatcgt ctacgcgtac cgcttcatct9960cgccgcaggt cttctacgat gagctgcgcc ccttctacga accgattcga gtcgggggcc10020agagctacct cggccccggt gccgtagaga tgcccctctt cgtgctggag cacgtcctct10080ggggctcgca atcggacgac caaacttatc gagaattcaa agagacgtac ctgccctatg10140tgcttcccgc gtacagggcg gtctacgctc ggttctccgg ggagccggcg ctcatcgacc10200gcgcgctcga cgaggcgcga gcggtcggta cgcgggacga gcacgtccgg gctgggctga10260cagccctcga gcgggtcttc aaggtcctgc tgcgcttccg ggcgcctcac ctcaaattgg10320cggagcgggc gtacgaagtc gggcaaagcg gccccgaaat cggcagcggg gggtacgcgc10380ccagcatgct cggtgagctg ctcacgctga cgtatgccgc gcggtcccgc gtccgcgccg10440cgctcgacga atcctgaagc ttggatcccc cgaatttccc cgatcgttca aacatttggc10500aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atctaatttc10560tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat10620gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat10680agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatccgggaa 10740ttgggtaccg gatttggagc caagtctcat aaacgccatt gtggaagaaa gtcttgagtt 10800ggtggtaatg taacagagta gtaagaacag agaagagaga gagtgtgaga tacatgaatt 10860gtcgggcaac aaaaatcctg aacatcttat tttagcaaag agaaagagtt ccgagtctgt 10920agcagaagag tgaggagaaa tttaagctct tggacttgtg aattgttccg cctcttgaat 10980acttcttcaa tcctcatata ttcttcttct atgttacctg aaaaccggca tttaatctcg 11040cgggtttatt ccggttcaac attttttttg ttttgagtta ttatctgggc ttaataacgc 11100aggcctgaaa taaattcaag gcccaactgt tttttttttt aagaagttgc tgttaaaaaa 11160aaaaaaaggg aattaacaac aacaacaaaa aaagataaag aaaataataa caattacttt 11220aattgtagac taaaaaaaca tagattttat catgaaaaaa agagaaaaga aataaaaact 11280tggatcaaaa aaaaaaacat acagatcttc taattattaa cttttcttaa aaattaggtc 11340ctttttccca acaattaggt ttagagtttt ggaattaaac caaaaagatt gttctaaaaa 11400atactcaaat ttggtagata agtttcctta ttttaattag tcaatggtag atactttttt 11460ttcttttctt tattagagta gattagaatc ttttatgcca agttttgata aattaaatca 11520agaagataaa ctatcataat caacatgaaa ttaaaagaaa aatctcatat atagtattag 11580tattctctat atatattatg attgcttatt cttaatgggt tgggttaacc aagacatagt 11640cttaatggaa agaatctttt ttgaactttt tccttattga ttaaattctt ctatagaaaa 11700gaaagaaatt atttgaggaa aagtatatac aaaaagaaaa atagaaaaat gtcagtgaag 11760cagatgtaat ggatgaccta atccaaccac caccatagga tgtttctact tgagtcggtc 11820ttttaaaaac gcacggtgga aaatatgaca cgtatcatat gattccttcc tttagtttcg 11880tgataataat cctcaactga tatcttcctt tttttgtttt ggctaaagat attttattct 11940cattaataga aaagacggtt ttgggctttt ggtttgcgat ataaagaaga ccttcgtgtg 12000gaagataata attcatcctt tcgtcttttt ctgactcttc aatctctccc aaagcctaaa 12060gcgatctctg caaatctctc gcgactctct ctttcaaggt atattttctg attctttttg 12120tttttgattc gtatctgatc tccaattttt gttatgtgga ttattgaatc ttttgtataa 12180attgcttttg acaatattgt tcgtttcgtc aatccagctt ctaaattttg tcctgattac 12240taagatatcg attcgtagtg tttacatctg tgtaatttct tgcttgattg tgaaattagg 12300attttcaagg acgatctatt caatttttgt gttttctttg ttcgattctc tctgttttag 12360gtttcttatg tttagatccg tttctctttg gtgttgtttt gatttctctt acggcttttg 12420atttggtata tgttcgctga ttggtttcta cttgttctat tgttttattt caggtggatc 12480caccatgaac gacattcaat tggatcaagc gagcgtcaag aagcgtccct cgggcgcgta 12540cgacgcaacc acgcgcctgg ccgcgagctg gtacgtcgcg atgcgctcca acgagctcaa 12600ggacaagccg accgagttga cgctcttcgg ccgtccgtgc gtggcgtggc gcggagccac 12660ggggcgggcc gtggtgatgg accgccactg ctcgcacctg ggcgcgaacc tggctgacgg 12720gcggatcaag gacgggtgca tccagtgccc gtttcaccac tggcggtacg acgaacaggg 12780ccagtgcgtt cacatccccg gccataacca ggcggtgcgc cagctggagc cggtgccgcg 12840cggggcgcgt cagccgacgt tggtcaccgc cgagcgatac ggctacgtgt gggtctggta 12900cggctccccg ctgccgctgc acccgctgcc cgaaatctcc gcggccgatg tcgacaacgg 12960cgactttatg cacctgcact tcgcgttcga gacgaccacg gcggtcttgc ggatcgtcga 13020gaacttctac gacgcgcagc acgcaacccc ggtgcacgca ctcccgatct cggccttcga 13080actcaagctc ttcgacgatt ggcgccagtg gccggaggtt gagtcgctgg ccctggcggg 13140cgcgtggttc ggtgccggga tcgacttcac cgtggaccgg tacttcggcc ccctcggcat 13200gctgtcacgc gcgctcggcc tgaacatgtc gcagatgaac ctgcacttcg atggctaccc 13260cggcgggtgc gtcatgaccg tcgccctgga cggagacgtc aaatacaagc tgctccagtg 13320tgtgacgccg gtgagcgaag gcaagaacgt catgcacatg ctcatctcga tcaagaaggt 13380gggcggcatc ctgctccgcg cgaccgactt cgtgctgttc gggctgcaga ccaggcaggc 13440cgcggggtac gacgtcaaaa tctggaacgg aatgaagccg gacggcggcg gcgcgtacag 13500caagtacgac aagctcgtgc tcaagtaccg ggcgttctat cgaggctggg tcgaccgcgt 13560cgcaagtgag cggtgaagct tggatccccc gaatttcccc gatcgttcaa acatttggca 13620ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tctaatttct 13680gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg 13740ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata 13800gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atccgggaat 13860tccggaccgc gctctagtgc gatcgctttt ttaattaatt ttaagctttc tagaggccgg 13920ccgcggccgc ctcgaggtac catcaggata ttcttgctta agatgttgaa ctctatggag 13980gtttgtatga actgatgatc taggaccgga taagttccct tcttcatagc gaacttattc 14040aaagaatgtt ttgtgtatca ttcttgttac attgttatta atgaaaaaat attattggtc 14100attggactga acacgagtgt taaatatgga ccaggcccca aataagatcc attgatatat 14160gaattaaata acaagaataa atcgagtcac caaaccactt gcctttttta acgagacttg 14220ttcaccaact tgatacaaaa gtcattatcc tatgcaaatc aataatcata caaaaatatc 14280caataacact aaaaaattaa aagaaatgga taatttcaca atatgttata cgataaagaa 14340gttacttttc caagaaattc actgatttta taagcccact tgcattagat aaatggcaaa 14400aaaaaacaaa aaggaaaaga aataaagcac gaagaattct agaaaatacg aaatacgctt 14460caatgcagtg ggacccacgg ttcaattatt gccaattttc agctccaccg tatatttaaa 14520aaataaaacg ataatgctaa aaaaatataa atcgtaacga tcgttaaatc tcaacggctg 14580gatcttatga cgaccgttag aaattgtggt tgtcgacgag tcagtaataa acggcgtcaa 14640agtggttgca gccggcacac acgagtcgtg tttatcaact caaagcacaa atacttttcc 14700tcaacctaaa aataaggcaa ttagccaaaa acaactttgc gtgtaaacaa cgctcaatac 14760acgtgtcatt ttattattag ctattgcttc accgccttag