專利名稱：經(jīng)過特異性滅活丁子香酚和阿魏酸分解代謝的基因構(gòu)建制備取代酚的生產(chǎn)菌株的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及生產(chǎn)菌株的構(gòu)建且涉及制備取代的甲氧基苯酚，特別是香草醛的方法。
DE-A4227076(制備取代的甲氧基苯酚的方法，及適用于該目的的微生物)描述了使用一種新的假單胞菌屬種類制備取代的甲氧基苯酚。在該文中起始材料是丁子香酚且產(chǎn)物是阿魏酸、香草酸、松柏醇和松柏醛。
由Rosazza等撰寫(阿魏酸一種豐富的芳香族天然產(chǎn)物J.Ind.Microbiol.15:457-471)的使用阿魏酸可能的生物轉(zhuǎn)化的綜述也于1995年出版。
在EP-A0845532中描述了來自假單胞菌屬種類的合成松柏醇，松柏醛，阿魏酸，香草酸和香草醛的基因和酶。
英國Norwich郡食品研究所在WO97/35999中描述了將反式-阿魏酸轉(zhuǎn)化成反式-阿魏酰-SCoA酯并隨后轉(zhuǎn)化成香草醛的酶及裂解該酯的基因。1998年，該專利的內(nèi)容以科技文獻的形式出版(Gasson等，1998，阿魏酸代謝成香草醛。生物學(xué)化學(xué)雜志，273:4163-4170:Narbad和Gasson 1998。在新分離的熒光假單胞菌菌株中使用新的CoA-依賴型途徑經(jīng)香草醛代謝阿魏酸。微生物學(xué)，144:1397-1405)。
DE-A-19532317描述了使用擬無枝酸菌屬種類經(jīng)發(fā)酵從阿魏酸以高產(chǎn)量獲得香草醛。
已知方法的缺點在于它們僅能獲得極低產(chǎn)量的香草醛或者使用相當(dāng)昂貴的起始化合物。盡管最后提到的方法(DE-A19532317)確實實現(xiàn)了高產(chǎn)量，但使用假單胞菌屬種類HR199和擬無枝酸菌屬種類HR167將丁子香酚生物轉(zhuǎn)化成香草醛需要進行2步發(fā)酵，因此導(dǎo)致相當(dāng)昂貴和費時。
因此本發(fā)明的目的是構(gòu)建能以一步方法相當(dāng)廉價地將原材料丁子香酚轉(zhuǎn)化成香草醛的生物。
借助于構(gòu)建單細(xì)胞的生產(chǎn)菌株或多細(xì)胞的生物實現(xiàn)該目的，該菌株的特征在于丁子香酚和/或阿魏酸分解代謝的酶被滅活使得積累中間產(chǎn)物松柏醇，松柏醛，阿魏酸，香草醛和/或香草酸。
該生產(chǎn)菌株可以是單細(xì)胞或多細(xì)胞。因此，本發(fā)明涉及微生物，植物或動物。而且也可使用從生產(chǎn)品種獲得的提取物。根據(jù)本發(fā)明，優(yōu)選使用單細(xì)胞生物。這些所述的生物可以是微生物或動物或植物細(xì)胞。根據(jù)本發(fā)明，特別優(yōu)選使用真菌和細(xì)菌。最優(yōu)選的是細(xì)菌種類。在其丁子香酚和/或阿魏酸分解代謝被改變后特別可使用的細(xì)菌是紅球菌屬，假單胞菌屬和埃希氏桿菌屬的種類。
在最簡單的情況下，已知的常規(guī)微生物學(xué)方法可用于分離根據(jù)本發(fā)明使用的生物。
因此，可使用酶抑制劑改變丁子香酚和/或阿魏酸分解代謝中涉及的蛋白質(zhì)的酶活性。而且，經(jīng)過突變編碼這些蛋白質(zhì)的基因可改變丁子香酚和/或阿魏酸分解代謝中涉及的蛋白質(zhì)的酶活性。該突變可借助于傳統(tǒng)方法以隨機方式產(chǎn)生，例如經(jīng)過使用紫外照射或誘導(dǎo)突變的化學(xué)劑。
諸如缺失，插入和/或核苷酸交換的重組DNA方法同樣適合于分離新生物。因此，例如可使用其它的DNA元件(Ω元件)滅活該生物的基因。合適的載體可用于以改變和/或滅活的基因結(jié)構(gòu)取代完整基因。在本文中，待滅活的基因，和用于滅活的DNA元件可借助于傳統(tǒng)克隆技術(shù)或借助于聚合酶鏈?zhǔn)椒磻?yīng)(PCR)獲得。
例如，在本發(fā)明的一個可能的實施方案中，可經(jīng)過插入Ω元件或?qū)肴笔нM入合適的基因中來改變丁子香酚分解代謝和阿魏酸分解代謝。在本文中，可使用上述重組DNA方法滅活編碼脫氫酶，合成酶，水合酶-醛縮酶，硫解酶或脫甲基酶的基因的功能以便阻斷相關(guān)酶的生產(chǎn)。優(yōu)選的是，該基因是編碼松柏醇脫氫酶，松柏醛脫氫酶，阿魏酰CoA合成酶，烯酰CoA水合酶-醛縮酶，β-酮硫解酶，香草醛脫氫酶或香草酸脫甲基酶的基因。非常特別優(yōu)選的是編碼在EP-A0845532中限定的氨基酸序列的基因和/或編碼其等位變異體的核苷酸序列。
因此本發(fā)明還涉及制備轉(zhuǎn)化生物和突變體的基因結(jié)構(gòu)。
優(yōu)選采用編碼脫氫酶，合成酶，水合酶-醛縮酶，硫解酶或脫甲基酶的核苷酸序列被滅活的基因結(jié)構(gòu)來分離該生物和突變體。特別優(yōu)選的是編碼松柏醇脫氫酶，松柏醛脫氫酶，阿魏酰CoA合成酶，烯酰CoA水合酶-醛縮酶，β-酮硫解酶，香草醛脫氫酶或香草酸脫甲基酶的核苷酸序列被滅活的基因結(jié)構(gòu)。極其優(yōu)選的基因結(jié)構(gòu)表現(xiàn)為

圖1a至1r給出的結(jié)構(gòu)，具有圖2a至2r所示的核苷酸序列和/或編碼其等位變異體的核苷酸序列。在本文中，特別優(yōu)選的是核苷酸序列1至18。
本發(fā)明還包含這些基因結(jié)構(gòu)的部分序列及其功能等同物。功能等同物的意思應(yīng)理解為該DNA的衍生物，其中個別的核苷酸堿基被交換(擺動交換(Wobbel austausche))而不改變其功能。在蛋白質(zhì)水平上也可交換氨基酸而不導(dǎo)致其功能改變。
在該基因結(jié)構(gòu)的上游和/或下游可插入一個或多個DNA序列。經(jīng)過克隆該基因結(jié)構(gòu)，可獲得適用于轉(zhuǎn)化和/或轉(zhuǎn)染生物和/或適用于接合轉(zhuǎn)移進生物的質(zhì)?；蜉d體。
本發(fā)明還涉及用于制備根據(jù)本發(fā)明轉(zhuǎn)化的生物和突變體的質(zhì)粒和/或載體。因而這些生物和突變體含有所述的基因結(jié)構(gòu)。因此本發(fā)明還涉及含有所說質(zhì)粒和/或載體的生物。
質(zhì)粒和/或載體的性質(zhì)取決于其應(yīng)用的對象。例如，為了用以Ω元件滅活的基因取代假單胞菌中丁子香酚和/或阿魏酸分解代謝的完整基因，所需的載體一方面可轉(zhuǎn)移進假單胞菌中(經(jīng)接合可轉(zhuǎn)移的質(zhì)粒)，但另一方面不能在這些生物中復(fù)制，因此在假單胞菌中是不穩(wěn)定的(所謂的自殺質(zhì)粒)。借助于該質(zhì)粒系統(tǒng)轉(zhuǎn)移進假單胞菌的DNA片段只有當(dāng)它們經(jīng)同源重組整合進細(xì)菌細(xì)胞基因組中時才能保留。
所述的基因結(jié)構(gòu)，載體和質(zhì)?？捎糜谥苽洳煌霓D(zhuǎn)化生物或突變體。所說的基因結(jié)構(gòu)可用于以改變的和/或滅活的基因結(jié)構(gòu)取代完整的核酸序列。在這種可經(jīng)轉(zhuǎn)化或轉(zhuǎn)染或接合獲得的細(xì)胞中，用改變的和/或滅活的基因結(jié)構(gòu)經(jīng)同源重組取代完整的基因，因此所得的細(xì)胞最終在其基因組中僅具有改變的和/或滅活的基因結(jié)構(gòu)。以這種方式，根據(jù)本發(fā)明優(yōu)選可改變和/或滅活基因，以便有關(guān)生物能產(chǎn)生松柏醇，松柏醛，阿魏酸，香草醛和/或香草酸。
在DE-A4227076和EP-A0845532中詳細(xì)描述的菌株假單胞菌屬種類HR199(DSM7063)的突變體是根據(jù)本發(fā)明以這種方式構(gòu)建的生產(chǎn)菌株的例子，其中相應(yīng)的基因結(jié)構(gòu)特別是來自圖1a至1r與圖2a至2r
1．假單胞菌屬種類HR199calAΩKm，含有ΩKm-滅活的calA基因代替編碼松柏醇脫氫酶的完整calA基因(圖1a；圖2a)。
2．假單胞菌屬種類HR199calAΩGm，含有ΩGm滅活的calA基因代替編碼松柏醇脫氫酶的完整calA基因(圖1b；圖2b)。
3．假單胞菌屬種類HR199calAΔ，含有缺失滅活的calA基因代替編碼松柏醇脫氫酶的完整calA基因(圖1c；圖2c)。
4．假單胞菌屬種類HR199calBΩKm，含有ΩKm滅活的calB基因代替編碼松柏醛脫氫酶的完整calB基因(圖1d；圖2d)。
5．假單胞菌屬種類HR199calBΩGm，含有ΩGm-滅活的calB基因代替編碼松柏醛脫氫酶的完整calB基因(圖1e；圖2e)。
6．假單胞菌屬種類HR199calBΔ，含有缺失滅活的calB基因代替編碼松柏醛脫氫酶的完整calB基因(圖1f；圖2f)。
7．假單胞菌屬種類HR199fcsΩKm，含有ΩKm-滅活的fcs基因代替編碼阿魏酰CoA合成酶的完整fcs基因(圖1g；圖2g)。
8．假單胞菌屬種類HR199fcsΩGm，含有ΩGm滅活的fcs基因代替編碼阿魏酰-CoA合成酶的完整fcs基因(圖1h；圖2h)。
9．假單胞菌屬種類HR199fcsΔ，含有缺失滅活的fcs基因代替編碼阿魏酰CoA合成酶的完整fcs基因(圖1i；圖2i)。
10．假單胞菌屬種類HR199echΩKm，含有ΩKm滅活的ech基因代替編碼烯酰-CoA水合酶-醛縮酶的完整ech基因(圖1j；圖2j)。
11．假單胞菌屬種類HR199echΩGm，含有ΩGm滅活的ech基因代替編碼烯酰-CoA水合酶-醛縮酶的完整ech基因(圖1k；圖2k)。
12．假單胞菌屬種類HR199echΔ，含有缺失滅活的ech基因代替編碼烯酰-CoA水合酶-醛縮酶的完整ech基因(圖11；圖21)。
13．假單胞菌屬種類HR199aatΩKm，含有ΩKm-滅活的aat基因代替編碼β-酮硫解酶的完整aat基因(圖1m，圖2m)。
14．假單胞菌屬種類HR199aatΩGm，含有ΩGm-滅活的aat基因代替編碼β-酮硫解酶的完整aat基因(圖1n；圖2n)。
15．假單胞菌屬種類HR199aatΔ，含有缺失滅活的aat基因代替編碼β-酮硫解酶的完整aat基因(圖10；20)。
16．假單胞菌屬種類HR199vdhΩKm，含有ΩKm-滅活的vdh基因代替編碼香草醛脫氫酶的完整vdh基因(圖1p；圖2p)。
17．假單胞菌屬種類HR199vdhΩGm，含有ΩGm-滅活的vdh基因代替編碼香草醛脫氫酶的完整vdh基因(圖1q；圖2q)。
18．假單胞菌屬種類HR199vdhΔ，含有缺失滅活的vdh基因代替編碼香草醛脫氫酶的完整vdh基因(圖1r；圖2r)。
19．假單胞菌屬種類HR199vdhBΩKm，含ΩKm滅活的vdhB基因代替編碼香草醛脫氫酶Ⅱ的完整vdhB基因。
20．假單胞菌屬種類HR199vdhBΩGm，含有ΩGm-滅活的vdhB基因代替編碼香草醛脫氫酶Ⅱ的完整vdhB基因。
21．假單胞菌屬種類HR199vdhBΔ，含有缺失滅活的vdhB基因代替編碼香草醛脫氫酶Ⅱ的完整vdhB基因。
22．假單胞菌屬種類HR199adHΩKm，含有ΩKm滅活的adh基因代替編碼乙醇脫氫酶的完整adH基因。
23．假單胞菌屬種類HR199adhΩGm，含有ΩGm-滅活的adh基因代替編碼乙醇脫氫酶的完整adh基因。
24．假單胞菌屬種類HR199adhΔ，含有缺失滅活的adh基因代替編碼乙醇脫氫酶的完整adh基因。
25．假單胞菌屬種類HR199vanAΩKm，含有ΩKm滅活的vanA基因代替編碼香草酸脫甲基酶α-亞基的完整vanA基因。
26．假單胞菌屬種類HR199vanAΩGm，含有ΩGm-滅活的vanA基因代替編碼香草酸脫甲基酶α-亞基的完整vanA基因。
27．假單胞菌屬種類HR199vanAΔ，含有缺失滅活的vanA基因代替編碼香草酸脫甲基酶α-亞基的完整vanA基因。
28．假單胞菌屬種類HR199vanBΩKm，含有ΩKm滅活的vanB基因代替編碼香草酸脫甲基酶β-亞基的完整vanB基因。
29．假單胞菌屬種類HR199vanBΩGm，含有ΩGm-滅活的vanB基因代替編碼香草酸脫甲基酶β-亞基的完整vanB基因。
30．假單胞菌屬種類HR199vanBΔ，含有缺失滅活的vanB基因代替編碼香草酸脫甲基酶β-亞基的完整vanB基因。
本發(fā)明還涉及有機化合物的生物技術(shù)制備方法。特別是該方法可用于制備醇，醛和有機酸。后者優(yōu)選是松柏醇，松柏醛，阿魏酸，香草醛和香草酸。
上述生物用于該新方法。特別優(yōu)選的生物包括細(xì)菌，特別是假單胞菌屬種類。具體地說，上述假單胞菌屬種類優(yōu)選用于下面的方法1．假單胞菌屬種類HR199calAΩkm，假單胞菌屬種類HR199calAΩGm和假單胞菌屬種類HR199calAΔ用于從丁子香酚制備松柏醇。
2．假單胞菌屬種類HR199calBΩKm，假單胞菌屬種類HR199calBΩGm和假單胞菌屬種類HR199calBΔ用于從丁子香酚或松柏醇制備松柏醛。
3．假單胞菌屬種類HR199fcsΩKm，假單胞菌屬種類HR199fcsΩGm，假單胞菌屬種類HR199fcsΔ，假單胞菌屬種類HR199echΩKm，假單胞菌屬種類HR199echΩGm和假單胞菌屬種類HR199echΔ用于從丁子香酚或松柏醇或松柏醛制備阿魏酸。
4．假單胞菌屬種類HR199vdhΩKm，假單胞菌屬種類HR199vdhΩGm，假單胞菌屬種類HR199vdhΔ，假單胞菌屬種類HR199vdhΩGmvdhBΩKm，假單胞菌屬種類HR199vdhΩKmvdhBΩGm，假單胞菌屬種類HR199vdhΔvdhBΩGm和假單胞菌屬種類HR199vdKΔvdhBΩKm用于從丁子香酚或松柏醇或松柏醛或阿魏酸制備香草醛。
5．假單胞菌屬種類HR199vanAΩKm，假單胞菌屬種類HR199vanAΩGm，假單胞菌屬種類HR199vanAΔ，假單胞菌屬種類HR199vanBΩKm，假單胞菌屬種類HR199vanBΩGm和假單胞菌屬種類HR199vanBΔ用于從丁子香酚或松柏醇或松柏醛或阿魏酸或香草醛制備香草酸。
丁子香酚是優(yōu)選的底物。然而，巴可加入其它底物或甚至用其它底物代替丁子香酚。
根據(jù)本發(fā)明采用的用于該生物的合適營養(yǎng)培養(yǎng)基是合成的，半合成的或復(fù)合培養(yǎng)基。這些培養(yǎng)基可包括含碳和含氮化合物，無機鹽、其中包括合適的痕量元素和維生素。
合適的含碳化合物是碳水化合物，碳?xì)浠衔锘蛴袡C標(biāo)準(zhǔn)化學(xué)劑?？蓛?yōu)選使用的化合物的例子是糖，醇或糖醇，有機酸或復(fù)合物混合物。
優(yōu)選的糖是葡萄糖。可優(yōu)選使用的有機酸是檸檬酸或乙酸。復(fù)合混合物的例子是麥芽提取物，酵母提取物，酪蛋白或酪蛋白水解產(chǎn)物。
無機化合物是合適的含氮物質(zhì)。其例子有硝酸鹽和銨鹽。也可使用有機氮源。這些氮源包括酵母提取物，大豆粉、酪蛋白，酪蛋白水解物和玉米漿。
可使用的無機鹽的例子是硫酸鹽，硝酸鹽，氯化物，碳酸鹽和磷酸鹽。所說的鹽所含的金屬優(yōu)選是鈉，鉀，鎂，錳，鈣，鋅和鐵。
培養(yǎng)溫度優(yōu)選范圍是50至100℃。特別優(yōu)選的范圍是從15至60℃，最優(yōu)選22至37℃培養(yǎng)基的pH優(yōu)選是2至12。特別優(yōu)選的范圍是4至8。
原則上，技術(shù)人員已知的任何生物反應(yīng)器均可用于實施該新方法。優(yōu)先考慮的是適于浸沒方法的任何裝置。這意味著根據(jù)本發(fā)明可采用具有或不具有機械混合裝置的容器。后者的例子是搖動裝置，泡罩塔反應(yīng)器或循環(huán)式反應(yīng)器。前者優(yōu)選包括所有已知的裝配有任何設(shè)計的攪拌器的裝置。
可連續(xù)或分批實施該新方法。達(dá)到最大產(chǎn)量所需的發(fā)酵時間取決于所用生物的具體特性。然而，原則上，發(fā)酵時間在2至200小時之間。
下面更詳細(xì)地解釋本發(fā)明，在提及的實施例中利用丁子香酚的菌株假單胞菌屬種類HR199(DSM7063)的突變體是借助于插入Ω元件或?qū)肴笔Ы?jīng)特異性滅活丁子香酚分解代謝的基因以靶向方式產(chǎn)生的。采用的Ω元件是編碼對抗生素卡那霉素(ΩKm)和慶大霉素(ΩGm)產(chǎn)生抗性的DNA片段。這些抗性基因是使用標(biāo)準(zhǔn)方法從Tn5和質(zhì)粒pBBR 1MCS-5分離出的。編碼松柏醇脫氫酶，松柏醛脫氫酶，阿魏酰-CoA合成酶，烯酰-CoA水合酶-醛縮酶，β-酮硫解酶，香草醛脫氫酶，乙醇脫氫酶，香草醛脫氫酶Ⅱ和香草酸脫甲基酶的基因calA,calB,fcs,ech,aat,vdh,adh,vdhB,vanA和vanB使用標(biāo)準(zhǔn)方法從菌株假單胞菌屬種類HR199的基因組DNA分離出并克隆進pBluescript SK-。借助于用合適的限制性核酸內(nèi)切酶消化，可從這些基因中去除DNA片段(缺失)或用Ω元件取代(插入)，導(dǎo)致各基因失活。以這種方式突變的基因重新克隆進可接合轉(zhuǎn)移的載體中并隨后導(dǎo)入菌株假單胞菌屬種類HR199。使用合適的選擇獲得用新導(dǎo)入的失活基因取代了各有功能的野生型基因的轉(zhuǎn)接合子(transkonju-ganten)。以這種方式獲得的插入和缺失突變體現(xiàn)在僅具有各失活的基因。使用這一方法可獲得僅具有一種缺陷基因的突變體以及用該方式滅活了幾種基因的多重突變體。這些突變體可用于進行下列生物轉(zhuǎn)化a)將丁子香酚轉(zhuǎn)化為松柏醇，松柏醛，阿魏酸，香草醛和/或香草酸；b)將松柏醇轉(zhuǎn)化成松柏醛，阿魏酸，香草醛和/或香草酸；c)將松柏醛轉(zhuǎn)化成阿魏酸，香草醛和/或香草酸；d)將阿魏酸轉(zhuǎn)化成香草醛和/或香草酸，和e)將香草醛轉(zhuǎn)化成香草酸。
材料和方法生長細(xì)菌的條件大腸桿菌的菌株在37℃下在Luria-Bertani(LB)或M9無機培養(yǎng)基中繁殖(J.Sambrook,E.F.Fritsch和T.Maniatis,1989。分子克?。粚嶒炇沂謨?，第2版，冷泉港實驗室出版，紐約冷泉港)。假單胞菌屬種類的菌株在營養(yǎng)肉湯(NB,0.8％,wt/vol)或無機培養(yǎng)基(MM)(H.G.Schlegel等，1961，微生物學(xué)報38:209-222)或HR無機培養(yǎng)基(HR-MM)(J.Rabenhorst,1996，應(yīng)用微生物學(xué)及生物技術(shù)，46:470-474)中30℃下繁殖。阿魏酸，香草醛，香草酸和原兒荼酸溶于二甲基甲砜并加入到各培養(yǎng)基中以得到0.1％(wt/vol)的終濃度。丁子香酚可直接加入到培養(yǎng)基中以得到0.1％(vol/vol)的終濃度或加入到MM瓊脂板蓋的濾紙(圓形濾紙595,Schleicher &Schuell,Dassel，德國)上。繁殖假單胞菌屬種類的轉(zhuǎn)接合子和突變體時，分別使用終濃度為25μg/ml,100μg/ml和7.5μg/ml的四環(huán)素，卡那霉素和慶大霉素。
培養(yǎng)上清液中代謝中間產(chǎn)物的定性和定量檢測。
用高壓液相色譜(Knauer HPLC)直接或用雙蒸水稀釋后分析培養(yǎng)上清液。在Nucleosil 100 C18(7μm,250×4mm)上進行色譜。0.1％(vol/vol)甲酸和乙腈用作溶劑。用于洗脫物質(zhì)的梯度過程如下00:00-06:30→26％乙腈06:30-08:00→100％乙腈
08:00-12:00→100％乙腈12:00-13:00→26％乙腈13:00-18:00→26％乙腈香草醛脫氫酶Ⅱ的純化。
在4℃下進行純化。
粗制提取物將在丁子香酚中繁殖的假單胞菌屬種類HRl99細(xì)胞在10mM磷酸鈉緩沖液pH6.0中洗滌，然后重懸于相同緩沖液中并在1000psi的壓力下通過弗氏壓碎器(Amicon,silver Spring，美國馬里蘭州)2次破碎細(xì)胞。對細(xì)胞勻漿物進行超速離心(1h,100,000xg,4℃)，所得的粗提物的可溶性部分從上清液中獲得。
在DEAE葡聚糖纖維素上進行陰離子交換色譜粗提物的可溶部分在10mM磷酸鈉緩沖液，pH6.0中透析過夜。將透析產(chǎn)物上樣到用10mM磷酸鈉緩沖液pH6.0平衡過的DEAE-Sephacel柱(2.6cm×35cm，柱容器[BV]:186ml)上，其流速為0.8ml/分。用2倍BV的10mM磷酸鈉緩沖液pH6.0沖洗柱。香草醛脫氫酶Ⅱ(VDHⅡ)用在10mM磷酸鈉緩沖液pH6.0(750ml)中從0到400mM NaCl的線性鹽梯度洗脫。收集10ml餾份。合并具有高VDHⅡ活性的餾份以形成DEAE合并液。
測定香草醛脫氫酶活性使用光學(xué)酶試驗在30℃下測定VDH活性。體積為1ml的反應(yīng)混合物含0.1mmol磷酸鉀(pH7.1),0.125μmol香草醛，0.5μmol NAD,1.2μmol丙酮酸(鈉鹽)，乳酸脫氫酶(IU；來自豬心)和酶溶液。在λ=340mm下監(jiān)測香草醛的氧化(ε香草醛=11.6cm2/μmol)。酶活性以單位(U)表示，1U相應(yīng)于每分鐘轉(zhuǎn)變1μmol香草醛的酶的量。使用Lowry等(O.H.Lowry.N.J.Rosebrough,A.L.Farr和R.J.Randall.1951，生物學(xué)化學(xué)雜志，193:265-275)的方法測定樣品中的蛋白質(zhì)濃度。
測定松柏醇脫氫酶活性根據(jù)Jaeger等(E.L.Jaeger,Eggeling和H.Sahm,1981.普通微生物學(xué).6:333-336)使用光學(xué)酶試驗在30℃下測定CADH活性。體積為1ml的反應(yīng)混合物含有0.2mmol tris/HCL(pH9.0),0.4μmol松柏醇，2μmol NAD,0.1mmol氨基脲和酶溶液。在λ=340nm下監(jiān)測NAD的減少(ε=6.3cm2/μmol)。酶活性用單位(U)來表示，lU相應(yīng)于每分鐘轉(zhuǎn)化1μmol底物的酶量。按Lowry等(0.H.Lowry,N.J.Rosebrough,A.L.Far和R.J.Randall,1951，生物學(xué)化學(xué)雜志，193:265-275)的方法測定樣品中的蛋白質(zhì)濃度。
測定松柏醛脫氫酶的活性使用光學(xué)酶試驗在30℃下測定CALDH活性。體積為1ml的反應(yīng)混合物含有0.1mmol的tris/HCL(pH8.8),0.08μmol的松柏醛，2.7μmol的NAD和酶溶液。在λ=400nm下監(jiān)測松柏醛氧化成阿魏酸(ε=34cm2/μmol)。酶活性以單位(U)表示，1U相應(yīng)于每分鐘轉(zhuǎn)變1μmol底物的酶量。按Lowry等(O.H.Lowry,N.J.Rosebrough,A.L.Far和Randall,1951，生物學(xué)化學(xué)雜志，193:265-275)的方法測定樣品中的蛋白質(zhì)濃度。
測定阿魏酰CoA合成酶(阿魏酸硫激酶)活性。
使用Zenk等(Zenk等，1980，生物化學(xué)年鑒，101:182-187)改良的光學(xué)酶試驗在30℃下測定FCS活性。體積為1ml的反應(yīng)混合物含有0.09mmol的磷酸鉀(pH7.0),2.1μmol的MgCl2,0.7μmol的阿魏酸，2μmol的ATP,0.4μmol的輔酶A和酶溶液。在λ=345nm下監(jiān)測從阿魏酸形成CoA酯(ε=10cm2/μmol)。酶活性以單位(U)表示，1U相應(yīng)于每分鐘轉(zhuǎn)化1μmol底物的酶量。使用Lowry等(O.H.Lowry,N.J.Rosebrough,A.L.Farr和R.J.Randall,1951，生物學(xué)化學(xué)雜志，193:265-275)的方法測定樣品中的蛋白質(zhì)濃度。
電泳方法使用Stegemann等(Stegemann等，1973,Z.Naturforsch.28c:722-732)的方法在7.4％(wt/vol)聚丙烯酰胺凝膠中天然條件下或者使用Laemmli(Laemmli,U.K.1970，自然(倫敦)227:680-685)的方法在11.5％(wt/vol)聚丙烯酰胺凝膠中在變性條件下分離含蛋白質(zhì)的提取物。Serva Blue R用于非特異性蛋白質(zhì)染色。為了特異性染色松柏醇脫氫酶，松柏醛脫氫酶和香草醛脫氫酶，將凝膠在100mM KP緩沖液(pH7.0)中重新緩沖20分鐘并隨后在加入了0.08％(wt/vol)NAD,0.04％(wt/vol)對硝基藍(lán)四唑氯，0.003％(wt/vol)吩嗪硫酸甲酯和1mM的各底物的相同緩沖液中30℃下溫育直到看見相應(yīng)的顏色帶。
將蛋白質(zhì)從聚丙烯酰胺凝膠上轉(zhuǎn)移到PVDF膜上。
使用半干快速印跡器具(B32/33,Biometra,Gttingen，德國)按照廠家說明將蛋白質(zhì)從SDS-聚丙烯酰胺凝膠轉(zhuǎn)移到PVDF膜(Waters-Millipore,Bedford,Mass,USA)上。
測定N端氨基酸序列使用蛋白質(zhì)肽測序儀(477A型，應(yīng)用生物系統(tǒng)，F(xiàn)oster City,USA)和PTH分析儀按照廠商說明測定N-端氨基酸序列。
分離和操作DNA使用Marmur的方法(J.Marmur,1961，分子生物學(xué)雜志，3:208-218)分離基因組DNA。使用標(biāo)準(zhǔn)方法(J.E.Sambrook,F.Fritsch和T.Maniatis,1989，分子克隆實驗室手冊，第2版，冷泉港實驗室出版，紐約冷泉港)分離和分析其它質(zhì)粒DNA和/或DNA限制性片段。
轉(zhuǎn)移DNA使用Hanahan的方法(D.Hanahan,1983，分子生物學(xué)雜志，166:557-580)制備和轉(zhuǎn)化感受態(tài)的大腸桿菌細(xì)胞。在含有質(zhì)粒的大腸桿菌S17-1菌株(供體)和假單胞菌屬種類菌株(受體)之間的接合質(zhì)粒轉(zhuǎn)移在NB瓊脂板上按Friedrich等的方法(B.Friedrich等，1981，細(xì)菌學(xué)雜志，147:198-205)進行或借助于“微量互補法”在含0.5％(wt/vol)葡萄糖酸作碳源及25μg四環(huán)素/ml或100μg卡那霉素/ml的MM瓊脂平板上進行。在這種情況下，將受體細(xì)胞以一個方向進行接種劃線。5分鐘后，將供體菌株的細(xì)胞進行接種劃線，這些劃線與受體接種劃線相交叉。在30℃培養(yǎng)48h后，轉(zhuǎn)接合子直接位于交叉點下方生長，而供體菌株和受體菌株都不能生長。
雜交實驗在0.8％(wt/vol)瓊脂糖凝膠上在50mM tris-50mM硼酸-1.25mM EDTA緩沖液(pH8.5)中電泳分離DNA限制性酶切片段(J.E.Sambrook,F.Fritsch和T.Maniatis.1989，分子克隆實驗室手冊。第2版，冷泉港實驗室出版，紐約冷泉港)。將變性的DNA從凝膠轉(zhuǎn)移到帶正電的尼龍膜(孔經(jīng)0.45μm,PallFiltrationstechnik,Dreieich，德國)上，隨后與生物素標(biāo)記的或洋地黃毒苷標(biāo)記的DNA探針的雜交，及這些DNA探針的制備均使用標(biāo)準(zhǔn)方法進行(J.E.Sambrook,F.Fritsch和T.Maniatis,1989，分子克隆實驗室手冊，第2版，冷泉港實驗室出版社，紐約冷泉港)。
DNA測序按Sanger等(Sanger等1977，美國科學(xué)院學(xué)報74:5463-5467)的雙脫氧鏈終止法使用“LI-COR”DNA測序儀400L型(LI-COR公司，生物技術(shù)部，Lincdn,NE,USA)并使用“含7-脫氮-2-脫氧鳥苷三磷酸的熱測序酶熒光標(biāo)記的引物循環(huán)測序試劑盒”(Amersham生命科學(xué)，Amersham International pls.,Little Chalfont,Buckinghamshire，英國)“非放射活性”測定核苷酸序列，在每種情況按廠商說明書進行。
按Strauss等(E.C.Strauss等，1986，生物化學(xué)年鑒，154:353-360)的“引物跳躍方法”使用合成寡核苷酸進行測序。
化學(xué)試劑，生化試劑和酶從C.F.Boehringer & Shne(Mannheim，德國)或從GIBCO/BRL(Eggenstein，德國)獲得限制性酶，T4 DNA連接酶，λDNA和酶及光學(xué)酶試驗的底物。[γ-32P]ATP來自Amersham/Buchler(Braunschweig，德國)。寡核苷酸從MWG-Btotech GmbH(Ebersberg，德國)獲得。NA型瓊脂糖從Pharmacia-LKB(Uppsala,Schweden)獲得。所有其它的化學(xué)試劑來自Haarmann &Reimer(Holzminden，德國)，E.Merch AG(Darmstadt，德國)，F(xiàn)lukaChemie(Buchs，瑞士)，Serva Feinbiochemica(Heidelberg，德國)或Sigma Chemie(Deisenhofen，德國)。
為了構(gòu)建ΩGm元件，在制備規(guī)模上分離出質(zhì)粒pBRIMCS-5的983bp EaeⅠ片段(M.E.Kovach,P.H.Elzer,D.S.Hill,G.T.Robertson,M.A.Farris,R.M.Roop和K.M.Peterson,1995，基因166:175-176)，然后用綠豆核酸酶處理(逐漸消化單鏈DNA分子末端)。然后將僅含慶大霉素抗性基因(編碼慶大霉素-3-乙酰轉(zhuǎn)移酶)的片段連接到SmaⅠ-裂解的pSKsym DNA(見上文)上?？勺鳛镾maⅠ片段，EcoRⅠ片段，HindⅢ片段或SalⅠ片段從所得質(zhì)粒重新分離出ΩGm元件。
實施例2從假單胞菌屬種類HRl99(DSM7063)克隆待插入Ω元件或缺失滅活的基因。
單獨從大腸桿菌S17-1菌株DSM10439和DSM10440克隆fcs,ech,vdh和aat基因并使用質(zhì)粒pE207和pE5-1(見EP-A-0845532)。從這些質(zhì)粒在制備規(guī)模上分離所述片段并按如下所述處理為了克隆fcs基因，將來自質(zhì)粒pE207的2350bp SalⅠ/EcoRⅠ片段和來自質(zhì)粒pE5-1的3700bp EcoRⅠ/SalⅠ片段一起在pBluescript SK-中克隆使得2片段借助于EcoRⅠ端連接在一起。從所得的雜種質(zhì)粒在制備規(guī)模上分離6050bp SalⅠ片段并經(jīng)過用Bal 31核酸酶處理縮短到大約2480bp。PstⅠ連接子隨后連接到該片段的末端，用PstⅠ消化后，將該片段克隆進pBluescript SK-(pSKfcs)。轉(zhuǎn)化大腸桿菌XL1 blue后，所得的克隆表達(dá)fcs基因并表現(xiàn)出0.2U/mg蛋白質(zhì)的Fcs活性。
為了克隆ech基因，在制備規(guī)模上從質(zhì)粒pE 207分離3800bpHindⅢ/EcoRⅠ片段并經(jīng)過用Bal 31核酸酶處理縮短到大約1470bp。然后，將EcoRⅠ連接子連接到該片段的末端，用EcoRⅠ消化后，將該片段克隆進pBluescript SK-(pSkech)。
為了克隆vdh基因，在制備規(guī)模上從質(zhì)粒pE207分離2350bp的SalⅠ/EcoRⅠ片段?？寺∵MpBluescript SK-后，使用外切核酸酶Ⅲ/綠豆核酸酶系統(tǒng)將該片段在一端截短大約1530bp。然后將EcoRⅠ連接子連接到該片段的末端，用EcoRⅠ消化后，將該片段克隆進pBluescript SK-(pSKvdh)。轉(zhuǎn)化大腸桿菌XL1 blue后，獲得的克隆表達(dá)VDH基因且表現(xiàn)出0.01U/mg蛋白質(zhì)的VDH活性。
為了克隆aat基因，在制備規(guī)模上從質(zhì)粒pE5-1分離3700bpEcoRⅠ/SalⅠ片段并經(jīng)過用Bal 31核酸酶處理縮短到大約1590bp。然然后將EcoRⅠ連接子連接到該片段的末端，用EcoRⅠ消化后，將該片段克隆進pBluescript SK-(pSKaat)。
實施例3經(jīng)過插入Ω元件或經(jīng)過缺失這些基因的部分區(qū)域滅活上述基因。
用BssHⅡ消化含fcs基因的質(zhì)粒pSKfcs，導(dǎo)致從fcs基因切割出1290bp的片段。隨后重新連接，在pBluescript SK-中以克隆的形式(pSKfcsΔ)獲得fcs基因的缺失衍生物(fcsΔ)(見圖1i和2i)。另外，切除該片段后，將Ω元件ΩKm和ΩGm代替其連接進去。這樣產(chǎn)生了fcs基因的Ω-滅活衍生物(fcsΩKm，見圖1g和2g)及(fcsΩGm，見圖1h和2h)，它們均在pBluescript SK-中以克隆的形式獲得(pSKfcsΩKm和pSKfcsΩGm)。在其雜交質(zhì)粒具有經(jīng)缺失或經(jīng)Ω元件插入而失活的fcs基因的大腸桿菌克隆粗提物中不可能檢測到任何FCS活性。
用NruⅠ消化含ech基因的質(zhì)粒pSKech，導(dǎo)致從ech基因切除了53bp的片段及430bp的片段。重新連接后，ech基因的缺失衍生物(echΔ，見圖11和21)在pBluescript SK-中以克隆的形式獲得(pSKechΔ)。另外，切除該片段后，將Ω元件ΩKm和ΩGm代替其連接進其中。結(jié)果產(chǎn)生ech基因的Ω-滅活衍生物(echΩKm和echΩGm)，它們在pBluescript SK-中以克隆的形式獲得(pSKechΩKm和pSKechΩGm)。
用BssHⅡ消化含vdh基因的質(zhì)粒pSKvdh，導(dǎo)致從vdh基因切除了210bp的片段。重新連接后，vdh基因的缺失衍生物(vdhΔ，見圖10和20)以克隆的形式在pBluescript SK-(pSK vdhΔ)中獲得。另外，切除該片段后，將Ω元件ΩKm和ΩGm代替其連接進入。結(jié)果產(chǎn)生vdh基因的Ω-滅活衍生物(vdhΩKm和vdhΩGm)，在pBluescript SK-中以克隆的形式獲得(pSKvdhΩKm，見圖1m和2m)及(pSKvdhΩGm，見圖1n和2n)。在所得的大腸桿菌克隆的粗提物中不能檢測到任何VDH活性，該克隆中的雜交質(zhì)粒具有經(jīng)缺失或Ω元件插入滅活的vdh基因。
用BssHⅡ消化含aat基因的質(zhì)粒pSKaat，導(dǎo)致從aat基因中切除了59bp的片段。重新連接后，aat基因的缺失衍生物(aatΔ，見圖1r和2r)在pBluescript SK-中以克隆的形式獲得(pSKaatΔ)。另外，切除該片段后，將Ω元件ΩKm和ΩGm代替其連接進入其中。結(jié)果產(chǎn)生aat基因的Ω-滅活衍生物(aatΩKm，見圖1p和2p)和(aatΩGm，見圖1q和2q)，在pBluescript SK-中以克隆的形式獲得(pSKaatΩKm和pSKaatΩGm)。
為了用Ω-元件滅活的基因取代假單胞菌屬種類HR199中的完整基因，需要一種載體，一方面該載體可轉(zhuǎn)移進假單胞菌中(可接合轉(zhuǎn)移的質(zhì)粒)，另一方面它在這些細(xì)菌中不能復(fù)制，因此在假單胞菌中不穩(wěn)定(“自殺質(zhì)?！?。使用這種質(zhì)粒系統(tǒng)轉(zhuǎn)移進假單胞菌中的DNA片段只有借助于同源重組(RecA-依賴性重組)整合進細(xì)菌細(xì)胞的基因組中才能保留。在這種情況下，使用“自殺質(zhì)?！眕SUP202(Simon等，1983，見A.Piihler，細(xì)菌-植物相互作用的分子遺傳學(xué)，SpringerVerlag,Berlin,Heidelberg，紐約，p98-106)。
用PstⅠ消化后，以質(zhì)粒pSKfcsΩKm和pSKfcsΩGm分離滅活的基因fcsΩKm和fcsΩGm并連接到PstⅠ-裂解的pSUP202DNA上。將連接混合物轉(zhuǎn)化進大腸桿菌S17-1。在分別還含卡那霉素或慶大霉素的含四環(huán)素LB培養(yǎng)基中進行選擇。獲得其雜交質(zhì)粒(pSUPfcsΩKm)含滅活基因fcsΩKm的卡那霉素抗性轉(zhuǎn)化子。慶大霉素抗性轉(zhuǎn)化子的相應(yīng)雜交質(zhì)粒(pSUPfcsΩGm)含滅活的基因fcsΩGm。
EcoRⅠ消化后，從質(zhì)粒pSKechΩKm和pSKechΩGm分離滅活的基因echΩKm和echΩGm并連接到EcoRⅠ裂解的pSUP202 DNA上。將連接混合物轉(zhuǎn)化進大腸桿菌S17-1。在分別還含卡那霉素或慶大霉素的含四環(huán)素LB培養(yǎng)基上進行選擇。獲得其雜交質(zhì)粒(pSUPechΩKm)含滅活基因echΩKm的卡那霉素抗性轉(zhuǎn)化子。慶大霉素抗性轉(zhuǎn)化子的相應(yīng)雜交質(zhì)粒(pSUPechΩGm)含滅活的基因echΩGm。
EcoRⅠ消化后，從質(zhì)粒pSKvdhΩKm和pSKvdhΩGm分離滅活的基因vdhΩKm和vdhΩGm并連接到EcoRⅠ-裂解的pSUP202DNA上。將連接混合物轉(zhuǎn)化進大腸桿菌S17-1。在分別還含卡那霉素或慶大霉素的含四環(huán)素LB培養(yǎng)基上進行選擇。獲得其雜交質(zhì)粒(pSUPvdhΩKm)含滅活基因vdhΩKm的卡那霉素抗性轉(zhuǎn)化子。慶大霉素抗性轉(zhuǎn)化子的相應(yīng)雜交換粒(pSUPvdhΩGm)含滅活基因vdhΩGm。
EcoRⅠ消化后，從質(zhì)粒pSKaatΩKm和pSKaatΩGm分離滅活基因aatΩKm和aatΩGm并連接到EcoRⅠ裂解的pSUP202DNA上。將連接混合物轉(zhuǎn)化進大腸桿菌S17-1中。在分別還含卡那霉素或慶大霉素的含四環(huán)素LB培養(yǎng)基上進行選擇。獲得其雜交質(zhì)粒(pSUPaatΩKm)含滅活基因aatΩKm的卡那霉素抗性轉(zhuǎn)化子。慶大霉素抗性轉(zhuǎn)化子的相應(yīng)雜交質(zhì)粒(pSUPaatΩGm)含滅活的基因aatΩGm。
實施例5將缺失滅活的基因亞克隆進具有“sacB選擇系統(tǒng)”的可接合轉(zhuǎn)移“自殺質(zhì)?！盤HE55中為了用缺失滅活的基因取代假單胞菌屬種類HR199中的完整基因，需要具有在pSUP202的例子中已描述過的特性的載體。在缺失失活基因的情況下，由于不存在選擇假單胞菌屬種類HR199中基因成功取代的可能性，與Ω元件滅活基因相反，使用了另一選擇系統(tǒng)。在“sacB選擇系統(tǒng)”中，取代的缺失失活基因被克隆進一種質(zhì)粒中，該質(zhì)粒除抗生素抗性基因外還具有sacB基因。將該雜交質(zhì)粒接合轉(zhuǎn)移進假單胞菌后，在完整基因在基因組中所處的位點處經(jīng)同源重組整合質(zhì)粒(第一次交換)。這種產(chǎn)生的“雜基因型”菌株同時具有完整基因和缺失失活基因，且被pHE55 DNA將這些基因互相分隔開。這些菌株表現(xiàn)出由載體編碼的抗性且也具有活性sacB基因。隨后的目的是借助于第二次同源重組事件將pHE55 DNA與完整基因一起分離出基因組DNA(第二次交換)。這次重組事件產(chǎn)生的菌株僅具有失活的基因。而且，pHE55編碼的抗生素抗性和sacB基因均已丟失。如果將菌株在含蔗糖的培養(yǎng)基上劃線培養(yǎng)，見表達(dá)sacB基因的菌株的生長受抑制，因為該基因產(chǎn)物將蔗糖轉(zhuǎn)變成聚合物，而該聚合物積累于細(xì)胞周質(zhì)中。由于發(fā)生了第二次重組事件而不再攜帶sacB基因的細(xì)胞的生長因此不受抑制。為了具有在表型上選擇缺失滅活基因整合的可能性，該基因不與完整基因交換，相反，使用的菌株待替換的基因已通過插入Ω元件而被“標(biāo)記”。當(dāng)發(fā)生成功替換時，所得的菌株喪失由Ω元件編碼的抗生素抗性。
