CN101189342A - 具有改良生长特性的植物及其制备方法 - Google Patents
具有改良生长特性的植物及其制备方法 Download PDFInfo
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Abstract
本发明涉及通过增加至少部分富含亮氨酸重复受体样激酶(RKS11、RKS4或它们的直系同源物)的表达来改良植物生长特性的方法。一类这样的方法包括向植物中引入富含亮氨酸重复受体样激酶(RKS11或RKS4或它们的直系同源物)的编码核酸分子。本发明还涉及引入了富含亮氨酸重复受体样激酶(RKS11或RKS4或它们的直系同源物)编码核酸或其变体的转基因植物,所述植物相对于相应的野生型植物具有改良的生长特性。本发明也涉及在本发明方法中有用的构建体。
Description
发明领域
本发明一般地涉及分子生物学领域,并涉及改良植物生长特性的方法。更具体地,本发明涉及通过增加植物中至少部分富含亮氨酸重复受体样激酶(Leucine Rich Repeat Receptor-Like Kinase)(RKS11或RKS4或它们的直系同源物)的表达来改良植物生长特性尤其是产率的方法。本发明还涉及具有增加的至少部分富含亮氨酸重复受体样激酶(RKS11或RKS4或它们的直系同源物)表达的植物,所述植物相对于相应的野生型植物具有改良的生长特性。本发明还提供在发明方法中有用的构建体。
发明背景
鉴于世界人口不断增长而农业可耕种土地面积逐渐缩小,激起提高农业效率方面的研究。作物和园艺改良的常规方法是利用选育技术来鉴定具有期望特性的植物。然而,这样的选育技术有几个缺点,即这些技术通常是劳动密集型的,并且产生通常含有异源遗传组分(包括不期望的性状)的植物,这些遗传组分并非总是产生从亲本植物传递的期望性状。分子生物学的进展已经允许人类改造动物和植物的种质(germplasm)。植物遗传工程需要分离和操作遗传物质(通常为DNA或RNA的形式)及随后将该遗传物质引入植物。这种技术能够提供具有多种改良的经济、农业或园艺性状的植物。
具有特别经济利益的性状是产率,并且对于许多植物而言是种子产率。产率通常被定义为来自作物经济价值的可测量产出。其可以根据数量和/或质量来定义。产率直接依赖于几种因素,例如:器官的数量和大小,植物构造(例如分枝数),种子产量及更多的因素。根的发育,营养吸收和胁迫耐受性也是决定产率的重要因素。因此优化上述因素之一可以有助于增加作物的产率。诸如谷物、稻、小麦、芸苔和大豆的作物占人类总卡路里摄取量的一半以上,不论是通过种子本身的直接消耗,还是通过由加工的种子所饲养的肉类产品的消耗。它们也是工业加工所用的糖类、油类和多类代谢物的来源。种子含有胚芽和胚乳,前者为种子萌发后新的芽和根的来源,后者为在萌发和幼苗早期生长过程中胚芽生长的营养源。种子的发育涉及许多基因,并且需要代谢物自根、叶和茎转移至正在生长的种子。特别是胚乳,吸收糖类、油类和蛋白质的代谢前体,将其合成为贮存高分子,以使谷粒长大。增加植物种子产率的能力,无论是增加种子数量、种子生物量、种子发育、种子饱满性状或任何其他种子相关性状,将在农业中具有许多应用,而且甚至具有许多非农业用途,例如诸如药物、抗体或疫苗等物质的生物技术生产。
受体样激酶(RLK)参与将胞外信号传递至细胞。RLK蛋白具有始于N-端带有被加工的分泌信号的模块结构、胞外结构域、单个跨膜结构域和胞质激酶结构域。受体样激酶据推测形成同二聚体或两种相关激酶的异二聚体,类似于动物受体激酶(Torii,Curr.Opin.Plant Biol.3,361-367,2000)。动物受体样激酶大多具有酪氨酸激酶活性,而植物RLK都具有Ser/Thr激酶特异性,或者有时候可能具有双重特异性。在动物中,大多数RLK作为生长因子受体起作用,而植物受体样激酶可能在多种过程中发挥功能,包括发育、激素感知或病原体反应。有关植物受体样激酶的发育功能如分生组织发育、花粉-雌蕊相互作用、激素信号传导、配子体发育、细胞形态发生和分化、器官形态、器官脱落和体细胞胚胎发生等的综述,由Becraft(Annu.Rev.Cell Dev.Biol.,18,163-192,2002)提供。
或者,受体样激酶可以依据其胞外结构域的结构进行分类(Shiu和Bleecker,Proc.Natl.Acad.Sci.USA 98,10763-10768,2001)。最大的一类是含有富含亮氨酸重复(LRR)的RLK;其又可以基于RLK胞外部分LRR结构域的组织而分为13个亚组(LRRI至LRRXIII)。LRR单位可以以可变的数目存在,且可以作为连续或中断的重复排布。
RKS(SERK样受体激酶)蛋白所具有的模块结构对应于LRR II亚家族的LRR-RLK。从N-端至C-端,其结构域排布为:信号序列、多个亮氨酸拉链基序、一对保守的半胱氨酸、4个或5个LRR结构域,接下来是另一对保守的半胱氨酸、跨膜结构域和胞内激酶结构域。拟南芥属(Arabidopsis)RKS基因构成14个成员的基因家族,并且与SERK(体细胞胚胎发生受体激酶)相关。SERK最初在胡萝卜中表征(Schmidt等,Development 124,2049-2069,1997),并且在胚胎发生细胞中特异性表达。SERK同源物也见于其他植物物种(拟南芥属(Hecht等,Plant Physiol.127,803-816,2001)或向日葵属(Helianthus)以及单子叶植物如玉米或鸭茅(Dactylis glomerata)(Somleva等,Plant Cell Rep.19,718-726,2000))。在拟南芥(Arabidopsis thaliana)中过表达SERK增加拟南芥培养物的胚胎发生潜能,证实了其增加胚胎发生能力的推测功能。在拟南芥中,AtSERK1仅表达于发育中的胚珠(特别是胚囊),并且在受精后在胚乳和胚芽中表达,直至心期。组成型表达AtSERK1的转基因植物据报道不改变植物表型(Hecht等,2001)。蒺藜苜蓿(Medicago truncatula)MtSERK1的表征表明,至少是在豆科植物中,SERK在发育方面可能起着比单独胚胎发生更为广泛的用途(Nolan等Plant Physiol.133,218-230,2003)。
WO 2004/007712描述并表征了许多拟南芥RKS基因。据推测,修饰RKS基因的表达将导致修饰油菜素甾醇类(brassinosteroid)信号传导路径。数据表明,依据特定RKS基因和表达类型(与野生型相比上调或下调表达)的不同,会产生多种表型。例如,RKS4和RKS10据报道刺激细胞分裂。过表达RKS4基因导致细胞分裂增加以及植物表型改变,而调节RKS10的确改变细胞数量,但不改变植物或器官的大小。过表达RKS10还导致形成许多生殖分生组织,其在正常发育的花中不终止。RKS10的过表达以及表达下调对于花粉形成均具有强烈的负效应。根长度受到RKS10过表达的负面影响,而侧根的起始和长出得到促进。抑制RKS1表达可以获得对于根生长的相同效应。同样,过表达RKS3、RKS4或RKS6基因对于根长度具有正效应。增加的茎尖分生组织(apical shoot meristem)形成和长出不仅可以通过过表达RKS0而且可以通过下调RKS3、RKS4、RKS8或RKS10的表达来实现。RKS4过表达据报道导致更大的种子大小,但是并不导致更高的种子产率;对于RKS11基因未进行功能分析。然而,在该篇文献中,仅仅研究了全长RKS蛋白。
本领域已知,表达截短的受体样激酶通常导致功能缺失表型。例如,Shpak等(Plant Cell 15,1095-1110,2003)描述了缺乏胞质激酶结构域的截短ERECTA蛋白(LRR XIII亚家族的LRR-RLK)。ERECTA调控器官形态和花序结构。表达截短ERECTA蛋白的转基因植物花序紧凑、长角果短而钝圆;此为功能缺失型erecta突变植物的特有表型。CLAVATA是归类为亚家族XI的另一LRR-RLK,控制植物中的茎尖和花分生组织。两种clavata1突变体即clv1-6和clv1-7缺乏全部激酶结构域,但与激酶结构域内的其他突变相比,其突变表型相当温和(Clark等Cell 89,575-585,1997;Torii,2000)。
现已惊奇地发现,与对照植物相比,增加至少部分亚家族II富含亮氨酸重复受体样激酶(LRR-RLK)(优选RKS11或RKS4或它们的直系同源物)的表达,使植物具有改良的生长特性。
发明内容
因此本发明提供了改良植物生长特性的方法,包括增加亚家族IILRR-RLK(优选RKS11或RKS4或它们的直系同源物)或其部分(所述部分在下文称作LRR-II-RLP,表示亚家族II富含亮氨酸重复受体样蛋白的部分)的表达;前提是所述改良的生长特性不包括经增加RKS4(SEQ IDNO:12)表达后增加的种子大小。
选择合适的对照植物是实验设置的常规部分,并且包括相应的野生型植物或不含目的基因的相应植物。对照植物通常为相同的植物物种,或者甚至与待比较植物为同一品种。对照植物还可以是待比较植物的无效合子(nullizygote)。如本文所用的“对照植物”不仅指完整植物,而且指植物部分,包括种子和种子部分。
增加亚家族II LRR-RLK或LRR-II-RLP编码核酸表达的优选方法是在植物中引入并表达分离的亚家族II LRR-RLK或LRR-II-RLP编码核酸。
待引入植物(因此可用于实施本发明方法)的核酸是任意编码亚家族II LRR-RLK的核酸,优选RKS11或RKS4或它们的直系同源物的编码核酸,更优选核酸编码下文所述类型的LRR-II-RLP。
如本文所用的术语“RKS11”或“RKS11多肽”是指如SEQ ID NO:2所示的多肽。如本文所用的术语“RKS4”或“RKS4多肽”是指如SEQ ID NO:12所示的多肽。RKS11和RKS4多肽均包含如下结构域(从N-端至C-端):(i)信号序列,(ii)亮氨酸拉链基序,具有被6个其他氨基酸分隔开的3个Leu残基,(iii)具有2个保守半胱氨酸残基的基序,(iv)4个富含亮氨酸重复单位,各约23个氨基酸残基,(v)富含丝氨酸和脯氨酸残基的结构域,(vi)单个跨膜结构域,和(vii)激酶结构域,两端均侧接功能未知的结构域。
术语“结构域”表示在进化相关蛋白质序列比对(如下文所述进行)中的特定位置上保守的一组氨基酸。虽然同源物之间在其他位置上的氨基酸可变,在特定位置上高度保守的氨基酸意味着对于蛋白质的结构、稳定性或活性必不可少的氨基酸。由于这些氨基酸是因其在家族蛋白质同源物比对序列中的高度保守性鉴定的,它们可用作为标识符以确定具有新测定序列的多肽是否属于以前鉴定的多肽家族。
RKS11的信号肽据预测长31氨基酸。RKS11中具有保守半胱氨酸残基的基序由Diévart&Clark(2003)描述,Cys残基位于SEQ ID NO:2中第 66和73位。LRR结构域串联置于氨基酸102至196。另外,RKS11蛋白在第4个LRR结构域与TM区之间含有包括34%脯氨酸和丝氨酸的氨基酸链。跨膜(TM)区据推测跨越氨基酸240至262。激酶结构域既与Pfam PF00069型又与Pfam PF07714型的激酶结构域相对应,这可能表明其具有双重特异性活性,而且包含氨基酸303至572。激酶催化结构域的N-末端包含富含甘氨酸残基的链,邻近一个赖氨酸残基,已证明参与ATP结合。激酶催化结构域的中心部分具有保守的天冬氨酸残基,其对于RKS11蛋白的激酶活性很重要。在跨膜结构域和激酶结构域之间发现功能未知的结构域(RELH结构域),其特征在于存在“RELHXXTDG”基序(SEQID NO:5)。X可代表任意氨基酸,优选首个X为疏水非极性氨基酸,更优选缬氨酸或丙氨酸。在激酶结构域C-端发现另一功能未知的结构域(EGD结构域),始于“EGDGLA”基序(SEQ ID NO:6),并止于“ELSGPR”基序(SEQ ID NO:7)。
RKS4(SEQ ID NO:12)为RKS11的旁系同源物,并与RKS11高度同源。通过比对RKS11和RKS4蛋白的序列,可以容易地鉴定出RKS4中相应的Leu拉链基序、保守的半胱氨酸残基、LRR、跨膜及激酶结构域。RKS11和RKS4还共享FNVAGNPLIC基序(SEQ ID NO:8),且两蛋白的最后20个氨基酸均含有7个Asp或Glu残基。RKS4和RKS11彼此之间的不同之处在例如Leu拉链基序中:RKS11蛋白的拉链基序中含有3个Leu残基,而RKS4具有2个Leu残基。而且,两蛋白在具有保守半胱氨酸的基序之间还存在两个残基的差异。
如本文所用的术语“LRR-II-RLP”涵盖截短形式的属于亚家族II的富含亮氨酸重复受体样激酶(LRR-RLK)(如Shiu和Bleeker,2001所定义的拟南芥那样,或如Diévart和Clark,Curr.Opin.Plant Biol.6,507-516,2003所列的那样),其中所述截短定位于激酶结构域。术语“LRR-II-RLP”也涵盖激酶结构域突变了的亚家族II LRR-RLK激酶,所述突变体与野生型蛋白质相比激酶活性降低,但优选没有激酶活性。由于亚家族II LRR-RLK胞外结构域在结构上及其相似,任何亚家族II LRR-RLK都可以用于本发明的方法;优选这样的LRR-RLK为来自拟南芥的RKS11(At4g30520)或RKS4(At2g23950)。或其中之一的直系同源物,更优选LRR-RLK为如SEQID NO:2所示的拟南芥RKS11。术语LRR-II-RLP还涵盖天然存在的截短形式的亚家族II LRR-RLK激酶,且其中不存在活性激酶结构域。这类天然截短的受体样激酶的实例有SEQ ID NO:14(GenBank登录号BX827036,RKS11的截短同源物)或稻序列SEQ ID NO:15(BAD68256),其为截短形式的SEQ ID NO:16(BAD68255)。优选可用于本发明方法的LRR-II-RLP蛋白为截短形式的亚家族II富含亮氨酸重复受体样激酶(LRR-RLK),其中在蛋白质的C-端一半具有缺失,从而至少降低、优选基本上失活该受体激酶的活性,但是更优选缺失基本上全部的激酶结构域。而且优选LRR-II-RLP蛋白为截短形式的RKS11或RKS4或它们的直系同源物;最优选LRR-II-RLP为如SEQ ID NO:10所示的RKS11trunc,或如SEQ ID NO:14所示的序列。
亚家族II的LRR-RLK蛋白不仅涵盖Diévart和Clark(2003)所列的拟南芥蛋白质,而且还涵盖其同源物,只要这些同源物落入如Shiu和Bleeker(2001)所定义的亚家族II LRR-RLK范围内即可。优选的同源物为RKS11(SEQ ID NO:2)和RKS4(SEQ ID NO:12)的直系同源物和旁系同源物。
“旁系同源物”是相同物种内部通过祖先基因复制而起源基因,而“直系同源物”是通过物种形成而起源的来自不同生物体的基因。可以通过进行所谓的交互(reciprocal)BLAST搜索容易地找到直系同源物和旁系同源物。这可以通过一次BLAST实现:在任何序列数据库如公众可获得的NCBI数据库中针对查询序列(例如SEQ ID NO:1或SEQ ID NO:2)进行BLAST。当从核苷酸序列开始时可使用BLASTN或TBLASTX(使用标准缺省值),而当从蛋白质序列开始时可使用BLASTP或TBLASTN(使用标准缺省值)。可以任选过滤BLAST结果。然后,在查询序列所源自的生物体的序列中针对过滤结果或者未过滤结果的全长序列进行二次BLAST搜索(反向BLAST)(当查询序列为SEQ ID NO:1或SEQ ID NO:2时,二次BLAST必将在拟南芥序列中进行)。然后比较第一次和第二次BLAST的结果。如果一次BLAST的高分命中事件(high-ranking hit)来自查询序列所源自的相同物种,随之反向BLAST结果理想地将查询序列作为最高命中事件,则鉴定到旁系同源物;如果一次BLAST的高分命中事件不来自查询序列所源自的相同物种,且优选经反向BLAST结果是查询序列作为最高命中事件,则鉴定到直系同源物。优选的直系同源物是RKS11(SEQID NO:2)、RKS4(SEQ ID NO:12)或截短形式RKS11(SEQ ID NO:10)的直系同源物。高分命中事件是那些E值低的命中事件。E值越低,得分的显著性越高(或者换言之,偶然发现命中事件的概率越低)。E值的计算是本领域众所周知的。在大家族的情况下,可以使用Clustal W,继之以邻近连接树来辅助观察相关基因的聚类,以鉴定直系同源物和旁系同源物。除了E值之外,还对比较进行同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。
RKS11或RKS4受体样激酶的直系同源物优选包含信号肽、Leu拉链基序以及具有两个保守半胱氨酸残基的基序、包括四个LRR重复的LRR结构域、跨膜结构域,还优选包含与Pfam数据库中所定义的Pfam PF00069和/或Pfam PF07714型激酶结构域相对应的激酶结构域。信号肽序列和跨膜结构域的推测为本领域公知,而上文定义的LRR和激酶结构域高度保守。同样,保守的半胱氨酸残基和亮氨酸拉链基序可以通过与SEQ ID NO:2或SEQ ID NO:12比较而容易地鉴定,由此所属领域的技术人员能够容易地鉴定落入上文定义的直系同源物序列。优选地,当以Needleman和Wunsch算法使用空位开放罚分11、空位延伸罚分1进行比较时,可用于本发明的直系同源物与SEQ ID NO:2具有至少59%的序列同一性。而且,可用于本发明的直系同源物优选在最后一个LRR结构域和跨膜结构域之间包含富含丝氨酸和/或脯氨酸的结构域(对应于SEQ ID NO:2的氨基酸197至240),所述Ser/Pro富含结构域包含至少23%丝氨酸和/或脯氨酸残基,且包含FNV(A/V)GNP(L/M)IC基序(SEQ ID NO:8)。还优选位于激酶结构域N-端功能未知的结构域包含如上文所定义的RELHXXTDG基序。而且优选可用于本发明的直系同源物包含至少长60个氨基酸的EGD结构域。
落入“RKS11或其直系同源物”定义的植物衍生多肽的实例如SEQ IDNO:18所示(稻(Oryza sativa),GenBank登录号BAD10034)。
下表显示了基于整体全局序列比对,RKS11同源多肽序列与SEQ IDNO:2所示氨基酸序列相比的序列同一性和相似性百分比。同一性和相似性百分比以Needleman和Wunsch算法使用空位开放罚分11、空位延伸罚分1进行计算。
表1:RKS4和RKS11蛋白序列与SEQ ID NO:2基于整体全局序列比对的同源性
RKS同源物 | SEQ ID NO: | 同一性%/相似性% |
拟南芥RKS4 | SEQ ID NO:12 | 82.4/89.7 |
稻RKS11 | SEQ ID NO:18 | 59.9/71.7 |
根据本发明优选的方面,直系同源物与SEQ ID NO:2所示的氨基酸序列具有至少59%的序列同一性。
可以通过序列比对容易地确定多肽与SEQ ID NO:2所示的氨基酸序列是否具有至少59%的同一性。为比较而进行序列比对的方法是本领域众所周知的,此类方法包括GAP、BESTFIT、BLAST、FASTA和TFASTA。GAP使用Needleman和Wunsch的算法(J.Mol.Biol.48:443-453,1970)来寻找两完整序列之间匹配数最大化且空位数最小化的比对。BLAST算法计算序列同一性百分比,并对两序列之间的相似性进行统计学分析。执行BLAST分析的软件可通过美国国家生物技术信息中心公开地获得。与SEQ ID NO:2所示的氨基酸具有至少59%同一性的RKS11多肽或其直系同源物可通过将查询序列(优选为蛋白质序列(全长或无分泌信号序列的成熟形式))与已知的RKS11直系同源物蛋白质序列进行比对而容易地鉴定。同样,对于RKS4多肽而言,序列同一性可通过将查询序列与已知的RKS4直系同源物蛋白质序列进行比对来确立。例如,此类同源物可以使用获自http://clustalw.genome.jp/sit-bin/nph-ClustalW的ClustalW多重序列比对算法(1.83版),采用缺省的双比对参数以及百分比的记分方法而容易地鉴定。可以进行微小的人工编辑以优化保守基序之间的比对,这对于所属领域的技术人员而言将是显而易见的。然而,当搜索合适的LRR-II-RLP蛋白或者鉴定合适的亚家族II LRR-RLK以产生这样的LRR-II-RLP时,优选仅利用蛋白质的胞外结构域(即跨膜结构域的N-端)来确定序列同源性。优选的同源物是与SEQ ID NO:10具有最高序列同一性的那些同源物。
落入LRR-II-RLP范畴的合适突变体涵盖那些其激酶活性降低(与野生型蛋白质相比)或完全失活的突变体。本领域众所周知如何引入突变从而抑制转磷酸作用或自我磷酸化作用。转磷酸作用或自我磷酸化作用的缺乏产生不稳定的蛋白质复合物,从而配体不能够结合,或者不可能进行信号传导。例如,可在激酶结果于的活性位点上或其附近引入突变,以降低或抑制激酶活性,其他突变也可能有用,例如,在ATPA结合位点上突变由此阻止ATP结合,或者在自我磷酸化的情况下,通过改变正常被磷酸化的氨基酸。
为确定受体样激酶的激酶活性,可以利用多种测定法且为本领域公知(例如Current Protocols in Molecular Biology,卷1和2,Ausubel等(1994),Current Protocols)。简言之,激酶测定法通常包括:(1)使激酶蛋白质与含待磷酸化靶位点的底物多肽进行接触;(2)在适宜的条件下在适宜的激酶缓冲液中进行靶位点的磷酸化;(3)在合适的反应阶段之后,从非磷酸化的底物中分离磷酸化的产物。激酶活性的存在与否通过磷酸化靶标的存在与否来确定。另外,可进行定量测量。可利用纯化的受体样激酶、或者含有或富集受体样激酶的细胞提取物作为激酶蛋白质的来源。或者,可使用Zhao等的方法(Plant Mol.Biol.26,791-803,1994),其中在大肠杆菌中表达稻受体样激酶的胞质结构域,并测定激酶活性。小肽作为底物尤为适宜。肽的磷酸化位点基序中必需包含一个或多个丝氨酸、苏氨酸或酪氨酸残基。汇编的磷酸化位点可见Biochimica et Biophysica Acta 1314,191-225,(1996)。另外,肽底物最好可以具有净正电荷,以便于与磷酸纤维素滤膜结合(能够从非磷酸化的肽中分离磷酸化的肽,以检测磷酸化的肽)。如果磷酸化位点基序未知,可使用一般的酪氨酸激酶底物。例如,“Src相关肽”(RRLIEDAEYAARG)为许多受体和非受体酪氨酸激酶的底物)。为确定合成肽磷酸化的动力学参数,需要一系列范围的肽浓度。对于最初的反应,可使用0.7-1.5mM的肽浓度。对于每一种激酶而言,确定活性的最佳缓冲液、离子强度和pH非常重要。标准5×激酶缓冲液通常含有5mg/mlBSA(牛血清白蛋白,防止激酶吸附于测定管),150mM Tris-Cl(pH7.5),100mM MgCl2。大多数酪氨酸激酶需要二价阳离子,虽然有些酪氨酸激酶(例如,胰岛素-、IGF-1-和PDGF受体激酶)需要MnCl2代替MgCl2(或除MgCl2之外还需要MnCl2)。每一种蛋白质激酶的二价阳离子最佳浓度必需凭经验确定。一种常用的磷供体是放射性标记的[γ-32P]ATP(终浓度通常为0.2mM)。掺入到肽中的32P量可以通过用闪烁计数器测量硝酸纤维素上干燥垫片(pad)的活性来确定。
而且,LRR-II-RLP多肽的活性可以通过在稻种子特异性启动子的控制下在稻类植物尤其是稻类品种日本晴(Nipponbare)中表达LRR-II-RLP多肽进行测定,这将产生与相应的对照植物相比产率增加的植物。产率的增加可以例如通过如下一项或多项进行衡量:饱满种子数的增加、种子总重量的增加、收获指数的增加和/或种子氨基酸水平的提高。
蛋白质突变体(以及同源物)涵盖肽、寡肽、多肽、蛋白质和酶,其相对于所讨论的未修饰蛋白质具有氨基酸取代、缺失和/或插入,并且(在同源物的情况下,以及对于某些突变体而言)与其源自的未修饰形式蛋白质具有相似的生物学活性和功能活性。为了生产这样的同源物,蛋白质的氨基酸可以由具有相似性质(如相似的疏水性、亲水性、抗原性、形成或打破α螺旋结构或β片层结构的倾向)的其他氨基酸替换。保守取代表是本领域众所周知的(例如见Creighton(1984)Proteins.W.H.Freeman andCompany以及表2)。
表2:保守氨基酸取代的实例
残基 | 保守取代 | 残基 | 保守取代 |
Ala | Ser | Leu | Ile;Val |
Arg | Lys | Lys | Arg;Gln |
Asn | Gln;His | Met | Leu;Ile |
Asp | Glu | Phe | Met;Leu;Tyr |
残基 | 保守取代 | 残基 | 保守取代 |
Gln | Asn | Ser | Thr;Gly |
Cys | Ser | Thr | Ser;Val |
Glu | Asp | Trp | Tyr |
Gly | Pro | Tyr | Trp;Phe |
His | Asn;Gln | Val | Ile;Leu |
Ile | Leu,Val |
突变体可以是蛋白质的“取代变体”的形式,即在氨基酸序列中至少有一个残基被去除,并且在其位置上插入不同的残基。氨基酸取代通常是单个残基的取代但是视施加于多肽的功能性限制而定也可能是成簇取代;插入通常在1到10个氨基酸残基的数量级。优选地,氨基酸取代包括保守的氨基酸取代,除非希望改变蛋白质的功能或结构性质。
突变体也可以是蛋白质的“插入变体”的形式,即在蛋白质的预定位点引入一个或多个氨基酸残基。插入可以包括氨基端和/或羧基端的融合,以及单个或多个氨基酸的内部序列插入。一般氨基酸序列内部的插入将小于氨基或羧基端的融合,数量级约1到10个残基。氨基或羧基端融合蛋白质或肽的实例包括在酵母双杂交系统中应用的转录激活因子的结合结构域或激活结构域、噬菌体外壳蛋白质、(组氨酸)6标签、谷胱甘肽S-转移酶标签、蛋白质A、麦芽糖结合蛋白、二氢叶酸还原酶、Tag·100表位、c-myc表位、表位、lacZ、CMP(钙调蛋白结合肽)、HA表位、蛋白质C表位和VSV表位。特别是,有用的LRR-II-RLP多肽可通过在胞外结构域中插入一个或多个富含亮氨酸重复结构域建立,或者通过在天然存在蛋白质的C-末端融合类似于亚家族II LRR-RLK优选RKS11、RKS4或它们的直系同源物(如At3g43740(SEQ ID NO:44)或At5g21090(SEQ ID NO:46)所编码的蛋白质)的胞外结构域的跨膜结构域。
蛋白质“缺失变体”形式的突变体特征在于从蛋白质中除去一个或多个氨基酸。优选的缺失突变体是那些缺失部分激酶结构域的突变体,从而残留部分的转磷酸化或自我磷酸化活性至少降低,优选活性完全丧失。更优选的突变体是那些基本上缺失了全部激酶结构域的突变体。此外更优选的突变体是那些与SEQ ID NO:10比对基本上缺乏相同部分的激酶结构域的突变体。最优选突变体是SEQ ID NO:10所示的RKS11trunc。
可通过本领域众所周知的肽合成技术,如固相肽合成法等,或通过重组DNA操作容易地得到蛋白质的氨基酸变体。用于产生蛋白质的取代、插入或缺失变体的DNA序列操作方法是本领域众所周知的。例如,本领域的技术人员熟知在DNA预定位点产生取代突变的技术,包括M13诱变、T7-Gen体外诱变(USB,Cleveland,OH)、QuickChange定点诱变(Stratagene,San Diego,CA)、PCR介导的定点诱变或其他定点诱变方案。所有这些技术可用于产生适用于本发明方法的LRR-II-RLP。
RKS11或RKS4多肽可以分别是SEQ ID NO:2、SEQ ID NO:12的衍生物。“衍生物”包括肽、寡肽、多肽,与天然存在形式的蛋白质如SEQID NO:2或SEQ ID NO:12所示的氨基酸序列相比,其可以包括以非天然存在氨基酸残基取代氨基酸、或添加非天然存在的氨基酸残基。衍生物SEQ ID NO:10、SEQ ID NO:14和SEQ ID NO:18是可以适用于产生LRR-II-RLP以用于本发明方法的其他实例。
蛋白质的“衍生物”也涵盖肽、寡肽、多肽,与天然存在形式多肽的氨基酸序列相比,其可以包括天然存在的改变的(糖基化、酰基化、泛素化、异戊烯化、磷酸化、豆蔻酰化、硫酸化等)或非天然改变的氨基酸残基。衍生物与其源自的氨基酸序列相比,还可以包括一个或多个非氨基酸取代基或添加,例如共价或非共价地结合于氨基酸序列的报告分子或其他配体,例如与之结合有利于衍生物检测的报告分子,以及相对于天然存在蛋白质的氨基酸序列而言非天然存在的氨基酸残基。
应当理解落入“RKS11多肽或其直系同源物”或“RKS4多肽或其直系同源物”定义的序列并不局限于SEQ ID NO:2、SEQ ID NO:12及SEQID NO:18所示的序列,而是满足如下标准的任何多肽:包含(i)信号序列,(ii)亮氨酸拉链基序、具有被6个其他氨基酸分隔开的2个或3个Leu残基(iii)具有2个保守半胱氨酸残基的基序,(iv)4个富含亮氨酸重复单位,各约23个氨基酸残基,(v)富含丝氨酸和脯氨酸残基的结构域,(vi)单个跨膜结构域和(vii)激酶结构域两端均侧接功能未知的结构域(RELH-和EGD-结构域);而且,作为如SEQ ID NO:2所示RKS11的直系同源物可适用于产生LRR-II-RLP蛋白以用于本发明的方法。
SEQ ID NO:10的LRR-II-RLP蛋白以前不为人知。本发明因此提供选自下组的新的分离的LRR-II-RLP蛋白:
a)无激酶活性的多肽,其包含(i)信号序列,(ii)亮氨酸拉链基序,具有被6个其他氨基酸分隔开的2个或3个Leu残基,(iii)具有2个保守半胱氨酸残基的基序,(iv)4个富含亮氨酸重复单位,各约23个氨基酸残基,(v)富含丝氨酸和脯氨酸残基的结构域,(vi)单个跨膜结构域,和(vii)部分或完整的RELH结构域;
b)基本上缺乏整个激酶结构域的亚家族II富含亮氨酸重复受体样激酶;
c)如SEQ ID NO:10所示的多肽;
d)具有与SEQ ID NO:10所示的一个或多个氨基酸序列具有至少90%序列同一性、优选95%、96%、97%、98%或99%序列同一性的氨基酸序列的多肽,
前提是所述LRR-II-RLP蛋白不是SEQ ID NO:14所示的蛋白。
SEQ ID NO:9所示的序列作为LRR-II-RLP编码核酸迄今为止不为人知。本发明因此还提供选自下组的分离的核酸:
i)如SEQ ID NO:9所示的核酸序列或其互补链;
ii)编码SEQ ID NO:10所示氨基酸序列的核酸序列;
iii)能够在严谨条件下与上述(i)或(ii)中核酸序列杂交的核酸序列,所述杂交序列编码LRR-II-RLP蛋白;
iv)编码如上文(a)至(d)中所述蛋白的核酸;
v)上述(i)至(iii)中任一核酸序列的一部分,所述部分编码LRR-II-RLP蛋白,
前提是所述LRR-II-RLP编码核酸并非如SEQ ID NO:13所示或者并不编码SEQ ID NO:14的蛋白。
编码RKS11多肽、RKS4多肽或它们的直系同源物的核酸适用于产生LRR-II-RLP蛋白以用于本发明的方法,并且可以是任何天然或合成的核酸。如上文所定义的RKS11多肽或其直系同源物由RKS11核酸/基因编码。因此如本文所定义的术语“RKS11核酸/基因”是编码如上文所定义的RKS11多肽或其直系同源物的任何核酸/基因。RKS11核酸的实例包括SEQ ID NO:1和SEQ ID NO:17所示的那些核酸。RKS11核酸/基因及其变体可适用于产生LRR-II-RLP蛋白的编码核酸以用于本发明的方法。变体RKS11核酸/基因包括RKS11核酸/基因的部分和/或能够与RKS11核酸/基因杂交的核酸,前提是这些杂交序列编码全部或部分的RKS11或其直系同源物。
如上文所定义的RKS4多肽或其直系同源物由RKS11核酸/基因编码。因此如本文所定义的术语“RKS4核酸/基因”是编码如上文所定义的RKS4多肽(如SEQ ID NO:11)或其直系同源物的任何核酸/基因。RKS4核酸/基因及其变体可适用于实施本发明的方法。变体RKS4核酸/基因包括RKS4核酸/基因的部分和/或能够与RKS4核酸/基因杂交的核酸,前提是这些杂交序列编码全部或部分的RKS4或其直系同源物。
本文定义的术语“部分”指包括至少编码这样的蛋白质的足够核苷酸的DNA片段,所述蛋白质包含(i)信号序列,(ii)亮氨酸拉链基序,具有被6个其他氨基酸分隔开的2个或3个Leu残基,(iii)具有2个保守半胱氨酸残基的基序,(iv)4个富含亮氨酸重复单位,各约23个氨基酸残基,(v)富含丝氨酸和脯氨酸残基的结构域,(vi)单个跨膜结构域,和(vii)部分或完整的激酶结构域,所述部分源自RKS11、RKS4或它们的直系同源物。例如,可以通过在RKS11或RKS4核酸中产生一个或多个缺失来制备部分。部分可以以分离的形式应用,或者它们可以与其他编码(或非编码)序列融合以,例如,产生组合数种活性的蛋白质。当与其他编码序列融合时,翻译产生的多肽可以大于预测的RKS11或RKS4片断。优选功能性部分是SEQ IDNO:1、SEQ ID NO:11和SEQ ID NO:17中任一所示核酸的部分。
另一类变体RKS11或RKS4核酸/基因是在降低的严谨条件下,优选在严谨条件下能够分别与上文所定义的RKS11核酸/基因或RKS4核酸/基因杂交的核酸,所述杂交序列编码的多肽包含:(i)信号序列,(ii)亮氨酸拉链基序,具有被6个其他氨基酸分隔开的2个或3个Leu残基,(iii)具有2个保守半胱氨酸残基的基序,(iv)4个富含亮氨酸重复单位,各约23个氨基酸残基,(v)富含丝氨酸和脯氨酸残基的结构域,(vi)单个跨膜结构域,和(vii)部分或完整的RELH结构域,且所述杂交序列编码全部或部分的RKS11、RKS4或它们的直系同源物。优选所述杂交序列能够与SEQ ID NO:1、SEQ ID NO:11和SEQ ID NO:17中任一所示核酸杂交、或与任一上文所定义的前述序列的部分杂交。
本文定义的术语“杂交”指其中基本同源互补的核苷酸序列彼此退火的过程。杂交过程能够完全在溶液中发生,即两互补的核酸均在溶液中。杂交过程也可以如此进行,即互补核酸之一固定于基质如磁珠、琼脂糖珠或任何其他树脂上。此外,杂交过程也可以如此进行,即其中互补核酸之一固定在固相支持物如硝酸纤维素或尼龙膜上,或者例如通过照相平板印刷固定在例如硅质玻璃支持物上(后者称之为核酸阵列或微阵列,或称之为核酸芯片)。为了使杂交发生,通常使核酸分子热变性或化学变性,以使双链解链成两条单链,和/或除去单链核酸中的发夹结构或其他二级结构。
术语“严谨性”是指杂交发生的条件。杂交的严谨性受诸如温度、盐浓度、离子强度和杂交缓冲液组成等条件的影响。通常,对于在确定的离子强度和pH值时的特定序列,选择比热解链温度(Tm)低约30℃的低严谨条件。中等严谨条件是温度比Tm低20℃,而高严谨条件是温度比Tm低10℃。高严谨杂交条件通常用于分离与靶核酸序列具有高序列相似性的杂交序列。不过,由于遗传密码的简并性,核酸可以在序列上有差异而依然编码基本上相同的多肽。因此,有时候可能需要中等严谨杂交条件以鉴定此类核酸分子。
Tm是在确定的离子强度和pH值时,50%的靶序列与完全匹配的探针杂交的温度。Tm依赖于溶液条件和探针的碱基组成及长度。例如,较长的序列在较高温度下特异性杂交。在低于Tm值16℃到32℃获得最大杂交率。在杂交溶液中存在一价阳离子会减少两核酸链之间的静电排斥作用,从而促进杂合体形成;当钠浓度高达0.4M时,这一作用明显(至于更高的浓度,此作用可以忽略)。每个百分点的甲酰胺可使DNA-DNA和DNA-RNA双链体的解链温度降低0.6至0.7℃,加入50%甲酰胺使杂交在30至45℃完成,尽管这将降低杂交率。碱基对错配降低杂交率和双链体的热稳定性。平均而言,对于大的探针,每个百分点碱基错配使Tm值下降约1℃。Tm值可以根据杂合体类型使用下列方程式计算:
1)DNA-DNA杂合体(Meinkoth和Wahl,Anal.Biochem.,138:267-284,1984):
Tm=81.5℃+16.6×log10[Na+]a+0.41×%[G/Cb]-500×[Lc]-1-0.61×%甲酰胺
2)DNA-RNA或RNA-RNA杂合体:
Tm=79.8+18.5(log10[Na+]a)+0.58(%G/Cb)+11.8(%G/Cb)2-820/Lc
3)寡DNA或寡RNAd杂合体:
<20个核苷酸:Tm=2(ln)
20-35个核苷酸:Tm=22+1.46(ln)
a或对于其他一价阳离子,但是仅在0.01-0.4M范围内精确。
b仅对于在30%到75%范围内的%GC精确。
cL=双链体的碱基对长度。
d寡,寡核苷酸;ln,引物的有效长度=2×(G/C数)+(A/T数)。
可以通过许多已知技术中的任一来控制非特异性结合,例如用含蛋白质的溶液封闭膜,在杂交缓冲液中添加异源RNA、DNA和SDS,以及用RNA酶处理。对于非同源探针,可以通过改变(i)逐步降低退火温度(例如从68℃至42℃)或(ii)逐步降低甲酰胺浓度(例如从50%至0%)中之一进行系列杂交。熟练的技术人员知晓多种参数可以在杂交过程中进行改变,从而保持或者改变严谨条件。
例如,长于50个核苷酸的DNA杂合体的典型的高严谨杂交条件包括在1×SSC中于65℃杂交或者在1×SSC和50%甲酰胺中于42℃杂交,接着在0.3×SSC中于65℃洗涤。长于50个核苷酸的DNA杂合体的中等严谨杂交条件的实例包括在4×SSC中于50℃杂交或者在6×SSC和50%甲酰胺中于40℃杂交,接着在2×SSC中于50℃洗涤。杂合体的长度是杂交核酸的预期长度。当已知序列的核酸进行杂交时,杂合体的长度可以通过比对序列并鉴定本文所述的保守区域进行确定。1×SSC是0.15M NaCl和15mM柠檬酸钠;杂交和洗涤可以另外地包括5×Denhardt′s试剂、0.5-1.0%SDS、100μg/ml片段化的变性鲑精DNA、0.5%焦磷酸钠。
为了定义严谨性水平,可以方便地参照Sambrook等(2001)《分子克隆:实验室手册》第三版,冷泉港实验室出版,冷泉港,纽约,或者CurrentProtocols in Molecular Biology,John Wiley&Sons,N.Y.(1989以及年度更新)。
RKS11或RKS4核酸或其变体可以来自任何天然或人工的来源。核酸/基因或其变体可以分离自微生物来源,如细菌、酵母或真菌,或分离自植物、藻类或动物(包括人类)来源。可以通过精细的人为操作在组成和/或基因组环境方面修饰所述核酸的天然形式,为了产生合适的LRR-II-RLP尤为如此。优选植物来源的核酸,无论来源于同一植物物种(例如对于其待引入的物种而言)或来源于不同植物物种。可以从双子叶物种,优选从十字花科(Brassicaceae),更优选从拟南芥分离所述核酸。更优选地,从拟南芥中分离的RKS11核酸如SEQ ID NO:1所述,而RKS11氨基酸序列如SEQ IDNO:2所示。此外还优选,从拟南芥中分离的RKS4核酸如SEQ ID NO:11所述,而RKS4氨基酸序列如SEQ ID NO:12所示。
