CN101951755A - 来自藻类的二酰基甘油酰基转移酶2基因及其编码的蛋白质 - Google Patents
来自藻类的二酰基甘油酰基转移酶2基因及其编码的蛋白质 Download PDFInfo
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Abstract
本发明公开内容涉及从藻类中分离、纯化以及鉴定一种二酰基甘油酰基转移酶2(DGAT2),以及编码DGAT2的基因。DGAT2可以参与超长链的多不饱和脂肪酸向三酰基甘油转化的过程,且效率远高于DGAT1。本发明公开内容涉及使用DGAT2基因调节种子油含量,脂肪酸合成,以及脂肪酸组成的方法,还涉及了使用该基因转化的组织和植物。本发明公开内容还涉及了具有包含导入的本公开内容中的DNA序列的转基因植物,植物组织以及植物种子,以及生产所述植物及植物种子的方法。
Description
优先权要求
本申请要求申请号为60/008,752,申请日为2007年12月21日,名称为“来自藻类的二酰基甘油酰基转移酶2基因及其编码的蛋白质”的美国临时专利申请的优先权。
技术领域
本公开内容大体上涉及生物技术领域,更特别地,涉及对植物特性进行基因操纵的有用的基因。在一些特定的具体实施方式中,本公开涉及分离和/或纯化的编码二酰基甘油酰基转移酶2(DGAT2)的多肽和核酸以及其应用方法。
背景技术
油料种子作物是重要的农业经济作物。植物种子油是从人类饮食中获得必要的多不饱和脂肪酸的主要来源,并且是化学工业中的可再生原料。在发育的种子的质体中,脂肪酸合酶的复杂体系中的酶类负责脂肪酸的生物合成,这些脂肪酸被引导入胞浆酰基辅酶A池内,以维持三酰基甘油的累积。三酰基甘油(TAG)的生物合成位于内质网上,使用3-磷酸甘油和脂肪酰辅酶A作为基本底物。有三种酰基转移酶参与植物的储存性的脂质的生物合成,其名称分别为甘油-3-磷酸酰基转移酶(GPAT,EC2.3.1.15),溶血磷脂酸酰基转移酶(LPAT,EC2.3.1.51)以及二酰基甘油酰基转移酶(DGAT,EC2.3.1.20)。这三种酰基转移酶催化甘油骨架的分步式的酰基化反应,并且最后一步是通过DGAT将sn-1,2-二酰甘油(DAG)酰基化形成TAG,这个生化过程通常被称为Kennedy途径。已经公开了DGAT介导的甘油骨架的酰基化反应生产TAG是植物脂质积累过程中的限速步骤。因此,DGAT是植物脂质生物合成中的遗传修饰的一个靶点。
发明内容
我们在此公开了具有与假微型海链藻(T.pseudonana)中二酰基甘油酰基转移酶2(DGAT)至少90%的序列同一性的一系列多肽。这些多肽可以用于改变植物中多不饱和脂肪酸的水平。同样公开了包含假微型海链藻(Thalassiosira pseudonana)DGAT2的催化性的二酰甘油转移酶结构域的多肽,以及具有与DGAT2的催化性的二酰甘油转移酶结构域至少90%的序列同一性的多肽。进一步描述了编码具有与假微型海链藻DGAT2至少90%的序列同一性的多肽的多聚核苷酸序列,以及编码具有与假微型海链藻DGAT2的催化性的二酰甘油转移酶结构域至少90%的序列同一性的多肽的多核苷酸。
在此我们公开了从假微型海链藻中分离和纯化出的二酰基甘油酰基转移酶2(DGAT)基因以及cDNA序列。同样公开了假微型海链藻中的DGAT2的cDNA的全长序列,并且该cDNA序列具有与DGAT2的cDNA至少80%的序列同一性。在一些具体实施方式中,这些cDNA序列可以包含于载体中。这些多核苷酸可用于修饰植物中的三酰基甘油的天然合成以用于提高商品化植物油的产量,或者用于修饰其组成来获得植物以及植物产品中特定的商品成分的改善。
同样公开了从假微型海链藻中分离及纯化的其它的DGAT2家族的基因和cDNA序列,以及来自其它藻类种属的DGAT2家族的基因和cDNA序列,这些藻类种属包括莱茵衣藻(Chlamydomonas reinhardtii),绿藻(Ostreococcuslucinarinus),Ostreococcus tauri,以及三角褐指藻(Phaeodactylumtricornutum)。这些多核苷酸也可以用于修饰植物中的三酰基甘油的天然合成以用于提高商品化植物油的产量,或者用于修饰其组成来获得植物以及植物产品中特定的商品成分的改善。
一种包含核酸构建体的转基因植物同样被公开。描述了转化细胞或植物的方法;该方法包括将分离的、纯化的或重组的核酸引入到细胞或植物中。生产基因转化植物种子的过程包括将核酸转入到植物种子中去。在一些具体实施方式中,这些方法可以用来修饰植物以改变其种子油含量。
更通常地说,一些实施例公开了分离、纯化和鉴定藻类中的DGAT2基因,以及DGAT2在超长链的多不饱和脂肪酸的生产中的应用。通过下文详细描述的一些实施例,其结合附图作为参考,上述的内容将更易被理解,
附图说明
图1描述了推导的氨基酸序列(SEQ ID NO:1),对应于假微型海链藻的DGAT2 cDNA全长序列(SEQ ID NO:2)。
图2描述了SEQ ID NO:1(TpDGA2)和gi:37182187、gi:50541689、gi:74623358、gi:74623359、gi:86279638以及gi:62825813的序列比对结果,上述均为2型二酰基甘油酰基转移酶。序列中的4个或更多共有的氨基酸用粗体表示。包括这些2型二酰基甘油酰基转移酶的催化性的二酰甘油转移酶结构域的氨基酸序列由如下残基组成:236-365(TpDGA2);79-208(gi:37182187);76-205(gi:50541689);76-205(gi:74623358);34-165(gi:74623359);33-165(gi:86279638);36-165(gi:62825813)。
图3A描述了与SEQ ID NO:1同源的多肽序列的一个例子(SEQ ID NO:3)。图3B描述了与SEQ ID NO:2具有至少90%序列同源性的多核苷酸序列的一部分,和编码SEQ ID NO:3的多肽的一部分;(SEQ ID NO:4)。
图4A描述了与SEQ ID NO:1同源的多肽序列的另一个例子;(SEQ IDNO:5)。图4B描述了与SEQ ID NO:2具有至少90%序列同源性的多核苷酸序列的一部分,和编码SEQ ID NO:5的多肽的一部分;(SEQ ID NO:6)。
图5A描述了与SEQ ID NO:1同源的多肽序列的另一个例子;(SEQ IDNO:7)。图5B描述了与SEQ ID NO:2具有至少90%序列同源性的多核苷酸序列的一部分,和编码SEQ ID NO:7的多肽的一部分;(SEQ ID NO:8)。
图6A描述了与SEQ ID NO:1同源的多肽序列的另一个例子;(SEQ IDNO:9)。图6B描述了与SEQ ID NO:2具有至少90%序列同源性的多核苷酸序列的一部分,和编码SEQ ID NO:9的多肽的一部分;(SEQ ID NO:10)。
图7A描述了与SEQ ID NO:1同源的多肽序列的另一个例子;(SEQ IDNO:11)。图7B描述了与SEQ ID NO:2具有至少90%序列同源性的多核苷酸序列的一部分,和编码SEQ ID NO:11的多肽的一部分;(SEQ ID NO:12)。
图8A描述了与SEQ ID NO:1同源的多肽序列的另一个例子;(SEQ IDNO:13)。图8B描述了与SEQ ID NO:2具有至少90%序列同源性的多核苷酸序列的一部分,和编码SEQ ID NO:13的多肽的一部分;(SEQ ID NO:14)。
图9描述了薄层层析法(TLC)对在酵母突变体H1246 MATα(DGAT、PDAT-、ASAT1-、ASAT2-,其为TAG合成缺陷型)中通过表达TpDGAT2和AtDGAT1产生的TAG的分析。泳道1显示了AtDGAT1的表达,泳道2-6显示了TpDGAT2的表达。可以在泳道2-6观察到清晰的TAG(三酰基甘油)条带。泳道8表示空载体(pYES2.1)对照,并且此泳道没有TAG(三酰基甘油)条带。泳道8的右侧泳道的上样为TAG标准品,其用作TAG标记物。
图10显示了用空载体(pYES2.1 Con;空白柱形),用假微型海链藻的DGAT2 cDNA(pYES:DGAT;斑点状柱形),用拟南芥(A.thaliana)DGAT1cDNA(纯黑柱形)转化的酵母突变体H1246 MATα中的DGAT活性。使用不同的14C标记的酰基辅酶A作为酰基供体,以及未标记的sn-1,2甘油二油酸酯作为受体,来检测通过诱导的酵母细胞裂解物制备得到的微粒体膜组分以测定DGAT的活性。此处相对DGAT活性用DPM(掺入合成到TAG中的14C标记的底物的数量)表示。结果显示了底物偏好性以及TpDGAT2和AtDGAT1的相对活性。
图11描述了TpDGAT2(TpDGAT2-1)与来自假微型海链藻(T.pseudonana)或来自其它藻类种属(Cr-莱茵衣藻(Cr-Chlamydomonasreinhardtii);Ol-绿藻(Ol-Ostreococcus lucimarinus);Ot-Ostreococcus tauri;Pt-三角褐指藻(Pt-Phaeodactylum tricornutum))的家族成员的氨基酸序列的同源性比较。TpDGA2(TpDGAT2-1)与其家族成员TpDGAT2-2、TpDGAT2-3、TpDGAT2-4分别拥有24%、25%以及17%的序列同一性。在这些不同的藻类种属中TpDGAT2(TpDGAT2-1)显示了与PtDGAT2-1的高序列相似性(48%序列同一性),以及与CrDGAT2-1、CrDGAT2-2、和CrDGAT2-3的相对高的相似性(分别为20%、23%以及24%)。
具体实施方式
Ⅰ一些具体实施方式的概述
此处公开了从假微型海链藻中分离及纯化的2型二酰基甘油酰基转移酶(DGAT2)。这种多肽令人惊讶的能力为在其它生物以及其它生物细胞中修饰超长链的多不饱和脂肪酸(VLCPUFA)的合成,这种能力可用来转化植物以及植物种子来产生包含所需的脂肪酸组成的转基因植物及植物种子。本公开内容中还包括与假微型海链藻的DGAT2的氨基酸序列具有至少90%序列同一性并且具有DGAT2活性的多肽。在一些特定的具体实施方式中,这些多肽序列包括,例如,SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11或SEQ ID NO:13。同样公开了与假微型海链藻DGAT2的催化性二酰基甘油酰基转移酶结构域具有至少90%序列同一性的序列的多肽。在一些特定的具体实施方式中,这些多肽序列包括,例如,SEQ ID NO:15、SEQ ID NO:17或SEQ ID NO:19。在图2中描述了假微型海链藻DGAT2的催化性二酰基甘油酰基转移酶结构域;其由假微型海链藻DGAT2完整公开的多肽序列中的236-365位氨基酸残基构成。
SEQ ID NO:15的多肽包括假微型海链藻DGAT2的催化性二酰基甘油酰基转移酶结构域。一些具体实施方式中涉及包括与分离或纯化的如SEQ ID NO:15所示的多肽具有至少约90%、91%、92%、93%、94%、95%、96%、97%、98%,、99%、99.5%、99.6%或99.7%序列同一性的分离或纯化的多肽,例如,SEQID NO:1。在特别的具体实施方式中,这些多肽具有二酰基甘油酰基转移酶活性。本领域技术人员可以通过,例如,体外酶活性测定方法,很容易测定二酰基甘油酰基转移酶的活性。该方法的具体细节在实施例4中进行了详述,并且该检测的典型的结果如图10所示。本领域技术人员很容易理解,本公开内容同样涉及从植物及藻类中得到的大幅度地同源的DNA序列,和编码包括与SEQ ID NO:15的推导的氨基酸序列具有90%氨基酸序列同源性或更高同源性的蛋白质。
其它的从藻类中分离或纯化的DGAT2家族的成员多肽,其包括与,例如SEQ IDNO:25(TpDGAT2-2)、SEQ ID NO:27(TpDGAT2-3)、SEQ ID NO:29(TpDGAT2-4)、SEQ ID NO:31(CrDGAT2-1)、SEQ ID NO:33(CrDGAT2-2)、SEQ ID NO:35(CrDGAT2-3)、SEQ ID NO:37(CrDGAT2-4)、SEQ ID NO:39(CrDGAT2-5)、SEQ IDNO:41(OlDGAT2-1)、SEQ ID NO:43(OlDGAT2-2)、SEQ ID NO:45(OlDGAT2-3)、SEQ ID NO:47(OlDGAT2-4)、SEQ ID NO:49(OtDGAT2-1)、SEQ ID NO:51(OtDGAT2-2)、SEQ ID NO:53(OtDGAT2-3)、SEQ ID NO:55(OtDGAT2-4)、SEQ IDNO:57(PtDGAT2-1)、SEQ ID NO:59(PtDGAT2-2)、SEQ ID NO:61(PtDGAT2-3)或SEQ ID NO:63(PtDGAT2-4)的氨基酸序列具有至少90%序列同源性的氨基酸序列。
一些具体实施方式涉及到编码上述的多肽的分离或纯化的核酸(多核苷酸)。这些多核苷酸序列可以包括,例如,SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:26(TpDGAT2-2)、SEQ ID NO:28(TpDGAT2-3)、SEQ ID NO:30(TpDGAT2-4)、SEQ ID NO:32(CrDGAT2-1)、SEQ IDNO:34(CrDGAT2-2)、SEQ ID NO:36(CrDGAT2-3)、SEQ ID NO:38(CrDGAT2-4)、SEQ ID NO:40(CrDGAT2-5)、SEQ ID NO:42(OlDGAT2-1)、SEQ ID NO:44(OlDGAT2-2)、SEQ ID NO:46(OlDGAT2-3)、SEQ ID NO:48(OlDGAT2-4)、SEQ IDNO:50(OtDGAT2-1)、SEQ ID NO:52(OtDGAT2-2)、SEQ ID NO:54(OtDGAT2-3)、SEQ ID NO:56(OtDGAT2-4)、SEQ ID NO:58(PtDGAT2-1)、SEQ ID NO:60(PtDGAT2-2)、SEQ ID NO:62(PtDGAT2-3)或SEQ ID NO:64(PtDGAT2-4)。在一些具体实施方式中,多核苷酸序列具有与编码公开的编码多肽的的公开的多核苷酸的碱基具有百分比为至少约80%、81%、82%、83%、84%、85%、,86%、,87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、99.5%、99.6%或99.7%的同源性。所述多核苷酸的一些例子为SEQ ID NOS:21-24。作为本领域技术人员容易理解的是,核酸序列上的轻微的改变不一定会改变其编码多肽的氨基酸序列。本领域技术人员很容易理解特定基因序列的同源性的改变可以改变其编码的氨基酸序列,可能会导致降低或提高基因的有效性,并且,在一些应用中(即,反义的引物、协同抑制、或RNAi),部分序列经常与全长形式一样有效地起作用。如何进行基因序列改变或缩短的方法是本领域技术人员熟知的,同样熟知的还有那些检测改变的基因的有效性的方法。在一些特定的具体实施方式中,有效性可能很容易地检测,通过,例如,常规的气象色谱法检测。在此将所有这样的基因的变体都引入作为本发明的一部分。
一些具体实施方式涉及一种载体,其包含分离或纯化的多核苷酸,所述多核苷酸与SEQ ID NO:2具有至少80%的同源性,例如,SEQ ID NO:2、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:20、SEQ IDNO:21-24。因此,提供了一种制备一种载体的方法,所述载体包括选自,例如SEQ IDNO:2,例如,SEQ ID NO:2、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:21-24或其片段的序列,将这些序列或其反向的部分序列,或其互补序列导入到植物细胞中。
特定的具体实施方式涉及包括与藻类中DGAT2家族成员具有至少80%同源性的序列的载体。这些载体可包括多核苷酸序列,例如,SEQ ID NO:26、SEQ IDNO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQED NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQID NO:58、SEQ ID NO:60、SEQ ID NO:62、或SEQ ID NO:64.
在一些具体实施方式中,分离和纯化的多核苷酸,以及包含这些分离和纯化的多核苷酸的载体,可以用于构建产生具有DGAT2活性的多肽的转基因植物。因此,一种具体实施方式涉及包括分离或纯化的多核苷酸的转基因植物以及植物种子,所述多核苷酸与SEQ ID NO:2具有至少80%同源性;例如,脱氧核糖核酸分子具有SEQEE NO:2、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:20、或SEQ ID NO:21-24的序列。其它的具体实施方式涉及包括分离或纯化的多核苷酸序列的转基因植物和植物种子,所述多核苷酸序列具有与藻类DGAT2家族的另一成员至少80%的同源性;例如,脱氧核糖核酸分子具有SEQ IDNO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、或SEQ ID NO:64的序列。与缺乏这种核酸构建体的植物相比,这些具体实施方式中的植物可以在种子中具有改变了的多不饱和脂肪酸的水平。这些植物中的脂肪酸可约有超过70%为多不饱和脂肪酸。
一种具体实施方式中包括生产所述植物和植物种子的方法。该方法包括构建一种具有编码多肽的多核苷酸的核酸构建体,所述多肽与选自SEQ ID NO:1、3、5、7、9、11、13、15、17和19的多肽具有至少90%的序列同一性,或者所述多肽与藻类DGAT2家族的多肽具有至少90%的序列同一性;例如,SEQ ID NO:25、SEQID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQID NO:57、SEQ ID NO:59、SEQ ID NO:61或SEQ ID NO:63;以及将该构建体导入植物中。该具体实施方式的方法可使用本领域普通技术人员熟知的任意方法完成,非限制性的例子为,农杆菌介导的转化。在具体的实施方式中,该方法还包括将编码具有Brusica丙酮酸脱氢酶激酶活性多肽的多核苷酸,编码具有二酰甘油乙酰转移酶活性多肽的多核苷酸,和/或具有编码3-磷酸-甘油脱氢酶活性多肽的多核苷酸,导入到植物中。该方法可能在选自如下的植物中实施,所述植物选自拟南芥(Arabidopsis thaliana)、琉璃苣(Borago spp.)、加拿大油料(Canola)、蓖麻(Ricinusspp.)、可可(Theobroma spp.)、玉蜀黍(Zea spp.)、棉(Gossypium spp)、海甘蓝(Crambe spp.)、萼距花(Cuphea spp.)、亚麻(Linum spp.)、雷斯克勒(Lesquerellaspp.)、鲸油草(Limnanthes spp.)、亚麻的突变株(Linola)、旱金莲(Tropaeolumspp.)、月见草(Oenothera spp.)、洋橄榄(Olea spp.)、油棕(Elaeis spp.)、花生(Arachis spp.)、油菜籽(rapeseed)、红花(Carthamus spp.)、大豆(Glycine spp.)、野生大豆(Soja spp.)、向日葵(Helianthus spp.)、烟草(Nicotiana spp.)、斑鸠菊(Vernonia spp.)、小麦(Triticum spp.)、大麦(Hordeum spp.)、稻(Oryza spp.)、燕麦(Avena spp.)、高粱(Sorghum spp.),、黑麦(Secale spp.)、十字花科植物(Brassicaceae),以及其它禾本科(Gramineae)植物。
在一些具体实施方式中,该方法还包括从包含引入的核酸构建体的植物中收获种子,以及从收获的种子中提取油。因此,其它的一些具体实施方式包括通过该方法产生的植物,以及从该方法产生的植物中提取的油。
一些可能使用DGAT2基因或其部分进行的操纵和传递,包括,但不限于,如下:提高或降低油含量的种子;具有提高的超长链的多不饱和脂肪酸含量的种子,以及显示了提高的或改变的富集超长链的多不饱和脂肪酸能力的植物。
Ⅱ缩写词
CaMV花椰菜花叶病毒
cDNA互补DNA
CERV香石竹蚀环病毒
CrDGAT2莱茵衣藻2型二酰甘油转移酶
DAG sn-1,2-甘油二酯
DGAT二酰基甘油酰基转移酶
DGAT2 2型二酰甘油转移酶
DHA二十二碳六烯酸
DNA脱氧核糖核酸
EPA二十碳五烯酸
GPAT甘油-3-磷酸酰基转移酶
LPAT溶血磷脂酸酰基转移酶
OlDGAT2绿藻(Ostreococcus lucimarinus)2型二酰甘油转移酶
OtDGAT2 Ostreococcus tauri 2型二酰甘油转移酶
PCR聚合酶链式反应
PtDGAT2三角褐指藻(Phaeodactylum tricornutum)2型二酰甘油转移酶
RNA核糖核酸
RNAi RNA干扰
RT-PCR逆转录PCR
T35S CaMV 35S终止子
TAG三酰基甘油
TLC薄层层析法
Tmas甘露碱合酶终止子
Tnos胭脂碱合酶终止子
TpDGAT2假微型海链藻(T.pseudonana)2型二酰甘油转移酶
TrbcS二磷酸核酮糖盐羟化酶小亚基终止子区域
VLCPUFA超长链多不饱和脂肪酸
Ⅲ术语
为了更容易阅读本公开内容中的各种不同的实施例,下文提供了各种专用名词的解释:
互补核酸序列:一条序列的“互补核酸序列”的含义为任意的DNA,其核酸序列互补于本公开内容中的序列,并且其方向为相反的(反向平行序列)。
序列同源性的程度或百分比:术语“序列同源性的程度或百分比”指的是两条序列最适的比对之后其序列同一性的程度或百分比。序列同一性百分比(或程度或同一性)定义为在比较窗口中最适排布下比较两条序列,在比较窗口中的两条序列最适排布下肽或多核苷酸序列的部分序列与参考序列(其不包括添加或缺失)相比可能包括添加或缺失(即,缺口)。所述百分比通过确定两条序列中存在的相同的氨基酸残基或核苷酸碱基产生的配对位置的数量来计算,将配对位置的数量除以比较窗口中总的位置总数,然后将结果乘以100即可获得序列同一性的百分比。
同源的分离和/或纯化序列:“同源的分离和/或纯化序列”是指分离和/或纯化的序列,其与核酸序列的碱基,或多肽序列的氨基酸,具有至少约80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、99.5%、99.6%或99.7%的同一性百分比。该百分比为纯统计学的,并且可能根据两条核酸序列在随机或全长上的不同进行归类。序列同一性可以通过,例如,设计的执行单个和多重序列比对的计算机程序进行确定。可以知道本领域任意普通技术人员都可以理解本发明包含密码子使用的简并性。此外,本领域技术人员可以理解在多肽氨基酸序列上可进行不破坏该多肽结构或功能的保守替代。本领域技术人员可以通过引入相似疏水性、极性、以及R-链长度的氨基酸替换另一个氨基酸来完成保守替代。另外,通过不同种属的同源蛋白质的序列间的比对,保守替代可能通过定位在种属间发生突变的氨基酸残基来确定,所述突变的氨基酸残基没有改变编码蛋白的基本功能。
分离:本领域技术人员容易理解地,“分离”指的是从其天然环境中“分离”获得的多肽。
核苷酸、多核苷酸、或核酸序列:“核苷酸,多核苷酸,或核酸序列”指的是单体和二聚的(所谓的串联)形式的双链或单链DNA以及所述DNA的转录产物。
序列同一性:当两条序列中的氨基酸序列或核苷酸残基如下文所述进行最大匹配度比对时两者相同,两条氨基酸或核酸序列被称为“同一的”。两条(或多条)多肽或多核苷酸序列比对一般通过比较两条最佳比对的序列过分割或“比较窗口”中确定并比较局部位置的序列相似性。序列最适比对的比较可以通过Smith和Waterman,Ad.App.Math 2:482(1981)的局部同源性算法,或者Neddlemanhe和Wunsch,J.Mol.Biol.48:443(1970)的同源性算法,或者Pearson和Lipman,Proc.Natl.Acad.Sci.(USA)85:2444(1988)的相似性检索,或者这些算法的计算机化的工具(WisconsinGenetics Software Package中的GAP、BESTFIT、FASTA、和TFASTA,GeneticsComputer Group(GCG),575 Science Dr.,Madison,Wis.),或者直接观察来进行。
上述给出的序列同一性的定义为本领域技术人员均可使用的定义。定义自身不需要任何算法的帮助,所述算法只有在获得序列的最适比对时,而非在计算序列同源性时使用。
从上述给出的定义,对应于最佳或最适比对的序列的获得的值,两条比较序列间有一个清晰可辨的并且唯一的序列同一性值。
在BLAST N或BLAST P“BLAST 2序列”中,软件可从网页http://worldwiseweb.ncbi.nlm.nih.gov/gorf/bl2.html上获得,发明人习惯性使用并且通常本领域技术人员用这些来比较和确定两条序列的同一性,依赖于比较的序列长度的断裂值可以直接在软件上选择(即,取代矩阵BLOSUM-62,长度>85,值为11.2)。
严格(条件下的)杂交:核酸序列的严格条件下的杂交的含义为在选择的温度和离子强度的条件下的杂交,所述条件使得两个互补DNA片段维持杂交。此处描述的从其他生物获得的DGAT2基因的同源性,例如,从植物中,可能通过扫描合适的同系物群来获得,其中扫描使用此处公开的特异的DGAT2基因的核酸序列,或其片段或探针来实行,或使用序列比对检索程序如BLAST、FASTA来进行序列同源性比对检索。
Ⅲ.从藻类获得的DGAT2修饰脂肪酸水平
A.概述
最近关于桐树以及蓖麻子中获得的DGAT2的研究显示了植物含有罕见的脂肪酸,DGAT2可以在罕见的脂肪酸到种子储存油的通路上有重要的作用。而DGAT2可以是植物油料种子中油脂生物合成基因修饰的潜在靶点,最近鉴定出的酶类分别利用共轭的脂肪酸桐油酸(桐树DGAT2)和蓖麻油酸(蓖麻子DGAT2)。任何一种酶(桐树DGAT2或蓖麻子DGAT2)都不参与三酰基甘油(TAG)中的商业上想要的长链ω-3多不饱和脂肪酸二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)。
海洋环纹硅藻(marine centric diatom algae)假微型海链藻可以生产和累积TAG中的长链ω-3多不饱和脂肪酸EPA和DHA,并且是高水平的超长链多不饱和脂肪酸(VLCPUFA)累积油类的来源。由于此原因,研究并鉴定了假微型海链藻中的二酰基甘油酰基转移酶(TpDGAT2)基因。令人惊奇的是,发现了TpDGAT2,其不像从桐树或蓖麻子中获得的DGAT2,可以有效地参与超长链多不饱和脂肪酸合成TAG。使用TpDGAT2基因来从假微型海链藻及相关藻类种属中检索多核苷酸序列,藻类中的其他DGAT2家族成员被确定。因此,确定藻类DGAT2基因在应用转基因工具中对于种子中的TAG组成和累积是有效的。
B.与具有2型二酰甘油转移酶活性的假微型海链藻DGAT2同源的多肽
使用搜索引擎,如BLAST,能够通过检索蛋白数据库(如NCBI蛋白质数据库)找到与全长的假微型海链藻DGAT2同源的蛋白质。其同样可以通过合理设计获得。合理设计的过程可以包括在所需获得的多肽长度中鉴别保守的氨基酸取代,并且在编码蛋白中制造这些取代。
使用假微型海链藻的DGAT2的全长氨基酸序列在NCBI蛋白质数据库(BLASTP)中检索时显示出了与TpDGAT2显著的序列同源性的多肽,其中在图2中示出了一些与TpDGAT2的比对结果。保守的2型二酰甘油转移酶结构域的比对如图2所示,由TpDGAT2的236-365位氨基酸残基以及与其他的DGAT2多肽相应的残基组成。保守的2型二酰甘油转移酶结构域在NCBI保守结构域数据库中有描述(http://worldwideweb.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml)。与该保守结构域同源的多肽序列赋予了包含该片段的蛋白质TpDGAT2的2型二酰甘油活性。
本领域普通技术人员可以理解,具有同源序列的多肽因其具有同源性可以设计为显示具有相同的结构和功能。本领域技术人员能够设计与那些在本公开内容图2序列比对中列出的具体的例子同源的多肽。这些同源多肽可以是那些本发明公开的多肽的保守取代的多肽,例如,SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、或SEQ ID NO:19的多肽。可以确定这些同源的多肽是否具有与TpDGAT2实质上相似的2型二酰甘油转移酶活性的简单的实验分析方法是本领域技术人员已知的。这些分析不需要过度费时,也不昂贵,也没有技术困难。例如,常规的气相色谱法可以用来检测由TpDGAT2产生的TAG。在下文详述的实施例中对这些分析试验进行了描述。
C.核酸分子应用于转化具有DGAT2活性
必须要理解公开的具体实施方式不包括在它们天然环境中的基因组核酸序列,也就是说,在假微型海链藻(T.pseudonana)、莱茵衣藻(hlamydomonas reinhardtii),绿藻(Ostreococcus ludmarinus)、Ostreococcus tauri、或三角褐指藻(Phaeodactylumtricornutum)的天然基因组中。一些具体实施方式涉及到已经能够从分离方法开始,如,离子交换色谱法,通过基于分子大小的排斥选择,或通过亲和性,或可替代地基于不同溶剂中的溶解度的分级分离技术进行分离、纯化或部分纯化的序列,或者从基因工程方法开始,如扩增,克隆,及亚克隆进行分离、纯化或部分纯化的序列,其可能是由载体携带序列。
进一步包括核酸分子,其可以杂交到上述公开的序列上。杂交条件可以是严格的,因此当其与编码公开的DGAT2分子的核酸分子具有至少90%,95%或97%序列同一性时才会发生杂交。严格条件可以包括那些用于已知的Southern杂交的条件,例如,在具有50%甲酰胺、5×SSC(150mM NaCl,15mM枸橼酸钠)、50mM磷酸钠(pH7.6)、5×Denhardt’s溶液、10%硫酸葡聚糖、以及20微克/毫升的变性、剪切的鲑鱼精DNA的溶液中42℃孵育过夜,随后用0.1×SSC在约65℃下清洗杂交支持物。其他广泛已知的杂交条件参见Sambrook等Molecular Cloning:A Laboratory Manual,Third Edition,Cold Spring Harbor,N.Y.(2001).
