本發(fā)明涉及一種殺蟲融合蛋白、該殺蟲融合的編碼基因，及上述融合蛋白的應用。(二)
背景技術(shù)：
：害蟲給全球農(nóng)業(yè)生產(chǎn)帶來巨大的損失，目前害蟲防治主要依靠化學農(nóng)藥，但是農(nóng)藥的使用加重了生產(chǎn)成本，并且農(nóng)藥殘留對人體健康帶來嚴重危害。因此，利用基因工程方法防治害蟲具有重大經(jīng)濟、環(huán)境和社會價值。獲得轉(zhuǎn)基因抗蟲作物的關(guān)鍵是克隆優(yōu)良殺蟲蛋白質(zhì)。殺蟲蛋白質(zhì)有很多，使用得最為廣泛的是蘇云金芽孢桿菌(Bacillusthuringiensis，簡稱Bt)在產(chǎn)胞期間所分泌一種伴胞晶體蛋白(insecticidalcrystalproteins，ICP)，如Cry1Ab，Cry1C等，這些蛋白對鱗翅目、雙翅目、鞘翅目等昆蟲(比如小菜蛾、玉米螟)有很強的殺傷作用(Schnepf,E.,Crickmore,N.,Van,R.J.,Lereclus,D.,Baum,J.,&Feitelson,J.,etal.1998,62(3),775-806.)，目前已被深入研究和廣泛使用，玉米、大豆、土豆、棉花等轉(zhuǎn)Bt基因作物已得到大規(guī)模商業(yè)化種植。蘇云金芽胞桿菌在營養(yǎng)生長期間，還會分泌一種與ICPs無氨基酸序列同源性、殺蟲機理完全不同的蛋白，即營養(yǎng)期殺蟲蛋白(vegetativeinsecticidalproteins，VIPs)，如Vip3A、Vip3B、Vip3C、Vip3D、Vip3H，對一些對ICPs不敏感的害蟲也有殺蟲活性，并且不會發(fā)生交叉抗性(Estruch,J.J.,Warren,G.W.,Mullins,M.A.,Nye,G.J.,Craig,J.A.,&Koziel,M.G.1996,ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,93(11),5389-94.)。Vip3蛋白作為一種新型的殺蟲蛋白，對目前使用最為廣泛的ICP蛋白從殺蟲機制、殺蟲譜到殺蟲活性上都是一個很好的補充。比如，ICPs對斜紋夜蛾和粘蟲的殺傷作用很弱，Vip3則對斜紋夜蛾和粘蟲有非常好的防治效果。盡管如此，在轉(zhuǎn)基因作物研發(fā)的實際操作中，仍然存在一些急需解決的技術(shù)性問題。例如，研究人員發(fā)現(xiàn)高表達單獨的Vip3基因的植株生長比較遲緩，容易出現(xiàn)白化現(xiàn)象。這可能是由于轉(zhuǎn)基因植物中直接表達Vip3蛋白后，該蛋白由于信號肽的分泌作用大量聚集并結(jié)合在植物細胞膜上并形成孔道，從而對細胞造成損壞，影響其生長發(fā)育。玉米是全世界產(chǎn)量最高的糧食作物，同時也面臨著諸多害蟲的威脅。而在這些害蟲當中，根螢葉甲的名字尤其讓人膽寒——它們是破壞力最強的玉米害蟲之一。根螢葉甲的幼蟲侵入寄主的根莖后，會吞食組織，導致根腐爛、植株倒伏，使得植株減產(chǎn)乃至死亡。而成蟲也會采食寄主的葉、花以及種子。在1955年以前，它們僅分布在美國有限的幾個州內(nèi)，但在這之后，它們侵入了本不太適合它們生存的美國北部。1990年，東歐激蕩，這種害蟲就在這個時候被意外引入塞爾維亞，于是，歐洲大陸尤其是東歐，也成了它們的地盤。而近些年也有蔓延至中國的境內(nèi)。根螢葉甲是根螢葉甲屬(Diabroticasp.)昆蟲的統(tǒng)稱。在美國，危害玉米的幾種根螢葉甲被稱作玉米根蟲(CornRootworm)，其中危害最大的三種被叫做“西方玉米根蟲”、“北方玉米根蟲”及“南方玉米根蟲”。目前發(fā)現(xiàn)的具有玉米根蟲殺滅效果的蛋白主要是來自蘇云金桿菌中的Cry蛋白和Vip蛋白。其中，mCry3A、Cry3Bb1、eCry3.1Ab和蛋白復合體Cry34Ab1/Cry35Ab1已經(jīng)在商業(yè)化的轉(zhuǎn)基因品系中應用，并且已經(jīng)產(chǎn)生了不同程度的抗性(LefkoSA,NowatzkiTM,ThompsonSD,etal.2008,JournalofAppliedEntomology,132(3),189–204；Zhao,J.Z.,Oneal,M.A.,Richtman,N.M.,Thompson,S.D.,Cowart,M.C.,&Nelson,M.E.,etal.2016,JournalofEconomicEntomology(3))。也有研究表明，RNAi技術(shù)可以用于玉米根蟲等鞘翅目害蟲的防治(Baum,J.A.,Bogaert,T.,Clinton,W.,Heck,G.R.,Feldmann,P.,&Ilagan,O.,etal.2007,NatureBiotechnology,25(11),1322-6.)。最近，杜邦公司從假單胞菌(Pseudomonas)中克隆的一個基因IPD072Aa具有殺滅玉米根蟲的效果(Schellenberger,U.,Oral,J.,Rosen,B.A.,Wei,J.Z.,Zhu,G.,&Xie,W.,etal.2016,Science,354(6312),635-637.)。第一代轉(zhuǎn)基因作物大多只具有抗鱗翅目害蟲的特性，而第二代轉(zhuǎn)基因作物正在向同時抗多種害蟲的復合性狀發(fā)展。獲得具有復合性狀的轉(zhuǎn)基因作物有多種方法，比如通過對具有單一性狀的轉(zhuǎn)基因作物的雜交；通過將多個基因表達框構(gòu)建在同一個表達框里面；通過共轉(zhuǎn)化，將多個含有單基因的農(nóng)桿菌混在一起，進行混合轉(zhuǎn)化，篩選出多個基因同時整合了兩個或多個質(zhì)粒T-DNA的轉(zhuǎn)基因植株。但是上述方法在實際操作和應用中都還存在一些問題。因此，獲得更為簡單高效的培育具有復合性狀的轉(zhuǎn)基因作物的方法是當下需要切實解決的問題。(三)技術(shù)實現(xiàn)要素：本發(fā)明目的是提供一種通過基因融合使得同一個殺蟲融合蛋白同時對鱗翅目和鞘翅目害蟲具有殺傷效果的方法。本發(fā)明采用的技術(shù)方案是：本發(fā)明提供一種殺蟲融合蛋白，所述殺蟲融合蛋白從N端到C端分別為IPD072蛋白和Vip3毒素。進一步，所述IPD072蛋白為IPD072Aa蛋白、IPD072Bb蛋白、IPD072Ca蛋白或IPD072Da蛋白之一；所述Vip毒素為Vip3A毒素、Vip3B毒素、Vip3C毒素、Vip3D毒素或Vip3H毒素中的一種。進一步，所述殺蟲融合蛋白由IPD072Aa蛋白和Vip3A毒素融合而成，氨基酸序列為SEQIDNO：2所示。進一步，所述殺蟲融合蛋白由IPD072Aa蛋白和Vip3H毒素融合而成，氨基酸序列為SEQIDNO：4所示。本發(fā)明還提供一種所述殺蟲融合蛋白的編碼基因，所述的基因從5’-3’依次為編碼IPD072蛋白的核苷酸序列和編碼Vip3毒素的核苷酸序列；且上述2個核苷酸序列位于同一個開放閱讀框內(nèi)。進一步，所述的編碼IPD072蛋白的基因為IPD072Aa、IPD072Bb、IPD072Ca、IPD072Da之一；所述編碼Vip3毒素的基因為Vip3A、Vip3B、Vip3C、Vip3D或Vip3H之一。更優(yōu)選，所述編碼基因的核苷酸序列為SEQIDNO：1或SEQIDNO：3所示。本發(fā)明還提供一種所述的殺蟲融合蛋白在制備轉(zhuǎn)基因抗蟲作物、轉(zhuǎn)基因殺蟲微生物或抗體中的應用。進一步，所述轉(zhuǎn)基因抗蟲作物是由融合蛋白聯(lián)合BT晶體毒素轉(zhuǎn)化制備而成。與現(xiàn)有技術(shù)相比，本發(fā)明有益效果主要體現(xiàn)在：本發(fā)明所設計的IPD072蛋白與一個Vip3毒素融合成的人工蛋白質(zhì)分子，與原先的IPD072蛋白和Vip3蛋白相比，具有如下優(yōu)點：殺蟲譜廣，能同時防治鱗翅目和鞘翅目中多種重要害蟲(如甜菜夜蛾、粘蟲、玉米根蟲)，殺蟲效率高達50％-100％；有利于具有上述不同功能的蛋白和其它抗蟲蛋白(如ICPs)聯(lián)合使用，進一步擴大殺蟲譜，延緩害蟲抗性產(chǎn)生。(四)附圖說明圖1是本發(fā)明的殺蟲融合蛋白的結(jié)構(gòu)圖，殺蟲融合蛋白中N端為IPD072蛋白，C端為Vip3毒素。