ctttctcgtg acctagtcgt 14820cctcgtcttt tcttccttct tcttctataa aacaataccc aaagagctct tcttcttcac 14880aattcagatt tcaatttctc aaaatcttaa aaactttctc tcaattctct ctaccgtgat 14940cggaaccatg acaaccttaa gctgtaaagt gacctcggta gaagctatca cggataccgt 15000atatcgtgtc cgcatcgtgc cagacgcggc cttttctttt cgtgctggtc agtatttgat 15060ggtagtgatg gatgagcgcg acaaacgtcc gttctcaatg gcttcgacgc cggatgaaaa 15120agggtttatc gagctgcata ttggcgcttc tgaaatcaac ctttacgcga aagcagtcat 15180ggaccgcatc ctcaaagatc atcaaatcgt ggtcgacatt ccccacggag aagcgtggct 15240gcgcgatgat gaagagcgtc cgatgatttt gattgcgggc ggcaccgggt tctcttatgc 15300ccgctcgatt ttgctgacag cgttggcgcg taacccaaac cgtgatatca ccatttactg 15360gggcgggcgt gaagagcagc atctgtatga tctctgcgag cttgaggcgc tttcgttgaa 15420gcatcctggt ctgcaagtgg tgccggtggt tgaacaaccg gaagcgggct ggcgtgggcg 15480tactggcacc gtgttaacgg cggtattgca ggatcacggt acgctggcag agcatgatat 15540ctatattgcc ggacgttttg agatggcgaa aattgcccgc gatctgtttt gcagtgagcg 15600taatgcgcgg gaagatcgcc tgtttggcga tgcgtttgca tttatctgag gatcccccga 15660atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 15720gtcttgcgat gattatcatc taatttctgt tgaattacgt taagcatgta ataattaaca 15780tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 15840tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 15900tgtcatctat gttactagat ccgggaatta gcggccgcct cgaggtaccg gatttggagc 15960caagtctcat aaacgccatt gtggaagaaa gtcttgagtt ggtggtaatg taacagagta 16020gtaagaacag agaagagaga gagtgtgaga tacatgaatt gtcgggcaac aaaaatcctg 16080aacatcttat tttagcaaag agaaagagtt ccgagtctgt agcagaagag tgaggagaaa 16140tttaagctct tggacttgtg aattgttccg cctcttgaat acttcttcaa tcctcatata 16200ttcttcttct atgttacctg aaaaccggca tttaatctcg cgggtttatt ccggttcaac 16260attttttttg ttttgagtta ttatctgggc ttaataacgc aggcctgaaa taaattcaag 16320gcccaactgt tttttttttt aagaagttgc tgttaaaaaa aaaaaaaggg aattaacaac 16380aacaacaaaa aaagataaag aaaataataa caattacttt aattgtagac taaaaaaaca 16440tagattttat catgaaaaaa agagaaaaga aataaaaact tggatcaaaa aaaaaaacat 16500acagatcttc taattattaa cttttcttaa aaattaggtc ctttttccca acaattaggt 16560ttagagtttt ggaattaaac caaaaagatt gttctaaaaa atactcaaat ttggtagata 16620agtttcctta ttttaattag tcaatggtag atactttttt ttcttttctt tattagagta 16680gattagaatc ttttatgcca agttttgata aattaaatca agaagataaa ctatcataat 16740caacatgaaa ttaaaagaaa aatctcatat atagtattag tattctctat atatattatg 16800attgcttatt cttaatgggt tgggttaacc aagacatagt cttaatggaa agaatctttt 16860ttgaactttt tccttattga ttaaattctt ctatagaaaa gaaagaaatt atttgaggaa 16920aagtatatac aaaaagaaaa atagaaaaat gtcagtgaag cagatgtaat ggatgaccta 16980atccaaccac caccatagga tgtttctact tgagtcggtc ttttaaaaac gcacggtgga 17040aaatatgaca cgtatcatat gattccttcc tttagtttcg tgataataat cctcaactga 17100tatcttcctt tttttgtttt ggctaaagat attttattct cattaataga aaagacggtt 17160ttgggctttt ggtttgcgat ataaagaaga ccttcgtgtg gaagataata attcatcctt 17220tcgtcttttt ctgactcttc aatctctccc aaagcctaaa gcgatctctg caaatctctc 17280gcgactctct ctttcaaggt atattttctg attctttttg tttttgattc gtatctgatc 17340tccaattttt gttatgtgga ttattgaatc ttttgtataa attgcttttg acaatattgt 17400tcgtttcgtc aatccagctt ctaaattttg tcctgattac taagatatcg attcgtagtg 17460tttacatctg tgtaatttct tgcttgattg tgaaattagg attttcaagg acgatctatt 17520caatttttgt gttttctttg ttcgattctc tctgttttag gtttcttatg tttagatccg 17580tttctctttg gtgttgtttt gatttctctt acggcttttg atttggtata tgttcgctga 17640ttggtttcta cttgttctat tgttttattt caggtggatc tgttggggat ctaccatgag 17700cccagaacga cgcccggccg acatccgccg tgccaccgag gcggacatgc cggcggtctg 17760caccatcgtc aaccactaca tcgagacaag cacggtcaac ttccgtaccg agccgcagga 17820accgcaggag tggacggacg acctcgtccg tctgcgggag cgctatccct ggctcgtcgc 17880cgaggtggac ggcgaggtcg ccggcatcgc ctacgcgggc ccctggaagg cacgcaacgc 17940ctacgactgg acggccgagt cgaccgtgta cgtctccccc cgccaccagc ggacgggact 18000gggctccacg ctctacaccc acctgctgaa gtccctggag gcacagggct tcaagagcgt 18060ggtcgctgtc atcgggctgc ccaacgaccc gagcgtgcgc atgcacgagg cgctcggata 18120tgccccccgc ggcatgctgc gggcggccgg cttcaagcac gggaactggc atgacgtggg 18180tttctggcag ctggacttca gcctgccggt accgccccgt ccggtcctgc ccgtcaccga 18240gatcccccga atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat 18300cctgttgccg gtcttgcgat gattatcatc taatttctgt tgaattacgt taagcatgta 18360ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg 18420caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta 18480tcgcgcgcgg tgtcatctat gttactagat ccgggaattg ggtacccaat tccggaccgc 18540tgctctagag gcgcgcccct agggagcttc tgcagacgcg tcgacgtcat atggatccga 18600tctgttgccc gtctcactgg tgaaaagaaa aaccacccca gtacattaaa aacgtccgca 18660atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag 18720ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc 18780catcagtccg ggacggtcga cctgcaggca tgcaagctca cgtagtgtac gtaatcgatt 18840tcgaagggcc ccctagtcca tgggcttttt ctcctcgtgc tcgtaaacgg acccgaacat 18900ctctggagct ttcttcaggg ccgacaatcg gatctcgcgg aaatcctgca cgtcggccgc 18960tccaagccgt cgaatctgag ccttaatcac aattgtcaat tttaatcctc tgtttatcgg 19020cagttcgtag agcgcgccgt gcgtcccgag cgatactgag cgaagcaagt gcgtcgagca 19080gtgcccgctt gttcctgaaa tgccagtaaa gcgctggctg ctgaaccccc agccggaact 19140gaccccacaa ggccctagcg tttgcaatgc accaggtcat cattgaccca ggcgtgttcc 19200accaggccgc tgcctcgcaa ctcttcgcag gcttcgccga cctgctcgcg ccacttcttc 19260acgcgggtgg aatccgatcc gcacatgagg cggaaggttt ccagcttgag cgggtacggc 19320tcccggtgcg agctgaaata gtcgaacatc cgtcgggccg tcggcgacag cttgcggtac 19380ttctcccata tgaatttcgt gtagtggtcg ccagcaaaca gcacgacgat ttcctcgtcg 19440atcaggacct ggcaacggga cgttttcttg ccacggtcca ggacgcggaa gcggtgcagc 19500agcgacaccg attccaggtg cccaacgcgg tcggacgtga agcccatcgc cgtcgcctgt 19560aggcgcgaca ggcattcctc ggccttcgtg taataccggc cattgatcga ccagcccagg 19620tcctggcaaa gctcgtagaa cgtgaaggtg atcggctcgc cgataggggt gcgcttcgcg 