已经很好地建立了DNA的分离和克隆。相似的,编码分离酶类的DNA可以被插入到载体中,用常规的方法转入到酵母细胞。然而,因为没有可有效利用VLCPUFA的DGAT2基因已经被克隆过,所以还不可能通过调整DGAT2的活性来进行定位遗传修饰。我们确定了DGAT2参与TAG合成并且比DGAT更有效地利用VLCPUFA。
编码DGAT2的核酸分子,例如与SEQ ID NO:2、SEQ ID NO:16、SEQ IDNO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQID NO:55、SEQ ED NO:57、SEQ ID NO:59或SEQ ID NO:61具有至少80%同一性的序列可以转入一种生物体中,例如一种植物。这些同源序列为例如SEQ IDNO:21-24。本领域已知的,有许多方法将基因和基因构建体导入生物体,如植物中,并且转化和组织培养技术的结合已经成功地整合以实现创造转基因生物体的有效的策略,例如作物植物。这些方法已经在大量文献中被描述过(Potrykus,1991;Vasil,1994;Walden和Wingender,1995;Songstad等,1995),其为本领域技术人员所熟知。例如,本领域技术人员可以确定地知道,农杆菌介导的拟南芥转化可以通过真空浸染(Bechtold等,1993)或有伤接种(Katavic等,1994),同样,使用农杆菌Ti质粒介导的转化(如,下胚轴(DeBlock等,1989)或子叶叶柄(Moloney等,1989)伤口侵染),粒子轰击/基因枪法(Sanford等,1987;Nehra等,1994;Becker等,1994)或聚乙二醇辅助的原生质体转化(Rhodes等,1988;Shimamoto等,1989)方法,能够转化其他植物或作物种属。
在植物中通过遗传工程转化新的基因或改变已有基因的表达修饰植物的代谢有许多成功的例子。目前通常将基因导入到许多农学上重要的植物种属中来改善作物生长状况(如,植物油或块茎淀粉量/组成;膳食改善;除草剂、疾病或害虫抗性;重金属耐受等)(MacKenzie和Jain,1997;Budziszewski等,1996;Somerville,1993;Kishore和Somerville,1993)。
对本领域技术人员来说同样明显的是,和其他地方的说明(Meyer,1995;Dada等,1997),其可能利用植物启动子直接预期上调或下调转基因的表达(如,基于CaMV35S),或通过使用启动子其可使基因在特定的细胞、组织中(如,napin启动子在发育的种子子叶中的转基因表达)、器官(如,根)中,或者在一个特定的发育的阶段,或特定的外界刺激(如,热激)下表达。
此处使用的启动子可以是可诱导型的、组成型的、或组织特异型的、或具有这些特征的不同的组合。有效的启动子包括,但不限于,组成型启动子,如,香石竹蚀环病毒(CERV),花椰菜花叶病毒(CaMV)35S启动子,或更特别的双倍增强花椰菜花叶病毒启动子,其包括串联的两个CaMV 35S启动子(被称为“双35S”启动子)。
在特定情况中可能希望使用组织特异型或生长发育调节启动子代替组成型启动子。组织特异性启动子允许在特定的组织中过表达而不影响其他的组织中的表达。举例来说,在种子组织中过表达酶的启动子为ACP启动子,在PCT国际公布文本WO92/18634,公开日为1992年10月29日的文件中有描述。
启动子和终止调节区可以在宿主细胞中具有功能,并且可以与植物细胞和基因是异源的(是指,非自然发生)或同源的(从植物宿主物种中获得)。可能用到的合适的启动子如上文所述。
终止调节区域可以从获得启动子的基因的3′区获得,或来自其他基因。可用的合适的终止区是本领域熟知的,包括根癌农杆菌胭脂碱合酶终止子(Tnos),根癌农杆菌甘露碱合酶终止子(Tmas)和CaMV 35S终止子(T35S),豌豆二磷酸核酮糖盐小亚基终止子区(TrbcS),或Tnos终止子区。这些基因构建体可以适于筛选通过农杆菌转化宿主细胞的活性和筛选提高的类异戊二烯水平。
合适的,基因的核苷酸序列可以从GenBank(美国卫生与公众服务部的注册商标)核苷酸数据库中获得,并且可以搜索不会酶切该核苷酸的限制性内切酶。这些限制性酶切位点可以通过常规方法,如在PCR引物中加入酶切位点或通过亚克隆添加到基因上。
优选的,此处使用的DNA构建体包含在一个载体中,最合适的是一个适于在合适宿主(植物)细胞中表达的表达载体。可以理解的是可以产生包括导入DNA序列的植物的任何载体都可以。
合适的载体对于本领域技术人员来说是众所周知的,并且在一般技术参考文献如Pouwels等,Cloning Vectors。A Laboratory Manual,Elsevier,Amsterdam(1986)中进行了描述。特别适合的载体包括Ti质粒载体。
将DNA构建体导入宿主细胞的转化技术是本领域已知的,其包括如下方法,如微量注射、使用聚乙二醇、电穿孔、高速率冲击穿透、或农杆菌介导的转化。在转化植物细胞或植物之后,那些插入了所需的DNA的植物细胞或植物可以通过如抗生素抗药性、除草剂抗性、氨基酸类似物耐受性、或使用表现型标记来筛选。
可以用各种试验分析来确定植物细胞是否显示了增加的基因表达,例如,Northern杂交或定量反转录PCR(RT-PCR)。通过常规的方法,整个转基因植物可从转化细胞再生成植株。所述转基因植物具有提高的类异戊二烯水平,可以继续繁殖和自花授粉来产生纯合子系。所述植物生产的种子包括引入的特定的基因,并且可以生长至生成植物,该植物产生选择的表型。
根据本发明公开内容特别优选的用于修饰的植物选自拟南芥(Arabidopsisthaliana)、琉璃苣(Borago spp.)、加拿大油料(Canola)、蓖麻(Ricinuscommunis)(Ricinus spp.)、可可豆(Theobroma cacao)(Theobroma spp.)、玉蜀黍(Zeamays)(Zea spp.)、棉(Gossypium spp)、海甘蓝(Crambe spp.)、萼距花(Cuphea spp.)、亚麻(Linum spp.)、雷斯克勒(Lesquerella spp.)、沼花(Limnanthes spp.)、亚麻的突变株(Linola)、旱金莲(Tropaeolum spp.)、月见草(Oenothera spp.)、洋橄榄(Olea spp.)、油棕(Elaeis spp.)、花生(Arachis spp.)、油菜籽(rapeseed)、红花(Carthamus spp.)、大豆(Glycine spp.和Soja spp.)、向日葵(Helianthus spp.)、烟草(Nicotiana spp.)、斑鸠菊(Vernonia spp.)、小麦(Triticum spp.)、大麦(Hordeum spp.)、稻(Oryza spp.)、燕麦(Avena spp.)、高粱(Sorghum spp.)、黑麦(Secale spp.)以及其它禾本科(Gramineae)植物。
一些具体实施方式用来改变油料种子作物产生的油料种子的产量或组成。油料种子作物为那些具有产生在商业上重要收益的可食用的或工业用油的植物品种,并且包括许多上述所列的植物品种。这些油料作物对于本领域技术人员来说是已知的。
在一个实施例中,培养用编码DGAT2的核苷酸序列转化的植物。收获转基因植物的种子,提取种子中的脂肪酸。提取的脂肪酸用于随后添加到组合物中,例如药物组合物,营养食品组合物,或食品组合物。
在特定的具体实施方式中,提高或改变油类产品的其他方法还可能以用于待转化的植物(如,为了在植物中表达,包含一核酸序列,该核酸序列选自包括以下核酸的组:编码例如,具有甘蓝型油菜丙酮酸脱氢酶激酶活性的多肽的核酸(参见,如,Manila等的美国专利7,214,859(2007年5月8日),Zou等的美国专利6,500,670(2002年12月),以及Randall等的美国专利6,256,636(2001年7月)),编码具有二酰基甘油酰基转移酶活性的多肽的核酸(参见,如,Zou等的美国专利7,015,373和美国专利6,500,670(2002年12月)),以及编码具有3-磷酸-甘油脱氢酶活性多肽的核酸(参见,如,美国专利7,112,724以及其组合)。
除了那些此处详细描述的具体实施例外,具体实施方式易受到各种修饰和改变形式的影响。因此,具体实施方式不限于所公开的特定的形式。一般来讲,公开内容包含的所有的改变,等同体或替代均落入随后附上的权利要求中。
实施例
实施例1:DNA操作
DNA制备、质粒扩增及分离使用标准的方法和步骤(Sambrook等,1999)。测序在使用Taq DyeDeoxyTM Terminator Cycle Sequencing试剂盒(Applied Biosystems,Inc.)的Applied Biosystems Model 373A DNA测序系统上进行。核苷酸与推导的氨基酸序列使用BLAST程序(ALtschulet等,1990)在数据库中进行比较。在NCBI核苷酸与蛋白质数据库中基于和其它脂肪酸二酰基甘油酰基转移酶基因的同源性鉴定DGAT2基因,这是本领域已知的。
实施例2:在酵母转化体体内表达TpDGAT2来形成三酰基甘油(TAG)
将DGAT2基因插入到pYES2.1中(Invitrogen)。构建体通过测序确认,并且将pYES2.1/TpDGAT2用于转化酿酒酵母菌株H1246 MAT-α。该突变株为四倍体突变株(DGAT-、PDAT-、ASAT-、ASAT2-)。从推定的转化体内分离质粒DNA,并通过Southern分析来确定pYES2.1/TpDGAT2的存在。只包括载体(pYES2.1)的H1246MAT-α转化体作为对照。包括拟南芥DGAT1的H1246 MAT-α转化体作为阳性对照。
在20mL的SD培养基(合成葡萄糖培养基,含有葡萄糖,不含尿嘧啶,Ausubel等,1995,Vol.2,p.13.1.3中描述)中过夜培养单个克隆,28℃摇床培养(270rpm)。从过夜培养物中收集细胞团,重悬于50mL培养基中用于诱导表达(含有半乳糖但不含尿嘧啶的SD培养基)。细胞于28℃,转速270rpm下重新孵育,4到6小时后收获。GAL诱导的酵母转化体5000rpm,5分钟离心收获,重悬于100mM Hepes NaOH溶液中,pH7.4,含有1mM的EDTA和1mM的DTT。
参考图9,空载体转化体和阴性对照(泳道8)中没有显示TAG条带,而阳性对照(泳道1)中有TAG条带。含有载体的每个DGAT2(泳道2-6)都显示了TAG条带,其证实了DGAT2具有合成TAG的能力。泳道8右侧的泳道的条带为TAG标准品,其用作TAG标记物。
实施例3:TpDGAT2的底物偏好性
使用酸洗玻璃珠制备细胞裂解液,如Ausubel等(1995)描述的那样。酵母裂解物中的蛋白质使用Brdford(1976)分析方法测定,每个裂解物中的蛋白质水平均为标准化且等分的(250μg蛋白质),用于DGAT2的活性分析。
DGAT分析在pH7.4,100转/分的水浴中30℃下进行10分钟。测定混合物(终体积0.5mL)包括100μg裂解物蛋白质,90mM HEPES-NaOH,20μM sn-1,2甘油二油酸酯,以及18μM 14C酰基辅酶A(放射性比度2nCi/nmol)作为酰基供体。14C标记的TAG通过硅胶G盘上的己烷∶二乙基乙醚∶乙酸(70∶30∶1 v/v/v)的TLC进行分离,放射标记的TAG条带通过在Bioscan AR-2000 radio-TLC扫描仪上使用Win-Scan 2D软件进行观测(Bioscan Inc.,Washington DC,USA),并且按照Taylor等(1991)所述进行条带刮下与条带定量。
实施例4:TpDAGT2转化体中的脂肪酸组成
酿酒酵母菌株H1246 MAT-α用拟南芥/pYES 2.1或假微型海链藻/pYES 2.1进行转化。转化体在28℃生长3天并使用半乳糖诱导。转化体不用任何东西处理(对照),用50μMDHA处理或用150μMDHA处理。三种包含AtDGAT1/pYES2.1的转化体以及三种包含TpDGAT2/pYES2.1的转化体的基于常规气相色谱法的脂肪酸分布如表1所示。
脂肪酸被确定为16:0、16:1、18:0、18:1(油酸),以及22:6(DHA);以及每种的组成以总脂肪酸的百分比表示。DHA的表达从对照菌株中的0提高到150μMTpDGAT2/pYES2.1中的6.01%,并且是150μM AtDGAT/pYES2.1的两倍多。(表1)这些结果进一步证实了TpDGAT2利用DHA脂肪酸的效率高于DGAT1。
脂肪酸组成的方式中,包含DGAT2 cDNA的突变系显示了总的饱和脂肪酸的下降,以及不饱和脂肪酸的升高,如表1所示。这些变化指向了“更健康的”油类,并且可以直接应用到加拿大油料,其他十字花科的油料种子以及其他的食用油作物来生产相似的结构组成的改良型油类。
表1.在酵母突变菌株H1246 MAT-α中由DGAT2和DGAT1表达的TAG的脂肪酸组成
脂肪酸组成
% % % % %
处理 16:0 16:1 18:0 18:1 22:6 %饱和 %不饱和
AtDGAT/pYES2.1-不补给 13.93 35.71 17.84 32.51 0.00 31.78 68.22
AtDGAT/pYES2.1-50μM DHA 19.68 27.54 16.51 34.82 1.46 36.18 62.36
AtDGAT/pYES2.1-150μM DHA 19.24 27.83 15.21 35.08 2.63 34.45 62.91
TpDGAT2/pYES2.1-不补给 10.03 30.23 13.83 45.90 0.00 23.87 76.13
TpDGAT2/pYES2.1-50μM DHA 6.43 35.39 8.49 45.07 4.62 14.92 80.47
TpDGAT2/pYES2.1-150μM DHA 5.77 31.73 11.57 44.93 6.01 17.34 76.66
实施例5:野生型拟南芥中DGAT2 cDNA的过表达
将全长的DGAT2 cDNA用作PCR扩增的模板。片段通过限制性内切酶消化切割,然后连入到载体的相应位点。通过测序来确定构建体的完整性。
载体转入到农杆菌中,用于转化野生型拟南芥,并且进行结果分析。
实施例6:用于种子特异性表达的DGAT2 cDNA植物转化载体的构建
将全长的DGAT2 cDNA用作PCR扩增模板,使用在序列各个末端提供了新的酶切位点的引物。PCR程序如下:94℃1分钟;94℃30秒,55℃30秒,72℃1分钟,30个循环;和72℃5分钟。然后将PCR产物连接入PCR2.1载体(Invitrogen)。片段被切割然后连接到载体的对应位点。通过测序来确定构建体的完整性。
实施例7:用植物DGAT2载体构建体转化农杆菌
电转化农杆菌细胞,GV3101(pMP90)菌株,按照如下方法制备:农杆菌培养物在2YT中生长24到48小时,当600nm处吸光度达到0.5到0.7时,细胞置于冰上冷却通过离心(5,000×g,GSA转子,4℃,10分钟)收集。收集物用1、0.5、0.02体积的冷的10%的无菌甘油清洗,重悬于0.01体积的冷的10%甘油。电转化细胞随后在液氮中速冻然后储存于-70℃。用20-50ng转化DNA通过电穿孔转入农杆菌细胞,按照制造商的说明书执行,然后将农杆菌细胞置于选择培养基(加入50μg的卡那霉素的LB)中,28℃培养过夜。单独的转化细胞在5mL添加50μg/mL卡那霉素以及25μg/mL庆大霉素的LB培养基中生长过夜(28℃,225r.p.m)。进行DNA提取和纯化。构建体的保真度在转化植物前通过DNA测序再次检查。
实施例8:转化拟南芥
拟南芥的种子在荧光灯照明条件下(120μE·m-2S-1),16小时有光/8小时黑暗的条件下,22℃生长。4到6株植物于潮湿的TERRA-LITE REDI-EATRH的10cm2的盆中培养(W.R.Grace & Co.Canada Ltd.Ajax,ON,Canada)。为了防止盆中的土壤混合物落入接种培养基,土壤成耸起的平面,种子播种于顶部,整个盆用尼龙纱窗纱覆盖,并用橡皮筋固定。在开始开花时用农杆菌浸润植物真空转化。
为了培养农杆菌,5mL悬液在含有50μg/mL的卡那霉素和25μg/mL的庆大霉素的LB培养基中28℃培养过夜。在浸染前一天,将“种子培养物”分为4瓶,每瓶包括250mL补充了50μg/mL的卡那霉素和25μg/mL的庆大霉素的LB培养基。这些培养物在28℃下培养过夜。在检测600nm处吸光度(大约=1.0)的第二天早晨,通过离心收集细胞(5,000×g,GSA转子,室温,10分钟),并且重悬于浸染培养基(蔗糖5%;Silwet-77 0.005%水溶液)中以获得600nm处0.8的光密度。
将农杆菌悬液倒入烧杯中,并且将盆栽植物插入烧杯中使花和实(bolt)完全浸润。将烧杯放置于大的玻璃钟罩中,并使用真空泵抽真空,直到茎表面的气泡形成以及溶液开始轻微地产生气泡,然后迅速地释放真空。必要的时间和压力每个实验室间都有差异,但是好的浸染是可以根据均匀的黑暗水浸组织很明显地判断出来。将盆从烧杯中移出,侧面放置于塑料浅箱上,并且使用塑料圆顶覆盖,以保持湿度。第二天,不覆盖植物,直立放置,在生长箱内使其生长大约四周,条件为连续光照,如Katavic等(1995)所描述。当果实成熟并干燥后,收集种子并选择阳性转化子。
实施例9:转化甘蓝型油菜
转化基本上按照Moleney等,1989,Plant Cell Reports 8:238-242描述的方法执行。
农杆菌菌株GV301/pMP90(Koncz C.& Schell,J.,1986,Mol.Gen.Genet.204:383-396)用于转化研究。从LB肉汤培养基(Difco,USA)(100mL)中收获稳定期细菌培养物,重悬于10mL新鲜的添加1%DMSO(二甲基亚砜)作为冷冻保护剂的LB肉汤培养基中。每等分200μl储存于-20℃,直到用于转化,将每等分加入到2mL包含2%蔗糖,50μM乙酰丁香酮的脑、心脏浸剂肉汤(Difco,USA)中,pH5.6,并且在28℃培养过夜。细菌细胞密度大约为每毫升1×109个细胞。
子叶外植体暴露于包含植物转化载体的农杆菌中,按照Moloney等(1989),Plant Cell Rep.8:238-242的方法进行。外植体的叶柄切面短暂地浸没在细菌培养物中。将外植体插入到共培养基中使得切面与培养基接触。每张100×15mm的培养皿上放置10个外植体。共培养平板用STRTCHT’N SEALTM塑料膜封口。平板在如上述的温度和光周期条件下的培养箱中培养3天,使得种子萌发。随后将外植体转移到选择培养基上。
在选择培养基上培养3到4周后,剪下再生的绿色芽(推定的转化体),转移至新鲜的选择培养基上继续培养。当芽达到1.5-2.0cm长时,将其转移至生根培养基。推定的转基因芽基本上按照Jefferson,R.A(1997)描述的方法进行gus基因扫描。有蓝色的染色存在被认为是转化的证据。
通过在卡那霉素上筛选、Southern杂交、PCR(聚合酶链式反应)以及后代分析来确认转化体。
实施例10:推定的转化体(转基因植物)的筛选以及转基因植物分析
大量收获每个构建体的种子。种子用含有20%漂白剂以及0.01%Trion X-100的溶液浸泡来进行表面消毒20分钟,随后用无菌水漂洗3次。室温条件下,无菌的种子重新置于悬于灭菌的0.1%植物琼脂中倒的平板(每500-1000种子约1mL植物琼脂)上,室温条件下,将包含2,000-4,000种子的体积置于150×15mm的卡那霉素选择平板上。平板在冷的无光条件下培养两天,然后在可控环境下(22℃日光灯荧光灯(120μE·m-2s-1)照射16小时光照/8小时黑暗周期)生长7到10天。选择培养基包括1/2MSG培养基、0.8%植物琼脂、3%蔗糖,、50μg/mL卡那霉素和50μg/mL特美汀。培养皿和盖子用MicroporeTM外科带(3M Canada,London,ON,Canada)封口。在7到10天后,对长出绿色叶子和在培养基中有稳定根的药物抗性植株进行转化体鉴定,在3-5叶片阶段,筛选的转化体移栽到充分湿润的土壤混合物的平面。转化体生长至成熟并从个体植株中收获产生的种子(T2代,如Katavic等(1994)所定义),进一步进行分析。
从个体T1植物中分离基因组DNA。分别在T1转化体中使用PCR扩增来确认cDNA或基因的存在。用Southern分析来选择包含单个拷贝片段插入的转化体。用限制性酶酶切DNA样品,在1%的琼脂糖凝胶电泳上分离,用尼龙膜((Hybond-N+,Amersham)进行Southern杂交。DGAT2 cDNA片段,用α-[32P]dCTP(NEN/DuPont)标记后用作探针。杂交在60℃进行。杂交膜随后曝光于Kodak X-OMAT-AR胶片。
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序列表
<110>加拿大国家研究委员会,植物生物技术研究所和陶氏益农公司
邹继涛
徐靖宇
郑志富
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Asp Asp Asp His Thr Ile Ile Glu Thr Asn Pro His Ser Ala Ile Pro
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Asn Gln Glu Glu Phe Asp Ile His Thr Ser Pro Ser Ile Phe Ala Phe
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Val Pro His Gly Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro
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Asp Asp Asp His Thr Ile Ile Glu Thr Asn Pro His Ser Ala Ile Pro
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Val Pro His Gly Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro
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Glu Arg Gly His Glu Gln Thr Trp Gly Leu Phe Arg Pro Val Val Ala
245 250 255
Thr Ala Thr Lys Leu Phe Pro Leu Val Arg Thr Phe Ile Ser Trp Met
260 265 270
Asn Gly Val Asp Ala Ser Asp Ser Ala Val Ser Arg Ala Leu Ala Pro
275 280 285
Pro Tyr Thr Ser Asp His Pro Gly Arg Val Gly Val Ser Pro Gly Gly
290 295 300
Ile Ala Glu Met Phe Glu Thr Tyr Pro Lys Pro Gly Phe His Pro Asn
305 310 315 320
Asp Glu Ala Ala Leu Leu Lys Asp Arg Asn Gly Leu Phe Lys Leu Ala
325 330 335
Met Lys His Lys Leu Pro Ile Val Pro Val Tyr Cys Phe Gly Ala Thr
340 345 350
Lys Met Leu Arg Arg Val Gln Leu Pro Ala Phe Val Glu Thr Leu Ser
355 360 365
Arg Met Leu Lys Ile Ser Leu Cys Leu Phe Phe Gly Lys Leu Gly Leu
370 375 380
Pro Ile Pro Phe Arg Gln Arg Leu Met Tyr Val Met Gly Lys Thr Leu
385 390 395 400
Phe Pro Pro Leu Pro Arg Asp Gly Val Asn Thr Ser Met Met Glu Gly
405 410 415
Gly Glu Glu Phe Asp Gly Arg Val Gln Glu Met His Asp Ala Phe Cys
420 425 430
Asn Glu Ile Thr Arg Ile Phe Glu Arg Asn Lys Asp His Tyr Gly Trp
435 440 445
Gly Asn Lys Asn Leu Arg Leu Val
450 455
<210>6
<211>1432
<212>DNA
<213>Artificial
<220>
<223>F224Y mutant
<400>6
atgacaacaa agaagcgtcc actaccccgt catctgcacc ttccacctgg agtagaagta 60
ctcgtctctc caccacccta cgaagtatgc acgctcgtcg acagattgtt ggtctacgcc 120
tcgtcgttga ttgtcgttgg atctcccgtt tggttctacg gaggcatcat ttatttttac 180
aggaagtgga agaagtatcg ttctcttgct gctgctactg aggctgcgag acatgagagt 240
ggtggcggtg gtgcatcgtc aacggttcgt tgcagaggta cacgtcaacg tacatcgtct 300
gatgacggca actacacatc gtcaactggc gaaagctcgc aagaaatgaa cgaacaagag 360
acacaaacac aatcacatcg acaacaaaca gagcaataca acaactacaa acgattagca 420
acaagatacg gagtagcact cgctgcaatc attctcatat ccatctgggg gcctcatcgt 480
gacaagcgtg taggagaatg gctcggtgtc aagaagtgga gattgtggga tgcatggttg 540
aactatgttg gattcactgt actaaaggac aatggagatg atgaccacac aataatacaa 600
acgaatccac actcagcaat acccaatcaa gaagagtttg acatacacac atctccatca 660
atcttcgcat acgtacccca cggcatcttt cctttcggac tcgccttttc atgtctaccc 720
gaacgaggac acgaacaaac atggggtctc ttccgaccag tcgttgcaac agccaccaaa 780
ctctttccgc tggtacgaac cttcatttct tggatgaacg gagtggatgc ttcgcgttcg 840
gcggtgtctc gtgctcttgc tcctccgtat accagtgatc atccgggaag agtgggagtt 900
tcgcccggtg gtattgccga gatgtttgag acgtatccaa agccggggtt tcatcctaat 960
gacgaggcag cattgttaaa agatcggaat ggattgttca agcttgcgat gaaacacaag 1020
ctgccgattg ttccggtgta ctgctttgga gctacaaaga tgttgagacg agtgcaatta 1080
cctgcgtttg tggagacgtt gagcagaatg ctcaagatca gtctttgttt attctttgga 1140
aagcttgggt tgcctattcc tttccgacag cggctgatgt atgtcatggg caagacgttg 1200
tttcctcctc tgccgagaga tggcgtgaac acttctatga tggaaggagg agaagaattt 1260
gatgaacgag tgcaagagat gcatgatgca ttctgcaatg agataactcg catcttcgag 1320
cgaaacaaag accactacgg ttggggtaac aaaaacttga gactcgtatg agagtgtgag 1380
tgatattcat atgcaactct taacttaaag ccacagacca cacaggcaca aa 1432
<210>7
<211>456
<212>PRT
<213>Artificial
<220>
<223>V298I mutant
<400>7
Met Thr Thr Lys Lys Arg Pro Lys Pro Arg His Lys His Leu Pro Pro
1 5 10 15
Gly Val Glu Val Leu Val Ser Pro Pro Pro Tyr Glu Val Cys Thr Leu
20 25 30
Val Asp Arg Leu Leu Val Tyr Ala Ser Ser Leu Ile Val Val Gly Ser
35 40 45
Pro Val Trp Phe Tyr Gly Gly Ile Ile Tyr Phe Tyr Arg Lys Trp Lys
50 55 60
Leu Tyr Arg Ser Lys Ala Ala Ala Thr Phe Ala Ala Arg His Glu Ser
65 70 75 80
Gly Gly Gly Gly Ala Ser Ser Thr Val Arg Cys Arg Gly Thr Arg Gln
85 90 95
Arg Thr Ser Ser Asp Asp Gly Asn Tyr Thr Ser Ser Thr Gly Glu Ser
100 105 110
Ser Gln Glu Met Asn Glu Gln Glu Thr Gln Thr Gln Ser His Arg Gln
115 120 125
Gln Thr Glu Gln Tyr Asn Asn Tyr Lys Arg Leu Ala Thr Arg Tyr Gly
130 135 140
Val Ala Lys Ala Ala Ile Ile Lys Ile Ser Ile Trp Gly Pro His Arg
145 150 155 160
Asp Lys Arg Val Gly Glu Trp Leu Gly Val Lys Lys Trp Arg Leu Trp
165 170 175
Asp Ala Trp Leu Asn Tyr Val Gly Phe Thr Val Leu Lys Asp Asn Gly
180 185 190
Asp Asp Asp His Thr Ile Ile Glu Thr Asn Pro His Ser Ala Ile Pro
195 200 205
Asn Gln Glu Glu Phe Asp Ile His Thr Ser Pro Ser Ile Phe Ala Phe
210 215 220
Val Pro His Gly Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro
225 230 235 240
Glu Arg Gly His Glu Gln Thr Trp Gly Leu Phe Arg Pro Val Val Ala
245 250 255
Thr Ala Thr Lys Leu Phe Pro Leu Val Arg Thr Phe Ile Ser Trp Met
260 265 270
Asn Gly Val Asp Ala Ser Asp Ser Ala Val Ser Arg Ala Leu Ala Pro
275 280 285
Pro Tyr Thr Ser Asp His Pro Gly Arg Ile Gly Val Ser Pro Gly Gly
290 295 300
Ile Ala Glu Met Phe Glu Thr Tyr Pro Lys Pro Gly Phe His Pro Asn
305 310 315 320
Asp Glu Ala Ala Leu Leu Lys Asp Arg Asn Gly Leu Phe Lys Leu Ala
325 330 335
Met Lys His Lys Leu Pro Ile Val Pro Val Tyr Cys Phe Gly Ala Thr
340 345 350
Lys Met Leu Arg Arg Val Gln Leu Pro Ala Phe Val Glu Thr Leu Ser
355 360 365
Arg Met Leu Lys Ile Ser Leu Cys Leu Phe Phe Gly Lys Leu Gly Leu