圖2是本發(fā)明中將殺蟲融合蛋白基因?qū)胫参镏械谋磉_框結(jié)構(gòu)圖；pUBI為玉米泛素啟動子，殺蟲融合蛋白基因為IPD072-Vip3毒素融合基因。圖3是本發(fā)明中將殺蟲融合蛋白和ICPs聯(lián)合導入植物中的表達框結(jié)構(gòu)圖；pUBI為玉米泛素啟動子，殺蟲融合蛋白基因為IPD072-Vip3毒素融合基因，p35S為花椰菜花葉病毒35S啟動子，ICPs為伴胞晶體蛋白編碼基因。(五)具體實施方式下面結(jié)合具體實施例對本發(fā)明進行進一步描述，但本發(fā)明的保護范圍并不僅限于此：實施例1、IPD072Aa-Vip3A殺蟲融合蛋白表達載體的構(gòu)建IPD072Aa和Vip3A殺蟲蛋白的基因均由上海生工合成，其DNA序列分別為SEQIDNO：5和中國專利200610049611.0中的SEQIDNO：7，并克隆在pET28a表達載體的限制性內(nèi)切酶BamHI和SacI位點之間。構(gòu)建好的載體分別命名為pET28a-IPD072Aa和pET28a-Vip3A。IPD072Aa-Vip3A合成具體步驟如下：1、以Vip3A(中國專利200610049611.0中的SEQIDNO：7)作為模板進過PCR獲得Vip3A基因片段。引物為：Vip3A-F：5’CCCGGGAAGGGTGGAGGAATGAACAAGAACAACACCAAG和Vip3A-R：5’CGAGCTCCTACTTGATGCTCACGTCGTAGAACTTCACGA。這兩個引物分別包含限制性內(nèi)切酶位點XmalI和ScaI的位點。2、用XmalI和ScaI處理PCR產(chǎn)物，并與用相同的酶處理過的pET28a-IPD072Aa載體連接。3、轉(zhuǎn)入大腸桿菌，獲得質(zhì)粒為pET28a-IPD072Aa-Vip3A。融合蛋白基因IPD072Aa-Vip3A(SEQIDNO：1)被克隆到了pET28a表達載體中，融合蛋白基因編碼的融合蛋白IPD072Aa-Vip3A(SEQIDNO：2)分子量大約為98kD。實施例2、IPD072Aa-Vip3H融合蛋白表達載體的構(gòu)建IPD072Aa和Vip3H殺蟲蛋白的基因均由上海生工合成，其DNA序列分別為SEQIDNO：5和SEQIDNO：6，并克隆在pET28a表達載體的限制性內(nèi)切酶BamHI和SacI位點之間。構(gòu)建好的載體分別命名為pET28a-IPD072Aa和pET28a-Vip3H。IPD072Aa-Vip3H合成具體步驟如下：4、以Vip3H(SEQIDNO：6)作為模板進過PCR獲得Vip3A基因片段。引物為：Vip3H-F：5’CCCGGGAAGGGTGGAGGAATGAACAAGAACAACAGTAAG和Vip3H-R：5’CGAGCTCCTACTTGATGCTGAAGTCCCTGAAGGTGATGT。這兩個引物分別包含限制性內(nèi)切酶位點XmalI和ScaI的位點。5、用XmalI和ScaI處理PCR產(chǎn)物，并與用相同的酶處理過的pET28a-IPD072Aa載體連接。6、轉(zhuǎn)入大腸桿菌，獲得質(zhì)粒為pET28a-IPD072Aa-Vip3H。融合蛋白基因IPD072Aa-Vip3H(SEQIDNO：3)被克隆到了pET28a表達載體中，融合蛋白基因編碼的融合蛋白IPD072Aa-Vip3H(SEQIDNO：4)分子量大約為98kD。實施例3、殺蟲蛋白的表達利用通用標準方法將上述含融合蛋白基因的表達載體(pET28a-IPD072Aa-Vip3A、pET28a-IPD072Aa-Vip3H、pET28a-IPD072Aa、pET28a-Vip3A和pET28a-Vip3H)分別轉(zhuǎn)入大腸桿菌BL21star。挑取單克隆菌落接種到100mlLB細菌培養(yǎng)液中，37℃震動培養(yǎng)至OD600＝0.6，加入IPTG到0.5mM，繼續(xù)在相同條件下培育4小時。收集培養(yǎng)液5000g離心10分鐘沉淀大腸桿菌細胞，然后棄上清收集沉淀。沉淀中加30毫升pH7.5、20mMTris-HCL緩沖液，超聲粉碎后即可用于殺蟲活性的測定。實施例4、IPD072Aa-Vip3A和IPD072Aa-Vip3H對多種鱗翅目和鞘翅目害蟲具有殺傷作用實施例3所獲得的殺蟲蛋白(IPD072Aa-Vip3A、IPD072Aa-Vip3H、IPD072Aa、Vip3A和Vip3H)分別涂在昆蟲人工飼料的表面，新生一齡幼蟲用來進行殺蟲性測定。陰性對照為pET28a空載體，準備方法與實施例3相同。殺蟲活性結(jié)果如表1所示：表1、融合蛋白的殺蟲效率粘蟲甜菜夜蛾西方玉米根蟲IPD072Aa0％0％100％Vip3A100％100％0％Vip3H100％100％0％IPD072Aa-Vip3A100％100％100％IPD072Aa-Vip3H100％100％100％陰性對照0％0％0％實施例5、IPD072Aa-Vip3A植物轉(zhuǎn)化載體構(gòu)建以IPD072Aa-Vip3A(SEQIDNO：1)作為模板經(jīng)過PCR獲得IPD072Aa-Vip3A基因片段。引物為：IVA-F：5’AGGATCCAACAATGGGCATCACCGTGACCAAC和IVA-R：5’CGAGCTCCTACTTGATGCTCACGTCGTAGAACTTC。這兩個引物分別包含限制性內(nèi)切酶位點BamH1和ScaI的位點。人工合成終止子序列ter(SEQIDNO：8)，5’端和3’端分別設置有ScaI和KpnI位點。把用BamH1和ScaI酶切后的IPD072Aa-Vip3A和用ScaI和KpnI酶切后的終止子連入經(jīng)BamH1和KpnI酶切的pCambia1300載體中，獲得pCambia1300-IPD072Aa-Vip3A-ter。pUBI為玉米泛素蛋白啟動子，通過PCR獲得。以商業(yè)玉米品種鄭單958的基因組DNA為模板，通過PCR擴增獲得pUBI。PCR反應條件為：95℃3分鐘；95℃15秒，68℃15秒，72℃2分鐘，重復32個循環(huán)；然后72℃10分鐘。將獲得的大約2.0Kb的PCR產(chǎn)物克隆到T-載體pMD19中。獲得序列正確的克隆用于下面的試驗。PCR引物為pUBI-F(5’GAAGCTTGCATGCCTACAGTGCAGCGTGACCC)和pUBI-R(5’GGGTGGATCCTCTAGAGTCGACCTGCAGAAGTAAC)，引物上分別設置了HindIII和BamHI限制性內(nèi)切酶酶切位點。用HindIII和BamHI酶切pUBI，把酶切后的片段連入用相同酶切的pCambia1300-IPD072Aa-Vip3A-ter載體中，獲得終載體。這個載體命名為：pCambia1300-pUBI-IPD072Aa-Vip3A。最后，通過電轉(zhuǎn)的方法把上述T-DNA質(zhì)粒轉(zhuǎn)入農(nóng)桿菌LBA4404中，通過含有15μg/ml四環(huán)素和50μg/mL的卡那霉素的YEP固體培養(yǎng)基篩選出陽性克隆，并保菌，用于接下來的植物轉(zhuǎn)化。實施例6、IPD072Aa-Vip3H植物轉(zhuǎn)化載體構(gòu)建以IPD072Aa-Vip3H(SEQIDNO：3)作為模板經(jīng)過PCR獲得IPD072Aa-Vip3H基因片段。引物為：IVH-F：5’AGGATCCAACAATGGGCATCACCGTGACCAAC和IVH-R：5’CGAGCTCCTACTTGATGCTGAAGTCCCTGAAGG。這兩個引物分別包含限制性內(nèi)切酶位點BamH1和ScaI的位點。人工合成終止子序列ter(SEQIDNO：8)，5’端和3’端分別設置有ScaI和KpnI位點。把用BamH1和ScaI酶切后的IPD072Aa-Vip3H和用ScaI和KpnI酶切后的終止子連入經(jīng)BamH1和KpnI酶切的pCambia1300載體中，獲得pCambia1300-IPD072Aa-Vip3H-ter。pUBI為玉米泛素蛋白啟動子的獲得方法同實施例5。用HindIII和BamHI酶切pUBI，把酶切后的片段連入用相同酶切的pCambia1300-IPD072Aa-Vip3H-ter載體中，獲得終載體。這個載體命名為：pCambia1300-pUBI-IPD072Aa-Vip3H。