用PstⅠ消化后，從質(zhì)粒pSKfcsΔ分離失活基因fcsΔ并連接到PstⅠ-裂解的pHE55 DNA上。將連接混合物轉(zhuǎn)化進大腸桿菌S17-1。在含四環(huán)素的LB培養(yǎng)基上進行選擇。獲得四環(huán)素抗性轉(zhuǎn)化子，其雜交質(zhì)粒(pHEfcsΔ)含滅活基因fcsΔ。
用EcoRⅠ消化后，從質(zhì)粒pSKechΔ分離失活基因echΔ并用綠豆核酸酶處理(產(chǎn)生鈍末端)。將該片段連接到BamHⅠ-裂解的且用綠豆核酸酶處理的pHE55 DNA上。將連接混合物轉(zhuǎn)化進大腸桿菌S17-1。在含四環(huán)素的LB培養(yǎng)基上進行選擇。獲得四環(huán)素抗性轉(zhuǎn)化子，其雜交質(zhì)粒(pHEechΔ)含有失活基因echΔ。
用EcoRⅠ消化后，從質(zhì)粒pSKvdhΔ分離失活基因vdhΔ并用綠豆核酸酶處理。將該片段連接到BamHⅠ裂解的且用綠豆核酸酶處理過的pHE55 DNA上。將連接混合物轉(zhuǎn)化進大腸桿菌S17-1。在含四環(huán)素的LB培養(yǎng)基上進行選擇。獲得四環(huán)素抗性轉(zhuǎn)化子，其雜交質(zhì)粒(pHEvdhΔ)含有滅活的基因vdhΔ。
用EcoRⅠ消化后，從質(zhì)粒pSKaatΔ分離出滅活基因aatΔ并用綠豆核酸酶處理。將該片段連接到BamHⅠ裂解的并用綠豆核酸酶處理過的pHE55 DNA上。將連接混合物轉(zhuǎn)化進大腸桿菌S17-1。在含四環(huán)素的LB培養(yǎng)基上進行選擇。獲得四環(huán)素抗性轉(zhuǎn)化子，其雜交質(zhì)粒(phEaatΔ)含有滅活的基因aatΔ。
在接合實驗中菌株假單胞菌屬種類HR199用作受體，其中下述含有pSUP202雜交質(zhì)粒的菌株大腸桿菌S17-1用作供體。轉(zhuǎn)接合子在含有相應(yīng)于Ω元件之抗生素的含葡糖酸無機培養(yǎng)基上選擇?？筛鶕?jù)pSUP202-編碼的四環(huán)素抗性區(qū)分“同基因子”(通過2次交換用Ω元件插入失活基因取代完整基因)和“雜基因子”(借助于單次交換將雜交質(zhì)粒整合進基因組)轉(zhuǎn)接合子。
將假單胞菌屬種類HR199分別與大腸桿菌S17-1(pSUPfcsΩKm)和大腸桿菌S17-1(pSUPfcsΩGm)接合后獲得突變的假單胞菌屬種類HR199fcsΩKm和假單胞菌屬種類HR199fcsΩGm。經(jīng)過DNA測序證實完整fcs基因被ΩKm-滅活的或ΩGm-滅活的基因(分別為fcsΩKm和fcsΩGm)取代。
將假單胞菌屬種類HR199分別與大腸桿菌S17-1(pSUPechΩKm)和大腸桿菌S17-1(pSUPechΩGm)接合后獲得突變的假單胞菌屬種類HR199echΩKm假單胞菌屬種類HR199echΩGm。經(jīng)過DNA測序證實完整的ech基因被ΩKm-滅活的或ΩGm-滅活的基因(分別為echΩkm和echΩGm)取代。
將假單胞菌屬種類HR199分別與大腸桿菌S17-1(pSUPvdhΩKm)和大腸桿菌S17-1(pSUPvdhΩGm)接合后獲得突變的假單胞菌屬種類HR199vdhΩKm和假單胞菌屬種類HR199vdhΩGm。經(jīng)過DNA測序證實完整的vdh基因被ΩKm-滅活的或ΩGm-滅活的基因(分別為vdhΩKm和vdhΩGm)取代。
將假單胞菌屬種類HR199分別與大腸桿菌S17-1(pSUPaatΩKm)和大腸桿菌S17-1(pSUPaatΩGm)接合后獲得突變的假單胞菌屬種類HR199aatΩKm和假單胞菌屬種類HR199aatΩGm。經(jīng)過DNA測序證實完整的aat基因被ΩKm-滅活的或ΩGm-滅活的基因(分別是aatΩKm和aatΩGm)取代。
將假單胞菌屬種類HR199fcsΩ Km與大腸桿菌S17-1(pSUPvdhΩGm)接合后獲得突變的假單胞菌屬種類HR199fcsΩKm vdhΩGm。經(jīng)過DNA測序證實完整的vdh基因被ΩGm滅活的基因(vdhΩGm)取代。
將假單胞菌屬種類HR199vdhΩKm大腸桿菌S17-1(pSUPaatΩGm)接合后獲得突變的假單胞菌屬種類HR199vdh ΩKmaatΩGm。經(jīng)過DNA測序證實完整的aat基因被ΩGm-滅活的基因(aatΩGm)取代。
將假單胞菌屬種類HR199vdhΩ Km與大腸桿菌S17-1(pSUPechΩGm)接合后獲得突變的假單胞菌屬種類HR199vdhΩKmechΩGm。經(jīng)過DNA測序證實完整的ech基因被ΩGm滅活的基因(echΩGm)取代。
實施例7產(chǎn)生菌株假單胞菌屬種類HR199的突變體，其中經(jīng)過缺失部分區(qū)域特異性滅活丁子香酚分解代謝的基因。
在接合實驗中，菌株假單胞菌屬種類HR199fcsΩKm，假單胞菌屬種類HR199echΩKm，假單胞菌屬種類HR199vdhΩKm和假單胞菌屬種類HR199aatΩKm用作受體，下述含有pHE55雜交質(zhì)粒的大腸桿菌S17-1菌株用作供體。在除四環(huán)素(pHE55編碼的抗性)外還含有相應(yīng)于Ω元件的抗生素的含葡糖酸無機培養(yǎng)基上選擇“雜基因子”轉(zhuǎn)接合子。在含蔗糖的無機培養(yǎng)基上劃線培養(yǎng)后，獲得的轉(zhuǎn)接合子經(jīng)第二次重組事件(第二次交換)刪除了載體DNA。經(jīng)過在不含抗生素或含有相應(yīng)于Ω元件的抗生素的無機培養(yǎng)基中劃線培養(yǎng)，可鑒定出用缺失滅活的基因(無抗生素抗性)取代了Ω元件滅活的基因的突變體。
將假單胞菌屬種類HR199fcsΩKm與大腸桿菌S17-1(pHEfcsΔ)接合后獲得突變的假單胞菌屬種類HR199fcsΔ。經(jīng)DNA測序證實ΩKm滅活的基因(fcsΩKm)被缺失滅活的基因(fcsΔ)取代。
將假單胞菌屬種類HR199echΩKm與大腸桿菌S17-1(pHEechΔ)接合后獲得突變的假單胞菌屬種類HR199echΔ。經(jīng)DNA測序證實ΩKm滅活的基因(echΩKm)被缺失滅活基因(echΔ)取代。
將假單胞菌屬種類HR199vdhΩKm與大腸桿菌S17-1(pHEvdhΔ)接合后獲得突變的假單胞菌屬種類HR199vdhΔ。經(jīng)DNA測序證實ΩKm滅活的基因(vdhΩKm)被缺失滅活的基因(vdhΔ)取代。
將假單胞菌屬種類HR199aatΩKm與大腸桿菌S17-1(pHEaatΔ)接合后獲得突變的假單胞菌屬種類HR199aatΔ。經(jīng)過DNA測序證實ΩKm-滅活的基因(aatΩKm)被缺失滅活的基因(aatΔ)取代。
實施例8使用突變的假單胞菌屬種類HR199vdhΩKm將丁子香酚生物轉(zhuǎn)化成香草醛。
菌株假單胞菌屬種類HR199vdhΩKm在50ml含6mM丁子香酚的HR-MM中繁殖到光密度為大約OD600nm=0.6。17小時后，在培養(yǎng)上清中可檢測到2.9mM的香草醛，1.4mM阿魏酸和0.4mM香草酸。
實施例9使用突變的假單胞菌屬種類HR199vdhΩGmaatΩKm將丁子香酚生物轉(zhuǎn)化成阿魏酸菌株假單胞菌屬種類HR199vdhΩGmaatΩkm在50ml含6mM丁子香酚的HR-MM中繁殖到光密度為大約OD600nm=0.6。18小時后，在培養(yǎng)上清中可檢測到1.9mM香草醛，2.4mM阿魏酸和0.6mM香草酸。
實施例10使用突變假單胞菌屬種類HR199vdhΩGmaatΩKm將丁子香酚生物轉(zhuǎn)化成松柏醇。
菌株假單胞菌屬種類HR199vdhΩGmaatΩKm在50ml含60mM丁子香酚的HR-MM中繁殖到光密度大約為OD600nm=0.4。15小時后，在培養(yǎng)物上清中可檢測到1.7mM松柏醇，1.4mM香草醛，1.4mM阿魏酸和0.2mM香草酸。
實施例11使用突變的假單胞菌屬種類HR199vdhΩKm在10 l發(fā)酵罐中從丁子香酚發(fā)酵生產(chǎn)天然香草醛。
用100ml培養(yǎng)了24小時的預(yù)培養(yǎng)物接種生產(chǎn)發(fā)酵罐，其中該預(yù)培養(yǎng)物在調(diào)至pH7.0且由12.5g甘油/l,10g酵母提取物/l和0.37g乙酸/l組成的培養(yǎng)基中在搖床(120rpm)上32℃繁殖過。發(fā)酵罐含有9.9 l下列組成的培養(yǎng)基1.5g酵母提取物/l,1.6g KH2PO4/l,0.2g NaCl/l,0.2g MgSO4/l。用氫氧化鈉溶液將pH調(diào)節(jié)至pH7.0。滅菌后，向培養(yǎng)基中加入4g丁子香酚。溫度為32℃，通氣為3NL/分，攪拌速度為600rpm。用氫氧化鈉溶液將pH維持在pH6.5。
在接種后4小時，開始連續(xù)加入丁子香酚使得在65小時后發(fā)酵終止時，向培養(yǎng)物中加入了255g的丁子香酚。在發(fā)酵期間還加入40g酵母提取物。發(fā)酵終止時，丁子香酚的濃度為0.2g/l。香草醛的含量為2.6g/l。也存在3.4g的阿魏酸/l。
用諸如色譜，蒸餾和/或提取的已知物理方法分離以這種方式獲得的香草醛并用于制備天然調(diào)味品。
附圖的說明圖1a至1r分離生物和突變體的基因結(jié)構(gòu)cclA*松柏醇脫氫酶的部分失活基因calB*松柏醛脫氫酶的部分失活基因fcs*阿魏酰-CoA合成酶的部分失活基因ech*烯酰-CoA水合酶-醛縮酶的部分失活基因vdh*香草醛脫氫酶的部分失活基因aat*β-酮硫解酶的部分失活基因盡管標(biāo)記“*”的限制性酶裂解位點用于構(gòu)建，但它們在所得的構(gòu)建體上不再具有功能。
圖2a:calAΩKm基因結(jié)構(gòu)的核苷酸序列圖2b:calAΩGm基因結(jié)構(gòu)的核苷酸序列圖2c:calAΔ基因結(jié)構(gòu)的核苷酸序列圖2d:calBΩKm基因結(jié)構(gòu)的核苷酸序列圖2e:calBΩGm基因結(jié)構(gòu)的核苷酸序列圖2f:calBΔ基因結(jié)構(gòu)的核苷酸序列圖2g:fcsΩKm基因結(jié)構(gòu)的核苷酸序列圖2h:fcsΩGm基因結(jié)構(gòu)的核苷酸序列圖2i:fcsΔ基因結(jié)構(gòu)的核苷酸序列圖2j:echΩKm基因結(jié)構(gòu)的核苷酸序列圖2k:echΩGm基因結(jié)構(gòu)的核苷酸序列圖2l:echΔ基因結(jié)構(gòu)的核苷酸序列圖2m:vdhΩKm基因結(jié)構(gòu)的核苷酸序列圖2n:vdhΩGm基因結(jié)構(gòu)的核苷酸序列圖2o:vdhΔ基因結(jié)構(gòu)的核苷酸序列圖2p:aatΩKm基因結(jié)構(gòu)的核苷酸序列圖2q:aatΩGm基因結(jié)構(gòu)的核苷酸序列圖2r:aatΔ基因結(jié)構(gòu)的核苷酸序列序列CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420AAAGAGCATG CAA CTG ACC AAC AAG AAA ATC GTC GTC ACC GGA GTG TCC TCC 472Met Gln Leu Thr Asn Lys Lys Ile Val Val Thr Gly Val Ser Ser1 5 10 15GGT ATC GGT GCC GAA ACT GCC CGC GTT CTG CGC TCT CAC GGC GCC ACA520Gly Ile Gly Ala Glu Thr Ala Arg Val Leu Arg Ser His Gly Ala Thr20 25 30GTG ATT GGC GTA GAT CGC AAC ATG CCG AGC CTG ACT CTG GAT GCT TTC568Val Ile Gly Val Asp Arg Asn Met Pro Ser Leu Thr Leu Asp Ala Phe35 40 45GTT CAG GCT GAC CTG AGC CAT CCT GAA GGC ATC GAT AAG GCC ATC GGG616Val Gln Ala Asp Leu Ser His Pro Glu Gly Ile Asp Lys Ala Ile50 55 60 62ACAGCAAGCG AACCGGAATT GCCAGCTGGG GCGCCCTCTG GTAAGGTTGG GAAGCCCTGC 676AAAGTAAACT GGATGGCTTT CTTGCCGCCA AGGATCTGAT GGCGCAGGGG ATCAAGATCT 736GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATG ATT GAA CAA GAT GGA TTG CAC 790Met Ile Glu Gln Asp Gly Leu His1 5GCA GGT TCT CCG GCC GCT TGG GTG GAG AGG CTA TTC GGC TAT GAC TGG838Ala Gly Ser Pro Ala Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp10 15 20GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG TTC CGG CTG TCA886Ala Gln Gln Thr Ile Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser25 30 35 40GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT GCC934Ala Gln Gly Arg Pro Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala45 50 55CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG TGG CTG GCC ACG982Leu Asn Glu Leu Gln Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr60 65 70ACG GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC ACT GAA GCG GGA 1030Thr Gly Val Pro Cys Ala Ala Val Leu Asp Val Val Thr Glu Ala Gly75 80 85AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG TCA 1078Arg Asp Trp Leu Leu Leu Gly Glu Val Pro Gly Gln Asp Leu Leu Ser90 95 100TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA ATG 1126Ser His Leu Ala Pro Ala Glu Lys Val Ser Ile Met Ala Asp Ala Met105 110 115 120CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC CAA 1174Arg Arg Leu His Thr Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gln125 130 135GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT CTT 1222Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met Glu Ala Gly Leu140 145 150GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA GCC 1270Val Asp Gln Asp Asp Leu Asp Glu Glu His Gln Gly Leu Ala Pro Ala155 160 165GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT CTC 1318Glu Leu Phe Ala Arg Leu Lys Ala Arg Met Pro Asp Gly Glu Asp Leu170 175 180GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT 1366Val Val Thr His Gly Asp Ala Cys Leu Pro Asn Ile Met Val Glu Asn185 190 195 200GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG GAC 1414Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu Gly Val Ala Asp205 210 215CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG CTT 1462Arg Tyr Gln Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu220 225 230GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC GCT 1510Gly Gly Glu Trp Ala Asp Arg Phe Leu Val Leu Tyr Gly Ile Ala Ala235 240 245CCC GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT GAC GAG TTC TTC 1558Pro Asp Ser Gln Arg Ile Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe250 255 260 264TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCTG GCC GCG GTG1613Ala Ala Val225ATT GCA TTC ATG TGT GCT GAG GAG TCA CGT TGG ATC AAC GGC ATA AAT 1661Ile Ala Phe Met Cys Ala Glu Glu Ser Arg Trp Ile Asn Gly Ile Asn230 235 240ATT CCA GTG GAC GGA GGT TTG GCA TCG ACC TAC GTG TAA GTTCGTGGAC1710Ile Pro Val Asp Gly Gly Leu Ala Ser Thr Tyr Val245 250 255GCCCTTTGCA CGCGCACTAT ATCTCTATGC AGCAGCTGAA AGCAGCTTTG GTTTTGATCG 1770GAGGTAGCGG GCGGAAAGGT GCAGAATGTC TAAATAATAA AGGATTCTTG TGAAGCTTTA 1830GTTGTCCGTA AACGAAAATA AAAATAAAGA GGAATGATAT GAAAGCAAGT AGATCAGTCT 1890GCACTTTCAA AATAGCTACC CTGGCAGGCG CCATTTATGC AGCGCTGCCA ATGTCAGCTG 1950CAAACTCGAT GCAGCTGGAT GTAGGTAGCT CGGATTGGAC GGTGCGTTGG GGACAACACC 2010CTCAAGTATA GCCTTGCCTC TCGCCTGAAT GAGCAAGACT CAAGTCTGAC AAATGCGCCG 2070ACTGTCAATG GTTATATCCG GATATTCAAA GTCAGGGTGA TCGTAACTTT GACCGGGGGC 2130TTGGTATCCA ATCGTCTCGA TATTCTGGCT GCAG 2164圖2aCTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420AAAGAGCATG CAA CTG ACC AAC AAG AAA ATC GTC GTC ACC GGA GTG TCC TCC 472Met Gln Leu Thr Asn Lys Lys Ile Val Val Thr Gly Val Ser Ser1 5 10 15GGT ATC GGT GCC GAA ACT GCC CGC GTT CTG CGC TCT CAC GGC GCC ACA520Gly Ile Gly Ala Glu Thr Ala Arg Val Leu Arg Ser His Gly Ala Thr20 25 30GTG ATT GGC GTA GAT CGC AAC ATG CCG AGC CTG ACT CTG GAT GCT TTC568Val Ile Gly Val Asp ArG Asn Met Pro Ser Leu Thr Leu Asp Ala Phe35 40 45GTT CAG GCT GAC CTG AGC CAT CCT GAGGGGAGAG GCGGTTTGCG TATTGGGCGC 622Val Gln Ala Asp Leu Ser His Pro50 55ATGCATAAAA ACTGTTGTAA TTCATTAAGC ATTCTGCCGA CATGGAAGCC ATCACAAACG 682GCATGATGAA CCTGAATCGC CAGCGGCATC AGCACCTTGT CGCCTTGCGT ATAATATTTG 742CCCATGGACG CACACCGTGG AAACGGATGA AGGCACGAAC CCAGTTGACA TAAGCCTGTT 802CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG CAACTGGTCC AGAACCTTGA 862CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT GGCTTGTTAT GACTGTTTTT 922TTGTACAGTC TATGCCTCGG GCATCCAAGC AGCAAGCGCG TTACGCCGTG GGTCGATGTT 982TGATGTTATG GAGCAGCAAC G ATG TTA CGC AGC AGC AAC GAT GTT ACG CAG 1033Met Leu ArG Ser Ser Asn Asp Val Thr Gln1 5 10CAG GGC AGT CGC CCT AAA ACA AAG TTA GGT GGC TCA AGT ATG GGC ATC 1081Gln Gly Ser Arg Pro Lys Thr Lys Leu Gly Gly Ser Ser Met Gly Ile15 20 25ATT CGC ACA TGT AGG CTC GGC CCT GAC CAA GTC AAA TCC ATG CGG GCT 1129Ile Arg Thr Cys Arg Leu Gly Pro Asp Gln Val Lys Ser Met Arg Ala30 35 40GCT CTT GAT CTT TTC GGT CGT GAG TTC GGA GAC GTA GCC ACC TAC TCC 1177Ala Leu Asp Leu Phe Gly Arg Glu Phe Gly Asp Val Ala Thr Tyr Ser45 50 55CAA CAT CAG CCG GAC TCC GAT TAC CTC GGG AAC TTG CTC CGT AGT AAG 1225Gln His Gln Pro Asp Ser Asp Tyr Leu Gly Asn Leu Leu Arg Ser Lys60 65 70ACA TTC ATC GCG CTT GCT GCC TTC GAC CAA GAA GCG GTT GTT GGC GCT 1273Thr Phe Ile Ala Leu Ala Ala Phe Asp Gln Glu Ala Val Val Gly Ala75 80 85 90CTC GCG GCT TAC GTT CTG CCC AGG TTT GAG CAG CCG CGT AGT GAG ATC 1321Leu Ala Ala Tyr Val Leu Pro Arg Phe Glu Gln Pro Arg Ser Glu Ile95 100 105TAT ATC TAT GAT CTC GCA GTC TCC GGC GAG CAC CGG AGG CAG GGC ATT 1369Tyr Ile Tyr Asp Leu Ala Val Ser Gly Glu His Arg Arg Gln Gly Ile110 115 120GCC ACC GCG CTC ATC AAT CTC CTC AAG CAT GAG GCC AAC GCG CTT GGT 1417Ala Thr Ala Leu Ile Asn Leu Leu Lys His Glu Ala Asn Ala Leu Gly125 130 135GCT TAT GTG ATC TAC GTG CAA GCA GAT TAC GGT GAC GAT CCC GCA GTG 1465Ala Tyr Val Ile Tyr Val Gln Ala Asp Tyr Gly Asp Asp Pro Ala Val140 145 150GCT CTC TAT ACA AAG TTG GGC ATA CGG GAA GAA GTG ATG CAC TTT GAT 1513Ala Leu Tyr Thr Lys Leu Gly Ile Arg Glu Glu Val Met His Phe Asp155 160 165 170ATC GAC CCA AGT ACC GCC ACC TAA CAATTCGTTC AAGCCGAGAT CGGCTTCCCT 1567Ile Asp Pro Ser Thr Ala Thr175 177G ATT GCA TTC ATG TGT GCT GAG GAG TCA CGT TGG ATC AAC GGC ATA AAT 1616Ile Ala Phe Met Cys Ala Glu Glu Ser Arg Trp Ile Asn Gly Ile Asn228 230 235 240ATT CCA GTG GAC GGA GGT TTG GCA TCG ACC TAC GTG TAA GTTCGTGGAC1665Ile Pro Val Asp Gly Gly Leu Ala Ser Thr Tyr Val245 250 255GCCCTTTGCA CGCGCACTAT ATCTCTATGC AGCAGCTGAA AGCAGCTTTG GTTTTGATCG 1725GAGGTAGCGG GCGGAAAGGT GCAGAATGTC TAAATAATAA AGGATTCTTG TGAAGCTTTA 1785GTTGTCCGTA AACGAAAATA AAAATAAAGA GGAATGATAT GAAAGCAAGT AGATCAGTCT 1845GCACTTTCAA AATAGCTACC CTGGCAGGCG CCATTTATGC AGCGCTGCCA ATGTCAGCTG 1905CAAACTCGAT GCAGCTGGAT GTAGGTAGCT CGGATTGGAC GGTGCGTTGG GGACAACACC 1965CTCAAGTATA GCCTTGCCTC TCGCCTGAAT GAGCAAGACT CAAGTCTGAC AAATGCGCCG 2025ACTGTCAATG GTTATATCCG GATATTCAAA GTCAGGGTGA TCGTAACTTT GACCGGGGGC 2085TTGGTATCCA ATCGTCTCGA TATTCTGGCT GCAG 2119圖2bCTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420AAAGAGCATG CAA CTG ACC AAC AAG AAA ATC GTC GTC ACC GGA GTG TCC TCC 472Met Gln Leu Thr Asn Lys Lys Ile Val Val Thr Gly Val Ser Ser1 5 10 15GGT ATC GGT GCC GAA ACT GCC CGC GTT CTG CGC TCT CAC GGC GCC ACA520Gly Ile Gly Ala Glu Thr Ala Arg Val Leu Arg Ser His Gly Ala Thr20 25 30GTG ATT GGC GTA GAT CGC AAC ATG CCG AGC CTG ACT CTG GAT GCT TTC568Val Ile Gly Val Asp Arg Asn Met Pro Ser Leu Thr Leu Asp Ala Phe35 40 45GTT CAG GCT GAC CTG AGC CAT CCT GAA GGC ATC GATC AAC GGC ATA AAT 617Val Gln Ala Asp Leu Ser His Pro Glu Gly Ile Asn Gly Ile Asn50 55 58 240ATT CCA GTG GAC GGA GGT TTG GCA TCG ACC TAC GTG TAA GTTCGTGGAC 666Ile Pro Val Asp Gly Gly Leu Ala Ser Thr Tyr Val245 250 255GCCCTTTGCA CGCGCACTAT ATCTCTATGC AGCAGCTGAA AGCAGCTTTG GTTTTGATCG 726GAGGTAGCGG GCGGAAAGGT GCAGAATGTC TAAATAATAA AGGATTCTTG TGAAGCTTTA 786GTTGTCCGTA AACGAAAATA AAAATAAAGA GGAATGATAT GAAAGCAAGT AGATCAGTCT 846GCACTTTCAA AATAGCTACC CTGGCAGGCG CCATTTATGC AGCGCTGCCA ATGTCAGCTG 906CAAACTCGAT GCAGCTGGAT GTAGGTAGCT CGGATTGGAC GGTGCGTTGG GGACAACACC 966CTCAAGTATA GCCTTGCCTC TCGCCTGAAT GAGCAAGACT CAAGTCTGAC AAATGCGCCG 1026ACTGTCAATG GTTATATCCG GATATTCAAA GTCAGGGTGA TCGTAACTTT GACCGGGGGC 1086TTGGTATCCA ATCGTCTCGA TATTCTGGCT GCAG 1120圖2cGAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC60GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420AGAATAACAA TTGACTCCTC AGGAGGTCAG CG ATG AGC ATT CTT GGT TTG AAT 473Met Ser Ile Leu Gly Leu Asn1 5GGT GCC CCG GTC GGA GCT GAG CAG CTG GGC TCG GCT CTT GAT CGC ATG 521Gly Ala Pro Val Gly Ala Glu Gln Leu Gly Ser Ala Leu Asp Arg Met10 15 20AAG AAG GCG CAC CTG GAG CAG GGG CCT GCA AAC TTG GAG CTG CGT CTG 569Lys Lys Ala His Leu Glu Gln Gly Pro Ala Asn Leu Glu Leu ArG Leu25 30 35AGT AGG CTG GAT CGT GCG ATT GCA ATG CTT CTG GAA AAT CGT GAA GCA 617Ser Arg Leu Asp Arg Ala Ile Ala Met Leu Leu Glu Asn Arg Glu Ala40 45 50 55ATT GCC GAC GCG GTT TCT GCT GAC TTT GGC AAT CGC AGC CGT GAG CAA 665Ile Ala Asp Ala Val Ser Ala Asp Phe Gly Asn Arg Ser Arg Glu Gln60 65 70ACA CTG CTT TGC GAC ATT GCT GGC TCG GTG GCA AGC CTG AAG GAT AGC 713Thr Leu Leu Cys Asp Ile Ala Gly Ser Val Ala Ser Leu Lys Asp Ser75 80 85CGC GAG CAC GTG GCC AAA TGG ATG GAG CCC GAA CAT CAC AAG GCG ATG 761ArG Glu His Val Ala Lys Trp Met Glu Pro Glu His His Lys Ala Met90 95 100TTT CCA GGG GCG GAG GCA CGC GTT GAG TTT CAG CCG CTG GGT GTC GTT 809Phe Pro Gly Ala Glu Ala Arg Val Glu Phe Gln Pro Leu Gly Val Val105 110 115GGG GTC ATT AGT CCC TGG AAC TTC CCT ATC GTA CTG GCC TTT GGG CCG 857Gly Val Ile Ser Pro Trp Asn Phe Pro Ile Val Leu Ala Phe Gly Pro120 125 130 135CTG GCC GGC ATA TTC GCA GCA GGT AAT CGC GCC ATG CTC AAG CCG TCC 905Leu Ala Gly Ile Phe Ala Ala Gly Asn Arg Ala Met Leu Lys Pro Ser140 145 150GAG CTT ACC CCG CGG ACT TCT GCC CTG CTT GCG GAG CTA ATT GCT CGT 953Glu Leu Thr Pro Arg Thr Ser Ala Leu Leu Ala Glu Leu Ile Ala Arg155 160 165TAC TTC GAT GAA ACT GAG CTG ACT ACA GTG CTG GGC GAC GCT GAA GTC 1001Tyr Phe Asp Glu Thr Glu Leu Thr Thr Val Leu Gly Asp Ala Glu Val170 175 180GGT GCG CTG TTC AGT GCT CAG CCT TTC GAT CAT CTG ATC TTC ACC GGC 1049Gly Ala Leu Phe Ser Ala Gln Pro Phe Asp His Leu Ile Phe Thr Gly185 190 195GGC ACT GCC GTG GCC AAG CAC ATC ATG CGT GCC GCG GCG GAT AAC CTA 1097Gly Thr Ala Val Ala Lys His Ile Met Arg Ala Ala Ala Asp Asn Leu200 205 210 215GTG CCC GTT ACC CTG GAA TTG GGT GGC AAA TCG CCG GTG ATC GTT TCC 1145Val Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Val Ile Val Ser220 225 230CGC AGT GCA GAT ATG GCG GAC GTT GCA CAA CGG GTG TTG ACG GTG AAA 1193Arg Ser Ala Asp Met Ala Asp Val Ala Gln ArG Val Leu Thr Val Lys235 240 245ACC TTC AAT GCC GGG CAA ATC TGT CTG GCA CCG GAC TAT GTG CTG CTG 1241Thr Phe Asn Ala Gly Gln Ile Cys Leu Ala Pro Asp Tyr Val Leu Leu250 255 260CCG GAA GGGACAGCAA GCGAACCGGA ATTGCCAGCT GGGGCGCCCT CTGGTAAGGT 1297Pro Glu265TGGGAAGCCC TGCAAAGTAA ACTGGATGGC TTTCTTGCCG CCAAGGATCT GATGGCGCAG1357GGGATCAAGA TCTGATCAAG AGACAGGATG AGGATCGTTT CGC ATG ATT GAA CAA 1412Met Ile Glu Gln1GAT GGA TTG CAC GCA GGT TCT CCG GCC GCT TGG GTG GAG AGG CTA TTC 1460Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val Glu Arg Leu Phe5 10 15 20GGC TAT GAC TGG GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG 1508Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser Asp Ala Ala Val25 30 35TTC CGG CTG TCA GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC 1556Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe Val Lys Thr Asp40 45 50CTG TCC GGT GCC CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG 1604Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala Ala Arg Leu Ser55 60 65TGG CTG GCC ACG ACG GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC 1652Trp Leu Ala Thr Thr Gly val Pro Cys Ala