370 375 380
Pro Ile Pro Phe Arg Gln Arg Leu Met Tyr Val Met Gly Lys Thr Leu
385 390 395 400
Phe Pro Pro Leu Pro Arg Asp Gly Val Asn Thr Ser Met Met Glu Gly
405 410 415
Gly Glu Glu Phe Asp Gly Arg Val Gln Glu Met His Asp Ala Phe Cys
420 425 430
Asn Glu Ile Thr Arg Ile Phe Glu Arg Asn Lys Asp His Tyr Gly Trp
435 440 445
Gly Asn Lys Asn Leu Arg Leu Val
450 455
<210>8
<211>1432
<212>DNA
<213>Artificial
<220>
<223>V298I mutant
<400>8
atgacaacaa agaagcgtcc actaccccgt catctgcacc ttccacctgg agtagaagta 60
ctcgtctctc caccacccta cgaagtatgc acgctcgtcg acagattgtt ggtctacgcc 120
tcgtcgttga ttgtcgttgg atctcccgtt tggttctacg gaggcatcat ttatttttac 180
aggaagtgga agaagtatcg ttctcttgct gctgctactg aggctgcgag acatgagagt 240
ggtggcggtg gtgcatcgtc aacggttcgt tgcagaggta cacgtcaacg tacatcgtct 300
gatgacggca actacacatc gtcaactggc gaaagctcgc aagaaatgaa cgaacaagag 360
acacaaacac aatcacatcg acaacaaaca gagcaataca acaactacaa acgattagca 420
acaagatacg gagtagcact cgctgcaatc attctcatat ccatctgggg gcctcatcgt 480
gacaagcgtg taggagaatg gctcggtgtc aagaagtgga gattgtggga tgcatggttg 540
aactatgttg gattcactgt actaaaggac aatggagatg atgaccacac aataatacaa 600
acgaatccac actcagcaat acccaatcaa gaagagtttg acatacacac atctccatca 660
atcttcgcat tcgtacccca cggcatcttt cctttcggac tcgccttttc atgtctaccc 720
gaacgaggac acgaacaaac atggggtctc ttccgaccag tcgttgcaac agccaccaaa 780
ctctttccgc tggtacgaac cttcatttct tggatgaacg gagtggatgc ttcgcgttcg 840
gcggtgtctc gtgctcttgc tcctccgtat accagtgatc atccgggaag aatcggagtt 900
tcgcccggtg gtattgccga gatgtttgag acgtatccaa agccggggtt tcatcctaat 960
gacgaggcag cattgttaaa agatcggaat ggattgttca agcttgcgat gaaacacaag 1020
ctgccgattg ttccggtgta ctgctttgga gctacaaaga tgttgagacg agtgcaatta 1080
cctgcgtttg tggagacgtt gagcagaatg ctcaagatca gtctttgttt attctttgga 1140
aagcttgggt tgcctattcc tttccgacag cggctgatgt atgtcatggg caagacgttg 1200
tttcctcctc tgccgagaga tggcgtgaac acttctatga tggaaggagg agaagaattt 1260
gatgaacgag tgcaagagat gcatgatgca ttctgcaatg agataactcg catcttcgag 1320
cgaaacaaag accactacgg ttggggtaac aaaaacttga gactcgtatg agagtgtgag 1380
tgatattcat atgcaactct taacttaaag ccacagacca cacaggcaca aa 1432
<210>9
<211>456
<212>PRT
<213>Artificial
<220>
<223>I343L mutant
<400>9
Met Thr Thr Lys Lys Arg Pro Lys Pro Arg His Lys His Leu Pro Pro
1 5 10 15
Gly Val Glu Val Leu Val Ser Pro Pro Pro Tyr Glu Val Cys Thr Leu
20 25 30
Val Asp Arg Leu Leu Val Tyr Ala Ser Ser Leu Ile Val Val Gly Ser
35 40 45
Pro Val Trp Phe Tyr Gly Gly Ile Ile Tyr Phe Tyr Arg Lys Trp Lys
50 55 60
Leu Tyr Arg Ser Lys Ala Ala Ala Thr Phe Ala Ala Arg His Glu Ser
65 70 75 80
Gly Gly Gly Gly Ala Ser Ser Thr Val Arg Cys Arg Gly Thr Arg Gln
85 90 95
Arg Thr Ser Ser Asp Asp Gly Asn Tyr Thr Ser Ser Thr Gly Glu Ser
100 105 110
Ser Gln Glu Met Asn Glu Gln Glu Thr Gln Thr Gln Ser His Arg Gln
115 120 125
Gln Thr Glu Gln Tyr Asn Asn Tyr Lys Arg Leu Ala Thr Arg Tyr Gly
130 135 140
Val Ala Lys Ala Ala Ile Ile Lys Ile Ser Ile Trp Gly Pro His Arg
145 150 155 160
Asp Lys Arg Val Gly Glu Trp Leu Gly Val Lys Lys Trp Arg Leu Trp
165 170 175
Asp Ala Trp Leu Asn Tyr Val Gly Phe Thr Val Leu Lys Asp Asn Gly
180 185 190
Asp Asp Asp His Thr Ile Ile Glu Thr Asn Pro His Ser Ala Ile Pro
195 200 205
Asn Gln Glu Glu Phe Asp Ile His Thr Ser Pro Ser Ile Phe Ala Phe
210 215 220
Val Pro His Gly Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro
225 230 235 240
Glu Arg Gly His Glu Gln Thr Trp Gly Leu Phe Arg Pro Val Val Ala
245 250 255
Thr Ala Thr Lys Leu Phe Pro Leu Val Arg Thr Phe Ile Ser Trp Met
260 265 270
Asn Gly Val Asp Ala Ser Asp Ser Ala Val Ser Arg Ala Leu Ala Pro
275 280 285
Pro Tyr Thr Ser Asp His Pro Gly Arg Val Gly Val Ser Pro Gly Gly
290 295 300
Ile Ala Glu Met Phe Glu Thr Tyr Pro Lys Pro Gly Phe His Pro Asn
305 310 315 320
Asp Glu Ala Ala Leu Leu Lys Asp Arg Asn Gly Leu Phe Lys Leu Ala
325 330 335
Met Lys His Lys Leu Pro Leu Val Pro Val Tyr Cys Phe Gly Ala Thr
340 345 350
Lys Met Leu Arg Arg Val Gln Leu Pro Ala Phe Val Glu Thr Leu Ser
355 360 365
Arg Met Leu Lys Ile Ser Leu Cys Leu Phe Phe Gly Lys Leu Gly Leu
370 375 380
Pro Ile Pro Phe Arg Gln Arg Leu Met Tyr Val Met Gly Lys Thr Leu
385 390 395 400
Phe Pro Pro Leu Pro Arg Asp Gly Val Asn Thr Ser Met Met Glu Gly
405 410 415
Gly Glu Glu Phe Asp Gly Arg Val Gln Glu Met His Asp Ala Phe Cys
420 425 430
Asn Glu Ile Thr Arg Ile Phe Glu Arg Asn Lys Asp His Tyr Gly Trp
435 440 445
Gly Asn Lys Asn Leu Arg Leu Val
450 455
<210>10
<211>1432
<212>DNA
<213>Artificial
<220>
<223>I343L mutant
<400>10
atgacaacaa agaagcgtcc actaccccgt catctgcacc ttccacctgg agtagaagta 60
ctcgtctctc caccacccta cgaagtatgc acgctcgtcg acagattgtt ggtctacgcc 120
tcgtcgttga ttgtcgttgg atctcccgtt tggttctacg gaggcatcat ttatttttac 180
aggaagtgga agaagtatcg ttctcttgct gctgctactg aggctgcgag acatgagagt 240
ggtggcggtg gtgcatcgtc aacggttcgt tgcagaggta cacgtcaacg tacatcgtct 300
gatgacggca actacacatc gtcaactggc gaaagctcgc aagaaatgaa cgaacaagag 360
acacaaacac aatcacatcg acaacaaaca gagcaataca acaactacaa acgattagca 420
acaagatacg gagtagcact cgctgcaatc attctcatat ccatctgggg gcctcatcgt 480
gacaagcgtg taggagaatg gctcggtgtc aagaagtgga gattgtggga tgcatggttg 540
aactatgttg gattcactgt actaaaggac aatggagatg atgaccacac aataatacaa 600
acgaatccac actcagcaat acccaatcaa gaagagtttg acatacacac atctccatca 660
atcttcgcat tcgtacccca cggcatcttt cctttcggac tcgccttttc atgtctaccc 720
gaacgaggac acgaacaaac atggggtctc ttccgaccag tcgttgcaac agccaccaaa 780
ctctttccgc tggtacgaac cttcatttct tggatgaacg gagtggatgc ttcgcgttcg 840
gcggtgtctc gtgctcttgc tcctccgtat accagtgatc atccgggaag agtgggagtt 900
tcgcccggtg gtattgccga gatgtttgag acgtatccaa agccggggtt tcatcctaat 960
gacgaggcag cattgttaaa agatcggaat ggattgttca agcttgcgat gaaacacaag 1020
ctgccgcttg ttccggtgta ctgctttgga gctacaaaga tgttgagacg agtgcaatta 1080
cctgcgtttg tggagacgtt gagcagaatg ctcaagatca gtctttgttt attctttgga 1140
aagcttgggt tgcctattcc tttccgacag cggctgatgt atgtcatggg caagacgttg 1200
tttcctcctc tgccgagaga tggcgtgaac acttctatga tggaaggagg agaagaattt 1260
gatgaacgag tgcaagagat gcatgatgca ttctgcaatg agataactcg catcttcgag 1320
cgaaacaaag accactacgg ttggggtaac aaaaacttga gactcgtatg agagtgtgag 1380
tgatattcat atgcaactct taacttaaag ccacagacca cacaggcaca aa 1432
<210>11
<211>456
<212>PRT
<213>Artificial
<220>
<223>E365K mutant
<400>11
Met Thr Thr Lys Lys Arg Pro Lys Pro Arg His Lys His Leu Pro Pro
1 5 10 15
Gly Val Glu Val Leu Val Ser Pro Pro Pro Tyr Glu Val Cys Thr Leu
20 25 30
Val Asp Arg Leu Leu Val Tyr Ala Ser Ser Leu Ile Val Val Gly Ser
35 40 45
Pro Val Trp Phe Tyr Gly Gly Ile Ile Tyr Phe Tyr Arg Lys Trp Lys
50 55 60
Leu Tyr Arg Ser Lys Ala Ala Ala Thr Phe Ala Ala Arg His Glu Ser
65 70 75 80
Gly Gly Gly Gly Ala Ser Ser Thr Val Arg Cys Arg Gly Thr Arg Gln
85 90 95
Arg Thr Ser Ser Asp Asp Gly Asn Tyr Thr Ser Ser Thr Gly Glu Ser
100 105 110
Ser Gln Glu Met Asn Glu Gln Glu Thr Gln Thr Gln Ser His Arg Gln
115 120 125
Gln Thr Glu Gln Tyr Asn Asn Tyr Lys Arg Leu Ala Thr Arg Tyr Gly
130 135 140
Val Ala Lys Ala Ala Ile Ile Lys Ile Ser Ile Trp Gly Pro His Arg
145 150 155 160
Asp Lys Arg Val Gly Glu Trp Leu Gly Val Lys Lys Trp Arg Leu Trp
165 170 175
Asp Ala Trp Leu Asn Tyr Val Gly Phe Thr Val Leu Lys Asp Asn Gly
180 185 190
Asp Asp Asp His Thr Ile Ile Glu Thr Asn Pro His Ser Ala Ile Pro
195 200 205
Asn Gln Glu Glu Phe Asp Ile His Thr Ser Pro Ser Ile Phe Ala Phe
210 215 220
Val Pro His Gly Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro
225 230 235 240
Glu Arg Gly His Glu Gln Thr Trp Gly Leu Phe Arg Pro Val Val Ala
245 250 255
Thr Ala Thr Lys Leu Phe Pro Leu Val Arg Thr Phe Ile Ser Trp Met
260 265 270
Asn Gly Val Asp Ala Ser Asp Ser Ala Val Ser Arg Ala Leu Ala Pro
275 280 285
Pro Tyr Thr Ser Asp His Pro Gly Arg Val Gly Val Ser Pro Gly Gly
290 295 300
Ile Ala Glu Met Phe Glu Thr Tyr Pro Lys Pro Gly Phe His Pro Asn
305 310 315 320
Asp Glu Ala Ala Leu Leu Lys Asp Arg Asn Gly Leu Phe Lys Leu Ala
325 330 335
Met Lys His Lys Leu Pro Ile Val Pro Val Tyr Cys Phe Gly Ala Thr
340 345 350
Lys Met Leu Arg Arg Val Gln Leu Pro Ala Phe Val Lys Thr Leu Ser
355 360 365
Arg Met Leu Lys Ile Ser Leu Cys Leu Phe Phe Gly Lys Leu Gly Leu
370 375 380
Pro Ile Pro Phe Arg Gln Arg Leu Met Tyr Val Met Gly Lys Thr Leu
385 390 395 400
Phe Pro Pro Leu Pro Arg Asp Gly Val Asn Thr Ser Met Met Glu Gly
405 410 415
Gly Glu Glu Phe Asp Gly Arg Val Gln Glu Met His Asp Ala Phe Cys
420 425 430
Asn Glu Ile Thr Arg Ile Phe Glu Arg Asn Lys Asp His Tyr Gly Trp
435 440 445
Gly Asn Lys Asn Leu Arg Leu Val
450 455
<210>12
<211>1432
<212>DNA
<213>Artificial
<220>
<223>E365K mutant
<400>12
atgacaacaa agaagcgtcc actaccccgt catctgcacc ttccacctgg agtagaagta 60
ctcgtctctc caccacccta cgaagtatgc acgctcgtcg acagattgtt ggtctacgcc 120
tcgtcgttga ttgtcgttgg atctcccgtt tggttctacg gaggcatcat ttatttttac 180
aggaagtgga agaagtatcg ttctcttgct gctgctactg aggctgcgag acatgagagt 240
ggtggcggtg gtgcatcgtc aacggttcgt tgcagaggta cacgtcaacg tacatcgtct 300
gatgacggca actacacatc gtcaactggc gaaagctcgc aagaaatgaa cgaacaagag 360
acacaaacac aatcacatcg acaacaaaca gagcaataca acaactacaa acgattagca 420
acaagatacg gagtagcact cgctgcaatc attctcatat ccatctgggg gcctcatcgt 480
gacaagcgtg taggagaatg gctcggtgtc aagaagtgga gattgtggga tgcatggttg 540
aactatgttg gattcactgt actaaaggac aatggagatg atgaccacac aataatacaa 600
acgaatccac actcagcaat acccaatcaa gaagagtttg acatacacac atctccatca 660
atcttcgcat tcgtacccca cggcatcttt cctttcggac tcgccttttc atgtctaccc 720
gaacgaggac acgaacaaac atggggtctc ttccgaccag tcgttgcaac agccaccaaa 780
ctctttccgc tggtacgaac cttcatttct tggatgaacg gagtggatgc ttcgcgttcg 840
gcggtgtctc gtgctcttgc tcctccgtat accagtgatc atccgggaag agtgggagtt 900
tcgcccggtg gtattgccga gatgtttgag acgtatccaa agccggggtt tcatcctaat 960
gacgaggcag cattgttaaa agatcggaat ggattgttca agcttgcgat gaaacacaag 1020
ctgccgattg ttccggtgta ctgctttgga gctacaaaga tgttgagacg agtgcaatta 1080
cctgcgtttg tgaagacgtt gagcagaatg ctcaagatca gtctttgttt attctttgga 1140
aagcttgggt tgcctattcc tttccgacag cggctgatgt atgtcatggg caagacgttg 1200
tttcctcctc tgccgagaga tggcgtgaac acttctatga tggaaggagg agaagaattt 1260
gatgaacgag tgcaagagat gcatgatgca ttctgcaatg agataactcg catcttcgag 1320
cgaaacaaag accactacgg ttggggtaac aaaaacttga gactcgtatg agagtgtgag 1380
tgatattcat atgcaactct taacttaaag ccacagacca cacaggcaca aa 1432
<210>13
<211>456
<212>PRT
<213>Artificial
<220>
<223>L400I mutant
<400>13
Met Thr Thr Lys Lys Arg Pro Lys Pro Arg His Lys His Leu Pro Pro
1 5 10 15
Gly Val Glu Val Leu Val Ser Pro Pro Pro Tyr Glu Val Cys Thr Leu
20 25 30
Val Asp Arg Leu Leu Val Tyr Ala Ser Ser Leu Ile Val Val Gly Ser
35 40 45
Pro Val Trp Phe Tyr Gly Gly Ile Ile Tyr Phe Tyr Arg Lys Trp Lys
50 55 60
Leu Tyr Arg Ser Lys Ala Ala Ala Thr Phe Ala Ala Arg His Glu Ser
65 70 75 80
Gly Gly Gly Gly Ala Ser Ser Thr Val Arg Cys Arg Gly Thr Arg Gln
85 90 95
Arg Thr Ser Ser Asp Asp Gly Asn Tyr Thr Ser Ser Thr Gly Glu Ser
100 105 110
Ser Gln Glu Met Asn Glu Gln Glu Thr Gln Thr Gln Ser His Arg Gln
115 120 125
Gln Thr Glu Gln Tyr Asn Asn Tyr Lys Arg Leu Ala Thr Arg Tyr Gly
130 135 140
Val Ala Lys Ala Ala Ile Ile Lys Ile Ser Ile Trp Gly Pro His Arg
145 150 155 160
Asp Lys Arg Val Gly Glu Trp Leu Gly Val Lys Lys Trp Arg Leu Trp
165 170 175
Asp Ala Trp Leu Asn Tyr Val Gly Phe Thr Val Leu Lys Asp Asn Gly
180 185 190
Asp Asp Asp His Thr Ile Ile Glu Thr Asn Pro His Ser Ala Ile Pro
195 200 205
Asn Gln Glu Glu Phe Asp Ile His Thr Ser Pro Ser Ile Phe Ala Phe
210 215 220
Val Pro His Gly Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro
225 230 235 240
Glu Arg Gly His Glu Gln Thr Trp Gly Leu Phe Arg Pro Val ValAla
245 250 255
Thr Ala Thr Lys Leu Phe Pro Leu Val Arg Thr Phe Ile Ser Trp Met
260 265 270
Asn Gly Val Asp Ala Ser Asp Ser Ala Val Ser Arg Ala Leu Ala Pro
275 280 285
Pro Tyr Thr Ser Asp His Pro Gly Arg Val Gly Val Ser Pro Gly Gly
290 295 300
Ile Ala Glu Met Phe Glu Thr Tyr Pro Lys Pro Gly Phe His Pro Asn
305 310 315 320
Asp Glu Ala Ala Leu Leu Lys Asp Arg Asn Gly Leu Phe Lys Leu Ala
325 330 335
Met Lys His Lys Leu Pro Ile Val Pro Val Tyr Cys Phe Gly Ala Thr
340 345 350
Lys Met Leu Arg Arg Val Gln Leu Pro Ala Phe Val Glu Thr Leu Ser
355 360 365
Arg Met Leu Lys Ile Ser Leu Cys Leu Phe Phe Gly Lys Leu Gly Leu
370 375 380
Pro Ile Pro Phe Arg Gln Arg Leu Met Tyr Val Met Gly Lys Thr Ile
385 390 395 400
Phe Pro Pro Leu Pro Arg Asp Gly Val Asn Thr Ser Met Met Glu Gly
405 410 415
Gly Glu Glu Phe Asp Gly Arg Val Gln Glu Met His Asp Ala Phe Cys
420 425 430
Asn Glu Ile Thr Arg Ile Phe Glu Arg Asn Lys Asp His Tyr Gly Trp
435 440 445
Gly Asn Lys Asn Leu Arg Leu Val
450 455
<210>14
<211>1432
<212>DNA
<213>Artificial
<220>
<223>L400I mutant
<400>14
atgacaacaa agaagcgtcc actaccccgt catctgcacc ttccacctgg agtagaagta 60
ctcgtctctc caccacccta cgaagtatgc acgctcgtcg acagattgtt ggtctacgcc 120
tcgtcgttga ttgtcgttgg atctcccgtt tggttctacg gaggcatcat ttatttttac 180
aggaagtgga agaagtatcg ttctcttgct gctgctactg aggctgcgag acatgagagt 240
ggtggcggtg gtgcatcgtc aacggttcgt tgcagaggta cacgtcaacg tacatcgtct 300
gatgacggca actacacatc gtcaactggc gaaagctcgc aagaaatgaa cgaacaagag 360
acacaaacac aatcacatcg acaacaaaca gagcaataca acaactacaa acgattagca 420
acaagatacg gagtagcact cgctgcaatc attctcatat ccatctgggg gcctcatcgt 480
gacaagcgtg taggagaatg gctcggtgtc aagaagtgga gattgtggga tgcatggttg 540
aactatgttg gattcactgt actaaaggac aatggagatg atgaccacac aataatacaa 600
acgaatccac actcagcaat acccaatcaa gaagagtttg acatacacac atctccatca 660
atcttcgcat tcgtacccca cggcatcttt cctttcggac tcgccttttc atgtctaccc 720
gaacgaggac acgaacaaac atggggtctc ttccgaccag tcgttgcaac agccaccaaa 780
ctctttccgc tggtacgaac cttcatttct tggatgaacg gagtggatgc ttcgcgttcg 840
gcggtgtctc gtgctcttgc tcctccgtat accagtgatc atccgggaag agtgggagtt 900
tcgcccggtg gtattgccga gatgtttgag acgtatccaa agccggggtt tcatcctaat 960
gacgaggcag cattgttaaa agatcggaat ggattgttca agcttgcgat gaaacacaag 1020
ctgccgattg ttccggtgta ctgctttgga gctacaaaga tgttgagacg agtgcaatta 1080
cctgcgtttg tggagacgtt gagcagaatg ctcaagatca gtctttgttt attctttgga 1140
aagcttgggt tgcctattcc tttccgacag cggctgatgt atgtcatggg caagacgatc 1200
tttcctcctc tgccgagaga tggcgtgaac acttctatga tggaaggagg agaagaattt 1260
gatgaacgag tgcaagagat gcatgatgca ttctgcaatg agataactcg catcttcgag 1320
cgaaacaaag accactacgg ttggggtaac aaaaacttga gactcgtatg agagtgtgag 1380
tgatattcat atgcaactct taacttaaag ccacagacca cacaggcaca aa 1432
<210>15
<211>124
<212>PRT
<213>Artificial
<220>
<223>Catalytic domain
<400>15
Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro Glu Arg Gly His
1 5 10 15