RKS11或RKS4多肽或其同源物可以由RKS11或RKS4核酸/基因的可选剪接变体编码。本文所用的术语“可选剪接变体”涵盖其中选择的内含子和/或外显子已被切除、替换或添加的核酸序列变体。这样的变体保留了蛋白质的生物活性,这可以通过选择性地保留蛋白质的功能性区段来实现。这样的剪接变体可以是天然的或人工的。产生这类剪接变体的方法是本领域众所周知的。优选的剪接变体是如SEQ ID NO:1和SEQ ID NO:17所示RKS11核酸的剪接变体;且优选的RKS4剪接变体是如SEQ ID NO:11所示序列的剪接变体。SEQ ID NO:1的剪接变体的实例是如SEQ ID NO:48所示的序列。更优选编码多肽的剪接变体,所述多肽包含:(i)信号序列,(ii)亮氨酸拉链基序,具有被6个其他氨基酸分隔开的2个或3个Leu残基,(iii)具有2个保守半胱氨酸残基的基序,(iv)4个富含亮氨酸重复单位,各约23个氨基酸残基,(v)富含丝氨酸和脯氨酸残基的结构域,(vi)单个跨膜结构域,和(vii)激酶结构域,两端均侧接功能未知的结构域(RELH-和EGD-结构域),所述剪接变体可用于产生合适的LRR-II-RLP蛋白。
同源物还可以由RKS11、RKS4多肽或它们的直系同源物的编码核酸的等位基因变体所编码,优选由SEQ ID NO:1、SEQ ID NO:11和SEQ IDNO:17所示核酸的等位基因变体编码。更优选由等位基因变体编码的多肽包含:(i)信号序列,(ii)亮氨酸拉链基序,具有被6个其他氨基酸分隔开的2个或3个Leu残基,(iii)具有2个保守半胱氨酸残基的基序,(iv)4个富含亮氨酸重复单位,各约23个氨基酸残基,(v)富含丝氨酸和脯氨酸残基的结构域,(vi)单个跨膜结构域,和(vii)激酶结构域,两端均侧接功能未知的结构域(RELH-和EGD-结构域)。等位基因变体天然存在,并且这些天然等位基因用于产生合适的LRR-II-RLP蛋白的用途涵盖在本发明的方法中。等位基因变体包括单核苷酸多态性(SNP),以及小型插入/缺失多态性(INDEL)。INDEL的大小通常小于100bp。SNP和INDEL在大多数生物体天然存在的多态性品系中形成最大的一组序列变体。
有利地,实施本发明的方法使植物具有多种改良的生长特性,尤其是增加的产率,特别是种子产率。
本文所定义术语“增加的产率”意指植物的一个或多个部分增加的生物量(重量),这可以包括地上(可收获)部分和/或地下(可收获)部分。
特别地,这样的可收获部分为种子,并且实施本发明的方法使植物的种子产率相对于合适对照植物的种子产率而言增加。
增加的种子产率可以表现为以下的一项或多项:a)种子生物量(种子总重量)的增加,可以是以单个种子和/或每株植物和/或每公顷或每英亩为基础的增加;b)每株植物花朵数量的增加;c)增加的(饱满)种子数量;d)种子饱满率(其表达为饱满种子数与种子总数的比值)的增加;e)增加的收获指数,其表达为可收获部分如种子的产率与总生物量的比值;和f)千粒重(TKW)的增加,其是从所计的饱满种子数和它们的总重量推算出的。TKW的增加可以源自种子大小和/或种子重量的增加,并且还可以源自胚芽和/或胚乳大小的增加。
种子产率的增加也可以表现为种子大小和/或种子体积的增加。不过,应当指出,“增加的种子产率”不包括RKS4(SEQ ID NO:12)过表达时增加的种子大小。此外,种子产率的增加也可以表现为种子面积和/或种子长度和/或种子宽度和/或种子周长的增加。增加的种子产率也涵盖种子中氨基酸和/或其他代谢物改良的组成,优选增加的氨基酸水平。增加的产率也可能产生改变的构造,或者可以作为改变的构造的结果而发生。
以玉米为例,产率的增加可以表现为以下的一项或多项:每公顷或每英亩植物数量的增加,每株植物穗数的增加,行数、行粒数、粒重、千粒重、穗长度/直径的增加,种子饱满率(其为饱满种子数除以种子总数再乘以100)的增加,等等。以稻为例,产率的增加可以表现为以下一项或多项的增加:每公顷或每英亩的植物数量,每株植物的圆锥花序数量,每个圆锥花序的小穗数量,每个圆锥花序的花朵(小花)数量(其表达为饱满种子数与初级圆锥花序数的比值),种子饱满率的增加(其为饱满种子数除以种子总数再乘以100)的增加,千粒重的增加,等等。
由于本发明的转基因植物具有增加的产率,相对于相应野生型植物在它们生命周期相应阶段的生长速率而言,这些植物可能呈现增加的生长速率(在至少它们部分的生命周期中)。增加的生长速率可能对植物的一个或多个部分(包括种子)是特异性的,或者可能基本上遍及整个植物。具有增加的生长速率的植物甚至可能呈现早期开花。生长速率的增加可能出现在植物生命周期的一个或多个阶段,或者出现在基本上整个植物生命周期的过程中。在植物生命周期的早期阶段,生长速率的增长可能反映为增强的活力(增强的幼苗突出活力)。生长速率的增加可以改变植物的收获周期,使植物能够比其它可能的情况更晚播种和/或更快收获。如果生长速率充分增加,可以允许播种同种植物物种更多的种子(例如播种和收获稻类植物,随后在一个常规的生长期播种和收获更多的稻类植物)。同样的,如果生长速率充分地增长,可以允许播种不同植物物种更多的种子(例如播种和收获稻类植物,随后,如播种和任选的收获大豆、马铃薯或任何其它适合的植物)。在一些作物植物的情况下也可能从同一根茎收获增加的次数。改变植物的收获周期可能会导致每英亩年生物量产量的增加(这是由于(比方说在一年中)任何特定植物可以生长和收获次数的增加)。与野生型对应物相比,生长速率的增加还可能使能够在更广阔的地域栽培转基因植物,因为种植作物的地域限制通常由种植时(早季)或收获时(晚季)不利的环境条件所决定。如果缩短收获周期,就可以避免这类不利条件。可以根据生长曲线获取多种参数来确定生长速率,这类参数可以是:T-Mid(植物达到其最大大小的50%所需时间)和T-90(植物达到其最大大小90%所需时间),等等。
术语“代谢物”是指合成代谢和分解代谢中产生的中间物质,优选低分子量的中间物质,换言之,是指在代谢过程中产生或消耗的物质,如氨基酸。术语代谢物“改进的组成”是指这些代谢物浓度期望的变化。根据代谢物类型的不同,所述变化可以是浓度的增加或减小。优选相对于合适的对照植物测量代谢物浓度/水平的变化。本发明中优选的代谢为氨基酸,特别是色氨酸、苯丙氨酸、酪氨酸、异亮氨酸、缬氨酸中的一种或多种。可以在整个植物或在某些植物部分、器官、组织或细胞中改进代谢物水平。在优选的实施方案中,改进种子中的代谢物水平。
根据本发明优选的方面,相对于对照植物而言,执行本发明的方法赋予植物增加的生长速率,特别是在植物发育的早期阶段(通常为发芽后三周),产生早期活力。本发明因此提供了增加植物生长速率的方法,所述方法包括增加植物中LRR-II-RLP蛋白编码核酸的表达。本发明因此还提供了获得相对于对照植物而言具有早期活力的植物的方法,所述方法包括调节、优选增加植物中LRR-II-RLP蛋白编码核酸的表达。
本发明的方法有利地适用于任何植物。
本文所用术语“植物”涵盖整个植物、植物的祖先和后代、以及植物的部分,包括种子、芽、茎、叶、根(包括块茎)、花、以及组织和器官。术语“植物”还涵盖植物细胞、悬浮培养物、愈伤组织、胚、分生区、配子体、孢子体、花粉和小孢子,同样,其中上述每一种含有目的基因/核酸。
尤其可用于本发明方法的植物包括属于植物界(Viridiplantae)超家族的所有植物,尤其是单子叶植物和双子叶植物,包括选自如下清单的饲料或饲料豆科植物、观赏植物、粮食作物、乔木或灌木:槭树属物种(Acerspp.)、猕猴桃属物种(Actinidia spp.)、秋葵属物种(Abelmoschus spp.)、冰草属物种(Agropyron spp.)、葱芹属物种(Allium spp.)、苋属物种(Amaranthus spp.)、凤梨(Ananas comosus)、番荔枝属物种(Annona spp.)、芹菜(Apium graveolens)、拟南芥、落花生属物种(Arachis spp.)、木波罗属物种(Artocarpus spp.)、石刁柏(Asparagus officinalis)、燕麦(Avena sativa)、阳桃(Averrhoa carambola)、冬瓜(Benincasa hispida)、巴西栗(Bertholletiaexcelsea)、甜菜(Beta vulgaris)、芸苔属物种(Brassica spp.)、Cadabafarinosa、大叶茶(Camellia sinensis)、美人蕉(Canna indica)、辣椒属物种(Capsicum spp.)、苔草(Carex elata)、番木瓜(Carica papaya)、大果假虎刺(Carissa macrocarpa)、山核桃属物种(Carya spp.)、红花(Carthamustinctorius)、栗属物种(Castanea spp.)、苦苣(Cichorium endivia)、樟属物种(Cinnamomum spp.)、西瓜(Citrullus lanatus)、柑橘属物种(Citrus spp.)、椰子属物种(Cocos spp.)、咖啡属物种(Coffea spp.)、芋(Colocasia esculenta)、可拉属(Cola spp.)、芫荽(Coriandrum sativum)、榛属物种(Corylus spp.)、山楂属物种(Crataegus spp.)、番红花(Crocus sativus)、南瓜属物种(Cucurbita spp.)、香瓜属物种(Cucumis spp.)、菜蓟属物种(Cynara spp.)、胡萝卜(Daucus carota)、山马蟥属物种(Desmodium spp.)、龙眼(Dimocarpuslongan)、薯蓣属物种(Dioscorea spp.)、柿树属物种(Diospyros spp.)、稗属物种(Echinochloa spp.)、穇子(Eleusine coracana)、枇杷(Eriobotryajaponica)、红仔果(Eugenia uniflora)、荞麦属物种(Fagopyrum spp.)、山毛榉属物种(Fagus spp.)、无花果(Ficus carica)、金桔属物种(Fortunellaspp.)、草莓属物种(Fragaria spp.)、银杏(Ginkgo biloba)、大豆属物种(Glycine spp.)、陆地棉(Gossypium hirsutum)、向日葵属物种(Helianthusspp.)、萱草(Hemerocallis fulva)、木槿属物种(Hibiscus spp.)、大麦属物种(Hordeum spp.)、甘薯(Ipomoea batatas)、核桃属物种(Juglans spp.)、莴苣(Lactuca sativa)、山黧豆属物种(Lathyrus spp.)、兵豆(Lens culinaris)、亚麻(Linum usitatissimum)、荔枝(Litchi chinensis)、百脉根属物种(Lotusspp.)、棱角丝瓜(luffa acutangula)、羽扇豆属物种(Lupinus spp.)、地杨梅(Luzula sylvatica)、硬皮豆属物种(Macrotyloma spp.)、苹果属物种(Malusspp.)、西印度樱桃(Malpighia emarginata)、曼密苹果(Mammeaamericana)、芒果(Mangifera indica)、木薯属物种(Manihot spp.)、人心果(Manilkara zapota)、紫苜蓿(Medicago sativa)、草木樨属物种(Melilotusspp.)、薄荷属物种(Mentha spp.)、苦瓜属物种(Momordica spp.)、黑桑(Morusnigra)、芭蕉属物种(Musa spp.)、烟草属物种(Nicotianum spp.)、木犀榄属物种(Olea spp.)、仙人掌属物种(Opuntia spp.)、Ornithopus spp.、稻属物种(Oryza spp.)、黍属物种(Panicum sp)、鸡蛋果(Passiflora edulis)、欧防风(Pastinaca sativa)、鳄梨属物种(Persea spp.)、香芹(Petroselinum crispum)、菜豆属物种(Phaseolus spp.)、刺葵属物种(Phoenix spp.)、酸浆属物种(Physalis spp.)、松属物种(Pinus spp.)、阿月浑子(Pistacia vera)、豌豆属物种(Pisum spp.)、早熟禾属物种(Poa spp.)、杨属物种(Populus spp.)、牧豆树属物种(Prosopis spp.)、李属物种(Prunus spp.)、番石榴属物种(Psidiumspp.)、石榴(Punica granatum)、西洋梨(Pyrus communis)、栎属物种(Quercusspp.)、萝卜(Raphanus sativus)、波叶大黄(Rheum rhabarbarum)、茶藨子属物种(Ribes spp.)、悬钩子属物种(Rubus spp.)、甘蔗属物种(Saccharumspp.)、接骨木属物种(Sambucus spp.)、黑麦(Secale cereale)、胡麻属物种(Sesamum spp.)、白芥属物种(Sinapis sp.)、茄属物种(Solanum spp.)、两色蜀黍(Sorghum bicolor)、菠菜属物种(Spinacia spp.)、蒲桃属物种(Syzygiumspp.)、酸豆(Tamarindus indica)、可可树(Theobroma cacao)、车轴草属物种(Trifolium spp.)、小黑麦(Triticosecale rimpaui)、小麦属物种(Triticumspp.)、小金莲花(Tropaeolum minus)、旱金莲(Tropaeolum majus)、越桔属物种(Vaccinium spp.)、野豌豆属物种(Vicia spp.)、豇豆属物种(Vigna spp.)、香堇菜(Viola odorata)、葡萄属物种(Vitis spp.)、玉蜀黍(Zea mays)、北美洲野生稻(Zizania palustris)、枣属物种(Ziziphus spp.)等等。根据本发明优选的实施方案,植物为作物植物,如大豆、向日葵、芸苔、苜蓿、油菜籽、棉花、番茄、马铃薯或烟草。还优选植物是单子叶植物,如甘蔗。更优选植物是谷类,如稻、玉米、小麦、大麦、粟、黑麦、高粱或燕麦。
根据本发明优选的实施方案,植物为作物植物。作物植物的实例包括大豆、向日葵、芸苔、苜蓿、油菜籽、棉花、番茄、马铃薯和烟草。还优选植物是单子叶植物。单子叶植物的实例包括甘蔗。更优选植物是谷类。谷类的实例包括稻、玉米、小麦、大麦、粟、黑麦、高粱和燕麦。
可以通过增加LRR-II-RLP多肽的水平来增加所述蛋白的活性。备选地,当LRR-II-RLP多肽水平没有改变,或者甚至当LRR-II-RLP多肽水平降低的时候,其活性也可以增加。这种情况出现在多肽固有特性发生改变的时候,例如,通过制备比野生型多肽更具活性的突变体形式。
可以通过引入遗传修饰(优选在RKS11基因座、RKS4基因座或者编码天然截短形式RKS11或RKS4的基因座处)来增加可用于本发明的LRR-II-RLP多肽的活性。本文所定义的基因座意指基因组区,其包括目的基因和编码区上游或下游的10KB。
例如,可以通过任意一种(或多种)如下方法引入遗传修饰:TDNA激活、TILLING、定点诱变、转座子诱变、定向进化及同源重组,或通过在植物细胞中引入和表达编码LRR-II-RLP多肽的核酸。引入遗传修饰之后的步骤是选择活性增加的LRR-II-RLP多肽,所述活性的增加使植物具有改良的生长特性。
T-DNA激活标记(Hayashi等Science(1992)1350-1353)包括将通常含有启动子(也可以是翻译增强子或内含子)的T-DNA插入在目的基因的基因组区或基因编码区上游或下游10KB,从而在构型上使启动子能够指导靶基因的表达。通常破坏天然启动子对靶基因表达的调控,基因由新引入的启动子控制。启动子一般嵌入T-DNA中。例如,通过农杆菌(Agrobacterium)感染将此T-DNA随机插入植物基因组,并导致在插入T-DNA附近的基因过表达。得到的转基因植物由于引入启动子附近基因的过表达而表现出显性表型。引入的启动子可以是任何能够在期望生物体内(在本案中是植物)指导基因表达的启动子。例如,组成型、组织偏好型、细胞类型偏好型和诱导型启动子都适用于T-DNA激活。
也可以利用TILLING(定向诱导的基因组局部突变)技术将遗传修饰引入RKS11或RKS4基因座或天然LRR-II-RLP基因座。这是一种诱变技术,用于产生和/或鉴定以及最后分离诱变的能分别呈现LRR-II-RLP活性(即与相应的野生型植物相比增加转基因植物产率的效应,其中增加的产率包括至少如下之一:种子总重量、饱满种子数和收获指数)的RKS11或RKS4核酸变体。TILLING还允许选择携带此类突变变体的植物。这些突变变体甚至可能比其天然形式基因呈现更高的LRR-II-RLP活性。TILLING将高密度诱变和高通量筛选方法结合在一起。TILLING一般遵循的步骤有:(a)EMS诱变(Redei和Koncz,1992;Feldmann等,1994;Lightner和Caspar,1998);(b)DNA制备和个体合并;(c)目的区域的PCR扩增;(d)变性和退火以形成异源双链体;(e)DHPLC,其中库中存在的异源双链体会在色谱图上检测到额外的峰;(f)突变个体的鉴定;和(g)突变PCR产物的测序。进行TILLING的方法是本领域众所周知的(McCallumNat Biotechnol.2000年4月;18(4):455-7,由Stemple 2004综述(TILLING-a high-throughput harvest for functional genomics.Nat RevGenet.2004年2月;5(2):145-50))。
定点诱变可用于产生RKS11或RKS4核酸或其部分的变体(如编码LRR-II-RLP蛋白的那些变体)。可以通过若干方法来完成定点诱变,最常见的是基于PCR的方法(Current Potocols in Molecular Biology.Wiley编辑)。
转座子诱变是以基因中的转座子插入为基础的诱变技术,常常导致截短或基因敲除。此技术已经用于若干植物物种,包括稻(Greco等,PlantPhysiol,125,1175-1177,2001)、玉米(McCarty等,Plant J.44,52-61,2005)和拟南芥(Parinov和Sundaresan,Curr.Opin.Biotechnol.11,157-161,2000)。
结构域改组或定向进化也可以用于产生RKS11或RKS4核酸或其部分的变体、或者LRR-II-RLP活性增加的LRR-II-RLP蛋白的编码核酸的变体。定向进化包括DNA改组的重复,继之以适当筛选和/或选择(Castle等,(2004)Science 304(5674):1151-4;美国专利5,811,238和6,395,547)。
TDNA激活、TILLING、定点诱变、转座子诱变和定向进化是能产生新的RKS11、RKS4或LRR-II-RLP蛋白编码核酸的等位基因和变体的技术的实例。
同源重组允许向基因组中的指定选择位置引入所选的核酸。同源重组是生物科学中常规使用的标准技术,其用于低等生物体如酵母或小立碗藓(physcomitrella)。在植物中执行同源重组的方法已经不仅在模式植物中描述(Offringa等Extrachromosomal homologous recombination andgene targeting in plant cells after Agrobacterium-mediated transformation.1990 EMBO J.1990年10月9(10):3077-84),而且也在作物植物如稻中描述(Terada R,Urawa H,Inagaki Y,Tsugane K,Iida S.Efficient genetargeting by homologous recombination in rice.Nat Biotechnol.2002.Iida和Terada:A tale of two integrations,transgene and T-DNA:genetargeting by homologous recombination in rice.Curr Opin Biotechnol.2004 Apr;15(2):132-8)。所靶向的核酸(其可以是上文定义的RKS11或RKS4核酸或其变体)不需分别靶向RKS11或RKS4基因座,但是可以被引入例如高表达的区域。所靶向的核酸可以是改良的等位基因,其用于替换内源基因或者额外引入到内源基因中。
根据本发明的优选实施方案,通过在植物中引入和表达分离的编码LRR-II-RLP蛋白的核酸,可以改良植物的生长特性。优选LRR-II-RLP蛋白源自拟南芥RKS11或RKS4,或源自上文所述它们的直系同源物,更优选LRR-II-RLP蛋白为截短的拟南芥RKS11或RKS4,或截短的上文所述的它们的直系同源物,最优选LRR-II-RLP蛋白如SEQ ID NO:10或SEQ ID NO:14所示。
根据本发明的优选方面,设想LRR-II-RLP编码核酸分子的表达有所提高或增加。获得提高或增加的基因或基因产物表达的方法在本领域有充分的记录,其包括,例如由适当的启动子驱动的过表达、转录增强子或翻译增强子的使用。可以将用作启动子或增强子元件的分离的核酸引入到非异源形式多核苷酸的适当位置(一般是上游),从而上调RKS11或RKS4核酸或其变体的表达。例如,可以通过突变、缺失和/或取代,在体内改变内源启动子(见Kmiec,美国专利No.5,565,350;Zarling等,PCT/US93/03868),或者将分离的启动子在本发明基因的适当方向和距离引入植物细胞中,从而控制基因的表达。
如果期望多肽表达,通常要在多核苷酸编码区的3’末端纳入多腺苷酸化区域。多腺苷酸化区域可以源自天然基因、多种其他植物基因或T-DNA。例如,加入的3’末端序列可以源自胭脂碱合酶或章鱼碱合酶基因、或可选地源自其他植物基因、或次优选地源自任何其他真核基因。
也可以在5’非翻译区或部分编码序列的编码序列中加入内含子序列,来增加在细胞溶质中累积的成熟信使的量。已显示,纳入植物和动物表达构建体转录单元中的可剪接内含子均可以在mRNA和蛋白质水平使基因表达提高至高达1000倍,Buchman和Berg,Mol.Cell biol.8:4395-4405(1988);Callis等,Genes Dev.1:1183-1200(1987)。通常这类内含子放置在转录单元5’末端附近时,其提高基因表达的作用最大。玉米内含子Adh1-S内含子1、2和6以及Bronze-1内含子的使用是本领域公知的。通常见TheMaize Handbook,116章,Freeling和Walbot编辑,Springer,N.Y.(1994)。
其他控制序列(除启动子、增强子、沉默子、内含子序列、3′UTR和/或5′UTR区域之外)可以是蛋白质和/或RNA稳定元件。
本发明还提供遗传构建体和载体,以促进用于本发明方法中的核苷酸序列的引入和/或表达。
因此,提供的基因构建体包含:
(i)LRR-II-RLP编码核酸;
(ii)一个或多个能驱动(i)中核酸序列表达的控制序列;和任选的
(iii)转录终止序列。
可以使用本领域技术人员熟知的重组DNA技术构建用于本发明方法的构建体。可以将基因构建体插入载体中,所述载体可商购获得,适合于转化进入植物并在转化的细胞中表达目的基因。
使用包含目的序列(即LRR-II-RLP编码核酸)的载体转化植物。将目的序列有效连接于一个或多个控制序列(至少连接于启动子)。术语“调控元件”、“控制序列”和“启动子”在本文都可互换使用,从广义上是指能够影响其连接序列表达的调控核酸序列。上述术语包括源自典型真核生物基因组基因的转录调控序列(包括具有或没有CCAAT盒序列的TATA盒,其对于精确的转录起始是必需的),以及另外的调控元件(即上游激活序列、增强子和沉默子),其通过应答发育刺激和/或外部刺激或通过组织特异性的方式改变基因表达。该术语还涵盖经典原核生物基因的转录调控序列,在此情况下可以包括-35盒序列和/或-10盒转录调控序列。术语“调控元件”也涵盖合成的融合分子或衍生物,其赋予、激活或提高细胞、组织或器官中核酸分子的表达。本文所用的术语“有效连接”指启动子序列和目的基因之间的功能性连接,以使启动子序列能起始目的基因的转录。
有利地,可以使用任何类型的启动子驱动核酸序列的表达。术语“启动子”是指位于基因转录起点上游的核酸控制序列,其参与RNA聚合酶和其他蛋白质的识别和结合,由此指导有效连接的核酸进行转录。启动子可以是组成型启动子,是指在生长发育的大多数但不必是所有阶段中、并且在大多数环境条件下、在至少一种细胞、组织或器官中转录激活的启动子。可选地,启动子可以是诱导型启动子,即响应化学、环境或物理刺激,具有诱导的或增加的转录起始。诱导型启动子的实例是胁迫诱导型启动子,即当植物接触多种胁迫条件时激活的启动子;或者是病原体诱导型启动子。另外或备选的,启动子可以是组织特异性的启动子,即能够在某些组织,如在叶、根、种子等组织中优先起始转录的启动子;或者可以是泛素(ubiquitous)启动子,基本上在生物体的所有组织或细胞中激活;或者启动子可以是发育调控型的,从而在某些发育阶段或在发生发育改变的植物部分激活。能够仅在某些组织中起始转录的启动子在文中称为“组织特异性”启动子,与此类似,能够仅在某些细胞中起始转录的启动子在文中称为“细胞特异性”启动子。
优选的,LRR-II-RLP编码核酸有效连接于种子特异性启动子。种子特异性启动子主要在种子组织中转录激活,但不必仅在种子组织中激活(渗漏表达的情况下)。种子特异性启动可以在种子发育和/或萌发期间激活。种子特异性启动在本领域众所周知。优选种子特异性启动是如SEQ ID NO:19所示的启动子,或者具有相似长度和/或相似表达模式的启动子如WO2004/070039中的PRO0058。例如,可以通过将启动子偶联于报告基因,并检查报告基因在植物组织中的功能,来分析相似长度和/或相似表达模式。一种众所周知的报告基因是β-葡糖醛酸糖苷酶,并使用比色GUS染色法观察植物组织中β-葡糖醛酸糖苷酶的活性。应当清楚,本发明的应用不局限于SEQ ID NO:10所示的LRR-II-RLP编码核酸,也不局限于SEQID NO:14,本发明的应用也不局限于受种子特异性启动子驱动的LRR-II-RLP编码核酸的表达。同样可用于驱动LRR-II-RLP编码核酸表达的其他种子特异性启动子的实例如下表3所示。
表3:种子特异性启动子的实例
基因来源 | 表达模式 | 参考文献 |
种子特异性基因 | 种子 | Simon等,Plant Mol.Biol.5:191,1985;Scofield等,J.Biol.Chem.262:12202,1987;Baszczynski等,Plant Mol.Biol.14:633,1990. |
巴西果白蛋白 | 种子 | Pearson等,Plant Mol.Biol.18:235-245,1992. |
豆球蛋白 | 种子 | Ellis等,Plant Mol.Biol.10:203-214,1988. |
谷蛋白(稻) | 种子 | Takaiwa等,Mol.Gen.Genet.208:15-22,1986;Takaiwa等,FEBS Letts.221:43-47,1987. |
玉米醇溶蛋白 | 种子 | Matzke等Plant Mol Biol,14(3):323-321990 |
napA | 种子 | Stalberg等,Planta 199:515-519,1996. |
小麦LMW和HMW麦谷蛋白-1 | 胚乳 | Mol Gen Genet 216:81-90,1989;NAR17:461-2,1989 |
小麦SPA | 种子 | Albani等,Plant Cell,9:171-184,1997 |
小麦a、b、g-小麦醇溶蛋白 | 胚乳 | EMBO J.3:1409-15,1984 |
大麦Itr1启动子 | 胚乳 | |
大麦B1、C、D大麦醇溶蛋白 | 胚乳 | Theor Appl Gen 98:1253-62,1999;Plant J4:343-55,1993;Mol Gen Genet 250:750-60,1996 |
大麦DOF | 胚乳 | Mena等,The Plant Journal,116(1):53-62,1998 |
blz2 | 胚乳 | EP99106056.7 |
合成启动子 | 胚乳 | Vicente-Carbajosa等,Plant J.13:629-640,1998. |
稻醇溶谷蛋白NRP33 | 胚乳 | Wu等,Plant Cell Physiology 39(8)885-889,1998 |
稻a-球蛋白Glb-1 | 胚乳 | Wu等,Plant Cell Physiology 39(8)885-889,1998 |
稻OSH1 | 胚 | Sato等,Proc.Natl.Acad.Sci.USA,93:8117-8122,1996 |
稻a-球蛋白REB/OHP-1 | 胚乳 | Nakase等Plant Mol.Biol.33:513-522,1997 |
稻ADP-葡萄糖PP | 胚乳 | Trans Res 6:157-68,1997 |
玉米ESR基因家族 | 胚乳 | Plant J12:235-46,1997 |
高粱g-高梁醇溶蛋白 | 胚乳 | PMB 32:1029-35,1996 |
KNOX | 胚 | Postma-Haarsma等,Plant Mol.Biol.39:257-71,1999 |
稻油质蛋白 | 胚和糊粉 | Wu等,J.Biochem.,123:386,1998 |
向日葵油质蛋白 | 种子(胚和干种子) | Cummins等,Plant Mol.Biol.19:873-876,1992 |
PRO0117,推定的稻40S核糖体蛋白 | 胚乳中弱表达 | WO2004/070039 |
PRO0136,稻丙氨酸转氨酶 | 胚乳中弱表达 | |
PRO0147,胰蛋白酶抑制剂ITR1(大麦) | 胚乳中弱表达 | |
PRO0151,稻WSI18 | 胚+胁迫 | WO 2004/070039 |
PRO0175,稻RAB21PRO0058 | 胚+胁迫种子 | WO2004/070039WO 2004/070039 |
任选的,还可以在引入植物的构建体中使用一个或多个终止子序列。术语“终止子”包括控制序列,其为位于转录单元末端的DNA序列,传递信号引发初级转录物的3’加工和多腺苷酸化以及转录的终止。另外的调控元件可以包括转录及翻译增强子。本领域技术人员将知道适合用于实施本发明的终止子和增强子的序列。这类序列为本领域技术人员所公知,或者可以容易地获得。
本发明的遗传构建体还可以包括复制起点序列,这是在特定细胞类型中维持和/或复制所必需的。一个实例是当需要将遗传构建体作为附加型遗传元件(如质粒或粘粒分子)在细菌细胞中维持时。优选的复制起点包括(但不限于)f1-ori和colE1。
遗传构建体可以任选地包括可选择的标记基因。如本文所用,术语“可选择的标记基因”包括赋予细胞表型的任何基因,该基因在细胞中的表达有利于鉴定和/或选择经本发明的核酸构建体转染或转化的细胞。适当的标记可以选自赋予抗生素或除草剂抗性、引入新的代谢性状或允许可视选择的标记。可选择的标记基因的实例包括赋予抗生素抗性的基因(例如磷酸化新霉素和卡那霉素的npt II,或磷酸化潮霉素的hpt),赋予除草剂抗性的基因(例如bar提供对Basta的抗性;aroA或gox提供对草甘膦的抗性),或提供代谢特性的基因(如允许植物使用甘露糖作为唯一碳源的manA)。可视标记基因致使形成颜色(例如β-葡糖醛酸糖苷酶,GUS)、发光(例如荧光素酶)或荧光(绿色荧光蛋白GFP及其衍生物)。
本发明还涵盖可由本发明方法获得的植物。本发明因此提供可由本发明方法获得的植物,所述植物中引入了LRR-II-RLP编码核酸。
本发明还提供产生具有改良生长特性的转基因植物的方法,包括在植物细胞中引入和表达LRR-II-RLP编码核酸。
更具体地,本发明提供产生具有改良生长特性的转基因植物的方法,所述方法包括:
(i)向植物细胞中引入和表达LRR-II-RLP编码核酸;和
(ii)在促进植物生长和发育的条件下培养植物细胞。
可以将核酸直接引入植物细胞或植物本身(包括引入组织、器官或植物的任何其他部分)。根据本发明的优选方面,优选通过转化将核酸引入植物。
本文所提及的术语“转化”包含将外源多核苷酸转移进宿主细胞,不考虑转移所用的方法。无论是通过器官发生还是胚胎发生,能够随即克隆增殖的植物组织都可以使用本发明的遗传构建体转化,并从其再生整个植物。具体的组织选择将因可提供和最适于转化的具体物种的克隆增殖系统而改变。示例性的靶组织包括叶盘、花粉、胚、子叶、下胚轴、雌配子、愈伤组织、既有的分生组织(例如顶端分生组织、腋芽和根分生组织)以及诱导的分生组织(例如子叶分生组织和下胚轴分生组织)。可以将多核苷酸瞬时地或稳定地引入宿主细胞,并且可以保持非整合的状态,例如作为质粒。备选地,其可以整合进入宿主基因组。得到的转化植物细胞可以接着用于以本领域技术人员公知的方式再生为转化的植物。
植物物种的转化目前是一种相当常规的技术。有利地,可以使用若干转化方法的任一向适当的祖先细胞引入目的基因。转化方法包括利用脂质体、电穿孔、增强游离DNA摄取的化学物质、直接向植物注射DNA、粒子枪轰击、用病毒或花粉转化和显微投影(microprojection)。方法可以选自原生质体的钙/聚乙二醇方法(Krens,F.A.等,1882,Nature 296,72-74;Negrutiu I.等,1987年6月,Plant Mol.Biol.8,363-373);原生质体的电穿孔法(Shillito R.D.等,1985 Bio/Technol 3,1099-1102);植物材料的显微注射(Crossway A.等,1986,Mol.Gen Genet 202,179-185);DNA或RNA包被的粒子轰击(Klein T.M.等,1987,Nature 327,70);(非整合型)病毒感染等等。优选使用任何众所周知的稻转化方法通过农杆菌介导的转化,来产生表达LRR-II-RLP编码核酸的转基因稻类植物,例如在任何以下文献中描述的方法:公开的欧洲专利申请EP 1198985 A1,Aldemita和Hodges(Planta,199,612-617,1996);Chan等(Plant Mol.Biol.22(3)491-506,1993),Hiei等(Plant J.6(2)271-282,1994),其公开的内容如同其陈述的全部内容那样并入本文作为参考。至于谷物转化优选的方法如Ishida等(Nat.Biotechnol.1996年6月,14(6):745-50)或Frame等(Plant Physiol.2002年5月,129(1):13-22)中所述,其公开的内容如同其陈述的全部内容那样并入本文作为参考。
通常在转化以后,选出存在一个或多个标记的植物细胞或细胞群,所述标记由与目的基因共转移的植物可表达基因编码,继之转化的材料再生成整个植物。
DNA转移和再生之后,可评估推定的转化植物,例如用Southern分析目的基因的存在、拷贝数和/或基因组结构。可选的或额外的,可用Northern和/或Western分析监测新引入DNA的表达水平,这两种技术都是本领域普通技术人员熟知的。
产生的转化植物可以通过多种方式繁殖,如用克隆繁殖或经典的育种技术。例如,第一代(或T1)转化植物可自交得到纯合的第二代(或T2)转化体,T2植物进一步通过经典育种技术繁殖。
产生的转化生物体可以有多种形式。例如,它们可以是转化细胞和非转化细胞的嵌合体;克隆的转化体(例如所有细胞经过转化以包含表达盒);转化和非转化组织的嫁接体(例如在植物中,转化的根茎嫁接到非转化的接穗上)。
本发明显然延及由本文所述方法产生的任何植物细胞或植物,以及所有的植物部分和其繁殖体。本发明还涵盖由任意上述方法产生的原代转化或转染的细胞、组织、器官或整个植物的后代,所述后代的唯一要求是与本发明方法产生的亲本呈现同样的基因型和/或表型特性。本发明也包括含有分离的LRR-II-RLP蛋白编码核酸的宿主细胞。本发明优选的宿主细胞是植物细胞。
本发明也延及植物可收获的部分,例如,但不限于种子、叶、果实、花、茎培养物、根茎、块茎和球茎。此外本发明还涉及由这样的植物可收获部分直接衍生的产品,如干粒或干粉、油、脂肪和脂肪酸、淀粉或蛋白质。
本发明还涵盖LRR-II-RLP编码核酸的用途。
一种这样的用途涉及改良植物的生长特性,特别是提高产率,尤其是种子产率。种子产率可以包括如下一项或多项:增加的饱满种子数、增加的种子重量(种子总重量)、收获指数、改进的代谢物组成等等。
可以在育种程序中使用LRR-II-RLP编码核酸,或者LRR-II-RLP多肽,其中鉴定可以遗传地连接于LRR-II-RLP编码核酸的DNA标记。可以使用LRR-II-RLP编码核酸/基因,或者LRR-II-RLP多肽界定分子标记。接着可以将此DNA或蛋白质标记在育种程序中使用,以选择具有改良的生长特性的植物。例如,LRR-II-RLP编码基因或其变体可以是如SEQ IDNO:9和SEQ ID NO:13所示的核酸。
LRR-II-RLP编码核酸/基因的等位基因变体也可以用于标记辅助的育种程序。这类育种程序有时需要使用,例如EMS诱变,通过植物的诱变处理引入等位基因变异;可选的,此程序可以以收集无意产生的所谓“天然”起源的等位基因变体开始。然后通过例如PCR进行等位基因变体的鉴定。随后是选择步骤,用以选择所讨论序列的较好等位基因变体,所述等位基因变体赋予植物改良的生长特性。一般通过监测含有所研究序列的不同等位基因变体植物的生长行为来进行选择,例如SEQ ID NO:9和SEQ ID NO:13中任一的不同等位基因变体。可以在温室或田地中监测生长行为。更多任选的步骤包括,将经鉴定含有较好等位基因变体的植物与另一植物杂交。例如,可使用这种方法产生目的表型特征的组合。
LRR-II-RLP编码核酸还可以作为探针,用于对其为基因一部分的基因进行遗传和物理作图,以及作为那些基因连锁性状的标记。这些信息可以在植物育种中使用,以得到具有期望表型的品系。LRR-II-RLP编码核酸的这类应用仅需要至少15个核苷酸长的核酸序列。LRR-II-RLP编码核酸也可以用作限制性片段长度多态性(RFLP)标记。可用LRR-II-RLP编码核酸对限制酶切消化的植物基因组DNA的Southern印迹进行探测。随后使用计算机程序如MapMaker(Lander等,(1987)Genomics 1,174-181)对产生的带型进行遗传分析,以构建遗传图谱。此外,可以使用核酸对含有一组个体的限制性内切酶处理的基因组DNA的Southern印迹进行探测,所述一组个体为代表明确的遗传杂交的亲本和子代的一组个体。记录DNA多态性的分离,并用于计算在先前用此群体获得的遗传图谱中LRR-II-RLP编码核酸或其变体的位置(Botstein等(1980)Am.J.Hum.Genet.32:314-331)。