Ala Val Leu Asp Val Val70 75 80ACT GAA GCG GGA AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG 1700Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu Val Pro Gly Gln85 90 95 100GAT CTC CTG TCA TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG 1748Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys Val Ser Ile Met105 110 115GCT GAT GCA ATG CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA 1796Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro Ala Thr Cys Pro120 125 130TTC GAC CAC CAA GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG 1844Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met135 140 145GAA GCC GGT CTT GTC GAT CAGGAT GAT CTG GAC GAA GAG CAT CAG GGG 1892Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu Glu His Gln Gly150 155 160CTC GCG CCA GCC GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC 1940Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala Arg Met Pro Asp165 170 175 180GGC GAG GAT CTC GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC 1988Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys Leu Pro Asn Ile185 190 195ATG GTG GAA AAT GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG 2036Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu200 205 210GGT GTG GCG GAC CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT 2084Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala Thr Arg Asp Ile215 220 225GCT GAA GAG CTT GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC 2132Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe Leu Val Leu Tyr230 235 240GGT ATC GCC GCT CCC GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT 2180Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe Tyr Arg Leu Leu245 250 255 260GAC GAG TTC TTC TGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC 2235Asp Glu Phe Phe264CGC CAT GCC AAG CCT GTT CTC GTG CAA AGT CCT GTG GGT GAG TCG AAC 2283His Ala Lys Pro Val Leu Val Gln Ser Pro Val Gly Glu Ser Asn444 445 450 455TTG GCG ATG CGC GCA CCC TAC GGA GAA GCG ATC CAC GGA CTG CTC TCT 2331Leu Ala Met Arg Ala Pro Tyr Gly Glu Ala Ile His Gly Leu Leu Ser460 465 470GTC CTC CTT TCA ACG GAG TGT TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA 2385Va1 Leu Leu Ser Thr Glu Cys475 480 481GGAGCATGCG CTTCTGGGCC CGTTTCTTGA GTATTCATTG GATAGTCACG CGTGGTAGCT 2445TCGAGCCTGC ACAGCTGATG AGCACCCTGG AAGGCGCGCT GTACGCGGAC GACTGGGTTC 2505ATCTTCGCCA TTCATGACGG AACTCCGTTC CCCAGTACCG CGATGACTAT TTTGCCTCTT 2565CCGATGTCCG ATTCCACGCC GCCTGACGCT AAGCGGGGGC GGGGGCGCCC GCATCCCAGC 2625CCAGACAGCA ACAAATGAGT AGGCTCTTGG ATGCCGCGGC GGCTGAGATT GGTAACGGCA 2685ATTTCGTCAA TGTGACGATG GATTCGATTG CCCGTGCTGC CGGCGTCTCA AAAAAAACGC 2745TGTACGTCTT GGTGGCGAGC AAGGAAGAAC TCATTTCCCG GTTAGTGGCT CGAGACATGT 2805CCAACCTTGA GGAATTC2822圖2dGAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420AGAATAACAA TTGACTCCTC AGGAGGTCAG CG ATG AGC ATT CTT GGT TTG AAT 473Met Ser Ile Leu Gly Leu Asn1 5GGT GCC CCG GTC GGA GCT GAG CAG CTG GGC TCG GCT CTT GAT CGC ATG 521Gly Ala Pro Val Gly Ala Glu Gln Leu Gly Ser Ala Leu Asp ArG Met10 15 20AAG AAG GCG CAC CTG GAG CAG GGG CCT GCA AAC TTG GAG CTG CGT CTG 569Lys Lys Ala His Leu Glu Gln Gly Pro Ala Asn Leu Glu Leu Arg Leu25 30 35AGT AGG CTG GAT CGT GCG ATT GCA ATG CTT CTG GAA AAT CGT GAA GCA 617Ser Arg Leu Asp Arg Ala Ile Ala Met Leu Leu Glu Asn Arg Glu Ala40 45 50 55ATT GCC GAC GCG GTT TCT GCT GAC TTT GGC AAT CGC AGC CGT GAG CAA 665Ile Ala Asp Ala Val Ser Ala Asp Phe Gly Asn Arg Ser Arg Glu Gln60 65 70ACA CTG CTT TGC GAC ATT GCT GGC TCG GTG GCA AGC CTG AAG GAT AGC 713Thr Leu Leu Cys Asp Ile Ala Gly Ser Val Ala Ser Leu Lys Asp Ser75 80 85CGC GAG CAC GTG GCC AAA TGG ATG GAG CCC GAA CAT CAC AAG GCG ATG 761Arg Glu His Val Ala Lys Trp Met Glu Pro Glu His His Lys Ala Met90 95 100TTT CCA GGG GCG GAG GCA CGC GTT GAG TTT CAG CCG CTG GGT GTC GTT 809Phe Pro Gly Ala Glu Ala Arg Val Glu Phe Gln Pro Leu Gly Val Val105 110 115GGG GTC ATT AGT CCC TGG AAC TTC CCT ATC GTA CTG GCC TTT GGG CCG 857Gly Val Ile Ser Pro Trp Asn Phe Pro Ile Val Leu Ala Phe Gly Pro120 125 130 135CTG GCC GGC ATA TTC GCA GCA GGT AAT CGC GCC ATG CTC AAG CCG TCC 905Leu Ala Gly Ile Phe Ala Ala Gly Asn Arg Ala Met Leu Lys Pro Ser140 145 150GAG CTT ACC CCG CGG ACT TCT GCC CTG CTT GCG GAG CTA ATT GCT CGT 953Glu Leu Thr Pro Arg Thr Ser Ala Leu Leu Ala Glu Leu Ile Ala Arg155 160 165TAC TTC GAT GAA ACT GAG CTG ACT ACA GTG CTG GGC GAC GCT GAA GTC 1001Tyr Phe Asp Glu Thr Glu Leu Thr Thr Val Leu Gly Asp Ala Glu Val170 175 180GGT GCG CTG TTC AGT GCT CAG CCT TTC GAT CAT CTG ATC TTC ACC GGC 1049Gly Ala Leu Phe Ser Ala Gln Pro Phe Asp His Leu Ile Phe Thr Gly185 190 195GGC ACT GCC GTG GCC AAG CAC ATC ATG CGT GCC GCG GCG GAT AAC CTA 1097Gly Thr Ala Val Ala Lys His Ile Met Arg Ala Ala Ala Asp Asn Leu200 205 210 215GTG CCC GTT ACC CTG GAA TTG GGT GGC AAA TCG CCG GTG ATC GTT TCC 1145Val Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Val Ile Val Ser220 225 230CGC AGT GCA GAT ATG GCG GAC GTT GCA CAA CGG GTG TTG ACG GTG AAA 1193Arg Ser Ala Asp Met Ala Asp Val Ala Gln Arg Val Leu Thr Val Lys235 240 245ACC TTC AAT GCC GGG CAA ATC TGT CTG GCA CCG GAC TAT GTG CTG GGG 1241Thr Phe Asn Ala Gly Gln Ile Cys Leu Ala Pro Asp Tyr Val Leu250 255 260 262GAGAGGCGGT TTGCGTATTG GGCGCATGCA TAAAAACTGT TGTAATTCAT TAAGCATTCT 1301GCCGACATGG AAGCCATCAC AAACGGCATG ATGAACCTGA ATCGCCAGCG GCATCAGCAC 1361CTTGTCGCCT TGCGTATAAT ATTTGCCCAT GGACGCACAC CGTGGAAACG GATGAAGGCA 1421CGAACCCAGT TGACATAAGC CTGTTCGGTT CGTAAACTGT AATGCAAGTA GCGTATGCGC 1481TCACGCAACT GGTCCAGAAC CTTGACCGAA CGCAGCGGTG GTAACGGCGC AGTGGCGGTT 1541TTCATGGCTT GTTATGACTG TTTTTTTGTA CAGTCTATGC CTCGGGCATC CAAGCAGCAA 1601GCGCGTTACG CCGTGGGTCG ATGTTTGATG TTATGGAGCA GCAACG ATG TTA CGC1656Met Leu Arg1AGC AGC AAC GAT GTT ACG CAG CAG GGC AGT CGC CCT AAA ACA AAG TTA 1704Ser Ser Asn Asp Val Thr Gln Gln Gly Ser Arg Pro Lys Thr Lys Leu5 10 15GGT GGC TCA AGT ATG GGC ATC ATT CGC ACA TGT AGG CTC GGC CCT GAC 1752Gly Gly Ser Ser Met Gly Ile Ile Arg Thr Cys Arg Leu Gly Pro Asp20 25 30 35CAA GTC AAA TCC ATG CGG GCT GCT CTT GAT CTT TTC GGT CGT GAG TTC 1800Gln Val Lys Ser Met Arg Ala Ala Leu Asp Leu Phe Gly Arg Glu Phe40 45 50GGA GAC GTA GCC ACC TAC TCC CAA CAT CAG CCG GAC TCC GAT TAC CTC 1848Gly Asp Val Ala Thr Tyr Ser Gln His Gln Pro Asp Ser Asp Tyr Leu55 60 65GGG AAC TTG CTC CGT AGT AAG ACA TTC ATC GCG CTT GCT GCC TTC GAC 1896Gly Asn Leu Leu Arg Ser Lys Thr Phe Ile Ala Leu Ala Ala Phe Asp70 75 80CAA GAA GCG GTT GTT GGC GCT CTC GCG GCT TAC GTT CTG CCC AGG TTT 1944Gln Glu Ala Val Val Gly Ala Leu Ala Ala Tyr Val Leu Pro Arg Phe85 90 95GAG CAG CCG CGT AGT GAG ATC TAT ATC TAT GAT CTC GCA GTC TCC GGC 1992Glu Gln Pro Arg Ser Glu Ile Tyr Ile Tyr Asp Leu Ala Val Ser Gly100 105 110 115GAG CAC CGG AGG CAG GGC ATT GCC ACC GCG CTC ATC AAT CTC CTC AAG 2040Glu His Arg Arg Gln Gly Ile Ala Thr Ala Leu Ile Asn Leu Leu Lys120 125 130CAT GAG GCC AAC GCG CTT GGT GCT TAT GTG ATC TAC GTG CAA GCA GAT 2088His Glu Ala Asn Ala Leu Gly Ala Tyr Val Ile Tyr Val Gln Ala Asp135 140 145TAC GGT GAC GAT CCC GCA GTG GCT CTC TAT ACA AAG TTG GGC ATA CGG 2136Tyr Gly Asp Asp Pro Ala Val Ala Leu Tyr Thr Lys Leu Gly Ile Arg150 155 160GAA GAA GTG ATG CAC TTT GAT ATC GAC CCA AGT ACC GCC ACC TAA CAA 2184Glu Glu Val Met His Phe Asp Ile Asp Pro Ser Thr Ala Thr165 170 175 177TTCGTTCAAG CCGAGATCGG CTTCCCTG CAA AGT CCT GTG GGT GAG TCG AAC2236Gln Ser Pro Val Gly Glu Ser Asn451 455TTG GCG ATG CGC GCA CCC TAC GGA GAA GCG ATC CAC GGA CTG CTC TCT 2284Leu Ala Met Arg Ala Pro Tyr Gly Glu Ala Ile His Gly Leu Leu Ser460 465 470GTC CTC CTT TCA ACG GAG TGT TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA 2338Val Leu Leu Ser Thr Glu Cys475 480 481GGAGCATGCG CTTCTGGGCC CGTTTCTTGA GTATTCATTG GATAGTCACG CGTGGTAGCT 2398TCGAGCCTGC ACAGCTGATG AGCACCCTGG AAGGCGCGCT GTACGCGGAC GACTGGGTTC 2458ATCTTCGCCA TTCATGACGG AACTCCGTTC CCCAGTACCG CGATGACTAT TTTGCCTCTT 2518CCGATGTCCG ATTCCACGCC GCCTGACGCT AAGCGGGGGC GGGGGCGCCC GCATCCCAGC 2578CCAGACAGCA ACAAATGAGT AGGCTCTTGG ATGCCGCGGC GGCTGAGATT GGTAACGGCA 2638ATTTCGTCAA TGTGACGATG GATTCGATTG CCCGTGCTGC CGGCGTCTCA AAAAAAACGC 2698TGTACGTCTT GGTGGCGAGC AAGGAAGAAC TCATTTCCCG GTTAGTGGCT CGAGACATGT 2758CCAACCTTGA GGAATTC2775圖2eGAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420AGAATAACAA TTGACTCCTC AGGAGGTCAG CG ATG AGC ATT CTT GGT TTG AAT 473Met Ser Ile Leu Gly Leu Asn1 5GGT GCC CCG GTC GGA GCT GAG CAG CTG GGC TCG GCT CTT GAT CGC ATG 521Gly Ala Pro Val Gly Ala Glu Gln Leu Gly Ser Ala Leu Asp Arg Met10 15 20AAG AAG GCG CAC CTG GAG CAG GGG CCT GCA AAC TTG GAG CTG CGT CTG 569Lys Lys Ala His Leu Glu Gln Gly Pro Ala Asn Leu Glu Leu Arg Leu25 30 35AGT AGG CTG GAT CGT GCG ATT GCA ATG CTT CTG GAA AAT CGT GAA GCA 617Ser Arg Leu Asp Arg Ala Ile Ala Met Leu Leu Glu Asn Arg Glu Ala40 45 50 55ATT GCC GAC GCG GTT TCT GCT GAC TTT GGC AAT CGC AGC CGT GAG CAA 665Ile Ala Asp Ala Val Ser Ala Asp Phe Gly Asn Arg Ser Arg Glu Gln60 65 70ACA CTG CTT TGC GAC ATT GCT GGC TCG GTG GCA AGC CTG AAG GAT AGC 713Thr Leu Leu Cys Asp Ile Ala Gly Ser Val Ala Ser Leu Lys Asp Ser75 80 85CGC GAG CAC GTG GCC AAA TGG ATG GAG CCC GAA CAT CAC AAG GCG ATG 761Arg Glu His Val Ala Lys Trp Met Glu Pro Glu His His Lys Ala Met90 95 100TTT CCA GGG GCG GAG GCA CGC GTT GAG TTT CAG CCG CTG GGT GTC GTT 809Phe Pro Gly Ala Glu Ala Arg Val Glu Phe Gln Pro Leu Gly Val Val105 110 115GGG GTC ATT AGT CCC TGG AAC TTC CCT ATC GTA CTG GCC TTT GGG CCG 857Gly Val Ile Ser Pro Trp Asn Phe Pro Ile Val Leu Ala Phe Gly Pro120 125 130 135CTG GCC GGC ATA TTC GCA GCA GGT AAT CGC GCC ATG CTC AAG CCG TCC 905Leu Ala Gly Ile Phe Ala Ala Gly Ash Arg Ala Met Leu Lys Pro Ser140 145 150GAG CTT ACC CCG CGG ACT TCT GCC CTG CTT GCG GAG CTA ATT GCT CGT 953Glu Leu Thr Pro Arg Thr Ser Ala Leu Leu Ala Glu Leu Ile Ala Arg155 160 165TAC TTC GAT GAA ACT GAG CTG ACT ACA GTG CTG GGC GAC GCT GAA GTC 1001Tyr Phe Asp Glu Thr Glu Leu Thr Thr Val Leu Gly Asp Ala Glu Val170 175 180GGT GCG CTG TTC AGT GCT CAG CCT TTC GAT CAT CTG ATC TTC ACC GGC 1049Gly Ala Leu Phe Ser Ala Gln Pro Phe Asp His Leu Ile Phe Thr Gly185 190 195GGC ACT GCC GTG GCC AAG CAC ATC ATG CGT GCC GCG GCG GAT AAC CTA 1097Gly Thr Ala Val Ala Lys His Ile Met Arg Ala Ala Ala Asp Asn Leu200 205 210 215GTG CCC GTT ACC CTG GAA TTG GGT GGC AAA TCG CCG GTG ATC GTT TCC 1145Val Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Val Ile Val Ser220 225 230CGC AGT GCA GAT ATG GCG GAC GTT GCA CAA CGG GTG TTG ACG GTG AAA 1193Arg Ser Ala Asp Met Ala Asp Val Ala Gln Arg Val Leu Thr Val Lys235 240 245ACC TTC AAT GCC GGG CAA ATC TGT CTG GCA CC GTG GGT GAG TCG AAC1240Thr Phe Asn Ala Gly Gln Ile Cys Leu AlaVal Gly Glu Ser Asn250 255 257454 455TTG GCG ATG CGC GCA CCC TAC GGA GAA GCG ATC CAC GGA CTG CTC TCT 1288Leu Ala Met ArG Ala Pro Tyr Gly Glu Ala Ile His Gly Leu Leu Ser460 465 470GTC CTC CTT TCA ACG GAG TGT TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA 1342Val Leu Leu Ser Thr Glu Cys475 480 481GGAGCATGCG CTTCTGGGCC CGTTTCTTGA GTATTCATTG GATAGTCACG CGTGGTAGCT 1402TCGAGCCTGC ACAGCTGATG AGCACCCTGG AAGGCGCGCT GTACGCGGAC GACTGGGTTC 1462ATCTTCGCCA TTCATGACGG AACTCCGTTC CCCAGTACCG CGATGACTAT TTTGCCTCTT 1522CCGATGTCCG ATTCCACGCC GCCTGACGCT AAGCGGGGGC GGGGGCGCCC GCATCCCAGC 1582CCAGACAGCA ACAAATGAGT AGGCTCTTGG ATGCCGCGGC GGCTGAGATT GGTAACGGCA 1642ATTTCGTCAA TGTGACGATG GATTCGATTG CCCGTGCTGC CGGCGTCTCA AAAAAAACGC 1702TGTACGTCTT GGTGGCGAGC AAGGAAGAAC TCATTTCCCG GTTAGTGGCT CGAGACATGT 1762CCAACCTTGA GGAATTC 1779圖2fCTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG G ATG CGT TCT CTC GAG 356Met Arg Ser Leu Glu1 5GCG CTT CTT CCC TTC CCG GGT CGA ATT CTT GAG CGT CTC GAG CAT TGG 404Ala Leu Leu Pro Phe Pro Gly Arg Ile Leu Glu Arh Leu glu His Trp10 15 20GCT AAG ACC CGT CCA GAA CAA ACC TGC GTT GCT GCC AGG GCG GCA AAT 452Ala Lys Thr Arg Pro Glu Gln Thr Cys Val Ala Ala Arg Ala Ala Asn25 30 35GGG GAA TGG CGT CGT ATC AGC TAC GCG GAA ATG TTC CAC AAC GTC CGC 500Gly Glu Trp Arg Arg Ile Ser Tyr Ala Glu Met Phe His Asn Val Arg40 45 50GCC ATC GCA CAG AGC TTG CTT CCT TAC GGA CTA TCG GCA GAG CGT CCG 548Ala Ile Ala Gln Ser Leu Leu Pro Tyr Gly Leu Ser Ala Glu Arg Pro55 60 65CTG CTT ATC GTC TCT GGA AAT GAC CTG GAA CAT CTT CAG CTG GCA TTT 596Leu Leu Ile Val Ser Gly Asn Asp Leu Glu His Leu Gln Leu Ala Phe70 75 80 85GGG GCT ATG TAT GCG GGC ATT CCC TAT TGC CCG GTG TCT CCT GCT TAT 644Gly Ala Met Tyr Ala Gly Ile Pro Tyr Cys Pro Val Ser Pro Ala Tyr90 95 100TCA CTG CTG TCG CAA GAT TTG GCG AAG CTG CGT CAC ATC GTA GGT CTT 692Ser Leu Leu Ser Gln Asp Leu Ala Lys Leu Arg HisIle Val Gly Leu105 110115CTG CAA CCG GGA CTG GTC TTT GCT GCC GAT GCA GCA CCT TTC CAG GGG 740Leu Gln Pro Gly Leu Val Phe Ala Ala Asp Ala A la Pro Phe Gln120 125 130 132ACAGCAAGCG AACCGGAATT GCCAGCTGGG GCGCCCTCTG GTAAGGTTGG GAAGCCCTGC 800AAAGTAAACT GGATGGCTTT CTTGCCGCCA AGGATCTGAT GGCGCAGGGG ATCAAGATCT 860GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATG ATT GAA CAA GAT GGA TTG CAC 914Met Ile Glu Gln Asp Gly Leu His1 5GCA GGT TCT CCG GCC GCT TGG GTG GAG AGG CTA TTC GGC TAT GAC TGG 962Ala Gly Ser Pro Ala Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp10 15 20GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG TTC CGG CTG TCA 1010Ala Gln Gln Thr Ile Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser25 30 35 40GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT GCC 1058Ala Gln Gly Arg Pro Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala45 50 55CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG TGG CTG GCC ACG 1106Leu Asn Glu Leu Gln Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr60 65 70ACG GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC ACT GAA GCG GGA 1154Thr Gly Val Pro Cys Ala Ala Val Leu Asp Val Val Thr Glu Ala Gly75 80 85AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG TCA 1202Arg Asp Trp Leu Leu Leu Gly Glu Val Pro Gly Gln Asp Leu Leu Ser90 95 100TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA ATG 1250Ser His Leu Ala Pro Ala Glu Lys Val Ser Ile Met Ala Asp Ala Met105 110 115 120CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC CAA 1298Arg Arg Leu His Thr Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gln125 130 135GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT CTT 1346Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met Glu Ala Gly Leu140 145 150GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA GCC 1394Val Asp Gln Asp Asp Leu Asp Glu Glu His Gln Gly Leu Ala Pro Ala155 160 165GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT CTC 1442Glu Leu Phe Ala Arg Leu Lys Ala Arg Met Pro Asp Gly Glu Asp Leu170 175 180GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT 1490Val Val Thr His Gly Asp Ala Cys Leu Pro ASn Ile Met Val Glu Asn185 190 195 200GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG GAC 1538Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu Gly Val Ala Asp205 210 215CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG CTT 1586Arg Tyr Gln Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu220 225 230GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC GCT 1634Gly Gly Glu Trp Ala Asp Arg Phe Leu Val Leu Tyr Gly Ile Ala Ala235 240 245CCC GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT GAC GAG TTC TTC 1682Pro Asp Ser Gln Arg Ile Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe250 255 260 264TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCCT GTT TTG CAA1737Val Leu Gln563 565TGG CGG TCG GCG AAA GTT GAT GCG CTG TAT CGT GGT GAA GAT CAA TCC 1785Trp Arg Ser Ala Lys Val Asp Ala Leu Tyr Arg Gly Glu Asp Gln Ser570 575 580ATG CTG CGT GAC GAG GCC ACA CTG TGA GTTGGTCAGG GGGGGCTTAC 1832Met Leu Arg Asp Glu Ala Thr Leu585 589TCGGCGTTTT CCGACACTGC GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG 1892GGTGCCCTGT CGCTGGTGTC GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG 1952ATGCGTGCGT CGCTTGAACC ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA 2012GCAAGCTTTG ATGCTTACCT GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG 2072TCGGTTCCGG CCTTGGGGGT GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG 2132GCCGGCGAGC AGATTTCCCA AGGCGCTGAT CACGTGCTGT GTGTCGCGGG CTGCAG 2188圖2gCTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG G ATG CGT TCT CTC GAG 356Met Arg Ser Leu Glu1 5GCG CTT CTT CCC TTC CCG GGT CGA ATT CTT GAG CGT CTC GAG CAT TGG 404Ala Leu Leu Pro Phe Pro Gly Arg Ile Leu Glu Arg Leu Glu His Trp10 15 20GCT AAG ACC CGT CCA GAA CAA ACC TGC GTT GCT GCC AGG GCG GCA AAT 452Ala Lys Thr Arg Pro Glu Gln Thr Cys Val Ala Ala Arg Ala Ala Asn25 30 35GGG GAA TGG CGT CGT ATC AGC TAC GCG GAA ATG TTC CAC AAC GTC CGC 500Gly Glu Trp Arg Arg Ile Ser Tyr Ala Glu Met Phe His Asn Val Arg40 45 50GCC ATC GCA CAG AGC TTG CTT CCT TAC GGA CTA TCG GCA GAG CGT CCG 548Ala Ile Ala Gln Ser Leu Leu Pro Tyr Gly Leu Ser Ala Glu Arg Pro55 60 65CTG CTT ATC GTC TCT GGA AAT GAC CTG GAA CAT CTT CAG CTG GCA TTT 596Leu Leu Ile Val Ser Gly Asn Asp Leu Glu His Leu Gln Leu Ala Phe70 75 80 85GGG GCT ATG TAT GCG GGC ATT CCC TAT TGC CCG GTG TCT CCT GCT TAT 644Gly Ala Met Tyr Ala Gly Ile Pro Tyr Cys Pro Val Ser Pro Ala Tyr90 95 100TCA CTG CTG TCG CAA GAT TTG GCG AAG CTG CGT CAC ATC GTA GGT CTT 692Ser Leu Leu Ser Gln Asp Leu Ala Lys Leu Arg His Ile Val Gly Leu105 110 115CTG CAA CCG GGA CTG GTC TTT GCT GCC GAT GCA GCA CCT TTC CAG GGG 740Leu Gln Pro Gly Leu Val Phe Ala Ala Asp Ala Ala Pro Phe Gln120 125 130 132GAGAGGCGGT TTGCGTATTG GGCGCATGCA TAAAAACTGT TGTAATTCAT TAAGCATTCT 800GCCGACATGG AAGCCATCAC AAACGGCATG ATGAACCTGA