Glu Gln Thr Trp Gly Leu Phe Arg Pro Val Val Ala Thr Ala Thr Lys
20 25 30
Leu Phe Pro Leu Val Arg Thr Phe Ile Ser Trp Met Asn Gly Val Asp
35 40 45
Ala Ser Asp Ser Ala Val Ser Arg Ala Leu Ala Pro Pro Tyr Thr Ser
50 55 60
Asp His Pro Gly Arg Val Gly Val Ser Pro Gly Gly Ile Ala Glu Met
65 70 75 80
Phe Glu Thr Tyr Pro Lys Pro Gly Phe His Pro Asn Asp Glu Ala Ala
85 90 95
Leu Leu Lys Asp Arg Asn Gly Leu Phe Lys Leu Ala Met Lys His Lys
100 105 110
Leu Pro Ile Val Pro Val Tyr Cys Phe Gly Ala Thr
115 120
<210>16
<211>372
<212>DNA
<213>Artificial
<220>
<223>Catalytic domain
<400>16
atctttcctt tcggactcgc cttttcatgt ctacccgaac gaggacacga acaaacatgg 60
ggtctcttcc gaccagtcgt tgcaacagcc accaaactct ttccgctggt acgaaccttc 120
atttcttgga tgaacggagt ggatgcttcg cgttcggcgg tgtctcgtgc tcttgctcct 180
ccgtatacca gtgatcatcc gggaagagtg ggagtttcgc ccggtggtat tgccgagatg 240
tttgagacgt atccaaagcc ggggtttcat cctaatgacg aggcagcatt gttaaaagat 300
cggaatggat tgttcaagct tgcgatgaaa cacaagctgc cgattgttcc ggtgtactgc 360
tttggagcta ca 372
<210>17
<211>124
<212>PRT
<213>Artificial
<220>
<223>V298I mutant catalytic domain
<400>17
Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro Glu Arg Gly His
1 5 10 15
Glu Gln Thr Trp Gly Leu Phe Arg Pro Val Val Ala Thr Ala Thr Lys
20 25 30
Leu Phe Pro Leu Val Arg Thr Phe Ile Ser Trp Met Asn Gly Val Asp
35 40 45
Ala Ser Asp Ser Ala Val Ser Arg Ala Leu Ala Pro Pro Tyr Thr Ser
50 55 60
Asp His Pro Gly Arg Ile Gly Val Ser Pro Gly Gly Ile Ala Glu Met
65 70 75 80
Phe Glu Thr Tyr Pro Lys Pro Gly Phe His Pro Asn Asp Glu Ala Ala
85 90 95
Leu Leu Lys Asp Arg Asn Gly Leu Phe Lys Leu Ala Met Lys His Lys
100 105 110
Leu Pro Ile Val Pro Val Tyr Cys Phe Gly Ala Thr
115 120
<210>18
<211>372
<212>DNA
<213>Artificial
<220>
<223>V298I mutant catalytic domain
<400>18
atctttcctt tcggactcgc cttttcatgt ctacccgaac gaggacacga acaaacatgg 60
ggtctcttcc gaccagtcgt tgcaacagcc accaaactct ttccgctggt acgaaccttc 120
atttcttgga tgaacggagt ggatgcttcg cgttcggcgg tgtctcgtgc tcttgctcct 180
ccgtatacca gtgatcatcc gggaagaatc ggagtttcgc ccggtggtat tgccgagatg 240
tttgagacgt atccaaagcc ggggtttcat cctaatgacg aggcagcatt gttaaaagat 300
cggaatggat tgttcaagct tgcgatgaaa cacaagctgc cgattgttcc ggtgtactgc 360
tttggagcta ca 372
<210>19
<211>124
<212>PRT
<213>Artificial
<220>
<223>I343L mutant catalytic domain
<400>19
Ile Phe Pro Phe Gly Leu Ala Phe Ser Cys Leu Pro Glu Arg Gly His
1 5 10 15
Glu Gln Thr Trp Gly Leu Phe Arg Pro Val Val Ala Thr Ala Thr Lys
20 25 30
Leu Phe Pro Leu Val Arg Thr Phe Ile Ser Trp Met Asn Gly Val Asp
35 40 45
Ala Ser Asp Ser Ala Val Ser Arg Ala Leu Ala Pro Pro Tyr Thr Ser
50 55 60
Asp His Pro Gly Arg Val Gly Val Ser Pro Gly Gly Ile Ala Glu Met
65 70 75 80
Phe Glu Thr Tyr Pro Lys Pro Gly Phe His Pro Asn Asp Glu Ala Ala
85 90 95
Leu Leu Lys Asp Arg Asn Gly Leu Phe Lys Leu Ala Met Lys His Lys
100 105 110
Leu Pro Leu Val Pro Val Tyr Cys Phe Gly Ala Thr
115 120
<210>20
<211>372
<212>DNA
<213>Artificial
<220>
<223>I343L mutant catalytic domain
<400>20
atctttcctt tcggactcgc cttttcatgt ctacccgaac gaggacacga acaaacatgg 60
ggtctcttcc gaccagtcgt tgcaacagcc accaaactct ttccgctggt acgaaccttc 120
atttcttgga tgaacggagt ggatgcttcg cgttcggcgg tgtctcgtgc tcttgctcct 180
ccgtatacca gtgatcatcc gggaagagtg ggagtttcgc ccggtggtat tgccgagatg 240
tttgagacgt atccaaagcc ggggtttcat cctaatgacg aggcagcatt gttaaaagat 300
cggaatggat tgttcaagct tgcgatgaaa cacaagctgc cgcttgttcc ggtgtactgc 360
tttggagcta ca 372
<210>21
<211>1432
<212>DNA
<213>Artificial
<220>
<223>80%nucleotide sequence homology to TpDGA2
<400>21
atgactacta aaaaacgacc tcttccgcga catctccacc tacctccagg tgttgaagtt 60
ctggtgtcac ctcctccgta cgaagtttgc accctggtgg acagattgtt ggtgtacgcg 120
tcctccttga tagtggtagg ttcaccggta tggttctacg gtgggatcat atatttttac 180
aggaaatgga aaaaatatcg atcactagca gcagcaacag aggcagccag acatgagagt 240
ggagggggag gagcttcctc taccgtacga tgcagaggaa ctcgacagcg aacttcctca 300
gatgacggga actacacttc ctctacaggg gaaagctccc aggaaatgaa cgaacaggag 360
actcagactc agtctcatcg tcagcagact gagcagtaca acaactacaa gcgtttagct 420
actagatacg gtgttgctct ggcagctatc atactgattt cgatctgggg cccacatcga 480
gacaaacgag ttggtgaatg gctgggagtg aaaaaatgga gattgtggga tgcttggttg 540
aactatgtag gtttcacagt tcttaaagac aatggtgatg atgaccacac tattattcag 600
accaatcctc actctgctat tccgaatcag gaagagtttg acattcacac ttcaccttct 660
atcttcgctt tcgttccgca cgggatcttt ccattcggtc tggcgttttc ttgtcttccg 720
gaacgtggtc acgaacagac ttggggactg ttccgtcctg tggtagctac tgcgacgaag 780
ctgtttcccc tcgttcgtac gttcatatca tggatgaacg gtgtcgatgc atcccgatcc 840
gccgtctcac gagcactagc accaccctat acgagtgatc atcccggtag agtcggtgta 900
tccccgggag gaatagcgga gatgtttgag acctatccta aacccggctt tcatccaaat 960
gacgaggctg ctttgttaaa ggatcgcaat ggtttgttca aactagccat gaagcacaaa 1020
ctccccatag tacccgtcta ctgctttggt gcaactaaaa tgttgagacg tgtccagtta 1080
ccagcctttg tcgagacctt gagcagaatg ctgaaaatca gtctatgttt attctttggt 1140
aaactaggct tgccaatacc attccgtcaa cgcctcatgt atgtgatggg gaaaaccttg 1200
tttccaccac tccccagaga tggggtcaac acatcaatga tggaaggtgg tgaagaattt 1260
gatgaacgtg tccaggagat gcatgatgct ttctgcaatg agattacacg gatcttcgag 1320
cgtaacaagg accactacgg atggggaaac aagaacttga gactggtttg agagtgtgag 1380
tgatattctt atgctacact aaacttaaaa cctcaaacga ctcaagctca ga 1432
<210>22
<211>1432
<212>DNA
<213>Artificial
<220>
<223>85%nucleotide sequence homology to TpDGA2
<400>22
atgacaacta aaaaacgacc tcttccgcgt catctccacc tacctcctgg tgttgaagtt 60
ctcgtgtcac ctccaccgta cgaagtttgc accctcgtgg acagattgtt ggtgtacgcg 120
tcgtccttga tagtggttgg ttcaccggtt tggttctacg gtgggatcat atatttttac 180
aggaaatgga aaaaatatcg atctctagca gcagctacag aggcagccag acatgagagt 240
ggtgggggag gagcatcctc taccgttcga tgcagaggaa ctcgtcagcg aacttcgtca 300
gatgacggga actacacttc gtctacaggg gaaagctcgc aggaaatgaa cgaacaggag 360
acacagactc agtctcatcg acagcagaca gagcagtaca acaactacaa gcgtttagct 420
acaagatacg gtgttgctct cgcagctatc atactcattt cgatctgggg ccctcatcga 480
gacaaacgag taggtgaatg gctgggagtc aaaaaatgga gattgtggga tgcttggttg 540
aactatgttg gtttcacagt tctaaaagac aatggtgatg atgaccacac tataattcag 600
accaatccac actctgctat tcccaatcag gaagagtttg acattcacac ttctccttct 660
atcttcgctt tcgtaccgca cgggatcttt ccattcggac tggcgttttc ttgtctaccg 720
gaacgtggac acgaacagac ttggggactg ttccgacctg tggtagcaac tgcgaccaag 780
ctgtttcccc tcgtacgtac gttcatatct tggatgaacg gtgtcgatgc atcgcgatcc 840
gccgtgtcac gagcacttgc accaccctat accagtgatc atcccggtag agtcggagta 900
tccccgggtg gaatagcgga gatgtttgag acgtatccta aacccgggtt tcatccaaat 960
gacgaggctg ctttgttaaa cgatcgcaat ggtttgttca aacttgccat gaagcacaaa 1020
ctgcccatag taccggtcta ctgctttggt gcaacaaaaa tgttgagacg tgtgcagtta 1080
ccagcctttg tcgagacgtt gagcagaatg ctgaaaatca gtctatgttt attctttgga 1140
aaactaggct tgcctatacc attccgacaa cgcctcatgt atgtgatggg caaaaccttg 1200
tttccaccgc tccccagaga tggggtgaac acatcaatga tggaaggtgg agaagaattt 1260
gatgaacgtg tccaggagat gcatgatgca ttctgcaatg agattacacg gatcttcgag 1320
cgaaacaagg accactacgg atggggtaac aagaacttga gactggtttg agagtgtgag 1380
tgatattctt atgcaacact aaacttaaaa ccacaaacga cacaagctca ga 1432
<210>23
<211>1432
<212>DNA
<213>Artificial
<220>
<223>90%nucleotide sequence homology to TpDGA2
<400>23
atgacaacta aaaaacgtcc tcttccgcgt catctgcacc tacctcctgg agtagaagtt 60
ctcgtgtcac caccaccgta cgaagtttgc accctcgtcg acagattgtt ggtgtacgcg 120
tcgtcgttga ttgtggttgg ttcacccgtt tggttctacg gtggcatcat atatttttac 180
aggaaatgga aaaaatatcg atctcttgct gcagctacag aggcagcgag acatgagagt 240
ggtgggggag gagcatcgtc taccgttcgt tgcagaggta ctcgtcagcg aacatcgtca 300
gatgacggga actacacttc gtcaactggc gaaagctcgc aggaaatgaa cgaacaggag 360
acacagactc agtcacatcg acagcagaca gagcagtaca acaactacaa gcgattagct 420
acaagatacg gtgtagcact cgcagctatc attctcattt cgatctgggg ccctcatcgt 480
gacaaacgag taggtgaatg gctcggtgtc aaaaaatgga gattgtggga tgcatggttg 540
aactatgttg gtttcacagt tctaaaagac aatggagatg atgaccacac tataattcag 600
acgaatccac actcagctat acccaatcag gaagagtttg acattcacac ttctccatct 660
atcttcgctt tcgtaccgca cggcatcttt ccattcggac tcgcgttttc atgtctaccg 720
gaacgtggac acgaacagac atggggactg ttccgacctg tcgtagcaac agcgaccaag 780
ctctttcccc tcgtacgaac gttcatatct tggatgaacg gtgtggatgc atcgcgttcc 840
gcggtgtcac gagcacttgc tccaccctat accagtgatc atcccggaag agtcggagtt 900
tcccccggtg gaatagcgga gatgtttgag acgtatccaa aacccgggtt tcatccaaat 960
gacgaggcag ctttgttaaa cgatcggaat ggtttgttca aacttgcgat gaagcacaaa 1020
ctgccgatag taccggtcta ctgctttgga gctacaaaaa tgttgagacg tgtgcagtta 1080
cctgcctttg tcgagacgtt gagcagaatg ctcaaaatca gtctatgttt attctttgga 1140
aaacttggct tgcctattcc attccgacaa cggctcatgt atgtgatggg caaaacgttg 1200
tttccaccgc tcccgagaga tggggtgaac acttcaatga tggaaggagg agaagaattt 1260
gatgaacgtg tccaggagat gcatgatgca ttctgcaatg agataacacg gatcttcgag 1320
cgaaacaagg accactacgg ttggggtaac aagaacttga gactcgtttg agagtgtgag 1380
tgatattcat atgcaacact taacttaaaa ccacaaacca cacaagcaca ga 1432
<210>24
<211>1432
<212>DNA
<213>Artificial
<220>
<223>95%nucleotide sequence homology to TpDGA2
<400>24
atgacaacaa aaaaacgtcc actaccgcgt catctgcacc ttccacctgg tgtagaagta 60
ctcgtgtctc caccacccta cgaagtttgc acgctcgtcg acagattgtt ggtctacgcc 120
tcgtccttga ttgtcgttgg atctcccgtt tggttctacg gaggcatcat atatttttac 180
aggaaatgga aaaaatatcg ttctcttgca gcagcaacag aggcagcgag acatgagagt 240
ggtgggggtg gtgcttcgtc tacggttcgt tgcagaggta cacgtcagcg aacatcgtct 300
gatgacggca actacacatc gtcaactggc gaaagctcgc aggaaatgaa cgaacaggag 360
acacagacac agtcacatcg acagcagaca gagcagtaca acaactacaa gcgattagca 420
acaagatacg gagtagcact cgcagcaatc attctcatat ccatctgggg ccctcatcgt 480
gacaaacgtg taggagaatg gctcggtgtc aaaaaatgga gattgtggga tgcatggttg 540
aactatgttg gattcactgt actaaaagac aatggagatg atgaccacac aataatacag 600
acgaatccac actcagctat acccaatcag gaagagtttg acatacacac atctccatca 660
atcttcgcat tcgtaccgca cggcatcttt cctttcggac tggccttttc atgtctaccg 720
gaacgtggac acgaacagac atggggtctc ttccgaccag tcgtagcaac agccaccaag 780
ctctttccgc tggtacgaac gttcatttct tggatgaacg gtgtggatgc atcgcgttcc 840
gcggtgtctc gtgcacttgc acctccctat accagtgatc atccgggaag agtgggagta 900
tcgcccggtg gtatagccga gatgtttgag acgtatccaa aaccggggtt tcatcctaat 960
gacgaggcag ctttgttaaa cgatcggaat ggattgttca aacttgcgat gaaccacaaa 1020
ctgccgattg ttccggtcta ctgctttgga gcaacaaaaa tgttgagacg agtgcagtta 1080
cctgcgtttg tggagacgtt gagcagaatg ctcaaaatca gtctttgttt attctttgga 1140
aaacttgggt tgcctattcc tttccgacaa cggctgatgt atgtcatggg caaaacgttg 1200
tttcctccgc tgccgagaga tggggtgaac acttcaatga tggaaggagg agaagaattt 1260
gatgaacgag tgcaggagat gcatgatgca ttctgcaatg agataactcg catcttcgag 1320
cgaaacaagg accactacgg ttggggtaac aagaacttga gactcgtatg agagtgtgag 1380
tgatattcat atgcaactct taacttaaaa ccacaaacca cacaagcaca ga 1432
<210>25
<211>309
<212>PRT
<213>T.pseudonana
<400>25
Leu Ala Val Thr Leu Trp Leu Gly Trp Asn Gly Ile Val Val Cys Ile
1 5 10 15
Ala Val Tyr Leu Leu Phe Ile Ala Asn Asn Ile Glu Arg Met Val Ile
20 25 30
Ile Gly Leu Ala Thr Met Ser Leu Ile Leu Pro Ala His Phe Pro Gly
35 40 45
Ala Leu Gly Tyr Lys Ile Gly Asp Trp Ile Met Arg Gln Ala Glu Lys
50 55 60
Tyr Phe Gly Leu Lys Thr Val Ile Glu Asp Glu Glu Asp Leu Ile Arg
65 70 75 80
His Ala Asn Glu Asn Lys Ala Val Ile Phe Ala Phe Asn Pro His Asp
85 90 95
Met Leu Pro Tyr Ala Val Phe Ala Phe Ala Pro Thr Leu Lys Arg Leu
100 105 110
Pro Gly Lys Ile Gly Lys Asp Gly Thr Cys Leu Met Ser Ser Ala Ile
115 120 125
Phe Asn Ile Pro Phe Leu Arg Gln Val Tyr Thr Trp Val Asn Ser Leu
130 135 140
Pro Val Asp Lys Lys Thr Phe Leu Gly Arg Leu Lys Arg Gly Gln Ser
145 150 155 160
Phe Ala Phe Val Pro Gly Gly Val Gln Glu Val Ile Met Leu Asp Pro
165 170 175
Asn Gln Pro Lys Asp Val Val Leu Tyr Leu Lys Asn Arg Lys Gly Phe
180 185 190
Val Lys Leu Ala Leu Ala Thr Gly Ser Pro Ile Val Pro Val Phe Gly
195 200 205
Phe His Leu Asp Gly Ser Tyr Gly Tyr Trp Leu Pro Lys Gly Lys Leu
210 215 220
Val Glu Arg Leu Ser Arg Thr Leu Gly Phe Leu Pro Leu Leu Phe Trp
225 230 235 240
Gly Arg Trp Met Ile Pro Phe Gly Ile Pro His Pro Lys Lys Ile His
245 250 255
Val Val Val Gly Ser Ala Ile Asp Val Pro Asn Glu Gly Glu Asp Val
260 265 270
Ser Gln Glu Ser Ile Glu Lys Tyr His Ala Ile Phe Leu Lys Glu Leu
275 280 285
Glu Ala Leu Phe Glu Arg His Lys Glu Glu Ala Gly Tyr Gly His Arg
290 295 300
Gln Leu Lys Ile Val
305
<210>26
<211>930
<212>DNA
<213>T.pseudonana
<400>26
ctggcagtca cattgtggtt ggggtggaat gggatcgtgg tgtgtatcgc agtctacctc 60
ttgttcattg ccaacaatat tgaacgaatg gtgattattg gattggcaac gatgtcgttg 120
atactgcctg ctcactttcc aggagctttg ggctacaaga ttggggattg gattatgcgt 180
caggcagaga agtactttgg attaaagacg gtgattgaag acgaagagga tttgattcgg 240
catgccaatg agaacaaagc agtgatattt gccttcaatc cacatgatat gttgccgtat 300
gcagtatttg cattcgctcc tacattgaag agactaccgg gtaagatcgg gaaggatgga 360
acgtgcctca tgtcatcggc aatcttcaac attccttttt tgagacaagt gtatacgtgg 420
gtgaacagcc ttccagtaga caagaaaaca tttctgggga ggctgaagag agggcaaagc 480
tttgcttttg ttcctggggg agtgcaagag gtcattatgc ttgatccgaa ccagccaaaa 540
gatgtggtgc tatatctcaa gaaccgcaaa ggattcgtga agctggcgtt ggcgacaggc 600
tcgcccatcg tgcccgtgtt tggctttcat ctggatggaa gctatggcta ttggctgccg 660
aaagggaaac tggtcgagag actttcacga acattgggct ttcttcctct tctcttttgg 720
gggcgttgga tgataccttt cggcatacca caccccaaaa agattcacgt tgtcgttgga 780
tcagcaatag atgtaccgaa cgagggagaa gatgtctcac aagagtcaat tgaaaagtac 840
catgccatct ttctgaagga gcttgaagca ttgtttgaga ggcacaagga agaagcggga 900
tacggacatc ggcaattgaa gattgtctaa 930
<210>27
<211>158
<212>PRT
<213>T.pseudonana
<400>27
Met Glu Asp Tyr Leu Lys Asn Gly Glu Asp Val Ala Leu Pro Pro Gly
1 5 10 15
Gly Phe Glu Glu Ala Thr Leu Thr Cys Thr Thr Gln Asp Arg Val Phe
20 25 30
Ile Lys Lys Arg Tyr Gly Phe Val Arg Leu Cys Leu Lys Tyr Gly Val
35 40 45
Ala Ile Arg Pro Val Tyr Val Phe Gly Glu Gly Arg Leu Phe Gly Asn
50 55 60
Val Gln Gly Met Trp Lys Thr Arg Leu Ala Leu Asn Arg Trp Gly Ile
65 70 75 80
Pro Thr Ile Leu Val Trp Gly Ser Trp Phe Phe Pro Leu Leu Pro Lys
85 90 95
Lys Gly Val Asn Leu His Ile Val Val Gly Lys Pro Leu Ile Val Pro
100 105 110
Lys Ile Asp Asn Pro Thr Lys Glu Glu Val Ile Ala Trp His Glu Lys
115 120 125
Tyr Ile Thr Glu Leu Lys Arg Ile Tyr Glu Glu Tyr Lys Glu Val Ala
130 135 140
Tyr Gly Asn Glu Asp Gly Lys Val Ala Lys Leu Glu Val Trp
145 150 155
<210>28
<211>477
<212>DNA
<213>T.pseudonana
<400>28
atggaagact atctgaaaaa tggagaggat gtggcacttc ctcctggggg atttgaggag 60
gctaccctga cttgtacaac gcaggatagg gtgtttatca agaagcggta tggctttgtg 120
aggctgtgtt tgaagtatgg agtggcgata cgaccagtct atgtgtttgg agagggaaga 180
ttgtttggca acgtacaagg aatgtggaag acaagacttg ccttgaatcg atggggcatt 240