19680tactccaaca cctgctgcca caccagttcg tcatcgtcgg cccgcagctc gacgccggtg 19740taggtgatct tcacgtcctt gttgacgtgg aaaatgacct tgttttgcag cgcctcgcgc 19800gggattttct tgttgcgcgt ggtgaacagg gcagagcggg ccgtgtcgtt tggcatcgct 19860cgcatcgtgt ccggccacgg cgcaatatcg aacaaggaaa gctgcatttc cttgatctgc 19920tgcttcgtgt gtttcagcaa cgcggcctgc ttggcctcgc tgacctgttt tgccaggtcc 19980tcgccggcgg tttttcgctt cttggtcgtc atagttcctc gcgtgtcgat ggtcatcgac 20040ttcgccaaac ctgccgcctc ctgttcgaga cgacgcgaac gctccacggc ggccgatggc 20100gcgggcaggg cagggggagc cagttgcacg ctgtcgcgct cgatcttggc cgtagcttgc 20160tggaccatcg agccgacgga ctggaaggtt tcgcggggcg cacgcatgac ggtgcggctt 20220gcgatggttt cggcatcctc ggcggaaaac cccgcgtcga tcagttcttg cctgtatgcc 20280ttccggtcaa acgtccgatt cattcaccct ccttgcggga ttgccccgac tcacgccggg 20340gcaatgtgcc cttattcctg atttgacccg cctggtgcct tggtgtccag ataatccacc 20400ttatcggcaa tgaagtcggt cccgtagacc gtctggccgt ccttctcgta cttggtattc 20460cgaatcttgc cctgcacgaa taccagcgac cccttgccca aatacttgcc gtgggcctcg 20520gcctgagagc caaaacactt gatgcggaag aagtcggtgc gctcctgctt gtcgccggca 20580tcgttgcgcc acatctaggt actaaaacaa ttcatccagt aaaatataat attttatttt 20640ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc 20700gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc 20760gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc 20820tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc 20880atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc 20940ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt 21000atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc 21060gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc 21120ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac 21180aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt 21240ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt 21300atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag 21360ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta 21420cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc 21480cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg 21540gcgtataaca tagtatcgac ggagccgatt ttgaaaccac aattatgggt gatgctgcca 21600acttactgat ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc tgtgtctatc 21660agctgtccct cctgttcagc tactgacggg gtggtgcgta acggcaaaag caccgccgga 21720catcagcgct atctctgctc tcactgccgt aaaacatggc aactgcagtt cacttacacc 21780gcttctcaac ccggtacgca ccagaaaatc attgatatgg ccatgaatgg cgttggatgc 21840cgggcaacag cccgcattat gggcgttggc ctcaacacga ttttacgtca cttaaaaaac 21900tcaggccgca gtcggtaacc tcgcgcatac agccgggcag tgacgtcatc gtctgcgcgg 21960aaatggacga acagtggggc tatgtcgggg ctaaatcgcg ccagcgctgg ctgttttacg 22020cgtatgacag tctccggaag acggttgttg cgcacgtatt cggtgaacgc actatggcga 22080cgctggggcg tcttatgagc ctgctgtcac cctttgacgt ggtgatatgg atgacggatg 22140gctggccgct gtatgaatcc cgcctgaagg gaaagctgca cgtaatcagc aagcgatata 22200cgcagcgaat tgagcggcat aacctgaatc tgaggcagca cctggcacgg ctgggacgga 22260agtcgctgtc gttctcaaaa tcggtggagc tgcatgacaa agtcatcggg cattatctga 22320acataaaaca ctatcaataa gttggagtca ttacccaatt atgatagaat ttacaagcta 22380taaggttatt gtcctgggtt tcaagcatta gtccatgcaa gtttttatgc tttgcccatt 22440ctatagatat attgataagc gcgctgccta tgccttgccc cctgaaatcc ttacatacgg 22500cgatatcttc tatataaaag atatattatc ttatcagtat tgtcaatata ttcaaggcaa 22560tctgcctcct catcctcttc atcctcttcg tcttggtagc tttttaaata tggcgcttca 22620tagagtaatt ctgtaaaggt ccaattctcg ttttcatacc tcggtataat cttacctatc 22680acctcaaatg gttcgctggg tttatcgcac ccccgaacac gagcacggca cccgcgacca 22740ctatgccaag aatgcccaag gtaaaaattg ccggccccgc catgaagtcc gtgaatgccc 22800cgacggccga agtgaagggc aggccgccac ccaggccgcc gccctcactg cccggcacct 22860ggtcgctgaa tgtcgatgcc agcacctgcg gcacgtcaat gcttccgggc gtcgcgctcg 22920ggctgatcgc ccatcccgtt actgccccga tcccggcaat ggcaaggact gccagcgctg 22980ccatttttgg ggtgaggccg ttcgcggccg aggggcgcag cccctggggg gatgggaggc 23040ccgcgttagc gggccgggag ggttcgagaa gggggggcac cccccttcgg cgtgcgcggt 23100cacgcgcaca gggcgcagcc ctggttaaaa acaaggttta taaatattgg tttaaaagca 23160ggttaaaaga caggttagcg gtggccgaaa aacgggcgga aacccttgca aatgctggat 23220tttctgcctg tggacagccc ctcaaatgtc aataggtgcg cccctcatct gtcagcactc 23280tgcccctcaa gtgtcaagga tcgcgcccct catctgtcag tagtcgcgcc cctcaagtgt 23340caataccgca gggcacttat ccccaggctt gtccacatca tctgtgggaa actcgcgtaa 23400aatcaggcgt tttcgccgat ttgcgaggct ggccagctcc acgtcgccgg ccgaaatcga 23460gcctgcccct catctgtcaa cgccgcgccg ggtgagtcgg cccctcaagt gtcaacgtcc 23520gcccctcatc tgtcagtgag ggccaagttt tccgcgaggt atccacaacg ccggcggccg 23580cggtgtctcg cacacggctt cgacggcgtt tctggcgcgt ttgcagggcc atagacggcc 23640gccagcccag cggcgagggc aaccagcccg gtgagcgtcg caaaggcgct cggtcttgcc 23700ttgctcgtcg gtgatgtact tcaccagctc cgcgaagtcg ctcttcttga tggagcgcat 23760ggggacgtgc ttggcaatca cgcgcacccc ccggccgttt tagcggctaa aaaagtcatg 23820gctctgccct cgggcggacc acgcccatca tgaccttgcc aagctcgtcc tgcttctctt 23880cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa ccgcgccgtg cgcgggtcgt 23940cggtgagcca gagtttcagc aggccgccca ggcggcccag gtcgccattg atgcgggcca 24000gctcgcggac gtgctcatag tccacgacgc ccgtgatttt gtagccctgg ccgacggcca 24060gcaggtaggc cgacaggctc atgccggccg ccgccgcctt ttcctcaatc gctcttcgtt 24120cgtctggaag gcagtacacc ttgataggtg ggctgccctt cctggttggc ttggtttcat 24180cagccatccg cttgccctca tctgttacgc cggcggtagc cggccagcct cgcagagcag 24240gattcccgtt gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa cacccgctcg 24300cgggtgggcc tacttcacct atcctgcccg gctgacgccg ttggatacac caaggaaagt 24360ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga tataccgaaa 24420aaatcgctat aatgaccccg aagcagggtt atgcagcgga aaagcgccac gcttcccgaa 24480gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 24540gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 