在遗传作图中使用的植物基因衍生探针的产生和用途描述于Bematzky和Tanksley(1986)Plant Mol.Biol.Reporter 4:37-41中。很多出版物中描述过用上述方法或其变通形式对特定cDNA克隆进行遗传作图。例如,可以使用F2杂交群体、回交群体、随机交配群体、近亲同基因系和其他个体集合作图。这类方法是本领域技术人员众所周知的。
核酸探针也可以用于物理作图(即在物理图谱上安置序列;见Hoheisel等In:Non-mammalian Genomic Analysis:A Practical Guide,Academicpress 1996,319-346页,及其中引用的参考文献)。
在另一个实施方案中,核酸探针用于直接荧光原位杂交(FISH)作图(Trask(1991)Trends Genet.7:149-154)。尽管目前FISH作图的方法倾向于使用大的克隆(几个到几百个KB;见Laan等(1995)Genome Res.5:13-20),但是灵敏度的提高允许在FISH作图中应用较短的探针。
用于遗传和物理作图的多种基于核酸扩增的方法可以使用所述核酸进行。实例包括等位基因特异性扩增(Kazazian(1989)J.Lab.Clin.Med 11,95-96)、PCR扩增片段的多态性(CAPS;Sheffield等(1993)Genomics 16,325-332)、等位基因特异性连接(Landegren等(1988)Science 241,1077-1080)、核苷酸延伸反应(Sokolov(1990)Nucleic Acid Res.18,3671)、放射杂交作图(Walter等(1997)Nat.Genet.7,22-28)和Happy作图(Dear和Cook(1989)Nucleic Acid Res.17,6795-6807)。为实施这些方法,使用核酸序列设计和产生引物对,用于扩增反应或引物延伸反应。这类引物的设计是本领域技术人员众所周知的。使用基于PCR的遗传作图的方法,可能需要鉴定跨越相应于本发明核酸序列区域作图的亲本之间DNA序列的差异。不过,这对作图方法通常不是必要的。
根据本发明的方法产生如前所述具有改良生长特性的植物。这些有利的生长特性还可以组合其他经济上有利的性状,如其他提高产率的性状、对多种胁迫的耐受性、改变多种构造特征和/或生化和/或生理特征的性状。
附图说明
现参考以下附图描述本发明,其中:
图1为RKS11和RKS4受体激酶蛋白之间共有结构域的图形概览。显示了N-端的信号序列;此外还显示了4个LRR结构域、跨膜结构域和激酶结构域。
图2显示了用于在稻中转化和表达拟南芥RKS11trunc编码序列(内参CDS3142,p074,图2a)的二元载体,该基因处于稻糊粉和胚特异性启动子(内参PRO0218,SEQ ID NO:19)的控制之下。
图3为全长RKS11 RLK(SEQ ID NO:2)、实施例部分所用的截短形式(RKS11trunc,SEQ ID NO:10)以及天然形式的LRR-II-RLP蛋白(SEQ IDNO:14,Genbank BX827036)的多重比对。比对结果显示在LRR-II-RLP蛋白的C-端容许一定的序列可变性。
图4详述了用于实施本发明方法的序列实例。
实施例
现参考以下实施例描述本发明,所述实施例仅意在举例说明。
DNA操作:除非另外说明,重组DNA技术根据描述于(Sambrook(2001)《分子克隆:实验室手册》第三版,冷泉港实验室出版,冷泉港,纽约或者Ausubel等(1994),Current Protocols in Molecular Biology,Current Protocols卷1和2)的标准方法实施。植物分子操作的标准材料和方法由R.D.D.Croy描述于Plant Molecular Biology Labfase(1993),由BIOS Scientific Publications Ltd(UK)和Blackwell Scientific Publications(UK)出版。
实施例1:本发明方法所用核酸序列相关序列的鉴定
利用数据库序列搜索工具如基本局部比对工具(BLAST)(Altschul等(1990)J.Mol.Biol.215:403-410;和Altschul等(1997)Nucleic Acids Res.25:3389-3402),在美国国家生物技术信息中心(NCBI)(http://www.ncbi.nlm.nih.gov)Entrez核苷酸数据库保持的序列中鉴定本发明方法所用核酸序列的相关序列(全长cDNA、EST或基因组)。BLAST程序通过将核酸或多肽序列与序列数据库进行比较,以及通过计算匹配的统计学显著性,用于寻找序列之间的局部相似性。例如,对本发明核酸所编码的多肽运用TBLASTN算法,使用缺省设置,开启过滤器,以忽略低复杂度序列。分析输出视窗为两两比较,并根据概率分值(E值)排序,其中分值反映特定比对偶然发生的概率(E值越低,命中事件的显著性越高)。除了E值之外,还对比较进行同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在有些情况下,可以调整缺省参数以更改搜索的严格度。例如,可以提高E值以显示次严格的匹配。以这种方式可以鉴定短的几乎精确的匹配。上表1提供了本发明方法所用核酸序列的几个相关核酸序列。下表4给出了拟南芥亚家族II LRR-RLK序列的概览,这可以利用胞外结构域序列作为查询序列而容易地鉴定。
表4
常用名 | “染色体定位” | SEQ ID NO核酸/蛋白质 |
RKS8 | At1g34210 | 20/21 |
RKS1 | At1g60800 | 22/23 |
RKS0 | At1g71830 | 24/25 |
RKS13 | At2g13790 | 26/27 |
RKS12 | At2g13800 | 28/29 |
RKS4 | At2g23950 | 11/12 |
RKS14 | At3g25560 | 30/31 |
RKS11 | At4g30520 | 1/2 |
RKS10 | At4g33430 | 32/33 |
常用名 | “染色体定位” | SEQ ID NO核酸/蛋白质 |
RKS6 | At5g10290 | 34/35 |
RKS7 | At5g16000 | 36/37 |
RKS5 | At5g45780 | 38/39 |
RKS3 | At5g63710 | 40/41 |
RKS2 | At5g65240 | 42/43 |
实施例2:基因克隆
使用拟南芥幼苗cDNA文库(Invitrogen,Paisley,UK)作为模板通过PCR扩增拟南芥RKS11(内部代码CDS3142,SEQ ID NO:1)。从幼苗提取的RNA经反转录后,将cDNA克隆进pCMV Sport6.0中。该库平均插入物大小为1.5kb,并且原始克隆数为1.59×107cfu。在6×1011cfu/ml的第一次扩增之后,确定原始滴度为9.6×105cfu/ml。提取质粒之后,将200ng模板用于50μl PCR混合物中。PCR扩增RKS11编码序列所用引物为Prm06771(SEQ ID NO 3,有义)和Prm06772(SEQ ID NO 4,反向互补),其包括Gateway重组的AttB位点。在标准条件下使用Hifi Taq DNA聚合酶进行PCR。同样用标准方法扩增和纯化2020bp的RKS11 PCR片段(带有attB位点)。接着进行Gateway操作的第一步,BP反应,在此期间将PCR片段与pDONR201质粒体内重组以产生(根据术语)“进入克隆”,p424。作为技术一部分的质粒pDONR201购自Invitrogen。
实施例3:载体构建和稻转化
接着,使用进入克隆p424和用于稻转化的指定载体p0831一起进行LR反应。此载体在T-DNA边界内包含以下部分作为功能性元件:植物可选择的标记;可视的标记表达盒;旨在与已克隆到进入克隆中的目的序列进行LR体内重组的Gateway表达盒。用于胚和糊粉特异性表达的稻启动子(SEQ ID NO:19)位于此Gateway盒的上游。
LR重组步骤之后,将产生的表达载体p074(图2)转化进入农杆菌菌株LBA4044,随后转化进入稻植物。使转化的稻类植物生长,随后检测实施例4中描述的参数。
实施例4:转化体评估:生长测量
大约产生了15到20个独立的T0转化体。初级转化体由组织培养室转移到温室生长并收获T1种子。5个事件得以保留,其中T1后代发生转基因存在/缺乏的3∶1分离。通过可视标记筛选,在每一事件中选出10个含有转基因(杂合子和纯合子)的T1幼苗,和10个缺乏转基因(无效合子)的T1幼苗。将所选择的T1植物转移到温室中。每个植物给予一个独特的条形标记,以将表型数据与对应植物明确联系起来。所选择的T1植物在10cm直径花盆的土壤中在下列环境情况下生长:光周期=11.5小时、日光强度=30,000勒克斯或以上、日间温度=28℃、夜间温度=22℃、相对湿度=60-70%。转基因植物和相应的无效合子在随机位置上并排生长。从播种期到成熟期,使植物几次通过数字成像箱。在每个时间点上对每株植物从至少6个不同的角度取得数字图像(2048×1536像素,1600万色)。
收获成熟的初级圆锥花序、装袋并贴上条形码标记,然后在37℃烤箱中干燥三天。随后将圆锥花序脱粒并且收集所有的种子。使用鼓风装置将饱满谷壳和空壳分开。在分离后,使用可商购的计数仪器对两批种子进行计数。弃去空壳。在分析天平上称重饱满的谷壳,并使用数字成像测量种子的截面积。此方法得到一组下面描述的种子相关参数。
这些参数是使用图像分析软件,以自动方式从数字图像中得到并且进行统计分析的。将对不平衡设计进行修正的双因素ANOVA(方差分析)用作统计模型,对植物表型特征进行全面评估。对用本发明基因转化的所有植株的所有事件的所有测量参数进行F测验。进行F检验以检查基因对所有转化事件的效应,并检验基因的总效应,亦称为“整体基因效应”。如果F检验的值显示数据具有显著性,那么结论是有“基因”效应,这意味着不仅仅是基因的存在或定位引起了效应。真实整体基因效应的显著性阈值设置为F检验的5%概率水平。
为了检查基因在事件中的效应,即品系特异性效应,在每一事件中使用来自转基因植物和相应无效植物的数据集进行t检验。“无效植物”或“无效分离子”或“无效合子”是以与转基因植物相同的方式处理、但是转基因已经从中分离的植物。也可以将无效植物描述为纯合的阴性转化植物。将t检验显著性的阈值设定为10%概率水平。一些事件的结果可以高于此阈值或低于此阈值。这是基于这种假设,即基因可以仅在基因组中的某些位置中具有效应,且这种位置依赖性效应的发生不是罕见的。本文此类基因效应也称为“基因的品系效应(line effect of the gene)”。通过将t值与t分布比较得到p值,或者通过将F值与F分布比较得到p值。p值给出了无效假设(即不存在转基因效应)正确的概率。
在第一个实验中得到的RKS11数据在使用T2植物的第二个实验中得到证实。选择了具有正确表达模式的4个品系用于进一步分析。通过监控标记表达来筛选T1中来自阳性植物(杂合子和纯合子)的一批种子。对于每一个所选事件,随后保存几批杂合种子用于进行T2评估。在每批种子中,在温室中种植等量的阳性和阴性植物用于评估。
在T2代中评估了总计120个RKS11转化植物,即每个事件的30个植物中,有15个为转基因阳性而15个为阴性。
由于进行的两个实验具有重叠事件,因此进行组合分析。这可用于检查两个实验中效应的一致性,并且如果情况果真如此,其可用于从两个实验中收集证据从而增加结论的可信性。使用的方法是考虑到数据的多级结构(即实验-事件-分离子)的混合模型方法。通过对比卡方分布的似然比测试来获得p值。
实施例5:RKS11转化体的评估:产率相关参数的测量
当如上所述对种子进行分析时,发明人发现,与缺乏RKS11转基因的植物相比,用RKS11基因构建体转化的植物具有更高的种子产率,表达为饱满种子数、种子总重量和收获指数。饱满种子数通过计数在分离步骤之后剩下的饱满谷壳数来测定。种子总重量通过称重从植物收获的所有饱满谷壳来测量。收获指数在本发明中定义为种子总产率与地上面积(以mm2计)之间的比值,乘以106的系数。
T1代植物所获得的结果总结于表5:
表5
%差异 | p值 | |
饱满种子数 | +29 | 0.0194 |
种子总重量 | +28 | 0.0248 |
收获指数 | +27 | 0.0181 |
T2代再次获得这些阳性结果。表6的数据显示了由T2代个体品系的数据计算得到的饱满种子数、种子总重量和收获指数的整体增长百分比,以及相应的p值。在与T1代结果的组合分析中,对这些T2数据进行了重新评估,并且获得的p值显示所观察到的效应具有显著性。
表6
与之相似,用含有处于PRO0058(SEQ ID NO:50)控制之下的CDS3142(SEQ ID NO:1)的载体转化稻,获得了增加的种子产率。
实施例6:转化植物的代谢分析
如实施例4所述在温室中培养用RKS11转化的植物(如实施例2所述)。根据本发明就多种代谢物而言的改进的组成根据如下方法进行测定。
a)样品匀浆化
将10至30颗稻粒移至塑料管(Eppendorf,Safe-Lock,2mL)中,在液氮冷却条件下,用不锈钢球在球磨仪(Retsch)中进行匀浆化。
b)冻干
在实验过程中,小心将样品保持在深度冷冻状态(低于-40℃),或者直到首次与溶剂接触之前,通过冻干匀浆材料使之不含水分。将样品转移稻预冷的(-40℃)冷冻干燥器中。主要干燥阶段的初始温度为-35℃,而压力为0.120mbar。在干燥过程中,按照压力和温度程序来改变参数。12小时后的最终温度为+30℃,而最终压力为0.001至0.004mbar。在关闭真空泵和冷却机器之后,向系统中冲入空气(经干燥管干燥)或氩。
c)萃取
在向冻干设备中充气后,即刻牢固密封装有冻干植物材料的管,以保护材料免受空气湿度的影响。为进行萃取,在玻璃纤维萃取套管中称重部分50mg干燥的匀浆化植物材料,并转移至ASE装置(快速溶剂萃取仪ASE200,配有溶剂控制器和AutoASE软件(DIONEX))的5ml萃取柱中。向ASE装置(快速溶剂萃取仪ASE 200,配有溶剂控制器和AutoASE软件(DIONEX))的24个样品点填满植物材料,包括一些质量控制试验的样品。
用大约10ml甲醇/水(80/20,v/v)于70℃在140bar的压力下,以5分钟的加热阶段,1分钟的静态萃取来萃取极性物质。用大约10ml甲醇/二氯甲烷(40/60,v/v)于70℃在140bar的压力下,以5分钟的加热阶段,1分钟的静态萃取来萃取更为亲脂性的物质。将两种溶剂混合物汇集到相同的玻璃管(离心管,50ml,配有螺帽和可穿透的隔膜,以用于ASE(DIONEX))中。向溶液中补加可商购的内参如核醣醇、L-甘氨酸-2,2-d2、L丙氨酸-2,3,3,3-d4、甲硫氨酸-d3、精氨酸(13C)、色氨酸-d5、α-甲基吡喃葡糖苷十九烷酸甲酯、十一烷酸甲酯、十三烷酸甲酯、十五烷酸甲酯和二十九烷酸甲酯。将总萃取物与8ml水混合。弃去植物样品固体残留物和萃取套管。振荡萃取物,然后以至少1400g离心5至10分钟,以加速相分离。取1ml上清甲醇/水相(“极性相”,无色)进行气相色谱(GC)分析,并取1ml进行液相色谱(LC)分析。弃去剩余的甲醇/水相。与此类似,取0.75ml有机相(“液相”,深绿色)进行进一步的GC分析,并取0.75ml进行LC分析。所有这些样品经IR Dancer红外真空干燥机(Hettich)蒸发干燥。蒸发过程中最高温度不超过40℃。设备中的压力为10mbar或以下。
d)处理液相和极性相以进行LC/MS或LC/MS/MS分析
将经蒸发干燥的液相萃取物和极性萃取物吸入流动相进行LC分析。
e)LC-MS分析
LC部分在可由Agilent Technologies,USA商购的LC/MS系统中进行。将10μl极性萃取物以200μl/min的流速注入系统。色谱期间分离柱(反相C18)维持在15℃。对于液相萃取物,将5μl以200μl/min的流速注入系统。分离柱(反相C18)维持在30℃。以梯度洗脱进行HPLC。质谱分析在配有turbo喷雾离子源的Applied Biosystems API 4000三级四极仪器中进行。对于极性萃取物,仪器以负离子模式通过全扫描模式在100-1000amu测量;而对于液相萃取物,仪器以正离子模式通过全扫描模式在100-1000amu测量。
f)衍生液相以进行GC/MS分析
向蒸发干燥的萃取物中添加140μl氯仿、37μl盐酸(以重量计37%的HCl水溶液)、320μl甲醇和20μl甲苯的混合物,以进行甲醇分解转移作用(transmethanolysis)。牢固密封容器,并于100℃加热2小时,同时振荡。随后对溶液进行蒸发直到残留物彻底干燥。通过与甲氧胺盐酸盐(5mg/ml吡啶溶液,100ml于60℃进行1.5小时)在牢固密封的容器中反应来进行羰基的甲氧化作用。加入20μl奇数碳的直链脂肪酸溶液(7至25碳原子的脂肪酸各0.3mg/mL而27、29和31碳原子的脂肪酸各0.6mg/mL的3/7(v/v)吡啶/甲苯溶液)作为时间标准。最后,还是在牢固密封的容器中于60℃用100μlN-甲基-N-(三甲基硅烷基)-2,2,2-三氟乙酰胺(MSTFA)衍生30分钟。注入GC之前的终体积为220μl。
g)衍生极性相以进行GC/MS分析
通过与甲氧胺盐酸盐(5mg/ml吡啶溶液,50ml于60℃进行1.5小时)在牢固密封的容器中反应来进行羰基的甲氧化作用。加入10μl奇数碳的直链脂肪酸溶液(7至25碳原子的脂肪酸各0.3mg/mL而27、29和31碳原子的脂肪酸各0.6mg/mL的3/7(v/v)吡啶/甲苯溶液)作为时间标准。最后,还是在牢固密封的容器中于60℃用50μl N-甲基-N-(三甲基硅烷基)-2,2,2-三氟乙酰胺(MSTFA)衍生30分钟。注入GC之前的终体积为110μl。
h)GC-MS分析
GC-MS系统包括与Agilent 5973 MSD偶联的Agilent 6890 GC。自动取样器为CompiPal或GCPal from CTC。为进行分析,根据待分析的样品材料和相分离步骤级分的不同,采用可商购的毛细管分离柱(30m×0.25mm×0.25μm),使用含0%至35%芳香族部分的具有不同聚甲基硅氧烷的固定相(例如:DB-1ms、HP-5ms、DB-XLB、DB-35ms、AgilentTechnolo-gies)。根据样品材料和相分离步骤级分的不同,采用不同的加热速率在70℃至340℃的烘箱温度梯度以不分流(splitless)模式注射多达1μL的终体积,以实现充分的色谱分离以及各分析物峰内的多次扫描。使用平常的GC-MS标准条件,例如恒速额定的1至1.7ml/min,并以氦作为流动相。通过在70eV电子撞击进行离子化,m/z扫描范围为15至600,扫描速率为2.5至3次扫描/秒,标准频率条件。
i)多种植物样品的分析
分别对独立的系列20个植物样品进行样品测量。实验中,每个系列含有至少3份重复的各转基因品系,外加至少3株相应的无效分离品系植物作为对照。各分析物的峰面积调整为确立的植物干重面积(标准化面积)。通过相对于对照进一步标准化来计算比值。实验中,通过用标准化面积除以同系列中对照组相应数据的均值来计算比值。所获得的数值称为对照比(ratio_by_control)。这些数值在系列中是可比较的,并表明转基因植物中的分析物浓度与对照组有多大差异,对照组为给定系列中相应无效分离品系的植物。合适对照是预先选好的,且证明载体和转化方法本身对于植物的代谢组成没有显著的影响。
不同植物分析的结果见下表7:
表7:RKS11转化体种子分析的结果,最小_比率和最大_比率相对于对照植物而言
代谢物 | 最小_比率 | 最大_比率 | 方法 |
色氨酸 | 1.61163522 | 1.855345912 | LC |
苯丙氨酸 | 1.558558559 | 2.207207207 | LC |
酪氨酸 | 1.342697685 | 1.574832386 | GC |
异亮氨酸 | 1.567605011 | 1.788026211 | GC |
缬氨酸 | 1.384151826 | 1.667510183 | GC |
第1栏显示了所分析的代谢物。第2和3栏显示了最小和最大比率,从中可以得出转基因植物与其相应的野生型无效分离对照品系相比,见于各独立实验的所分析代谢物的增长范围。第4栏显示了分析方法。
序列表
<110>克罗普迪塞恩股份有限公司
<120>具有改良生长特性的植物及其制备方法
<130>CD-132-PCT
<150>EP 05104980.7
<151>2005-06-08
<150>US 60/690,483
<151>2005-06-15
<160>50
<170>PatentIn version 3.3
<210>1
<211>2189
<212>DNA
<213>拟南芥(Arabidopsis thaliana)
<400>1
aaagtaatgc tgtctctctt ctcttcaaaa ttattgttaa cctctcgtaa ctaaaatctt 60
ccatggtagt agtaacaaag aagaccatga agattcaaat tcatctcctt tactcgttct 120
tgttcctctg tttctctact ctcactctat cttctgagcc cagaaaccct gaagttgagg 180
cgttgataag tataaggaac aatttgcatg atcctcatgg agctttgaac aattgggacg 240
agttttcagt tgatccttgt agctgggcta tgatcacttg ctctcccgac aacctcgtca 300
ttggactagg agcgccgagc cagtctctct cgggaggttt atctgagtct atcggaaatc 360
tcacaaatct ccgacaagtg tcattgcaaa ataacaacat ctccggcaaa attccaccgg 420
agctcggttt tctacccaaa ttacaaacct tggatctttc caacaaccga ttctccggtg 480
acatccctgt ttccatcgac cagctaagca gccttcaata tctgagactc aacaacaact 540
ctttgtctgg gcccttccct gcttctttgt cccaaattcc tcacctctcc ttcttggact 600
tgtcttacaa caatctcagt ggccctgttc ctaaattccc agcaaggact ttcaacgttg 660
ctggtaatcc tttgatttgt agaagcaacc cacctgagat ttgttctgga tcaatcaatg 720
caagtccact ttctgtttct ttgagctctt catcaggacg caggtctaat agattggcaa 780
tagctcttag tgtaagcctt ggctctgttg ttatactagt ccttgctctc gggtcctttt 840
gttggtaccg aaagaaacaa agaaggctac tgatccttaa cttaaacgat aaacaagagg 900
aagggcttca aggacttggg aatctaagaa gcttcacatt cagagaactc catgtttata 960
cagatggttt cagttccaag aacattctcg gcgctggtgg attcggtaat gtgtacagag 1020
gcaagcttgg agatgggaca atggtggcag tgaaacggtt gaaggatatt aatggaacct 1080
caggggattc acagtttcgt atggagctag agatgattag cttagctgtt cataagaatc 1140
tgcttcggtt aattggttat tgcgcaactt ctggtgaaag gcttcttgtt tacccttaca 1200
tgcctaatgg aagcgtcgcc tctaagctta aatctaaacc ggcattggac tggaacatga 1260
ggaagaggat agcaattggt gcagcgagag gtttgttgta tctacatgag caatgtgatc 1320
ccaagatcat tcatagagat gtaaaggcag ctaatattct cttagacgag tgctttgaag 1380
ctgttgttgg tgactttgga ctcgcaaagc tccttaacca tgcggattct catgtcacaa 1440
ctgcggtccg tggtacggtt ggccacattg cacctgaata tctctccact ggtcagtctt 1500
ctgagaaaac cgatgtgttt gggttcggta tactattgct cgagctcata accggactga 1560
gagctcttga gtttggtaaa accgttagcc agaaaggagc tatgcttgaa tgggtgagga 1620
aattacatga agagatgaaa gtagaggaac tattggatcg agaactcgga actaactacg 1680
ataagattga agttggagag atgttgcaag tggctttgct atgcacacaa tatctgccag 1740
ctcatcgtcc taaaatgtct gaagttgttt tgatgcttga aggcgatgga ttagccgaga 1800
gatgggctgc ttcgcataac cattcacatt tctaccatgc caatatctct ttcaagacaa 1860
tctcttctct gtctactact tctgtctcaa ggcttgacgc acattgcaat gatccaactt 1920
atcaaatgtt tggatcttcg gctttcgatg atgacgatga tcatcagcct ttagattcct 1980
ttgccatgga actatccggt ccaagataac acaatgaaag aaagatatca tttttacgat 2040
ggatcaaaca atccaatgaa aaaagctcta cacttttata atatagacat gtatatggtg 2100
gtgaaaattg atgaaaaata tctctacagt ttgagattat gtgttcgtta tgttgatgat 2160
gtatatatta acttttaatt gtgagtttc 2189
<210>2
<211>648
<212>PRT
<213>拟南芥
<400>2
Met Val Val Val Thr Lys Lys Thr Met Lys Ile Gln Ile His Leu Leu
1 5 10 15
Tyr Ser Phe Leu Phe Leu Cys Phe Ser Thr Leu Thr Leu Ser Ser Glu
20 25 30
Pro Arg Asn Pro Glu Val Glu Ala Leu Ile Ser Ile Arg Asn Asn Leu
35 40 45
His Asp Pro His Gly Ala Leu Asn Asn Trp Asp Glu Phe Ser Val Asp
50 55 60
Pro Cys Ser Trp Ala Met Ile Thr Cys Ser Pro Asp Asn Leu Val Ile
65 70 75 80
Gly Leu Gly Ala Pro Ser Gln Ser Leu Ser Gly Gly Leu Ser Glu Ser
85 90 95
Ile Gly Asn Leu Thr Asn Leu Arg Gln Val Ser Leu Gln Asn Asn Asn
100 105 110
Ile Ser Gly Lys Ile Pro Pro Glu Leu Gly Phe Leu Pro Lys Leu Gln
115 120 125
Thr Leu Asp Leu Ser Asn Asn Arg Phe Ser Gly Asp Ile Pro Val Ser
130 135 140
Ile Asp Gln Leu Ser Ser Leu Gln Tyr Leu Arg Leu Asn Asn Asn Ser
145 150 155 160
Leu Ser Gly Pro Phe Pro Ala Ser Leu Ser Gln Ile Pro His Leu Ser
165 170 175
Phe Leu Asp Leu Ser Tyr Asn Asn Leu Ser Gly Pro Val Pro Lys Phe
180 185 190
Pro Ala Arg Thr Phe Asn Val Ala Gly Asn Pro Leu Ile Cys Arg Ser
195 200 205
Asn Pro Pro Glu Ile Cys Ser Gly Ser Ile Asn Ala Ser Pro Leu Ser
210 215 220
Val Ser Leu Ser Ser Ser Ser Gly Arg Arg Ser Asn Arg Leu Ala Ile
225 230 235 240
Ala Leu Ser Val Ser Leu Gly Ser Val Val Ile Leu Val Leu Ala Leu
245 250 255
Gly Ser Phe Cys Trp Tyr Arg Lys Lys Gln Arg Arg Leu Leu Ile Leu
260 265 270
Asn Leu Asn Asp Lys Gln Glu Glu Gly Leu Gln Gly Leu Gly Asn Leu
275 280 285
Arg Ser Phe Thr Phe Arg Glu Leu His Val Tyr Thr Asp Gly Phe Ser
290 295 300
Ser Lys Asn Ile Leu Gly Ala Gly Gly Phe Gly Asn Val Tyr Arg Gly
305 310 315 320
Lys Leu Gly Asp Gly Thr Met Val Ala Val Lys Arg Leu Lys Asp Ile
325 330 335
Asn Gly Thr Ser Gly Asp Ser Gln Phe Arg Met Glu Leu Glu Met Ile
340 345 350
Ser Leu Ala Val His Lys Asn Leu Leu Arg Leu Ile Gly Tyr Cys Ala
355 360 365
Thr Ser Gly Glu Arg Leu Leu Val Tyr Pro Tyr Met Pro Asn Gly Ser
370 375 380
Val Ala Ser Lys Leu Lys Ser Lys Pro Ala Leu Asp Trp Asn Met Arg
385 390 395 400
Lys Arg Ile Ala Ile Gly Ala Ala Arg Gly Leu Leu Tyr Leu His Glu
405 410 415
Gln Cys Asp Pro Lys Ile Ile His Arg Asp Val Lys Ala Ala Asn Ile
420 425 430
Leu Leu Asp Glu Cys Phe Glu Ala Val Val Gly Asp Phe Gly Leu Ala
435 440 445
Lys Leu Leu Asn His Ala Asp Ser His Val Thr Thr Ala Val Arg Gly
450 455 460
Thr Val Gly His Ile Ala Pro Glu Tyr Leu Ser Thr Gly Gln Ser Ser
465 470 475 480
Glu Lys Thr Asp Val Phe Gly Phe Gly Ile Leu Leu Leu Glu Leu Ile
485 490 495
Thr Gly Leu Arg Ala Leu Glu Phe Gly Lys Thr Val Ser Gln Lys Gly
500 505 510
Ala Met Leu Glu Trp Val Arg Lys Leu His Glu Glu Met Lys Val Glu
515 520 525
Glu Leu Leu Asp Arg Glu Leu Gly Thr Asn Tyr Asp Lys Ile Glu Val
530 535 540
Gly Glu Met Leu Gln Val Ala Leu Leu Cys Thr Gln Tyr Leu Pro Ala
545 550 555 560
His Arg Pro Lys Met Ser Glu Val Val Leu Met Leu Glu Gly Asp Gly
565 570 575
Leu Ala Glu Arg Trp Ala Ala Ser His Asn His Ser His Phe Tyr His
580 585 590
Ala Asn Ile Ser Phe Lys Thr Ile Ser Ser Leu Ser Thr Thr Ser Val
595 600 605
Ser Arg Leu Asp Ala His Cys Asn Asp Pro Thr Tyr Gln Met Phe Gly
610 615 620
Ser Ser Ala Phe Asp Asp Asp Asp Asp His Gln Pro Leu Asp Ser Phe
625 630 635 640
Ala Met Glu Leu Ser Gly Pro Arg
645
<210>3
<211>59
<212>DNA
<213>人工序列
<220>
<223>引物:prm06771
<400>3
ggggacaagt ttgtacaaaa aagcaggctt aaacaatggt agtagtaaca aagaagacc 59
<210>4
<211>50
<212>DNA
<213>人工序列
<220>
<223>引物:prm06772
<400>4
ggggaccact ttgtacaaga aagctgggtt tcattgtgtt atcttggacc 50
<210>5
<21l>9
<212>PRT
<213>人工序列
<220>
<223>″RELHXXTDG″基序
<220>
<221>misc_feature
<222>(5)..(6)
<223>Xaa可以是任何天然存在的氨基酸
<400>5
Arg Glu Leu His Xaa Xaa Thr Asp Gly
1 5
<210>6
<211>6
<212>PRT
<213>人工序列
<220>
<223>″EGDGLA″基序
<400>6
Glu Gly Asp Gly Leu Ala
1 5
<210>7
<211>6
<212>PRT
<213>人工序列
<220>
<223>″ELSGPR″基序
<400>7
Glu Leu Ser Gly Pro Arg
1 5
<210>8
<211>10
<212>PRT
<213>人工序列
<220>
<223>FNV(A/V)GNP(L/M)IC基序
<220>
<221>MISC_FEATURE
<222>(4)..(4)
<223>Xaa可以是Ala或Val
<220>
<221>MISC_FEATURE
<222>(8)..(8)
<223>Xaa可以是Leu或Met
<400>8
Phe Asn Val Xaa Gly Asn Pro Xaa Ile Cys
1 5 10
<210>9
<211>987
<212>DNA
<213>拟南芥
<400>9
atggtagtag taacaaagaa gaccatgaag attcaaattc atctccttta ctcgttcttg 60
ttcctctgtt tctctactct cactctatct tctgagccca gaaaccctga agttgaggcg 120
ttgataagta taaggaacaa tttgcatgat cctcatggag ctttgaacaa ttgggacgag 180
ttttcagttg atccttgtag ctgggctatg atcacttgct ctcccgacaa cctcgtcatt 240
ggactaggag cgccgagcca gtctccctcg ggaggtttat ctgagtctat cggaaatctc 300
acaaatctcc gacaagtgtc attgcaaaat aacaacatct ccggcaaaat tccaccggag 360
ctcggttttc tacccaaatt acaaaccttg gatctttcca acaaccgatt ctccggtgac 420
atccctgttt ccatcgacca gctaagcagc cttcaatatc tgagactcaa caacaactct 480
ttgtctgggc ccttccctgc ttctttgtcc caaattcctc acctctcctt cttggacttg 540
tcttacaaca atctcagtgg ccctgttcct aaattcccag caaggacttt caacgttgct 600
ggtaatcctt tgatttgtag aagcaaccca cctgagattt gttctggatc aatcaatgca 660
agtccacttt ctgtttcttt gagctcttca tcaggacgca ggtctaatag attggcaata 720
gctcttagtg taagccttgg ctctgttgtt atactagtcc ttgctctcgg gtccttttgt 780
tggtaccgaa agaaacaaag aaggctactg atccttaact taaacgataa acaagaggaa 840
gggcttcaag gacttgggaa tctaagaagc ttcacattca gagaactcca tgtttataca 900