ATCGCCAGCG GCATCAGCAC 860CTTGTCGCCT TGCGTATAAT ATTTGCCCAT GGACGCACAC CGTGGAAACG GATGAAGGCA 920CGAACCCAGT TGACATAAGC CTGTTCGGTT CGTAAACTGT AATGCAAGTA GCGTATGCGC 980TCACGCAACT GGTCCAGAAC CTTGACCGAA CGCAGCGGTG GTAACGGCGC AGTGGCGGTT1040TTCATGGCTT GTTATGACTG TTTTTTTGTA CAGTCTATGC CTCGGGCATC CAAGCAGCAA1100GCGCGTTACG CCGTGGGTCG ATGTTTGATG TTATGGAGCA GCAACG ATG TTA CGC1155Met Leu Arg1AGC AGC AAC GAT GTT ACG CAG CAG GGC AGT CGC CCT AAA ACA AAG TTA 1203Ser Ser Asn Asp Val Thr Gln Gln Gly Ser Arg Pro Lys Thr Lys Leu5 10 15GGT GGC TCA AGT ATG GGC ATC ATT CGC ACA TGT AGG CTC GGC CCT GAC 1251Gly Gly Ser Ser Met Gly Ile Ile Arg Thr Cys Arg Leu Gly Pro Asp20 25 30 35CAA GTC AAA TCC ATG CGG GCT GCT CTT GAT CTT TTC GGT CGT GAG TTC 1299Gln Val Lys Ser Met Arg Ala Ala Leu Asp Leu Phe Gly Arg Glu Phe40 45 50GGA GAC GTA GCC ACC TAC TCC CAA CAT CAG CCG GAC TCC GAT TAC CTC 1347Gly Asp Val Ala Thr Tyr Ser Gln His Gln Pro Asp Ser Asp Tyr Leu55 60 65GGG AAC TTG CTC CGT AGT AAG ACA TTC ATC GCG CTT GCT GCC TTC GAC 1395Gly Asn Leu Leu Arg Ser Lys Thr Phe Ile Ala Leu Ala Ala Phe Asp70 75 80CAA GAA GCG GTT GTT GGC GCT CTC GCG GCT TAC GTT CTG CCC AGG TTT 1443Gln Glu Ala Val Val Gly Ala Leu Ala Ala Tyr Val Leu Pro Arg Phe85 90 95GAG CAG CCG CGT AGT GAG ATC TAT ATC TAT GAT CTC GCA GTC TCC GGC 1491Glu Gln Pro Arg Ser Glu Ile Tyr Ile Tyr Asp Leu Ala Val Ser Gly100 105 110 115GAG CAC CGG AGG CAG GGC ATT GCC ACC GCG CTC ATC AAT CTC CTC AAG 1539Glu His Arg Arg Gln Gly Ile Ala Thr Ala Leu Ile Asn Leu Leu Lys120 125 130CAT GAG GCC AAC GCG CTT GGT GCT TAT GTG ATC TAC GTG CAA GCA GAT 1587His Glu Ala Asn Ala Leu Gly Ala Tyr Val Ile Tyr Val Gln Ala Asp135 140 145TAC GGT GAC GAT CCC GCA GTG GCT CTC TAT ACA AAG TTG GGC ATA CGG 1635Tyr Gly Asp Asp Pro Ala Val Ala Leu Tyr Thr Lys Leu Gly Ile Arg150 155 160GAA GAA GTG ATG CAC TTT GAT ATC GAC CCA AGT ACC GCC ACC TAA CAA 1683Glu Glu Val Met His Phe Asp Ile Asp Pro Ser Thr Ala Thr165 170 175 177TTCGTTCAAG CCGAGATCGG CTTCCCCT GTT TTG CAA TGG CGG TCG GCG AAA1735Val Leu Gln Trp Arg Ser Ala Lys563 565 570GTT GAT GCG CTG TAT CGT GGT GAA GAT CAA TCC ATG CTG CGT GAC GAG 1783Val Asp Ala Leu Tyr Arg Gly Glu Asp Gln Ser Met Leu Arg Asp Glu575 580 585GCC ACA CTG TGA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC 1835Ala Thr Leu589GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 1895GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 1955ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT 2015GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 2075GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 2135AGGCGCTGAT CACGTGCTGT GTGTCGCGGG CTGCAG 2171圖2hCTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG G ATG CGT TCT CTC GAG 356Met Arg Ser Leu Glu1 5GCG CTT CTT CCC TTC CCG GGT CGA ATT CTT GAG CGT CTC GAG CAT TGG 404Ala Leu Leu Pro Phe Pro Gly Arg Ile Leu Glu Arg Leu Glu His Trp10 15 20GCT AAG ACC CGT CCA GAA CAA ACC TGC GTT GCT GCC AGG GCG GCA AAT 452Ala Lys Thr Arg Pro Glu Gln Thr Cys Val Ala Ala Arg Ala Ala Asn25 30 35GGG GAA TGG CGT CGT ATC AGC TAC GCG GAA ATG TTC CAC AAC GTC CGC 500Gly Glu Trp Arg Arg Ile Ser Tyr Ala Glu Met Phe His Asn Val Arg40 45 50GCC ATC GCA CAG AGC TTG CTT CCT TAC GGA CTA TCG GCA GAG CGT CCG 548Ala Ile Ala Gln Ser Leu Leu Pro Tyr Gly Leu Ser Ala Glu Arg Pro55 60 65CTG CTT ATC GTC TCT GGA AAT GAC CTG GAA CAT CTT CAG CTG GCA TTT 596Leu Leu Ile Val Ser Gly Asn Asp Leu Glu His Leu Gln Leu Ala Phe70 75 80 85GGG GCT ATG TAT GCG GGC ATT CCC TAT TGC CCG GTG TCT CCT GCT TAT 644Gly Ala Met Tyr Ala Gly Ile Pro Tyr Cys Pro Val Ser Pro Ala Tyr90 95 100TCA CTG CTG TCG CAA GAT TTG GCG AAG CTG CGT CAC ATC GTA GGT CTT 692Ser Leu Leu Ser Gln Asp Leu Ala Lys Leu Arg His Ile Val Gly Leu105 110 115CTG CAA CCG GGA CTG GTC TTT GCT GCC GAT GCA GCA CCT TTC CAG CGC 740Leu Gln Pro Gly Leu Val Phe Ala Ala Asp Ala Ala Pro Phe Gln Arg120 125 130 133GCT GTT TTG CAA TGG CGG TCG GCG AAA GTT GAT GCG CTG TAT CGT GGT 788Ala Val Leu Gln Trp Arg Ser Ala Lys Val Asp Ala Leu Tyr Arg Gly562 565 570 575GAA GAT CAA TCC ATG CTG CGT GAC GAG GCC ACA CTG TGA GTTGGTCAGG837Glu Asp Gln Ser Met Leu Arg Asp Glu Ala Thr Leu580 585 589GGGGGCTTAC TCGGCGTTTT CCGACACTGC GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT 897TGATTGCGGG GGTGCCCTGT CGCTGGTGTC GCCTATCGAC TTAGGGGTAA AGGTCGCTCG 957CGAAGTTCTG ATGCGTGCGT CGCTTGAACC ACAAATGGTC GATAGCGTAC TCGCAGGCTC 1017TATGGCTCAA GCAAGCTTTG ATGCTTACCT GCTCCCGCGG CACATTGGCT TGTACAGCGG 1077TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT GCAGCGCATT TGCGGCACAG GCTTCGAACT 1137GCTTCGGCAG GCCGGCGAGC AGATTTCCCA AGGCGCTGAT CACGTGCTGT GTGTCGCGGG 1197CTGCAG1203圖2iGAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240GGTGCACG ATG AAT AGC TAC GAT GGC CGT TGG TCT ACC GTT GAT GTG AAG 290Met Asn Ser Tyr Asp Gly Arg Trp Ser Thr Val Asp Val Lys1 5 10GTT GAA GAA GGT ATC GCT TGG GTC ACG CTG AAC CGC CCG GAG AAG CGC 338Val Glu Glu Gly Ile Ala Trp Val Thr Leu Asn Arg Pro Glu Lys Arg15 20 25 30AAC GCA ATG AGC CCA ACT CTC AAT CGA GAG ATG GTC GAG GTT CTG GAG 386Asn Ala Met Ser Pro Thr Leu Asn Arg Glu Met Val Glu Val Leu Glu35 40 45GTG CTG GAG CAG GAC GCA GAT GCT CGC GTG CTT GTT CTG ACT GGT GCA 434Val Leu Glu Gln Asp Ala Asp Ala Arg Val Leu Val Leu Thr Gly Ala50 55 60GGC GAA TCC TGG ACC GCG GGC ATG GAC CTG AAG GAG TAT TTC CGC GAG 482Gly Glu Ser Trp Thr Ala Gly Met Asp Leu Lys Glu Tyr Phe Arg Glu65 70 75ACC GAT GCT GGC CCC GAA ATT CTG CAA GAG AAG ATT CGT CGGGGACAGC531Thr Asp Ala Gly Pro Glu Ile Leu Gln Glu Lys Ile Arg80 85 90 91AAGCGAACCG GAATTGCCAG CTGGGGCGCC CTCTGGTAAG GTTGGGAAGC CCTGCAAAGT 591AAACTGGATG GCTTTCTTGC CGCCAAGGAT CTGATGGCGC AGGGGATCAA GATCTGATCA 651AGAGACAGGA TGAGGATCGT TTCGC ATG ATT GAA CAA GAT GGA TTG CAC GCA 703Met Ile Glu Gln Asp Gly Leu His Ala1 5GGT TCT CCG GCC GCT TGG GTG GAG AGG CTA TTC GGC TAT GAC TGG GCA 751Gly Ser Pro Ala Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala10 15 20 25CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG TTC CGG CTG TCA GCG 799Gln Gln Thr Ile Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser Ala30 35 40CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT GCC CTG 847Gln Gly Arg Pro Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala Leu45 50 55AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG TGG CTG GCC ACG ACG 895ASn Glu Leu Gln Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr60 65 70GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC ACT GAA GCG GGA AGG943Gly Val Pro Cys Ala Ala Val Leu Asp Val Val Thr Glu Ala Gly Arg75 80 85GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG TCA TCT991Asp Trp Leu Leu Leu Gly Glu Val Pro Gly Gln Asp Leu Leu Ser Ser90 95 100 105CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA ATG CGG 1039His Leu Ala Pro Ala Glu Lys Val Ser Ile Met Ala Asp Ala Met Arg110 115 120CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC CAA GCG 1087Arg Leu His Thr Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gln Ala125 130 135AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT CTT GTC 1135Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met Glu Ala Gly Leu Val140 145 150GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA GCC GAA 1183Asp Gln Asp Asp Leu Asp Glu Glu His Gln Gly Leu Ala Pro Ala Glu155 160 165CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT CTC GTC 1231Leu Phe Ala Arg Leu Lys Ala Arg Met Pro Asp Gly Glu Asp Leu Val170 175 180 185GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT GGC 1279Val Thr His Gly Asp Ala Cys Leu Pro Asn Ile Met Val Glu Asn Gly190 195 200CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG GAC CGC 1327Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu Gly Val Ala Asp Arg205 210 215TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG CTT GGC 1375Tyr Gln Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu Gly220 225 230GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC GCT CCC 1423Gly Glu Trp Ala Asp Arg Phe Leu Val Leu Tyr Gly Ile Ala Ala Pro235 240 245GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT GAC GAG TTC TTC TGA 1471Asp Ser Gln Arg Ile Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe250 255 260 264GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CC GAG CAG GGC ATG1525Glu Gln Gly Met255AAG CAG TTC CTT GAC GAG AAA AGC ATC AAG CCG GGC TTG CAG ACC TAC 1573Lys Gln Phe Leu Asp Glu Lys Ser Ile Lys Pro Gly Leu Gln Thr Tyr260 265 270AAG CGC TGA TAAATGCGCC GGGGCCCTCG CTGCGCCCCC GGCCTTCCAA TAATGACAAT1632Lys Arg275 276AATGAGGAGT GCCCAATGTT TCACGTGCCC CTGCTTATTG GTGGTAAGCC TTGTTCAGCA 1692TCTGATGAGC GCACCTTCGA GCGTCGTAGC CCGCTGACCG GAGAAGTGGT ATCGCGCGTC 1752GCTGCTGCCA GTTTGGAAGA TGCGGACGCC GCAGTGGCCG CTGCACAGGC TGCGTTTCCT 1812GAATGGGCGG CGCTTGCTCC GAGCGAACGC CGTGCCCGAC TGCTGCGAGC GGCGGATCTT 1872CTAGAGGACC GTTCTTCCGA GTTCACCGCC GCAGCGAGTG AAACTGGCGC AGCGGGAAAC 1932TGGTATGGGT TTAACGTTTA CCTGGCGGCG GGCATGTTGC GGGGAATTC 1981圖2jGAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240GGTGCACG ATG AAT AGC TAC GAT GGC CGT TGG TCT ACC GTT GAT GTG AAG 290Met Asn Ser Tyr Asp Gly Arg Trp Ser Thr Val Asp Val Lys1 5 10GTT GAA GAA GGT ATC GCT TGG GTC ACG CTG AAC CGC CCG GAG AAG CGC 338Val Glu Glu Gly Ile Ala Trp val Thr Leu Asn Arg Pro Glu Lys Arg15 20 25 30AAC GCA ATG AGC CCA ACT CTC AAT CGA GAG ATG GTC GAG GTT CTG GAG 386Asn Ala Met Ser Pro Thr Leu Asn Arg Glu Met Val Glu Val Leu Glu35 40 45GTG CTG GAG CAG GAC GCA GAT GCT CGC GTG CTT GTT CTG ACT GGT GCA 434Val Leu Glu Gln Asp Ala Asp Ala Arg Val Leu Val Leu Thr Gly Ala50 55 60GGC GAA TCC TGG ACC GCG GGC ATG GAC CTG AAG GAG TAT TTC CGC GAG 482Gly Glu Ser Trp Thr Ala Gly Met Asp Leu Lys Glu Tyr Phe Arg Glu65 70 75ACC GAT GCT GGC CCC GAA ATT CTG CAA GAG AAG ATT CGT CGGGGGAGAG531Thr Asp Ala Gly Pro Glu Ile Leu Gln Glu Lys Ile Arg80 85 90 91GCGGTTTGCG TATTGGGCGC ATGCATAAAA ACTGTTGTAA TTCATTAAGC ATTCTGCCGA 591CATGGAAGCC ATCACAAACG GCATGATGAA CCTGAATCGC CAGCGGCATC AGCACCTTGT 651CGCCTTGCGT ATAATATTTG CCCATGGACG CACACCGTGG AAACGGATGA AGGCACGAAC 711CCAGTTGACA TAAGCCTGTT CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG 771CAACTGGTCC AGAACCTTGA CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT 831GGCTTGTTAT GACTGTTTTT TTGTACAGTC TATGCCTCGG GCATCCAAGC AGCAAGCGCG 891TTACGCCGTG GGTCGATGTT TGATGTTATG GAGCAGCAAC G ATG TTA CGC AGC AGC 947Met Leu Arg Ser Ser1 5AAC GAT GTT ACG CAG CAG GGC AGT CGC CCT AAA ACA AAG TTA GGT GGC 995Asn Asp Val Thr Gln Gln Gly Ser Arg Pro Lys Thr Lys Leu Gly Gly10 15 20TCA AGT ATG GGC ATC ATT CGC ACA TGT AGG CTC GGC CCT GAC CAA GTC 1043Ser Ser Met Gly Ile Ile Arg Thr Cys Arg Leu Gly Pro Asp Gln Val25 30 35AAA TCC ATG CGG GCT GCT CTT GAT CTT TTC GGT CGT GAG TTC GGA GAC 1091Lys Ser Met Arg Ala Ala Leu Asp Leu Phe Gly Arg Glu Phe Gly Asp40 45 50GTA GCC ACC TAC TCC CAA CAT CAG CCG GAC TCC GAT TAC CTC GGG AAC 1139Val Ala Thr Tyr Ser Gln His Gln Pro Asp Ser Asp Tyr Leu Gly Asn55 60 65TTG CTC CGT AGT AAG ACA TTC ATC GCG CTT GCT GCC TTC GAC CAA GAA 1187Leu Leu Arg Ser Lys Thr Phe Ile Ala Leu Ala Ala Phe Asp Gln Glu70 75 80 85GCG GTT GTT GGC GCT CTC GCG GCT TAC GTT CTG CCC AGG TTT GAG CAG 1235Ala Val Val Gly Ala Leu Ala Ala Tyr Val Leu Pro Arg Phe Glu Gln90 95 100CCG CGT AGT GAG ATC TAT ATC TAT GAT CTC GCA GTC TCC GGC GAG CAC 1283Pro Arg Ser Glu Ile Tyr Ile Tyr Asp Leu Ala Val Ser Gly Glu His105 110 115CGG AGG CAG GGC ATT GCC ACC GCG CTC ATC AAT CTC CTC AAG CAT GAG 1331Arg Arg Gln Gly Ile Ala Thr Ala Leu Ile Asn Leu Leu Lys His Glu120 125 130GCC AAC GCG CTT GGT GCT TAT GTG ATC TAC GTG CAA GCA GAT TAC GGT 1379Ala Asn Ala Leu Gly Ala Tyr Val Ile Tyr Val Gln Ala Asp Tyr Gly135 140 145GAC GAT CCC GCA GTG GCT CTC TAT ACA AAG TTG GGC ATA CGG GAA GAA 1427Asp Asp Pro Ala Val Ala Leu Tyr Thr Lys Leu Gly Ile Arg Glu Glu150 155 160 165GTG ATG CAC TTT GAT ATC GAC CCA AGT ACC GCC ACC TAA CAATTCGTTC1476Val Met His Phe Asp Ile Asp Pro Ser Thr Ala Thr170 175 177AAGCCGAGAT CGGCTTCCCC GAG CAG GGC ATG AAG CAG TTC CTT GAC GAG 1526Glu Gln Gly Met Lys Gln Phe Leu Asp Glu255 260AAA AGC ATC AAG CCG GGC TTG CAG ACC TAC AAG CGC TGA TAAATGCGCC1575Lys Ser Ile Lys Pro Gly Leu Gln Thr Tyr Lys Arg265 270 275 276GGGGCCCTCG CTGCGCCCCC GGCCTTCCAA TAATGACAAT AATGAGGAGT GCCCAATGTT 1635TCACGTGCCC CTGCTTATTG GTGGTAAGCC TTGTTCAGCA TCTGATGAGC GCACCTTCGA 1695GCGTCGTAGC CCGCTGACCG GAGAAGTGGT ATCGCGCGTC GCTGCTGCCA GTTTGGAAGA 1755TGCGGACGCC GCAGTGGCCG CTGCACAGGC TGCGTTTCCT GAATGGGCGG CGCTTGCTCC 1815GAGCGAACGC CGTGCCCGAC TGCTGCGAGC GGCGGATCTT CTAGAGGACC GTTCTTCCGA 1875GTTCACCGCC GCAGCGAGTG AAACTGGCGC AGCGGGAAAC TGGTATGGGT TTAACGTTTA 1935CCTGGCGGCG GGCATGTTGC GGGGAATTC 1964圖2kGAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240GGTGCACG ATG AAT AGC TAC GAT GGC CGT TGG TCT ACC GTT GAT GTG AAG 290Met Asn Ser Tyr Asp Gly Arg Trp Ser Thr Val Asp Val Lys1 5 10GTT GAA GAA GGT ATC GCT TGG GTC ACG CTG AAC CGC CCG GAG AAG CGC 338Val Glu Glu Gly Ile Ala Trp Val Thr Leu Asn Arg Pro Glu Lys Arg15 20 25 30AAC GCA ATG AGC CCA ACT CTC AAT CGA GAG ATG GTC GAG GTT CTG GAG 386Asn Ala Met Ser Pro Thr Leu Asn Arg Glu Met Val Glu Val Leu Glu35 40 45GTG CTG GAG CAG GAC GCA GAT GCT CGC GTG CTT GTT CTG ACT GGT GCA 434Val Leu Glu Gln Asp Ala Asp Ala Arg Val Leu Val Leu Thr Gly Ala50 55 60GGC GAA TCC TGG ACC GCG GGC ATG GAC CTG AAG GAG TAT TTC CGC GAG 482Gly Glu Ser Trp Thr Ala Gly Met Asp Leu Lys Glu Tyr Phe Arg Glu65 70 75ACC GAT GCT GGC CCC GAA ATT CTG CAA GAG AAG ATT CGT CGC GAG CAG 530Thr Asp Ala Gly Pro Glu Ile Leu Gln Glu Lys Ile Arg Arg Glu Gln80 85 90 92 255GGC ATG AAG CAG TTC CTT GAC GAG AAA AGC ATC AAG CCG GGC TTG CAG 578Gly Met Lys Gln Phe Leu Asp Glu Lys Ser Ile Lys Pro Gly Leu Gln260 265 270ACC TAC AAG CGC TGA TAAATGCGCC GGGGCCCTCG CTGCGCCCCC GGCCTTCCAA 633Thr Tyr Lys Arg275 276TAATGACAAT AATGAGGAGT GCCCAATGTT TCACGTGCCC CTGCTTATTG GTGGTAAGCC 693TTGTTCAGCA TCTGATGAGC GCACCTTCGA GCGTCGTAGC CCGCTGACCG GAGAAGTGGT 753ATCGCGCGTC GCTGCTGCCA GTTTGGAAGA TGCGGACGCC GCAGTGGCCG CTGCACAGGC 813TGCGTTTCCT GAATGGGCGG CGCTTGCTCC GAGCGAACGC CGTGCCCGAC TGCTGCGAGC 873GGCGGATCTT CTAGAGGACC GTTCTTCCGA GTTCACCGCC GCAGCGAGTG AAACTGGCGC 933AGCGGGAAAC TGGTATGGGT TTAACGTTTA CCTGGCGGCG GGCATGTTGC GGGGAATTC 992圖2lGAATTCCAAT AATGACAATA ATGAGGAGTG CCCA ATG TTT CAC GTG CCC CTG CTT 55Met Phe His Val Pro Leu Leu1 5ATT GGT GGT AAG CCT TGT TCA GCA TCT GAT GAG CGC ACC TTC GAG CGT 103Ile Gly Gly Lys Pro Cys Ser Ala Ser Asp Glu Arg Thr Phe Glu Arg10 15 20CGT AGC CCG CTG ACC GGA GAA GTG GTA TCG CGC GTC GCT GCT GCC AGT 151Arg Ser Pro Leu Thr Gly Glu Val Val Ser Arg Val Ala Ala Ala Ser25 30 35TTG GAA GAT GCG GAC GCC GCA GTG GCC GCT GCA CAG GCT GCG TTT CCT 199Leu Glu Asp Ala Asp Ala Ala Val Ala Ala Ala Gln Ala Ala Phe Pro40 45 50 55GAA TGG GCG GCG CTT GCT CCG AGC GAA CGC CGT GCC CGA CTG CTG CGA 247Glu Trp Ala Ala Leu Ala Pro Ser Glu Arg Arg Ala Arg Leu Leu Arg60 65 70GCG GCG GAT CTT CTA GAG GAC CGT TCT TCC GAG TTC ACC GCC GCA GCG 295Ala Ala Asp Leu Leu Glu Asp Arg Ser Ser Glu Phe Thr Ala Ala Ala75 80 85AGT GAA ACT GGC GCA GCG GGA AAC TGG TAT GGG TTT AAC GTT TAC CTG 343Ser Glu Thr Gly Ala Ala Gly Asn Trp Tyr Gly Phe Asn Val Tyr Leu90 95 100GCG GCG GGC ATG TTG CGG GAA GCC GCG GCC ATG ACC ACA CAG ATT CAG 391Ala Ala Gly Met Leu Arg Glu Ala Ala Ala Met Thr Thr Gln Ile Gln105 110 115GGC GAT GTC ATT CCG TCC AAT GTG CCC GGT AGC TTT GCC ATG GCG GTT 439Gly Asp Val Ile Pro Ser Asn Val Pro Gly Ser Phe Ala Met Ala Val120 125 130 135CGA CAG CCA TGT GGC GTG GTG CTC GGT ATT GCG CCT TGG AAT GCT CCG 487Arg Gln Pro Cys Gly Val Val Leu Gly Ile Ala Pro Trp Asn Ala Pro140 145 150GTA ATC CTT GGC GTA CGG GCT GTT GCG ATG CCG TTG GCA TGC GGC AAT 535Val Ile Leu Gly Val ArG Ala Val Ala Met Pro Leu Ala Cys Gly Asn155 160 165ACC GTG GTG TTG AAA AGC TCT GAG CTG AGT CCC TTT ACC CAT CGC CTG 583Thr Val Val Leu Lys Ser Ser Glu Leu Ser Pro phe Thr His Arg Leu170 175 180ATT GGT CAG GTG TTG CAT GAT GCT GGT CTG GGG GAT GGC GTG GTG AAT 631Ile Gly Gln Val Leu His Asp Ala Gly Leu Gly Asp Gly Val Val Asn185 190 195GTC ATC AGC AAT GCC CCG CAA GAC GCT CCT GCG GTG GTG GAG CGA CTG 679Val Ile Ser Asn Ala Pro Gln Asp Ala Pro Ala Val Val Glu ArG Leu200 205 210 215ATT GCA AAT CCT GCG GTA CGT CGA GTG AAC TTC ACC GGT TCG ACC CAC 727Ile Ala Asn Pro Ala Val Arg Arg Val Asn Phe Thr Gly Ser Thr His220 225 230GTT GGA CGG ATC ATT GGT GAG CTG TCT GCG CGT CAT CTG AAG CCT GCT 775Val Gly Arg Ile Ile Gly Glu Leu Ser Ala Arg His Leu Lys Pro Ala235 240 245GTG CTG GAA TTA GGT GGT AAG GCT CCG TTC TTG GTC TTG GAC GAT GCC 823Val Leu Glu Leu Gly Gly Lys Ala Pro Phe Leu Val Leu Asp Asp Ala250 255 260GAC CTC GAT GCG GCG GTC GAA GCG GCG GCC TTT GGT GCC TAC TTC AAT 871Asp Leu Asp Ala Ala Val Glu Ala Ala Ala Phe Gly Ala Tyr Phe Asn265 270 275CAG GGT CAA ATC TGC ATG TCC ACT GAG CGT CTG ATT GTG ACA GCA GTC 919Gln Gly Gln Ile Cys Met Ser Thr Glu Arg Leu Ile Val Thr Ala Val280 285 290 295GCA GAC GCC TTT GTT GAA AAG CTG GCG AGG AAG GTC GCC ACA CTG CGT 967Ala Asp Ala Phe Val Glu Lys Leu Ala Arg Lys Val Ala Thr Leu Arg300 305 310GCT GGC GAT CCT AAT GAT CCG CAA TCG GTC TTG GGT TCG TTG ATT GAT 1015Ala Gly Asp Pro Asn Asp Pro Gln Ser Val Leu Gly Ser Leu Ile Asp315 320 325GCC AAT GCA GGT CAA CGC ATC CAG GTT CTG GTC GAT GAT GCG CTC GGG 1063Ala Asn Ala Gly Gln Arg Ile Gln Val Leu Val Asp Asp Ala Leu330 335 340 342GACAGCAAGC GAACCGGAAT TGCCAGCTGG GGCGCCCTCT GGTAAGGTTG GGAAGCCCTG1123CAAAGTAAAC TGGATGGCTT TCTTGCCGCC AAGGATCTGA TGGCGCAGGG GATCAAGATC1183TGATCAAGAG ACAGGATGAG GATCGTTTCG C ATG ATT GAA CAA GAT GGA TTG 1235Met Ile Glu Gln Asp Gly Leu1 5CAC GCA