ccgactatat tggtatgggg tagttggttc tttcccttgc ttccgaagaa gggtgtcaac 300
ctacatattg ttgttggaaa gcctttgatt gtgccaaaga ttgacaatcc aacaaaggaa 360
gaggttattg cgtggcatga aaagtatatt accgagttga agaggattta tgaagagtac 420
aaggaggttg cgtacggtaa cgaggacgga aaggttgcaa agcttgaggt ttggtga 477
<210>29
<211>376
<212>PRT
<213>T.pseudonana
<400>29
Met Lys Tyr Arg Lys Leu Asn Leu Ala Thr Leu Pro Asp Glu Leu Phe
1 5 10 15
Thr Lys Gln Thr Asp Val Lys Glu Trp Met Ala Leu Thr Thr Thr Ser
20 25 30
Ala Glu Gly Gly Phe Pro Pro Ser Ser Leu Asn Lys Val Leu Pro Val
35 40 45
Lys Arg Asp Ile Glu Ile Glu Arg Ile Ile Gly Thr Ser Tyr Tyr Met
50 55 60
Ile Gly Asn Thr Val Pro Phe Ala Val Pro Leu Leu Leu Ala Ala Ser
65 70 75 80
Tyr Phe Ser Ala Val Gly Ala Leu Ile Phe Lys ValTyr Met Val Tyr
85 90 95
Phe Thr Thr Leu Phe Val ValPhe Thr Tyr Tyr Phe Tyr Pro Lys Tyr
100 105 110
Met Lys Arg Tyr Asn Arg Pro Lys Ser Met Ser Lys Thr Asp Ile Lys
115 120 125
Asp Asn Gln Tyr Leu Tyr Thr Glu Arg His Thr Gln Lys Tyr Leu Ser
130 135 140
Met Gln Phe Val Trp Pro Glu Ser Ile Gln Arg Pro Ala Leu Asn Asp
145 150 155 160
Gln Pro Val Ile Phe Ala Ala Ile Pro His Gly Leu Ser Pro Leu Gly
165 170 175
Ile Thr Ala Tyr Pro Met Trp Ser Lys Leu Phe Asn Asp Lys Leu Cys
180 185 190
His Trp Thr Cys Ala Pro Val Val Leu Lys Leu Pro Leu Ile Ser Ser
195 200 205
Phe Met Lys Ala Ile Gly Tyr Ile Pro Ala Lys Ala Lys Asn Ile Thr
210 215 220
Asp Thr Leu Ile Lys Lys Glu Glu Asn Val Gly Ile Ile Leu Asp Gly
225 230 235 240
Ile Ala Gly Met Phe Gln Ala His Asp Glu Val Ala His Val Lys Arg
245 250 255
Arg Lys Gly Ile Ile Lys Ile Ala Leu Arg Ala Gly Ala Ala Val Val
260 265 270
Pro Val Tyr Gly Phe Gly His Thr Ser Leu Trp Lys Ile Val Val Asp
275 280 285
Pro Phe Gly Phe Leu Glu Trp Leu Ser Thr Lys Ser Asp Val Ser Val
290 295 300
Thr Pro Phe Phe Gly Arg Phe Asn Trp Phe Leu Gly Pro Pro Lys Arg
305 310 315 320
Val Ala Val Cys Val Cys Met Gly Asp Ala Ile Lys Cys Pro Lys Ile
325 330 335
Glu Glu Pro Thr Gln Gln Asp Ile Asp Lys Tyr His Gly Leu Leu Leu
340 345 350
Lys Gly Tyr Asp Gln Leu Phe Glu Gln His Lys Val Ala Tyr Gly Trp
355 360 365
Gly Asp Lys Lys Leu Gln Phe Val
370 375
<210>30
<211>1131
<212>DNA
<213>T.pseudonana
<400>30
atgaagtatc gcaaactcaa cctagccacc ctaccagacg aactcttcac caaacagaca 60
gatgtgaaag agtggatggc gcttaccaca acgtcagcgg aaggaggctt tccaccatcg 120
tcattaaaca aagtgttgcc tgtgaagaga gacatcgaga ttgaacgtat catcggcact 180
tcctactata tgattgggaa tacggttcca tttgcagttc cgcttctgtt ggctgcatcg 240
tactttagtg cagttggtgc tctgattttc aaggtgtaca tggtgtactt taccacactg 300
ttcgtagtgt tcacgtatta tttctaccca aagtacatga aacgatacaa tcgtccgaag 360
tccatgtcta agactgacat caaggacaac caatatctgt acacagaacg tcacacccaa 420
aagtatctct ccatgcaatt cgtgtggcca gaatcaatcc aaagaccagc tcttaacgat 480
caaccagtaa tctttgcagc cattccacat ggattaagcc cgttaggaat cacggcatat 540
ccaatgtggt caaagttgtt caatgataaa ctttgccatt ggacttgtgc accagtggtg 600
ttgaagttgc ctttgatatc ttcgtttatg aaggctattg gttacattcc agcgaaagca 660
aagaatatca cggacacact gatcaagaag gaagagaatg ttggtatcat tcttgatgga 720
atcgctggaa tgtttcaggc tcatgatgaa gtggcacacg tgaagagaag gaaggggatt 780
atcaagattg cattgagggc cggagccgca gttgtacctg tgtacggttt cggtcatact 840
tcgttgtgga aaatcgtcgt tgatcccttt ggattcttgg aatggctgag tacaaaatcg 900
gatgtctctg tcacaccttt cttcgggagg ttcaactggt ttttgggtcc tccgaaacga 960
gttgctgtct gtgtctgcat gggagacgca ataaagtgtc ctaagatcga ggaaccgacg 1020
caacaagaca ttgataagta tcatggactc ttattgaaag gatacgatca actatttgaa 1080
cagcacaaag tagcatacgg atggggtgat aagaaactgc agtttgttta a 1131
<210>31
<211>346
<212>PRT
<213>Chlamydomonas reinhardtiiv
<400>31
Met Ala Gly Gly Lys Ser Asn Gly Thr Gly Ala Ala Asp Ala His Val
1 5 10 15
Arg Thr Ser His Leu Thr Leu Lys Ala Gly Glu Asp Pro Pro Pro Asn
20 25 30
Val Arg Ile Tyr Ser Asp Gly Ile Lys Pro Asp Ala Arg Gln Asn Leu
35 40 45
Leu Val Gln Ile Leu Ala Gly Ile Thr Met Ser Ile Tyr Val Gly Phe
50 55 60
Met Asn Tyr Phe Met Leu Leu Val Val Leu Ser Tyr Trp Ser Arg Ile
65 70 75 80
Cys Arg Tyr Val Val Leu Ala Leu Leu Gly Thr Leu Ala Leu Pro Cys
85 90 95
Lys Pro Val Leu Trp Pro Ala Phe Asn Lys Leu Trp Ile Phe Lys Thr
100 105 110
Trp Arg His Tyr Phe His Tyr Ser Phe Leu Ile Glu Glu Pro Leu Asp
115 120 125
Pro Asn Lys Arg Tyr Ile Phe Val Glu Phe Pro His Gly Ala Phe Pro
130 135 140
Ile Gly Pro Ile Val Ala Gly Thr Leu Met Gln Thr Leu Phe Pro His
145 150 155 160
Met Met Ile Tyr Ser Val Ala Ala Ser Val Val Phe Tyr Ile Pro Phe
165 170 175
Trp Arg His Phe Ile Thr Trp Ile Gly Ser Val Pro Ala Thr Pro Gly
180 185 190
Asn Phe Lys Arg Leu Leu Lys Lys Gly Ser Val Ala Val Val Val Gly
195 200 205
Gly Ile Ala Glu Met Tyr Met Gly Asn Lys Lys Lys Glu Arg Ile Lys
210 215 220
Leu Val Gly Arg Arg Gly Phe Ala Arg Ile Ala Leu Glu Glu Gln Val
225 230 235 240
Asp Gly Ile Val Cys Val Tyr Tyr Phe Gly Gln Ser Gln Val Leu Asp
245 250 255
Phe Gly Pro Ser Trp Leu Ala Asp Phe Ser Arg Arg Met Arg Thr Ser
260 265 270
Phe Gly Tyr Leu Thr Gly Trp Met Gly Leu Pro Val Pro Arg Pro Ile
275 280 285
Pro Ile Tyr Met Val Asn Gly Lys Pro Ile Pro Val Pro Lys Val Ala
290 295 300
Arg Asp Ser Pro Glu Phe Asp Lys Glu Val Asp Lys Leu Leu Asp Ala
305 310 315 320
Thr Ile Thr Glu Leu Gly Glu Met Tyr Asn Arg His Arg Gly Glu Tyr
325 330 335
Gly Trp Gly Asp Arg Pro Leu Ser Ile Glu
340 345
<210>32
<211>1041
<212>DNA
<213>Chlamydomonas reinhardtii
<400>32
atggcaggtg gaaagtcaaa cggcacgggc gcggcggacg cgcacgtgcg tacctcgcac 60
ttgaccctga aagctgggga ggacccgccc ccgaatgttc gcatctacag tgacggcatc 120
aagccggacg cgcggcagaa cctgcttgtt cagatcctgg ccggcatcac gatgtcgatt 180
tatgtaggct tcatgaacta tttcatgctg ctggtggtgc tctcctactg gagccgcatc 240
tgccgctatg tggtcctggc gctgctaggc acactggcgc tgccctgcaa gcccgtgctg 300
tggcctgcct tcaacaagct gtggatcttc aagacctggc gtcactactt ccactacagt 360
ttcctgattg aggagccgct tgaccccaac aagcgctaca tctttgtcga gttcccgcac 420
ggcgcgttcc ccattggtcc catcgtggcg ggcacgctca tgcagactct gttcccgcac 480
atgatgatct acagcgtggc cgcctccgtc gtgttctaca tccccttctg gcgccatttc 540
atcacgtgga tcggctcggt gcccgcaacg cccggcaact tcaagcggct gctgaagaag 600
ggcagtgtgg cggtggtggt gggcggcatt gccgagatgt acatgggcaa caagaagaag 660
gagcgcatta agctagtggg ccgccgcggc ttcgcacgca tcgcgctgga ggagcaggtg 720
gacggcattg tgtgcgtgta ctacttcggt cagagccaag tgctggactt cgggccctcc 780
tggctggcgg actttagccg ccgcatgcgc accagcttcg gctacctcac gggatggatg 840
gggctgccgg tgccgcggcc catccccatc tacatggtga atgggaagcc catcccggtg 900
cccaaggtgg ctcgtgactc gcccgagttc gacaaggagg tggataagct gcttgacgcc 960
accatcacgg agctgggcga gatgtacaac aggcacagag gcgagtacgg ctggggcgac 1020
cgcccgctgt ccatcgagta g 1041
<210>33
<211>413
<212>PRT
<213>Chlamydomonas reinhardtii
<400>33
Met Thr Pro Arg Asp Pro Pro Val Pro Arg Pro Pro Pro Gly Val Arg
1 5 10 15
Gln Tyr Thr Asp Gly Arg Ser Ala Ser Tyr Val Leu Pro Leu Pro Tyr
20 25 30
Arg Leu Leu Ala Gln Leu Thr Leu Gly Leu Tyr Val Gly Phe Pro Tyr
35 40 45
Ile Leu Leu Gly Leu Leu Leu Gly Thr Ala Ala Gly Ser Arg Ala Ala
50 55 60
Ala Ala Ala Leu Ala Leu Thr Leu Gly Ser Leu Leu Val Pro Ala Pro
65 70 75 80
Pro His Ile Arg Gln Gly Met Leu Asp Ser Ala Leu Phe Arg Leu Trp
85 90 95
Arg Ala Tyr Phe Asn Tyr Ser Tyr Ala Tyr Asp Gln Leu Pro Asp Phe
100 105 110
Asn Arg Pro His Ile Phe Val Asn Ser Pro His Gly Ala Phe Pro Leu
115 120 125
Ser Gln Ile Leu Cys Ile Ser Leu Ser Asn Ile Val Trp Pro Gly Phe
130 135 140
Pro Val His Ser Leu Ala Ala Ser Val Leu Trp Tyr Ile Pro Leu Trp
145 150 155 160
Arg His Met Lys Ala Ala Leu Gly Ala Ala Pro Ala Ser Arg Asp Asn
165 170 175
Ala Arg Met Leu Leu Arg His Arg Gly Ser Val Ala Val Leu Ala Gly
180 185 190
Gly Ile Ala Glu Met Tyr Thr Ser Ser Pro Ser Arg Ala Ala Ala Ala
195 200 205
Thr Glu Pro Asp Glu Ala Ala Ala Ala Gly Gly Ala Ile Asp Thr Thr
210 215 220
Glu Ala Ala Gly Ala Thr Gly Ser Ser Ser Thr Thr Thr Ser Pro Pro
225 230 235 240
Gln Pro Lys Glu Gln Gln Arg Asp Gly Glu Gln Arg Gln Gly Pro Arg
245 250 255
Lys Gly Leu Lys Gly Leu Leu Lys Gly Pro Lys Asp Asp Pro Asp Pro
260 265 270
Ala Ala Glu Glu Glu Gln Gly Leu Gly Leu Ala Pro Glu Arg Ile Lys
275 280 285
Leu Leu Gly Arg Arg Gly Phe Val Arg Leu Ala Val Glu Met Gly Val
290 295 300
Pro Ile Val Pro Ile Tyr His Met Gly Asn Ser Lys Ile Leu Thr Phe
305 310 315 320
Gly Pro Gln Ser Leu Gln Gln Leu Ser Arg Arg Leu Arg Met Ala Leu
325 330 335
Gly Ala Val Phe Gly Val Trp Gly Leu Pro Val Pro Arg Pro Gln Pro
340 345 350
Leu Met Met Cys Val Gly Ser Pro Ile Pro Val Pro Tyr Val Asp Pro
355 360 365
Ala Ala Glu Pro Glu Arg Phe Glu Ala Val Val Ala Ala Val His Gly
370 375 380
Gln Val Val Ala Ala Phe Gln Asp Leu Tyr Asn Arg Tyr Arg Val Gln
385 390 395 400
Tyr Gly Cys Gly Trp Glu Arg Arg Pro Leu Glu Val Cys
405 410
<210>34
<211>1241
<212>DNA
<213>Chlamydomonas reinhardtii
<400>34
atgaccccgc gggatccgcc ggtgccgcgg ccgccgccgg gcgtacggca gtacactgac 60
ggccggtcgg cgtcgtacgt actgccgctg ccgtatcgcc tgctggccca gctgactctg 120
ggtttgtacg tgggctttcc ctacatcctg ctggggttgt tgctgggcac ggctgccggc 180
tcgcgcgccg ccgccgccgc cctggctctg acgctgggca gcctgctggt gccggcccca 240
ccgcacatcc ggcagggcat gctggactcg gcactgttca ggctgtggcg cgcctacttc 300
aactacagct acgcctacga ccaactgccc gacttcaacc gcccacacat ctttgtcaac 360
agcccgcacg gcgccttccc gctgtcgcag atcctgtgca tctccctgtc caacatcgtg 420
tggccgggct tccccgtgca cagcctggcg gcctcggtgc tgtggtacat accgctgtgg 480
cgccacatga aggcggcgct gggggccgcg cccgccagcc gggacaacgc gcgcatgctg 540
ctgaggcacc gcgggtcggt ggcggtgctg gcgggcggca ttgcggagat gtacacgtca 600
tcgccctccc gcgccgccgc tgccaccgaa ccagatgagg ctgcggctgc gggtggggcg 660
atcgacacga ctgaagccgc cggcgccacc ggctcaagca gcaccaccac tagcccgccg 720
cagccaaagg agcagcagcg cgatggggag cagcgccagg ggccgcgcaa ggggctgaag 780
gggctgctga aaggcccgaa ggacgatccc gatccggcgg cggaggagga gcagggcctc 840
gggttggcac ctgaacgcat caagctgctg ggccggcgcg gcttcgtgcg gctggcggtg 900
gagatgggtg tgcccattgt acccatatac cacatgggca acagcaagat cctgaccttc 960
gggccgcagt cactgcagca gctgtcgcgc cgcctgcgca tggcgctggg cgccgtgttc 1020
ggcgtgtggg gcctgcctgt gccgcgcccc cagccgctca tgatgtgtgt gggcagcccc 1080
attcccgtgc cgtacgtgga tccagccgcc gagccggagc gcttcgaggc cgtggtggcg 1140
gcggtgcacg ggcaggtggt ggcggccttt caggatctgt acaacaggta ccgcgtgcag 1200
tacggctgcg gttgggagcg ccggccgctg gaggtgtgct g 1241
<210>35
<211>320
<212>PRT
<213>Chlamydomonas reinhardtii
<400>35
Met Gln Ser Lys Arg Cys Ala Glu Leu Ala Ser Gly Ala Leu Trp Pro
1 5 10 15
Met Asp Arg Asp Gln Met Arg Asp Arg Asp Pro Trp Lys Leu Arg Asp
20 25 30
Arg Ala Ile Ser Gln Ala Trp Val Trp Pro Leu Leu Ile Gly Thr Leu
35 40 45
Leu Tyr Val Gln Ser Thr Thr Leu Thr Ile Ala Phe Leu Leu Trp His
50 55 60
Ile Trp Lys Val Met Ala Ser Tyr Phe Pro Gly Ala Arg Leu Ile Lys
65 70 75 80
Thr Ala Asp Leu Asp Pro Ala Gly Arg Tyr Ile Phe Val Ser His Pro
85 90 95
His Gly Val Ile Ala Ile Ser Asp Trp Leu Ala Phe Ala Thr Glu Ala
100 105 110
Leu Gly Phe Ser Lys Leu Phe Pro Gly Leu Asp Leu Arg Cys Ala Thr
115 120 125
Leu Ala Ser Asn Phe Trp Val Pro Gly Leu Arg Glu Tyr Ile Leu Ser
130 135 140
His Gly Met Cys Gly Val Gly Arg Asp Thr Leu Ala Arg Val Leu Thr
145 150 155 160
Gly Lys Pro Gly Arg Ala Val Val Leu Val Val Gly Gly Ala Ser Glu
165 170 175
Ala Leu Leu Ala Ala Glu Gly Thr Tyr Asp Leu Val Leu Arg Asn Arg
180 185 190
Lys Gly Phe Val Arg Leu Ala Leu Gln Thr Gly Ala Ser Leu Val Pro
195 200 205
Val Leu Ser Tyr Gly Glu Thr Asp Thr Phe His Thr Tyr Ile Pro Pro
210 215 220
Pro Cys Ser Arg Ala Ala Ala Val Met Lys Val Leu Lys Gln Val Phe
225 230 235 240
Gly Phe Ser Thr Pro Leu Cys Trp Gly Thr Gly Leu Phe Gly Gly Trp
245 250 255
Gly Met Leu Ala Leu Gln Val Pro Leu Thr Val Val Val Gly Ala Pro
260 265 270
Ile Gln Val Asp Lys Val Ser Ser Pro Thr Glu Ala Glu Val Ala Ala
275 280 285
Leu His Lys Thr Tyr Thr Glu Ala Leu Gln Lys Leu Trp Asp Asp Thr
290 295 300
Val Asp Lys Tyr Gly Lys Gly Val Lys Arg Pro Leu Ala Ile Val Gln
305 310 315 320
<210>36
<211>963
<212>DNA
<213>Chlamydomonas reinhardtii
<400>36
atgcaaagta agcgttgtgc agagctggcc tctggggctc tgtggcccat ggaccgcgac 60
cagatgcgcg accgcgaccc atggaagctg cgcgaccgag ctataagcca agcatgggtg 120
tggcctctgc tcatcggcac attgctttac gtgcagagca ccacgctcac aattgccttc 180
ctgctgtggc atatctggaa ggttatggcc tcttacttcc ccggcgcccg cctgattaag 240
accgccgacc tggatccggc tggccgctat atattcgtga gccacccgca cggcgtcatc 300
gccatttccg actggctggc atttgccaca gaggcgctgg gcttctccaa actgttccca 360
ggcctggacc tgcgctgcgc cacgctggct tcaaacttct gggtgcctgg tttgcgtgag 420
tacatcctat cgcacggcat gtgcggcgtg gggcgagaca ctctggcgcg cgtgctgaca 480
ggaaagccgg gccgtgcggt tgtgttggtg gtgggcggcg cgtctgaggc gctgttggcg 540
gcggagggaa cttatgacct ggtgctgcgc aaccgcaagg gctttgtgcg cctggcgctg 600
cagaccggcg ccagtctggt gccggtgctg tcgtacggtg agacagacac cttccacacc 660
tacatcccgc cgccctgcag ccgggcggcc gcggtcatga aggtgctgaa gcaggtgttt 720
ggcttctcca cgcccctgtg ctggggcacc ggactgttcg ggggctgggg catgctagcg 780
ctgcaggtgc cgctcactgt ggtggtgggg gcacccatac aggtggacaa ggtgtccagt 840
cccacggagg ctgaggtggc ggcgctgcat aagacctaca cggaggcact gcagaagctg 900
tgggatgaca cagtggacaa gtacggcaag ggtgtcaagc ggccgctggc catcgtgcaa 960
tga 963
<210>37
<211>324
<212>PRT
<213>Chlamydomonas reinhardtii
<400>37
Met Ala Ile Asp Lys Ala Pro Thr Asn Val Arg Ile Trp Ser Asp Gly
1 5 10 15
Val Thr Glu Lys Gly Lys Gln Ser Ile Phe Ser Ser Leu Val Ala Met
20 25 30
Leu Thr Leu Phe Ile Tyr Cys Gly Trp Met His Val Leu Leu Ala Leu
35 40 45
Val Ile Leu Ser Phe Trp Tyr Arg Trp Ala Leu Val Thr Val Leu Leu
50 55 60
Leu Tyr Ser Thr Leu Leu Leu Pro Pro Lys Pro Val Leu Trp Gly Pro
65 70 75 80
Val Cys Arg Ser Trp Ile Phe Gln Thr Trp Arg Glu Tyr Phe Lys Phe
85 90 95
Ser Tyr Val Phe Asp Glu Val Leu Asp Ser Lys Lys Lys Tyr Ile Phe
100 105 110
Ala Glu Phe Pro His Gly Val Phe Pro Met Gly Pro Leu Ile Gly Ala
115 120 125
Thr Glu Cys Gln Ile Met Phe Pro Gly Phe Asp Ile Phe Gly Leu Ala
130 135 140
Ala Asn Val Val Phe Thr Val Pro Phe Trp Arg His Phe Val Ala Trp
145 150 155 160
Leu Gly Ser Val Pro Ala Thr Thr Arg Asp Phe Lys Arg Val Leu Lys
165 170 175
Gln Gly Ser Val Ala Val Ile Val Gly Gly Ile Ala Glu Met Tyr Met
180 185 190
Gln Ser Pro Thr Lys Glu Gln Ile Met Leu Lys Asp Arg Lys Gly Phe
195 200 205
Val Arg Val Ala Val Glu Glu Gly Val Asp Gly Gly Ile Val Pro Val
210 215 220
Tyr His Phe Gly Asn Ser Gln Val Leu Asp Phe Gly Pro Gln Ala Met
225 230 235 240
Ala Ser Val Ser Arg Arg Leu Arg Ala Ala Leu Gly Phe Leu Tyr Gly
245 250 255
Val Ala Tyr Leu Pro Leu Pro Arg Arg Arg Asn Ile Tyr Met Val Cys
260 265 270
Gly Lys Pro Val Pro Val Thr Arg Thr Ala Arg Asp Asp Pro Lys Phe
275 280 285
Glu Glu Val Val Asp Ala Thr His Ala Ala Val Met Ala Ala Leu Gln