gatggtttca gttccaagaa cattctcggc gctggtggat tcggtaatgt gtacagaggc 960
aagctggaga tgggacaatg gtggcag 987
<210>10
<211>329
<212>PRT
<213>拟南芥
<400>10
Met Val Val Val Thr Lys Lys Thr Met Lys Ile Gln Ile His Leu Leu
1 5 10 15
Tyr Ser Phe Leu Phe Leu Cys Phe Ser Thr Leu Thr Leu Ser Ser Glu
20 25 30
Pro Arg Asn Pro Glu Val Glu Ala Leu Ile Ser Ile Arg Asn Asn Leu
35 40 45
His Asp Pro His Gly Ala Leu Asn Asn Trp Asp Glu Phe Ser Val Asp
50 55 60
Pro Cys Ser Trp Ala Met Ile Thr Cys Ser Pro Asp Asn Leu Val Ile
65 70 75 80
Gly Leu Gly Ala Pro Ser Gln Ser Pro Ser Gly Gly Leu Ser Glu Ser
85 90 95
Ile Gly Asn Leu Thr Asn Leu Arg Gln Val Ser Leu Gln Asn Asn Asn
100 105 110
Ile Ser Gly Lys Ile Pro Pro Glu Leu Gly Phe Leu Pro Lys Leu Gln
115 120 125
Thr Leu Asp Leu Ser Asn Asn Arg Phe Ser Gly Asp Ile Pro Val Ser
130 135 140
Ile Asp Gln Leu Ser Ser Leu Gln Tyr Leu Arg Leu Asn Asn Asn Ser
145 150 155 160
Leu Ser Gly Pro Phe Pro Ala Ser Leu Ser Gln Ile Pro His Leu Ser
165 170 175
Phe Leu Asp Leu Ser Tyr Asn Asn Leu Ser Gly Pro Val Pro Lys Phe
180 185 190
Pro Ala Arg Thr Phe Asn Val Ala Gly Asn Pro Leu Ile Cys Arg Ser
195 200 205
Asn Pro Pro Glu Ile Cys Ser Gly Ser Ile Asn Ala Ser Pro Leu Ser
210 215 220
Val Ser Leu Ser Ser Ser Ser Gly Arg Arg Ser Asn Arg Leu Ala Ile
225 230 235 240
Ala Leu Ser Val Ser Leu Gly Ser Val Val Ile Leu Val Leu Ala Leu
245 250 255
Gly Ser Phe Cys Trp Tyr Arg Lys Lys Gln Arg Arg Leu Leu Ile Leu
260 265 270
Asn Leu Asn Asp Lys Gln Glu Glu Gly Leu Gln Gly Leu Gly Asn Leu
275 280 285
Arg Ser Phe Thr Phe Arg Glu Leu His Val Tyr Thr Asp Gly Phe Ser
290 295 300
Ser Lys Asn Ile Leu Gly Ala Gly Gly Phe Gly Asn Val Tyr Arg Gly
305 310 315 320
Lys Leu Glu Met Gly Gln Trp Trp Gln
325
<210>11
<211>2334
<212>DNA
<213>拟南芥
<400>11
tgctttctct cctctctgtt ttttctagct ttctctctca caaaatcaaa actttttttc 60
tgtgttcatt atcatctcct taaattcata acaatacact ctcataattc ttctctgcgt 120
aggagagaca ttgcagaaat aaaagagttt ttaatagacc caagaaaaag agaaaaaaag 180
cattcatttt tttcttgctt tgttgctttg cttttgcttt tctgtttctc ttcttttgtg 240
ggcacaacaa aacatcaaca aagtaatgat tttgtgttct tccttctcct tctggtaatc 300
taatctaaag cttttcatgg tggtgatgaa gttaataaca atgaagatat tctctgttct 360
gttactacta tgtttcttcg ttacttgttc tctctcttct gaacccagaa accctgaagt 420
ggaggcgttg ataaacataa agaacgagtt acatgatcca catggtgttt tcaaaaactg 480
ggatgagttt tctgttgatc cttgtagctg gactatgatc tcttgttctt cagacaacct 540
cgtaattggc ttaggagctc caagtcagtc tctttcagga actttatctg ggtctattgg 600
aaatctcact aatcttcgac aagtgtcatt acagaacaat aacatctccg gtaaaatccc 660
accggagatt tgttctcttc ccaaattaca gactctggat ttatccaata accggttctc 720
cggtgaaatc cccggttctg ttaaccagct gagtaatctc caatatctga ggttgaacaa 780
caactcatta tctgggccct ttcctgcttc tctgtctcaa atccctcacc tctctttctt 840
agacttgtct tataacaatc tcagaggtcc tgttcctaaa tttcctgcaa ggacattcaa 900
tgttgctggg aaccctttga tttgtaaaaa cagcctaccg gagatttgtt caggatcaat 960
cagtgcaagc cctctttctg tctctttacg ttcttcatca ggacgtagaa ccaacatatt 1020
agcagttgca cttggtgtaa gccttggctt tgctgttagt gtaatcctct ctctcgggtt 1080
catttggtat cgaaagaaac aaagacggtt aacgatgctt cgcattagtg acaagcaaga 1140
ggaagggtta cttgggttgg gaaatctaag aagcttcaca ttcagggaac ttcatgtagc 1200
tacggatggt tttagttcca agagtattct tggtgctggt gggtttggta atgtctacag 1260
aggaaaattc ggggatggga cagtggttgc agtgaaacga ttgaaagatg tgaatggaac 1320
ctccgggaac tcacagtttc gtactgagct tgagatgatc agcttagctg ttcataggaa 1380
tttgcttcgg ttaatcggtt attgtgcgag ttctagcgaa agacttcttg tttaccctta 1440
catgtccaat ggcagcgtcg cctctaggct caaagctaag ccagcgttgg actggaacac 1500
aaggaagaag atagcgattg gagctgcaag agggttgttt tatctacacg agcaatgcga 1560
tcccaagatt attcaccgag atgtcaaggc agcaaacatt ctcctagatg agtattttga 1620
agcagttgtt ggggattttg gactagcaaa gctactcaac cacgaggatt cacatgtcac 1680
aaccgcggtt agaggaactg ttggtcacat tgcacctgag tatctctcca ccggtcagtc 1740
atctgagaaa accgatgtct ttgggttcgg tatacttttg ctagagctca tcacaggaat 1800
gagagctctc gagtttggca agtctgttag ccagaaagga gctatgctag aatgggtgag 1860
gaagctacac aaggaaatga aagtagagga gctagtagac cgagaactgg ggacaaccta 1920
cgatagaata gaagttggag agatgctaca agtggcactg ctctgcactc agtttcttcc 1980
agctcacaga cccaaaatgt ctgaagtagt tcagatgctt gaaggagatg gattagctga 2040
gagatgggct gcttcacatg accattcaca tttctaccat gccaacatgt cttacaggac 2100
tattacctct actgatggca acaaccaaac caaacatctg tttggctcct caggatttga 2160
agatgaagat gataatcaag cgttagattc attcgccatg gaactatctg gtccaaggta 2220
gtaaatcttg gacacagaaa gaaacagata taatatcccc atgacttcaa tttttgtttt 2280
tgagatgata tagacatgta aatggttatc aaacctttga aaaaattgag attc 2334
<210>12
<211>634
<212>PRT
<213>拟南芥
<400>12
Met Val Val Met Lys Leu Ile Thr Met Lys Ile Phe Ser Val Leu Leu
1 5 10 15
Leu Leu Cys Phe Phe Val Thr Cys Ser Leu Ser Ser Glu Pro Arg Asn
20 25 30
Pro Glu Val Glu Ala Leu Ile Asn Ile Lys Asn Glu Leu His Asp Pro
35 40 45
His Gly Val Phe Lys Asn Trp Asp Glu Phe Ser Val Asp Pro Cys Ser
50 55 60
Trp Thr Met Ile Ser Cys Ser Ser Asp Asn Leu Val Ile Gly Leu Gly
65 70 75 80
Ala Pro Ser Gln Ser Leu Ser Gly Thr Leu Ser Gly Ser Ile Gly Asn
85 90 95
Leu Thr Asn Leu Arg Gln Val Ser Leu Gln Asn Asn Asn Ile Ser Gly
100 105 110
Lys Ile Pro Pro Glu Ile Cys Ser Leu Pro Lys Leu Gln Thr Leu Asp
115 120 125
Leu Ser Asn Asn Arg Phe Ser Gly Glu Ile Pro Gly Ser Val Asn Gln
130 135 140
Leu Ser Asn Leu Gln Tyr Leu Arg Leu Asn Asn Asn Ser Leu Ser Gly
145 150 155 160
Pro Phe Pro Ala Ser Leu Ser Gln Ile Pro His Leu Ser Phe Leu Asp
165 170 175
Leu Ser Tyr Asn Asn Leu Arg Gly Pro Val Pro Lys Phe Pro Ala Arg
180 185 190
Thr Phe Asn Val Ala Gly Asn Pro Leu Ile Cys Lys Asn Ser Leu Pro
195 200 205
Glu Ile Cys Ser Gly Ser Ile Ser Ala Ser Pro Leu Ser Val Ser Leu
210 215 220
Arg Ser Ser Ser Gly Arg Arg Thr Asn Ile Leu Ala Val Ala Leu Gly
225 230 235 240
Val Ser Leu Gly Phe Ala Val Ser Val Ile Leu Ser Leu Gly Phe Ile
245 250 255
Trp Tyr Arg Lys Lys Gln Arg Arg Leu Thr Met Leu Arg Ile Ser Asp
260 265 270
Lys Gln Glu Glu Gly Leu Leu Gly Leu Gly Asn Leu Arg Ser Phe Thr
275 280 285
Phe Arg Glu Leu His Val Ala Thr Asp Gly Phe Ser Ser Lys Ser Ile
290 295 300
Leu Gly Ala Gly Gly Phe Gly Asn Val Tyr Arg Gly Lys Phe Gly Asp
305 310 315 320
Gly Thr Val Val Ala Val Lys Arg Leu Lys Asp Val Asn Gly Thr Ser
325 330 335
Gly Asn Ser Gln Phe Arg Thr Glu Leu Glu Met Ile Ser Leu Ala Val
340 345 350
His Arg Asn Leu Leu Arg Leu Ile Gly Tyr Cys Ala Ser Ser Ser Glu
355 360 365
Arg Leu Leu Val Tyr Pro Tyr Met Ser Asn Gly Ser Val Ala Ser Arg
370 375 380
Leu Lys Ala Lys Pro Ala Leu Asp Trp Asn Thr Arg Lys Lys Ile Ala
385 390 395 400
Ile Gly Ala Ala Arg Gly Leu Phe Tyr Leu His Glu Gln Cys Asp Pro
405 410 415
Lys Ile Ile His Arg Asp Val Lys Ala Ala Asn Ile Leu Leu Asp Glu
420 425 430
Tyr Phe Glu Ala Val Val Gly Asp Phe Gly Leu Ala Lys Leu Leu Asn
435 440 445
His Glu Asp Ser His Val Thr Thr Ala Val Arg Gly Thr Val Gly His
450 455 460
Ile Ala Pro Glu Tyr Leu Ser Thr Gly Gln Ser Ser Glu Lys Thr Asp
465 470 475 480
Val Phe Gly Phe Gly Ile Leu Leu Leu Glu Leu Ile Thr Gly Met Arg
485 490 495
Ala Leu Glu Phe Gly Lys Ser Val Ser Gln Lys Gly Ala Met Leu Glu
500 505 510
Trp Val Arg Lys Leu His Lys Glu Met Lys Val Glu Glu Leu Val Asp
515 520 525
Arg Glu Leu Gly Thr Thr Tyr Asp Arg Ile Glu Val Gly Glu Met Leu
530 535 540
Gln Val Ala Leu Leu Cys Thr Gln Phe Leu Pro Ala His Arg Pro Lys
545 550 555 560
Met Ser Glu Val Val Gln Met Leu Glu Gly Asp Gly Leu Ala Glu Arg
565 570 575
Trp Ala Ala Ser His Asp His Ser His Phe Tyr His Ala Asn Met Ser
580 585 590
Tyr Arg Thr Ile Thr Ser Thr Asp Gly Asn Asn Gln Thr Lys His Leu
595 600 605
Phe Gly Ser Ser Gly Phe Glu Asp Glu Asp Asp Asn Gln Ala Leu Asp
610 615 620
Ser Phe Ala Met Glu Leu Ser Gly Pro Arg
625 630
<210>13
<211>2292
<212>DNA
<213>拟南芥
<400>13
caaactcatc tcctctgaga gagactgttt gtttgagaag aagacgaacg taaagacttg 60
tttactaaac ccgagatttc tttctttctt gttttcaaaa taaaaaagca gagatctctt 120
ctccttcttc ttctttttgc tttgtttgct tttgctttct cttctttttc tatgtgtgtt 180
tgtggtcact accccgaaag gaaataaagt aatgctgtct ctcttctctt caaaattatt 240
gttaacctct cgtaactaag atgttccatg gtagtagtaa caaagaagac catgaagatt 300
caaattcatc tcctttactc gttcttgttc ctctgtttct ctactctcac tctatcttct 360
gagcccagaa accctgaagt tgaggcgttg ataagtataa ggaacaattt gcatgatcct 420
catggagctt tgaacaattg ggacgagttt tcagttgatc cttgtagctg ggctatgatc 480
acttgctctc ccgacaacct cgtcattgga ctaggagcgc caagccagtc tctctcggga 540
ggtttatctg agtctatcgg aaatctcaca aatctccgac aagtgtcatt gcaaaataac 600
aacatctccg gcaaaattcc accggagctc ggttttctac ccaaattaca aaccttggat 660
ctttccaaca accgattctc cggtgacatc cctgtttcca tcgaccagct aagcagcctt 720
caatatctga gactcaacaa caactctttg tctgggccct tccctgcttc tttgtcccaa 780
attcctcacc tctccttctt ggacttgtct tacaacaatc tcagtggccc tgttcctaaa 840
ttcccagcaa ggactttcaa cgttgctggt aatcctttga tttgtagaag caacccacct 900
gagatttgtt ctggatcaat caatgcaagt ccactttctg tttctttgag ctcttcatca 960
ggacgcaggt ctaatagatt ggcaatagct cttagtgtaa gccttggctc tgttgttata 1020
ctagtccttg ctctcgggtc cttttgttgg taccgaaaga aacaaagaag gctactgatc 1080
cttaacttaa acgataaaca agaggaaggg cttcaaggac ttgggaatct aagaagcttc 1140
acattcagag aactccatgt ttatacagat ggtttcagtt ccaagaacat tctcggcgct 1200
ggtggattcg gtaatgtgta cagaggcaag cttggagatg ggacaatggt ggcagtgaaa 1260
cggttgaagg atattaatgg aacctcaggg gattcacagt ttcgtatgga gctagagatg 1320
attagcttag ctgttcataa gaatctgctt cggttaattg gttattgcgc aacttctggt 1380
gaaaggcttc ttgtttaccc ttacatgcct aatggaagcg tcgcctctaa gcttaaatct 1440
aaaccgcatt ggactggaac atgaggaaga ggatagcaat tggtgcagcg agatgtttgt 1500
tgtatctaca tgagcaatgt gatcccaaga tcattcatag agatgtaaag gcagctaata 1560
ttctcttaga cgagtgcttt gaagctgttg ttggtgactt tgcactcgca aagctcctta 1620
accatgcgga ttctcatgta acaactgcgg tccgtggtac ggttggccac attgcacctg 1680
aatatctctc cactggtcag tcttctgaga aaaccgatgt gtttgggttc ggtatactat 1740
tgctcgagct cataaccgta ttttttgttc ttgagtttgg taaaaccgtt agccagaaag 1800
gagctatgct tgaatgggtg aggaaattac atgaagagat gaaagtagag gaactattgg 1860
atcgagaact cggaactaac tacgataaga ttgaagttgg agagatgttg caagtggctt 1920
tgctatgcac acaatatctg ccagctcatc gtcctaaaat gtctgaagtt gttttgatgc 1980
ttgaaggcga tggattagcc gagagatggg ctgtcgcata accattcaca tttctaccat 2040
gccaatatct ctttcaagac aatctcttct ctgtctacta cttctgtctc aaggctttac 2100
gcacattgca atgatccaac ttatcaaatg tttggatctt cggctttcga tgatgacgat 2160
gatcatcagc ctttagattc ctttgccatg gaactatccg gtccaagata acacaatgaa 2220
agaaagatat gatttttacg atggatcaaa caatccaatg aaaaaagctc tacacttcca 2280
taatatacac at 2292
<210>14
<211>398
<212>PRT
<213>拟南芥
<400>14
Met Val Val Val Thr Lys Lys Thr Met Lys Ile Gln Ile His Leu Leu
1 5 10 15
Tyr Ser Phe Leu Phe Leu Cys Phe Ser Thr Leu Thr Leu Ser Ser Glu
20 25 30
Pro Arg Asn Pro Glu Val Glu Ala Leu Ile Ser Ile Arg Asn Asn Leu
35 40 45
His Asp Pro His Gly Ala Leu Asn Asn Trp Asp Glu Phe Ser Val Asp
50 55 60
Pro Cys Ser Trp Ala Met Ile Thr Cys Ser Pro Asp Asn Leu Val Ile
65 70 75 80
Gly Leu Gly Ala Pro Ser Gln Ser Leu Ser Gly Gly Leu Ser Glu Ser
85 90 95
Ile Gly Asn Leu Thr Asn Leu Arg Gln Val Ser Leu Gln Asn Asn Asn
100 105 110
Ile Ser Gly Lys Ile Pro Pro Glu Leu Gly Phe Leu Pro Lys Leu Gln
115 120 125
Thr Leu Asp Leu Ser Asn Asn Arg Phe Ser Gly Asp Ile Pro Val Ser
130 135 140
Ile Asp Gln Leu Ser Ser Leu Gln Tyr Leu Arg Leu Asn Asn Asn Ser
145 150 155 160
Leu Ser Gly Pro Phe Pro Ala Ser Leu Ser Gln Ile Pro His Leu Ser
165 170 175
Phe Leu Asp Leu Ser Tyr Asn Asn Leu Ser Gly Pro Val Pro Lys Phe
180 185 190
Pro Ala Arg Thr Phe Asn Val Ala Gly Asn Pro Leu Ile Cys Arg Ser
195 200 205
Asn Pro Pro Glu Ile Cys Ser Gly Ser Ile Asn Ala Ser Pro Leu Ser
210 215 220
Val Ser Leu Ser Ser Ser Ser Gly Arg Arg Ser Asn Arg Leu Ala Ile
225 230 235 240
Ala Leu Ser Val Ser Leu Gly Ser Val Val Ile Leu Val Leu Ala Leu
245 250 255
Gly Ser Phe Cys Trp Tyr Arg Lys Lys Gln Arg Arg Leu Leu Ile Leu
260 265 270
Asn Leu Asn Asp Lys Gln Glu Glu Gly Leu Gln Gly Leu Gly Asn Leu
275 280 285
Arg Ser Phe Thr Phe Arg Glu Leu His Val Tyr Thr Asp Gly Phe Ser
290 295 300
Ser Lys Asn Ile Leu Gly Ala Gly Gly Phe Gly Asn Val Tyr Arg Gly
305 310 315 320
Lys Leu Gly Asp Gly Thr Met Val Ala Val Lys Arg Leu Lys Asp Ile
325 330 335
Asn Gly Thr Ser Gly Asp Ser Gln Phe Arg Met Glu Leu Glu Met Ile
340 345 350
Ser Leu Ala Val His Lys Asn Leu Leu Arg Leu Ile Gly Tyr Cys Ala
355 360 365
Thr Ser Gly Glu Arg Leu Leu Val Tyr Pro Tyr Met Pro Asn Gly Ser
370 375 380
Val Ala Ser Lys Leu Lys Ser Lys Pro His Trp Thr Gly Thr
385 390 395
<210>15
<211>418
<212>PRT
<213>稻(Oryza sativa)
<400>15
Met Arg Met Arg Trp Trp Ala Ala Pro Leu Ala Ala Val Leu Ala Val
1 5 10 15
Ile Leu Leu Pro Ser Ser Thr Ala Thr Leu Ser Pro Ala Gly Ile Asn
20 25 30
Tyr Glu Val Val Ala Leu Met Ala Ile Lys Thr Glu Leu Gln Asp Pro
35 40 45
Tyr Asn Val Leu Asp Asn Trp Asp Ile Asn Ser Val Asp Pro Cys Ser
50 55 60
Trp Arg Met Val Thr Cys Ser Ala Asp Gly Tyr Val Ser Ala Leu Gly
65 70 75 80
Leu Pro Ser Gln Ser Leu Ser Gly Lys Leu Ser Pro Gly Ile Gly Asn
85 90 95
Leu Thr Arg Leu Gln Ser Val Leu Leu Gln Asn Asn Ala Ile Ser Gly
100 105 110
Thr Ile Pro Ala Ser Ile Gly Arg Leu Gly Met Leu Gln Thr Leu Asp
115 120 125
Met Ser Asp Asn Gln Ile Thr Gly Ser Ile Pro Ser Ser Ile Gly Asp
130 135 140
Leu Lys Asn Leu Asn Tyr Leu Lys Leu Asn Asn Asn Ser Leu Ser Gly
145 150 155 160
Val Leu Pro Asp Ser Leu Ala Ala Ile Asn Gly Leu Ala Leu Val Asp
165 170 175
Leu Ser Phe Asn Asn Leu Ser Gly Pro Leu Pro Lys Ile Ser Ser Arg
180 185 190
Thr Phe Asn Ile Val Gly Asn Pro Met Ile Cys Gly Val Lys Ser Gly
195 200 205
Asp Asn Cys Ser Ser Val Ser Met Asp Pro Leu Ser Tyr Pro Pro Asp
210 215 220
Asp Leu Lys Thr Gln Pro Gln Gln Gly Ile Ala Arg Ser His Arg Ile
225 230 235 240
Ala Ile Ile Cys Gly Val Thr Val Gly Ser Val Ala Phe Ala Thr Ile
245 250 255
Ile Val Ser Met Leu Leu Trp Trp Arg His Arg Arg Asn Gln Gln Ile
260 265 270
Phe Phe Asp Val Asn Asp Gln Tyr Asp Pro Glu Val Cys Leu Gly His
275 280 285
Leu Lys Arg Tyr Ala Phe Lys Glu Leu Arg Ala Ala Thr Asn Asn Phe
290 295 300
Asn Ser Lys Asn Ile Leu Gly Glu Gly Gly Tyr GlyIle Val Tyr Lys
305 310 315 320
Gly Phe Leu Arg Asp Gly Ala Ile Val Ala Val Lys Arg Leu Lys Asp
325 330 335
Tyr Asn Ala Val Gly Gly Glu Val Gln Phe Gln Thr Glu Val Glu Val
340 345 350
Ile Ser Leu Ala Val His Arg Asn Leu Leu Arg Leu Ile Gly Phe Cys
355 360 365
Thr Thr Glu Asn Glu Arg Leu Leu Val Tyr Pro Tyr Met Pro Asn Gly
370 375 380
Ser Val Ala Ser Gln Leu Arg Glu Leu Val Asn Gly Lys Pro Ala Leu
385 390 395 400
Asp Trp Ser Arg Arg Arg Arg Met Phe Leu Gly Leu Glu Phe Cys Trp
405 410 415
Leu Ser
<210>16
<211>628
<212>PRT
<213>稻
<400>16
Met Arg Met Arg Trp Trp Ala Ala Pro Leu Ala Ala Val Leu Ala Val
1 5 10 15
Ile Leu Leu Pro Ser Ser Thr Ala Thr Leu Ser Pro Ala Gly Ile Asn
20 25 30
Tyr Glu Val Val Ala Leu Met Ala Ile Lys Thr Glu Leu Gln Asp Pro
35 40 45
Tyr Asn Val Leu Asp Asn Trp Asp Ile Asn Ser Val Asp Pro Cys Ser
50 55 60
Trp Arg Met Val Thr Cys Ser Ala Asp Gly Tyr Val Ser Ala Leu Gly
65 70 75 80
Leu Pro Ser Gln Ser Leu Ser Gly Lys Leu Ser Pro Gly Ile Gly Asn
85 90 95
Leu Thr Arg Leu Gln Ser Val Leu Leu Gln Asn Asn Ala Ile Ser Gly
100 105 110
Thr Ile Pro Ala Ser Ile Gly Arg Leu Gly Met Leu Gln Thr Leu Asp
115 120 125
Met Ser Asp Asn Gln Ile Thr Gly Ser Ile Pro Ser Ser Ile Gly Asp
130 135 140
Leu Lys Asn Leu Asn Tyr Leu Lys Leu Asn Asn Asn Ser Leu Ser Gly
145 150 155 160
Val Leu Pro Asp Ser Leu Ala Ala Ile Asn Gly Leu Ala Leu Val Asp
165 170 175
Leu Ser Phe Asn Asn Leu Ser Gly Pro Leu Pro Lys Ile Ser Ser Arg
180 185 190
Thr Phe Asn Ile Val Gly Asn Pro Met Ile Cys Gly Val Lys Ser Gly
195 200 205
Asp Asn Cys Ser Ser Val Ser Met Asp Pro Leu Ser Tyr Pro Pro Asp
210 215 220
Asp Leu Lys Thr Gln Pro Gln Gln Gly Ile Ala Arg Ser His Arg Ile
225 230 235 240
Ala Ile Ile Cys Gly Val Thr Val Gly Ser Val Ala Phe Ala Thr Ile
245 250 255
Ile Val Ser Met Leu Leu Trp Trp Arg His Arg Arg Asn Gln Gln Ile
260 265 270
Phe Phe Asp Val Asn Asp Gln Tyr Asp Pro Glu Val Cys Leu Gly His
275 280 285
Leu Lys Arg Tyr Ala Phe Lys Glu Leu Arg Ala Ala Thr Asn Asn Phe
290 295 300
Asn Ser Lys Asn Ile Leu Gly Glu Gly Gly Tyr Gly Ile Val Tyr Lys
305 310 315 320
Gly Phe Leu Arg Asp Gly Ala Ile Val Ala Val Lys Arg Leu Lys Asp
325 330 335
Tyr Asn Ala Val Gly Gly Glu Val Gln Phe Gln Thr Glu Val Glu Val
340 345 350
Ile Ser Leu Ala Val His Arg Asn Leu Leu Arg Leu Ile Gly Phe Cys
355 360 365
Thr Thr Glu Asn Glu Arg Leu Leu Val Tyr Pro Tyr Met Pro Asn Gly
370 375 380
Ser Val Ala Ser Gln Leu Arg Glu Leu Val Asn Gly Lys Pro Ala Leu
385 390 395 400
Asp Trp Ser Arg Arg Lys Arg Ile Ala Leu Gly Thr Ala Arg Gly Leu
405 410 415
Leu Tyr Leu His Glu Gln Cys Asp Pro Lys Ile Ile His Arg Asp Val
420 425 430
Lys Ala Ser Asn Val Leu Leu Asp Glu Tyr Phe Glu Ala Ile Val Gly
435 440 445
Asp Phe Gly Leu Ala Lys Leu Leu Asp His Arg Glu Ser His Val Thr
450 455 460
Thr Ala Val Arg Gly Thr Val Gly His Ile Ala Pro Glu Tyr Leu Ser
465 470 475 480
Thr Gly Gln Ser Ser Glu Lys Thr Asp Val Phe Gly Phe Gly Val Leu
485 490 495