24600cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 24660aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 24720gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 24780cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccag 24840aaggccgcca gagaggccga gcgcggccgt gaggcttgga cgctagggca gggcatgaaa 24900aagcccgtag cgggctgcta cgggcgtctg acgcggtgga aagggggagg ggatgttgtc 24960tacatggctc tgctgtagtg agtgggttgc gctccggcag cggtcctgat caatcgtcac 25020cctttctcgg tccttcaacg ttcctgacaa cgagcctcct tttcgccaat ccatcgacaa 25080tcaccgcgag tccctgctcg aacgctgcgt ccggaccggc ttcgtcgaag gcgtctatcg 25140cggcccgcaa cagcggcgag agcggagcct gttcaacggt gccgccgcgc tcgccggcat 25200cgctgtcgcc ggcctgctcc tcaagcacgg ccccaacagt gaagtagctg attgccatca 25260gcgcattgac ggcgtccccg gccgaaaaac ccgcctcgca gaggaagcga agctgcgcgt 25320cggccgtttc catctgcggt gcgcccggtc gcgtgccggc atggatgcgc gcgccatcgc 25380ggtaggcgag cagcgcctgc ctgaagctgc gggcattccc gatcagaaat gagcgccagt 25440cgtcgtcggc tctcggcacc gaatgcgtat gattctccgc cagcatggct tcggccagtg 25500cgtcgagcag cgcccgcttg ttcctgaagt gccagtaaag cgccggctgc tgaaccccca 25560accgttccgc cagtttgcgt gtcgtcagac cgtctacgcc gacctcgttc aacaggtcca 25620gggcggcacg gatcactgta ttcggctgca actttgtcat gcttgacact ttatcactga 25680taaacataat atgtccacca acttatcagt gataaagaat ccgcgcgttc aatcggacca 25740gcggaggctg gtccggaggc cagacgtgaa acccaacata cccctgatcg taattctgag 25800cactgtcgcg ctcgacgctg tcggcatcgg cctgattatg ccggtgctgc cgggcctcct 25860gcgcgatctg gttcactcga acgacgtcac cgcccactat ggcattctgc tggcgctgta 25920tgcgttggtg caatttgcct gcgcacctgt gctgggcgcg ctgtcggatc gtttcgggcg 25980gcggccaatc ttgctcgtct cgctggccgg cgccagatc 26019<210>35<2ll>20119<212>DNA<213>合成<220><22l>啟動子<222>(229)..(1960)<220><22l>exon<222>(1962)..(2160)<220><221>misc_feature<222>(2160)..(2228)<223>plastid targeting domain<220><221>基大<222>(2260)..(3960)<223>prnC<220><221>終止子<222>(3966)..(4241)<220><22l>啟動子<222>(4245)..(5971)<220><221>exon<222>(5984)..(6182)<220><221>misc_feature<222>(6183)..(6251)<223>plastid targeting domain<220><221>基因<222>(6282)..(7373)<223>prnD<220><22l>終止子<222>(7379)..(7639)<220><22l>啟動子<222>(7739)..(8739)<220><221>exon<222>(8745)..(8943)<220><221>misc_feature<222>(8944)..(9012)<223>plastid targeting domain<220><221>基因<222>(9037)..(9743)<223>fre<220><221>終止子<222>(9755)..(10036)<220><221>啟動子<222>(10049)..(11385)<220><22l>內(nèi)含子<222>(11383)..(11779)<220><221>基因<222>(11796)..(12338)<220><221>終子<222>(12345)..(12626)<400>35tggggaaccc tgtggttggc atgcacatac aaatggacga acggataaac cttttcacgc 60ccttttaaat atccgattat tctaataaac gctcttttct cttaggttta cccgccaata 120tatcctgtca aacactgata gtttaaactg aaggcgggaa acgacaatct gatctatcgt 180tctagtcgta cgttttgcga tcgcactaga gcggccgcct cgaggtaccg gatttggagc 240caagtctcat aaacgccatt gtggaagaaa gtcttgagtt ggtggtaatg taacagagta 300gtaagaacag agaagagaga gagtgtgaga tacatgaatt gtcgggcaac aaaaatcctg 360aacatcttat tttagcaaag agaaagagtt ccgagtctgt agcagaagag tgaggagaaa 420tttaagctct tggacttgtg aattgttccg cctcttgaat acttcttcaa tcctcatata 480ttcttcttct atgttacctg aaaaccggca tttaatctcg cgggtttatt ccggttcaac 540attttttttg ttttgagtta ttatctgggc ttaataacgc aggcctgaaa taaattcaag 600gcccaactgt tttttttttt aagaagttgc tgttaaaaaa aaaaaaaggg aattaacaac 660aacaacaaaa aaagataaag aaaataataa caattacttt aattgtagac taaaaaaaca 720tagattttat catgaaaaaa agagaaaaga aataaaaact tggatcaaaa aaaaaaacat 780acagatcttc taattattaa cttttcttaa aaattaggtc ctttttccca acaattaggt 840ttagagtttt ggaattaaac caaaaagatt gttctaaaaa atactcaaat ttggtagata 900agtttcctta ttttaattag tcaatggtag atactttttt ttcttttctt tattagagta 960gattagaatc ttttatgcca agttttgata aattaaatca agaagataaa ctatcataat1020caacatgaaa ttaaaagaaa aatctcatat atagtattag tattctctat atatattatg1080attgcttatt cttaatgggt tgggttaacc aagacatagt cttaatggaa agaatctttt1140ttgaactttt tccttattga ttaaattctt ctatagaaaa gaaagaaatt atttgaggaa1200aagtatatac aaaaagaaaa atagaaaaat gtcagtgaag cagatgtaat ggatgaccta1260atccaaccac caccatagga tgtttctact tgagtcggtc ttttaaaaac gcacggtgga1320aaatatgaca cgtatcatat gattccttcc tttagtttcg tgataataat cctcaactga1380tatcttcctt tttttgtttt ggctaaagat attttattct cattaataga aaagacggtt1440ttgggctttt ggtttgcgat ataaagaaga ccttcgtgtg gaagataata attcatcctt1500tcgtcttttt ctgactcttc aatctctccc aaagcctaaa gcgatctctg caaatctctc1560gcgactctct ctttcaaggt atattttctg attctttttg tttttgattc gtatctgatc1620tccaattttt gttatgtgga ttattgaatc ttttgtataa attgcttttg acaatattgt1680tcgtttcgtc aatccagctt ctaaattttg tcctgattac taagatatcg attcgtagtg1740tttacatctg tgtaatttct tgcttgattg tgaaattagg attttcaagg acgatctatt1800caatttttgt gttttctttg ttcgattctc tctgttttag gtttcttatg tttagatccg1860tttctctttg gtgttgtttt gatttctctt acggcttttg atttggtata tgttcgctga1920ttggtttcta cttgttctat tgttttattt caggtggatc a gtc aca caa aga gta1976Val Thr Gln Arg Val1 5aag aag aac aat ggc ttc ctc tat gct ctc ttc cgc tac tat ggt tgc 2024Lys Lys Asn Asn Gly Phe Leu Tyr Ala Leu Phe Arg Tyr Tyr Gly Cys10 15 20ctc tcc ggc tca ggc cac tat ggt cgc tcc ttt caa cgg act taa gtc 2072Leu Ser Gly Ser Gly His Tyr Gly Arg Ser Phe Gln Arg Thr Val25 30 35ctc cgc tgc ctt ccc agc cac ccg caa ggc taa caa cga cat tac ttc 2120Leu Arg Cys Leu Pro Ser His Pro Gln Gly Gln Arg His Tyr Phe40 45 50cat cac aag caa cgg cgg aag agt taa ctg cat gca ggt g tggcctccga 2170His His Lys Gln Arg Arg Lys Ser Leu His Ala Gly55 60ttggaaagaa gaagtttgag actctctctt accttcctga ccttaccgat tctgcaggag2230gtcgcgtcaa ctgcatgcag gctagcatga ctcagaagag ccccgcgaac gaacacgata2290gcaatcactt cgacgtaatc atcctcggct cgggcatgtc cggcacccag atgggggcca2350tcttggccaa acaacagttt cgcgtgctga tcatcgagga gtcgtcgcac ccgcggttca2410cgatcggcga atcgtcgatc cccgagacgt ctcttatgaa ccgcatcatc gctgatcgct2470acggcattcc ggagctcgac cacatcacgt cgttttattc gacgcaacgt tacgtcgcgt2530cgagcacggg cattaagcgc