290 295 300
Glu Ala Tyr Asp Arg His Lys Thr Glu Tyr Gly Trp Ala Asp Arg Pro
305 310 315 320
Leu Val Ile Ser
<210>38
<211>975
<212>DNA
<213>Chlamydomonas reinhardtii
<400>38
atggcgattg ataaagcacc gacaaatgtg cgaatttgga gcgatggcgt cacggagaag 60
ggcaagcaaa gcatcttctc atcgctggtg gctatgttga cgctcttcat ctactgtggc 120
tggatgcatg tgctgctggc gcttgtgatc ctgtccttct ggtaccgctg ggcgctggtg 180
acggtgctgc tgctgtactc caccctgctg ctgccgccta agccggtgct gtggggaccg 240
gtctgtcgct cctggatctt ccagacctgg cgggagtact tcaagttctc ttacgtgttt 300
gatgaggtgc tggactcgaa gaagaagtac atcttcgcgg agttcccgca cggtgtcttc 360
cccatgggcc cactcattgg cgccacagaa tgccagatca tgtttcccgg ctttgacatc 420
ttcgggctgg cggcgaatgt ggtgttcacg gtccccttct ggcggcattt cgtggcgtgg 480
ctgggctccg tgccggccac cacacgcgac ttcaagcggg tgctgaagca aggaagcgtg 540
gcggtcatcg tgggaggcat cgcagagatg tacatgcaga gccccacgaa ggagcagatc 600
atgttgaagg accgcaaggg ctttgttcgt gtggcggtgg aggagggcgt ggatggcggc 660
atcgtgccgg tctaccactt tggcaactct caggtgctgg acttcggccc ccaggccatg 720
gccagtgtgt cccgccggct gcgtgcggcc ctgggcttcc tgtacggagt ggcctacctg 780
cccctgccca ggcgccgcaa catttacatg gtgtgcggca agcccgttcc cgtcacgcgc 840
accgcccgcg acgaccccaa gtttgaggag gtggttgacg ccactcacgc cgctgtgatg 900
gcggccctgc aggaggccta cgaccgccac aagaccgagt acggctgggc cgaccgaccg 960
ctggtcatca gctga 975
<210>39
<211>327
<212>PRT
<213>Chlamydomonas reinhardtii
<400>39
Met Pro Leu Ala Lys Leu Arg Asn Val Val Leu Glu Tyr Ala Ala Ile
1 5 10 15
Ala Ile Tyr Val Ser Ala Ile Tyr Thr Ser Val Val Leu Leu Pro Ser
20 25 30
Ala Leu Ala Leu Phe Tyr Leu Phe Gly Ala Thr Ser Pro Ser Ala Trp
35 40 45
Leu Leu Leu Ala Ala Phe Leu Ala Leu Thr Phe Thr Pro Leu Gln Leu
50 55 60
Thr Thr Gly Ala Leu Ser Glu Arg Phe Val Gln Phe Ser Val Ala Arg
65 70 75 80
Ala Ala Ala Tyr Phe Pro Thr Arg Val Val Val Thr Asp Pro Glu Ala
85 90 95
Phe Arg Thr Asp Arg Gly Tyr Leu Phe Gly Phe Cys Pro His Ser Ala
100 105 110
Leu Pro Ile Ala Leu Pro Ile Ala Phe Ala Thr Thr Ser Pro Leu Leu
115 120 125
Pro Lys Glu Leu Arg Gly Arg Thr His Gly Leu Ala Ser Ser Val Cys
130 135 140
Phe Ser Ala Pro Ile Val Arg Gln Leu Tyr Trp Trp Leu Gly Val Arg
145 150 155 160
Pro Ala Thr Arg Gln Ser Ile Ser Gly Leu Leu Arg Ala Arg Lys Val
165 170 175
Ala Val Leu Val Pro Gly Gly Val Gln Glu Val Leu Asn Met Glu His
180 185 190
Gly Lys Glu Val Ala Tyr Leu Ser Ser Arg Thr Gly Phe Val Arg Leu
195 200 205
Ala Val Gln His Gly Ala Pro Leu Val Pro Val Trp Ala Phe Gly Gln
210 215 220
Thr Arg Ala Tyr Ser Trp Phe Arg Pro Gly Pro Pro Leu Val Pro Thr
225 230 235 240
Trp Leu Val Glu Arg Ile Ser Arg Ala Ala Gly Ala Val Pro Ile Gly
245 250 255
Met Phe Gly Gln Tyr Gly Thr Pro Met Pro His Arg Glu Pro Leu Thr
260 265 270
Ile Val Val Gly Arg Pro Ile Pro Val Pro Glu Leu Ala Pro Gly Gln
275 280 285
Leu Glu Pro Glu Pro Glu Val Leu Ala Ala Leu Leu Lys Arg Phe Thr
290 295 300
Asp Asp Leu Gln Ala Leu Tyr Asp Lys His Lys Ala Gln Phe Gly Lys
305 310 315 320
Gly Glu Glu Leu Val Ile Met
325
<210>40
<211>984
<212>DNA
<213>Chlamydomonas reinhardtii
<400>40
atgccgctcg caaagctgcg aaacgtggtg ctggagtacg cggccatagc catctacgtc 60
agcgccatct acacctcggt ggtgctgctg ccctcggcgc tcgcgctgtt ctacctgttt 120
ggggccacca gcccctcggc ctggctgctg ctagccgcct tcctggccct caccttcacg 180
ccgctgcagc tgaccaccgg tgcgctgtcg gagcggttcg tgcagttcag tgtggcgcgg 240
gcggcggcct acttccccac ccgcgtggtg gtcacggacc cggaggcctt ccgcactgac 300
cgcggctact tgttcggatt ctgcccgcac tcggctctgc ccatcgcact gcccatcgcc 360
ttcgccacca cctcgccgct gctgcccaag gagctgcgcg gccgcacaca cggcttggcg 420
tcgtccgtgt gcttcagcgc gcccatagtg cggcagctgt actggtggct gggcgtgcgg 480
cccgccacgc ggcagagcat cagcggcctg ttgcgggcgc gcaaggtggc ggtgctggtg 540
ccggggggcg tgcaggaggt gctcaacatg gagcacggca aggaggtggc ctacctctcc 600
agccgcaccg gcttcgtgcg actggccgtg cagcacggcg cgccgctggt gccagtgtgg 660
gcgttcggcc agacgcgcgc gtacagctgg ttccggccgg ggccgccgct cgtgcccacg 720
tggctcgtgg agcgcatctc acgtgccgcc ggcgccgtac ccatcggcat gtttgggcag 780
tacggcacgc ccatgccgca ccgcgagccc ctcaccattg tggtgggtcg ccccatcccg 840
gtgccggagc tggcgccggg ccagctcgag cccgagcccg aggtgctggc ggcgctcctc 900
aagcgcttca cggacgacct gcaggcgctg tacgacaagc acaaggcgca gttcggcaag 960
ggcgaggagc tggtcataat gtag 984
<210>41
<211>469
<212>PRT
<213>Ostreococcus lucimarinus
<400>41
Met Arg Ala Pro Ala Asp Ala Ala Ile Asp Trp Arg Ala Pro Ser Ala
1 5 10 15
Gly Ala Leu Ala Cys Leu Leu Ala Val Ala Ile Thr Asn Phe Gly Val
20 25 30
Gly Gly Ala Leu Phe Gly Gly Arg Gly Trp Arg Ala Trp Gln Pro Phe
35 40 45
Ala Arg Gly Arg Ser Trp Arg Phe Thr Leu Ala Gln Ala Ile Gly Trp
50 55 60
Thr Leu Ala Ser Ala Ser Leu Ala Cys Val Met Ala Cys Gly Thr Leu
65 70 75 80
Val Trp Arg Asp Ala Arg Asp Ala Arg Asp Asp Gly Thr Arg Glu Arg
85 90 95
Arg Leu Ala Val Ala Ala Leu Ser Trp Thr Ser Leu Gly Leu Ser Val
100 105 110
Ala Ser Glu Ala Ala Val Ala Ala Ser Leu Ala Phe Phe Asp Val Gln
115 120 125
Asp Glu Gly Glu Ala Gly Arg Gly Ala Ala Arg Gly Arg Glu Gly Leu
130 135 140
Asp Phe Arg Asp Val Ala Arg Val Ala Thr Leu Leu Ser Ile Ala His
145 150 155 160
Val Leu His Ala Pro His Ala Val Ile Phe Ala Thr Leu Ala Thr Val
165 170 175
Tyr Ala Leu Gly Ser Ser Gly Ala Leu Ala Ser Ile Val Val Leu Tyr
180 185 190
Ala Ser Thr Tyr Phe Leu Gln Arg Asp Leu Glu Arg Gly Arg Arg Lys
195 200 205
Trp Asp Ala Phe Arg Ala Trp Ser Ser Arg Trp Ile Glu Gly Ala Ala
210 215 220
Lys Ala Trp His Gly Ser Val Arg Met Ile His Asp Gly Ala His Gly
225 230 235 240
Ala Gly Ser Thr Pro Leu Val Phe Ala Tyr His Pro His Ser Leu Ile
245 250 255
Pro Ala Gly Ala Val Trp Phe His Phe Leu Pro Gln Phe Gly Arg Arg
260 265 270
Phe Glu Asn Val Lys Pro Val Thr Leu Ala Ala Ser Val Leu Phe Lys
275 280 285
Pro Pro Phe Val Arg Glu Leu Ala Ala Trp Leu Gly Val Arg Ser Val
290 295 300
Ser Gln Glu Ile Phe Arg Ser Thr Leu Arg His Glu Arg Ala Val Val
305 310 315 320
Val Cys Pro Gly Gly Gln Gly Glu Met Cys Glu His Val Gly Gly Leu
325 330 335
Lys Glu Glu Thr Ile Thr Leu Cys Thr Lys His Arg Gly Phe Val Arg
340 345 350
Leu Ala Ile Glu Glu Lys Ala Arg Leu Val Pro Val Val Cys Phe Gly
355 360 365
Glu Ser Ser Ser Trp Arg Asn Leu Leu Arg His Pro Gly Arg Tyr Leu
370 375 380
Tyr Arg Arg Phe Arg Val Ala Thr Pro Leu Leu Ala Val Gly Tyr Leu
385 390 395 400
Gly Ile Leu Pro Ile Pro Ala Arg Val Pro Leu Thr Phe Val Val Gly
405 410 415
Asp Pro Met Ser Leu Pro Glu Pro Asp Asp Ala Gly Arg Ala Arg Glu
420 425 430
Ser Asp Val Glu Ile Ala His Ala Ala Tyr Tyr Arg Glu Val Ala Arg
435 440 445
Leu Phe Ala Lys His Lys Gly Ala Ser Gly Phe Pro Asn Leu Asn Leu
450 455 460
Lys Leu Leu His Glu
465
<210>42
<211>1410
<212>DNA
<213>Ostreococcus lucimarinus
<400>42
atgcgcgcgc ccgcggacgc ggcgatcgat tggcgcgcgc cgtccgcggg cgcgctcgcg 60
tgcctgctcg cggtggcgat cacgaacttt ggcgtcggtg gcgcgctgtt cggaggacga 120
ggatggcgcg cgtggcaacc gttcgcgcga ggacgatcgt ggaggttcac gctggcgcaa 180
gcgatcggat ggacgctggc gagcgcgtcg ctggcgtgcg tgatggcgtg cgggacgctg 240
gtgtggcgag acgcgaggga cgcgagggac gacgggacgc gcgagcggcg actggcggtg 300
gcggcgctga gttggacgag cctgggactg agcgtcgcga gcgaggcggc ggtggcggcg 360
tcgctggcgt tcttcgacgt ccaggacgag ggcgaggcgg ggcgaggcgc ggcgagggga 420
agggaaggtt tggattttag agacgtcgcg cgcgtcgcga cgctgttgtc catcgcgcac 480
gtgttgcacg cgccgcacgc ggtgattttc gcgacgctgg cgacggtgta cgcgctcggg 540
tcgtcgggag cgctggcgtc catcgttgtt ctgtacgcga gcacgtattt cttgcagcgc 600
gatctcgagc gcgggcgccg gaagtgggac gcgtttcgag cgtggtcgtc gcgatggata 660
gagggcgcgg cgaaggcgtg gcacgggagc gtgcgcatga ttcacgacgg cgcgcacggc 720
gcgggctcga cgcctctcgt ctttgcctat cacccgcact cgctcattcc ggcgggcgcc 780
gtgtggtttc actttttacc tcagtttggt cgtcgctttg aaaacgtcaa gcccgtgacg 840
ttggccgcga gcgttctttt caagccgccg ttcgtgcgag agctcgccgc gtggttgggc 900
gtgcgcagcg tgtcgcaaga aatatttcgt tcgacgcttc gtcacgagcg cgcggtcgtc 960
gtgtgtccgg gcggtcaggg cgagatgtgc gagcacgtcg gcggattgaa ggaggagacc 1020
atcacgctct gcacgaaaca tcgcgggttc gttcgactcg ccatcgaaga aaaagcgcgt 1080
ctcgtgcccg tcgtgtgttt cggcgagagt agcagctggc gcaatctctt gcggcacccc 1140
ggtcgatatt tgtacagacg ctttcgcgtc gcgacgccgc ttttagcggt gggctacctc 1200
ggcattctcc cgattccggc ccgagtgccg ctcacgttcg tcgtcggcga cccgatgtcg 1260
cttcccgagc ccgatgacgc gggacgagcg agggagagcg acgttgagat cgctcacgcg 1320
gcgtattacc gcgaggtggc gcgcttgttc gcaaagcaca agggcgcgag cggatttccg 1380
aatttaaact tgaaattgct gcacgagtga 1410
<210>43
<211>316
<212>PRT
<213>Ostreococcus lucimarinus
<400>43
Met Phe Ala Trp Leu Gly Leu Ile His Val Asp Val Ala Val Thr Ala
1 5 10 15
Leu Ala Val Trp Thr Leu Pro Ser Ala Met Ala Val Thr Ala Leu Ala
20 25 30
Thr Leu Val Ala Ala Ala Ala Ile Pro Arg Thr Val Ala Thr Pro Arg
35 40 45
Trp Gly Ala Arg Leu Ala Arg Ala Val Thr Arg Thr Ala Thr Ala Tyr
50 55 60
Phe Pro Thr Arg Leu Glu Phe Glu Asp Glu Glu Ala Tyr Leu Arg Ala
65 70 75 80
Val Arg Asn Glu Glu Ala Cys Val Ile Gly Leu Glu Pro His Gly Val
85 90 95
Leu Pro Leu Ser Val Ile Ser Phe Ala Glu Tyr Phe Met His Asp Glu
100 105 110
Glu Gly Ala Arg Arg Arg Gly Leu Thr Pro Ala Ala Arg Arg Gly Ala
115 120 125
Arg Ala Leu Ala Ser Ala Ala Ile Phe Lys Val Pro Leu Val Lys His
130 135 140
Leu Trp Thr Trp Leu Gly Leu Asp Pro Ile Ser Lys Ala Cys Met Leu
145 150 155 160
Arg Met Leu Arg Ala Gly Lys Thr Ala Val Ile Ile Pro Gly Gly Val
165 170 175
Ala Glu Cys Met Ala Met Glu Arg Gly Val Glu Thr Leu Tyr Leu Arg
180 185 190
Lys Arg Tyr Gly Phe Val Lys Ile Ala Ile Val Thr Gly Ala Lys Leu
195 200 205
Ile Pro Ala Tyr Thr Phe Gly Gln Ser Arg Thr Tyr Gly Tyr Trp Arg
210 215 220
Leu Gly Pro Pro Ile Val Pro Lys Phe Val Ala Asp Trp Ile Gly Lys
225 230 235 240
Thr Phe Ser Phe Ala Pro Ile Ile Phe Trp Gly Lys Phe Cys Thr Pro
245 250 255
Ile Pro Tyr Ala Thr Ala Leu Asn Thr Val Val Gly Lys Pro Ile Glu
260 265 270
Val Glu Lys Asn Pro Asp Pro Ser Lys Glu Glu Val Gln Ala Lys Leu
275 280 285
Asp Glu Phe Ile Asp Ala Met Arg Ser Leu Tyr Asp Ser His Lys Ala
290 295 300
Arg Phe Gly Tyr Glu Asp Val Arg Leu Val Ile Cys
305 310 315
<210>44
<211>951
<212>DNA
<213>Ostreococcus lucimarinus
<400>44
atgttcgcgt ggctcggatt gattcacgtc gacgtcgcgg tgacggcgct ggcggtgtgg 60
acgctgccga gcgcgatggc ggtgacggcg ctcgcgacgc tggtcgcggc ggcggcgatc 120
ccgcggacgg tggcgacgcc gaggtggggc gcgcggttgg cgcgcgcggt gacgaggacg 180
gcgacggcgt actttccgac gcgattggaa ttcgaagacg aggaggcgta tctgcgagcg 240
gtgaggaacg aagaggcgtg cgtgatagga ctggaaccgc acggggtgct gccgctgagc 300
gtgatatcgt tcgcggagta ttttatgcac gatgaggagg gggcgcggcg acgaggattg 360
acgccggcgg cgagacgagg cgcgcgggcg ctggcgagcg cggcgatatt taaagtcccg 420
ctcgtgaaac atctgtggac gtggttgggg ttggatccga tctcgaaggc gtgcatgctg 480
aggatgctgc gagcggggaa gacggcggtg atcattcccg gcggcgtcgc cgagtgcatg 540
gcgatggaac gcggcgtgga gacgttgtat ttgcgcaagc gatacggatt cgtgaagatc 600
gccatcgtga ccggagcgaa actgattccc gcgtacacgt tcggacaaag tcggacgtac 660
gggtactggc ggctcgggcc gccgatcgtg ccgaaattcg tggcggattg gatcgggaag 720
acgttttcgt tcgcgccgat tattttctgg gggaaatttt gcacgcccat cccgtacgcg 780
acggcgctca acacggtcgt gggcaaaccg atcgaggttg aaaaaaaccc agatccgagc 840
aaggaagagg ttcaggcgaa attggacgag tttatcgacg ccatgcgttc gctgtacgac 900
agtcacaagg cgagattcgg ttacgaagac gttcgactcg tgatttgtta g 951
<210>45
<211>338
<212>PRT
<213>Ostreococcus lucimarinus
<400>45
Met Ile Tyr Ala Trp Ile Leu Ser Ala Ile Phe Val Tyr Pro Ala Tyr
1 5 10 15
Cys Val Phe Gly Pro Ser Met Trp Leu Lys Asn Phe Phe Leu Gly Tyr
20 25 30
Ile Ala Trp Tyr Ala Thr Leu Asp Arg Lys Thr Ala Ser Ser Gly Lys
35 40 45
Arg Phe Ala Arg Trp Ser Arg Arg Leu Pro Phe Trp Arg Ile Leu Ala
50 55 60
Glu Tyr Phe Pro Val Arg Leu His Val Ser Ala Lys Leu Asp Pro Ser
65 70 75 80
Gly Asn Tyr Leu Phe Gly Tyr His Pro His Gly Val Ile Gly Val Gly
85 90 95
Ala Leu Leu Thr Phe Ala Thr Glu Ala Thr Gly Phe Tyr Glu Ala Phe
100 105 110
Pro Gly Leu Asp Leu Arg Leu Leu Thr Leu Ser Met Asn Phe Lys Phe
115 120 125
Pro Phe Thr Arg Glu Val Leu Met Gly Leu Gly Ile Asn Ser Val Thr
130 135 140
Lys Ser Ser Val Glu Thr Asn Leu Thr Arg Ala Pro Gly Ala Ser Val
145 150 155 160
Ala Ile Val Ile Gly Gly Ala Ser Glu Ala Leu Asp Ala Arg Pro Gly
165 170 175
Trp Ala Thr Leu Thr Leu Ala Arg Arg Lys Gly Phe Val Lys Met Ala
180 185 190
Leu Arg Thr Gly Ala Ser Leu Val Pro Val Phe Ala Phe Gly Glu Asn
195 200 205
Asp Ile Phe Glu Gln Val Glu Asn Pro Glu Gly Gly Arg Leu Arg Asn
210 215 220
Phe Gln Met Tyr Ile Lys Gln Leu Ile Gly Ile Thr Pro Pro Ala Phe
225 230 235 240
Tyr Gly Arg Ser Leu Ser Arg Gly Met Trp Arg Arg Ile Phe Gly Arg
245 250 255
Lys Gly Val Leu Pro Lys Arg Glu Pro Ile Glu Val Val Val Gly Asn
260 265 270
Pro Ile Ala Val Pro Lys Val Val Asp Pro Ser Asn Glu Ile Ile Asp
275 280 285
Lys Tyr His Ala Leu Tyr Thr Glu Ser Leu Lys Glu Leu Tyr Glu Leu
290 295 300
His Arg Arg Gln Phe His Arg Leu Asn Arg Gly Gly Ser Ser Asp Asp
305 310 315 320
Leu Leu Ser Asp Leu Leu Thr Arg Gln Gly Lys Leu Gln Asn Met Gln
325 330 335
Phe Lys
<210>46
<211>1017
<212>DNA
<213>Ostreococcus lucimarinus
<400>46
atgatatacg cgtggatact gagcgcgata ttcgtatatc ccgcgtattg cgtgttcggg 60
ccgtcgatgt ggttgaagaa tttcttcctg gggtacatcg cgtggtacgc gacgctcgac 120
aggaagacgg cgagctcggg gaagcgtttc gctcgatggt ctcggcggtt accgttttgg 180
aggattttgg cggagtattt tccggtgcga ttacacgtga gcgcgaagtt agatccgagc 240
ggtaattact tgttcggtta tcatccgcac ggcgtcatcg gcgtcggcgc gttgttgacg 300
ttcgccaccg aggcgacggg attttacgaa gcatttccgg gactcgattt gcgtcttctc 360
acgctgtcca tgaacttcaa gttcccgttc actcgcgagg tgttgatggg gctcgggatc 420
aatagcgtga ccaagtcgag cgtggagacg aatctgacgc gcgcgccggg ggcgtccgtc 480
gccatagtca tcggcggcgc ctccgaagcg ttggacgcgc gtccgggctg ggccacgctc 540
acgctcgcca gacgcaaggg gttcgtaaag atggctcttc gcacaggagc atcgctcgtg 600
cccgtgttcg cgttcggcga gaatgacatc ttcgaacaag tggaaaatcc cgaaggcgga 660
cgattgagga atttccaaat gtacatcaaa caactcattg gcatcacgcc gcctgctttt 720
tacgggcggt cgctcagtcg aggcatgtgg cgtcgaatct ttggtcgcaa aggtgtgctt 780
ccgaagcgtg agccgatcga agtcgtcgtg ggcaatccca tcgccgtgcc caaagtcgtc 840
gatccgtcaa acgaaatcat cgacaagtac cacgccctgt acaccgaatc tttgaaagag 900
ctttacgagt tacatcgtcg acagtttcat agactcaatc gcggggggtc gtcggatgat 960
cttctgagcg atctcctgac tcggcaagga aaactgcaaa acatgcagtt caagtag 1017
<210>47
<211>350
<212>PRT
<213>Ostreococcus lucimarinus
<400>47
Met Gly Ser Asn Ala Gln Arg Gly Ala Leu Trp Arg Glu His Arg Ala
1 5 10 15
Val Glu Ala Ala Thr Ile Ala Ala Met Arg Ala Arg Gly Val Arg Asp
20 25 30
Val Pro Trp Ser Ser Ala Lys Arg Met Leu Ala Val Leu Cys Val Ser
35 40 45
Ala Ile Tyr Thr Ser Trp Ile Leu Ser Pro Val Val Ser Ala Val Ala
50 55 60
Val Ile Leu Ile Pro Ser Leu Arg Ala Tyr Val Gly Cys Tyr Leu Phe
65 70 75 80
Ala Ser Tyr Ala Leu Gly Val Arg Val Pro Met Asn Gly Leu Tyr Lys
85 90 95