Leu Val Glu Leu Ile Thr Gly Gln Lys Ala Leu Asp Phe Gly Arg Leu
500 505 510
Ala Asn Gln Lys Gly Gly Val Leu Asp Trp Val Lys Lys Leu His Gln
515 520 525
Glu Lys Gln Leu Ser Met Met Val Asp Lys Asp Leu Gly Ser Asn Tyr
530 535 540
Asp Arg Val Glu Leu Glu Glu Met Val Gln Val Ala Leu Leu Cys Thr
545 550 555 560
Gln Tyr Tyr Pro Ser His Arg Pro Arg Met Ser Glu Val Ile Arg Met
565 570 575
Leu Glu Gly Asp Gly Leu Ala Glu Lys Trp Glu Ala Ser Gln Asn Val
580 585 590
Asp Thr Pro Lys Ser Val Ser Ser Glu Leu Leu Pro Pro Lys Phe Met
595 600 605
Asp Phe Ala Ala Asp Glu Ser Ser Leu Gly Leu Glu Ala Met Glu Leu
610 615 620
Ser Gly Pro Arg
625
<210>17
<211>2037
<212>DNA
<213>稻
<400>17
atggcctcca acctcttcct cctcctcttc ttcctcgtcg tctcctacgc gccgttcctc 60
gccttctcct ccgagcccct caaccctgaa gtggaggcgc tgatcgccat caggcagggg 120
ctggtcgacc cgcacggcgt gctgaacaac tgggacgagg actccgtcga cccctgcagc 180
tgggccatgg tcacctgctc cgcccacaac ctcgtcatcg gcctgggagc gcccagccag 240
ggattgtcgg ggaccctgtc cggcaggatc gccaacctca ccaatcttga acaagtgctg 300
ctgcagaaca acaacatcac cggccggctg ccgccggagc tgggcgcgct gccgaggctg 360
cagacgctcg acctctccaa caaccgcttc tccggccgcg tccccgacac gctcggccgc 420
ctctccaccc tccgatacct gaggctaaac aacaacagct tgtccggggc gttcccgtcg 480
tcgctggcca agatcccaca gctctccttc ctggacttgt cctacaacaa cctcactggc 540
cctgttcctc acttccccac aagaacattc aacgtcgtgg gcaatccaat gatatgcggg 600
agcagcagcg gcagccatgc ggggaacgcg aacgcagcgg agtgcgccac cgtggtcgcc 660
ccggtcaccg tgccattccc gctggactcc actccgagca gcagcagcag ggcggcagcg 720
gcagcggtgg ggaggtcaaa gggtggagga ggcgccgcgc ggttgccgat cggagtaggg 780
acaagccttg gcgcctccgc gcttgtgctc ctcgccgtct cctgctttct ctggaggcgc 840
aggcgccggc accgctgcct cctctcgggc ccctcctccg tcctcggcat cctcgagaag 900
gggagagacg tggaggatgg gggaggaggg gaggtgatgg cgaggctggg gaacgtgagg 960
cagttcgggc tgagggagct gcacgcggcg acggacgggt tcagcgcgag gaacatactg 1020
gggaaaggag ggttcgggga cgtgtaccgg gggaggctct ccgacggcac ggtcgtggcg 1080
gtgaagcggc tcaaggaccc gaccgcgtcc ggggaggcgc agttccggac ggaggtggag 1140
atgatcagcc tcgccgtgca ccgccacctc ctccgcctcg tcggcttctg cgccgcggcc 1200
tccggcgagc gcctcctcgt ctacccctac atgcccaacg gcagcgtcgc ctcccgcctt 1260
cgagggaagc cgccgctgga ctggcagacg aggaagcgga tcgcggtggg gacggcgagg 1320
ggattgctgt acctgcacga gcagtgcgac ccaaagatca tccaccgcga cgtgaaggcc 1380
gcgaacgtgc tgctggacga gtgccacgag gccgtcgtcg gcgacttcgg gctcgccaag 1440
ctgctcgacc acggcgactc ccacgtcacc acggcggtgc gcggcacggt ggggcacatc 1500
gcgccggagt acctctccac ggggcagtcg tcggagaaga ccgacgtgtt cggcttcggc 1560
atcctgctgc tcgagctcgt caccggccag cgcgcgctcg aggtcggcaa gggctccggc 1620
gtcatccagc accagaaggg cgtcatgctc gattgggtga ggaaggtgca ccaagagaag 1680
ctgcatgact tgctagtgga ccaagatttg gggcctcact acgacaggat agaggtggcg 1740
gagatggtgc aggtggcgct gctctgcacc cagttccagc cgtctcaccg gccgaggatg 1800
tcggaggtgg tccggatgct ggagggagac gggctcgccg agaaatggga ggccaaccac 1860
cggccggcgg cgatggcggc ggcggcggcg ccccatgagc tcggctacga ccaccgcaac 1920
gactccaacg gctccgtctt cttcaacgac ttccacgaca acgacagcag ccttagcagc 1980
gacgaggtgc ggtccatcga catggtagag gagatggagc tgtcagggcc aaggtag 2037
<210>18
<211>678
<212>PRT
<213>稻
<400>18
Met Ala Ser Asn Leu Phe Leu Leu Leu Phe Phe Leu Val Val Ser Tyr
1 5 10 15
Ala Pro Phe Leu Ala Phe Ser Ser Glu Pro Leu Asn Pro Glu Val Glu
20 25 30
Ala Leu Ile Ala Ile Arg Gln Gly Leu Val Asp Pro His Gly Val Leu
35 40 45
Asn Asn Trp Asp Glu Asp Ser Val Asp Pro Cys Ser Trp Ala Met Val
50 55 60
Thr Cys Ser Ala His Asn Leu Val Ile Gly Leu Gly Ala Pro Ser Gln
65 70 75 80
Gly Leu Ser Gly Thr Leu Ser Gly Arg Ile Ala Asn Leu Thr Asn Leu
85 90 95
Glu Gln Val Leu Leu Gln Asn Asn Asn Ile Thr Gly Arg Leu Pro Pro
100 105 110
Glu Leu Gly Ala Leu Pro Arg Leu Gln Thr Leu Asp Leu Ser Asn Asn
115 120 125
Arg Phe Ser Gly Arg Val Pro Asp Thr Leu Gly Arg Leu Ser Thr Leu
130 135 140
Arg Tyr Leu Arg Leu Asn Asn Asn Ser Leu Ser Gly Ala Phe Pro Ser
145 150 155 160
Ser Leu Ala Lys Ile Pro Gln Leu Ser Phe Leu Asp Leu Ser Tyr Asn
165 170 175
Asn Leu Thr Gly Pro Val Pro His Phe Pro Thr Arg Thr Phe Asn Val
180 185 190
Val Gly Asn Pro Met Ile Cys Gly Ser Ser Ser Gly Ser His Ala Gly
195 200 205
Asn Ala Asn Ala Ala Glu Cys Ala Thr Val Val Ala Pro Val Thr Val
210 215 220
Pro Phe Pro Leu Asp Ser Thr Pro Ser Ser Ser Ser Arg Ala Ala Ala
225 230 235 240
Ala Ala Val Gly Arg Ser Lys Gly Gly Gly Gly Ala Ala Arg Leu Pro
245 250 255
Ile Gly Val Gly Thr Ser Leu Gly Ala Ser Ala Leu Val Leu Leu Ala
260 265 270
Val Ser Cys Phe Leu Trp Arg Arg Arg Arg Arg His Arg Cys Leu Leu
275 280 285
Ser Gly Pro Ser Ser Val Leu Gly Ile Leu Glu Lys Gly Arg Asp Val
290 295 300
Glu Asp Gly Gly Gly Gly Glu Val Met Ala Arg Leu Gly Asn Val Arg
305 310 315 320
Gln Phe Gly Leu Arg Glu Leu His Ala Ala Thr Asp Gly Phe Ser Ala
325 330 335
Arg Asn Ile Leu Gly Lys Gly Gly Phe Gly Asp Val Tyr Arg Gly Arg
340 345 350
Leu Ser Asp Gly Thr Val Val Ala Val Lys Arg Leu Lys Asp Pro Thr
355 360 365
Ala Ser Gly Glu Ala Gln Phe Arg Thr Glu Val Glu Met Ile Ser Leu
370 375 380
Ala Val His Arg His Leu Leu Arg Leu Val Gly Phe Cys Ala Ala Ala
385 390 395 400
Ser Gly Glu Arg Leu Leu Val Tyr Pro Tyr Met Pro Asn Gly Ser Val
405 410 415
Ala Ser Arg Leu Arg Gly Lys Pro Pro Leu Asp Trp Gln Thr Arg Lys
420 425 430
Arg Ile Ala Val Gly Thr Ala Arg Gly Leu Leu Tyr Leu His Glu Gln
435 440 445
Cys Asp Pro Lys Ile Ile His Arg Asp Val Lys Ala Ala Asn Val Leu
450 455 460
Leu Asp Glu Cys His Glu Ala Val Val Gly Asp Phe Gly Leu Ala Lys
465 470 475 480
Leu Leu Asp His Gly Asp Ser His Val Thr Thr Ala Val Arg Gly Thr
485 490 495
Val Gly His Ile Ala Pro Glu Tyr Leu Ser Thr Gly Gln Ser Ser Glu
500 505 510
Lys Thr Asp Val Phe Gly Phe Gly Ile Leu Leu Leu Glu Leu Val Thr
515 520 525
Gly Gln Arg Ala Leu Glu Val Gly Lys Gly Ser Gly Val Ile Gln His
530 535 540
Gln Lys Gly Val Met Leu Asp Trp Val Arg Lys Val His Gln Glu Lys
545 550 555 560
Leu His Asp Leu Leu Val Asp Gln Asp Leu Gly Pro His Tyr Asp Arg
565 570 575
Ile Glu Val Ala Glu Met Val Gln Val Ala Leu Leu Cys Thr Gln Phe
580 585 590
Gln Pro Ser His Arg Pro Arg Met Ser Glu Val Val Arg Met Leu Glu
595 600 605
Gly Asp Gly Leu Ala Glu Lys Trp Glu Ala Asn His Arg Pro Ala Ala
610 615 620
Met Ala Ala Ala Ala Ala Pro His Glu Leu Gly Tyr Asp His Arg Asn
625 630 635 640
Asp Ser Asn Gly Ser Val Phe Phe Asn Asp Phe His Asp Asn Asp Ser
645 650 655
Ser Leu Ser Ser Asp Glu Val Arg Ser Ile Asp Met Val Glu Glu Met
660 665 670
Glu Leu Ser Gly Pro Arg
675
<210>19
<211>1236
<212>DNA
<213>稻
<400>19
ggtcagccaa tacattgatc cgttgccaat catgcaaagt attttggctg tggccgagtg 60
ccggaattga taattgtgtt ctgactaaat taaatgacca gaagtcgcta tcttccaatg 120
tatccgaaac ctggattaaa caatcctgtt ctgttctcta gcccctcctg catggccgga 180
ttgttttttt gacatgtttt cttgactgag gcctgtttgt tctaaacttt ttcttcaaac 240
ttttaacttt ttcatcacat cagaactttt ctacacatat aaacttttaa cttttccgtc 300
acatcgttcc aatttcaatc aaactttcaa ttttggcgtg aactaaacac accctgagtc 360
ttttattgct cctccgtacg ggttggctgg ttgagaatag gtattttcag agagaaaatc 420
tagatattgg gaggaacttg gcatgaatgg ccactatatt tagagcaatt ctacggtcct 480
tgaggaggta ccatgaggta ccaaaatttt agtgtaaatt ttagtatctc attataacta 540
ggtattatga ggtaccaaat ttacaataga aaaaatagta cttcatggta ctttcttaag 600
taccgtaaaa ttgctcctat atttaagggg atgtttatat ctatccatat ccataatttg 660
attttgataa gaaaaaatgt gagcacacca agcatgtcca tgaccttgca ctcttggctc 720
actcgtcaac tgtgaagaac ctcaaaaatg ctcaatatag ctacaggtgc ctgaaaaaat 780
aactttaaag ttttgaacat cgatttcact aaacaacaat tattatctcc ctctgaaaga 840
tgatagttta gaactctaga atcattgtcg gcggagaaag taaattattt tccccaaatt 900
tccagctatg aaaaaaccct caccaaacac catcaaacaa gagttcacca aaccgcccat 960
gcggccatgc tgtcacgcaa cgcaccgcat tgcctgatgg ccgctcgatg catgcatgct 1020
tccccgtgca catatccgac agacgcgccg tgtcagcgag ctcctcgacc gacctgtgta 1080
gcccatgcaa gcatccaccc ccgccacgta caccccctcc tcctccctac gtgtcaccgc 1140
tctctccacc tatatatgcc cacctggccc ctctcctccc atctccactt cacccgatcg 1200
cttcttcttc ttcttcgttg cattcatctt gctagc 1236
<210>20
<211>3461
<212>DNA
<213>拟南芥
<400>20
acttttgtag tgactagtga gtagagtagg cttttagaga gagagagaga gagacggctg 60
ttgaaagata accacagaac acaaaaactc attcattaag aatgagaaag aaagtcccaa 120
aaaccttttt tgctctgaaa aagcaacgca aagttttgaa aaatctcaca cctttttcac 180
ttctctgttt gtagctgtta ccacttgtgt ttcccctttg gcatttttct cggttgtcat 240
taatgagagt aaaatcatca tcaagtgtaa acttctctct ctctttctct atctctatct 300
caaagctctc aactttggag agatcatggt ttgtgtttga tttctcaagt tttttttttt 360
ttaccctctt ggaggatctg ggaggagaaa tttgcttttt tttggtaaat ggggagaaaa 420
aagtttgaag cttttggttt tgtctgctta atctcactgc ttcttctgtt taattcgtta 480
tggcttgcct cttctaacat ggaaggtgat gcactgcaca gtttgagagc taatctagtt 540
gatccaaata atgtcttgca aagctgggat cctacgcttg ttaatccgtg tacttggttt 600
cacgtaacgt gtaacaacga gaacagtgtt ataagagtcg atcttgggaa tgcagacttg 660
tctggtcagt tggttcctca gctaggtcag ctcaagaact tgcagtactt ggagctttat 720
agtaataaca taaccgggcc ggttccaagc gatcttggga atctgacaaa cttagtgagc 780
ttggatcttt acttgaacag cttcactggt ccaattccag attctctagg aaagctattc 840
aagcttcgct ttcttcggct caacaataac agtctcaccg gaccaattcc catgtcattg 900
actaatatca tgacccttca agttttggat ctgtcgaaca accgattatc cggatctgtt 960
cctgataatg gttccttctc gctcttcact cccatcagtt ttgctaacaa cttggatcta 1020
tgcggcccag ttactagccg tccttgtcct ggatctcccc cgttttctcc tccaccacct 1080
tttataccac ctcccatagt tcctacacca ggtgggtata gtgctactgg agccattgcg 1140
ggaggagttg ctgctggtgc tgctttacta tttgctgccc ctgctttagc ttttgcttgg 1200
tggcgtagaa gaaaacctca agaattcttc tttgatgttc ctgccgaaga ggaccctgag 1260
gttcacttgg ggcagcttaa gcggttctct ctacgggaac ttcaagtagc aactgatagc 1320
ttcagcaaca agaacatttt gggccgaggt gggttcggaa aagtctacaa aggccgtctt 1380
gctgatggaa cacttgttgc agtcaaacgg cttaaagaag agcgaacccc aggtggcgag 1440
ctccagtttc agacagaagt ggagatgata agcatggccg ttcacagaaa tctcctcagg 1500
ctacgcggtt tctgtatgac ccctaccgag agattgcttg tttatcctta catggctaat 1560
ggaagtgtcg cttcctgttt gagagaacgt ccaccatcac agttgcctct agcctggtca 1620
ataagacagc aaatcgcgct aggatcagcg aggggtttgt cttatcttca tgatcattgc 1680
gaccccaaaa ttattcaccg tgatgtgaaa gctgctaata ttctgttgga cgaggaattt 1740
gaggcggtgg taggtgattt cgggttagct agacttatgg actataaaga tactcatgtc 1800
acaacggctg tgcgtgggac tattggacac attgctcctg agtatctctc aactggaaaa 1860
tcttcagaga aaactgatgt ttttggctac gggatcatgc ttttggaact gattacaggt 1920
cagagagctt ttgatcttgc aagactggcg aatgacgatg acgttatgct cctagattgg 1980
gtgaaagggc ttttgaagga gaagaagctg gagatgcttg tggatcctga cctgcaaagc 2040
aattacacag aagcagaagt agaacagctc atacaagtgg ctcttctctg cacacagagc 2100
tcacctatgg aacgacctaa gatgtctgag gttgttcgaa tgcttgaagg tgacggttta 2160
gcggagaaat gggacgagtg gcagaaagtg gaagttctca ggcaagaagt ggagctctct 2220
tctcacccca cctctgactg gatccttgat tcgactgata atcttcatgc tatggagttg 2280
tctggtccaa gataaacgac attgtaattt gcctaacaga aaagagaaag aacagagaaa 2340
tattaagaga atcacttctc tgtattcttt atttctttgg tagaaaaata atgtagtctc 2400
taatcaaatc ttattccatc tatcagcatt cttcattcat ttcttgtgaa aaccaaggcc 2460
tttaaattaa acataatcac aaacaccaag ttctatacat tacatatgtc ttccactgga 2520
taaagaggaa gaaaaggcta ttccaaaaaa cattttgagc tctttgttcc gcaagagaag 2580
gaacagcaca agtgaaacac actgaaaaac accaagcttg cattataaca tatgcaggta 2640
agaatcacga atcatgggcg ggtcttgtat tgtctgagaa cgaatgagag ccagatgagg 2700
gatcatgctt ctgcgatgtg aagagattag ggtatgagta atagggatca tcttccattg 2760
aaggcagtct catttgctga tggtcctggt cagggctgag tctgtcaagt ggtgaagtct 2820
ctggcctttg gagctctggc agatcgccat agctgctgac tgatgagttc tgatgatcaa 2880
actgcggttt ctccattgac gggaatctat gtggtgtaag atcgtcatag ttgctgcctg 2940
atgagttctg atgatcaaac tgcggtttct ccaatgacga gaatctctgt ggtgtaagat 3000
cgtcatagtt gctgcctgat gagttctgat gatcaaactg tggtttttcc attgaatgga 3060
atctctgtgg ggtaagatcg ccatagctac tgaatgatga gttctgacga tcaaactgcg 3120
gtttttccat tgaagagtgt gtctgtggct gttggttgtt ctgctcagag ttgaaaagtg 3180
acgaagtttc tgctctctgg agctctgtaa gatctccgct actgctggcc gaagaattct 3240
gatgatcaaa ttgaaggttt cccattgagg gagcatggaa agggttctca gacggacttt 3300
ctggatactg atcagaagtc ttcctggtaa gctcgttgat tctgagttgt gccaggcttg 3360
cagctgagcg ggcagctgat gctgcacgtt ctgctgagtc agcagcagct tgagcagcca 3420
ttaagacatc ctgcaagtct ccatcggata ttttccttgg a 3461
<210>21
<211>628
<212>PRT
<213>拟南芥
<400>21
Met Gly Arg Lys Lys Phe Glu Ala Phe Gly Phe Val Cys Leu Ile Ser
1 5 10 15
Leu Leu Leu Leu Phe Asn Ser Leu Trp Leu Ala Ser Ser Asn Met Glu
20 25 30
Gly Asp Ala Leu His Ser Leu Arg Ala Asn Leu Val Asp Pro Asn Asn
35 40 45
Val Leu Gln Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe
50 55 60
His Val Thr Cys Asn Asn Glu Asn Ser Val Ile Arg Val Asp Leu Gly
65 70 75 80
Asn Ala Asp Leu Ser Gly Gln Leu Val Pro Gln Leu Gly Gln Leu Lys
85 90 95
Asn Leu Gln Tyr Leu Glu Leu Tyr Ser Asn Asn Ile Thr Gly Pro Val
100 105 110
Pro Ser Asp Leu Gly Asn Leu Thr Asn Leu Val Ser Leu Asp Leu Tyr
115 120 125
Leu Asn Ser Phe Thr Gly Pro Ile Pro Asp Ser Leu Gly Lys Leu Phe
130 135 140
Lys Leu Arg Phe Leu Arg Leu Asn Asn Asn Ser Leu Thr Gly Pro Ile
145 150 155 160
Pro Met Ser Leu Thr Asn Ile Met Thr Leu Gln Val Leu Asp Leu Ser
165 170 175
Asn Asn Arg Leu Ser Gly Ser Val Pro Asp Asn Gly Ser Phe Ser Leu
180 185 190
Phe Thr Pro Ile Ser Phe Ala Asn Asn Leu Asp Leu Cys Gly Pro Val
195 200 205
Thr Ser Arg Pro Cys Pro Gly Ser Pro Pro Phe Ser Pro Pro Pro Pro
210 215 220
Phe Ile Pro Pro Pro Ile Val Pro Thr Pro Gly Gly Tyr Ser Ala Thr
225 230 235 240
Gly Ala Ile Ala Gly Gly Val Ala Ala Gly Ala Ala Leu Leu Phe Ala
245 250 255
Ala Pro Ala Leu Ala Phe Ala Trp Trp Arg Arg Arg Lys Pro Gln Glu
260 265 270
Phe Phe Phe Asp Val Pro Ala Glu Glu Asp Pro Glu Val His Leu Gly
275 280 285
Gln Leu Lys Arg Phe Ser Leu Arg Glu Leu Gln Val Ala Thr Asp Ser
290 295 300
Phe Ser Asn Lys Asn Ile Leu Gly Arg Gly Gly Phe Gly Lys Val Tyr
305 310 315 320
Lys Gly Arg Leu Ala Asp Gly Thr Leu Val Ala Val Lys Arg Leu Lys
325 330 335
Glu Glu Arg Thr Pro Gly Gly Glu Leu Gln Phe Gln Thr Glu Val Glu
340 345 350
Met Ile Ser Met Ala Val His Arg Asn Leu Leu Arg Leu Arg Gly Phe
355 360 365
Cys Met Thr Pro Thr Glu Arg Leu Leu Val Tyr Pro Tyr Met Ala Asn
370 375 380
Gly Ser Val Ala Ser Cys Leu Arg Glu Arg Pro Pro Ser Gln Leu Pro
385 390 395 400
Leu Ala Trp Ser Ile Arg Gln Gln Ile Ala Leu Gly Ser Ala Arg Gly
405 410 415
Leu Ser Tyr Leu His Asp His Cys Asp Pro Lys Ile Ile His Arg Asp
420 425 430
Val Lys Ala Ala Asn Ile Leu Leu Asp Glu Glu Phe Glu Ala Val Val
435 440 445
Gly Asp Phe Gly Leu Ala Arg Leu Met Asp Tyr Lys Asp Thr His Val
450 455 460
Thr Thr Ala Val Arg Gly Thr Ile Gly His Ile Ala Pro Glu Tyr Leu
465 470 475 480
Ser Thr Gly Lys Ser Ser Glu Lys Thr Asp Val Phe Gly Tyr Gly Ile
485 490 495
Met Leu Leu Glu Leu Ile Thr Gly Gln Arg Ala Phe Asp Leu Ala Arg
500 505 510
Leu Ala Asn Asp Asp Asp Val Met Leu Leu Asp Trp Val Lys Gly Leu
515 520 525
Leu Lys Glu Lys Lys Leu Glu Met Leu Val Asp Pro Asp Leu Gln Ser
530 535 540
Asn Tyr Thr Glu Ala Glu Val Glu Gln Leu Ile Gln Val Ala Leu Leu
545 550 555 560
Cys Thr Gln Ser Ser Pro Met Glu Arg Pro Lys Met Ser Glu Val Val
565 570 575
Arg Met Leu Glu Gly Asp Gly Leu Ala Glu Lys Trp Asp Glu Trp Gln
580 585 590
Lys Val Glu Val Leu Arg Gln Glu Val Glu Leu Ser Ser His Pro Thr
595 600 605
Ser Asp Trp Ile Leu Asp Ser Thr Asp Asn Leu His Ala Met Glu Leu
610 615 620
Ser Gly Pro Arg
625
<210>22
<211>2573
<212>DNA
<213>拟南芥
<400>22
accaaagtca taaaaatgat ataaaatata aaccaagaag cactattagt atcactcatc 60
actcaatagc tttttttttt tcagatctct aggaaaattt ctcagacttt cagagcattt 120
tggtgctttt gttttgcttg gtttctcggg aaaattctct tcacactttt agagttttct 180
ctctcttctc tatctcttgt cacagctcgt ttagcattca ttctcttaat cgtttcttca 240
ttcaaaaatt ctctctttct aagctgagta acttgcaagt cgtcattttt caattggggt 300
cttctccttt ttacttcctt tgaattgaaa tcacagagaa agagaggttc cattgctgga 360
aaatggattg ctcaaattag tttctgtgtc taaataaaga cagagaagaa gaaaagtaga 420
caaaagctaa gttttttttt cagatatggg agagagaaaa atgagttggt gggttctttg 480
aaacttgttt ttgttcagac caaagttgat tgctttaaga agggatatgg aaggtgtgag 540
atttgtggtg tggagattag gatttctggt ttttgtatgg ttctttgata tctcttctgc 600
tacactttct cctactggtg taaactatga agtgacagct ttggttgctg tgaagaatga 660
attgaatgat ccgtacaaag ttcttgagaa ttgggatgtg aattcagttg atccttgtag 720
ctggagaatg gtttcttgca ctgatggcta tgtctcttca ctggatcttc ctagccaaag 780
cttgtctggt acattgtctc ctagaatcgg aaacctcacc tatttacaat cagtggtgtt 840
gcaaaacaat gcaatcactg gtccaattcc ggaaacgatt gggaggttgg agaagcttca 900
gtcacttgat ctttcgaaca attcattcac cggggagata ccggcctcac ttggagaact 960
caagaacttg aattacttgc ggttaaacaa taacagtctt ataggaactt gccctgagtc 1020
tctatccaag attgagggac tcactctagt cgacatttcg tataacaatc ttagtggttc 1080
gctgccaaaa gtttctgcca gaactttcaa ggtaattggt aatgcgttaa tctgtggccc 1140
aaaagctgtt tcaaactgtt ctgctgttcc cgagcctctc acgcttccac aagatggtcc 1200
agatgaatca ggaactcgta ccaatggcca tcacgttgct cttgcatttg ccgcaagctt 1260
cagtgcagca ttttttgttt tctttacaag cggaatgttt ctttggtgga gatatcgccg 1320
taacaagcaa atattttttg acgttaatga acaatatgat ccagaagtga gtttagggca 1380
cttgaagagg tatacattca aagagcttag atctgccacc aatcatttca actcgaagaa 1440