aacttcggct tcgtgttcca caagcccggc caggagcacg2590acccgaagga gttcacccag tgcgtcattc ccgagctgcc gtgggggccg gagagccatt2650attaccggca agacgtcgac gcctacttgt tgcaagccgc cattaaatac ggctgcaagg2710tccaccagaa aactaccgtg accgaatacc acaccgataa agacggcgtc gcggtgacca2770ccgcccaggg cgaacggttc accggccggt acatgatcga ctgcggagga cctcgcgcgc2830cgctcgcgac caagttcagg ctccgcgaag aaccgtgtcg cttcaagacg cactcgcgca2890gcctctacac gcacatgctc ggggtcaagc cgttcgacga catcttcaag gtcaaggggc2950agcgctggcg ctggcacgag gggaccttgc accacatgtt cgagggcggc tggctctggg3010tgattccgtt caacaaccac ccgcggtcga ccaacaacct ggtgagcgtc ggcctgcagc3070tcgacccgcg tgtctacccg aaaaccgaca tctccgcaca gcaggaattc gatgagttcc3130tcgcgcggtt cccgagcatc ggggctcagt tccgggacgc cgtgccggtg cgcgactggg3190tcaagaccga ccgcctgcaa ttctcgtcga acgcctgcgt cggcgaccgc tactgcctga3250tgctgcacgc gaacggcttc atcgacccgc tcttctcccg ggggctggaa aacaccgcgg3310tgaccatcca cgcgctcgcg gcgcgcctca tcaaggcgct gcgcgacgac gacttctccc3370ccgagcgctt cgagtacatc gagcgcctgc agcaaaagct tttggaccac aacgacgact3430tcgtcagctg ctgctacacg gcgttctcgg acttccgcct atgggacgcg ttccacaggc3490tgtgggcggt cggcaccatc ctcgggcagt tccggctcgt gcaggcccac gcgaggttcc3550gcgcgtcgcg caacgagggc gacctcgatc acctcgacaa cgaccctccg tatctcggat3610acctgtgcgc ggacatggag gagtactacc agttgttcaa cgacgccaaa gccgaggtcg3670aggccgtgag tgccgggcgc aagccggccg atgaggccgc ggcgcggatt cacgccctca3730ttgacgaacg agacttcgcc aagccgatgt tcggcttcgg gtactgcatc accggggaca3790agccgcagct caacaactcg aagtacagcc tgctgccggc gatgcggctg atgtactgga3850cgcaaacccg cgcgccggca gaggtgaaaa agtacttcga ctacaacccg atgttcgcgc3910tgctcaaggc gtacatcacg acccgcatcg gcctggcgct gaagaagtag ggatcccccg3970aatttccccg atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc4030ggtcttgcga tgattatcat ctaatttctg ttgaattacg ttaagcatgt aataattaac4090atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac4150atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg4210gtgtcatcta tgttactaga tccgggaatt ccgctcgagg taccggattt ggagccaagt4270ctcataaacg ccattgtgga agaaagtctt gagttggtgg taatgtaaca gagtagtaag4330aacagagaag agagagagtg tgagatacat gaattgtcgg gcaacaaaaa tcctgaacat4390cttattttag caaagagaaa gagttccgag tctgtagcag aagagtgagg agaaatttaa4450gctcttggac ttgtgaattg ttccgcctct tgaatacttc ttcaatcctc atatattctt4510cttctatgtt acctgaaaac cggcatttaa tctcgcgggt ttattccggt tcaacatttt4570ttttgttttg agttattatc tgggcttaat aacgcaggcc tgaaataaat tcaaggccca4630actgtttttt tttttaagaa gttgctgtta aaaaaaaaaa aagggaatta acaacaacaa4690caaaaaaaga taaagaaaat aataacaatt actttaattg tagactaaaa aaacatagat4750tttatcatga aaaaaagaga aaagaaataa aaacttggat caaaaaaaaa aacatacaga4810tcttctaatt attaactttt cttaaaaatt aggtcctttt tcccaacaat taggtttaga4870gttttggaat taaaccaaaa agattgttct aaaaaatact caaatttggt agataagttt4930ccttatttta attagtcaat ggtagatact tttttttctt ttctttatta gagtagatta4990gaatctttta tgccaagttt tgataaatta aatcaagaag ataaactatc ataatcaaca5050tgaaattaaa agaaaaatct catatatagt attagtattc tctatatata ttatgattgc5110ttattcttaa tgggttgggt taaccaagac atagtcttaa tggaaagaat cttttttgaa5170ctttttcctt attgattaaa ttcttctata gaaaagaaag aaattatttg aggaaaagta5230tatacaaaaa gaaaaataga aaaatgtcag tgaagcagat gtaatggatg acctaatcca5290accaccacca taggatgttt ctacttgagt cggtctttta aaaacgcacg gtggaaaata5350tgacacgtat catatgattc cttcctttag tttcgtgata ataatcctca actgatatct5410tccttttttt gttttggcta aagatatttt attctcatta atagaaaaga cggttttggg5470cttttggttt gcgatataaa gaagaccttc gtgtggaaga taataattca tcctttcgtc5530tttttctgac tcttcaatct ctcccaaagc ctaaagcgat ctctgcaaat ctctcgcgac5590tctctctttc aaggtatatt ttctgattct ttttgttttt gattcgtatc tgatctccaa5650tttttgttat gtggattatt gaatcttttg tataaattgc ttttgacaat attgttcgtt5710tcgtcaatcc agcttctaaa ttttgtcctg attactaaga tatcgattcg tagtgtttac5770atctgtgtaa tttcttgctt gattgtgaaa ttaggatttt caaggacgat ctattcaatt5830tttgtgtttt ctttgttcga ttctctctgt tttaggtttc ttatgtttag atccgtttct5890ctttggtgtt gttttgattt ctcttacggc ttttgatttg gtatatgttc gctgattggt5950ttctacttgt tctattgttt tatttcaggt gga tca gtc aca caa aga gta aag 6004
Ser Val Thr Gln Arg Val Lys65 70aag aac aat ggc ttc ctc tat gct ctc ttc cgc tac tat ggt tgc ctc 6052Lys Asn Asn Gly Phe Leu Tyr Ala Leu Phe Arg Tyr Tyr Gly Cys Leu75 80 85tcc ggc tca ggc cac tat ggt cgc tcc ttt caa cgg act taa gtc ctc 6100Ser Gly Ser Gly His Tyr Gly Arg Ser Phe Gln Arg Thr Val Leu90 95 100cgc tgc ctt ccc agc cac ccg caa ggc taa caa cga cat tac ttc cat 6148Arg Cys Leu Pro Ser His Pro Gln Gly Gln Arg His Tyr Phe His105 110 115cac aag caa cgg cgg aag agt taa ctg cat gca g gtgtggcctc 6192His Lys Gln Arg Arg Lys Ser Leu His Ala120 125cgattggaaa gaagaagttt gagactctct cttaccttcc tgaccttacc gattctgcag 6252gaggtcgcgt caactgcatg caggctagca tgaacgacat tcaattggat caagcgagcg 6312tcaagaagcg tccctcgggc gcgtacgacg caaccacgcg cctggccgcg agctggtacg 6372tcgcgatgcg ctccaacgag ctcaaggaca agccgaccga gttgacgctc ttcggccgtc 6432cgtgcgtggc gtggcgcgga gccacggggc gggccgtggt gatggaccgc cactgctcgc 6492acctgggcgc gaacctggct gacgggcgga tcaaggacgg gtgcatccag tgcccgtttc 6552accactggcg gtacgacgaa cagggccagt gcgttcacat ccccggccat aaccaggcgg 6612tgcgccagct ggagccggtg ccgcgcgggg cgcgtcagcc gacgttggtc accgccgagc 6672gatacggcta cgtgtgggtc tggtacggct ccccgctgcc gctgcacccg ctgcccgaaa 6732tctccgcggc cgatgtcgac aacggcgact ttatgcacct gcacttcgcg ttcgagacga 6792ccacggcggt cttgcggatc gtcgagaact tctacgacgc gcagcacgca accccggtgc 6852acgcactccc gatctcggcc ttcgaactca agctcttcga cgattggcgc cagtggccgg 6912aggttgagtc gctggccctg gcgggcgcgt ggttcggtgc cgggatcgac ttcaccgtgg 6972accggtactt cggccccctc agcatgctgt cacgcgcgct cggcctgaac atgtcgcaga 7032tgaacctgca cttcgatggc taccccggcg ggtgcgtcat gaccgtcgcc ctggacggag 7092acgtcaaata caagctgctc cagtgtgtga cgccggtgag cgaaggcaag aacgtcatgc 7152acatgctcat ctcgatcaag aaggtgggcg gcatcctgcg ccgcgcgacc gacttcgtgc 7212tgttcgggct gcagaccagg caggccgcgg ggtacgacgt caaaatctgg aacggaatga 7272agccggacgg cggcggcgcg tacagcaagt acgacaagct cgtgctcaag taccgggcgt 7332tctatcgagg ctgggtcgac cgcgtcgcaa gtgagcggtg aggatccccc gaatttcccc 7392gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 7452atgattatca tctaatttct gttgaattac gttaagcatg taataattaa catgtaatgc7512atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac7572gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct7632atgttactag atccgggaat tccggaccgc tgctctagtg agaccgatcg cttttttaat7692taattttaag ctttctagag gccggccgcg gccgcctcga ggtaccatca ggatattctt7752gcttaagatg ttgaactcta tggaggtttg tatgaactga tgatctagga ccggataagt7812tcccttcttc atagcgaact tattcaaaga atgttttgtg tatcattctt gttacattgt7872tattaatgaa aaaatattat tggtcattgg actgaacacg agtgttaaat atggaccagg7932ccccaaataa gatccattga tatatgaatt aaataacaag aataaatcga gtcaccaaac7992cacttgcctt ttttaacgag acttgttcac caacttgata caaaagtcat tatcctatgc8052aaatcaataa tcatacaaaa atatccaata acactaaaaa attaaaagaa atggataatt8112tcacaatatg ttatacgata aagaagttac ttttccaaga aattcactga ttttataagc8172ccacttgcat tagataaatg gcaaaaaaaa acaaaaagga aaagaaataa agcacgaaga8232attctagaaa atacgaaata cgcttcaatg cagtgggacc cacggttcaa ttattgccaa8292ttttcagctc caccgtatat ttaaaaaata aaacgataat gctaaaaaaa tataaatcgt8352aacgatcgtt aaatctcaac ggctggatct tatgacgacc gttagaaatt gtggttgtcg8412acgagtcagt aataaacggc gtcaaagtgg ttgcagccgg cacacacgag tcgtgtttat8472caactcaaag cacaaatact tttcctcaac ctaaaaataa ggcaattagc caaaaacaac8532tttgcgtgta aacaacgctc aatacacgtg tcattttatt attagctatt gcttcaccgc8592cttagctttc tcgtgaccta gtcgtcctcg tcttttcttc cttcttcttc tataaaacaa8652tacccaaaga gctcttcttc ttcacaattc agatttcaat ttctcaaaat cttaaaaact8712ttctctcaat tctctctacc gtgatcggat ca gtc aca caa aga gta aag aag 8765Val Thr Gln Arg Val Lys Lys130aac aat ggc ttc ctc tat gct ctc ttc cgc tac tat ggt tgc ctc tcc 8813Asn Asn Gly Phe Leu Tyr Ala Leu Phe Arg Tyr Tyr Gly Cys Leu Ser135 140 145ggc tca ggc cac tat ggt cgc tcc ttt caa cgg act taa gtc ctc cgc 8861Gly Ser Gly His Tyr Gly Arg Ser Phe Gln Arg Thr Val Leu Arg150 155 160tgc ctt ccc agc cac ccg caa ggc taa caa cga cat tac ttc cat cac 8909Cys Leu Pro Ser His Pro Gln Gly Gln Arg His Tyr Phe His His165 170 175aag caa cgg cgg aag agt taa ctg cat gca ggt g tggcctccga 8953Lys Gln Arg Arg Lys Ser Leu His Ala Gly180 185ttggaaagaa gaagtttgag actctctctt accttcctga ccttaccgat tctgcaggag9013gtcgcgtcaa ctgcatgcag gctagcacat gacaacctta agctgtaaag tgacctcggt9073agaagctatc acggataccg tatatcgtgt ccgcatcgtg ccagacgcgg ccttttcttt9133tcgtgctggt cagtatttga tggtagtgat ggatgagcgc gacaaacgtc cgttctcaat9193ggcttcgacg ccggatgaaa aagggtttat cgagctgcat attggcgctt ctgaaatcaa9253cctttacgcg aaagcagtca tggaccgcat cctcaaagat catcaaatcg tggtcgacat9313tccccacgga gaagcgtggc tgcgcgatga tgaagagcgt ccgatgattt tgattgcggg9373cggcaccggg ttctcttatg cccgctcgat tttgctgaca gcgttggcgc gtaacccaaa9433ccgtgatatc accatttact ggggcgggcg tgaagagcag catctgtatg atctctgcga9493gcttgaggcg ctttcgttga agcatcctgg tctgcaagtg gtgccggtgg ttgaacaacc9553ggaagcgggc tggcgtgggc gtactggcac cgtgttaacg gcggtattgc aggatcacgg9613tacgctggca gagcatgata tctatattgc cggacgtttt gagatggcga aaattgcccg9673cgatctgttt tgcagtgagc gtaatgcgcg ggaagatcgc ctgtttggcg atgcgtttgc9733atttatctga gctagcggat cccccgaatt tccccgatcg ttcaaacatt tggcaataaa9793gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatctaa tttctgttga9853attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt9913ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg9973caaactagga taaattatcg cgcgcggtgt catctatgtt actagatccg ggaattagcg 10033gccgcctcga ggtaccggat ttggagccaa gtctcataaa cgccattgtg gaagaaagtc 10093ttgagttggt ggtaatgtaa cagagtagta agaacagaga agagagagag tgtgagatac 10153atgaattgtc gggcaacaaa aatcctgaac atcttatttt agcaaagaga aagagttccg 10213agtctgtagc agaagagtga ggagaaattt aagctcttgg acttgtgaat tgttccgcct 10273cttgaatact tcttcaatcc tcatatattc ttcttctatg ttacctgaaa accggcattt 10333aatctcgcgg gtttattccg gttcaacatt ttttttgttt tgagttatta tctgggctta 10393ataacgcagg cctgaaataa attcaaggcc caactgtttt tttttttaag aagttgctgt 10453taaaaaaaaa aaaagggaat taacaacaac aacaaaaaaa gataaagaaa ataataacaa 10513ttactttaat tgtagactaa aaaaacatag attttatcat gaaaaaaaga gaaaagaaat 10573aaaaacttgg atcaaaaaaa aaaacataca gatcttctaa ttattaactt ttcttaaaaa 10633ttaggtcctt tttcccaaca attaggttta gagttttgga attaaaccaa aaagattgtt 10693ctaaaaaata ctcaaatttg gtagataagt ttccttattt taattagtca atggtagata 10753cttttttttc ttttctttat tagagtagat tagaatcttt tatgccaagt tttgataaat 10813taaatcaaga agataaacta tcataatcaa catgaaatta aaagaaaaat ctcatatata 10873gtattagtat tctctatata tattatgatt gcttattctt aatgggttgg gttaaccaag 10933acatagtctt aatggaaaga atcttttttg aactttttcc ttattgatta aattcttcta 10993tagaaaagaa agaaattatt tgaggaaaag tatatacaaa aagaaaaata gaaaaatgtc 11053agtgaagcag atgtaatgga tgacctaatc caaccaccac cataggatgt ttctacttga 11113gtcggtcttt taaaaacgca cggtggaaaa tatgacacgt atcatatgat tccttccttt 11173agtttcgtga taataatcct caactgatat cttccttttt ttgttttggc taaagatatt 11233ttattctcat taatagaaaa gacggttttg ggcttttggt ttgcgatata aagaagacct 11293tcgtgtggaa gataataatt catcctttcg tctttttctg actcttcaat ctctcccaaa 11353gcctaaagcg atctctgcaa atctctcgcg actctctctt tcaaggtata ttttctgatt 11413ctttttgttt ttgattcgta tctgatctcc aatttttgtt atgtggatta ttgaatcttt 11473tgtataaatt gcttttgaca atattgttcg tttcgtcaat ccagcttcta aattttgtcc 11533tgattactaa gatatcgatt cgtagtgttt acatctgtgt aatttcttgc ttgattgtga 11593aattaggatt ttcaaggacg atctattcaa tttttgtgtt ttctttgttc gattctctct 11653gttttaggtt tcttatgttt agatccgttt ctctttggtg ttgttttgat ttctcttacg 11713gcttttgatt tggtatatgt tcgctgattg gtttctactt gttctattgt tttatttcag 11773gtggatctgt tggggatcta ccatgagccc agaacgacgc ccggccgaca tccgccgtgc 11833caccgaggcg