最后，通過電轉(zhuǎn)的方法把上述T-DNA質(zhì)粒轉(zhuǎn)入農(nóng)桿菌LBA4404中，通過含有15μg/ml四環(huán)素和50μg/mL的卡那霉素的YEP固體培養(yǎng)基篩選出陽性克隆，并保菌，用于接下來的植物轉(zhuǎn)化。實施例7、IPD072Aa-Vip3A和ICPs蛋白植物轉(zhuǎn)化載體構(gòu)建以Cry1Ab(SEQIDNO：7)殺蟲蛋白的基因由上海生工合成，5’端和3’端分別設置有BamH1和ScaI位點。人工合成終止子序列ter(SEQIDNO：8)，5’端和3’端分別設置ScaI和EcoRI位點。把用BamHI和ScaI酶切后的Cry1Ab和用ScaI和EcoRI酶切后的終止子連入經(jīng)BamH1和EcoRI酶切的pCambia1300載體中，獲得pCambia1300-Cry1Ab-ter。p35S啟動子為花椰菜花葉病毒CaMV35S啟動子，由上海生工合成，序列如SEQIDNO：9，5’端和3’端分別設置有KpnI和BamHI位點。用BamH1和EcoRI酶切pCambia1300-Cry1Ab-ter，用KpnI和BamHI酶切p35S，把上述兩個片段和經(jīng)KpnI和EcoRI酶切的載體pCambia1300-IPD072Aa-Vip3A-ter進行三段連接，獲得終載體。這個載體命名為：pCambia1300-pUBI-IPD072Aa-Vip3A-p35S-Cry1Ab。最后，通過電轉(zhuǎn)的方法把上述T-DNA質(zhì)粒轉(zhuǎn)入農(nóng)桿菌LBA4404中，通過含有15μg/ml四環(huán)素和50μg/mL的卡那霉素的YEP固體培養(yǎng)基篩選出陽性克隆，并保菌，用于接下來的植物轉(zhuǎn)化。實施例8、IPD072Aa-Vip3H和ICPs蛋白植物轉(zhuǎn)化載體構(gòu)建以Cry1Ab(SEQIDNO：7)殺蟲蛋白的基因由上海生工合成，5’端和3’端分別設置有BamH1和ScaI位點。人工合成終止子序列ter(SEQIDNO：8)，5’端和3’端分別設置ScaI和EcoRI位點。把用BamHI和ScaI酶切后的Cry1Ab和用ScaI和EcoRI酶切后的終止子連入經(jīng)BamH1和EcoRI酶切的pCambia1300載體中，獲得pCambia1300-Cry1Ab-ter。p35S啟動子為花椰菜花葉病毒CaMV35S啟動子，由上海生工合成，序列如SEQIDNO：9，5’端和3’端分別設置有KpnI和BamHI位點。用BamH1和EcoRI酶切pCambia1300-Cry1Ab-ter，用KpnI和BamHI酶切p35S，把上述兩個片段和經(jīng)KpnI和EcoRI酶切的載體pCambia1300-IPD072Aa-Vip3H-ter進行三段連接，獲得終載體。這個載體命名為：pCambia1300-pUBI-IPD072Aa-Vip3H-p35S-Cry1Ab。最后，通過電轉(zhuǎn)的方法把上述T-DNA質(zhì)粒轉(zhuǎn)入農(nóng)桿菌LBA4404中，通過含有15μg/ml四環(huán)素和50μg/mL的卡那霉素的YEP固體培養(yǎng)基篩選出陽性克隆，并保菌，用于接下來的植物轉(zhuǎn)化。實施例9、轉(zhuǎn)基因玉米玉米的轉(zhuǎn)化技術(shù)已經(jīng)比較成熟。參考文獻如:VladimirSidorov&DavidDuncan(inM.PaulScott(ed.),MethodsinMolecularBiology:TransgenicMaize,vol:526；YujiIshida,YukohHiei&ToshihikoKomari(2007)Agrobacterium-mediatedtransformationofmaize.NatureProtocols2:1614-1622?；痉椒ㄈ缦拢喝∈诜酆?-10天的Hi-II玉米穗，收集所有的未成熟胚(大小為1.0-1.5mm)。將實施例3中構(gòu)建的含有T-DNA載體轉(zhuǎn)化農(nóng)桿菌獲得的重組農(nóng)桿菌與未成熟胚在共培養(yǎng)培養(yǎng)基上(MS+2mg/L2,4-D+30g/L蔗糖+3g/L瓊脂(sigma7921)+40mg/L乙酰丁香酮)共培養(yǎng)2-3天(22℃)。轉(zhuǎn)移未成熟胚到愈傷誘導培養(yǎng)基上(MS+2mg/L2,4-D+30g/L蔗糖+2.5g/Lgelrite+5mg/LAgNO3+200mg/L乙酰丁香酮)，28℃暗培養(yǎng)10-14天。將所有的愈傷轉(zhuǎn)到帶有2mM草甘膦的篩選培養(yǎng)基(與愈傷誘導培養(yǎng)基相同)上，28℃暗培養(yǎng)2-3周。轉(zhuǎn)移所有的組織到新鮮含2mM草甘膦的篩選培養(yǎng)基上，28℃暗培養(yǎng)2-3周。然后，轉(zhuǎn)移所有篩選后成活的胚性組織到再生培養(yǎng)基(MS+30g/L蔗糖+0.5mg/Lkinetin+2.5g/Lgelrite+200mg/L乙酰丁香酮)上，28℃暗培養(yǎng)10-14天，每皿一個株系。轉(zhuǎn)移胚性組織到新鮮的再生培養(yǎng)基上，26℃光照培養(yǎng)10-14天。轉(zhuǎn)移所有發(fā)育完全的植株到生根培養(yǎng)基(1/2MS+20g/L蔗糖+2.5g/Lgelrite+200mg/L乙酰丁香酮)上，26℃光照培養(yǎng)直到根發(fā)育完全。獲得含上述轉(zhuǎn)化載體的轉(zhuǎn)基因玉米植株。實施例10、轉(zhuǎn)基因玉米可以殺蟲將實施例9制備的轉(zhuǎn)基因玉米植株的T0代植株移栽到溫室中，用商業(yè)化品種“鄭丹958”的母本，“鄭58”(Z58)的花粉進行授粉，收獲T0代種子。然后將這些品系與商業(yè)化品種“鄭丹958”的母本，“鄭58”(Z58)進行回交轉(zhuǎn)育，獲得Z58近等位基因系。再對這些近等位基因系的除草劑抗性進行比較分析。我們對獲得的98個轉(zhuǎn)pCambia1300-IPD072Aa-Vip3A-ter載體的轉(zhuǎn)基因株系(命名為IV3A)和105個轉(zhuǎn)pCambia1300-IPD072Aa-Vip3H-ter載體的轉(zhuǎn)基因株系(命名為IV3H)進行抗蟲性測定，其殺蟲效果效果如表2所示：表2*：*注：表2為對播種后30天的玉米葉片提取物進行不同害蟲的殺蟲效果檢測。每個實驗都至少設置3個重復。對我們獲得的69個轉(zhuǎn)pCambia1300-IPD072Aa-Vip3A-p35S-Cry1Ab載體的轉(zhuǎn)基因株系(命名為IV3A1Ab)和73個轉(zhuǎn)pCambia1300-IPD072Aa-Vip3H-p35S-Cry1Ab載體的轉(zhuǎn)基因株系(命名為IV3H1Ab)進行抗蟲性測定，其殺蟲效果效果如表3所示：表3*：害蟲殺蟲率玉米螟100％棉鈴蟲100％粘蟲100％甜菜夜蛾100％西方玉米根蟲100％*注：表3為對播種后30天的玉米葉片提取物進行不同害蟲的殺蟲效果檢測。每個實驗都至少設置3個重復。最后，還需要注意的是，以上列舉的僅是本發(fā)明的具體實施例。顯然，本發(fā)明不限于以上實施例，還可以有許多變形。本領(lǐng)域的普通技術(shù)人員能從本發(fā)明公開的內(nèi)容直接導出或聯(lián)想到的所有變形，均應認為是本發(fā)明的保護范圍。