GGT TCT CCG GCC GCT TGG GTG GAG AGG CTA TTC GGC TAT GAC 1283His Ala Gly Ser Pro Ala Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp10 15 20TGG GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG TTC CGG CTG 1331Trp Ala Gln Gln Thr Ile Gly Cys Ser Asp Ala Ala Val Phe Arg Leu25 30 35TCA GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT 1379Ser Ala Gln Gly Arg Pro Val Leu Phe Val Lys Thr Asp Leu Ser Gly40 45 50 55GCC CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG TGG CTG GCC 1427Ala Leu Asn Glu Leu Gln Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala60 65 70ACG ACG GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC ACT GAA GCG 1475Thr Thr Gly Val Pro Cys Ala Ala Val Leu Asp Val Val Thr Glu Ala75 80 85GGA AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG 1523Gly Arg Asp Trp Leu Leu Leu Gly Glu Val Pro Gly Gln Asp Leu Leu90 95 100TCA TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA 1571Ser Ser His Leu Ala Pro Ala Glu Lys Val Ser Ile Met Ala Asp Ala105 110 115ATG CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC 1619Met Arg Arg Leu His Thr Leu Asp Pro Ala Thr Cys Pro Phe Asp His120 125 130 135CAA GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT 1667Gln Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met Glu Ala Gly140 145 150CTT GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA 1715Leu Val Asp Gln Asp Asp Leu Asp Glu Glu His Gln Gly Leu Ala Pro155 160 165GCC GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT 1763Ala Glu Leu Phe Ala Arg Leu Lys Ala Arg Met Pro Asp Gly Glu Asp170 175 180CTC GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA 1811Leu Val Val Thr His Gly Asp Ala Cys Leu Pro Asn Ile Met Val Glu185 190 195AAT GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG 1859Asn Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu Gly Val Ala200 205 210 215GAC CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG 1907Asp Arg Tyr Gln Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu220 225 230CTT GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC 1955Leu Gly Gly Glu Trp Ala Asp Arg Phe Leu Val Leu Tyr Gly Ile Ala235 240 245GCT CCC GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT GAC GAG TTC 2003Ala Pro Asp Ser Gln Arg Ile Ala Phe Tyr Arg Leu Leu Asp Glu Phe250 255 260TTC TGA GCGGGACTCT GGGGTTCGAAATGACCGACC AAGCGACGCC CG GCC CAG 2057phe Ala Gln264 421CGC GTC GAT TCG GGC ATT TGC CAT ATC AAT GGA CCG ACT GTG CAT GAC 2105Arg Val Asp Ser Gly Ile Cys His Ile Asn Gly Pro Thr Val His Asp425 430 435GAG GCT CAG ATG CCA TTC GGT GGG GTG AAG TCC AGC GGC TAC GGC AGC 2153Glu Ala Gln Met Pro Phe Gly Gly Val Lys Ser Ser Gly Tyr Gly Ser440 445 450TTC GGC AGT CGA GCA TCG ATT GAG CAC TTT ACC CAG CTG CGC TGG CTG 2201Phe Gly Ser Arg Ala Ser Ile Glu His Phe Thr Gln Leu Arg Trp Leu455 460 465 470ACC ATT CAG AAT GGC CCG CGG CAC TAT CCA ATC TAA ATCGATCTTC2247Thr Ile Gln Asn Gly Pro Arg His Tyr Pro Ile475 480 481GGGCGCCGCG GGCATCATGC CCGCGGCGCT CGCCTCATTT CAATCTCTAA CTTGATAAAA 2307ACAGAGCTGT TCTCCGGTCT TGGTGGATCA AGGCCAGTCG CGGAGAGTCT CGAAGAGGAG 2367AGTACAGTGA ACGCCGAGTC CACATTGCAA CCGCAGGCAT CATCATGCTC TGCTCAGCCA 2427CGCTACCGCA GTGTGTCGAT TGGTCATCCT CCGGTTGAGG TTACGCAAGA CGCTGGAGGT 2487ATTGTCCGGA TGCGTTCTCT CGAGGCGCTT CTTCCCTTCC CGGGTGGAAT TC 2539圖2mGAATTCCAAT AATGACAATA ATGAGGAGTG CCCA ATG TTT CAC GTG CCC CTG CTT 55Met Phe His Val Pro Leu Leu1 5ATT GGT GGT AAG CCT TGT TCA GCA TCT GAT GAG CGC ACC TTC GAG CGT 103Ile Gly Gly Lys Pro Cys Ser Ala Ser Asp Glu Arg Thr Phe Glu Arg10 15 20CGT AGC CCG CTG ACC GGA GAA GTG GTA TCG CGC GTC GCT GCT GCC AGT 151Arg Ser Pro Leu Thr Gly Glu Val Val Ser Arg Val Ala Ala Ala Ser25 30 35TTG GAA GAT GCG GAC GCC GCA GTG GCC GCT GCA CAG GCT GCG TTT CCT 199Leu Glu Asp Ala Asp Ala Ala Val Ala Ala Ala Gln Ala Ala Phe Pro40 45 50 55GAA TGG GCG GCG CTT GCT CCG AGC GAA CGC CGT GCC CGA CTG CTG CGA 247Glu Trp Ala Ala Leu Ala Pro Ser Glu Arg Arg Ala Arg Leu Leu Arg60 65 70GCG GCG GAT CTT CTA GAG GAC CGT TCT TCC GAG TTC ACC GCC GCA GCG 295Ala Ala Asp Leu Leu Glu Asp Arg Ser Ser Glu Phe Thr Ala Ala Ala75 80 85AGT GAA ACT GGC GCA GCG GGA AAC TGG TAT GGG TTT AAC GTT TAC CTG 343Ser Glu Thr Gly Ala Ala Gly Asn Trp Tyr Gly Phe Asn Val Tyr Leu90 95 100GCG GCG GGC ATG TTG CGG GAA GCC GCG GCC ATG ACC ACA CAG ATT CAG 391Ala Ala Gly Met Leu Arg Glu Ala Ala Ala Met Thr Thr Gln Ile Gln105 110 115GGC GAT GTC ATT CCG TCC AAT GTG CCC GGT AGC TTT GCC ATG GCG GTT 439Gly Asp Val Ile Pro Ser Asn Val Pro Gly Ser Phe Ala Met Ala Val120 125 130 135CGA CAG CCA TGT GGC GTG GTG CTC GGT ATT GCG CCT TGG AAT GCT CCG 487Arg Gln Pro Cys Gly Val Val Leu Gly Ile Ala Pro Trp Asn Ala Pro140 145 150GTA ATC CTT GGC GTA CGG GCT GTT GCG ATG CCG TTG GCA TGC GGC AAT 535Val Ile Leu Gly Val Arg Ala Val Ala Met Pro Leu Ala Cys Gly Asn155 160 165ACC GTG GTG TTG AAA AGC TCT GAG CTG AGT CCC TTT ACC CAT CGC CTG 583Thr Val Val Leu Lys Ser Ser Glu Leu Ser Pro Phe Thr His Arg Leu170 175 180ATT GGT CAG GTG TTG CAT GAT GCT GGT CTG GGG GAT GGC GTG GTG AAT 631Ile Gly Gln Val Leu His Asp Ala Gly Leu Gly Asp Gly Val Val Asn185 190 195GTC ATC AGC AAT GCC CCG CAA GAC GCT CCT GCG GTG GTG GAG CGA CTG 679Val Ile Ser Asn Ala Pro Gln Asp Ala Pro Ala Val Val Glu Arg Leu200 205 210 215ATT GCA AAT CCT GCG GTA CGT CGA GTG AAC TTC ACC GGT TCG ACC CAC 727Ile Ala Asn Pro Ala Val Arg Arg Val Asn Phe Thr Gly Ser Thr His220 225 230GTT GGA CGG ATC ATT GGT GAG CTG TCT GCG CGT CAT CTG AAG CCT GCT 775Val Gly Arg Ile Ile Gly Glu Leu Ser Ala Arg His Leu Lys Pro Ala235 240 245GTG CTG GAA TTA GGT GGT AAG GCT CCG TTC TTG GTC TTG GAC GAT GCC 823Val Leu Glu Leu Gly Gly Lys Ala Pro Phe Leu Val Leu Asp Asp Ala250 255 260GAC CTC GAT GCG GCG GTC GAA GCG GCG GCC TTT GGT GCC TAC TTC AAT 871Asp Leu Asp Ala Ala Val Glu Ala Ala Ala Phe Gly Ala Tyr Phe Asn265 270 275CAG GGT CAA ATC TGC ATG TCC ACT GAG CGT CTG ATT GTG ACA GCA GTC 919Gln Gly Gln Ile Cys Met Ser Thr Glu Arg Leu Ile Val Thr Ala Val280 285 290 295GCA GAC GCC TTT GTT GAA AAG CTG GCG AGG AAG GTC GCC ACA CTG CGT 967Ala Asp Ala Phe Val Glu Lys Leu Ala Arg Lys Val Ala Thr Leu Arg300 305 310GCT GGC GAT CCT AAT GAT CCG CAA TCG GTC TTG GGT TCG TTG ATT GAT 1015Ala Gly Asp Pro Asn Asp Pro Gln Ser Val Leu Gly Ser Leu Ile Asp315 320 325GCC AAT GCA GGT CAA CGC ATC CAG GTGGGGAGAG GCGGTTTGCG TATTGGGCGC 1069Ala Asn Ala Gly Gln Arg Ile Gln330 335ATGCATAAAA ACTGTTGTAA TTCATTAAGC ATTCTGCCGA CATGGAAGCC ATCACAAACG1129GCATGATGAA CCTGAATCGC CAGCGGCATC AGCACCTTGT CGCCTTGCGT ATAATATTTG1189CCCATGGACG CACACCGTGG AAACGGATGA AGGCACGAAC CCAGTTGACA TAAGCCTGTT1249CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG CAACTGGTCC AGAACCTTGA1309CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT GGCTTGTTAT GACTGTTTTT1369TTGTACAGTC TATGCCTCGG GCATCCAAGC AGCAAGCGCG TTACGCCGTG GGTCGATGTT1429TGATGTTATG GAGCAGCAAC G ATG TTA CGC AGC AGC AAC GAT GTT ACG CAG 1480Met Leu Arg Ser Ser Ash Asp Val Thr Gln1 5 10CAG GGC AGT CGC CCT AAA ACA AAG TTA GGT GGC TCA AGT ATG GGC ATC 1528Gln Gly Ser Arg Pro Lys Thr Lys Leu Gly Gly Ser Ser Met Gly Ile15 20 25ATT CGC ACA TGT AGG CTC GGC CCT GAC CAA GTC AAA TCC ATG CGG GCT 1576Ile Arg Thr Cys Arg Leu Gly Pro Asp Gln Val Lys Ser Met Arg Ala30 35 40GCT CTT GAT CTT TTC GGT CGT GAG TTC GGA GAC GTA GCC ACC TAC TCC 1624Ala Leu Asp Leu Phe Gly Arg Glu Phe Gly Asp Val Ala Thr Tyr Ser45 50 55CAA CAT CAG CCG GAC TCC GAT TAC CTC GGG AAC TTG CTC CGT AGT AAG 1672Gln His Gln Pro Asp Ser Asp Tyr Leu Gly Asn Leu Leu Arg Ser Lys60 65 70ACA TTC ATC GCG CTT GCT GCC TTC GAC CAA GAA GCG GTT GTT GGC GCT 1720Thr Phe Ile Ala Leu Ala Ala Phe Asp Gln Glu Ala Val Val Gly Ala75 80 85 90CTC GCG GCT TAC GTT CTG CCC AGG TTT GAG CAG CCG CGT AGT GAG ATC 1768Leu Ala Ala Tyr Val Leu Pro Arg Phe Glu Gln Pro Arg Ser Glu Ile95 100105TAT ATC TAT GAT CTC GCA GTC TCC GGC GAG CAC CGG AGG CAG GGC ATT 1816Tyr Ile Tyr Asp Leu Ala Val Ser Gly Glu His Arg Arg Gln Gly Ile110 115 120GCC ACC GCG CTC ATC AAT CTC CTC AAG CAT GAG GCC AAC GCG CTT GGT 1864Ala Thr Ala Leu Ile Asn Leu Leu Lys His Glu Ala Asn Ala Leu Gly125 130 135GCT TAT GTG ATC TAC GTG CAA GCA GAT TAC GGT GAC GAT CCC GCA GTG 1912Ala Tyr Val Ile Tyr Val Gln Ala Asp Tyr Gly Asp Asp Pro Ala Val140 145 150GCT CTC TAT ACA AAG TTG GGC ATA CGG GAA GAA GTG ATG CAC TTT GAT 1960Ala Leu Tyr Thr Lys Leu Gly Ile Arg Glu Glu Val Met His Phe Asp155 160 165 170ATC GAC CCA AGT ACC GCC ACC TAA CAATTCGTTC AAGCCGAGAT CGGCTTCCCA 2014Ile Asp Pro Ser Thr Ala Thr175 177A TTG GCC CAG CGC GTC GAT TCG GGC ATT TGC CAT ATC AAT GGA CCG ACT 2063Leu Ala Gln Arg Val Asp Ser Gly Ile Cys His Ile Asn Gly Pro Thr420 425 430 435GTG CAT GAC GAG GCT CAG ATG CCA TTC GGT GGG GTG AAG TCC AGC GGC 2111Val His Asp Glu Ala Gln Met Pro Phe Gly Gly Val Lys Ser Ser Gly440 445 450TAC GGC AGC TTC GGC AGT CGA GCA TCG ATT GAG CAC TTT ACC CAG CTG 2159Tyr Gly Ser Phe Gly Ser Arg Ala Ser Ile Glu His Phe Thr Gln Leu455 460 465CGC TGG CTG ACC ATT CAG AAT GGC CCG CGG CAC TAT CCA ATC TAA 2204Arg Trp Leu Thr Ile Gln Asn Gly Pro Arg His Tyr Pro Ile470 475 480 481ATCGATCTTC GGGCGCCGCG GGCATCATGC CCGCGGCGCT CGCCTCATTT CAATCTCTAA 2264CTTGATAAAA ACAGAGCTGT TCTCCGGTCT TGGTGGATCA AGGCCAGTCG CGGAGAGTCT 2324CGAAGAGGAG AGTACAGTGA ACGCCGAGTC CACATTGCAA CCGCAGGCAT CATCATGCTC 2384TGCTCAGCCA CGCTACCGCA GTGTGTCGAT TGGTCATCCT CCGGTTGAGG TTACGCAAGA 2444CGCTGGAGGT ATTGTCCGGA TGCGTTCTCT CGAGGCGCTT CTTCCCTTCC CGGGTGGAAT 2504TC2506圖2nGAATTCCAAT AATGACAATA ATGAGGAGTG CCCA ATG TTT CAC GTG CCC CTG CTT 55Met Phe His Val Pro Leu Leu1 5ATT GGT GGT AAG CCT TGT TCA GCA TCT GAT GAG CGC ACC TTC GAG CGT 103Ile Gly Gly Lys Pro Cys Ser Ala Ser Asp Glu Arg Thr Phe Glu Arg10 15 20CGT AGC CCG CTG ACC GGA GAA GTG GTA TCG CGC GTC GCT GCT GCC AGT 151Arg Ser Pro Leu Thr Gly Glu Val Val Ser Arg Val Ala Ala Ala Ser25 30 35TTG GAA GAT GCG GAC GCC GCA GTG GCC GCT GCA CAG GCT GCG TTT CCT 199Leu Glu Asp Ala Asp Ala Ala Val Ala Ala Ala Gln Ala Ala Phe Pro40 45 50 55GAA TGG GCG GCG CTT GCT CCG AGC GAA CGC CGT GCC CGA CTG CTG CGA 247Glu Trp Ala Ala Leu Ala Pro Ser Glu Arg Arg Ala Arg Leu Leu Arg60 65 70GCG GCG GAT CTT CTA GAG GAC CGT TCT TCC GAG TTC ACC GCC GCA GCG 295Ala Ala Asp Leu Leu Glu Asp Arg Ser Ser Glu Phe Thr Ala Ala Ala75 80 85AGT GAA ACT GGC GCA GCG GGA AAC TGG TAT GGG TTT AAC GTT TAC CTG 343Ser Glu Thr Gly Ala Ala Gly Asn Trp Tyr Gly Phe Asn Val Tyr Leu90 95 100GCG GCG GGC ATG TTG CGG GAA GCC GCG GCC ATG ACC ACA CAG ATT CAG 391Ala Ala Gly Met Leu Arg Glu Ala Ala Ala Met Thr Thr Gln Ile Gln105 110 115GGC GAT GTC ATT CCG TCC AAT GTG CCC GGT AGC TTT GCC ATG GCG GTT 439Gly Asp Val Ile Pro Ser Asn Val Pro Gly Ser Phe Ala Met Ala Val120 125 130 135CGA CAG CCA TGT GGC GTG GTG CTC GGT ATT GCG CCT TGG AAT GCT CCG 487Arg Gln Pro Cys Gly Val Val Leu Gly Ile Ala Pro Trp Asn Ala Pro140 145 150GTA ATC CTT GGC GTA CGG GCT GTT GCG ATG CCG TTG GCA TGC GGC AAT 535Val Ile Leu Gly Val Arg Ala Val Ala Met Pro Leu Ala Cys Gly Ash155 160 165ACC GTG GTG TTG AAA AGC TCT GAG CTG AGT CCC TTT ACC CAT CGC CTG 583Thr Val Val Leu Lys Ser Ser Glu Leu Ser Pro Phe Thr His Arg Leu170 175 180ATT GGT CAG GTG TTG CAT GAT GCT GGT CTG GGG GAT GGC GTG GTG AAT 631Ile Gly Gln Val Leu His Asp Ala Gly Leu Gly Asp Gly Val Val Asn185 190 195GTC ATC AGC AAT GCC CCG CAA GAC GCT CCT GCG GTG GTG GAG CGA CTG 679Val Ile Ser Asn Ala Pro Gln Asp Ala Pro Ala Val Val Glu Arg Leu200 205 210 215ATT GCA AAT CCT GCG GTA CGT CGA GTG AAC TTC ACC GGT TCG ACC CAC 727Ile Ala Asn Pro Ala Val Arg Arg val Asn Phe Thr Gly Ser Thr His220 225 230GTT GGA CGG ATC ATT GGT GAG CTG TCT GCG CGT CAT CTG AAG CCT GCT 775Val Gly Arg Ile Ile Gly Glu Leu Ser Ala Arg His Leu Lys Pro Ala235 240 245GTG CTG GAA TTA GGT GGT AAG GCT CCG TTC TTG GTC TTG GAC GAT GCC 823Val Leu Glu Leu Gly Gly Lys Ala Pro Phe Leu Val Leu Asp Asp Ala250 255 260GAC CTC GAT GCG GCG GTC GAA GCG GCG GCC TTT GGT GCC TAC TTC AAT 871Asp Leu Asp Ala Ala Val Glu Ala Ala Ala Phe Gly Ala Tyr Phe Asn265 270 275CAG GGT CAA ATC TGC ATG TCC ACT GAG CGT CTG ATT GTG ACA GCA GTC 919Gln Gly Gln Ile Cys Met Ser Thr Glu Arg Leu Ile Val Thr Ala Val280 285 290 295GCA GAC GCC TTT GTT GAA AAG CTG GCG AGG AAG GTC GCC ACA CTG CGT 967Ala Asp Ala Phe Val Glu Lys Leu Ala Arg Lys Val Ala Thr Leu Arg300 305 310GCT GGC GAT CCT AAT GAT CCG CAA TCG GTC TTG GGT TCG TTG ATT GAT 1015Ala Gly Asp Pro Asn Asp Pro Gln Ser Val Leu Gly Ser Leu Ile Asp315 320 325GCC AAT GCA GGT CAA CGC ATC CAG GTT CTG GTC GAT GAT GCG CTC GCA 1063Ala Asn Ala Gly Gln ArG Ile Gln Val Leu Val Asp Asp Ala Leu Ala330 335 340AAA GGC GCG CAATGGAA TTG GCC CAG CGC GTC GAT TCG GGC ATT TGC CAT 1113Lys Gly Ala Leu Ala Gln Arg Val Asp Ser Gly Ile Cys His345 346 420 425 430ATC AAT GGA CCG ACT GTG CAT GAC GAG GCT CAG ATG CCA TTC GGT GGG 1161Ile Asn Gly Pro Thr Val His Asp Glu Ala Gln Met Pro Phe Gly Gly435 440 445GTG AAG TCC AGC GGC TAC GGC AGC TTC GGC AGT CGA GCA TCG ATT GAG 1209Val Lys Ser Ser Gly Tyr Gly Ser Phe Gly Ser Arg Ala Ser Ile Glu450 455 460CAC TTT ACC CAG CTG CGC TGG CTG ACC ATT CAG AAT GGC CCG CGG CAC 1257His Phe Thr Gln Leu Arg Trp Leu Thr Ile Gln Asn Gly Pro Arg His465 470 475TAT CCA ATC TAAATCGATCTTC GGGCGCCGCG GGCATCATGC CCGCGGCGCT 1309Tyr Pro Ile480 481CGCCTCATTT CAATCTCTAA CTTGATAAAA ACAGAGCTGT TCTCCGGTCT TGGTGGATCA1369AGGCCAGTCG CGGAGAGTCT CGAAGAGGAG AGTACAGTGA ACGCCGAGTC CACATTGCAA1429CCGCAGGCAT CATCATGCTC TGCTCAGCCA CGCTACCGCA GTGTGTCGAT TGGTCATCCT 1489CCGGTTGAGG TTACGCAAGA CGCTGGAGGT ATTGTCCGGA TGCGTTCTCT CGAGGCGCTT 1549CTTCCCTTCC CGGGTGGAAT TC 1571圖2oGAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60GTGACGAGGC CACACT GTG AGT TGG TCA GGG GGG GCT TAC TCG GCG TTT TCC 112Met Ser Trp Ser Gly Gly Ala Tyr Ser Ala Phe Ser1 5 10GAC ACT GCG TTG GTT GCG GCA GTG CGC ACC CCC TGG ATT GAT TGC GGG 160Asp Thr Ala Leu Val Ala Ala Val Arg Thr Pro Trp Ile Asp Cys Gly15 20 25GGT GCC CTG TCG CTG GTG TCG CCT ATC GAC TTA GGG GTA AAG GTC GCT 208Gly Ala Leu Ser Leu Val Ser Pro Ile Asp Leu Gly Val Lys Val Ala30 35 40CGC GAA GTT CTG ATG CGT GCG TCG CTT GAA CCA CAA ATG GTC GAT AGC 256Arg Glu Val Leu Met Arg Ala Ser Leu Glu Pro Gln Met Val Asp Ser45 50 55 60GTA CTC GCA GGC TCT ATG GCT CAA GCA AGC TTT GAT GCT TAC CTG CTC 304Val Leu Ala Gly Ser Met Ala Gln Ala Ser Phe Asp Ala Tyr Leu Leu65 70 75CCG CGG CAC ATT GGC TTG TAC AGC GGT GTT CCC AAG TCG GTT CCG GCC 352Pro Arg His Ile Gly Leu Tyr Ser Gly Val Pro Lys Ser Val Pro Ala80 85 90TTG GGG GTG CAG CGC ATT TGC GGC ACA GGC TTC GAA CTG CTT CGG CAG 400Leu Gly Val Gln Arg Ile Cys Gly Thr Gly Phe Glu Leu Leu Arg Gln95 100 105GCC GGC GAG CAG ATT TCC CAA GGC GCT GAT CAC GTG CTG TGT GTC GCG 448Ala Gly Glu Gln Ile Ser Gln Gly Ala Asp His Val Leu Cys Val Ala110 115 120GCA GAG TCC ATG TCG CGT AAC CCC ATC GCG TCG TAT ACA CAC CGG GGC 496Ala Glu Ser Met Ser Arg Asn Pro Ile Ala Ser Tyr Thr His Arg Gly125 130 135 140GGG TTC CGC CTC GGT GCG CCC GTT GAG TTC AAG GAT TTT TTG TGG GAG 544Gly Phe Arg Leu Gly Ala Pro Val Glu Phe Lys Asp Phe Leu Trp Glu145 150 155GCA TTG TTT GAT CCT GCT CCA GGA CTC GAC ATG ATC GCT ACC GCA GAA 592Ala Leu Phe Asp Pro Ala Pro Gly Leu Asp Met Ile Ala Thr Ala Glu160 165 170AAC CTG GGGACAGCAA GCGAACCGGA ATTGCCAGCT GGGGCGCCCT CTGGTAAGGT648Asn Leu174TGGGAAGCCC TGCAAAGTAA ACTGGATGGC TTTCTTGCCG CCAAGGATCT GATGGCGCAG 708GGGATCAAGA TCTGATCAAG AGACAGGATG AGGATCGTTT CGC ATG ATT GAA CAA 763Met Ile Glu Gln1GAT GGA TTG CAC GCA GGT TCT CCG GCC GCT TGG GTG GAG AGG CTA TTC 811Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val Glu Arg Leu Phe5 10 15 20GGC TAT GAC TGG GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG 859Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser Asp Ala Ala Val25 30 35TTC CGG CTG TCA GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC 907Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe Val Lys Thr Asp40 45 50CTG TCC GGT GCC CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG 955Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala Ala Arg Leu Ser55 60 65TGG CTG GCC ACG ACG GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC 1003Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val Leu Asp Val Val70 75 80ACT GAA GCG GGA AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG 1051Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu Val Pro Gly Gln85 90 95 100GAT CTC CTG TCA TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG 1099Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys Val Ser Ile Met105 110 115GCT GAT GCA ATG CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA 1147Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro Ala Thr Cys Pro120 125 130TTC GAC CAC CAA GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG 1195Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met135 140 145GAA GCC GGT CTT GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG 1243Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu Glu His Gln Gly150 155 160CTC GCG CCA GCC GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC 1291Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala Arg Met Pro Asp165 170 175 180GGC GAG GAT CTC GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC 1339Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys Leu Pro Asn Ile185 190 195ATG GTG GAA AAT GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG 1387Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu200 205 210GGT GTG GCG GAC CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT 1435Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala Thr Arg Asp Ile215 220 225GCT GAA GAG CTT GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC 1483Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe Leu Val Leu Tyr230 235 240GGT ATC GCC GCT CCC GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT 1531Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe Tyr Arg Leu Leu245 250 255 260GAC GAG TTC TTC TGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC 1586Asp Glu phe Phe264CA TTG AGG GCG CAA GAG GAG AAA TGG ATT GAC CAA GAG ATC GTG GCT1633Leu Arg Ala Gln Glu Glu Lys Trp Ile Asp Gln Glu Ile Val Ala197 200 205 210GTT ACG GAT GAA CAG TTC GAT TTA GAG GGC TACAAC AGT CGA GCA ATT1681Val Thr Asp Glu Gln Phe Asp Leu Glu Gly Tyr Asn Ser Arg Ala Ile215 220 225GAA CTG CCT CGG AAG GCA AAA TTG TTG ATC GTG ACA GTC ATC CGC GGC 1729Glu Leu Pro Arg Lys Ala Lys Leu Leu Ile Val Thr Val Ile Arg Gly230 235 240CTA GCA GTC TTT GAA GCC CTT TCC CGA TTG AAG CCT GTT CAT TCT GGC 1777Leu Ala Val Phe Glu Ala Leu Ser Arg Leu Lys Pro Val His Ser Gly245 250 255GGG GTG CAG ACT GCG GGC AAC AGC TGT GCC GTA GTG GAC GGC GCC GCG 1825Gly Val Gln Thr Ala Gly Asn Ser Cys Ala Val Val Asp Gly Ala Ala260 265 270 275GCG GCT TTG GTG GCT CGA GAG TCG TCT GCG ACA CAG CCG GTC TTG GCT 1873Ala Ala Leu Val Ala Arg Glu Ser Ser Ala Thr Gln Pro Val Leu Ala280 285 290AGG ATA CTG GCT ACC TCC GTA GTC GGG ATC GAG CCC GAG CAT ATG GGG 1921Arg Ile Leu Ala Thr Ser Val Val Gly Ile Glu Pro Glu His Met Gly295 300 305CTC GGC CCT GCG CCC GCG ATT CGC CTG CTG CTT GCG CGT AGT GAT CTT 1969Leu Gly Pro Ala Pro Ala Ile Arg Leu Leu Leu Ala Arg Ser Asp Leu310 315 320AGT TTG AGG GAT ATC GAC CTC TTT GAG ATA AAC GAG GCG CAG GCC GCC 2017Ser Leu Arg Asp Ile Asp Leu Phe Glu Ile Asn Glu Ala Gln Ala Ala325 330 335CAA GTT CTA GCG GTA CAG CAT GAA TTG GGT ATT GAG CAC TCA AAA CTT 2065Gln Val Leu Ala Val Gln His Glu Leu Gly Ile Glu His Ser Lys Leu340 345 350 355AAT ATT TGG GGC GGG GCC ATT GCA CTT GGA CAC CCG CTT GCC GCG ACC 2113Asn Ile Trp Gly Gly Ala Ile Ala Leu Gly His Pro Leu Ala Ala Thr360 365 370GGA TTG CGT CTC TGC ATG ACC CTC GCT CAC CAA TTG CAA GCT AAT AAC 2161Gly Leu Arg Leu Cys Met Thr Leu Ala His Gln Leu Gln Ala Asn Asn375 380 385TTT CGA TAT GGA ATT GCC TCG GCA TGC ATT GGT GGG GGA CAG GGG ATG 2209Phe Arg Tyr Gly Ile Ala Ser Ala Cys Ile Gly Gly Gly Gln Gly Met390 395 400GCG GTT CTT TTA GAG AAT CCC CAC TTC GGT TCG TCC TCT GCA CGA AGT 2257Ala Val Leu Leu Glu Asn Pro His Phe Gly Ser Ser Ser Ala Arg Ser405 410 415TCG ATG ATT AAC AGA GTT GAC CAC TAT CCA CTG AGC TAA CGGGCATCTC2306Ser Met Ile Asn Arg Val Asp His Tyr Pro Leu Ser420 425 430 431CTTTGTTGCT TTGAGGTGGC GCACGAAGGA GGGCTCGAAA ATCTCTGCTA AAAACAAGAA2366GAAGGAACAG GGAACATGAT TAGTTTCGCT CGTATGGCAG AAAGTTTAGG AGTCCAGGCT2426AAACTTGCCC TTGCCTTCGC ACTCGTATTA TGTGTCGGGC TGATTGTTAC CGGCACGGGT2486TTCTACAGTG TACATACCTT GTCAGGGTTG GTGGGAATTC 2526圖2pGAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60GTGACGAGGC CACACT GTG AGT TGG TCA GGG GGG GCT TAC TCG GCG TTT TCC 112Met Ser Trp Ser Gly Gly Ala Tyr Ser Ala Phe Ser1 5 10GAC ACT GCG TTG GTT GCG GCA GTG CGC ACC CCC TGG ATT GAT TGC GGG 160Asp Thr Ala Leu Val Ala Ala Val Arg Thr Pro Trp Ile Asp Cys Gly15 20 25GGT GCC CTG TCG CTG GTG TCG CCT ATC GAC TTA GGG GTA AAG GTC GCT 208Gly Ala Leu Ser Leu Val Ser Pro Ile Asp Leu Gly VaI Lys Val Ala30 35 40CGC GAA GTT CTG ATG CGT GCG TCG CTT GAA CCA CAA ATG GTC GAT AGC 256Arg Glu Val Leu Met Arg Ala Ser Leu Glu Pro Gln Met Val Asp Ser45 50 55 60GTA CTC GCA GGC TCT ATG GCT CAA GCA AGC TTT GAT GCT TAC CTG CTC 304Val Leu Ala Gly Ser Met Ala Gln Ala Ser Phe Asp Ala Tyr Leu Leu65 70 75CCG CGG CAC ATT GGC TTG TAC AGC GGT GTT CCC AAG TCG GTT CCG GCC 352Pro Arg His Ile Gly Leu Tyr Ser Gly Val Pro Lys Ser Val Pro Ala80 85 90TTG GGG GTG CAG CGC ATT TGC GGC ACA GGC TTC GAA CTG CTT CGG CAG 400Leu Gly Val Gln Arg Ile Cys Gly Thr Gly Phe Glu Leu Leu Arg Gln95 100 105GCC GGC GAG CAG ATT TCC CAA GGC GCT GAT CAC GTG CTG TGT GTC GCG 448Ala Gly Glu Gln Ile Ser Gln Gly Ala Asp His Val Leu Cys Val Ala110 115 120GCA GAG TCC ATG TCG CGT AAC CCC ATC GCG TCG TAT ACA CAC CGG GGC 496Ala Glu Ser Met Ser Arg Asn Pro Ile Ala Ser Tyr Thr His Arg Gly125 130 135 140GGG TTC CGC CTC GGT GCG CCC GTT GAG TTC AAG GAT TTT TTG TGG GAG 544Gly Phe Arg Leu Gly Ala Pro Val Glu Phe Lys Asp Phe Leu Trp Glu145 150 155GCA TTG TTT GAT CCT GCT CCA GGA CTC GAC ATG ATC GCT ACC GCA GAA 592Ala Leu Phe Asp Pro Ala Pro Gly Leu Asp Met Ile Ala Thr Ala Glu160 165 170AAC CTG GGGGAGAGGC GGTTTGCGTA TTGGGCGCAT GCATAAAAAC TGTTGTAATT648Asn Leu174CATTAAGCAT TCTGCCGACA TGGAAGCCAT CACAAACGGC ATGATGAACC TGAATCGCCA 708GCGGCATCAG CACCTTGTCG CCTTGCGTAT AATATTTGCC CATGGACGCA CACCGTGGAA 768ACGGATGAAG GCACGAACCC AGTTGACATA AGCCTGTTCG GTTCGTAAAC TGTAATGCAA 828GTAGCGTATG CGCTCACGCA ACTGGTCCAG AACCTTGACC GAACGCAGCG GTGGTAACGG 888CGCAGTGGCG GTTTTCATGG CTTGTTATGA CTGTTTTTTT GTACAGTCTA TGCCTCGGGC 948ATCCAAGC AGCAAGCGCG TTACGCCGTG GGTCGATGTTTG ATGTTATGGA GCAGCAACG 1007ATG TTA CGC AGC AGC AAC GAT GTT ACG CAG CAG GGC AGT CGC CCT AAA 1055Met Leu Arg Ser Ser Asn Asp Val Thr Gln Gln Gly Ser Arg Pro Lys1 5 10 15ACA AAG TTA GGT GGC TCA AGT ATG GGC ATC ATT CGC ACA TGT AGG CTC 1103Thr Lys Leu Gly Gly Ser Ser Met Gly Ile Ile Arg Thr Cys Arg Leu20 25 30GGC CCT GAC CAA GTC AAA TCC ATG CGG GCT GCT CTT GAT CTT TTC GGT 1151Gly Pro Asp Gln Val Lys Ser Met Arg Ala Ala Leu Asp Leu Phe Gly35 40 45CGT GAG TTC GGA GAC GTA GCC ACC TAC TCC CAA CAT CAG CCG GAC TCC 1199Arg Glu Phe Gly Asp Val Ala Thr Tyr Ser Gln His Gln Pro Asp Ser50 55 60GAT TAC CTC GGG AAC TTG CTC CGT AGT AAG ACA TTC ATC GCG CTT GCT 1247Asp Tyr Leu Gly Asn Leu Leu Arg Ser Lys Thr Phe Ile Ala Leu Ala65 70 75 80GCC TTC GAC CAA GAA GCG GTT GTT GGC GCT CTC GCG GCT TAC GTT CTG 1295Ala Phe Asp Gln Glu Ala Val Val Gly Ala Leu Ala Ala Tyr Val Leu85 90 95CCC AGG TTT GAG CAG CCG CGT AGT GAG ATC TAT ATC TAT GAT CTC GCA 1343Pro Arg Phe Glu Gln Pro Arg Ser Glu Ile Tyr Ile Tyr Asp Leu Ala100 105 110GTC TCC GGC GAG CAC CGG AGG CAG GGC ATT GCC ACC GCG CTC ATC AAT 1391Val Ser Gly Glu His Arg Arg Gln Gly Ile Ala Thr Ala Leu Ile Asn115 120 125CTC CTC AAG CAT GAG GCC AAC GCG CTT GGT GCT TAT GTG ATC TAC GTG 1439Leu Leu Lys His Glu Ala Asn Ala Leu Gly Ala Tyr Val Ile Tyr Val130 135 140CAA GCA GAT TAC GGT GAC GAT CCC GCA GTG GCT CTC TAT ACA AAG TTG 1487Gln Ala Asp Tyr Gly Asp Asp Pro Ala Val Ala Leu Tyr Thr Lys Leu145 150 155 160GGC ATA CGG GAA GAA GTG ATG CAC TTT GAT ATC GAC CCA AGT ACC GCC 1535Gly Ile Arg Glu Glu Val Met His Phe Asp Ile Asp Pro Ser Thr Ala165 170 175ACC TAA CAATTCGTTC AAGCCGAGAT CGGCTTCCCA TTG AGG GCG CAA GAG GAG 1589Thr Leu Arg Ala Gln Glu Glu177 197 200AAA TGG ATT GAC CAA GAG ATC GTG GCT GTT ACG GAT GAA CAG TTC GAT 1637Lys Trp Ile Asp Gln Glu Ile Val Ala Val Thr Asp Glu Gln Phe Asp205 210 215TTA GAG GGC TAC AAC AGT CGA GCA ATT GAA CTG CCT CGG AAG GCA AAA 1685Leu Glu Gly Tyr Asn Ser Arg Ala Ile Glu Leu Pro Arg Lys Ala Lys220 225 230TTG TTG ATC GTG ACA GTC ATC CGC GGC CTA GCA GTC TTT GAA GCC CTT 1733Leu Leu Ile Val Thr Val Ile Arg Gly Leu Ala Val Phe Glu Ala Leu235 240 245 250TCC CGA TTG AAG CCT GTT CAT TCT GGC GGG GTG CAG ACT GCG GGC AAC 1781Ser Arg Leu Lys Pro Val His Ser Gly Gly Val Gln Thr Ala Gly Asn255 260 265AGC TGT GCC GTA GTG GAC GGC GCC GCG GCG GCT TTG GTG GCT CGA GAG 1829Ser Cys Ala Val Val Asp Gly Ala Ala Ala Ala Leu Val Ala Arg Glu270 275 280TCG TCT GCG ACA CAG CCG GTC TTG GCT AGG ATA CTG GCT ACC TCC GTA 1877Ser Ser Ala Thr Gln Pro Val Leu Ala Arg Ile Leu Ala Thr Ser Val285 290 295GTC GGG ATC GAG CCC GAG CAT ATG GGG CTC GGC CCT GCG CCC GCG ATT 1925Val Gly Ile Glu Pro Glu His Met Gly Leu Gly Pro Ala Pro Ala Ile300 305 310CGC CTG CTG CTT GCG CGT AGT GAT CTT AGT TTG AGG GAT ATC GAC CTC 1973Arg Leu Leu Leu Ala Arg Ser Asp Leu Ser Leu Arg Asp Ile Asp Leu315 320 325 330TTT GAG ATA AAC GAG GCG CAG GCC GCC CAA GTT CTA GCG GTA CAG CAT 2021Phe Glu Ile Asn Glu Ala Gln Ala Ala Gln Val Leu Ala Val Gln His335 340 345GAA TTG GGT ATT GAG CAC TCA AAA CTT AAT ATT TGG GGC GGG GCC ATT 2069Glu Leu Gly Ile Glu His Ser Lys Leu Asn Ile Trp Gly Gly Ala Ile350 355 360GCA CTT GGA CAC CCG CTT GCC GCG ACC GGA TTG CGT CTC TGC ATG ACC 2117Ala Leu Gly His Pro Leu Ala Ala Thr Gly Leu Arg Leu Cys Met Thr365 370 375CTC GCT CAC CAA TTG CAA GCT AAT AAC TTT CGA TAT GGA ATT GCC TCG 2165Leu Ala His Gln Leu Gln Ala Asn Asn Phe Arg Tyr Gly Ile Ala Ser380 385 390GCA TGC ATT GGT GGG GGA CAG GGG ATG GCG GTT CTT TTA GAG AAT CCC 2213Ala Cys Ile Gly Gly Gly Gln Gly Met Ala Val Leu Leu Glu Asn Pro395 400 405 410CAC TTC GGT TCG TCC TCT GCA CGA AGT TCG ATG ATT AAC AGA GTT GAC 2261His Phe Gly Ser Ser Ser Ala Arg Ser Ser Met Ile Asn ArG Val Asp415 420 425CAC TAT CCA CTG AGC TAA CGGGCATCTC CTTTGTTGCT TTGAGGTGGC 2309His Tyr Pro Leu Ser430 431GCACGAAGGA GGGCTCGAAA ATCTCTGCTA AAAACAAGAA GAAGGAACAG GGAACATGAT 2369TAGTTTCGCT CGTATGGCAG AAAGTTTAGG AGTCCAGGCT AAACTTGCCC TTGCCTTCGC 2429ACTCGTATTA TGTGTCGGGC TGATTGTTAC CGGCACGGGT TTCTACAGTG TACATACCTT 2489GTCAGGGTTG GTGGGAATTC 2509圖2qGAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60GTGACGAGGC CACACT GTG AGT TGG TCA GGG GGG GCT TAC TCG GCG TTT TCC 112Met Ser Trp Ser Gly Gly Ala Tyr Ser Ala Phe Ser15 10GAC ACT GCG TTG GTT GCG GCA GTG CGC ACC CCC TGG ATT GAT TGC GGG160Asp Thr Ala Leu Val Ala Ala Val Arg Thr Pro Trp Ile Asp Cys Gly15 20 25GGT GCC CTG TCG CTG GTG TCG CCT ATC GAC TTA GGG GTA AAG GTC GCT208Gly Ala Leu Ser Leu Val Ser Pro Ile Asp Leu Gly Val Lys Val Ala30 35 40CGC GAA GTT CTG ATG CGT GCG TCG CTT GAA CCA CAA ATG GTC GAT AGC256Arg Glu Val Leu Met Arg Ala Ser Leu Glu Pro Gln Met Val Asp Ser45 50 55 60GTA CTC GCA GGC TCT ATG GCT CAA GCA AGC TTT GAT GCT TAC CTG CTC304Val Leu Ala Gly Ser Met Ala Gln Ala Ser Phe Asp Ala Tyr Leu Leu65 70 75CCG CGG CAC ATT GGC TTG TAC AGC GGT GTT CCC AAG TCG GTT CCG GCC352Pro Arg His Ile Gly Leu Tyr Ser Gly Val Pro Lys Ser Val Pro Ala80 85 90TTG GGG GTG CAG CGC ATT TGC GGC ACA GGC TTC GAA CTG CTT CGG CAG400Leu Gly Val Gln Arg Ile Cys Gly Thr Gly Phe Glu Leu Leu Arg Gln95 100 105GCC GGC GAG CAG ATT TCC CAA GGC GCT GAT CAC GTG CTG TGT GTC GCG448Ala Gly Glu Gln Ile Ser Gln Gly Ala Asp His Val Leu Cys Val Ala110 115 120GCA GAG TCC ATG TCG CGT AAC CCC ATC GCG TCG TAT ACA CAC CGG GGC496Ala Glu Ser Met Ser Arg Asn Pro Ile Ala Ser Tyr Thr His Arg Gly125 130 135 140GGG TTC CGC CTC GGT GCG CCC GTT GAG TTC AAG GAT TTT TTG TGG GAG544Gly Phe Arg Leu Gly Ala Pro Val Glu Phe Lys Asp Phe Leu Trp Glu145 150 155GCA TTG TTT GAT CCT GCT CCA GGA CTC GAC ATG ATC GCT ACC GCA GAA592Ala Leu Phe Asp Pro Ala Pro Gly Leu Asp Met Ile Ala Thr Aia Glu160 165 170AAC CTG GCG CGC A TTG AGG GCG CAA GAG GAG AAA TGG ATT GAC CAA GAG 641Asn Leu Ala Arg Leu Arg Ala Gln Glu Glu Lys Trp Ile Asp Gln Glu175 176 197 200 205ATC GTG GCT GTT ACG GAT GAA CAG TTC GAT TTA GAG GGC TAC AAC AGT689Ile Val Ala Val Thr Asp Glu Gln Phe Asp Leu Glu Gly Tyr Asn Ser210 215 220CGA GCA ATT GAA CTG CCT CGG AAG GCA AAA TTG TTG ATC GTG ACA GTC737Arg Ala Ile Glu Leu Pro Arg Lys Ala Lys Leu Leu Ile Val Thr Val225 230 235 240ATC CGC GGC CTA GCA GTC TTT GAA GCC CTT TCC CGA TTG AAG CCT GTT 785Ile Arg Gly Leu Ala Val Phe Glu Ala Leu Ser Arg Leu Lys Pro Val245 250 255CAT TCT GGC GGG GTG CAG ACT GCG GGC AAC AGC TGT GCC GTA GTG GAC 833His Ser Gly Gly Val Gln Thr Ala Gly Asn Ser Cys Ala Val Val Asp260 265 270GGC GCC GCG GCG GCT TTG GTG GCT CGA GAG TCG TCT GCG ACA CAG CCG 881Gly Ala Ala Ala Ala Leu Val Ala Arg Glu Ser Ser Ala Thr Gln Pro275 280 285GTC TTG GCT AGG ATA CTG GCT ACC TCC GTA GTC GGG ATC GAG CCC GAG 929Val Leu Ala Arg Ile Leu Ala Thr Ser Val Val Gly Ile Glu Pro Glu290 295 300CAT ATG GGG CTC GGC CCT GCG CCC GCG ATT CGC CTG CTG CTT GCG CGT 977His Met Gly Leu Gly Pro Ala Pro Ala Ile Arg Leu Leu Leu Ala Arg305 310 315 320AGT GAT CTT AGT TTG AGG GAT ATC GAC CTC TTT GAG ATA AAC GAG GCG 1025Ser Asp Leu Ser Leu Arg Asp Ile Asp Leu Phe Glu Ile Asn Glu Ala325 330 335CAG GCC GCC CAA GTT CTA GCG GTA CAG CAT GAA TTG GGT ATT GAG CAC 1073Gln Ala Ala Gln Val Leu Ala Val Gln His Glu Leu Gly Ile Glu His340 345 350TCA AAA CTT AAT ATT TGG GGC GGG GCC ATT GCA CTT GGA CAC CCG CTT 1121Ser Lys Leu Asn Ile Trp Gly Gly Ala Ile Ala Leu Gly His Pro Leu355 360 365GCC GCG ACC GGA TTG CGT CTC TGC ATG ACC CTC GCT CAC CAA TTG CAA 1169Ala Ala Thr Gly Leu Arg Leu Cys Met Thr Leu Ala His Gln Leu Gln370 375 380GCT AAT AAC TTT CGA TAT GGA ATT GCC TCG GCA TGC ATT GGT GGG GGA 1217Ala Asn Asn Phe Arg Tyr Gly Ile Ala Ser Ala Cys Ile Gly Gly Gly385 390 395 400CAG GGG ATG GCG GTT CTT TTA GAG AAT CCC CAC TTC GGT TCG TCC TCT 1265Gln Gly Met Ala Val Leu Leu Glu Asn Pro His Phe Gly Ser Ser Ser405 410 415GCA CGA AGT TCG ATG ATT AAC AGA GTT GAC CAC TAT CCA CTG AGC TAA 1313Ala Arg Ser Ser Met Ile Asn Arg Val Asp His Tyr Pro Leu Ser420 425 430 431CGGGCATCTC CTTTGTTGCT TTGAGGTGGC GCACGAAGGA GGGCTCGAAA ATCTCTGCTA1373AAAACAAGAA GAAGGAACAG GGAACATGAT TAGTTTCGCT CGTATGGCAG AAAGTTTAGG1433AGTCCAGGCT AAACTTGCCC TTGCCTTCGC ACTCGTATTA TGTGTCGGGC TGATTGTTAC1493CGGCACGGGT TTCTACAGTG TACATACCTT GTCAGGGTTG GTGGGAATTC 1543圖2r
序列1CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420AAAGAGCATG CAACTGACCA ACAAGAAAAT CGTCGTCACC GGAGTGTCCT CCGGTATCGG 480TGCCGAAACT GCCCGCGTTC TGCGCTCTCA CGGCGCCACA GTGATTGGCG TAGATCGCAA 540CATGCCGAGC CTGACTCTGG ATGCTTTCGT TCAGGCTGAC CTGAGCCATC CTGAAGGCAT 600CGATAAGGCC ATCGGGACAG CAAGCGAACC GGAATTGCCA GCTGGGGCGC CCTCTGGTAA 660GGTTGGGAAG CCCTGCAAAG TAAACTGGAT GGCTTTCTTG CCGCCAAGGA TCTGATGGCG 720CAGGGGATCA AGATCTGATC AAGAGACAGG ATGAGGATCG TTTCGCATGA TTGAACAAGA 780TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC 840ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC 900GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT GAACTGCAGG ACGAGGCAGC 960GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC1020TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC1080TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC1140GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG AGCGAGCACG1200TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT1260CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGCGCATG CCCGACGGCG AGGATCTCGT1320CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG1380ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC1440CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG TGCTTTACGG1500TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG1560AGCGGGACTC TGGGGTTCGA AATGACCGAC CAAGCGACGC CCTGGCCGCG GTGATTGCAT1620TCATGTGTGC TGAGGAGTCA CGTTGGATCA ACGGCATAAA TATTCCAGTG GACGGAGGTT1680TGGCATCGAC CTACGTGTAA GTTCGTGGAC GCCCTTTGCA CGCGCACTAT ATCTCTATGC1740AGCAGCTGAA AGCAGCTTTG GTTTTGATCG GAGGTAGCGG GCGGAAAGGT GCAGAATGTC1800TAAATAATAA AGGATTCTTG TGAAGCTTTA GTTGTCCGTA AACGAAAATA AAAATAAAGA1860GGAATGATAT GAAAGCAAGT AGATCAGTCT GCACTTTCAA AATAGCTACC CTGGCAGGCG1920CCATTTATGC AGCGCTGCCA ATGTCAGCTG CAAACTCGAT GCAGCTGGAT GTAGGTAGCT1980CGGATTGGAC GGTGCGTTGG GGACAACACC CTCAAGTATA GCCTTGCCTC TCGCCTGAAT2040GAGCAAGACT CAAGTCTGAC AAATGCGCCG ACTGTCAATG GTTATATCCG GATATTCAAA2100GTCAGGGTGA TCGTAACTTT GACCGGGGGC TTGGTATCCA ATCGTCTCGA TATTCTGGCT2160GCAG 2164
序列2CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420AAAGAGCATG CAACTGACCA ACAAGAAAAT CGTCGTCACC GGAGTGTCCT CCGGTATCGG 480TGCCGAAACT GCCCGCGTTC TGCGCTCTCA CGGCGCCACA GTGATTGGCG TAGATCGCAA 540CATGCCGAGC CTGACTCTGG ATGCTTTCGT TCAGGCTGAC CTGAGCCATC CTGAGGGGAG 600AGGCGGTTTG CGTATTGGGC GCATGCATAA AAACTGTTGT AATTCATTAA GCATTCTGCC 660GACATGGAAG CCATCACAAA CGGCATGATG AACCTGAATC GCCAGCGGCA TCAGCACCTT 720GTCGCCTTGC GTATAATATT TGCCCATGGA CGCACACCGT GGAAACGGAT GAAGGCACGA 780ACCCAGTTGA CATAAGCCTG TTCGGTTCGT AAACTGTAAT GCAAGTAGCG TATGCGCTCA 840CGCAACTGGT CCAGAACCTT GACCGAACGC AGCGGTGGTA ACGGCGCAGT GGCGGTTTTC 900ATGGCTTGTT ATGACTGTTT TTTTGTACAG TCTATGCCTC GGGCATCCAA GCAGCAAGCG 960CGTTACGCCG TGGGTCGATG TTTGATGTTA TGGAGCAGCA ACGATGTTAC GCAGCAGCAA 1020CGATGTTACG CAGCAGGGCA GTCGCCCTAA AACAAAGTTA GGTGGCTCAA GTATGGGCAT 1080CATTCGCACA TGTAGGCTCG GCCCTGACCA AGTCAAATCC ATGCGGGCTG CTCTTGATCT 1140TTTCGGTCGT GAGTTCGGAG ACGTAGCCAC CTACTCCCAA CATCAGCCGG ACTCCGATTA 1200CCTCGGGAAC TTGCTCCGTA GTAAGACATT CATCGCGCTT GCTGCCTTCG ACCAAGAAGC 1260GGTTGTTGGC GCTCTCGCGG CTTACGTTCT GCCCAGGTTT GAGCAGCCGC GTAGTGAGAT 1320CTATATCTAT GATCTCGCAG TCTCCGGCGA GCACCGGAGG CAGGGCATTG CCACCGCGCT 1380CATCAATCTC CTCAAGCATG AGGCCAACGC GCTTGGTGCT TATGTGATCT ACGTGCAAGC 1440AGATTACGGT GACGATCCCG CAGTGGCTCT CTATACAAAG TTGGGCATAC GGGAAGAAGT 1500GATGCACTTT GATATCGACC CAAGTACCGC CACCTAACAA TTCGTTCAAG CCGAGATCGG 1560CTTCCCTGAT TGCATTCATG TGTGCTGAGG AGTCACGTTG GATCAACGGC ATAAATATTC 1620CAGTGGACGG AGGTTTGGCA TCGACCTACG TGTAAGTTCG TGGACGCCCT TTGCACGCGC 1680ACTATATCTC TATGCAGCAG CTGAAAGCAG CTTTGGTTTT GATCGGAGGT AGCGGGCGGA 1740AAGGTGCAGA ATGTCTAAAT AATAAAGGAT TCTTGTGAAG CTTTAGTTGT CCGTAAACGA 1800AAATAAAAAT AAAGAGGAAT GATATGAAAG CAAGTAGATC AGTCTGCACT TTCAAAATAG 1860CTACCCTGGC AGGCGCCATT TATGCAGCGC TGCCAATGTC AGCTGCAAAC TCGATGCAGC 1920TGGATGTAGG TAGCTCGGAT TGGACGGTGC GTTGGGGACA ACACCCTCAA GTATAGCCTT 1980GCCTCTCGCC TGAATGAGCA AGACTCAAGT CTGACAAATG CGCCGACTGT CAATGGTTAT 2040ATCCGGATAT TCAAAGTCAG GGTGATCGTA ACTTTGACCG GGGGCTTGGT ATCCAATCGT 2100CTCGATATTC TGGCTGCAG 2119