gacatgccgg cggtctgcac catcgtcaac cactacatcg agacaagcac 11893ggtcaacttc cgtaccgagc cgcaggaacc gcaggagtgg acggacgacc tcgtccgtct 11953gcgggagcgc tatccctggc tcgtcgccga ggtggacggc gaggtcgccg gcatcgccta 12013cgcgggcccc tggaaggcac gcaacgccta cgactggacg gccgagtcga ccgtgtacgt 12073ctccccccgc caccagcgga cgggactggg ctccacgctc tacacccacc tgctgaagtc 12133cctggaggca cagggcttca agagcgtggt cgctgtcatc gggctgccca acgacccgag 12193cgtgcgcatg cacgaggcgc tcggatatgc cccccgcggc atgctgcggg cggccggctt 12253caagcacggg aactggcatg acgtgggttt ctggcagctg gacttcagcc tgccggtacc 12313gccccgtccg gtcctgcccg tcaccgagat cccccgaatt tccccgatcg ttcaaacatt 12373tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatctaa 12433tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 12493agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 12553atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatccg 12613ggaattgggt acccaattcc ggaccgctgc tctagaggcg cgcccctagg gagcttctgc 12673agacgcgtcg acgtcatatg gatccgatct gttgcccgtc tcactggtga aaagaaaaac 12733caccccagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt 12793ttacaccaca atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca 12853caaaatcacc actcgataca ggcagcccat cagtccggga cggtcgacct gcaggcatgc 12913aagctcacgt agtgtacgta atcgatttcg aagggcccac tagtccatgg gctttttctc 12973ctcgtgctcg taaacggacc cgaacatctc tggagctttc ttcagggccg acaatcggat 13033ctcgcggaaa tcctgcacgt cggccgctcc aagccgtcga atctgagcct taatcacaat 13093tgtcaatttt aatcctctgt ttatcggcag ttcgtagagc gcgccgtgcg tcccgagcga 13153tactgagcga agcaagtgcg tcgagcagtg cccgcttgtt cctgaaatgc cagtaaagcg 13213ctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc 13273aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct 13333tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg 13393aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt 13453cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca 13513gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca 13573cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg 13633gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa 13693taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc 13753ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca 13813tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa 13873atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca 13933gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaac 13993aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg 14053gcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata 14113gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcgagacga 14173cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg 14233tcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg 14293cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc 14353gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct 14413tgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct 14473ggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc 14533tggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc 14593ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag 14653tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggtact aaaacaattc 14713atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa 14773aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc 14833aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt 14893gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc 14953ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc 15013ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc 15073ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa 15133gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata 15193ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg 15253ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc 15313cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata 15373taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc 15433ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc 15493catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat 15553aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca 15613gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg 15673aaaccacaat tatgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga 15733ggtgctccag tggcttctgt gtctatcagc tgtccctcct gttcagctac tgacggggtg 15793gtgcgtaacg gcaaaagcac cgccggacat cagcgctatc tctgctctca ctgccgtaaa 15853acatggcaac tgcagttcac ttacaccgct tctcaacccg gtacgcacca gaaaatcatt 15913gatatggcca tgaatggcgt tggatgccgg gcaacagccc gcattatggg cgttggcctc 15973aacacgattt tacgtcactt aaaaaactca ggccgcagtc ggtaacctcg cgcatacagc 16033cgggcagtga cgtcatcgtc tgcgcggaaa tggacgaaca gtggggctat gtcggggcta 16093aatcgcgcca gcgctggctg ttttacgcgt atgacagtct ccggaagacg gttgttgcgc 16153acgtattcgg tgaacgcact atggcgacgc tggggcgtct tatgagcctg ctgtcaccct 16213ttgacgtggt gatatggatg acggatggct ggccgctgta tgaatcccgc ctgaagggaa 16273agctgcacgt aatcagcaag cgatatacgc agcgaattga gcggcataac ctgaatctga 16333ggcagcacct ggcacggctg ggacggaagt cgctgtcgtt ctcaaaatcg gtggagctgc 16393atgacaaagt catcgggcat tatctgaaca taaaacacta tcaataagtt ggagtcatta 16453cccaattatg atagaattta caagctataa ggttattgtc ctgggtttca agcattagtc 16513catgcaagtt tttatgcttt gcccattcta tagatatatt gataagcgcg ctgcctatgc 16573cttgccccct gaaatcctta catacggcga tatcttctat ataaaagata tattatctta 16633tcagtattgt caatatattc aaggcaatct gcctcctcat cctcttcatc ctcttcgtct 16693tggtagcttt ttaaatatgg cgcttcatag agtaattctg taaaggtcca attctcgttt 16753tcatacctcg gtataatctt acctatcacc tcaaatggtt cgctgggttt atcgcacccc 16813cgaacacgag cacggcaccc gcgaccacta tgccaagaat gcccaaggta aaaattgccg 16873gccccgccat gaagtccgtg aatgccccga cggccgaagt gaagggcagg ccgccaccca 16933ggccgccgcc ctcactgccc ggcacctggt cgctgaatgt cgatgccagc acctgcggca 