SEQUENCELISTING<110>浙江大學<120>一種殺蟲融合蛋白、編碼基因及其應用<130><160>9<170>PatentInversion3.5<210>1<211>2652<212>DNA<213>unknown<220><223>人工序列<400>1atgggcatcaccgtgaccaacaacagcagcaacccgatcgaggtggccatcaaccactgg60ggcagcgacggcgacaccagcttcttcagcgtgggcaacggcaagcaggagacctgggac120aggagcgacagcaggggcttcgtgctgagcctgaagaagaacggcgcccagcacccgtac180tacgtgcaggccagcagcaagatcgaggtggacaacaacgccgtgaaggaccagggcagg240ctgatcgagccgctgagccgaggccccgggaagggtggaggaatgaacaagaacaacacc300aagctgagcaccagggccctgccgagcttcatcgactacttcaacggcatctacggcttc360gccaccggcatcaaggacatcatgaacatgatcttcaagaccgacaccggcggcgacctg420accctggacgagatcctgaagaaccagcagctgctgaacgacatcagcggcaagctggac480ggcgtgaacggcagcctgaacgacctgatcgcccagggcaacctgaacaccgagctgagc540aaggagatcctgaagatcgccaacgagcagaaccaggtgctgaacgacgtgaacaacaag600ctggacgccatcaacaccatgctgagggtgtacctgccgaagatcaccagcatgctgagc660gacgtgatgaagcagaactacgccctgagcctgcagatcgagtacctgagcaagcagctg720caggagatcagcgacaagctggacatcatcaacgtgaacgtgctgatcaacagcaccctg780accgagatcaccccggcctaccagaggatcaagtacgtgaacgagaagttcgaggagctg840accttcgccaccgagaccagcagcaaggtgaagaaggacggcagcccggccgacatcctg900gacgagctgaccgagctgaccggcctggccaagagcgtgccgaagaacgacgtggacggc960ttcgagttctacctgaacaccttccacgacgtgatggtgggcaacaacctgttcggcagg1020agcgccctgaagaccgccagcgagctgatcaccaaggagaacgtgaagaccagcggcagc1080gaggtgggcaacgtgtacaacagcctgatcgtgctgaccctgctgcaggccaaggccttc1140ctgaccctgaccacctgcaggaagctgctgggcctggccgacatcgactacaccagcatc1200atgaacgagcacctgaacaaggagaaggaggagttcagggtgaacatcccgccgaccctg1260agcaacaccttcagcaacccgaactacgccaaggtgaagggcagcgacgaggacgccaag1320atgatcgtggaggccaagccgggccacgccctggtgggcttcgagatcagcaacgacagc1380atcaccgtgctgaaggtgtacgaggccaagctgaagcagaactaccaggtggacaaggac1440agcctgagcgaggtgatctacggcgacatggacaagctgctgggcccggaccagagcggc1500ccgatctactacccgaacaacatcgtgttcccgaacgagtacgtgatcaccaagatcgac1560ttcaccaagaagatgaagaccctgaggtacgaggtgaccgccaacttctacgacagcagc1620accggcgagatcgacctgaacaagaagaaggtggagagcagcgaggccgagtacaggacc1680ctgagcgccaacgacgacggcgtgtacatgccgctgggcgtgatcagcgagaccttcctg1740accccgatcaacggcttcggcctgcaggccgacgagaacagcaggctgatcaccctgacc1800tgcaagagctacctgagggagctgctgctggccaccgacctgagcaacaaggagaccaag1860ctgatcgtgccgccgagcggcttcatcaagaacatcgtggagaacggcagcatcgaggag1920gacaacctggagccgtggaaggccaacaacaagaacgcctacgtggaccacaccggcggc1980gtgaacggcaccaaggccctgtacgtgcacaaggacggcggcatcagccagttcatcggc2040gacaagctgaagccgaagaccgagtacgtgatccagtacaccgtgaagggcaagccgagc2100atccacctgaaggacgagaacaccggctacatccactacgaggacaccaacaacaacctg2160gaggactaccagaccatcaccaagaggttcaccaccggcaccgacctgaagggcgtgtac2220ctgatcctgaagagccagaacggcgacgaggcctggggcgacaacttcatcatcctggag2280atcagcccgagcgagaagctgctgagcccggagctgatcaacaccaacaactggaccagc2340accggcagcaccaacatcagcggcaacaccctgaccctgtaccagggcggcaggggcatc2400ctgaagcagaacctgcagctggacagcttcagcacctacagggtgtacttcagcgtgagc2460ggcgacgccaacgtgaggatcaggaacagcagggaggtgctgttcgagaagaggtacatg2520agcggcgccaaggacgtgagcgagatcttcaccaccaagctgggcaaggacaacttctac2580atcgagctgagccagggcaacaacctgaacggcggcccgatcgtgaagttctacgacgtg2640agcatcaagtag2652<210>2<211>883<212>PRT<213>unknown<220><223>人工序列<400>2MetGlyIleThrValThrAsnAsnSerSerAsnProIleGluValAla151015IleAsnHisTrpGlySerAspGlyAspThrSerPhePheSerValGly202530AsnGlyLysGlnGluThrTrpAspArgSerAspSerArgGlyPheVal354045LeuSerLeuLysLysAsnGlyAlaGlnHisProTyrTyrValGlnAla505560SerSerLysIleGluValAspAsnAsnAlaValLysAspGlnGlyArg65707580LeuIleGluProLeuSerArgGlyProGlyLysGlyGlyGlyMetAsn859095LysAsnAsnThrLysLeuSerThrArgAlaLeuProSerPheIleAsp100105110TyrPheAsnGlyIleTyrGlyPheAlaThrGlyIleLysAspIleMet115120125AsnMetIlePheLysThrAspThrGlyGlyAspLeuThrLeuAspGlu130135140IleLeuLysAsnGlnGlnLeuLeuAsnAspIleSerGlyLysLeuAsp145150155160GlyValAsnGlySerLeuAsnAspLeuIleAlaGlnGlyAsnLeuAsn165170175ThrGluLeuSerLysGluIleLeuLysIleAlaAsnGluGlnAsnGln180185190ValLeuAsnAspValAsnAsnLysLeuAspAlaIleAsnThrMetLeu195200205ArgValTyrLeuProLysIleThrSerMetLeuSerAspValMetLys210215220GlnAsnTyrAlaLeuSerLeuGlnIleGluTyrLeuSerLysGlnLeu225230235240GlnGluIleSerAspLysLeuAspIleIleAsnValAsnValLeuIle245250255AsnSerThrLeuThrGluIleThrProAlaTyrGlnArgIleLysTyr260265270ValAsnGluLysPheGluGluLeuThrPheAlaThrGluThrSerSer275280285LysValLysLysAspGlySerProAlaAspIleLeuAspGluLeuThr290295300GluLeuThrGlyLeuAlaLysSerValProLysAsnAspValAspGly305310315320PheGluPheTyrLeuAsnThrPheHisAspValMetValGlyAsnAsn325330335LeuPheGlyArgSerAlaLeuLysThrAlaSerGluLeuIleThrLys340345350GluAsnValLysThrSerGlySerGluValGlyAsnValTyrAsnSer355360365LeuIleValLeuThrLeuLeuGlnAlaLysAlaPheLeuThrLeuThr370375380ThrCysArgLysLeuLeuGlyLeuAlaAspIleAspTyrThrSerIle385390395400MetAsnGluHisLeuAsnLysGluLysGluGluPheArgValAsnIle405410415ProProThrLeuSerAsnThrPheSerAsnProAsnTyrAlaLysVal420425430LysGlySerAspGluAspAlaLysMetIleValGluAlaLysProGly435440445HisAlaLeuValGlyPheGluIleSerAsnAspSerIleThrValLeu450455460LysValTyrGluAlaLysLeuLysGlnAsnTyrGlnValAspLysAsp465470475480SerLeuSerGluValIleTyrGlyAspMetAspLysLeuLeuGlyPro485490495AspGlnSerGlyProIleTyrTyrProAsnAsnIleValPheProAsn500505510GluTyrValIleThrLysIleAspPheThrLysLysMetLysThrLeu515520525ArgTyrGluValThrAlaAsnPheTyrAspSerSerThrGlyGluIle530535540AspLeuAsnLysLysLysValGluSerSerGluAlaGluTyrArgThr545550555560LeuSerAlaAsnAspAspGlyValTyrMetProLeuGlyValIleSer565570575GluThrPheLeuThrProIleAsnGlyPheGlyLeuGlnAlaAspGlu580585590AsnSerArgLeuIleThrLeuThrCysLysSerTyrLeuArgGluLeu595600605LeuLeuAlaThrAspLeuSerAsnLysGluThrLysLeuIleValPro610615620ProSerGlyPheIleLysAsnIleValGluAsnGlySerIleGluGlu625630635640AspAsnLeuGluProTrpLysAlaAsnAsnLysAsnAlaTyrValAsp645650655HisThrGlyGlyValAsnGlyThrLysAlaLeuTyrValHisLysAsp660665670GlyGlyIleSerGlnPheIleGlyAspLysLeuLysProLysThrGlu675680685TyrValIleGlnTyrThrValLysGlyLysProSerIleHisLeuLys690695700AspGluAsnThrGlyTyrIleHisTyrGluAspThrAsnAsnAsnLeu705710715720GluAspTyrGlnThrIleThrLysArgPheThrThrGlyThrAspLeu725730735LysGlyValTyrLeuIleLeuLysSerGlnAsnGlyAspGluAlaTrp740745750GlyAspAsnPheIleIleLeuGluIleSerProSerGluLysLeuLeu755760765SerProGluLeuIleAsnThrAsnAsnTrpThrSerThrGlySerThr770775780AsnIleSerGlyAsnThrLeuThrLeuTyrGlnGlyGlyArgGlyIle785790795800LeuLysGlnAsnLeuGlnLeuAspSerPheSerThrTyrArgValTyr805810815PheSerValSerGlyAspAlaAsnValArgIleArgAsnSerArgGlu820825830ValLeuPheGluLysArgTyrMetSerGlyAlaLysAspValSerGlu835840845IlePheThrThrLysLeuGlyLysAspAsnPheTyrIleGluLeuSer850855860GlnGlyAsnAsnLeuAsnGlyGlyProIleValLysPheTyrAspVal865870875880SerIleLys<210>3<211>2646<212>DNA<213>unknown<220><223>人工序列<400>3atgggcatcaccgtgaccaacaacagcagcaacccgatcgaggtggccatcaaccactgg60ggcagcgacggcgacaccagcttcttcagcgtgggcaacggcaagcaggagacctgggac120aggagcgacagcaggggcttcgtgctgagcctgaagaagaacggcgcccagcacccgtac180tacgtgcaggccagcagcaagatcgaggtggacaacaacgccgtgaaggaccagggcagg240ctgatcgagccgctgagccgaggccccgggaagggtggaggaatgaacaagaacaacagt300aagctctccacccgcgccctcccgtccttcatcgactacttcaacggcatctacggcttc360gccaccggcatcaaggacatcatgaacatgatcttcaagaccgacaccggcggcaacgtc420accctcgacgagatcctcaagaaccagcagctcctcaacgagatcagcggcaagctcgac480ggcgtgaacggctccctcaacgagctgatcgcccaggtcaacctcaacaccgagctgtcc540aaggagatcctcaagatctccaacgagcagaaccaggtgctcaacgacgtgaacaacaag600ctggacgccatcaacaccatgctgcacatctacctcccgaagatcacctccatgctctcc660gacgtgatgaagcagaactacgccctctccctccagatcgagtacctctccaagcagctc720caggagatcagcgacaagctcgacatcatcaacgtgaacgtgctcatcaactccaccctc780accgagatcaccccggcctaccagcgcatcaagtacgtgaacgagaagttcgaggagctg840accttcgccaccgagaccaccctcaaggtgaagaaggactcctccccggccgacatcctc900gacgagctgaccgagctgaccgagctggccaagtccgtgaccaagaacgacgtggacggc960ttcgagttctacctcaacaccttgcacgacgtgatggtgggcaacaacctcttcggccgc1020tccgccctcaagaccgcctccgagctgatcgccaaggagaacgtgaagacctccggctcc1080gaggtgggcaacgtgtacaacttcctcatcgtgctcaccgccctgcaggccaaggccttc1140ctcaccctcaccacctgccgcaagctcctcggcctcgccggcatcgactacacctccatc1200atgaacgagcacctcaacaaggagaaggaggagttccgcgtgaacatcctcccgaccctc1260tccaacaccttctccaacccgaactacgccaaggtgaagggctccgacgaggacgccaag1320atgatcgtggaggccaagccgggccacgccctcgtgggcttcgagatgtccaacgactcc1380atcaccgtgctcaaggtgtacgaggccaagctcaagcagaactaccaggtggacaaggac1440tccctctccgaggtgatctacggcgacaccgacaagctcttctgcccggaccagtccgag1500cagatatactacaccaacaacatcgtgttcccgaacgagtacgtgatcaccaagatcgac1560ttcaccaagaagatgaagaccctccgctacgaggtgaccgccaacttctacgactcctcc1620accggcgagatcgacctcaacaagaagaaggtggagtcctccgaggccgagtaccgcacc1680ctctccgccaacgacgacggcgtgtacatgccgctcggcgtgatctccgaaaccttcctc1740accccgatcaacggcttcggcctccaggccgacgagaactcccgcctcatcaccctcacc1800tgcaagtcctacctccgcgagctgctcctcgccaccgacctctccaacaaggagaccaag1860ctcatcgtgccgccgtccggcttcatctccaacatcgtggagaacggcggcatcgaggag1920gacaacctcgagccgtggaaggccaacaacaagaacgcctacgtggaccacaccggcggc1980gtgaacggcaccaaggccctctacgtgcacaaggacggcggcttctcccagttcatcggc2040gacaagctcaagccgaagaccgagtacgtgatccagtacaccgtgaagggcaaggccagc2100atctacctgaaggacgagaagaacaacgagggcatctacgaggagatcaacaacgacctg2160gaggacttccagaccgtgaccaagaggttcatcaccggcaccgacagcagcggcgtgcac2220ctgatcttcaccagccagaacggcgacgaggccttcggcggcaacttcatcatcagcgag2280atcaggagcagcgaggagctgctgagcccggagctgatcaagagcgacgcctgggtgggc2340agccagggcacctggatcagcggcaacagcctgaccatcaacagcaacgccaacggcacc2400ttcaggcagaacctgccgctggagagctacagcacctacagcatgaacttcaacgtgaac2460ggcttcgccaaggtgaccgtgaggaacagcagggaggtgctgttcgagaagaacttcagc2520cagctgagcccgaaggactacagcgagaagttcaccaccgccgccaacaacaccggcttc2580tacgtggagctgagcaggggcacccagggcggcaacatcaccttcagggacttcagcatc2640aagtag2646<210>4<211>881<212>PRT<213>unknown<220><223>人工序列<400>4MetGlyIleThrValThrAsnAsnSerSerAsnProIleGluValAla151015IleAsnHisTrpGlySerAspGlyAspThrSerPhePheSerValGly202530AsnGlyLysGlnGluThrTrpAspArgSerAspSerArgGlyPheVal354045LeuSerLeuLysLysAsnGlyAlaGlnHisProTyrTyrValGlnAla505560SerSerLysIleGluValAspAsnAsnAlaValLysAspGlnGlyArg65707580LeuIleGluProLeuSerArgGlyProGlyLysGlyGlyGlyMetAsn859095LysAsnAsnSerLysLeuSerThrArgAlaLeuProSerPheIleAsp100105110TyrPheAsnGlyIleTyrGlyPheAlaThrGlyIleLysAspIleMet115120125AsnMetIlePheLysThrAspThrGlyGlyAsnValThrLeuAspGlu130135140IleLeuLysAsnGlnGlnLeuLeuAsnGluIleSerGlyLysLeuAsp145150155160GlyValAsnGlySerLeuAsnGluLeuIleAlaGlnValAsnLeuAsn165170175ThrGluLeuSerLysGluIleLeuLysIleSerAsnGluGlnAsnGln180185190ValLeuAsnAspValAsnAsnLysLeuAspAlaIleAsnThrMetLeu195200205HisIleTyrLeuProLysIleThrSerMetLeuSerAspValMetLys210215220GlnAsnTyrAlaLeuSerLeuGlnIleGluTyrLeuSerLysGlnLeu225230235240GlnGluIleSerAspLysLeuAspIleIleAsnValAsnValLeuIle245250255AsnSerThrLeuThrGluIleThrProAlaTyrGlnArgIleLysTyr260265270ValAsnGluLysPheGluGluLeuThrPheAlaThrGluThrThrLeu275280285LysValLysLysAspSerSerProAlaAspIleLeuAspGluLeuThr290295300GluLeuThrGluLeuAlaLysSerValThrLysAsnAspValAspGly305310315320PheGluPheTyrLeuAsnThrLeuHisAspValMetValGlyAsnAsn325330335LeuPheGlyArgSerAlaLeuLysThrAlaSerGluLeuIleAlaLys340345350GluAsnValLysThrSerGlySerGluValGlyAsnValTyrAsnPhe355360365LeuIleValLeuThrAlaLeuGlnAlaLysAlaPheLeuThrLeuThr370375380ThrCysArgLysLeuLeuGlyLeuAlaGlyIleAspTyrThrSerIle385390395400MetAsnGluHisLeuAsnLysGluLysGluGluPheArgValAsnIle405410415LeuProThrLeuSerAsnThrPheSerAsnProAsnTyrAlaLysVal420425430LysGlySerAspGluAspAlaLysMetIleValGluAlaLysProGly435440445HisAlaLeuValGlyPheGluMetSerAsnAspSerIleThrValLeu450455460LysValTyrGluAlaLysLeuLysGlnAsnTyrGlnValAspLysAsp465470475480SerLeuSerGluValIleTyrGlyAspThrAspLysLeuPheCysPro485490495AspGlnSerGluGlnIleTyrTyrThrAsnAsnIleValPheProAsn500505510GluTyrValIleThrLysIleAspPheThrLysLysMetLysThrLeu515520525ArgTyrGluValThrAlaAsnPheTyrAspSerSerThrGlyGluIle530535540AspLeuAsnLysLysLysValGluSerSerGluAlaGluTyrArgThr545550555560LeuSerAlaAsnAspAspGlyValTyrMetProLeuGlyValIleSer565