Phe Phe Cys Gly Leu Glu Cys Gly Glu Glu Asn Gly Trp Glu Leu Val
100 105 110
Val Glu Asp Ala Thr Ala Gly Glu Lys Glu Ile Asp Cys Ser Lys Arg
115 120 125
Ala Tyr Leu Phe Ala Ala His Pro His Gly Leu Phe Ala Ser Gly Cys
130 135 140
Val Gly Asn Ile Val Leu Ser Asp Ala Ala Leu Arg Arg Phe Arg Ala
145 150 155 160
Arg His Val Arg Phe Phe Ile Asn Asn Leu Leu Ile Ser Val Phe Pro
165 170 175
Ile Ile Lys Asp Val Leu Ser Ser Leu Gly Phe Leu Pro Cys Thr Ala
180 185 190
Lys Met Met Arg Arg Val Leu Gly Arg Gly Glu Thr Gly Met Ile Val
195 200 205
Val Gly Gly Val Gln Glu Val Val Leu Thr Gly Asn Val Asp Val Glu
210 215 220
Glu Leu Tyr Leu Lys Asn Cys Phe Gly Phe Val Lys Val Ala Ile Gln
225 230 235 240
Val Gly Thr Pro Leu Val Pro Val Tyr Thr Phe Gly Glu Ser Leu Ala
245 250 255
Thr Gly Pro Asp Trp Val Pro Phe Arg Glu Ile Arg Lys Arg Leu Ser
260 265 270
Tyr Lys Phe Val Phe Pro Phe Arg Ser Leu Gly Ile Val His Arg Trp
275 280 285
Gly Phe Cys Phe Pro Arg Gly Lys Leu Thr Thr Val Val Gly Pro Pro
290 295 300
Ile Glu Val Lys Gln Asn Asp Arg Pro Ser Arg Glu Glu Val Ala Ala
305 310 315 320
Val His Ala Gln Tyr Cys Lys Ser Leu Leu Ala Leu Ile Glu Arg Asn
325 330 335
Lys Ala Ala Ala Gly Tyr Pro Thr Gln Val Thr Arg Leu Val
340 345 350
<210>48
<211>1053
<212>DNA
<213>Ostreococcus lucimarinus
<400>48
atgggctcga acgcgcagcg cggtgcgctc tggcgcgaac accgcgccgt ggaggcggcg 60
acgatcgcgg cgatgcgcgc gcgcggcgtg cgcgacgtgc cgtggagctc cgcgaagcgc 120
atgctcgcgg tgctgtgcgt cagcgcgatt tacacgtcgt ggatcctgtc cccggtcgtg 180
tccgcggtgg cggtgatttt gataccgtcg ttgcgagcgt acgtcgggtg ttatctcttc 240
gcgtcgtacg cgctcggggt gcgtgtgccc atgaatgggc tttataaatt cttttgcggg 300
ctcgagtgcg gagaagaaaa tggatgggaa ctcgtcgtcg aggacgcgac ggcgggcgag 360
aaagagattg attgctctaa gcgcgcgtat ttgttcgccg cgcacccgca cgggttattc 420
gcgtctggtt gcgtggggaa tatcgtattg agcgacgcgg cgttgaggcg attccgagcg 480
cggcacgtta gattcttcat caacaacttg ctcataagcg tgtttccgat catcaaagat 540
gtgctgtcgt cgcttgggtt cttaccatgc acagcaaaaa tgatgcgacg ggttttaggg 600
cgtggggaga ctgggatgat tgttgttggc ggcgttcaag aggtggtgct gacgggcaac 660
gtcgacgtcg aagagctata cttgaagaat tgtttcggat tcgtcaaggt agccatccaa 720
gtcggaacgc ccttagtgcc agtatacacg tttggcgagt ctctggctac tggtccggat 780
tgggtgccgt ttcgtgagat acgaaaacgc ctgagctata aatttgtatt tccgttccgc 840
tcgctcggca tcgtccatcg ttggggattc tgctttccgc gaggcaagct cacgacagtg 900
gtgggaccac ctattgaagt taagcagaac gatagaccgt cgcgcgagga agtggctgcg 960
gtgcatgcgc agtattgtaa gtcgcttttg gcactcattg aacgaaacaa agccgccgcg 1020
ggatacccaa cccaggtgac aagattggta tag 1053
<210>49
<211>421
<212>PRT
<213>Ostreococcus tauri
<220>
<221>misc_feature
<222>(58)..(65)
<223>Xaa can be any naturally occurring amino acid
<400>49
Met Ala Ala Ala Ala Asp Ala Lys Ala Ile Val Leu Ala Ser Thr Cys
1 5 10 15
Val Leu Val Ala Thr Cys Ala Thr His Phe Gly Ala Gly Arg Ala Leu
20 25 30
Trp Ser Asp Arg Gly Trp Arg Ala Trp Gln Pro Met Arg Gly Arg Arg
35 40 45
Ala Phe Val Ala Leu Gln Pro Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Gly Ala Gly Glu Pro Leu Val Gly Val Ser Leu Ala Phe Phe Asp
65 70 75 80
Glu Thr Arg Ala Gly Gly Arg Thr Ser Glu Thr Met Asp Ala Arg Asn
85 90 95
Val Ala Arg Val Val Gln Met Leu Thr Val Leu His Val Val His Ala
100 105 110
Pro His Met Ala Val Phe Ala Val Leu Gly Thr Ala Tyr Ala Leu Gly
115 120 125
Arg Trp Arg Thr Leu Ala Ala Val Val Ala Leu Tyr Ala Ser Thr Tyr
130 135 140
Val Thr Arg Gln Lys Ser Leu Glu Arg Gly Gln Arg Lys Trp Glu Gln
145 150 155 160
Phe Gln Thr Trp Thr Leu Arg Thr Val Glu Gly Ala Ala Lys Ser Trp
165 170 175
Tyr Gly Ser Val Arg Val Val His Asp Gly Lys Val Ser Glu Ala Ala
180 185 190
His Ser Ser Pro His Val Phe Ala Tyr His Pro His Ser Met Val Pro
195 200 205
Ala Gly Ala Val Trp Phe His Met Leu Pro Asp Phe Ser Ala Arg Phe
210 215 220
Arg Gly Ile Gln Pro Val Thr Leu Ala Ala Ser Val Leu Phe Lys Ala
225 230 235 240
Pro Ile Val Arg Glu Leu Ala Ala Trp Leu Gly Val Arg Ala Val Ser
245 250 255
Arg Glu Ile Phe Arg Ser Thr Leu Arg Glu Gln Gly Ala Val Val Val
260 265 270
Cys Pro Gly Gly Gln His Glu Met Gln Glu His Gly Gly Pro Met Glu
275 280 285
Glu Thr Ile Val Leu Cys Thr Lys His Lys Gly Phe Ile Arg Ile Ala
290 295 300
Ile Glu Glu Arg Ala Arg Val Val Pro Val Ile Cys Phe Gly Glu Ser
305 310 315 320
Lys Ser Trp Thr Asn Ile Met Ala Lys Pro Gly Arg Tyr Leu Tyr Arg
325 330 335
Arg Phe Arg Phe Gly Phe Thr Pro Leu Leu Ala Val Gly Tyr Leu Gly
340 345 350
Ile Leu Pro Leu Pro Arg Arg Val Pro Ile Thr Phe Val Ile Gly Glu
355 360 365
Pro Met Val Leu Pro Asp Pro Asp Ala Leu Thr Gly Leu Ala Lys Glu
370 375 380
Ser Asp Val Asp Ala Phe His Ala Ser Tyr Tyr Ser Gln Val Glu Arg
385 390 395 400
Leu Phe Asp Glu His Lys Ser Lys Ala Gly Phe Pro Glu Leu Cys Leu
405 410 415
Val Met Lys Asn Asp
420
<210>50
<211>1266
<212>DNA
<213>Ostreococcus tauri
<220>
<221>misc_feature
<222>(174)..(193)
<223>n is a,c,g,or t
<400>50
atggcggcgg cggcggacgc gaaagccatc gtcttggcct cgacgtgcgt cctcgtcgcg 60
acgtgcgcga cgcacttcgg cgcgggccga gcgctgtgga gcgatcgagg atggcgggcg 120
tggcagccga tgcgcggacg gcgcgcgttc gtggcgctgc agccggtggg gtgnnnnnnn 180
nnnnnnnnnn nnnggggtgc gggtgagccc ttggtggggg tgtcgctggc gttttttgat 240
gagacgcgcg cgggcgggcg gacgtcggag acgatggacg cgcgaaacgt cgcgcgcgtg 300
gtgcagatgt tgaccgtctt gcacgtcgtg cacgcgccgc acatggcggt gtttgcggtg 360
ttggggacgg cgtacgcgtt gggtcgatgg cgtacgctcg cggcggtggt cgcgttgtac 420
gcgtcgacgt acgtgacgag acagaagtcg ctcgagcggg gacagagaaa gtgggagcag 480
ttccagacgt ggacgcttcg gacggtggag ggggcggcga aatcgtggta cgggagcgtg 540
cgcgtggtgc acgacgggaa ggtttcggaa gcggcgcatt cgtcgccgca cgtcttcgcg 600
taccacccgc actcgatggt tcccgcgggc gccgtgtggt ttcacatgct cccggatttt 660
agcgcccgtt ttcgcgggat tcaacccgtg acgctcgcag cctcggtttt gtttaaggca 720
ccgatcgtcc gggagcttgc ggcgtggctt ggcgttcgtg cggtgagcag agagattttc 780
cgttcgacgc tacgagagca aggcgcggtt gtcgtgtgcc cgggaggaca gcacgagatg 840
caagagcacg gaggtccgat ggaggagacg atcgttttat gcaccaaaca taaaggattc 900
attcgaatag cgatcgagga gcgcgcgcgc gtcgtccccg tgatttgttt cggcgagagc 960
aagagttgga ccaacatcat ggcaaagccg ggccgttatc tctacagacg ctttcgattc 1020
ggtttcaccc cgttactagc cgtgggctac ctcggaattc ttccgctccc gagacgcgta 1080
cccatcacgt tcgtcatcgg cgagccgatg gtacttcctg atccggatgc cttgacggga 1140
ctggcgaagg aatcggatgt cgacgcgttc cacgcgtcgt attacagtca agtggagaga 1200
ctgttcgacg agcacaaatc caaagccggg ttccctgagc tttgcctcgt gatgaaaaac 1260
gattag 1266
<210>51
<211>325
<212>PRT
<213>Ostreococcus tauri
<400>51
Met Ser Arg Ser Ile Val Asp His Gly Val Leu Leu Val Trp Leu Gly
1 5 10 15
Leu Phe His Ala Leu Val Val Val Val Val Val Ala Ile Val Ala Leu
20 25 30
Glu Arg Arg Arg Ala Met Thr Val Leu Ala Ala Leu Met Ser Leu Ser
35 40 45
Val Val Pro Arg Arg Ile Arg Pro Arg Trp Gly Val Thr Leu Ala Arg
50 55 60
Ala Ile Thr Arg Thr Ala Lys Ser Tyr Phe Pro Cys Ala Leu Thr Phe
65 70 75 80
Glu Asn Glu Glu Ala Tyr Leu Lys Gly Ala Arg Lys Gly Val Gly Arg
85 90 95
Leu Val Gly Leu Glu Pro His Gly Ala Leu Pro Leu Ser Val Ile Ala
100 105 110
Phe Ala Asp Tyr Phe Met Phe Asp Glu Asp Gly Ile Glu Ala Arg Gly
115 120 125
Met Asn His Ala Ala Ser Met Asn Ser Arg Ala Leu Ala Ser Gly Ala
130 135 140
Ile Phe His Val Pro Leu Val Arg His Leu Trp Thr Trp Leu Gly Leu
145 150 155 160
Glu Pro Ile Ser Arg Arg Arg Met Thr Ser Met Leu Ser Asp Gly Ser
165 170 175
Thr Cys Val Ile Val Pro Gly Gly Val Ala Glu Cys Met Ala Met Glu
180 185 190
Arg Gly Val Glu Thr Leu Tyr Leu Lys Arg Arg Tyr Gly Phe Val Lys
195 200 205
Ile Ala Ile Gln Thr Gly Ala Ala Leu Val Pro Ala Tyr Thr Phe Gly
210 215 220
Gln Thr Arg Ala Tyr Lys Tyr Trp Arg Leu Gly Pro Pro Leu Val Pro
225 230 235 240
Thr Ser Val Ala Asn Trp Phe Ser Lys Thr Phe Ser Phe Ala Pro Met
245 250 255
Val Phe Trp Gly Lys Trp Phe Thr Pro Ile Pro Tyr Ala Thr Pro Leu
260 265 270
His Thr Val Val Gly Glu Leu Ile Glu Thr Thr Gln Asn Asp Asn Pro
275 280 285
Ser Arg Glu Glu Val Gln Ala Lys Leu Asp Glu Phe Ile Val Ala Met
290 295 300
Arg Ser Leu Tyr Asp Arg His Lys Ser Ala His Gly Tyr Ala Asp Val
305 310 315 320
Asp Leu Val Val Cys
325
<210>52
<211>978
<212>DNA
<213>Ostreococcus tauri
<400>52
atgtcgcgct cgatcgtcga tcacggcgtg ctgctcgtgt ggttggggtt gttccacgcc 60
ctggtcgtcg tcgtcgtcgt cgcgatcgtc gcgctcgagc gacgacgcgc gatgacggtg 120
ctcgccgcgt tgatgtcttt gagcgtcgtc ccgcggcgca tccgaccgcg ctggggcgtg 180
acgctggcgc gcgcgatcac gcgcacggcg aagtcgtatt tcccgtgcgc gttgacgttt 240
gagaacgagg aggcgtacct gaagggtgct cggaagggtg tcgggcggtt ggtgggtttg 300
gagccgcacg gagcgctgcc gctctcggtg atcgcgttcg cggattactt catgttcgat 360
gaagatggga tcgaggcgag ggggatgaat cacgctgcgt cgatgaattc gcgagcgttg 420
gcgtcggggg cgatatttca cgtcccgttg gtgcgacacc tgtggacgtg gttgggattg 480
gaaccgatat ctcgaaggcg gatgacgagt atgttaagcg acggttcgac gtgcgtgatc 540
gtgccgggtg gggtggcgga gtgcatggcg atggagagag gggttgagac gctgtatctc 600
aagcgaaggt acgggttcgt gaagattgcg atacagacgg gcgcggcact cgtgccggcg 660
tacacgtttg ggcagacgcg ggcgtacaag tactggcgac tcggtccgcc gttggtgccg 720
acgtccgttg caaattggtt ctcgaaaacg ttttctttcg cacccatggt tttttgggga 780
aagtggttca cgcccattcc gtacgctacc cctctgcaca cggtggttgg cgagctcatc 840
gagaccacgc aaaacgacaa tccgagtcgc gaggaggtgc aggcaaagct tgacgagttc 900
atcgtcgcta tgcgttcgct gtacgaccga cacaaatctg cacacgggta tgccgacgtc 960
gacctcgtcg tgtgctga 978
<210>53
<211>314
<212>PRT
<213>Ostreococcus tauri
<400>53
Met Arg Asn Ala Phe Leu Gly Tyr Ile Gly Trp Tyr Val Leu Leu Asp
1 5 10 15
Arg Arg Ser Asp Ser Ser Gly Thr Arg Phe Val Ala Trp Ser Arg Arg
20 25 30
Leu Pro Phe Trp Arg Ile Leu Ala Asp Tyr Phe Pro Val Arg Leu Tyr
35 40 45
Lys Ser Gly Glu Leu Asp Pro Lys Gly Asn Tyr Leu Phe Gly Tyr His
50 55 60
Pro His Gly Val Ile Gly Val Gly Ala Leu Met Thr Phe Ala Thr Glu
65 70 75 80
Ala Thr Gly Phe Tyr Glu Ala Phe Pro Gly Leu Asp Leu Arg Leu Leu
85 90 95
Thr Leu Ser Val Asn Phe Lys Phe Pro Phe Thr Arg Glu Val Leu Met
100 105 110
Ala Leu Gly Ile Asn Ser Val Thr Lys Ala Ser Val Met Thr Asn Leu
115 120 125
Thr Arg Ala Pro Gly Ala Ser Val Ala Ile Val Ile Gly Gly Ala Ala
130 135 140
Glu Ala Leu Asp Ala Arg Pro Gly Ser Ala Thr Leu Thr Leu Ala Arg
145 150 155 160
Arg Lys Gly Phe Val Lys Met Ala Leu Arg Thr Gly Ala Ser Leu Val
165 170 175
Pro Val Phe Ala Phe Gly Glu Asn Asp Ile Phe Glu Gln Val Glu Asn
180 185 190
Pro Asp Gly Gly Arg Leu Arg Lys Phe Gln Thr Tyr Ile Lys Gln Leu
195 200 205
Ile Gly Ile Ser Pro Pro Ala Phe Tyr Gly Arg Ser Leu Ser Arg Gly
210 215 220
Val Trp Arg Arg Ile Phe Gly Arg Lys Gly Val Leu Pro Lys Arg Glu
225 230 235 240
Pro Ile Glu Val Ile Ile Gly Asn Pro Ile His Val Pro Gln Val Asp
245 250 255
Asp Pro Ser Pro Asp Val Ile Asp Lys Tyr His Gln Leu Tyr Thr Val
260 265 270
Gly Leu Lys Glu Leu Tyr Glu Leu His Arg Arg Gln Phe His Gln Leu
275 280 285
Asn Arg Gly Gly Ser Ser Asp Asp Leu Leu Ser Asp Leu Ile Lys Arg
290 295 300
Lys Asn Asn Leu Gln Ala Met Thr Phe Lys
305 310
<210>54
<211>945
<212>DNA
<213>Ostreococcus tauri
<400>54
atgcggaacg cgttcttggg atacattggt tggtacgtgc tgttggaccg gcgctcggat 60
agctcgggca ctagattcgt ggcgtggtcg agacgtctac ctttttggcg catcctggct 120
gattacttcc cagttcgatt atacaagagc ggcgagctcg atccaaaagg gaattacttg 180
ttcgggtatc atccgcacgg cgtcatcggc gtcggggcgt tgatgacgtt tgccaccgaa 240
gcgacgggat tttacgaagc atttccagga ttggatttac ggctcttgac gttatcggtg 300
aacttcaagt ttccatttac gcgagaggtg ttgatggcgc tcgggattaa ctccgtcact 360
aaggcgagcg tcatgaccaa tcttacccgc gcaccaggcg cgagcgtcgc catcgtcatc 420
ggcggcgccg cagaggcgtt ggacgctcgt ccgggatcgg ccacgctcac gctggcgaga 480
cgtaaagggt tcgtgaaaat ggctctgcgc acgggtgcat cgctcgtgcc tgtttttgcc 540
ttcggggaga acgatatttt cgagcaagtc gagaatcccg acggcgggcg cctgcgcaag 600
tttcagacgt acatcaagca actcatcgga atctcaccgc cggcttttta cggccgctcg 660
ctcagtcggg gggtgtggcg tcgcattttt gggcgtaagg gagtgctgcc gaagcgtgaa 720
ccgatcgaag tgatcatcgg taatcccata cacgtccctc aggtggacga tccgtcgccc 780
gacgtcatcg acaagtatca tcaattgtac accgtgggac tcaaggaact ttacgagctg 840
catcgcagac aatttcacca gttgaatcgc ggaggttctt ccgacgatct gctaagcgat 900
ctgatcaagc gtaagaacaa cctccaagcc atgacattca aatag 945
<210>55
<211>354
<212>PRT
<213>Ostreococcus tauri
<400>55
Met Arg Thr Ser Ser Gly Ala Gly Gly Thr Arg Ala Arg Arg His Cys
1 5 10 15
Ala Thr Thr Asp Val Ala Arg Ser Phe Asp Ala Val Arg Arg Glu Met
20 25 30
Arg Glu Ala Arg Gly Ile Ala Asp Val Pro Trp Ser Ser Leu Lys Arg
35 40 45
Leu Leu Gly Val Ser Cys Val Ser Ala Ile Tyr Thr Ser Trp Ile Leu
50 55 60
Ser Pro Val Met Ser Ala Leu Ala Val Trp Arg Tyr Glu Trp Leu Arg
65 70 75 80
Ala Tyr Val Ala Cys Tyr Leu Phe Ala Ser Tyr Ala Leu Gly Val Ala
85 90 95
Met Pro Met Asn Ala Leu His Arg Phe Phe Cys Trp Leu Glu Thr Gly
100 105 110
Glu Glu Asn Gly Trp Gln Leu Val Val Glu Asp Asp Cys Asp Val Asp
115 120 125
Cys Ser Lys Arg Ala Tyr Leu Phe Thr Ala His Pro His Gly Leu Phe
130 135 140
Ala Ser Gly Cys Val Gly Asn Val Val Leu Ser Gly Arg Ala Leu Lys
145 150 155 160
Arg Phe Arg Ala Arg Arg Ile Trp Phe Phe Ile Asn Glu Leu Leu Ile
165 170 175
Arg Val Phe Pro Ile Ile Lys Asp Val Leu Ser Met Leu Gly Phe Val
180 185 190
Pro Cys Thr Ala Lys Met Met Lys Lys Val Leu Gly Arg Gly Glu Thr
195 200 205
Gly Leu Ile Val Val Gly Gly Val Gln Glu Val Val Leu Thr Gly Asn
210 215 220
Val Asp Glu Glu Glu Leu Tyr Leu Lys Asn Cys Phe Gly Phe Val Lys
225 230 235 240
Val Ala Met Gln Ala Gly Thr Pro Leu Val Pro Val Tyr Thr Phe Gly
245 250 255
Glu Ser Leu Ala Thr Gly Pro Asp Trp Val Pro Phe Arg Glu Leu Arg
260 265 270
Lys Arg Leu Ser Tyr Lys Phe Val Phe Pro Phe Arg Ser Leu Gly Ile
275 280 285
Ile His Arg Trp Gly Leu Cys Phe Pro Lys Ala Lys Leu Thr Thr Val
290 295 300
Val Gly Ala Pro Ile Glu Val Lys Gln Asn Pro Asn Pro Thr Arg Glu
305 310 315 320
Glu Val Ala Ala Val His Gln Gln Tyr Cys Asp Ala Leu Leu Ala Met
325 330 335
Ile Glu Arg Asn Lys Ala Arg Ala Gly Tyr Pro Thr Gln Arg Thr Lys
340 345 350
Leu Val
<210>56
<211>1065
<212>DNA
<213>Ostreococcus tauri
<400>56
atgcgaacct cctcgggcgc gggaggaacg cgcgcgcgtc gtcactgtgc gacgacggac 60
gtcgcgcgat cgttcgacgc cgttcgcagg gagatgcgag aggcgcgagg gatcgcggac 120
gtcccctgga gctcgctcaa gcgcttgctc ggtgtctcgt gcgtgagcgc gatttacacg 180
tcgtggatcc tctccccggt gatgagcgcg ctcgcggtgt ggcggtacga gtggttgaga 240
gcgtacgtcg cgtgctatct cttcgcgtcc tacgcgctcg gcgtggcgat gccgatgaac 300
gcgctgcatc ggttcttctg ttggctcgag acgggagagg aaaacgggtg gcagctcgtc 360
gtcgaggacg actgcgatgt ggactgctcg aagagggcgt acttgttcac ggcgcatccg 420
cacgggttgt tcgcgtcggg atgcgtcggg aacgtcgttt tgagcggacg cgcgctcaag 480
aggttccggg cgagacggat ttggttcttc atcaacgagc tgttaatccg agtgtttccg 540
atcatcaagg acgtgttgtc gatgctggga ttcgtgccgt gcacggcgaa aatgatgaag 600