cattctcgga agaggcggat acgggattgt gtacaaagga cacttaaacg atggaacttt 1500
ggtggctgtc aaacgtctca aggactgtaa cattgcgggt ggagaagtcc agtttcagac 1560
agaagtagag actataagtt tggctcttca tcgcaatctc ctccggctcc gcggtttctg 1620
tagtagcaac caggagagaa ttttagtcta cccttacatg ccaaatggga gtgtcgcatc 1680
acgcttaaaa gataatatcc gtggagagcc agcattagac tggtcgagaa ggaagaagat 1740
agcggttggg acagcgagag gactagttta cctacacgag caatgtgacc cgaagattat 1800
acaccgcgat gtgaaagcag ctaacattct gttagatgag gacttcgaag cagttgttgg 1860
tgattttggg ttagctaagc ttctagacca tagagactct catgtcacaa ctgcagtccg 1920
tggaactgtt ggccacattg cacctgagta cttatccacg ggtcagtcct cagagaagac 1980
tgatgtcttt ggctttggca tacttctcct tgagctcatt actggtcaga aagctcttga 2040
ttttggcaga tccgcacacc agaaaggtgt aatgcttgac tgggtgaaga agctgcacca 2100
agaagggaaa ctaaagcagt taatagacaa agatctaaat gacaagttcg atagagtaga 2160
actcgaagaa atcgttcaag ttgcgctact ctgcactcaa ttcaatccat ctcatcgacc 2220
gaaaatgtca gaagttatga agatgcttga aggtgacggt ttggctgaga gatgggaagc 2280
gacgcagaac ggtactggtg agcatcagcc accgccattg ccaccgggga tggtgagttc 2340
ttcgccgcgt gtgaggtatt actcggatta tattcaggaa tcgtctcttg tagtagaagc 2400
cattgagctc tcgggtcctc gatgattatg actcactgtt tttaaaaaat ttcttttctt 2460
gggtttgttt tttatttgtc gttttataat gttgatatag atgtgaagtt gagtgtgtaa 2520
tttttatgta aagaaaaaat atgaaatgca aaagaaaatg ttgattagcc tgc 2573
<210>23
<211>632
<212>PRT
<213>拟南芥
<400>23
Met Glu Gly Val Arg Phe Val Val Trp Arg Leu Gly Phe Leu Val Phe
1 5 10 15
Val Trp Phe Phe Asp Ile Ser Ser Ala Thr Leu Ser Pro Thr Gly Val
20 25 30
Asn Tyr Glu Val Thr Ala Leu Val Ala Val Lys Asn Glu Leu Asn Asp
35 40 45
Pro Tyr Lys Val Leu Glu Asn Trp Asp Val Asn Ser Val Asp Pro Cys
50 55 60
Ser Trp Arg Met Val Ser Cys Thr Asp Gly Tyr Val Ser Ser Leu Asp
65 70 75 80
Leu Pro Ser Gln Ser Leu Ser Gly Thr Leu Ser Pro Arg Ile Gly Asn
85 90 95
Leu Thr Tyr Leu Gln Ser Val Val Leu Gln Asn Asn Ala Ile Thr Gly
100 105 110
Pro Ile Pro Glu Thr Ile Gly Arg Leu Glu Lys Leu Gln Ser Leu Asp
115 120 125
Leu Ser Asn Asn Ser Phe Thr Gly Glu Ile Pro Ala Ser Leu Gly Glu
130 135 140
Leu Lys Asn Leu Asn Tyr Leu Arg Leu Asn Asn Asn Ser Leu Ile Gly
145 150 155 160
Thr Cys Pro Glu Ser Leu Ser Lys Ile Glu Gly Leu Thr Leu Val Asp
165 170 175
Ile Ser Tyr Asn Asn Leu Ser Gly Ser Leu Pro Lys Val Ser Ala Arg
180 185 190
Thr Phe Lys Val Ile Gly Asn Ala Leu Ile Cys Gly Pro Lys Ala Val
195 200 205
Ser Asn Cys Ser Ala Val Pro Glu Pro Leu Thr Leu Pro Gln Asp Gly
210 215 220
Pro Asp Glu Ser Gly Thr Arg Thr Asn Gly His His Val Ala Leu Ala
225 230 235 240
Phe Ala Ala Ser Phe Ser Ala Ala Phe Phe Val Phe Phe Thr Ser Gly
245 250 255
Met Phe Leu Trp Trp Arg Tyr Arg Arg Asn Lys Gln Ile Phe Phe Asp
260 265 270
Val Asn Glu Gln Tyr Asp Pro Glu Val Ser Leu Gly His Leu Lys Arg
275 280 285
Tyr Thr Phe Lys Glu Leu Arg Ser Ala Thr Asn His Phe Asn Ser Lys
290 295 300
Asn Ile Leu Gly Arg Gly Gly Tyr Gly Ile Val Tyr Lys Gly His Leu
305 310 315 320
Asn Asp Gly Thr Leu Val Ala Val Lys Arg Leu Lys Asp Cys Asn Ile
325 330 335
Ala Gly Gly Glu Val Gln Phe Gln Thr Glu Val Glu Thr Ile Ser Leu
340 345 350
Ala Leu His Arg Asn Leu Leu Arg Leu Arg Gly Phe Cys Ser Ser Asn
355 360 365
Gln Glu Arg Ile Leu Val Tyr Pro Tyr Met Pro Asn Gly Ser Val Ala
370 375 380
Ser Arg Leu Lys Asp Asn Ile Arg Gly Glu Pro Ala Leu Asp Trp Ser
385 390 395 400
Arg Arg Lys Lys Ile Ala Val Gly Thr Ala Arg Gly Leu Val Tyr Leu
405 410 415
His Glu Gln Cys Asp Pro Lys Ile Ile His Arg Asp Val Lys Ala Ala
420 425 430
Asn Ile Leu Leu Asp Glu Asp Phe Glu Ala Val Val Gly Asp Phe Gly
435 440 445
Leu Ala Lys Leu Leu Asp His Arg Asp Ser His Val Thr Thr Ala Val
450 455 460
Arg Gly Thr Val Gly His Ile Ala Pro Glu Tyr Leu Ser Thr Gly Gln
465 470 475 480
Ser Ser Glu Lys Thr Asp Val Phe Gly Phe Gly Ile Leu Leu Leu Glu
485 490 495
Leu Ile Thr Gly Gln Lys Ala Leu Asp Phe Gly Arg Ser Ala His Gln
500 505 510
Lys Gly Val Met Leu Asp Trp Val Lys Lys Leu His Gln Glu Gly Lys
515 520 525
Leu Lys Gln Leu Ile Asp Lys Asp Leu Asn Asp Lys Phe Asp Arg Val
530 535 540
Glu Leu Glu Glu Ile Val Gln Val Ala Leu Leu Cys Thr Gln Phe Asn
545 550 555 560
Pro Ser His Arg Pro Lys Met Ser Glu Val Met Lys Met Leu Glu Gly
565 570 575
Asp Gly Leu Ala Glu Arg Trp Glu Ala Thr Gln Asn Gly Thr Gly Glu
580 585 590
His Gln Pro Pro Pro Leu Pro Pro Gly Met Val Ser Ser Ser Pro Arg
595 600 605
Val Arg Tyr Tyr Ser Asp Tyr Ile Gln Glu Ser Ser Leu Val Val Glu
610 615 620
Ala Ile Glu Leu Ser Gly Pro Arg
625 630
<210>24
<211>2565
<212>DNA
<213>拟南芥
<400>24
aacaacacac taatcatagt ttctctggca ggcttgttgt tgcggcttaa taaaaagctc 60
ttttgttatt attacttcac gtagattttc cccaaaaagc tcttattttt ttgtttaaaa 120
aaaaaagttt catctttatt caacttttgt tttacagtgt gtgtgtgaga gagagagtgt 180
ggtttgattg aggaaagacg acgacgagaa cgccggagaa ttaggatttt gattttattt 240
tttactcttt gtttgtttta atgctaatgg gtttttaaaa gggttatcga aaaaatgagt 300
gagtttgtgt tgaggttgtc tctgtaaagt gttaatggtg gtgattttcg gaagttaggg 360
ttttctcgga tctgaagaga tcaaatcaag attcgaaatt tagcattgtt gtttgaaatg 420
gagtcgagtt atgtggtgtt tatcttactt tcactgatct tacttccgaa tcattcactg 480
tggcttgctt ctgctaattt ggaaggtgat gctttgcata ctttgagggt tactctagtt 540
gatccaaaca atgtcttgca gagctgggat cctacgctag tgaatccttg cacatggttc 600
catgtcactt gcaacaacga gaacagtgtc ataagagttg atttggggaa tgcagagtta 660
tctggccatt tagttccaga gcttggtgtg ctcaagaatt tgcagtattt ggagctttac 720
agtaacaaca taactggccc gattcctagt aatcttggaa atctgacaaa cttagtgagt 780
ttggatcttt acttaaacag cttctccggt cctattccgg aatcattggg aaagctttca 840
aagctgagat ttctccggct taacaacaac agtctcactg ggtcaattcc tatgtcactg 900
accaatatta ctacccttca agtgttagat ctatcaaata acagactctc tggttcagtt 960
cctgacaatg gctccttctc actcttcaca cccatcagtt ttgctaataa cttagaccta 1020
tgtggacctg ttacaagtca cccatgtcct ggatctcccc cgttttctcc tccaccacct 1080
tttattcaac ctcccccagt ttccaccccg agtgggtatg gtataactgg agcaatagct 1140
ggtggagttg ctgcaggtgc tgctttgctc tttgctgctc ctgcaatagc ctttgcttgg 1200
tggcgacgaa gaaagccact agatattttc ttcgatgtcc ctgccgaaga agatccagaa 1260
gttcatctgg gacagctcaa gaggttttct ttgcgggagc tacaagtggc gagtgatggg 1320
tttagtaaca agaacatttt gggcagaggt gggtttggga aagtctacaa gggacgcttg 1380
gcagacggaa ctcttgttgc tgtcaagaga ctgaaggaag agcgaactcc aggtggagag 1440
ctccagtttc aaacagaagt agagatgata agtatggcag ttcatcgaaa cctgttgaga 1500
ttacgaggtt tctgtatgac accgaccgag agattgcttg tgtatcctta catggccaat 1560
ggaagtgttg cttcgtgtct cagagagagg ccaccgtcac aacctccgct tgattggcca 1620
acgcggaaga gaatcgcgct aggctcagct cgaggtttgt cttacctaca tgatcactgc 1680
gatccgaaga tcattcaccg tgacgtaaaa gcagcaaaca tcctcttaga cgaagaattc 1740
gaagcggttg ttggagattt cgggttggca aagctaatgg actataaaga cactcacgtg 1800
acaacagcag tccgtggcac catcggtcac atcgctccag aatatctctc aaccggaaaa 1860
tcttcagaga aaaccgacgt tttcggatac ggaatcatgc ttctagaact aatcacagga 1920
caaagagctt tcgatctcgc tcggctagct aacgacgacg acgtcatgtt acttgactgg 1980
gtgaaaggat tgttgaagga gaagaagcta gagatgttag tggatccaga tcttcaaaca 2040
aactacgagg agagagaact ggaacaagtg atacaagtgg cgttgctatg cacgcaagga 2100
tcaccaatgg aaagaccaaa gatgtctgaa gttgtaagga tgctggaagg agatgggctt 2160
gcggagaaat gggacgaatg gcaaaaagtt gagattttga gggaagagat tgatttgagt 2220
cctaatccta actctgattg gattcttgat tctacttaca atttgcacgc cgttgagtta 2280
tctggtccaa ggtaaaaaaa aaaaacataa aattattgaa caataacaaa ttttacaagg 2340
taggtagttt ttttacccgt aagttttcgt tttttttaat tgttaatgta aaatgaaatc 2400
tagcattcaa agatttgtga ttttgtgcta tggttcgatt aaaagggaaa aaaattgtaa 2460
tctaaagatt tgtgtaagat tactgtctat tgtatgaagt atgaactatg aacacaatat 2520
atgtacatcc aaaaatacgt taaactaact ccgctgtttt gctac 2565
<210>25
<211>625
<212>PRT
<213>拟南芥
<400>25
Met Glu Ser Ser Tyr Val Val Phe Ile Leu Leu Ser Leu Ile Leu Leu
1 5 10 15
Pro Asn His Ser Leu Trp Leu Ala Ser Ala Asn Leu Glu Gly Asp Ala
20 25 30
Leu His Thr Leu Arg Val Thr Leu Val Asp Pro Asn Asn Val Leu Gln
35 40 45
Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val Thr
50 55 60
Cys Asn Asn Glu Asn Ser Val Ile Arg Val Asp Leu Gly Asn Ala Glu
65 70 75 80
Leu Ser Gly His Leu Val Pro Glu Leu Gly Val Leu Lys Asn Leu Gln
85 90 95
Tyr Leu Glu Leu Tyr Ser Asn Asn Ile Thr Gly Pro Ile Pro Ser Asn
100 105 110
Leu Gly Asn Leu Thr Asn Leu Val Ser Leu Asp Leu Tyr Leu Asn Ser
115 120 125
Phe Ser Gly Pro Ile Pro Glu Ser Leu Gly Lys Leu Ser Lys Leu Arg
130 135 140
Phe Leu Arg Leu Asn Asn Asn Ser Leu Thr Gly Ser Ile Pro Met Ser
145 150 155 160
Leu Thr Asn Ile Thr Thr Leu Gln Val Leu Asp Leu Ser Asn Asn Arg
165 170 175
Leu Ser Gly Ser Val Pro Asp Asn Gly Ser Phe Ser Leu Phe Thr Pro
180 185 190
Ile Ser Phe Ala Asn Asn Leu Asp Leu Cys Gly Pro Val Thr Ser His
195 200 205
Pro Cys Pro Gly Ser Pro Pro Phe Ser Pro Pro Pro Pro Phe Ile Gln
210 215 220
Pro Pro Pro Val Ser Thr Pro Ser Gly Tyr Gly Ile Thr Gly Ala Ile
225 230 235 240
Ala Gly Gly Val Ala Ala Gly Ala Ala Leu Leu Phe Ala Ala Pro Ala
245 250 255
Ile Ala Phe Ala Trp Trp Arg Arg Arg Lys Pro Leu Asp Ile Phe Phe
260 265 270
Asp Val Pro Ala Glu Glu Asp Pro Glu Val His Leu Gly Gln Leu Lys
275 280 285
Arg Phe Ser Leu Arg Glu Leu Gln Val Ala Ser Asp Gly Phe Ser Asn
290 295 300
Lys Asn Ile Leu Gly Arg Gly Gly Phe Gly Lys Val Tyr Lys Gly Arg
305 310 315 320
Leu Ala Asp Gly Thr Leu Val Ala Val Lys Arg Leu Lys Glu Glu Arg
325 330 335
Thr Pro Gly Gly Glu Leu Gln Phe Gln Thr Glu Val Glu Met Ile Ser
340 345 350
Met Ala Val His Arg Asn Leu Leu Arg Leu Arg Gly Phe Cys Met Thr
355 360 365
Pro Thr Glu Arg Leu Leu Val Tyr Pro Tyr Met Ala Asn Gly Ser Val
370 375 380
Ala Ser Cys Leu Arg Glu Arg Pro Pro Ser Gln Pro Pro Leu Asp Trp
385 390 395 400
Pro Thr Arg Lys Arg Ile Ala Leu Gly Ser Ala Arg Gly Leu Ser Tyr
405 410 415
Leu His Asp His Cys Asp Pro Lys Ile Ile His Arg Asp Val Lys Ala
420 425 430
Ala Asn Ile Leu Leu Asp Glu Glu Phe Glu Ala Val Val Gly Asp Phe
435 440 445
Gly Leu Ala Lys Leu Met Asp Tyr Lys Asp Thr His Val Thr Thr Ala
450 455 460
Val Arg Gly Thr Ile Gly His Ile Ala Pro Glu Tyr Leu Ser Thr Gly
465 470 475 480
Lys Ser Ser Glu Lys Thr Asp Val Phe Gly Tyr Gly Ile Met Leu Leu
485 490 495
Glu Leu Ile Thr Gly Gln Arg Ala Phe Asp Leu Ala Arg Leu Ala Asn
500 505 510
Asp Asp Asp Val Met Leu Leu Asp Trp Val Lys Gly Leu Leu Lys Glu
515 520 525
Lys Lys Leu Glu Met Leu Val Asp Pro Asp Leu Gln Thr Asn Tyr Glu
530 535 540
Glu Arg Glu Leu Glu Gln Val Ile Gln Val Ala Leu Leu Cys Thr Gln
545 550 555 560
Gly Ser Pro Met Glu Arg Pro Lys Met Ser Glu Val Val Arg Met Leu
565 570 575
Glu Gly Asp Gly Leu Ala Glu Lys Trp Asp Glu Trp Gln Lys Val Glu
580 585 590
Ile Leu Arg Glu Glu Ile Asp Leu Ser Pro Asn Pro Asn Ser Asp Trp
595 600 605
Ile Leu Asp Ser Thr Tyr Asn Leu His Ala Val Glu Leu Ser Gly Pro
610 615 620
Arg
625
<210>26
<211>2219
<212>DNA
<213>拟南芥
<400>26
ttcgaaactt ggtcaaatgt cgaatacgcg ttacaaagaa caaacctttc tctttatttc 60
gtttgtcttc gtcaacggct gaatcaacca aatggtccct ggaattaata aacctctaat 120
aataatggct ttgcttttac tctgatgaca agttcaaaaa tggaacaaag atcactcctt 180
tgcttccttt atctgctcct actattcaat ttcactctca gagtcgctgg aaacgctgaa 240
ggtgatgctt tgactcagct gaaaaacagt ttgtcatcag gtgaccctgc aaacaatgta 300
ctccaaagct gggatgctac tcttgttact ccatgtactt ggtttcatgt tacttgcaat 360
cctgagaata aagttactcg tgttgacctt gggaatgcaa aactatctgg aaagttggtt 420
ccagaacttg gtcagctttt aaacttgcag tacttggagc tttatagcaa taacattaca 480
ggggagatac ctgaggagct tggcgacttg gtggaactag taagcttgga tctttacgca 540
aacagcataa gcggtcccat cccttcgtct cttggcaaac taggaaaact ccggttcttg 600
cgtcttaaca acaatagctt atcaggggaa attccaatga ctttgacttc tgtgcagctg 660
caagttctgg atatctcaaa caatcggctc agtggagata ttcctgttaa tggttctttt 720
tcgctcttca ctcctatcag ttttgcgaat aatagcttaa cggatcttcc cgaacctccg 780
cctacttcta cctctcctac gccaccacca ccttcagggg ggcaaatgac tgcagcaata 840
gcagggggag ttgctgcagg tgcagcactt ctatttgctg ttccagccat tgcgtttgct 900
tggtggctca gaagaaaacc acaggaccac ttttttgatg tacctgctga agaagaccca 960
gaggttcatt taggacaact caaaaggttt accttgcgtg aactgttagt tgctactgat 1020
aactttagca ataaaaatgt attgggtaga ggtggttttg gtaaagtgta taaaggacgt 1080
ttagccgatg gcaatctagt ggctgtcaaa aggctaaaag aagaacgtac caagggtggg 1140
gaactgcagt ttcaaaccga agttgagatg atcagtatgg ccgttcatag gaacttgctt 1200
cggcttcgtg gcttttgcat gactccaact gaaagattac ttgtttatcc ctacatggct 1260
aatggaagtg ttgcttcttg tttaagagag cgtcctgaag gcaatccagc acttgattgg 1320
ccaaaaagaa agcatattgc tctgggatca gcaagggggc ttgcgtattt acatgatcat 1380
tgcgaccaaa aaatcattca ccgggatgtt aaagctgcta atatattgtt agatgaagag 1440
tttgaagctg ttgttggaga ttttgggctc gcaaaattaa tgaattataa tgactcccat 1500
gtgacaactg ctgtacgcgg tacaattggc catatagcgc ccgagtacct ctcgacagga 1560
aaatcttctg agaagactga tgtttttggg tacggggtca tgcttctcga gctcatcact 1620
ggacaaaagg ctttcgatct tgctcggctt gcaaatgatg atgatatcat gttactcgac 1680
tgggtgaaag aggttttgaa agagaagaag ttggaaagcc ttgtggatgc agaactcgaa 1740
ggaaagtacg tggaaacaga agtggagcag ctgatacaaa tggctctgct ctgcactcaa 1800
agttctgcaa tggaacgtcc aaagatgtca gaagtagtga gaatgctgga aggagatggt 1860
ttagctgaga gatgggaaga atggcaaaag gaggagatgc caatacatga ttttaactat 1920
caagcctatc ctcatgctgg cactgactgg ctcatcccct attccaattc ccttatcgaa 1980
aacgattacc cctcgggtcc aagataacct tttagaaagg gtcttttctt gtgggttctt 2040
caacaagtat atatatagat tggtgaagtt ttaagatgca aaaaaaaccc atgcactttt 2100
gaatatcaac tcctctataa gtagttttgt gtctcttgac gaataaagaa tatcattact 2160
ccacttgagc ataaagcaag atgtttacca accaataaag cttaacaata tttttccgt 2219
<210>27
<211>620
<212>PRT
<213>拟南芥
<400>27
Met Thr Ser Ser Lys Met Glu Gln Arg Ser Leu Leu Cys Phe Leu Tyr
1 5 10 15
Leu Leu Leu Leu Phe Asn Phe Thr Leu Arg Val Ala Gly Asn Ala Glu
20 25 30
Gly Asp Ala Leu Thr Gln Leu Lys Asn Ser Leu Ser Ser Gly Asp Pro
35 40 45
Ala Asn Asn Val Leu Gln Ser Trp Asp Ala Thr Leu Val Thr Pro Cys
50 55 60
Thr Trp Phe His Val Thr Cys Asn Pro Glu Asn Lys Val Thr Arg Val
65 70 75 80
Asp Leu Gly Asn Ala Lys Leu Ser Gly Lys Leu Val Pro Glu Leu Gly
85 90 95
Gln Leu Leu Asn Leu Gln Tyr Leu Glu Leu Tyr Ser Asn Asn Ile Thr
100 105 110
Gly Glu Ile Pro Glu Glu Leu Gly Asp Leu Val Glu Leu Val Ser Leu
115 120 125
Asp Leu Tyr Ala Asn Ser Ile Ser Gly Pro Ile Pro Ser Ser Leu Gly
130 135 140
Lys Leu Gly Lys Leu Arg Phe Leu Arg Leu Asn Asn Asn Ser Leu Ser
145 150 155 160
Gly Glu Ile Pro Met Thr Leu Thr Ser Val Gln Leu Gln Val Leu Asp
165 170 175
Ile Ser Asn Asn Arg Leu Ser Gly Asp Ile Pro Val Asn Gly Ser Phe
180 185 190
Ser Leu Phe Thr Pro Ile Ser Phe Ala Asn Asn Ser Leu Thr Asp Leu
195 200 205
Pro Glu Pro Pro Pro Thr Ser Thr Ser Pro Thr Pro Pro Pro Pro Ser
210 215 220
Gly Gly Gln Met Thr Ala Ala Ile Ala Gly Gly Val Ala Ala Gly Ala
225 230 235 240
Ala Leu Leu Phe Ala Val Pro Ala Ile Ala Phe Ala Trp Trp Leu Arg
245 250 255
Arg Lys Pro Gln Asp His Phe Phe Asp Val Pro Ala Glu Glu Asp Pro
260 265 270
Glu Val His Leu Gly Gln Leu Lys Arg Phe Thr Leu Arg Glu Leu Leu
275 280 285
Val Ala Thr Asp Asn Phe Ser Asn Lys Asn Val Leu Gly Arg Gly Gly
290 295 300
Phe Gly Lys Val Tyr Lys Gly Arg Leu Ala Asp Gly Asn Leu Val Ala
305 310 315 320
Val Lys Arg Leu Lys Glu Glu Arg Thr Lys Gly Gly Glu Leu Gln Phe
325 330 335
Gln Thr Glu Val Glu Met Ile Ser Met Ala Val His Arg Asn Leu Leu
340 345 350
Arg Leu Arg Gly Phe Cys Met Thr Pro Thr Glu Arg Leu Leu Val Tyr
355 360 365
Pro Tyr Met Ala Asn Gly Ser Val Ala Ser Cys Leu Arg Glu Arg Pro
370 375 380
Glu Gly Asn Pro Ala Leu Asp Trp Pro Lys Arg Lys His Ile Ala Leu
385 390 395 400
Gly Ser Ala Arg Gly Leu Ala Tyr Leu His Asp His Cys Asp Gln Lys
405 410 415
Ile Ile His Arg Asp Val Lys Ala Ala Asn Ile Leu Leu Asp Glu Glu
420 425 430
Phe Glu Ala Val Val Gly Asp Phe Gly Leu Ala Lys Leu Met Asn Tyr
435 440 445
Asn Asp Ser His Val Thr Thr Ala Val Arg Gly Thr Ile Gly His Ile
450 455 460
Ala Pro Glu Tyr Leu Ser Thr Gly Lys Ser Ser Glu Lys Thr Asp Val
465 470 475 480
Phe Gly Tyr Gly Val Met Leu Leu Glu Leu Ile Thr Gly Gln Lys Ala
485 490 495
Phe Asp Leu Ala Arg Leu Ala Asn Asp Asp Asp Ile Met Leu Leu Asp
500 505 510
Trp Val Lys Glu Val Leu Lys Glu Lys Lys Leu Glu Ser Leu Val Asp
515 520 525
Ala Glu Leu Glu Gly Lys Tyr Val Glu Thr Glu Val Glu Gln Leu Ile
530 535 540
Gln Met Ala Leu Leu Cys Thr Gln Ser Ser Ala Met Glu Arg Pro Lys
545 550 555 560
Met Ser Glu Val Val Arg Met Leu Glu Gly Asp Gly Leu Ala Glu Arg
565 570 575
Trp Glu Glu Trp Gln Lys Glu Glu Met Pro Ile His Asp Phe Asn Tyr
580 585 590
Gln Ala Tyr Pro His Ala Gly Thr Asp Trp Leu Ile Pro Tyr Ser Asn
595 600 605
Ser Leu Ile Glu Asn Asp Tyr Pro Ser Gly Pro Arg
610 615 620
<210>28
<211>1985
<212>DNA
<213>拟南芥
<400>28
ggaaaatgga acatggatca tcccgtggct ttatttggct gattctattt ctcgattttg 60
tttccagagt caccggaaaa acacaagttg atgctctcat tgctctaaga agcagtttat 120
catcaggtga ccatacaaac aatatactcc aaagctggaa tgccactcac gttactccat 180
gttcatggtt tcatgttact tgcaatactg aaaacagtgt tactcgtctt gacctgggga 240
gtgctaatct atctggagaa ctggtgccac agcttgctca gcttccaaat ttgcagtact 300
tggaactttt taacaataat attactgggg agatacctga ggagcttggc gacttgatgg 360
aactagtaag cttggacctt tttgcaaaca acataagcgg tcccatccct tcctctcttg 420
gcaaactagg aaaactccgc ttcttgcgtc tttataacaa cagcttatct ggagaaattc 480