570575GluThrPheLeuThrProIleAsnGlyPheGlyLeuGlnAlaAspGlu580585590AsnSerArgLeuIleThrLeuThrCysLysSerTyrLeuArgGluLeu595600605LeuLeuAlaThrAspLeuSerAsnLysGluThrLysLeuIleValPro610615620ProSerGlyPheIleSerAsnIleValGluAsnGlyGlyIleGluGlu625630635640AspAsnLeuGluProTrpLysAlaAsnAsnLysAsnAlaTyrValAsp645650655HisThrGlyGlyValAsnGlyThrLysAlaLeuTyrValHisLysAsp660665670GlyGlyPheSerGlnPheIleGlyAspLysLeuLysProLysThrGlu675680685TyrValIleGlnTyrThrValLysGlyLysAlaSerIleTyrLeuLys690695700AspGluLysAsnAsnGluGlyIleTyrGluGluIleAsnAsnAspLeu705710715720GluAspPheGlnThrValThrLysArgPheIleThrGlyThrAspSer725730735SerGlyValHisLeuIlePheThrSerGlnAsnGlyAspGluAlaPhe740745750GlyGlyAsnPheIleIleSerGluIleArgSerSerGluGluLeuLeu755760765SerProGluLeuIleLysSerAspAlaTrpValGlySerGlnGlyThr770775780TrpIleSerGlyAsnSerLeuThrIleAsnSerAsnAlaAsnGlyThr785790795800PheArgGlnAsnLeuProLeuGluSerTyrSerThrTyrSerMetAsn805810815PheAsnValAsnGlyPheAlaLysValThrValArgAsnSerArgGlu820825830ValLeuPheGluLysAsnPheSerGlnLeuSerProLysAspTyrSer835840845GluLysPheThrThrAlaAlaAsnAsnThrGlyPheTyrValGluLeu850855860SerArgGlyThrGlnGlyGlyAsnIleThrPheArgAspPheSerIle865870875880Lys<210>5<211>261<212>DNA<213>unknown<220><223>人工序列<400>5atgggcatcaccgtgaccaacaacagcagcaacccgatcgaggtggccatcaaccactgg60ggcagcgacggcgacaccagcttcttcagcgtgggcaacggcaagcaggagacctgggac120aggagcgacagcaggggcttcgtgctgagcctgaagaagaacggcgcccagcacccgtac180tacgtgcaggccagcagcaagatcgaggtggacaacaacgccgtgaaggaccagggcagg240ctgatcgagccgctgagctag261<210>6<211>2364<212>DNA<213>unknown<220><223>人工序列<400>6atgaacaagaacaacagtaagctctccacccgcgccctcccgtccttcatcgactacttc60aacggcatctacggcttcgccaccggcatcaaggacatcatgaacatgatcttcaagacc120gacaccggcggcaacgtcaccctcgacgagatcctcaagaaccagcagctcctcaacgag180atcagcggcaagctcgacggcgtgaacggctccctcaacgagctgatcgcccaggtcaac240ctcaacaccgagctgtccaaggagatcctcaagatctccaacgagcagaaccaggtgctc300aacgacgtgaacaacaagctggacgccatcaacaccatgctgcacatctacctcccgaag360atcacctccatgctctccgacgtgatgaagcagaactacgccctctccctccagatcgag420tacctctccaagcagctccaggagatcagcgacaagctcgacatcatcaacgtgaacgtg480ctcatcaactccaccctcaccgagatcaccccggcctaccagcgcatcaagtacgtgaac540gagaagttcgaggagctgaccttcgccaccgagaccaccctcaaggtgaagaaggactcc600tccccggccgacatcctcgacgagctgaccgagctgaccgagctggccaagtccgtgacc660aagaacgacgtggacggcttcgagttctacctcaacaccttgcacgacgtgatggtgggc720aacaacctcttcggccgctccgccctcaagaccgcctccgagctgatcgccaaggagaac780gtgaagacctccggctccgaggtgggcaacgtgtacaacttcctcatcgtgctcaccgcc840ctgcaggccaaggccttcctcaccctcaccacctgccgcaagctcctcggcctcgccggc900atcgactacacctccatcatgaacgagcacctcaacaaggagaaggaggagttccgcgtg960aacatcctcccgaccctctccaacaccttctccaacccgaactacgccaaggtgaagggc1020tccgacgaggacgccaagatgatcgtggaggccaagccgggccacgccctcgtgggcttc1080gagatgtccaacgactccatcaccgtgctcaaggtgtacgaggccaagctcaagcagaac1140taccaggtggacaaggactccctctccgaggtgatctacggcgacaccgacaagctcttc1200tgcccggaccagtccgagcagatatactacaccaacaacatcgtgttcccgaacgagtac1260gtgatcaccaagatcgacttcaccaagaagatgaagaccctccgctacgaggtgaccgcc1320aacttctacgactcctccaccggcgagatcgacctcaacaagaagaaggtggagtcctcc1380gaggccgagtaccgcaccctctccgccaacgacgacggcgtgtacatgccgctcggcgtg1440atctccgaaaccttcctcaccccgatcaacggcttcggcctccaggccgacgagaactcc1500cgcctcatcaccctcacctgcaagtcctacctccgcgagctgctcctcgccaccgacctc1560tccaacaaggagaccaagctcatcgtgccgccgtccggcttcatctccaacatcgtggag1620aacggcggcatcgaggaggacaacctcgagccgtggaaggccaacaacaagaacgcctac1680gtggaccacaccggcggcgtgaacggcaccaaggccctctacgtgcacaaggacggcggc1740ttctcccagttcatcggcgacaagctcaagccgaagaccgagtacgtgatccagtacacc1800gtgaagggcaaggccagcatctacctgaaggacgagaagaacaacgagggcatctacgag1860gagatcaacaacgacctggaggacttccagaccgtgaccaagaggttcatcaccggcacc1920gacagcagcggcgtgcacctgatcttcaccagccagaacggcgacgaggccttcggcggc1980aacttcatcatcagcgagatcaggagcagcgaggagctgctgagcccggagctgatcaag2040agcgacgcctgggtgggcagccagggcacctggatcagcggcaacagcctgaccatcaac2100agcaacgccaacggcaccttcaggcagaacctgccgctggagagctacagcacctacagc2160atgaacttcaacgtgaacggcttcgccaaggtgaccgtgaggaacagcagggaggtgctg2220ttcgagaagaacttcagccagctgagcccgaaggactacagcgagaagttcaccaccgcc2280gccaacaacaccggcttctacgtggagctgagcaggggcacccagggcggcaacatcacc2340ttcagggacttcagcatcaagtaa2364<210>7<211>1848<212>DNA<213>unknown<220><223>人工序列<400>7atggacaacaacccaaacatcaacgaatgcattccatacaactgcttgagtaacccagaa60gttgaagtacttggtggagaacgcattgaaaccggttacactcccatcgacatctccttg120tccttgacacagtttctgctcagcgagttcgtgccaggtgctgggttcgttctcggacta180gttgacatcatctggggtatctttggtccatctcaatgggatgcattcctggtgcaaatt240gagcagttgatcaaccagaggatcgaagagttcgccaggaaccaggccatctctaggttg300gaaggattgagcaatctctaccaaatctatgcagagagcttcagagagtgggaagccgat360cctactaacccagctctccgcgaggaaatgcgtattcaattcaacgacatgaacagcgcc420ttgaccacagctatcccattgttcgcagtccagaactaccaagttcctctcttgtccgtg480tacgttcaagcagctaatcttcacctcagcgtgcttcgagacgttagcgtgtttgggcaa540aggtggggattcgatgctgcaaccatcaatagccgttacaacgaccttactaggctgatt600ggaaactacaccgaccacgctgttcgttggtacaacactggcttggagcgtgtctggggt660cctgattctagagattggattagatacaaccagttcaggagagaattgaccctcacagtt720ttggacattgtgtctctcttcccgaactatgactccagaacctaccctatccgtacagtg780tcccaacttaccagagaaatctatactaacccagttcttgagaacttcgacggtagcttc840cgtggttctgcccaaggtatcgaaggctccatcaggagcccacacttgatggacatcttg900aacagcataactatctacaccgatgctcacagaggagagtattactggtctggacaccag960atcatggcctctccagttggattcagcgggcccgagtttacctttcctctctatggaact1020atgggaaacgccgctccacaacaacgtatcgttgctcaactaggtcagggtgtctacaga1080accttgtcttccaccttgtacagaagacccttcaatatcggtatcaacaaccagcaactt1140tccgttcttgacggaacagagttcgcctatggaacctcttctaacttgccatccgctgtt1200tacagaaagagcggaaccgttgattccttggacgaaatcccaccacagaacaacaatgtg1260ccacccaggcaaggattctcccacaggttgagccacgtgtccatgttccgttccggattc1320agcaacagttccgtgagcatcatcagagctcctatgttctcatggattcatcgtagtgct1380gagttcaacaatatcattccttcctctcaaatcacccaaatcccattgaccaagtctact1440aaccttggatctggaacttctgtcgtgaaaggaccaggcttcacaggaggtgatattctt1500agaagaacttctcctggccagattagcaccctcagagttaacatcactgcaccactttct1560caaagatatcgtgtcaggattcgttacgcatctaccacaaacttgcaattccacacctcc1620atcgacggaaggcctatcaatcagggtaacttctccgcaaccatgtcaagcggcagcaac1680ttgcaatccggcagcttcagaaccgtcggtttcactactcctttcaacttctctaacgga1740tcaagcgttttcacccttagcgctcatgtgttcaattctggcaatgaagtgtacattgac1800cgtattgagtttgtgcctgccgaagttaccttcgaggctgagtactag1848<210>8<211>208<212>DNA<213>unknown<220><223>人工序列<400>8gagctctagatgggccctgttctgcacaaagtggagtagtcagtcatcgatcaggaacca60gacaccagacttttattcatacagtgaagtgaagtgaagtgcagtgcagtgagttgctgg120tttttgtacaacttagtatgtatttgtatttgtaaaatacttctatcaataaaatttcta180attcctaaaaccaaaatccaggggtacc208<210>9<211>792<212>DNA<213>unknown<220><223>人工序列<400>9ggtacctggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcag60aagaccaaagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggat120tccattgcccagctatctgtcacttcatcaaaaggacagtagaaaaggaaggtggcacct180acaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtg240gtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaacca300cgtcttcaaagcaagtggattgatgtgataacatggtggagcacgacactctcgtctact360ccaagaatatcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaa420gggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcaaaa480ggacagtagaaaaggaaggtggcacctacaaatgccatcattgcgataaaggaaaggcta540tcgttcaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagca600tcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatct660ccactgacgtaagggatgacgcacaatcccactatccttcgcaagaccttcctctatata720aggaagttcatttcatttggagaggacacgctgaaatcaccagtctctctctacaaatct780atctctggatcc792當前第1頁1 2 3