aaggtgttgg gaaggggcga gaccggattg atcgtcgtcg gtggggttca ggaggttgtg 660
ttgactggta acgtcgacga agaggaactt tatctaaaaa attgtttcgg ctttgtcaaa 720
gtggccatgc aggccgggac gccgctcgtg cccgtataca cattcggcga atcgctagcc 780
accggcccgg actgggtacc gttcagagag ctgcgtaaac ggctgagcta caagtttgtg 840
ttcccgtttc gctcgcttgg cataatccat cgctgggggc tctgctttcc caaggcaaag 900
ctcacgaccg tggtaggcgc gccgattgag gtgaaacaaa acccaaatcc cacgcgcgag 960
gaagttgcgg cggtgcatca gcagtattgc gacgcactgc tggcgatgat tgagcgaaac 1020
aaggcccgcg cggggtatcc gacgcaacgc acaaagttgg tgtaa 1065
<210>57
<211>359
<212>PRT
<213>Phaeodactylum tricornutum
<400>57
Met Lys Glu Arg Arg Ser Gly Leu Asn Pro Ser Gly Ser Ser Val Tyr
1 5 10 15
Pro Leu His Pro Pro Asp Ser Arg Val Leu Val Arg Val Pro Ser Asp
20 25 30
Ile Ser Phe Leu Asp Arg Leu Ile Val Ala Gly Ser Ser Ile Phe Ile
35 40 45
Val Gly Ser Leu Val Trp Val Pro Leu Thr Ala Arg Trp Val Tyr Arg
50 55 60
Arg Trp Lys Gln Ala Lys Asp Lys Arg Lys Arg Ala Leu Tyr Ala Ser
65 70 75 80
Leu Leu Val Ile Leu Ala Val Leu Val Ile Gly Gly Pro His Arg Ser
85 90 95
Pro Arg Val Gly Lys Trp Leu Gln Val Arg Lys Trp Ser Leu Phe Gln
100 105 110
Ala Trp Val Lys Phe Ile Ala Met Glu Val Ile Leu Asp Gln Pro Lys
115 120 125
Gly Ile Thr Met Asp Val Gln Gln Asp Lys Ala Ile Phe Ala Phe Ala
130 135 140
Pro His Gly Ile Phe Pro Phe Ala Phe Ala Phe Gly Val Leu Pro Asp
145 150 155 160
Ile Ala Thr Gln Ser Phe Gly Tyr Val Arg Pro Val Val Ala Thr Ala
165 170 175
Thr Arg Leu Phe Pro Val Val Arg Asp Phe Ile Ser Trp Ala Asn Pro
180 185 190
Val Asp Ala Ser Lys Asp Ser Val Glu Arg Ala Leu Ala Leu Gly Asp
195 200 205
Arg Ile Ala Val Ile Pro Gly Gly Ile Ala Glu Ile Phe Glu Gly Tyr
210 215 220
Pro Lys Pro Asn Thr His Pro Asp Glu Glu Tyr Ala Ile Val Arg Ser
225 230 235 240
Gly Phe Leu Arg Leu Ala Ile Lys His Gly Ile Pro Val Ile Pro Val
245 250 255
Tyr Cys Phe Gly Ala Thr Lys Met Leu Lys Arg Leu Glu Leu Pro Gly
260 265 270
Leu Glu Gln Leu Ser Leu Phe Leu Arg Val Ser Ile Cys Leu Phe Phe
275 280 285
Gly Val Gly Gly Leu Pro Ile Pro Phe Arg Gln Arg Leu Ser Tyr Val
290 295 300
Met Gly Gln Pro Ile Leu Pro Pro Val Arg Thr Thr Gly Ser Asp Ile
305 310 315 320
Ser Asp Ala His Val Lys Glu Met Gln Asp Arg Phe Cys Ala Glu Val
325 330 335
Gln Arg Leu Phe Asp Arg His Lys Glu Ala Tyr Gly Trp Ser His Lys
340 345 350
Thr Leu Lys Leu Leu Glu Gln
355
<210>58
<211>1080
<212>DNA
<213>Phaeodactylum tricornutum
<400>58
atgaaagaaa gaagatctgg cctaaatccg tcaggatcct ccgtgtatcc attgcaccct 60
cctgacagtc gcgttctcgt tcgagtcccc tccgatattt cctttcttga tcgtctcatc 120
gtcgctggca gcagtatctt tattgtcggt tcgctagtat gggttccatt gaccgcaaga 180
tgggtctaca ggcggtggaa gcaagctaaa gataaacgaa agcgggcttt gtatgcctct 240
ctactcgtga ttctggcagt tctcgttatt ggcggacccc accgatctcc tcgtgtcggc 300
aaatggctcc aagtacgaaa gtggtccctc ttccaagcgt gggtaaagtt tattgctatg 360
gaagtgattt tggatcaacc gaaaggcatt actatggacg tccaacaaga caaggcgatt 420
tttgcattcg cgccacatgg aatctttccg tttgcgttcg cctttggagt gcttcccgat 480
attgccacac aatcgtttgg ctacgttcgt ccggtcgtgg caaccgccac aaggttgttt 540
cctgtagtcc gggatttcat ctcttgggcg aatccggtag acgcttccaa agattccgtt 600
gaacgtgctt tagcattggg cgatcgcatt gctgtaatac ctggaggaat tgcagaaatt 660
ttcgaaggat atccgaaacc gaacacgcat ccggatgaag agtacgctat cgtacggagt 720
ggatttttgc gtttggcaat aaaacacggt atcccagtga ttcccgtata ctgtttcggc 780
gctaccaaaa tgttgaagcg tctggagctt cctggcctgg agcaactgtc cctgtttcta 840
cgcgtgagca tttgcctctt ttttggagtc ggcgggttgc ccatcccttt ccgacaacga 900
ttgtcgtacg taatgggaca accaattttg ccacccgtaa ggacaacggg cagcgatatt 960
tcggacgcac acgtcaaaga aatgcaagat cgcttttgtg ctgaggtcca gcggctcttt 1020
gatcgacata aggaagctta tggttggtcc cacaaaacgc tgaaactatt ggaacagtga 1080
<210>59
<211>392
<212>PRT
<213>Phaeodactylum tricornutum
<400>59
Met Arg Glu Arg Ser Cys Ala Asn Ala Ser Asp Asp Asp Ser Ile His
1 5 10 15
Lys Gln Ser Pro Glu Leu Glu Ala Glu Phe Leu His Thr Ser Lys Leu
20 25 30
Thr Leu Ala Asp Met Arg Arg Leu Ala His Asp Pro Lys Asp Arg Arg
35 40 45
Leu Ala Thr Lys Pro Ala Ala Gln Ala Thr Lys Glu Asp Val Leu Thr
50 55 60
Val Gln Pro Met Ser Phe Val Glu His Thr Ala Cys Cys Leu Phe Leu
65 70 75 80
Ala Phe Gly Val Pro Asn Gly Ala Leu Thr Ile Pro Ile Ala Thr Trp
85 90 95
Leu Ile Gly Lys Phe Val Val Arg Asn Val Phe Leu Ala Phe Leu Leu
100 105 110
Ala Gly Cys Ile Leu Leu Pro Leu Ala Ile Leu Pro Gln Glu Tyr Val
115 120 125
Pro Ala Arg Leu Gln Ser Trp Leu Ala Leu Gln Ile Leu Lys Tyr Phe
130 135 140
Ser Phe Ser Leu Val Met Glu Glu Arg Pro Pro Thr Met Cys Thr Gly
145 150 155 160
Lys Gln Leu Ile Glu Gln Pro Ala Arg Pro Arg Ile Val Thr Ala Tyr
165 170 175
Pro His Gly Val Phe Pro Tyr Gly Asn Ala Leu Thr Val Val Thr Trp
180 185 190
Pro Leu Leu Thr Gly His His Ile Val Gly Leu Ala Ala Asn Ala Ala
195 200 205
Leu Arg Thr Pro Ile Phe Lys Gln Ile Leu Arg Ser Ile Gly Val Lys
210 215 220
Asp Ala Ser Arg Ala Ser Val Arg Asn Ala Leu Glu Thr Trp Pro Phe
225 230 235 240
Thr Val Gly Ile Ser Pro Gly Gly Val Ala Glu Val Phe Glu Thr Asn
245 250 255
His Phe Asn Glu His Ile Leu Leu Lys Glu Arg Ile Gly Val Ile Lys
260 265 270
Met Ala Ile Arg Thr Gly Ala Asp Leu Val Pro Gly Tyr Met Tyr Gly
275 280 285
Asn Thr Asn Leu Tyr Trp Cys Trp Thr Gly Glu Gly Ile Pro Gly Ala
290 295 300
Arg Trp Leu Leu Glu Tyr Val Ser Arg Lys Ile Leu Gly Phe Ala Leu
305 310 315 320
Val Pro Ile Ala Gly Arg Trp Gly Leu Pro Ile Pro Tyr Arg Thr Pro
325 330 335
Ile Leu Cys Val Val Gly Lys Pro Ile Pro Thr Ile His Leu Gln Thr
340 345 350
Glu Glu Pro Ser Met Glu Gln Ile Val Asp Ile Gln Glu Gln Leu Ser
355 360 365
Thr Glu Leu Lys Ser Met Phe Asp Arg Tyr Lys His Leu Tyr Gly Trp
370 375 380
Glu Asp Arg Met Leu Val Ile Thr
385 390
<210>60
<211>1179
<212>DNA
<213>Phaeodactylum tricornutum
<400>60
atgcgtgagc gaagctgcgc caacgcttct gacgatgaca gcattcacaa gcagtcgcca 60
gaattggagg ctgagtttct tcataccagc aagttgacct tagccgacat gcgacgattg 120
gcgcacgatc cgaaggatcg gaggttggca acaaaacctg cggcgcaagc tacgaaagaa 180
gacgtcttga cggtacaacc catgagtttc gtagaacaca ctgcttgctg tctgtttctc 240
gcgtttggag tgcccaatgg cgctctgacg attcccatag caacgtggct gatcggaaaa 300
ttcgtggtac gcaacgtttt cttggcgttt ctgttagcag gctgtatact tctaccgctt 360
gcgatactgc cgcaagaata tgtgcccgcc cgattgcaat cgtggcttgc tttgcagata 420
ctgaaatatt tttctttctc tttggtcatg gaggaacgcc ctccgacaat gtgtactggc 480
aagcagctga tcgagcagcc cgctcggcca cgaatcgtca cagcctatcc gcacggagtt 540
ttcccatacg gaaacgcgtt gactgtagtc acatggccgt tgttgacggg acaccatatt 600
gtgggtttgg cagcaaatgc cgctttgcgg acaccgatct ttaaacaaat cttgcggagc 660
attggcgtca aggacgcctc tcgagcgtcg gtacggaatg cgctggaaac atggcctttc 720
accgtcggga tttcgccagg tggcgtggcg gaagtttttg aaacaaacca cttcaatgag 780
cacattctgt tgaaagaacg tattggtgtc atcaagatgg ccattcgcac cggtgcggat 840
cttgtaccag gctatatgta tggtaatact aatctgtact ggtgctggac aggggaaggt 900
attcctggag ctcggtggct attggagtat gtttcgcgta aaatcctagg ttttgccctc 960
gtgcctatag cgggtagatg gggactacca ataccgtaca ggactccgat attgtgtgtc 1020
gtgggcaagc caataccaac cattcatttg caaaccgaag aaccatcaat ggagcaaatc 1080
gtggacattc aggaacaatt gtcaacagaa ttgaaatcaa tgttcgaccg ctataagcac 1140
ctgtacggat gggaagatcg aatgctagtg atcacataa 1179
<210>61
<211>329
<212>PRT
<213>Phaeodactylum tricornutum
<400>61
Met Glu Arg Thr Lys Ile Gln Asp Glu His Lys Ser Pro Pro Asn Pro
1 5 10 15
Ser Thr Phe Arg Trp Phe Leu Gly Leu Leu Val Ala Ser Thr Phe Ser
20 25 30
Met Val Tyr Phe Val Ala Pro Phe Tyr Met Leu Thr Val Val Phe Ala
35 40 45
Leu Val Phe Lys Tyr Pro Ser Val Glu Ile Ala Trp Met Tyr Ala Ile
50 55 60
Pro Met Ile Val Ser Ala Ile Leu Pro Pro Met Ala Ser Pro Leu Ala
65 70 75 80
Leu Arg Leu Ile Ser Pro Leu Ile Asp Tyr Phe Asp Tyr Glu Glu Ile
85 90 95
His Glu Thr Ser Pro Val Asp Val Gln Lys Glu Ile Leu Ser Asn Asn
100 105 110
Lys Asn Tyr Leu Leu Val Phe Gln Pro His Gly Ala Leu Ser Phe Thr
115 120 125
Gly Ile Thr Ser Met Val Thr Ala Pro Gln Ala Met Lys Gly Lys Leu
130 135 140
Pro Thr Ala Val Ala Asp Ala Leu Leu Tyr Thr Pro Ile Leu Lys His
145 150 155 160
Val Leu Gly Ile Phe Gly Leu Ile Ser Ala Ser Lys Ser Ser Met Ile
165 170 175
Arg Thr Leu Lys Lys Lys Gly Val Glu Gly Thr Ile Val Leu Tyr Val
180 185 190
Gly Gly Ile Ala Glu Leu Phe Leu Thr Asp Glu Thr Asp Glu Arg Leu
195 200 205
Tyr Leu Arg Lys Arg Lys Gly Phe Ile Lys Leu Ala Leu Gln Gln Gly
210 215 220
Val Asp Val Val Pro Val Tyr Leu Phe Gly Asn Thr Asn Ala Leu Ser
225 230 235 240
Val Leu Lys Thr Gly Phe Leu Ala Ala Ile Ser Arg Lys Leu Gln Ile
245 250 255
Ser Leu Thr Tyr Ile Trp Gly Lys Trp Tyr Leu Pro Ile Pro Arg Asp
260 265 270
Cys Lys Leu Leu Tyr Ala Ser Gly Gln Pro Leu Gly Met Pro His Ile
275 280 285
Leu Asp Pro Ser Gln Ala Asp Ile Asp Lys Trp His Glu Lys Tyr Cys
290 295 300
Ser Glu Val Met Arg Ile Phe Glu Lys Tyr Lys Glu Lys Val Pro Glu
305 310 315 320
Tyr Lys His Lys Lys Leu Glu Ile Ile
325
<210>62
<211>990
<212>DNA
<213>Phaeodactylum tricornutum
<400>62
atggagagaa caaagataca agacgagcac aaaagtcccc ctaatccgtc gacatttcga 60
tggttcctcg gccttctagt ggcgtcgacg ttttccatgg tctattttgt ggctcccttt 120
tacatgctta cagtcgtgtt tgcactagtt ttcaaatatc cttcggtaga aattgcatgg 180
atgtacgcta ttccgatgat tgtctcggcc attttgccac caatggcttc tccacttgcc 240
ttgcgactca tctccccgct cattgactac ttcgattacg aagagatcca cgaaacctca 300
ccggtggacg tccagaagga aatactaagc aacaacaaaa actatttgct agtctttcaa 360
ccgcatggag cactgtcgtt tacaggaatc acttcaatgg tgacagctcc acaagcaatg 420
aaaggcaaat tgccaacagc tgtggctgac gcactcttgt acacacctat actgaaacat 480
gtcttaggaa ttttcgggct gattagtgcc tccaaaagca gcatgatccg aactttaaaa 540
aagaagggtg tggaaggaac cattgttttg tacgttggtg ggattgccga gctctttttg 600
accgacgaga cggacgagcg cctctatctg cgaaagcgaa aagggtttat caaattagct 660
ctacaacagg gtgtcgatgt tgtacctgtg tatctatttg ggaacacaaa cgcgctgtcg 720
gtactaaaga cgggatttct cgcggcaatt tcgcgaaaat tacagatatc tctgacgtac 780
atttggggaa agtggtatct tccgattccc cgtgattgca aattgctgta tgcttccggt 840
cagccattag gaatgcctca tattttagac ccaagccaag ccgacattga taaatggcac 900
gaaaagtact gctccgaggt catgcggatc ttcgaaaaat acaaggaaaa ggttccggaa 960
tacaagcaca agaaattaga aattatttga 990
<210>63
<211>320
<212>PRT
<213>Phaeodactylum tricornutum
<400>63
Met Thr Arg Ser Lys Phe Ile Gly Ser Ala Gly Ala Ile Gly Leu Phe
1 5 10 15
Cys Leu Met Ile Ile Pro Asn Val Gly Ile Leu Ile Ala Thr Phe Leu
20 25 30
Tyr Pro Lys Val Leu Gly Phe Tyr Phe Leu Ile Pro Tyr Tyr Ala Tyr
35 40 45
Asn Leu Ser Ile Gly Lys His Glu Ala Arg Asp Gly Asn Gly Trp Asn
50 55 60
Trp Phe Ser Glu Asn Phe Phe Val Phe Asn Ile Val Arg Gly Tyr Leu
65 70 75 80
Asn Leu Lys Ile Glu Ala Asp Ser Glu Leu Lys Glu Ala Glu Ala Lys
85 90 95
Glu Gly Ala Gln Phe Val Phe Ala Val Ser Pro His Gly Thr Asn Ala
100 105 110
Asp Tyr Arg Val Phe Ile Asp Gly Met Leu His Glu Ala Leu Pro Gln
115 120 125
Thr Ala Ser Lys Ile Arg Thr Leu Ala Ala Thr Val Leu Phe His Ile
130 135 140
Pro Leu Val Arg Glu Ile Ala Leu Trp Thr Gly Cys Val Asp Ala Ser
145 150 155 160
Arg Ala Val Ala Val Glu Arg Leu Lys Glu Glu Gly Gly Ser Leu Leu
165 170 175
Val Ile Pro Gly Gly Gln Ala Glu Gln Met Tyr Thr Gln Tyr Gly Arg
180 185 190
Glu Arg Val Tyr Leu Lys Arg Arg Lys Gly Phe Leu Lys Leu Cys Leu
195 200 205
Lys Tyr Glu Ile Pro Val Val Pro Ala Tyr Val Phe Gly Val Ser Asp
210 215 220
Tyr Tyr Phe Thr Ser Ala Lys Leu Phe Gly Leu Arg Met Trp Leu Val
225 230 235 240
Gln Asn Leu Gly Ile Ala Leu Pro Leu Cys Trp Gly Arg Tyr Gly Leu
245 250 255
Pro Ile Cys Pro Arg Pro Val Asp Thr Thr Leu Val Phe Asp Lys Pro
260 265 270
Leu Tyr Leu Ser Cys Gln Asn Pro Ser Asn Pro Ser Glu Asp Glu Val
275 280 285
Asp Lys Ala His Leu Gln Phe Cys Gln Ala Leu Glu Lys Leu Phe Asp
290 295 300
Thr His Lys Glu Arg Leu Gly Tyr Gly Asp Arg Lys Leu Glu Ile Ile
305 310 315 320
<210>64
<211>963
<212>DNA
<213>Phaeodactylum tricornutum
<400>64
atgaccagat cgaagtttat aggaagtgct ggagctattg gcttattttg tttgatgatc 60
ataccgaatg tgggaattct gatcgcaaca tttctttatc ccaaagtact tgggttctac 120
tttctgattc cgtactacgc atacaacttg tccattggca aacacgaagc tcgagacggc 180
aacggctgga attggttcag cgagaatttc tttgtcttta acattgtgag gggatatcta 240
aatcttaaga ttgaagctga ctccgagctc aaggaagccg aagcgaaaga aggcgcccaa 300
tttgtgttcg ccgttagccc tcacggaacg aacgcagact atcgagtttt tattgacggt 360
atgctacatg aggcactccc acagactgca agcaagatca gaacactagc ggcgacagta 420
ctgttccaca ttcccttggt tcgtgaaatc gcactttgga caggatgtgt cgatgccagc 480
cgcgcagttg ctgtcgagag attaaaagaa gaaggtggtt cactgcttgt gattcccggt 540
ggccaagcag aacaaatgta cacccaatat ggacgtgaaa gagtatatct gaaacggcgc 600
aaaggatttt tgaagctttg cttgaagtac gagattccgg tcgtcccagc ttatgttttt 660
ggcgtatctg actattactt cacgtccgca aagctctttg gtctgcgaat gtggctcgtt 720
cagaatcttg gcattgctct tccactgtgc tggggaagat atggtctacc aatctgtcct 780
agaccagtcg ataccaccct tgtctttgac aaacctttat acctatcctg ccagaatccg 840
tcgaatccct cggaagacga ggttgacaag gctcatctgc aattttgcca agccctcgag 900
aagctgtttg atacacacaa agagaggctt gggtacggcg atcgaaagct ggaaataatt 960
tag 963
Claims (19)
1.一种分离或纯化的多肽,其包含与SEQ ID NO:15具有至少90%的序列同一性的序列。
2.根据权利要求1所述的分离或纯化的多肽,其特征在于,所述多肽与SEQ ID NO:1具有至少90%的序列同一性。
3.根据权利要求1所述的分离或纯化的多肽,其特征在于,所述多肽具有二酰基甘油酰基转移酶活性。
4.编码权利要求1中的分离或纯化的多肽的核酸序列。
5.根据权利要求4所述的核酸序列,其特征在于,所述核酸序列位于载体上。
6.一种包含权利要求4中所述的核酸序列的转基因植物。
7.一种包含权利要求4中所述的核酸序列的转基因植物,其特征在于,所述植物种子中与缺少该核酸序列的植物相比其多不饱和脂肪酸的水平发生了改变。
8.一种包含权利要求4中所述的核酸序列的转基因植物,其特征在于,所述植物中的脂肪酸约超过70%为多不饱和脂肪酸。
9.一种用权利要求4中的核酸序列转化的酵母细胞。
10.一种改变植物中超长链多不饱和脂肪酸水平的方法,所述方法包括:
向植物中导入包括编码多肽的多核苷酸的核酸分子,所述多肽与选自SEQ ID NO:1、15、25、27、28、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59和61中的多肽具有至少90%的序列同一性。
11.根据权利要求10所述的方法,其特征在于,所述核酸构建体是通过农杆菌介导的转化方法导入所述植物的。
12.根据权利要求10所述的方法,所述方法进一步包括:向所述植物中导入编码具有Brusica丙酮酸脱氢酶激酶活性多肽的多核苷酸。
13.根据权利要求10所述的方法,所述方法进一步包括:导入编码具有二酰甘油乙酰基转移酶活性多肽的多核苷酸。
14.根据权利要求10所述的方法,所述方法进一步包括:导入编码具有3-磷酸-甘油脱氢酶活性多肽的多核苷酸。
15.根据权利要求10所述的方法,其特征在于,所述植物选自拟南芥(Arabidopsis thaliana)、琉璃苣(Borago spp.)、加拿大油料(Canola)、蓖麻(Ricinus spp.)、可可(Theobroma spp.)、玉蜀黍(Zea spp.)、棉(Gossypiumspp)、海甘蓝(Crambe spp.)、萼距花(Cuphea spp.)、亚麻(Linum spp.)、雷斯克勒(Lesquerella spp.)、鲸油草(Limnanthes spp.)、亚麻突变株(Linola)、旱金莲(Tropaeolum spp.)、月见草(Oenothera spp.)、洋橄榄(Olea spp.)、油棕(Elaeis spp.)、花生(Arachis spp.)、油菜籽(rapeseed)、红花(Carthamus spp.)、大豆(Glycine spp.)、野生大豆(SoJa spp.)、向日葵(Helianthus spp.)、烟草(Nicotiana spp.)、斑鸠菊(Vernonia spp.)、小麦(Triticum spp.)、大麦(Hordeum spp.)、稻(Oryza spp.)、燕麦(Avenaspp.)、高粱(Sorghum spp.)、黑麦(Secale spp.)、十字花科植物(Brassicaceae),以及其它禾本科(Gramineae)植物。
16.一种根据权利要求10的方法生产的植物。
17.一种从根据权利要求10生产的植物中收获的种子。
18.从根据权利要求10生产的植物中提取的油。
19.根据权利要求10所述的方法,进一步包括:从包括核酸构建体的植物收获种子;以及从收获的种子中提取油类。
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CA2709067A1 (en) | 2009-07-09 |
EP2234474A4 (en) | 2011-05-04 |
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