caaggtcttt gactgctctg ccgctggatg ttcttgatat ctcaaacaat cggctcagtg 540
gagatattcc tgttaatggt tccttttcgc agttcacttc tatgagtttt gccaataata 600
aattaaggcc gcgacctgca tctccttcac catcaccttc aggaacgtct gcagcaatag 660
tagtgggagt tgctgcgggt gcagcacttc tatttgcgct tgcttggtgg ctgagaagaa 720
aactgcaggg tcactttctt gatgtacctg ctgaagaaga cccagaggtt tatttaggac 780
aatttaaaag gttctccttg cgtgaactgc tagttgctac agagaaattt agcaaaagaa 840
atgtattggg caaaggacgt tttggtatat tgtataaagg acgtttagct gatgacactc 900
tagtggctgt gaaacggcta aatgaagaac gtaccaaggg tggggaactg cagtttcaaa 960
ccgaagttga gatgatcagt atggccgttc ataggaactt gcttcggctt cgtggctttt 1020
gcatgactcc aactgaaaga ttacttgttt atccctacat ggctaatgga agtgttgctt 1080
cttgtttaag agagcgtcct gaaggcaatc cagcccttga ctggccaaaa agaaagcata 1140
ttgctctggg atcagcaagg gggctcgcat atttacacga tcattgcgac caaaagatca 1200
ttcacctgga tgtgaaagct gcaaatatac tgttagatga agagtttgaa gctgttgttg 1260
gagattttgg gctagcaaaa ttaatgaatt ataacgactc ccatgtgaca actgctgtac 1320
ggggtacgat tggccatata gcgcccgagt acctctcgac aggaaaatct tctgagaaga 1380
ctgatgtttt tgggtacggg gtcatgcttc tcgagctcat cactggacaa aaggctttcg 1440
atcttgctcg gcttgcaaat gatgatgata tcatgttact cgactgggtg aaagaggttt 1500
tgaaagagaa gaagttggaa agccttgtgg atgcagaact cgaaggaaag tacgtggaaa 1560
cagaagtgga gcagctgata caaatggctc tgctctgcac tcaaagttct gcaatggaac 1620
gtccaaagat gtcagaagta gtgagaatgc tggaaggaga tggtttagct gagagatggg 1680
aagaatggca aaaggaggag atgccaatac atgattttaa ctatcaagcc tatcctcatg 1740
ctggcactga ctggctcatc ccctattcca attcccttat cgaaaacgat tacccctcgg 1800
ggccaagata accttttaga aagggtcatt tcttgtgggt tcttcaacaa gtatatatat 1860
aggtagtgaa gttgtaagaa gcaaaacccc acattcacct ttgaatatca ctactctata 1920
atactaatca tatctactat actttctctc cacttccatt aagcaataaa aactattctt 1980
aaatc 1985
<210>29
<211>601
<212>PRT
<213>拟南芥
<400>29
Met Glu His Gly Ser Ser Arg Gly Phe Ile Trp Leu Ile Leu Phe Leu
1 5 10 15
Asp Phe Val Ser Arg Val Thr Gly Lys Thr Gln Val Asp Ala Leu Ile
20 25 30
Ala Leu Arg Ser Ser Leu Ser Ser Gly Asp His Thr Asn Asn Ile Leu
35 40 45
Gln Ser Trp Asn Ala Thr His Val Thr Pro Cys Ser Trp Phe His Val
50 55 60
Thr Cys Asn Thr Glu Asn Ser Val Thr Arg Leu Asp Leu Gly Ser Ala
65 70 75 80
Asn Leu Ser Gly Glu Leu Val Pro Gln Leu Ala Gln Leu Pro Asn Leu
85 90 95
Gln Tyr Leu Glu Leu Phe Asn Asn Asn Ile Thr Gly Glu Ile Pro Glu
100 105 110
Glu Leu Gly Asp Leu Met Glu Leu Val Ser Leu Asp Leu Phe Ala Asn
115 120 125
Asn Ile Ser Gly Pro Ile Pro Ser Ser Leu Gly Lys Leu Gly Lys Leu
130 135 140
Arg Phe Leu Arg Leu Tyr Asn Asn Ser Leu Ser Gly Glu Ile Pro Arg
145 150 155 160
Ser Leu Thr Ala Leu Pro Leu Asp Val Leu Asp Ile Ser Asn Asn Arg
165 170 175
Leu Ser Gly Asp Ile Pro Val Asn Gly Ser Phe Ser Gln Phe Thr Ser
180 185 190
Met Ser Phe Ala Asn Asn Lys Leu Arg Pro Arg Pro Ala Ser Pro Ser
195 200 205
Pro Ser Pro Ser Gly Thr Ser Ala Ala Ile Val Val Gly Val Ala Ala
210 215 220
Gly Ala Ala Leu Leu Phe Ala Leu Ala Trp Trp Leu Arg Arg Lys Leu
225 230 235 240
Gln Gly His Phe Leu Asp Val Pro Ala Glu Glu Asp Pro Glu Val Tyr
245 250 255
Leu Gly Gln Phe Lys Arg Phe Ser Leu Arg Glu Leu Leu Val Ala Thr
260 265 270
Glu Lys Phe Ser Lys Arg Asn Val Leu Gly Lys Gly Arg Phe Gly Ile
275 280 285
Leu Tyr Lys Gly Arg Leu Ala Asp Asp Thr Leu Val Ala Val Lys Arg
290 295 300
Leu Asn Glu Glu Arg Thr Lys Gly Gly Glu Leu Gln Phe Gln Thr Glu
305 310 315 320
Val Glu Met Ile Ser Met Ala Val His Arg Asn Leu Leu Arg Leu Arg
325 330 335
Gly Phe Cys Met Thr Pro Thr Glu Arg Leu Leu Val Tyr Pro Tyr Met
340 345 350
Ala Asn Gly Ser Val Ala Ser Cys Leu Arg Glu Arg Pro Glu Gly Asn
355 360 365
Pro Ala Leu Asp Trp Pro Lys Arg Lys His Ile Ala Leu Gly Ser Ala
370 375 380
Arg Gly Leu Ala Tyr Leu His Asp His Cys Asp Gln Lys Ile Ile His
385 390 395 400
Leu Asp Val Lys Ala Ala Asn Ile Leu Leu Asp Glu Glu Phe Glu Ala
405 410 415
Val Val Gly Asp Phe Gly Leu Ala Lys Leu Met Asn Tyr Asn Asp Ser
420 425 430
His Val Thr Thr Ala Val Arg Gly Thr Ile Gly His Ile Ala Pro Glu
435 440 445
Tyr Leu Ser Thr Gly Lys Ser Ser Glu Lys Thr Asp Val Phe Gly Tyr
450 455 460
Gly Val Met Leu Leu Glu Leu Ile Thr Gly Gln Lys Ala Phe Asp Leu
465 470 475 480
Ala Arg Leu Ala Asn Asp Asp Asp Ile Met Leu Leu Asp Trp Val Lys
485 490 495
Glu Val Leu Lys Glu Lys Lys Leu Glu Ser Leu Val Asp Ala Glu Leu
500 505 510
Glu Gly Lys Tyr Val Glu Thr Glu Val Glu Gln Leu Ile Gln Met Ala
515 520 525
Leu Leu Cys Thr Gln Ser Ser Ala Met Glu Arg Pro Lys Met Ser Glu
530 535 540
Val Val Arg Met Leu Glu Gly Asp Gly Leu Ala Glu Arg Trp Glu Glu
545 550 555 560
Trp Gln Lys Glu Glu Met Pro Ile His Asp Phe Asn Tyr Gln Ala Tyr
565 570 575
Pro His Ala Gly Thr Asp Trp Leu Ile Pro Tyr Ser Asn Ser Leu Ile
580 585 590
Glu Asn Asp Tyr Pro Ser Gly Pro Arg
595 600
<210>30
<211>2207
<212>DNA
<213>拟南芥
<400>30
ttttcttaaa aacctccaaa caaagaatcg aaaaaaagaa tatttcttat acaaaagaaa 60
taaacctcaa actctgcacc ttagagatta atactctcaa gaaaaacaag ttttgattcg 120
gacaaagatg ttgcaaggaa gaagagaagc aaaaaagagt tatgctttgt tctcttcaac 180
tttcttcttc ttctttatct gttttctttc ttcttcttct gcagaactca cagacaaagg 240
tgttaacttt gaagttgttg ccttaatagg aatcaaaagc tcactgactg atcctcatgg 300
agttctaatg aattgggatg acacagcagt tgatccatgt agctggaaca tgatcacttg 360
ttctgatggt tttgtcataa ggctagaagc tccaagccaa aacttatcag gaactctttc 420
atcaagtatt ggaaatttaa caaatcttca aactgtgtta ttgcagaaca attacataac 480
aggaaacatc cctcatgaga ttgggaaatt gatgaaactc aaaacacttg atctctctac 540
caataacttc actggtcaaa tcccattcac tctttcttac tccaaaaatc ttcagtactt 600
caggagggtt aataataaca gcctgacagg aacaattcct agctcattgg caaacatgac 660
ccaactcact tttttggatt tgtcgtataa taacttgagt ggaccagttc caagatcact 720
tgccaaaaca ttcaatgtta tgggcaattc tcagatttgt ccaacaggaa ctgagaaaga 780
ctgtaatggg actcagccta agccaatgtc aatcaccttg aacagttctc aaaataaatc 840
atctgatgga ggaactaaaa accggaaaat cgcggtagtc ttcggtgtaa gcttgacatg 900
tgtttgcttg ttgatcattg gctttggttt tcttctttgg tggagaagaa gacataacaa 960
acaagtatta ttctttgaca ttaatgagca aaacaaggaa gaaatgtgtc tagggaatct 1020
aaggaggttt aatttcaaag aacttcaatc cgcaactagt aacttcagca gcaagaatct 1080
ggtcggaaaa ggagggtttg gaaatgtgta taaaggttgt cttcatgatg gaagtatcat l140
cgcggtgaag agattaaagg atataaacaa tggtggtgga gaggttcagt ttcagacaga 1200
gcttgaaatg ataagccttg ccgtccaccg gaatctcctc cgcttatacg gtttctgtac 1260
tacttcctct gaacggcttc tcgtttatcc ttacatgtcc aatggcagtg tcgcttctcg 1320
tctcaaagct aaaccggtat tggattgggg cacaagaaag cgaatagcat taggagcagg 1380
aagagggttg ctgtatttgc atgagcaatg tgatccaaag atcattcacc gtgatgtcaa 1440
agctgcgaac atacttcttg acgattactt tgaagctgtt gtcggagatt tcgggttggc 1500
taagcttttg gatcatgagg agtcgcatgt gacaaccgcc gtgagaggaa cagtgggtca 1560
cattgcacct gagtatctct caacaggaca atcttctgag aagacagatg tgttcggttt 1620
cgggattctt cttctcgaat tgattactgg attgagagct cttgaattcg gaaaagcagc 1680
aaaccaaaga ggagcgatac ttgattgggt aaagaaacta caacaagaga agaagctaga 1740
acagatagta gacaaggatt tgaagagcaa ctacgataga atagaagtgg aagaaatggt 1800
tcaagtggct ttgctttgta cacagtatct tcccattcac cgtcctaaga tgtctgaagt 1860
tgtgagaatg cttgaaggcg atggtcttgt tgagaaatgg gaagcttctt ctcagagagc 1920
agaaaccaat agaagttaca gtaaacctaa cgagttttct tcctctgaac gttattcgga 1980
tcttacagat gattcctcgg tgctggttca agccatggag ttatcaggtc caagatgaca 2040
agagaaacta tatgaatggc tttgggtttg taaaaaacat atataagatt gtgtattttg 2100
ttgtatgctg tgatcttgta caggttttgg tatcagaaag acatattctc atgctttatc 2160
ccatgattag gaggaggtgg aatcaccgcc tccatttcgt agaaacg 2207
<210>31
<211>636
<212>PRT
<213>拟南芥
<400>31
Met Leu Gln Gly Arg Arg Glu Ala Lys Lys Ser Tyr Ala Leu Phe Ser
1 5 10 15
Ser Thr Phe Phe Phe Phe Phe Ile Cys Phe Leu Ser Ser Ser Ser Ala
20 25 30
Glu Leu Thr Asp Lys Gly Val Asn Phe Glu Val Val Ala Leu Ile Gly
35 40 45
Ile Lys Ser Ser Leu Thr Asp Pro His Gly Val Leu Met Asn Trp Asp
50 55 60
Asp Thr Ala Val Asp Pro Cys Ser Trp Asn Met Ile Thr Cys Ser Asp
65 70 75 80
Gly Phe Val Ile Arg Leu Glu Ala Pro Ser Gln Asn Leu Ser Gly Thr
85 90 95
Leu Ser Ser Ser Ile Gly Asn Leu Thr Asn Leu Gln Thr Val Leu Leu
100 105 110
Gln Asn Asn Tyr Ile Thr Gly Asn Ile Pro His Glu Ile Gly Lys Leu
115 120 125
Met Lys Leu Lys Thr Leu Asp Leu Ser Thr Asn Asn Phe Thr Gly Gln
130 135 140
Ile Pro Phe Thr Leu Ser Tyr Ser Lys Asn Leu Gln Tyr Phe Arg Arg
145 150 155 160
Val Asn Asn Asn Ser Leu Thr Gly Thr Ile Pro Ser Ser Leu Ala Asn
165 170 175
Met Thr Gln Leu Thr Phe Leu Asp Leu Ser Tyr Asn Asn Leu Ser Gly
180 185 190
Pro Val Pro Arg Ser Leu Ala Lys Thr Phe Asn Val Met Gly Asn Ser
195 200 205
Gln Ile Cys Pro Thr Gly Thr Glu Lys Asp Cys Asn Gly Thr Gln Pro
210 215 220
Lys Pro Met Ser Ile Thr Leu Asn Ser Ser Gln Asn Lys Ser Ser Asp
225 230 235 240
Gly Gly Thr Lys Asn Arg Lys Ile Ala Val Val Phe Gly Val Ser Leu
245 250 255
Thr Cys Val Cys Leu Leu Ile Ile Gly Phe Gly Phe Leu Leu Trp Trp
260 265 270
Arg Arg Arg His Asn Lys Gln Val Leu Phe Phe Asp Ile Asn Glu Gln
275 280 285
Asn Lys Glu Glu Met Cys Leu Gly Asn Leu Arg Arg Phe Asn Phe Lys
290 295 300
Glu Leu Gln Ser Ala Thr Ser Asn Phe Ser Ser Lys Asn Leu Val Gly
305 310 315 320
Lys Gly Gly Phe Gly Asn Val Tyr Lys Gly Cys Leu His Asp Gly Ser
325 330 335
Ile Ile Ala Val Lys Arg Leu Lys Asp Ile Asn Asn Gly Gly Gly Glu
340 345 350
Val Gln Phe Gln Thr Glu Leu Glu Met Ile Ser Leu Ala Val His Arg
355 360 365
Asn Leu Leu Arg Leu Tyr Gly Phe Cys Thr Thr Ser Ser Glu Arg Leu
370 375 380
Leu Val Tyr Pro Tyr Met Ser Asn Gly Ser Val Ala Ser Arg Leu Lys
385 390 395 400
Ala Lys Pro Val Leu Asp Trp Gly Thr Arg Lys Arg Ile Ala Leu Gly
405 410 415
Ala Gly Arg Gly Leu Leu Tyr Leu His Glu Gln Cys Asp Pro Lys Ile
420 425 430
Ile His Arg Asp Val Lys Ala Ala Asn Ile Leu Leu Asp Asp Tyr Phe
435 440 445
Glu Ala Val Val Gly Asp Phe Gly Leu Ala Lys Leu Leu Asp His Glu
450 455 460
Glu Ser His Val Thr Thr Ala Val Arg Gly Thr Val Gly His Ile Ala
465 470 475 480
Pro Glu Tyr Leu Ser Thr Gly Gln Ser Ser Glu Lys Thr Asp Val Phe
485 490 495
Gly Phe Gly Ile Leu Leu Leu Glu Leu Ile Thr Gly Leu Arg Ala Leu
500 505 510
Glu Phe Gly Lys Ala Ala Asn Gln Arg Gly Ala Ile Leu Asp Trp Val
515 520 525
Lys Lys Leu Gln Gln Glu Lys Lys Leu Glu Gln Ile Val Asp Lys Asp
530 535 540
Leu Lys Ser Asn Tyr Asp Arg Ile Glu Val Glu Glu Met Val Gln Val
545 550 555 560
Ala Leu Leu Cys Thr Gln Tyr Leu Pro Ile His Arg Pro Lys Met Ser
565 570 575
Glu Val Val Arg Met Leu Glu Gly Asp Gly Leu Val Glu Lys Trp Glu
580 585 590
Ala Ser Ser Gln Arg Ala Glu Thr Asn Arg Ser Tyr Ser Lys Pro Asn
595 600 605
Glu Phe Ser Ser Ser Glu Arg Tyr Ser Asp Leu Thr Asp Asp Ser Ser
610 615 620
Val Leu Val Gln Ala Met Glu Leu Ser Gly Pro Arg
625 630 635
<210>32
<211>2579
<212>DNA
<213>拟南芥
<400>32
aataattaaa attcgtcttc cttccttgct ctcggcgata acttggtttc tctcctctct 60
ctcatctctc tttgtttcga ccctttttta gtatatttcc aggaaatatc ttcttcctcc 120
tttcgttttc tctatctcag ttttctctct tctcagcatt aagtagtcaa cggtcagcga 180
tctcggcgtt ccttctaatc ggaaaagtct agcttcagtt tctttttttt ttgctttttt 240
ggtttccgcg attaatcgat ttgggtattt tgattttctc ttcaaattaa gtcaacgggt 300
ggatacgcgt tgagagggct tttctcgtat tctgcttcta atttcatcat cttggtatta 360
ccttgtgtgg gtggtagctt aatcgaagga ttcgagatcc cttttatcag gggttttaac 420
aatgatggat tttctctgat gagggatagt tctagggttt gtttttaatc tcttgaggat 480
aaaatggaac gaagattaat gatcccttgc ttcttttggt tgattctcgt tttggatttg 540
gttctcagag tctcgggcaa cgccgaaggt gatgctctaa gtgcactgaa aaacagttta 600
gccgacccta ataaggtgct tcaaagttgg gatgctactc ttgttactcc atgtacatgg 660
tttcatgtta cttgcaatag cgacaatagt gttacacgtg ttgaccttgg gaatgcaaat 720
ctatctggac agctcgtaat gcaacttggt cagcttccaa acttgcagta cttggagctt 780
tatagcaata acattactgg gacaatccca gaacagcttg gaaatctgac ggaattggtg 840
agcttggatc tttacttgaa caatttaagc gggcctattc catcaactct cggccgactt 900
aagaaactcc gtttcttgcg tcttaataac aatagcttat ctggagaaat tccaaggtct 960
ttgactgctg tcctgacgct acaagttctg gatctctcaa acaatcctct caccggagat 1020
attcctgtta atggttcctt ttcacttttc actccaatca gttttgccaa caccaagttg 1080
actccccttc ctgcatctcc accgcctcct atctctccta caccgccatc acctgcaggg 1140
agtaatagaa ttactggagc gattgcggga ggagttgctg caggtgctgc acttctattt 1200
gctgttccgg ccattgcact agcttggtgg cgaaggaaaa agccgcagga ccacttcttt 1260
gatgtaccag ctgaagagga cccagaagtt catttaggac aactgaagag gttttcattg 1320
cgtgaactac aagttgcttc ggataatttt agcaacaaga acatattggg tagaggtggt 1380
tttggtaaag tttataaagg acggttagct gatggtactt tagtggccgt taaaaggcta 1440
aaagaggagc gcacccaagg tggcgaactg cagttccaga cagaggttga gatgattagt 1500
atggcggttc acagaaactt gcttcggctt cgtggatttt gcatgactcc aaccgaaaga 1560
ttgcttgttt atccctacat ggctaatgga agtgttgcct cctgtttaag agaacgtccc 1620
gagtcccagc caccacttga ttggccaaag agacagcgta ttgcgttggg atctgcaaga 1680
gggcttgcgt atttacatga tcattgcgac ccaaagatta ttcatcgaga tgtgaaagct 1740
gcaaatattt tgttggatga agagtttgaa gccgtggttg gggattttgg acttgcaaaa 1800
ctcatggact acaaagacac acatgtgaca accgcagtgc gtgggacaat tggtcatata 1860
gcccctgagt acctttccac tggaaaatca tcagagaaaa ccgatgtctt tgggtatgga 1920
gtcatgcttc ttgagcttat cactggacaa agggcttttg atcttgctcg cctcgcgaat 1980
gatgatgatg tcatgttact agactgggtg aaagggttgt taaaagagaa gaaattggaa 2040
gcactagtag atgttgatct tcagggtaat tacaaagacg aagaagtgga gcagctaatc 2100
caagtggctt tactctgcac tcagagttca ccaatggaaa gacccaaaat gtctgaagtt 2160
gtaagaatgc ttgaaggaga tggtttagct gagagatggg aagagtggca aaaggaggaa 2220
atgttcagac aagatttcaa ctacccaacc caccatccag ccgtgtctgg ctggatcatt 2280
ggcgattcca cttcccagat cgaaaacgaa tacccctcgg gtccaagata agattcgaaa 2340
cacgaatgtt ttttctgtat tttgtttttc tctgtattta ttgagggttt tagcttctgc 2400
tgctccatat tattggttct taagtgaata catgaggatc agattgggtt tgtaagtgtt 2460
atatgatgaa aaaggatttg aatgttgttg aaagctaaaa cccaaacatg tcttaagctc 2520
accacttgag gattgttgcg cacttgattc acaaatatgt atcccatcaa ttattcttt 2579
<210>33
<211>615
<212>PRT
<213>拟南芥
<400>33
Met Glu Arg Arg Leu Met Ile Pro Cys Phe Phe Trp Leu Ile Leu Val
1 5 10 15
Leu Asp Leu Val Leu Arg Val Ser Gly Asn Ala Glu Gly Asp Ala Leu
20 25 30
Ser Ala Leu Lys Asn Ser Leu Ala Asp Pro Asn Lys Val Leu Gln Ser
35 40 45
Trp Asp Ala Thr Leu Val Thr Pro Cys Thr Trp Phe His Val Thr Cys
50 55 60
Asn Ser Asp Asn Ser Val Thr Arg Val Asp Leu Gly Asn Ala Asn Leu
65 70 75 80
Ser Gly Gln Leu Val Met Gln Leu Gly Gln Leu Pro Asn Leu Gln Tyr
85 90 95
Leu Glu Leu Tyr Ser Asn Asn Ile Thr Gly Thr Ile Pro Glu Gln Leu
100 105 110
Gly Asn Leu Thr Glu Leu Val Ser Leu Asp Leu Tyr Leu Asn Asn Leu
115 120 125
Ser Gly Pro Ile Pro Ser Thr Leu Gly Arg Leu Lys Lys Leu Arg Phe
130 135 140
Leu Arg Leu Asn Asn Asn Ser Leu Ser Gly Glu Ile Pro Arg Ser Leu
145 150 155 160
Thr Ala Val Leu Thr Leu Gln Val Leu Asp Leu Ser Asn Asn Pro Leu
165 170 175
Thr Gly Asp Ile Pro Val Asn Gly Ser Phe Ser Leu Phe Thr Pro Ile
180 185 190
Ser Phe Ala Asn Thr Lys Leu Thr Pro Leu Pro Ala Ser Pro Pro Pro
195 200 205
Pro Ile Ser Pro Thr Pro Pro Ser Pro Ala Gly Ser Asn Arg Ile Thr
210 215 220
Gly Ala Ile Ala Gly Gly Val Ala Ala Gly Ala Ala Leu Leu Phe Ala
225 230 235 240
Val Pro Ala Ile Ala Leu Ala Trp Trp Arg Arg Lys Lys Pro Gln Asp
245 250 255
His Phe Phe Asp Val Pro Ala Glu Glu Asp Pro Glu Val His Leu Gly
260 265 270
Gln Leu Lys Arg Phe Ser Leu Arg Glu Leu Gln Val Ala Ser Asp Asn
275 280 285
Phe Ser Asn Lys Asn Ile Leu Gly Arg Gly Gly Phe Gly Lys Val Tyr
290 295 300
Lys Gly Arg Leu Ala Asp Gly Thr Leu Val Ala Val Lys Arg Leu Lys
305 310 315 320
Glu Glu Arg Thr Gln Gly Gly Glu Leu Gln Phe Gln Thr Glu Val Glu
325 330 335
Met Ile Ser Met Ala Val His Arg Asn Leu Leu Arg Leu Arg Gly Phe
340 345 350
Cys Met Thr Pro Thr Glu Arg Leu Leu Val Tyr Pro Tyr Met Ala Asn
355 360 365
Gly Ser Val Ala Ser Cys Leu Arg Glu Arg Pro Glu Ser Gln Pro Pro
370 375 380
Leu Asp Trp Pro Lys Arg Gln Arg Ile Ala Leu Gly Ser Ala Arg Gly
385 390 395 400
Leu Ala Tyr Leu His Asp His Cys Asp Pro Lys Ile Ile His Arg Asp
405 410 415
Val Lys Ala Ala Asn Ile Leu Leu Asp Glu Glu Phe Glu Ala Val Val
420 425 430
Gly Asp Phe Gly Leu Ala Lys Leu Met Asp Tyr Lys Asp Thr His Val
435 440 445
Thr Thr Ala Val Arg Gly Thr Ile Gly His Ile Ala Pro Glu Tyr Leu
450 455 460
Ser Thr Gly Lys Ser Ser Glu Lys Thr Asp Val Phe Gly Tyr Gly Val
465 470 475 480
Met Leu Leu Glu Leu Ile Thr Gly Gln Arg Ala Phe Asp Leu Ala Arg
485 490 495
Leu Ala Asn Asp Asp Asp Val Met Leu Leu Asp Trp Val Lys Gly Leu
500 505 510
Leu Lys Glu Lys Lys Leu Glu Ala Leu Val Asp Val Asp Leu Gln Gly
515 520 525
Asn Tyr Lys Asp Glu Glu Val Glu Gln Leu Ile Gln Val Ala Leu Leu
530 535 540
Cys Thr Gln Ser Ser Pro Met Glu Arg Pro Lys Met Ser Glu Val Val
545 550 555 560
Arg Met Leu Glu Gly Asp Gly Leu Ala Glu Arg Trp Glu Glu Trp Gln
565 570 575
Lys Glu Glu Met Phe Arg Gln Asp Phe Asn Tyr Pro Thr His His Pro
580 585 590
Ala Val Ser Gly Trp Ile Ile Gly Asp Ser Thr Ser Gln Ile Glu Asn
595 600 605
Glu Tyr Pro Ser Gly Pro Arg
610 615
<210>34
<211>2206
<212>DNA
<213>拟南芥
<400>34
gagagtgata attgcgaaat tgccaaaaaa cgcaaagtct accactagac aagaaaatcg 60
aagcttttca ctttctcttt tttctgtttt gttgtctttg gttctactct ccgcactgaa 120
tctttcgatc agcgataatt gtttccttct tttgggattt tctccttgga tggaaccagc 180