序列3CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420AAAGAGCATG CAACTGACCA ACAAGAAAAT CGTCGTCACC GGAGTGTCCT CCGGTATCGG 480TGCCGAAACT GCCCGCGTTC TGCGCTCTCA CGGCGCCACA GTGATTGGCG TAGATCGCAA 540CATGCCGAGC CTGACTCTGG ATGCTTTCGT TCAGGCTGAC CTGAGCCATC CTGAAGGCAT 600CGATCAACGG CATAAATATT CCAGTGGACG GAGGTTTGGC ATCGACCTAC GTGTAAGTTC 660GTGGACGCCC TTTGCACGCG CACTATATCT CTATGCAGCA GCTGAAAGCA GCTTTGGTTT 720TGATCGGAGG TAGCGGGCGG AAAGGTGCAG AATGTCTAAA TAATAAAGGA TTCTTGTGAA 780GCTTTAGTTG TCCGTAAACG AAAATAAAAA TAAAGAGGAA TGATATGAAA GCAAGTAGAT 840CAGTCTGCAC TTTCAAAATA GCTACCCTGG CAGGCGCCAT TTATGCAGCG CTGCCAATGT 900CAGCTGCAAA CTCGATGCAG CTGGATGTAG GTAGCTCGGA TTGGACGGTG CGTTGGGGAC 960AACACCCTCA AGTATAGCCT TGCCTCTCGC CTGAATGAGC AAGACTCAAG TCTGACAAAT1020GCGCCGACTG TCAATGGTTA TATCCGGATA TTCAAAGTCA GGGTGATCGT AACTTTGACC1080GGGGGCTTGG TATCCAATCG TCTCGATATT CTGGCTGCAG 1120
序列4GAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420AGAATAACAA TTGACTCCTC AGGAGGTCAG CGATGAGCAT TCTTGGTTTG AATGGTGCCC 480CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540AGGGGCCTGC AAACTTGGAG CTGCGTCTGA GTAGGCTGGA TCGTGCGATT GCAATGCTTC 600TGGAAAATCG TGAAGCAATT GCCGACGCGG TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660AGCAAACACT GCTTTGCGAC ATTGCTGGCT CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720ACGTGGCCAA ATGGATGGAG CCCGAACATC ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840TACTGGCCTT TGGGCCGCTG GCCGGCATAT TCGCAGCAGG TAATCGCGCC ATGCTCAAGC 900CGTCCGAGCT TACCCCGCGG ACTTCTGCCC TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960ATGAAACTGA GCTGACTACA GTGCTGGGCG ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC1020AGCCTTTCGA TCATCTGATC TTCACCGGCG GCACTGCCGT GGCCAAGCAC ATCATGCGTG1080CCGCGGCGGA TAACCTAGTG CCCGTTACCC TGGAATTGGG TGGCAAATCG CCGGTGATCG1140TTTCCCGCAG TGCAGATATG GCGGACGTTG CACAACGGGT GTTGACGGTG AAAACCTTCA1200ATGCCGGGCA AATCTGTCTG GCACCGGACT ATGTGCTGCT GCCGGAAGGG ACAGCAAGCG1260AACCGGAATT GCCAGCTGGG GCGCCCTCTG GTAAGGTTGG GAAGCCCTGC AAAGTAAACT1320GGATGGCTTT CTTGCCGCCA AGGATCTGAT GGCGCAGGGG ATCAAGATCT GATCAAGAGA1380CAGGATGAGG ATCGTTTCGC ATGATTGAAC AAGATGGATT GCACGCAGGT TCTCCGGCCG1440CTTGGGTGGA GAGGCTATTC GGCTATGACT GGGCACAACA GACAATCGGC TGCTCTGATG1500CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG ACCGACCTGT1560CCGGTGCCCT GAATGAACTG CAGGACGAGG CAGCGCGGCT ATCGTGGCTG GCCACGACGG1620GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TCACTGAAGC GGGAAGGGAC TGGCTGCTAT1680TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CATCTCACCT TGCTCCTGCC GAGAAAGTAT1740CCATCATGGC TGATGCAATG CGGCGGCTGC ATACGCTTGA TCCGGCTACC TGCCCATTCG1800ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG GATGGAAGCC GGTCTTGTCG1860ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC AGCCGAACTG TTCGCCAGGC1920TCAAGGCGCG CATGCCCGAC GGCGAGGATC TCGTCGTGAC CCATGGCGAT GCCTGCTTGC1980CGAATATCAT GGTGGAAAAT GGCCGCTTTT CTGGATTCAT CGACTGTGGC CGGCTGGGTG2040TGGCGGACCG CTATCAGGAC ATAGCGTTGG CTACCCGTGA TATTGCTGAA GAGCTTGGCG2100GCGAATGGGC TGACCGCTTC CTCGTGCTTT ACGGTATCGC CGCTCCCGAT TCGCAGCGCA2160TCGCCTTCTA TCGCCTTCTT GACGAGTTCT TCTGAGCGGG ACTCTGGGGT TCGAAATGAC2220CGACCAAGCG ACGCCCGCCA TGCCAAGCCT GTTCTCGTGC AAAGTCCTGT GGGTGAGTCG2280AACTTGGCGA TGCGCGCACC CTACGGAGAA GCGATCCACG GACTGCTCTC TGTCCTCCTT2340TCAACGGAGT GTTAGAACCG TTGGTAGTGG TTTTGGACGG GCCCAGGAGC ATGCGCTTCT2400GGGCCCGTTT CTTGAGTATT CATTGGATAG TCACGCGTGG TAGCTTCGAG CCTGCACAGC2460TGATGAGCAC CCTGGAAGGC GCGCTGTACG CGGACGACTG GGTTCATCTT CGCCATTCAT2520GACGGAACTC CGTTCCCCAG TACCGCGATG ACTATTTTGC CTCTTCCGAT GTCCGATTCC2580ACGCCGCCTG ACGCTAAGCG GGGGCGGGGG CGCCCGCATC CCAGCCCAGA CAGCAACAAA2640TGAGTAGGCT CTTGGATGCC GCGGCGGCTG AGATTGGTAA CGGCAATTTC GTCAATGTGA2700CGATGGATTC GATTGCCCGT GCTGCCGGCG TCTCAAAAAA AACGCTGTAC GTCTTGGTGG2760CGAGCAAGGA AGAACTCATT TCCCGGTTAG TGGCTCGAGA CATGTCCAAC CTTGAGGAAT2820TC 2822
序列5GAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420AGAATAACAA TTGACTCCTC AGGAGGTCAG CGATGAGCAT TCTTGGTTTG AATGGTGCCC 480CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540AGGGGCCTGC AAACTTGGAG CTGCGTCTGA GTAGGCTGGA TCGTGCGATT GCAATGCTTC 600TGGAAAATCG TGAAGCAATT GCCGACGCGG TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660AGCAAACACT GCTTTGCGAC ATTGCTGGCT CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720ACGTGGCCAA ATGGATGGAG CCCGAACATC ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840TACTGGCCTT TGGGCCGCTG GCCGGCATAT TCGCAGCAGG TAATCGCGCC ATGCTCAAGC 900CGTCCGAGCT TACCCCGCGG ACTTCTGCCC TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960ATGAAACTGA GCTGACTACA GTGCTGGGCG ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC1020AGCCTTTCGA TCATCTGATC TTCACCGGCG GCACTGCCGT GGCCAAGCAC ATCATGCGTG1080CCGCGGCGGA TAACCTAGTG CCCGTTACCC TGGAATTGGG TGGCAAATCG CCGGTGATCG1140TTTCCCGCAG TGCAGATATG GCGGACGTTG CACAACGGGT GTTGACGGTG AAAACCTTCA1200ATGCCGGGCA AATCTGTCTG GCACCGGACT ATGTGCTGGG GGAGAGGCGG TTTGCGTATT1260GGGCGCATGC ATAAAAACTG TTGTAATTCA TTAAGCATTC TGCCGACATG GAAGCCATCA1320CAAACGGCAT GATGAACCTG AATCGCCAGC GGCATCAGCA CCTTGTCGCC TTGCGTATAA1380TATTTGCCCA TGGACGCACA CCGTGGAAAC GGATGAAGGC ACGAACCCAG TTGACATAAG1440CCTGTTCGGT TCGTAAACTG TAATGCAAGT AGCGTATGCG CTCACGCAAC TGGTCCAGAA1500CCTTGACCGA ACGCAGCGGT GGTAACGGCG CAGTGGCGGT TTTCATGGCT TGTTATGACT1560GTTTTTTTGT ACAGTCTATG CCTCGGGCAT CCAAGCAGCA AGCGCGTTAC GCCGTGGGTC1620GATGTTTGAT GTTATGGAGC AGCAACGATG TTACGCAGCA GCAACGATGT TACGCAGCAG1680GGCAGTCGCC CTAAAACAAA GTTAGGTGGC TCAAGTATGG GCATCATTCG CACATGTAGG1740CTCGGCCCTG ACCAAGTCAA ATCCATGCGG GCTGCTCTTG ATCTTTTCGG TCGTGAGTTC1800GGAGACGTAG CCACCTACTC CCAACATCAG CCGGACTCCG ATTACCTCGG GAACTTGCTC1860CGTAGTAAGA CATTCATCGC GCTTGCTGCC TTCGACCAAG AAGCGGTTGT TGGCGCTCTC1920GCGGCTTACG TTCTGCCCAG GTTTGAGCAG CCGCGTAGTG AGATCTATAT CTATGATCTC1980GCAGTCTCCG GCGAGCACCG GAGGCAGGGC ATTGCCACCG CGCTCATCAA TCTCCTCAAG2040CATGAGGCCA ACGCGCTTGG TGCTTATGTG ATCTACGTGC AAGCAGATTA CGGTGACGAT2100CCCGCAGTGG CTCTCTATAC AAAGTTGGGC ATACGGGAAG AAGTGATGCA CTTTGATATC2160GACCCAAGTA CCGCCACCTA ACAATTCGTT CAAGCCGAGA TCGGCTTCCC TGCAAAGTCC2220TGTGGGTGAG TCGAACTTGG CGATGCGCGC ACCCTACGGA GAAGCGATCC ACGGACTGCT2280CTCTGTCCTC CTTTCAACGG AGTGTTAGAA CCGTTGGTAG TGGTTTTGGA CGGGCCCAGG2340AGCATGCGCT TCTGGGCCCG TTTCTTGAGT ATTCATTGGA TAGTCACGCG TGGTAGCTTC2400GAGCCTGCAC AGCTGATGAG CACCCTGGAA GGCGCGCTGT ACGCGGACGA CTGGGTTCAT2460CTTCGCCATT CATGACGGAA CTCCGTTCCC CAGTACCGCG ATGACTATTT TGCCTCTTCC2520GATGTCCGAT TCCACGCCGC CTGACGCTAA GCGGGGGCGG GGGCGCCCGC ATCCCAGCCC2580AGACAGCAAC AAATGAGTAG GCTCTTGGAT GCCGCGGCGG CTGAGATTGG TAACGGCAAT2640TTCGTCAATG TGACGATGGA TTCGATTGCC CGTGCTGCCG GCGTCTCAAA AAAAACGCTG2700TACGTCTTGG TGGCGAGCAA GGAAGAACTC ATTTCCCGGT TAGTGGCTCG AGACATGTCC2760AACCTTGAGG AATTC 2775
序列6GAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420AGAATAACAA TTGACTCCTC AGGAGGTCAG CGATGAGCAT TCTTGGTTTG AATGGTGCCC 480CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540AGGGGCCTGC AAACTTGGAG CTGCGTCTGA GTAGGCTGGA TCGTGCGATT GCAATGCTTC 600TGGAAAATCG TGAAGCAATT GCCGACGCGG TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660AGCAAACACT GCTTTGCGAC ATTGCTGGCT CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720ACGTGGCCAA ATGGATGGAG CCCGAACATC ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840TACTGGCCTT TGGGCCGCTG GCCGGCATAT TCGCAGCAGG TAATCGCGCC ATGCTCAAGC 900CGTCCGAGCT TACCCCGCGG ACTTCTGCCC TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960ATGAAACTGA GCTGACTACA GTGCTGGGCG ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC1020AGCCTTTCGA TCATCTGATC TTCACCGGCG GCACTGCCGT GCCCAAGCAC ATCATGCGTG1080CCGCGGCGGA TAACCTAGTG CCCGTTACCC TGGAATTGGG TGGCAAATCG CCGGTGATCG1140TTTCCCGCAG TGCAGATATG GCGGACGTTG CACAACGGGT GTTGACGGTG AAAACCTTCA1200ATGCCGGGCA AATCTGTCTG GCACCGTGGG TGAGTCGAAC TTGGCGATGC GCGCACCCTA1260CGGAGAAGCG ATCCACGGAC TGCTCTCTGT CCTCCTTTCA ACGGAGTGTT AGAACCGTTG1320GTAGTGGTTT TGGACGGGCC CAGGAGCATG CGCTTCTGGG CCCGTTTCTT GAGTATTCAT1380TGGATAGTCA CGCGTGGTAG CTTCGAGCCT GCACAGCTGA TGAGCACCCT GGAAGGCGCG1440CTGTACGCGG ACGACTGGGT TCATCTTCGC CATTCATGAC GGAACTCCGT TCCCCAGTAC1500CGCGATGACT ATTTTGCCTC TTCCGATGTC CGATTCCACG CCGCCTGACG CTAAGCGGGG1560GCGGGGGCGC CCGCATCCCA GCCCAGACAG CAACAAATGA GTAGGCTCTT GGATGCCGCG1620GCGGCTGAGA TTGGTAACGG CAATTTCGTC AATGTGACGA TGGATTCGAT TGCCCGTGCT1680GCCGGCGTCT CAAAAAAAAC GCTGTACGTC TTGGTGGCGA GCAAGGAAGA ACTCATTTCC1740CGGTTAGTGG CTCGAGACAT GTCCAACCTT GAGGAATTC 1779
序列7CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG GATGCGTTCT CTCGAGGCGC 360TTCTTCCCTT CCCGGGTCGA ATTCTTGAGC GTCTCGAGCA TTGGGCTAAG ACCCGTCCAG 420AACAAACCTG CGTTGCTGCC AGGGCGGCAA ATGGGGAATG GCGTCGTATC AGCTACGCGG 480AAATGTTCCA CAACGTCCGC GCCATCGCAC AGAGCTTGCT TCCTTACGGA CTATCGGCAG 540AGCGTCCGCT GCTTATCGTC TCTGGAAATG ACCTGGAACA TCTTCAGCTG GCATTTGGGG 600CTATGTATGC GGGCATTCCC TATTGCCCGG TGTCTCCTGC TTATTCACTG CTGTCGCAAG 660ATTTGGCGAA GCTGCGTCAC ATCGTAGGTC TTCTGCAACC GGGACTGGTC TTTGCTGCCG 720ATGCAGCACC TTTCCAGGGG ACAGCAAGCG AACCGGAATT GCCAGCTGGG GCGCCCTCTG 780GTAAGGTTGG GAAGCCCTGC AAAGTAAACT GGATGGCTTT CTTGCCGCCA AGGATCTGAT 840GGCGCAGGGG ATCAAGATCT GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATGATTGAAC 900AAGATGGATT GCACGCAGGT TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT 960GGGCACAACA GACAATCGGC TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC1020GCCCGGTTCT TTTTGTCAAG ACCGACCTGT CCGGTGCCCT GAATGAACTG CAGGACGAGG1080CAGCGCGGCT ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG1140TCACTGAAGC GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT1200CATCTCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC1260ATACGCTTGA TCCGGCTACC TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG1320CACGTACTCG GATGGAAGCC GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG1380GGCTCGCGCC AGCCGAACTG TTCGCCAGGC TCAAGGCGCG CATGCCCGAC GGCGAGGATC1440TCGTCGTGAC CCATGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT1500CTGGATTCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG1560CTACCCGTGA TATTGCTGAA GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT1620ACGGTATCGC CGCTCCCGAT TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT1680TCTGAGCGGG ACTCTGGGGT TCGAAATGAC CGACCAAGCG ACGCCCCTGT TTTGCAATGG1740CGGTCGGCGA AAGTTGATGC GCTGTATCGT GGTGAAGATC AATCCATGCT GCGTGACGAG1800GCCACACTGT GAGTTGGTCA GGGGGGGCTT ACTCGGCGTT TTCCGACACT GCGTTGGTTG1860CGGCAGTGCG CACCCCCTGG ATTGATTGCG GGGGTGCCCT GTCGCTGGTG TCGCCTATCG1920ACTTAGGGGT AAAGGTCGCT CGCGAAGTTC TGATGCGTGC GTCGCTTGAA CCACAAATGG1980TCGATAGCGT ACTCGCAGGC TCTATGGCTC AAGCAAGCTT TGATGCTTAC CTGCTCCCGC2040GGCACATTGG CTTGTACAGC GGTGTTCCCA AGTCGGTTCC GGCCTTGGGG GTGCAGCGCA2100TTTGCGGCAC AGGCTTCGAA CTGCTTCGGC AGGCCGGCGA GCAGATTTCC CAAGGCGCTG2160ATCACGTGCT GTGTGTCGCG GGCTGCAG 2188
序列8CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG GATGCGTTCT CTCGAGGCGC 360TTCTTCCCTT CCCGGGTCGA ATTCTTGAGC GTCTCGAGCA TTGGGCTAAG ACCCGTCCAG 420AACAAACCTG CGTTGCTGCC AGGGCGGCAA ATGGGGAATG GCGTCGTATC AGCTACGCGG 480AAATGTTCCA CAACGTCCGC GCCATCGCAC AGAGCTTGCT TCCTTACGGA CTATCGGCAG 540AGCGTCCGCT GCTTATCGTC TCTGGAAATG ACCTGGAACA TCTTCAGCTG GCATTTGGGG 600CTATGTATGC GGGCATTCCC TATTGCCCGG TGTCTCCTGC TTATTCACTG CTGTCGCAAG 660ATTTGGCGAA GCTGCGTCAC ATCGTAGGTC TTCTGCAACC GGGACTGGTC TTTGCTGCCG 720ATGCAGCACC TTTCCAGGGG GAGAGGCGGT TTGCGTATTG GGCGCATGCA TAAAAACTGT 780TGTAATTCAT TAAGCATTCT GCCGACATGG AAGCCATCAC AAACGGCATG ATGAACCTGA 840ATCGCCAGCG GCATCAGCAC CTTGTCGCCT TGCGTATAAT ATTTGCCCAT GGACGCACAC 900CGTGGAAACG GATGAAGGCA CGAACCCAGT TGACATAAGC CTGTTCGGTT CGTAAACTGT 960AATGCAAGTA GCGTATGCGC TCACGCAACT GGTCCAGAAC CTTGACCGAA CGCAGCGGTG1020GTAACGGCGC AGTGGCGGTT TTCATGGCTT GTTATGACTG TTTTTTTGTA CAGTCTATGC1080CTCGGGCATC CAAGCAGCAA GCGCGTTACG CCGTGGGTCG ATGTTTGATG TTATGGAGCA1140GCAACGATGT TACGCAGCAG CAACGATGTT ACGCAGCAGG GCAGTCGCCC TAAAACAAAG1200TTAGGTGGCT CAAGTATGGG CATCATTCGC ACATGTAGGC TCGGCCCTGA CCAAGTCAAA1260TCCATGCGGG CTGCTCTTGA TCTTTTCGGT CGTGAGTTCG GAGACGTAGC CACCTACTCC1320CAACATCAGC CGGACTCCGA TTACCTCGGG AACTTGCTCC GTAGTAAGAC ATTCATCGCG1380CTTGCTGCCT TCGACCAAGA AGCGGTTGTT GGCGCTCTCG CGGCTTACGT TCTGCCCAGG1440TTTGAGCAGC CGCGTAGTGA GATCTATATC TATGATCTCG CAGTCTCCGG CGAGCACCGG1500AGGCAGGGCA TTGCCACCGC GCTCATCAAT CTCCTCAAGC ATGAGGCCAA CGCGCTTGGT1560GCTTATGTGA TCTACGTGCA AGCAGATTAC GGTGACGATC CCGCAGTGGC TCTCTATACA1620AAGTTGGGCA TACGGGAAGA AGTGATGCAC TTTGATATCG ACCCAAGTAC CGCCACCTAA1680CAATTCGTTC AAGCCGAGAT CGGCTTCCCC TGTTTTGCAA TGGCGGTCGG CGAAAGTTGA1740TGCGCTGTAT CGTGGTGAAG ATCAATCCAT GCTGCGTGAC GAGGCCACAC TGTGAGTTGG1800TCAGGGGGGG CTTACTCGGC GTTTTCCGAC ACTGCGTTGG TTGCGGCAGT GCGCACCCCC1860TGGATTGATT GCGGGGGTGC CCTGTCGCTG GTGTCGCCTA TCGACTTAGG GGTAAAGGTC1920GCTCGCGAAG TTCTGATGCG TGCGTCGCTT GAACCACAAA TGGTCGATAG CGTACTCGCA1980GGCTCTATGG CTCAAGCAAG CTTTGATGCT TACCTGCTCC CGCGGCACAT TGGCTTGTAC2040AGCGGTGTTC CCAAGTCGGT TCCGGCCTTG GGGGTGCAGC GCATTTGCGG CACAGGCTTC2100GAACTGCTTC GGCAGGCCGG CGAGCAGATT TCCCAAGGCG CTGATCACGT GCTGTGTGTC2160GCGGGCTGCA G 2171
序列9CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG GATGCGTTCT CTCGAGGCGC 360TTCTTCCCTT CCCGGGTCGA ATTCTTGAGC GTCTCGAGCA TTGGGCTAAG ACCCGTCCAG 420AACAAACCTG CGTTGCTGCC AGGGCGGCAA ATGGGGAATG GCGTCGTATC AGCTACGCGG 480AAATGTTCCA CAACGTCCGC GCCATCGCAC AGAGCTTGCT TCCTTACGGA CTATCGGCAG 540AGCGTCCGCT GCTTATCGTC TCTGGAAATG ACCTGGAACA TCTTCAGCTG GCATTTGGGG 600CTATGTATGC GGGCATTCCC TATTGCCCGG TGTCTCCTGC TTATTCACTG CTGTCGCAAG 660ATTTGGCGAA GCTGCGTCAC ATCGTAGGTC TTCTGCAACC GGGACTGGTC TTTGCTGCCG 720ATGCAGCACC TTTCCAGCGC GCTGTTTTGC AATGGCGGTC GGCGAAAGTT GATGCGCTGT 780ATCGTGGTGA AGATCAATCC ATGCTGCGTG ACGAGGCCAC ACTGTGAGTT GGTCAGGGGG 840GGCTTACTCG GCGTTTTCCG ACACTGCGTT GGTTGCGGCA GTGCGCACCC CCTGGATTGA 900TTGCGGGGGT GCCCTGTCGC TGGTGTCGCC TATCGACTTA GGGGTAAAGG TCGCTCGCGA 960AGTTCTGATG CGTGCGTCGC TTGAACCACA AATGGTCGAT AGCGTACTCG CAGGCTCTAT1020GGCTCAAGCA AGCTTTGATG CTTACCTGCT CCCGCGGCAC ATTGGCTTGT ACAGCGGTGT1080TCCCAAGTCG GTTCCGGCCT TGGGGGTGCA GCGCATTTGC GGCACAGGCT TCGAACTGCT1140TCGGCAGGCC GGCGAGCAGA TTTCCCAAGG CGCTGATCAC GTGCTGTGTG TCGCGGGCTG1200CAG 1203
序列10GAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240GGTGCACGAT GAATAGCTAC GATGGCCGTT GGTCTACCGT TGATGTGAAG GTTGAAGAAG 300GTATCGCTTG GGTCACGCTG AACCGCCCGG AGAAGCGCAA CGCAATGAGC CCAACTCTCA 360ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 420TTCTGACTGG TGCAGGCGAA TCCTGGACCG CGGGCATGGA CCTGAAGGAG TATTTCCGCG 480AGACCGATGC TGGCCCCGAA ATTCTGCAAG AGAAGATTCG TCGGGGACAG CAAGCGAACC 540GGAATTGCCA GCTGGGGCGC CCTCTGGTAA GGTTGGGAAG CCCTGCAAAG TAAACTGGAT 600GGCTTTCTTG CCGCCAAGGA TCTGATGGCG CAGGGGATCA AGATCTGATC AAGAGACAGG 660ATGAGGATCG TTTCGCATGA TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG 720GGTGGAGAGG CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC 780CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG 840TGCCCTGAAT GAACTGCAGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT 900TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG 960CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT1020CATGGCTGAT GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA1080CCAAGCGAAA CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA1140GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA1200GGCGCGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA1260TATCATGGTG GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC1320GGACCGCTAT CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA1380ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC1440CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA AATGACCGAC1500CAAGCGACGC CCCGAGCAGG GCATGAAGCA GTTCCTTGAC GAGAAAAGCA TCAAGCCGGG1560CTTGCAGACC TACAAGCGCT GATAAATGCG CCGGGGCCCT CGCTGCGCCC CCGGCCTTCC1620AATAATGACA ATAATGAGGA GTGCCCAATG TTTCACGTGC CCCTGCTTAT TGGTGGTAAG1680CCTTGTTCAG CATCTGATGA GCGCACCTTC GAGCGTCGTA GCCCGCTGAC CGGAGAAGTG1740GTATCGCGCG TCGCTGCTGC CAGTTTGGAA GATGCGGACG CCGCAGTGGC CGCTGCACAG1800GCTGCGTTTC CTGAATGGGC GGCGCTTGCT CCGAGCGAAC GCCGTGCCCG ACTGCTGCGA1860GCGGCGGATC TTCTAGAGGA CCGTTCTTCC GAGTTCACCG CCGCAGCGAG TGAAACTGGC1920GCAGCGGGAA ACTGGTATGG GTTTAACGTT TACCTGGCGG CGGGCATGTT GCGGGGAATT1980C1981
序列11GAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240GGTGCACGAT GAATAGCTAC GATGGCCGTT GGTCTACCGT TGATGTGAAG GTTGAAGAAG 300GTATCGCTTG GGTCACGCTG AACCGCCCGG AGAAGCGCAA CGCAATGAGC CCAACTCTCA 360ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 420TTCTGACTGG TGCAGGCGAA TCCTGGACCG CGGGCATGGA CCTGAAGGAG TATTTCCGCG 480AGACCGATGC TGGCCCCGAA ATTCTGCAAG AGAAGATTCG TCGGGGGAGA GGCGGTTTGC 540GTATTGGGCG CATGCATAAA AACTGTTGTA ATTCATTAAG CATTCTGCCG ACATGGAAGC 600CATCACAAAC GGCATGATGA ACCTGAATCG CCAGCGGCAT CAGCACCTTG TCGCCTTGCG 660TATAATATTT GCCCATGGAC GCACACCGTG GAAACGGATG AAGGCACGAA CCCAGTTGAC 720ATAAGCCTGT TCGGTTCGTA AACTGTAATG CAAGTAGCGT ATGCGCTCAC GCAACTGGTC 780CAGAACCTTG ACCGAACGCA GCGGTGGTAA CGGCGCAGTG GCGGTTTTCA TGGCTTGTTA 840TGACTGTTTT TTTGTACAGT CTATGCCTCG GGCATCCAAG CAGCAAGCGC GTTACGCCGT 900GGGTCGATGT TTGATGTTAT GGAGCAGCAA CGATGTTACG CAGCAGCAAC GATGTTACGC 960AGCAGGGCAG TCGCCCTAAA ACAAAGTTAG GTGGCTCAAG TATGGGCATC ATTCGCACAT1020GTAGGCTCGG CCCTGACCAA GTCAAATCCA TGCGGGCTGC TCTTGATCTT TTCGGTCGTG1080AGTTCGGAGA CGTAGCCACC TACTCCCAAC ATCAGCCGGA CTCCGATTAC CTCGGGAACT1140TGCTCCGTAG TAAGACATTC ATCGCGCTTG CTGCCTTCGA CCAAGAAGCG GTTGTTGGCG1200CTCTCGCGGC TTACGTTCTG CCCAGGTTTG AGCAGCCGCG TAGTGAGATC TATATCTATG1260ATCTCGCAGT CTCCGGCGAG CACCGGAGGC AGGGCATTGC CACCGCGCTC ATCAATCTCC1320TCAAGCATGA GGCCAACGCG CTTGGTGCTT ATGTGATCTA CGTGCAAGCA GATTACGGTG1380ACGATCCCGC AGTGGCTCTC TATACAAAGT TGGGCATACG GGAAGAAGTG ATGCACTTTG1440ATATCGACCC AAGTACCGCC ACCTAACAAT TCGTTCAAGC CGAGATCGGC TTCCCCGAGC1500AGGGCATGAA GCAGTTCCTT GACGAGAAAA GCATCAAGCC GGGCTTGCAG ACCTACAAGC1560GCTGATAAAT GCGCCGGGGC CCTCGCTGCG CCCCCGGCCT TCCAATAATG ACAATAATGA1620GGAGTGCCCA ATGTTTCACG TGCCCCTGCT TATTGGTGGT AAGCCTTGTT CAGCATCTGA1680TGAGCGCACC TTCGAGCGTC GTAGCCCGCT GACCGGAGAA GTGGTATCGC GCGTCGCTGC1740TGCCAGTTTG GAAGATGCGG ACGCCGCAGT GGCCGCTGCA CAGGCTGCGT TTCCTGAATG1800GGCGGCGCTT GCTCCGAGCG AACGCCGTGC CCGACTGCTG CGAGCGGCGG ATCTTCTAGA1860GGACCGTTCT TCCGAGTTCA CCGCCGCAGC GAGTGAAACT GGCGCAGCGG GAAACTGGTA1920TGGGTTTAAC GTTTACCTGG CGGCGGGCAT GTTGCGGGGA ATTC 1964