16993cgtcaatgct tccgggcgtc gcgctcgggc tgatcgccca tcccgttact gccccgatcc 17053cggcaatggc aaggactgcc agcgctgcca tttttggggt gaggccgttc gcggccgagg 17113ggcgcagccc ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaaggg 17173ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca 17233aggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaac 17293gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat 17353aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat 17413ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc 17473cacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc 17533cagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt 17593gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc 17653gcgaggtatc cacaacgccg gcggccgcgg tgtctcgcac acggcttcga cggcgtttct 17713ggcgcgtttg cagggccata gacggccgcc agcccagcgg cgagggcaac cagcccggtg 17773agcgtcgcaa aggcgctcgg tcttgccttg ctcgtcggtg atgtacttca ccagctccgc 17833gaagtcgctc ttcttgatgg agcgcatggg gacgtgcttg gcaatcacgc gcaccccccg 17893gccgttttag cggctaaaaa agtcatggct ctgccctcgg gcggaccacg cccatcatga 17953ccttgccaag ctcgtcctgc ttctcttcga tcttcgccag cagggcgagg atcgtggcat 18013caccgaaccg cgccgtgcgc gggtcgtcgg tgagccagag tttcagcagg ccgcccaggc 18073ggcccaggtc gccattgatg cgggccagct cgcggacgtg ctcatagtcc acgacgcccg 18133tgattttgta gccctggccg acggccagca ggtaggccga caggctcatg ccggccgccg 18193ccgccttttc ctcaatcgct cttcgttcgt ctggaaggca gtacaccttg ataggtgggc 18253tgcccttcct ggttggcttg gtttcatcag ccatccgctt gccctcatct gttacgccgg 18313cggtagccgg ccagcctcgc agagcaggat tcccgttgag caccgccagg tgcgaataag 18373ggacagtgaa gaaggaacac ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct 18433gacgccgttg gatacaccaa ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc 18493gtgcgaaaaa ggatggatat accgaaaaaa tcgctataat gaccccgaag cagggttatg 18553cagcggaaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 18613agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 18673agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 18733gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 18793tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 18853accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 18913gtgagcgagg aagcggaaga gcgccagaag gccgccagag aggccgagcg cggccgtgag 18973gcttggacgc tagggcaggg catgaaaaag cccgtagcgg gctgctacgg gcgtctgacg 19033cggtggaaag ggggagggga tgttgtctac atggctctgc tgtagtgagt gggttgcgct 19093ccggcagcgg tcctgatcaa tcgtcaccct ttctcggtcc ttcaacgttc ctgacaacga 19153gcctcctttt cgccaatcca tcgacaatca ccgcgagtcc ctgctcgaac gctgcgtccg 19213gaccggcttc gtcgaaggcg tctatcgcgg cccgcaacag cggcgagagc ggagcctgtt 19273caacggtgcc gccgcgctcg ccggcatcgc tgtcgccggc ctgctcctca agcacggccc 19333caacagtgaa gtagctgatt gtcatcagcg cattgacggc gtccccggcc gaaaaacccg 19393cctcgcagag gaagcgaagc tgcgcgtcgg ccgtttccat ctgcggtgcg cccggtcgcg 19453tgccggcatg gatgcgcgcg ccatcgcggt aggcgagcag cgcctgcctg aagctgcggg 19513cattcccgat cagaaatgag cgccagtcgt cgtcggctct cggcaccgaa tgcgtatgat 19573tctccgccag catggcttcg gccagtgcgt cgagcagcgc ccgcttgttc ctgaagtgcc 19633agtaaagcgc cggctgctga acccccaacc gttccgccag tttgcgtgtc gtcagaccgt 19693ctacgccgac ctcgttcaac aggtccaggg cggcacggat cactgtattc ggctgcaact 19753ttgtcatgct tgacacttta tcactgataa acataatatg tccaccaact tatcagtgat 19813aaagaatccg cgcgttcaat cggaccagcg gaggctggtc cggaggccag acgtgaaacc 19873caacataccc ctgatcgtaa ttctgagcac tgtcgcgctc gacgctgtcg gcatcggcct 19933gattatgccg gtgctgccgg gcctcctgcg cgatctggtt cactcgaacg acgtcaccgc 19993ccactatggc attctgctgg cgctgtatgc gttggtgcaa tttgcctgcg cacctgtgct 20053gggcgcgctg tcggatcgtt tcgggcggcg gccaatcttg ctcgtctcgc tggccggcgc 20113cagatc 20119
權(quán)利要求
1.以區(qū)域?qū)Ｒ坏姆绞綄Ⅺu素轉(zhuǎn)移至底物的方法，包括在存在氧化劑、鹵素供體、電子轉(zhuǎn)移酶、和還原劑時使底物接觸區(qū)域?qū)Ｒ恍喳u化酶，其中若轉(zhuǎn)移發(fā)生于體內(nèi)，則電子轉(zhuǎn)移酶是由異源核酸分子編碼的。
2.權(quán)利要求1的方法，還包含F(xiàn)AD或FMN成份。
3.權(quán)利要求2的方法，其中進(jìn)一步的成份是FAD。
4.權(quán)利要求2的方法，其中電子轉(zhuǎn)移酶是能夠催化電子由NADH或NADPH或鐵氧還蛋白轉(zhuǎn)移至FAD的酶。
5.權(quán)利要求2的方法，其中電子轉(zhuǎn)移酶是能夠催化電子由NADH或NADPH或鐵氧還蛋白轉(zhuǎn)移至區(qū)域?qū)Ｒ恍喳u化酶的酶。
6.權(quán)利要求2的方法，其中電子轉(zhuǎn)移酶是黃素還原酶、鐵氧還蛋白NADP還原酶、鐵氧還蛋白、心肌黃酶巰基還原酶或NADH-cyt-B5還原酶、NADPH-FMN還原酶、NADPH-cyt-p450還原酶、或硝酸還原酶。
7.權(quán)利要求6的方法，其中電子轉(zhuǎn)移酶包含與依照SEQ ID NO19、21、23、25、27、29、或31的任一氨基酸序列具有至少30％同一性的氨基酸序列。
8.權(quán)利要求7的方法，其中電子轉(zhuǎn)移酶包含SEQ ID NO19、21、23、25、29、或31任一的氨基酸序列。
9.權(quán)利要求1的方法，其中區(qū)域?qū)Ｒ恍喳u化酶是prnA、prnC、藤黃綠膿菌素鹵化酶pltA、pltD、和pltM、四環(huán)素鹵化酶cts4、水解酶a、或balhimycin鹵化酶bhaA。
10.權(quán)利要求9的方法，其中區(qū)域?qū)Ｒ恍喳u化酶包含SEQ ID NO1。
11.權(quán)利要求1 0的方法，其中區(qū)域?qū)Ｒ恍喳u化酶是包含依照SEQID NO3、5、7、9、11、13、15、或17任一的氨基酸結(jié)構(gòu)域的多肽。
12.表達(dá)與SEQ ID NO18、10、22、24、26、28、或30中的任一種基本相似的一種異源核酸和與SEQ ID N02、4、6、8、10、12、14、或16中的任一種基本相似的至少一種異源核酸的宿主細(xì)胞。
13.權(quán)利要求12的宿主細(xì)胞，其中宿主細(xì)胞是細(xì)菌、真菌、或植物細(xì)胞。
14.權(quán)利要求13的宿主細(xì)胞，其中宿主細(xì)胞是微生物細(xì)胞。
15.權(quán)利要求13的宿主細(xì)胞，其中宿主細(xì)胞還表達(dá)編碼prnB和prnD的核酸序列。
16.生成硝吡咯菌素的方法，包括培養(yǎng)權(quán)利要求15的宿主細(xì)胞。
17.保護(hù)植物免于病原體的危害的方法，包括用權(quán)利要求15的宿主細(xì)胞處理植物，由此宿主以抑制病原體的量生成硝吡咯菌素。
18.權(quán)利要求16的方法，還包括由宿主收集硝吡咯菌素。
19.包含權(quán)利要求14的宿主細(xì)胞的植物。
20.包含權(quán)利要求15的宿主細(xì)胞的植物。
21.保護(hù)植物免于病原體的危害的方法，包括培養(yǎng)權(quán)利要求20的植物，由此在植物中以抑制病原體的量生成硝吡咯菌素。
22.依照權(quán)利要求20的植物的種子。
23.預(yù)防農(nóng)作物上的真菌生長的方法，包括培養(yǎng)權(quán)利要求21的植物，其中植物是農(nóng)作物植物。
24.用于改進(jìn)宿主的鹵化底物生成的方法，包括在宿主中表達(dá)編碼電子轉(zhuǎn)移酶的異源核酸分子，其中宿主表達(dá)至少一種具有區(qū)域?qū)Ｒ恍喳u化酶活性的內(nèi)源多肽。
全文摘要
本發(fā)明描述了用于鹵化天然產(chǎn)物的生物合成的方法、轉(zhuǎn)基因植物、和轉(zhuǎn)基因微生物，其中鹵化具有底物和區(qū)域?qū)Ｒ恍?。具體而言，本發(fā)明涉及通過本發(fā)明方法生成的鹵化代謝物用于保護(hù)宿主生物體免于病原體的危害(更具體的說是保護(hù)植物免于植物病原體的危害)的用途。在這方面，本發(fā)明提供了對植物病原體的抵抗力增強(qiáng)的轉(zhuǎn)基因植物，和生物防治特性增強(qiáng)的生物防治生物體。
文檔編號C12P13/22GK1409759SQ00817150
公開日2003年4月9日 申請日期2000年12月7日 優(yōu)先權(quán)日1999年12月15日
發(fā)明者J·斯蒂芬斯, C·巴泰, J·M·迪茨, J·董, K·P·凱姆達(dá), S·希爾 申請人:辛根塔參與股份公司