序列12GAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240GGTGCACGAT GAATAGCTAC GATGGCCGTT GGTCTACCGT TGATGTGAAG GTTGAAGAAG 300GTATCGCTTG GGTCACGCTG AACCGCCCGG AGAAGCGCAA CGCAATGAGC CCAACTCTCA 360ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 420TTCTGACTGG TGCAGGCGAA TCCTGGACCG CGGGCATGGA CCTGAAGGAG TATTTCCGCG 480AGACCGATGC TGGCCCCGAA ATTCTGCAAG AGAAGATTCG TCGCGAGCAG GGCATGAAGC 540AGTTCCTTGA CGAGAAAAGC ATCAAGCCGG GCTTGCAGAC CTACAAGCGC TGATAAATGC 600GCCGGGGCCC TCGCTGCGCC CCCGGCCTTC CAATAATGAC AATAATGAGG AGTGCCCAAT 660GTTTCACGTG CCCCTGCTTA TTGGTGGTAA GCCTTGTTCA GCATCTGATG AGCGCACCTT 720CGAGCGTCGT AGCCCGCTGA CCGGAGAAGT GGTATCGCGC GTCGCTGCTG CCAGTTTGGA 780AGATGCGGAC GCCGCAGTGG CCGCTGCACA GGCTGCGTTT CCTGAATGGG CGGCGCTTGC 840TCCGAGCGAA CGCCGTGCCC GACTGCTGCG AGCGGCGGAT CTTCTAGAGG ACCGTTCTTC 900CGAGTTCACC GCCGCAGCGA GTGAAACTGG CGCAGCGGGA AACTGGTATG GGTTTAACGT 960TTACCTGGCG GCGGGCATGT TGCGGGGAAT TC 992
序列13GAATTCCAAT AATGACAATA ATGAGGAGTG CCCAATGTTT CACGTGCCCC TGCTTATTGG 60TGGTAAGCCT TGTTCAGCAT CTGATGAGCG CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120AGAAGTGGTA TCGCGCGTCG CTGCTGCCAG TTTGGAAGAT GCGGACGCCG CAGTGGCCGC 180TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240GCTGCGAGCG GCGGATCTTC TAGAGGACCG TTCTTCCGAG TTCACCGCCG CAGCGAGTGA 300AACTGGCGCA GCGGGAAACT GGTATGGGTT TAACGTTTAC CTGGCGGCGG GCATGTTGCG 360GGAAGCCGCG GCCATGACCA CACAGATTCA GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 420TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480TGCTCCGGTA ATCCTTGGCG TACGGGCTGT TGCGATGCCG TTGGCATGCG GCAATACCGT 540GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TACCCATCGC CTGATTGGTC AGGTGTTGCA 600TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGTCATCAGC AATGCCCCGC AAGACGCTCC 660TGCGGTGGTG GAGCGACTGA TTGCAAATCC TGCGGTACGT CGAGTGAACT TCACCGGTTC 720GACCCACGTT GGACGGATCA TTGGTGAGCT GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780GGAATTAGGT GGTAAGGCTC CGTTCTTGGT CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840CGAAGCGGCG GCCTTTGGTG CCTACTTCAATCAGGGTCAA ATCTGCATGT CCACTGAGCG 900TCTGATTGTG ACAGCAGTCG CAGACGCCTT TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960ACTGCGTGCT GGCGATCCTA ATGATCCGCA ATCGGTCTTG GGTTCGTTGA TTGATGCCAA1020TGCAGGTCAA CGCATCCAGG TTCTGGTCGA TGATGCGCTC GGGGACAGCA AGCGAACCGG1080AATTGCCAGC TGGGGCGCCC TCTGGTAAGG TTGGGAAGCC CTGCAAAGTA AACTGGATGG1140CTTTCTTGCC GCCAAGGATC TGATGGCGCA GGGGATCAAG ATCTGATCAA GAGACAGGAT1200GAGGATCGTT TCGCATGATT GAACAAGATG GATTGCACGC AGGTTCTCCG GCCGCTTGGG1260TGGAGAGGCT ATTCGGCTAT GACTGGGCAC AACAGACAAT CGGCTGCTCT GATGCCGCCG1320TGTTCCGGCT GTCAGCGCAG GGGCGCCCGG TTCTTTTTGT CAAGACCGAC CTGTCCGGTG1380CCCTGAATGA ACTGCAGGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG ACGGGCGTTC1440CTTGCGCAGC TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG CTATTGGGCG1500AAGTGCCGGG GCAGGATCTC CTGTCATCTC ACCTTGCTCC TGCCGAGAAA GTATCCATCA1560TGGCTGATGC AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA TTCGACCACC1620AAGCGAAACA TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT GTCGATCAGG1680ATGATCTGGA CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG1740CGCGCATGCC CGACGGCGAG GATCTCGTCG TGACCCATGG CGATGCCTGC TTGCCGAATA1800TCATGGTGGA AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG GGTGTGGCGG1860ACCGCTATCA GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT GGCGGCGAAT1920GGGCTGACCG CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG CGCATCGCCT1980TCTATCGCCT TCTTGACGAG TTCTTCTGAG CGGGACTCTG GGGTTCGAAA TGACCGACCA2040AGCGACGCCC GGCCCAGCGC GTCGATTCGG GCATTTGCCA TATCAATGGA CCGACTGTGC2100ATGACGAGGC TCAGATGCCA TTCGGTGGGG TGAAGTCCAG CGGCTACGGC AGCTTCGGCA2160GTCGAGCATC GATTGAGCAC TTTACCCAGC TGCGCTGGCT GACCATTCAG AATGGCCCGC2220GGCACTATCC AATCTAAATC GATCTTCGGG CGCCGCGGGC ATCATGCCCG CGGCGCTCGC2280CTCATTTCAA TCTCTAACTT GATAAAAACA GAGCTGTTCT CCGGTCTTGG TGGATCAAGG2340CCAGTCGCGG AGAGTCTCGA AGAGGAGAGT ACAGTGAACG CCGAGTCCAC ATTGCAACCG2400CAGGCATCAT CATGCTCTGC TCAGCCACGC TACCGCAGTG TGTCGATTGG TCATCCTCCG2460GTTGAGGTTA CGCAAGACGC TGGAGGTATT GTCCGGATGC GTTCTCTCGA GGCGCTTCTT2520CCCTTCCCGG GTGGAATTC 2539
序列14GAATTCCAAT AATGACAATA ATGAGGAGTG CCCAATGTTT CACGTGCCCC TGCTTATTGG 60TGGTAAGCCT TGTTCAGCAT CTGATGAGCG CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120AGAAGTGGTA TCGCGCGTCG CTGCTGCCAG TTTGGAAGAT GCGGACGCCG CAGTGGCCGC 180TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240GCTGCGAGCG GCGGATCTTC TAGAGGACCG TTCTTCCGAG TTCACCGCCG CAGCGAGTGA 300AACTGGCGCA GCGGGAAACT GGTATGGGTT TAACGTTTAC CTGGCGGCGG GCATGTTGCG 360GGAAGCCGCG GCCATGACCA CACAGATTCA GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 420TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480TGCTCCGGTA ATCCTTGGCG TACGGGCTGT TGCGATGCCG TTGGCATGCG GCAATACCGT 540GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TACCCATCGC CTGATTGGTC AGGTGTTGCA 600TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGTCATCAGC AATGCCCCGC AAGACGCTCC 660TGCGGTGGTG GAGCGACTGA TTGCAAATCC TGCGGTACGT CGAGTGAACT TCACCGGTTC 720GACCCACGTT GGACGGATCA TTGGTGAGCT GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780GGAATTAGGT GGTAAGGCTC CGTTCTTGGT CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840CGAAGCGGCG GCCTTTGGTG CCTACTTCAA TCAGGGTCAA ATCTGCATGT CCACTGAGCG 900TCTGATTGTG ACAGCAGTCG CAGACGCCTT TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960ACTGCGTGCT GGCGATCCTA ATGATCCGCA ATCGGTCTTG GGTTCGTTGA TTGATGCCAA1020TGCAGGTCAA CGCATCCAGG TGGGGAGAGG CGGTTTGCGT ATTGGGCGCA TGCATAAAAA1080CTGTTGTAAT TCATTAAGCA TTCTGCCGAC ATGGAAGCCA TCACAAACGG CATGATGAAC1140CTGAATCGCC AGCGGCATCA GCACCTTGTC GCCTTGCGTA TAATATTTGC CCATGGACGC1200ACACCGTGGA AACGGATGAA GGCACGAACC CAGTTGACAT AAGCCTGTTC GGTTCGTAAA1260CTGTAATGCA AGTAGCGTAT GCGCTCACGC AACTGGTCCA GAACCTTGAC CGAACGCAGC1320GGTGGTAACG GCGCAGTGGC GGTTTTCATG GCTTGTTATG ACTGTTTTTT TGTACAGTCT1380ATGCCTCGGG CATCCAAGCA GCAAGCGCGT TACGCCGTGG GTCGATGTTT GATGTTATGG1440AGCAGCAACG ATGTTACGCA GCAGCAACGA TGTTACGCAG CAGGGCAGTC GCCCTAAAAC1500AAAGTTAGGT GGCTCAAGTA TGGGCATCAT TCGCACATGT AGGCTCGGCC CTGACCAAGT1560CAAATCCATG CGGGCTGCTC TTGATCTTTT CGGTCGTGAG TTCGGAGACG TAGCCACCTA1620CTCCCAACAT CAGCCGGACT CCGATTACCT CGGGAACTTG CTCCGTAGTA AGACATTCAT1680CGCGCTTGCT GCCTTCGACC AAGAAGCGGT TGTTGGCGCT CTCGCGGCTT ACGTTCTGCC1740CAGGTTTGAG CAGCCGCGTA GTGAGATCTA TATCTATGAT CTCGCAGTCT CCGGCGAGCA1800CCGGAGGCAG GGCATTGCCA CCGCGCTCAT CAATCTCCTC AAGCATGAGG CCAACGCGCT1860TGGTGCTTAT GTGATCTACG TGCAAGCAGA TTACGGTGAC GATCCCGCAG TGGCTCTCTA1920TACAAAGTTG GGCATACGGG AAGAAGTGAT GCACTTTGAT ATCGACCCAA GTACCGCCAC1980CTAACAATTC GTTCAAGCCG AGATCGGCTT CCCAATTGGC CCAGCGCGTC GATTCGGGCA2040TTTGCCATAT CAATGGACCG ACTGTGCATG ACGAGGCTCA GATGCCATTC GGTGGGGTGA2100AGTCCAGCGG CTACGGCAGC TTCGGCAGTC GAGCATCGAT TGAGCACTTT ACCCAGCTGC2160GCTGGCTGAC CATTCAGAAT GGCCCGCGGC ACTATCCAAT CTAAATCGAT CTTCGGGCGC2220CGCGGGCATC ATGCCCGCGG CGCTCGCCTC ATTTCAATCT CTAACTTGAT AAAAACAGAG2280CTGTTCTCCG GTCTTGGTGG ATCAAGGCCA GTCGCGGAGA GTCTCGAAGA GGAGAGTACA2340GTGAACGCCG AGTCCACATT GCAACCGCAG GCATCATCAT GCTCTGCTCA GCCACGCTAC2400CGCAGTGTGT CGATTGGTCA TCCTCCGGTT GAGGTTACGC AAGACGCTGG AGGTATTGTC2460CGGATGCGTT CTCTCGAGGC GCTTCTTCCC TTCCCGGGTG GAATTC 2506
序列15GAATTCCAAT AATGACAATA ATGAGGAGTG CCCAATGTTT CACGTGCCCC TGCTTATTGG 60TGGTAAGCCT TGTTCAGCAT CTGATGAGCG CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120AGAAGTGGTA TCGCGCGTCG CTGCTGCCAG TTTGGAAGAT GCGGACGCCG CAGTGGCCGC 180TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240GCTGCGAGCG GCGGATCTTC TAGAGGACCG TTCTTCCGAG TTCACCGCCG CAGCGAGTGA 300AACTGGCGCA GCGGGAAACT GGTATGGGTT TAACGTTTAC CTGGCGGCGG GCATGTTGCG 360GGAAGCCGCG GCCATGACCA CACAGATTCA GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 420TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480TGCTCCGGTA ATCCTTGGCG TACGGGCTGT TGCGATGCCG TTGGCATGCG GCAATACCGT 540GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TACCCATCGC CTGATTGGTC AGGTGTTGCA 600TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGTCATCAGC AATGCCCCGC AAGACGCTCC 660TGCGGTGGTG GAGCGACTGA TTGCAAATCC TGCGGTACGT CGAGTGAACT TCACCGGTTC 720GACCCACGTT GGACGGATCA TTGGTGAGCT GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780GGAATTAGGT GGTAAGGCTC CGTTCTTGGT CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840CGAAGCGGCG GCCTTTGGTG CCTACTTCAA TCAGGGTCAA ATCTGCATGT CCACTGAGCG 900TCTGATTGTG ACAGCAGTCG CAGACGCCTT TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960ACTGCGTGCT GGCGATCCTA ATGATCCGCA ATCGGTCTTG GGTTCGTTGA TTGATGCCAA1020TGCAGGTCAA CGCATCCAGG TTCTGGTCGA TGATGCGCTC GCAAAAGGCG CGCAATGGAA1080TTGGCCCAGC GCGTCGATTC GGGCATTTGC CATATCAATG GACCGACTGT GCATGACGAG1140GCTCAGATGC CATTCGGTGG GGTGAAGTCC AGCGGCTACG GCAGCTTCGG CAGTCGAGCA1200TCGATTGAGC ACTTTACCCA GCTGCGCTGG CTGACCATTC AGAATGGCCC GCGGCACTAT1260CCAATCTAAA TCGATCTTCG GGCGCCGCGG GCATCATGCC CGCGGCGCTC GCCTCATTTC1320AATCTCTAAC TTGATAAAAA CAGAGCTGTT CTCCGGTCTT GGTGGATCAA GGCCAGTCGC1380GGAGAGTCTC GAAGAGGAGA GTACAGTGAA CGCCGAGTCC ACATTGCAAC CGCAGGCATC1440ATCATGCTCT GCTCAGCCAC GCTACCGCAG TGTGTCGATT GGTCATCCTC CGGTTGAGGT1500TACGCAAGAC GCTGGAGGTA TTGTCCGGAT GCGTTCTCTC GAGGCGCTTC TTCCCTTCCC1560GGGTGGAATT C 1571
序列16GAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60GTGACGAGGC CACACTGTGA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 420AGGCGCTGAT CACGTGCTGT GTGTCGCGGC AGAGTCCATG TCGCGTAACC CCATCGCGTC 480GTATACACAC CGGGGCGGGT TCCGCCTCGG TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540GGAGGCATTG TTTGATCCTG CTCCAGGACT CGACATGATC GCTACCGCAG AAAACCTGGG 600GACAGCAAGC GAACCGGAAT TGCCAGCTGG GGCGCCCTCT GGTAAGGTTG GGAAGCCCTG 660CAAAGTAAAC TGGATGGCTT TCTTGCCGCC AAGGATCTGA TGGCGCAGGG GATCAAGATC 720TGATCAAGAG ACAGGATGAG GATCGTTTCG CATGATTGAA CAAGATGGAT TGCACGCAGG 780TTCTCCGGCC GCTTGGGTGG AGAGGCTATT CGGCTATGAC TGGGCACAAC AGACAATCGG 840CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA 900GACCGACCTG TCCGGTGCCC TGAATGAACT GCAGGACGAG GCAGCGCGGC TATCGTGGCT 960GGCCACGACG GGCGTTCCTT GCGCAGCTGT GCTCGACGTT GTCACTGAAG CGGGAAGGGA1020CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA GGATCTCCTG TCATCTCACC TTGCTCCTGC1080CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG CATACGCTTG ATCCGGCTAC1140CTGCCCATTC GACCACCAAG CGAAACATCG CATCGAGCGA GCACGTACTC GGATGGAAGC1200CGGTCTTGTC GATCAGGATG ATCTGGACGA AGAGCATCAG GGGCTCGCGC CAGCCGAACT1260GTTCGCCAGG CTCAAGGCGC GCATGCCCGA CGGCGAGGAT CTCGTCGTGA CCCATGGCGA1320TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA TCGACTGTGG1380CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG ATATTGCTGA1440AGAGCTTGGC GGCGAATGGG CTGACCGCTT CCTCGTGCTT TACGGTATCG CCGCTCCCGA1500TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC TTCTGAGCGG GACTCTGGGG1560TTCGAAATGA CCGACCAAGC GACGCCCATT GAGGGCGCAA GAGGAGAAAT GGATTGACCA1620AGAGATCGTG GCTGTTACGG ATGAACAGTT CGATTTAGAG GGCTACAACA GTCGAGCAAT1680TGAACTGCCT CGGAAGGCAA AATTGTTGAT CGTGACAGTC ATCCGCGGCC TAGCAGTCTT1740TGAAGCCCTT TCCCGATTGA AGCCTGTTCA TTCTGGCGGG GTGCAGACTG CGGGCAACAG1800CTGTGCCGTA GTGGACGGCG CCGCGGCGGC TTTGGTGGCT CGAGAGTCGT CTGCGACACA1860GCCGGTCTTG GCTAGGATAC TGGCTACCTC CGTAGTCGGG ATCGAGCCCG AGCATATGGG1920GCTCGGCCCT GCGCCCGCGA TTCGCCTGCT GCTTGCGCGT AGTGATCTTA GTTTGAGGGA1980TATCGACCTC TTTGAGATAA ACGAGGCGCA GGCCGCCCAA GTTCTAGCGG TACAGCATGA2040ATTGGGTATT GAGCACTCAA AACTTAATAT TTGGGGCGGG GCCATTGCAC TTGGACACCC2100GCTTGCCGCG ACCGGATTGC GTCTCTGCAT GACCCTCGCT CACCAATTGC AAGCTAATAA2160CTTTCGATAT GGAATTGCCT CGGCATGCAT TGGTGGGGGA CAGGGGATGG CGGTTCTTTT2220AGAGAATCCC CACTTCGGTT CGTCCTCTGC ACGAAGTTCG ATGATTAACA GAGTTGACCA2280CTATCCACTG AGCTAACGGG CATCTCCTTT GTTGCTTTGA GGTGGCGCAC GAAGGAGGGC2340TCGAAAATCT CTGCTAAAAA CAAGAAGAAG GAACAGGGAA CATGATTAGT TTCGCTCGTA2400TGGCAGAAAG TTTAGGAGTC CAGGCTAAAC TTGCCCTTGC CTTCGCACTC GTATTATGTG2460TCGGGCTGAT TGTTACCGGC ACGGGTTTCT ACAGTGTACA TACCTTGTCA GGGTTGGTGG2520GAATTC 2526
序列17GAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60GTGACGAGGC CACACTGTGA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 420AGGCGCTGAT CACGTGCTGT GTGTCGCGGC AGAGTCCATG TCGCGTAACC CCATCGCGTC 480GTATACACAC CGGGGCGGGT TCCGCCTCGG TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540GGAGGCATTG TTTGATCCTG CTCCAGGACT CGACATGATC GCTACCGCAG AAAACCTGGG 600GGAGAGGCGG TTTGCGTATT GGGCGCATGC ATAAAAACTG TTGTAATTCA TTAAGCATTC 660TGCCGACATG GAAGCCATCA CAAACGGCAT GATGAACCTG AATCGCCAGC GGCATCAGCA 720CCTTGTCGCC TTGCGTATAA TATTTGCCCA TGGACGCACA CCGTGGAAAC GGATGAAGGC 780ACGAACCCAG TTGACATAAG CCTGTTCGGT TCGTAAACTG TAATGCAAGT AGCGTATGCG 840CTCACGCAAC TGGTCCAGAA CCTTGACCGA ACGCAGCGGT GGTAACGGCG CAGTGGCGGT 900TTTCATGGCT TGTTATGACT GTTTTTTTGT ACAGTCTATG CCTCGGGCAT CCAAGCAGCA 960AGCGCGTTAC GCCGTGGGTC GATGTTTGAT GTTATGGAGC AGCAACGATG TTACGCAGCA1020GCAACGATGT TACGCAGCAG GGCAGTCGCC CTAAAACAAA GTTAGGTGGC TCAAGTATGG1080GCATCATTCG CACATGTAGG CTCGGCCCTG ACCAAGTCAA ATCCATGCGG GCTGCTCTTG1140ATCTTTTCGG TCGTGAGTTC GGAGACGTAG CCACCTACTC CCAACATCAG CCGGACTCCG1200ATTACCTCGG GAACTTGCTC CGTAGTAAGA CATTCATCGC GCTTGCTGCC TTCGACCAAG1260AAGCGGTTGT TGGCGCTCTC GCGGCTTACG TTCTGCCCAG GTTTGAGCAG CCGCGTAGTG1320AGATCTATAT CTATGATCTC GCAGTCTCCG GCGAGCACCG GAGGCAGGGC ATTGCCACCG1380CGCTCATCAA TCTCCTCAAG CATGAGGCCA ACGCGCTTGG TGCTTATGTG ATCTACGTGC1440AAGCAGATTA CGGTGACGAT CCCGCAGTGG CTCTCTATAC AAAGTTGGGC ATACGGGAAG1500AAGTGATGCA CTTTGATATC GACCCAAGTA CCGCCACCTA ACAATTCGTT CAAGCCGAGA1560TCGGCTTCCC ATTGAGGGCG CAAGAGGAGA AATGGATTGA CCAAGAGATC GTGGCTGTTA 1620CGGATGAACA GTTCGATTTA GAGGGCTACA ACAGTCGAGC AATTGAACTG CCTCGGAAGG1680CAAAATTGTT GATCGTGACA GTCATCCGCG GCCTAGCAGT CTTTGAAGCC CTTTCCCGAT1740TGAAGCCTGT TCATTCTGGC GGGGTGCAGA CTGCGGGCAA CAGCTGTGCC GTAGTGGACG1800GCGCCGCGGC GGCTTTGGTG GCTCGAGAGT CGTCTGCGAC ACAGCCGGTC TTGGCTAGGA1860TACTGGCTAC CTCCGTAGTC GGGATCGAGC CCGAGCATAT GGGGCTCGGC CCTGCGCCCG1920CGATTCGCCT GCTGCTTGCG CGTAGTGATC TTAGTTTGAG GGATATCGAC CTCTTTGAGA1980TAAACGAGGC GCAGGCCGCC CAAGTTCTAG CGGTACAGCA TGAATTGGGT ATTGAGCACT2040CAAAACTTAA TATTTGGGGC GGGGCCATTG CACTTGGACA CCCGCTTGCC GCGACCGGAT2100TGCGTCTCTG CATGACCCTC GCTCACCAAT TGCAAGCTAA TAACTTTCGA TATGGAATTG2160CCTCGGCATG CATTGGTGGG GGACAGGGGA TGGCGGTTCT TTTAGAGAAT CCCCACTTCG2220GTTCGTCCTC TGCACGAAGT TCGATGATTA ACAGAGTTGA CCACTATCCA CTGAGCTAAC2280GGGCATCTCC TTTGTTGCTT TGAGGTGGCG CACGAAGGAG GGCTCGAAAA TCTCTGCTAA2340AAACAAGAAG AAGGAACAGG GAACATGATT AGTTTCGCTC GTATGGCAGA AAGTTTAGGA2400GTCCAGGCTA AACTTGCCCT TGCCTTCGCA CTCGTATTAT GTGTCGGGCT GATTGTTACC2460GGCACGGGTT TCTACAGTGT ACATACCTTG TCAGGGTTGG TGGGAATTC2509
序列18GAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60GTGACGAGGC CACACTGTGA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 420AGGCGCTGAT CACGTGCTGT GTGTCGCGGC AGAGTCCATG TCGCGTAACC CCATCGCGTC 480GTATACACAC CGGGGCGGGT TCCGCCTCGG TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540GGAGGCATTG TTTGATCCTG CTCCAGGACT CGACATGATC GCTACCGCAG AAAACCTGGC 600GCGCATTGAG GGCGCAAGAG GAGAAATGGA TTGACCAAGA GATCGTGGCT GTTACGGATG 660AACAGTTCGA TTTAGAGGGC TACAACAGTC GAGCAATTGA ACTGCCTCGG AAGGCAAAAT 720TGTTGATCGT GACAGTCATC CGCGGCCTAG CAGTCTTTGA AGCCCTTTCC CGATTGAAGC 780CTGTTCATTC TGGCGGGGTG CAGACTGCGG GCAACAGCTG TGCCGTAGTG GACGGCGCCG 840CGGCGGCTTT GGTGGCTCGA GAGTCGTCTG CGACACAGCC GGTCTTGGCT AGGATACTGG 900CTACCTCCGT AGTCGGGATC GAGCCCGAGC ATATGGGGCT CGGCCCTGCG CCCGCGATTC 960GCCTGCTGCT TGCGCGTAGT GATCTTAGTT TGAGGGATAT CGACCTCTTT GAGATAAACG1020AGGCGCAGGC CGCCCAAGTT CTAGCGGTAC AGCATGAATT GGGTATTGAG CACTCAAAAC1080TTAATATTTG GGGCGGGGCC ATTGCACTTG GACACCCGCT TGCCGCGACC GGATTGCGTC1140TCTGCATGAC CCTCGCTCAC CAATTGCAAG CTAATAACTT TCGATATGGA ATTGCCTCGG1200CATGCATTGG TGGGGGACAG GGGATGGCGG TTCTTTTAGA GAATCCCCAC TTCGGTTCGT1260CCTCTGCACG AAGTTCGATG ATTAACAGAG TTGACCACTA TCCACTGAGC TAACGGGCAT1320CTCCTTTGTT GCTTTGAGGT GGCGCACGAA GGAGGGCTCG AAAATCTCTG CTAAAAACAA1380GAAGAAGGAA CAGGGAACAT GATTAGTTTC GCTCGTATGG CAGAAAGTTT AGGAGTCCAG1440GCTAAACTTG CCCTTGCCTT CGCACTCGTA TTATGTGTCG GGCTGATTGT TACCGGCACG1500GGTTTCTACA GTGTACATAC CTTGTCAGGG TTGGTGGGAA TTC 154權(quán)利要求
1．轉(zhuǎn)化的和/或誘變的單細(xì)胞或多細(xì)胞生物，其特征在于丁子香酚和/或阿魏酸分解代謝的酶被滅活，使得中間產(chǎn)物松柏醇，松柏醛，阿魏酸，香草醛和/或香草酸積累。
2．根據(jù)權(quán)利要求1的生物，其特征在于經(jīng)過在相應(yīng)的基因插入Ω元件或?qū)肴笔Ц淖兌∽酉惴雍?或阿魏酸分解代謝。
3．根據(jù)權(quán)利要求1或2的生物，其特征在于編碼松柏醇脫氫酶，松柏醛脫氫酶，阿魏酰-CoA合成酶，烯酰-CoA水合酶-醛縮酶，β-酮硫解酶，香草醛脫氫酶或香草酸脫甲基酶的一個或多個基因被改變和/或被滅活。
4．根據(jù)權(quán)利要求1-3之一的生物，其特征在于它是單細(xì)胞的，優(yōu)選微生物或植物或動物細(xì)胞。
5．根據(jù)權(quán)利要求1-4之一的生物，其特征在于它是細(xì)菌，優(yōu)選假單胞菌屬種類。
6．編碼松柏醇脫氫酶，松柏醛脫氫酶，阿魏酰-CoA合成酶，烯酰-CoA水合酶-醛縮酶，β-酮硫解酶，香草醛脫氫酶或香草酸脫甲基酶，或兩個或多個該酶的核苷酸序列被改變和/或被滅活的基因結(jié)構(gòu)。
7．具有圖1a-1r所給序列的基因結(jié)構(gòu)。
8．具有圖2a-2r所給序列的基因結(jié)構(gòu)。
9．含有至少一個根據(jù)權(quán)利要求6-8之一的基因結(jié)構(gòu)的載體。
10．根據(jù)權(quán)利要求1-5之一的轉(zhuǎn)化的生物，其特征在于它含有至少一個根據(jù)權(quán)利要求9的載體。
11．根據(jù)權(quán)利要求1-5之一的生物，其特征在于它含有整合進基因組中代替各完整基因的至少一個根據(jù)權(quán)利要求6-8之一的基因結(jié)構(gòu)。
12．有機化合物，特別是醇，醛和有機酸的生物技術(shù)制備方法，其特征在于使用根據(jù)權(quán)利要求1-5或10-11之一的生物。
13．制備根據(jù)權(quán)利要求1-5之一的生物的方法，其特征在于借助于本身已知的微生物學(xué)培養(yǎng)方法實現(xiàn)改變丁子香酚和/或阿魏酸分解代謝。
14．制備根據(jù)權(quán)利要求1-5或10-11之一的生物的方法，其特征在于借助于重組DNA方法實現(xiàn)改變丁子香酚和/或阿魏酸分解代謝，和/或滅活相應(yīng)的基因。
15．根據(jù)權(quán)利要求1-5或10-11之一的生物在制備松柏醇，松柏醛，阿魏酸，香草醛和/或香草酸中的應(yīng)用。
16．根據(jù)權(quán)利要求6-8之一的基因結(jié)構(gòu)或根據(jù)權(quán)利要求9的載體在制備轉(zhuǎn)化的和/或誘變的生物中的應(yīng)用。
全文摘要
本發(fā)明涉及轉(zhuǎn)化的和/或誘變的單或多細(xì)胞生物體,其特征在于,丁子香酚和/或阿魏酸代謝的酶被滅活,使得中間產(chǎn)物松柏醛,松柏醇,阿魏酸,香草醛和/或香草酸被積累。
文檔編號C12N1/19GK1325444SQ99812907
公開日2001年12月5日 申請日期1999年10月20日 優(yōu)先權(quán)日1998年10月31日
發(fā)明者J·拉本霍爾斯特, A·斯坦比歇爾, H·普里菲爾特, J·奧維爾哈格 申請人:哈爾曼及賴默股份有限公司