tcaattaatg agatgagatg agaatgttca gcttgcagaa gatggctatg gcttttactc 240
tcttgttttt tgcctgttta tgctcatttg tgtctccaga tgctcaaggg gatgcactgt 300
ttgcgttgag gatctcctta cgtgcattac cgaatcagct aagtgactgg aatcagaacc 360
aagttaatcc ttgcacttgg tcccaagtta tttgtgatga caaaaacttt gtcacttctc 420
ttacattgtc agatatgaac ttctcgggaa ccttgtcttc aagagtagga atcctagaaa 480
atctcaagac tcttacttta aagggaaatg gaattacggg tgaaatacca gaagactttg 540
gaaatctgac tagcttgact agtttggatt tggaggacaa tcagctaact ggtcgtatac 600
catccactat cggtaatctc aagaaacttc agttcttgac cttgagtagg aacaaactta 660
atgggactat tccggagtca ctcactggtc ttccaaacct gttaaacctg ctgcttgatt 720
ccaatagtct cagtggtcag attcctcaaa gtctgtttga gatcccaaaa tataatttca 780
cgtcaaacaa cttgaattgt ggcggtcgtc aacctcaccc ttgtgtatcc gcggttgccc 840
attcaggtga ttcaagcaag cctaaaactg gcattattgc tggagttgtt gctggagtta 900
cagttgttct ctttggaatc ttgttgtttc tgttctgcaa ggataggcat aaaggatata 960
gacgtgatgt gtttgtggat gttgcaggtg aagtggacag gagaattgca tttggacagt 1020
tgaaaaggtt tgcatggaga gagctccagt tagcgacaga taacttcagc gaaaagaatg 1080
tacttggtca aggaggcttt gggaaagttt acaaaggagt gcttccggat aacaccaaag 1140
ttgctgtgaa gagattgacg gatttcgaaa gtcctggtgg agatgctgct ttccaaaggg 1200
aagtagagat gataagtgta gctgttcata ggaatctact ccgtcttatc gggttctgca 1260
ccacacaaac agaacgcctt ttggtttatc ccttcatgca gaatctaagt cttgcacatc 1320
gtctgagaga gatcaaagca ggcgacccgg ttctagattg ggagacgagg aaacggattg 1380
ccttaggagc agcgcgtggt tttgagtatc ttcatgaaca ttgcaatccg aagatcatac 1440
atcgtgatgt gaaagcagct aatgtgttac tagatgaaga ttttgaagca gtggttggtg 1500
attttggttt agccaagcta gtagatgtta gaaggactaa tgtgactact caagttcgag 1560
gaacaatggg tcacattgca ccagaatatt tatcaacagg gaaatcatca gagagaaccg 1620
atgttttcgg gtatggaatt atgcttcttg agcttgttac aggacaacgc gcaatagact 1680
tttcacgttt ggaggaagaa gatgatgtct tgttacttga ccacgtgaag aaactggaaa 1740
gagagaagag attaggagca atcgtagata agaatttgga tggagagtat ataaaagaag 1800
aagtagagat gatgatacaa gtggctttgc tttgtacaca aggttcacca gaagaccgac 1860
cagtgatgtc tgaagttgtg aggatgttag aaggagaagg gcttgcggag agatgggaag 1920
agtggcaaaa cgtggaagtc acgagacgtc atgagtttga acggttgcag aggagatttg 1980
attggggtga agattctatg cataaccaag atgccattga attatctggt ggaagatgac 2040
caaaaacatc aaaccttgag tttactgtaa agttgccaac ttcacttttt tgttttgttc 2100
ttcggtgaag aagtaaaatc agttgtataa atcttgtttt tgtttcatga tgtatctttt 2160
gactttaata aattctgtga atgaaaagaa ctatgatgtt ttgttg 2206
<210>35
<211>613
<212>PRT
<213>拟南芥
<400>35
Met Arg Met Phe Ser Leu Gln Lys Met Ala Met Ala Phe Thr Leu Leu
1 5 10 15
Phe Phe Ala Cys Leu Cys Ser Phe Val Ser Pro Asp Ala Gln Gly Asp
20 25 30
Ala Leu Phe Ala Leu Arg Ile Ser Leu Arg Ala Leu Pro Asn Gln Leu
35 40 45
Ser Asp Trp Asn Gln Asn Gln Val Asn Pro Cys Thr Trp Ser Gln Val
50 55 60
Ile Cys Asp Asp Lys Asn Phe Val Thr Ser Leu Thr Leu Ser Asp Met
65 70 75 80
Asn Phe Ser Gly Thr Leu Ser Ser Arg Val Gly Ile Leu Glu Asn Leu
85 90 95
Lys Thr Leu Thr Leu Lys Gly Asn Gly Ile Thr Gly Glu Ile Pro Glu
100 105 110
Asp Phe Gly Asn Leu Thr Ser Leu Thr Ser Leu Asp Leu Glu Asp Asn
115 120 125
Gln Leu Thr Gly Arg Ile Pro Ser Thr Ile Gly Asn Leu Lys Lys Leu
130 135 140
Gln Phe Leu Thr Leu Ser Arg Asn Lys Leu Asn Gly Thr Ile Pro Glu
145 150 155 160
Ser Leu Thr Gly Leu Pro Asn Leu Leu Asn Leu Leu Leu Asp Ser Asn
165 170 175
Ser Leu Ser Gly Gln Ile Pro Gln Ser Leu Phe Glu Ile Pro Lys Tyr
180 185 190
Asn Phe Thr Ser Asn Asn Leu Asn Cys Gly Gly Arg Gln Pro His Pro
195 200 205
Cys Val Ser Ala Val Ala His Ser Gly Asp Ser Ser Lys Pro Lys Thr
210 215 220
Gly Ile Ile Ala Gly Val Val Ala Gly Val Thr Val Val Leu Phe Gly
225 230 235 240
Ile Leu Leu Phe Leu Phe Cys Lys Asp Arg His Lys Gly Tyr Arg Arg
245 250 255
Asp Val Phe Val Asp Val Ala Gly Glu Val Asp Arg Arg Ile Ala Phe
260 265 270
Gly Gln Leu Lys Arg Phe Ala Trp Arg Glu Leu Gln Leu Ala Thr Asp
275 280 285
Asn Phe Ser Glu Lys Asn Val Leu Gly Gln Gly Gly Phe Gly Lys Val
290 295 300
Tyr Lys Gly Val Leu Pro Asp Asn Thr Lys Val Ala Val Lys Arg Leu
305 310 315 320
Thr Asp Phe Glu Ser Pro Gly Gly Asp Ala Ala Phe Gln Arg Glu Val
325 330 335
Glu Met Ile Ser Val Ala Val His Arg Asn Leu Leu Arg Leu Ile Gly
340 345 350
Phe Cys Thr Thr Gln Thr Glu Arg Leu Leu Val Tyr Pro Phe Met Gln
355 360 365
Asn Leu Ser Leu Ala His Arg Leu Arg Glu Ile Lys Ala Gly Asp Pro
370 375 380
Val Leu Asp Trp Glu Thr Arg Lys Arg Ile Ala Leu Gly Ala Ala Arg
385 390 395 400
Gly Phe Glu Tyr Leu His Glu His Cys Asn Pro Lys Ile Ile His Arg
405 410 415
Asp Val Lys Ala Ala Asn Val Leu Leu Asp Glu Asp Phe Glu Ala Val
420 425 430
Val Gly Asp Phe Gly Leu Ala Lys Leu Val Asp Val Arg Arg Thr Asn
435 440 445
Val Thr Thr Gln Val Arg Gly Thr Met Gly His Ile Ala Pro Glu Tyr
450 455 460
Leu Ser Thr Gly Lys Ser Ser Glu Arg Thr Asp Val Phe Gly Tyr Gly
465 470 475 480
Ile Met Leu Leu Glu Leu Val Thr Gly Gln Arg Ala Ile Asp Phe Ser
485 490 495
Arg Leu Glu Glu Glu Asp Asp Val Leu Leu Leu Asp His Val Lys Lys
500 505 510
Leu Glu Arg Glu Lys Arg Leu Gly Ala Ile Val Asp Lys Asn Leu Asp
515 520 525
Gly Glu Tyr Ile Lys Glu Glu Val Glu Met Met Ile Gln Val Ala Leu
530 535 540
Leu Cys Thr Gln Gly Ser Pro Glu Asp Arg Pro Val Met Ser Glu Val
545 550 555 560
Val Arg Met Leu Glu Gly Glu Gly Leu Ala Glu Arg Trp Glu Glu Trp
565 570 575
Gln Asn Val Glu Val Thr Arg Arg His Glu Phe Glu Arg Leu Gln Arg
580 585 590
Arg Phe Asp Trp Gly Glu Asp Ser Met His Asn Gln Asp Ala Ile Glu
595 600 605
Leu Ser Gly Gly Arg
610
<210>36
<211>2323
<212>DNA
<213>拟南芥
<400>36
atagagattt ggttttttga ttcttccaat ctcactctct ctgtctttct ctctccatca 60
aataccaaat tatctggaag ctgagtacat cttgttttct gctcattcct ctgtttcaac 120
aatggagagt actattgtta tgatgatgat gataacaaga tctttctttt gcttcttggg 180
atttttatgc cttctctgct cttctgttca cggattgctt tctcctaaag gtgttaactt 240
tgaagtgcaa gctttgatgg acataaaagc ttcattacat gatcctcatg gtgttcttga 300
taactgggat agagatgctg ttgatccttg tagttggaca atggtcactt gttcttctga 360
aaactttgtc attggcttag gcacaccaag tcagaattta tctggtacac tatctccaag 420
cattaccaac ttaacaaatc ttcggattgt gctgttgcag aacaacaaca taaaaggaaa 480
aattcctgct gagattggtc ggcttacgag gcttgagact cttgatcttt ctgataattt 540
cttccacggt gaaattcctt tttcagtagg ctatctacaa agcctgcaat atctgaggct 600
taacaacaat tctctctctg gagtgtttcc tctgtcacta tctaatatga ctcaacttgc 660
ctttcttgat ttatcataca acaatcttag tggtcctgtt ccaagatttg ctgcaaagac 720
gtttagcatc gttgggaacc cgctgatatg tccaacgggt accgaaccag actgcaatgg 780
aacaacattg atacctatgt ctatgaactt gaatcaaact ggagttcctt tatacgccgg 840
tggatcgagg aatcacaaaa tggcaatcgc tgttggatcc agcgttggga ctgtatcatt 900
aatcttcatt gctgttggtt tgtttctctg gtggagacaa agacataacc aaaacacatt 960
ctttgatgtt aaagatggga atcatcatga ggaagtttca cttggaaacc tgaggagatt 1020
tggtttcagg gagcttcaga ttgcgaccaa taacttcagc agtaagaact tattggggaa 1080
aggtggctat ggaaatgtat acaaaggaat acttggagat agtacagtgg ttgcagtgaa 1140
aaggcttaaa gatggaggag cattgggagg agagattcag tttcagacag aagttgaaat 1200
gatcagttta gctgttcatc gaaatctctt aagactctac ggtttctgca tcacacaaac 1260
tgagaagctt ctagtttatc cttatatgtc taatggaagc gttgcatctc gaatgaaagc 1320
aaaacctgtt cttgactgga gcataaggaa gaggatagcc ataggagctg caagagggct 1380
tgtgtatctc catgagcaat gtgatccgaa gattatccac cgcgatgtca aagcagcgaa 1440
tatacttctt gatgactact gtgaagctgt ggttggcgat tttggtttag ctaaactctt 1500
ggatcatcaa gattctcatg tgacaaccgc ggttagaggc acggtgggtc acattgctcc 1560
agagtatctc tcaactggtc aatcctctga gaaaacagat gtttttggct tcgggattct 1620
tcttcttgag cttgtaaccg gacaaagagc ttttgagttt ggtaaagcgg ctaaccagaa 1680
aggtgtgatg cttgattggg ttaaaaagat tcatcaagag aagaaacttg agctacttgt 1740
ggataaagag ttgttgaaga agaagagcta cgatgagatt gagttagacg aaatggtaag 1800
agtagctttg ttgtgcacac agtacctgcc aggacataga ccaaaaatgt ctgaagttgt 1860
tcgaatgctg gaaggagatg gacttgcaga gaaatgggaa gcttctcaaa gatcagacag 1920
tgtttcaaaa tgtagcaaca ggataaatga attgatgtca tcttcagaca gatactctga 1980
tcttaccgat gactctagtt tacttgtgca agcaatggag ctctctggtc ctagatgaaa 2040
tctatacatg aatctgaaga agaagaagaa catgcatctg tttcttgaat caagagggat 2100
tcttgttttt ttgtataata gagaggtttt ttggagggaa atgttgtgtc tctgtaactg 2160
tataggcttg ttgtgtaaga agttattact gcacttaggg ttaattcaaa gttctttaca 2220
taaaaaatga ttagttgcgt tgaatagagg gaacactttg ggagatttca tgtgtgaaat 2280
ttgggaattc atgtttgaga atgaaattta tcttattatt gga 2323
<210>37
<211>638
<212>PRT
<213>拟南芥
<400>37
Met Glu Ser Thr Ile Val Met Met Met Met Ile Thr Arg Ser Phe Phe
1 5 10 15
Cys Phe Leu Gly Phe Leu Cys Leu Leu Cys Ser Ser Val His Gly Leu
20 25 30
Leu Ser Pro Lys Gly Val Asn Phe Glu Val Gln Ala Leu Met Asp Ile
35 40 45
Lys Ala Ser Leu His Asp Pro His Gly Val Leu Asp Asn Trp Asp Arg
50 55 60
Asp Ala Val Asp Pro Cys Ser Trp Thr Met Val Thr Cys Ser Ser Glu
65 70 75 80
Asn Phe Val Ile Gly Leu Gly Thr Pro Ser Gln Asn Leu Ser Gly Thr
85 90 95
Leu Ser Pro Ser Ile Thr Asn Leu Thr Asn Leu Arg Ile Val Leu Leu
100 105 110
Gln Asn Asn Asn Ile Lys Gly Lys Ile Pro Ala Glu Ile Gly Arg Leu
115 120 125
Thr Arg Leu Glu Thr Leu Asp Leu Ser Asp Asn Phe Phe His Gly Glu
130 135 140
Ile Pro Phe Ser Val Gly Tyr Leu Gln Ser Leu Gln Tyr Leu Arg Leu
145 150 155 160
Asn Asn Asn Ser Leu Ser Gly Val Phe Pro Leu Ser Leu Ser Asn Met
165 170 175
Thr Gln Leu Ala Phe Leu Asp Leu Ser Tyr Asn Asn Leu Ser Gly Pro
180 185 190
Val Pro Arg Phe Ala Ala Lys Thr Phe Ser Ile Val Gly Asn Pro Leu
195 200 205
Ile Cys Pro Thr Gly Thr Glu Pro Asp Cys Asn Gly Thr Thr Leu Ile
2l0 215 220
Pro Met Ser Met Asn Leu Asn Gln Thr Gly Val Pro Leu Tyr Ala Gly
225 230 235 240
Gly Ser Arg Asn His Lys Met Ala Ile Ala Val Gly Ser Ser Val Gly
245 250 255
Thr Val Ser Leu Ile Phe Ile Ala Val Gly Leu Phe Leu Trp Trp Arg
260 265 270
Gln Arg His Asn Gln Asn Thr Phe Phe Asp Val Lys Asp Gly Asn His
275 280 285
His Glu Glu Val Ser Leu Gly Asn Leu Arg Arg Phe Gly Phe Arg Glu
290 295 300
Leu Gln Ile Ala Thr Asn Asn Phe Ser Ser Lys Asn Leu Leu Gly Lys
305 310 315 320
Gly Gly Tyr Gly Asn Val Tyr Lys Gly Ile Leu Gly Asp Ser Thr Val
325 330 335
Val Ala Val Lys Arg Leu Lys Asp Gly Gly Ala Leu Gly Gly Glu Ile
340 345 350
Gln Phe Gln Thr Glu Val Glu Met Ile Ser Leu Ala Val His Arg Asn
355 360 365
Leu Leu Arg Leu Tyr Gly Phe Cys Ile Thr Gln Thr Glu Lys Leu Leu
370 375 380
Val Tyr Pro Tyr Met Ser Asn Gly Ser Val Ala Ser Arg Met Lys Ala
385 390 395 400
Lys Pro Val Leu Asp Trp Ser Ile Arg Lys Arg Ile Ala Ile Gly Ala
405 410 415
Ala Arg Gly Leu Val Tyr Leu His Glu Gln Cys Asp Pro Lys Ile Ile
420 425 430
His Arg Asp Val Lys Ala Ala Asn Ile Leu Leu Asp Asp Tyr Cys Glu
435 440 445
Ala Val Val Gly Asp Phe Gly Leu Ala Lys Leu Leu Asp His Gln Asp
450 455 460
Ser His Val Thr Thr Ala Val Arg Gly Thr Val Gly His Ile Ala Pro
465 470 475 480
Glu Tyr Leu Ser Thr Gly Gln Ser Ser Glu Lys Thr Asp Val Phe Gly
485 490 495
Phe Gly Ile Leu Leu Leu Glu Leu Val Thr Gly Gln Arg Ala Phe Glu
500 505 510
Phe Gly Lys Ala Ala Asn Gln Lys Gly Val Met Leu Asp Trp Val Lys
515 520 525
Lys Ile His Gln Glu Lys Lys Leu Glu Leu Leu Val Asp Lys Glu Leu
530 535 540
Leu Lys Lys Lys Ser Tyr Asp Glu Ile Glu Leu Asp Glu Met Val Arg
545 550 555 560
Val Ala Leu Leu Cys Thr Gln Tyr Leu Pro Gly His Arg Pro Lys Met
565 570 575
Ser Glu Val Val Arg Met Leu Glu Gly Asp Gly Leu Ala Glu Lys Trp
580 585 590
Glu Ala Ser Gln Arg Ser Asp Ser Val Ser Lys Cys Ser Asn Arg Ile
595 600 605
Asn Glu Leu Met Ser Ser Ser Asp Arg Tyr Ser Asp Leu Thr Asp Asp
610 615 620
Ser Ser Leu Leu Val Gln Ala Met Glu Leu Ser Gly Pro Arg
625 630 635
<210>38
<211>1845
<212>DNA
<213>拟南芥
<400>38
atggagattt ctttgatgaa gtttctgttt ttaggaatct gggtttatta ttactctgtt 60
cttgactctg tttctgccat ggatagtctt ttatctccca agggtgttaa ctatgaagtg 120
gctgcgttaa tgtcagtgaa gaacaagatg aaagatgaga aagaggtttt gtctggttgg 180
gatattaact ctgttgatcc ttgtacttgg aacatggttg gttgttcttc tgaaggtttt 240
gtggtttctc tagagatggc tagtaaagga ttatcaggga tactatctac tagtattggg 300
gaattaactc atcttcatac tttgttactt cagaataatc agttaactgg tccgattcct 360
tctgagttag gccaactctc tgagcttgaa acgcttgatt tatcggggaa tcggtttagt 420
ggtgaaatcc cagcttcttt agggttctta actcacttaa actacttgcg gcttagcagg 480
aatcttttat ctgggcaagt ccctcacctc gtcgctggcc tctcaggtct ttctttcttg 540
gatctatctt tcaacaatct aagcggacca actccgaata tatcagcaaa agattacagg 600
attgtaggaa atgcatttct ttgtggtcca gcttcccaag agctttgctc agatgctaca 660
cctgtgagaa atgcgacggg tttgtctgaa aaggacaata gcaaacatca cagcttagtg 720
ctctcttttg catttggcat tgttgttgcc tttatcatct ccctaatgtt tctcttcttc 780
tgggtgcttt ggcatcgatc acgtctctca agatcacacg tgcagcaaga ctacgaattt 840
gaaatcggcc atctgaaaag gttcagtttt cgcgaaatac aaaccgcaac aagcaatttt 900
agtccaaaga acattttggg acaaggaggg tttgggatgg tttataaagg gtatctccca 960
aatggaactg tggtggcagt taaaagattg aaagatccga tttatacagg agaagttcag 1020
tttcaaaccg aagtagagat gattggctta gctgttcacc gtaacctttt acgcctcttt 1080
ggattctgta tgaccccgga agagagaatg cttgtgtatc cgtacatgcc aaatggaagc 1140
gtagctgatc gtctgagaga caattatgga gaaaagccgt ctctagattg gaatcggagg 1200
ataagcattg cactcggcgc agctcgagga cttgtttact tgcacgagca atgcaatcca 1260
aagattattc acagagacgt caaagctgca aatattctac ttgatgagag ctttgaagca 1320
atagttggcg attttggtct agcaaagctt ttagaccaga gagattcaca tgtcactacc 1380
gcagtccgag gaaccattgg acacatcgct cccgagtacc tttccactgg acagtcctca 1440
gagaaaaccg atgttttcgg attcggagta ctaatccttg aactcataac aggtcataag 1500
atgattgatc aaggcaatgg tcaagttcga aaaggaatga tattgagctg ggtaaggaca 1560
ttgaaagcag agaagagatt tgcagagatg gtggacagag atttgaaggg agagtttgat 1620
gatttggtgt tggaggaagt agtggaattg gctttgcttt gtacacagcc acatccgaat 1680
ctaagaccga ggatgtctca agtgttgaag gtactagaag gtttagtgga acagtgtgaa 1740
ggagggtatg aagctagagc tccaagtgtc tctaggaact acagtaatgg tcatgaagag 1800
cagtccttta ttattgaagc cattgagctc tctggaccac gatga 1845
<210>39
<211>614
<212>PRT
<213>拟南芥
<400>39
Met Glu Ile Ser Leu Met Lys Phe Leu Phe Leu Gly Ile Trp Val Tyr
1 5 10 15
Tyr Tyr Ser Val Leu Asp Ser Val Ser Ala Met Asp Ser Leu Leu Ser
20 25 30
Pro Lys Gly Val Asn Tyr Glu Val Ala Ala Leu Met Ser Val Lys Asn
35 40 45
Lys Met Lys Asp Glu Lys Glu Val Leu Ser Gly Trp Asp Ile Asn Ser
50 55 60
Val Asp Pro Cys Thr Trp Asn Met Val Gly Cys Ser Ser Glu Gly Phe
65 70 75 80
Val Val Ser Leu Glu Met Ala Ser Lys Gly Leu Ser Gly Ile Leu Ser
85 90 95
Thr Ser Ile Gly Glu Leu Thr His Leu His Thr Leu Leu Leu Gln Asn
100 105 110
Asn Gln Leu Thr Gly Pro Ile Pro Ser Glu Leu Gly Gln Leu Ser Glu
115 120 125
Leu Glu Thr Leu Asp Leu Ser Gly Asn Arg Phe Ser Gly Glu Ile Pro
130 135 140
Ala Ser Leu Gly Phe Leu Thr His Leu Asn Tyr Leu Arg Leu Ser Arg
145 150 155 160
Asn Leu Leu Ser Gly Gln Val Pro His Leu Val Ala Gly Leu Ser Gly
165 170 175
Leu Ser Phe Leu Asp Leu Ser Phe Asn Asn Leu Ser Gly Pro Thr Pro
180 185 190
Asn Ile Ser Ala Lys Asp Tyr Arg Ile Val Gly Asn Ala Phe Leu Cys
195 200 205
Gly Pro Ala Ser Gln Glu Leu Cys Ser Asp Ala Thr Pro Val Arg Asn
210 215 220
Ala Thr Gly Leu Ser Glu Lys Asp Asn Ser Lys His His Ser Leu Val
225 230 235 240
Leu Ser Phe Ala Phe Gly Ile Val Val Ala Phe Ile Ile Ser Leu Met
245 250 255
Phe Leu Phe Phe Trp Val Leu Trp His Arg Ser Arg Leu Ser Arg Ser
260 265 270
His Val Gln Gln Asp Tyr Glu Phe Glu Ile Gly His Leu Lys Arg Phe
275 280 285
Ser Phe Arg Glu Ile Gln Thr Ala Thr Ser Asn Phe Ser Pro Lys Asn
290 295 300
Ile Leu Gly Gln Gly Gly Phe Gly Met Val Tyr Lys Gly Tyr Leu Pro
305 310 315 320
Asn Gly Thr Val Val Ala Val Lys Arg Leu Lys Asp Pro Ile Tyr Thr
325 330 335
Gly Glu Val Gln Phe Gln Thr Glu Val Glu Met Ile Gly Leu Ala Val
340 345 350
His Arg Asn Leu Leu Arg Leu Phe Gly Phe Cys Met Thr Pro Glu Glu
355 360 365
Arg Met Leu Val Tyr Pro Tyr Met Pro Asn Gly Ser Val Ala Asp Arg
370 375 380
Leu Arg Asp Asn Tyr Gly Glu Lys Pro Ser Leu Asp Trp Asn Arg Arg
385 390 395 400
Ile Ser Ile Ala Leu Gly Ala Ala Arg Gly Leu Val Tyr Leu His Glu
405 410 415
Gln Cys Asn Pro Lys Ile Ile His Arg Asp Val Lys Ala Ala Asn Ile
420 425 430
Leu Leu Asp Glu Ser Phe Glu Ala Ile Val Gly Asp Phe Gly Leu Ala
435 440 445
Lys Leu Leu Asp Gln Arg Asp Ser His Val Thr Thr Ala Val Arg Gly
450 455 460
Thr Ile Gly His Ile Ala Pro Glu Tyr Leu Ser Thr Gly Gln Ser Ser
465 470 475 480
Glu Lys Thr Asp Val Phe Gly Phe Gly Val Leu Ile Leu Glu Leu Ile
485 490 495
Thr Gly His Lys Met Ile Asp Gln Gly Asn Gly Gln Val Arg Lys Gly
500 505 510
Met Ile Leu Ser Trp Val Arg Thr Leu Lys Ala Glu Lys Arg Phe Ala
515 520 525
Glu Met Val Asp Arg Asp Leu Lys Gly Glu Phe Asp Asp Leu Val Leu
530 535 540
Glu Glu Val Val Glu Leu Ala Leu Leu Cys Thr Gln Pro His Pro Asn
545 550 555 560
Leu Arg Pro Arg Met Ser Gln Val Leu Lys Val Leu Glu Gly Leu Val
565 570 575
Glu Gln Cys Glu Gly Gly Tyr Glu Ala Arg Ala Pro Ser Val Ser Arg
580 585 590
Asn Tyr Ser Asn Gly His Glu Glu Gln Ser Phe Ile Ile Glu Ala Ile
595 600 605
Glu Leu Ser Gly Pro Arg
610
<210>40
<211>2106
<212>DNA
<213>拟南芥
<400>40
catttctctc ttcaacccca tgttttcgtt ctcttccgtt tagagtgttt tcagctcctc 60
tatggctcac tcggggaacg gtgaaagttt ccatgatcct cttcgaggat tcattcaaag 120
aaattgcttt agatggaaca atcagaaatt gatcttacaa tgtttcatgg ccttagcttt 180
tgtgggaatc acttcgtcaa caactcaacc agatatcgaa ggaggagctc tgttgcagct 240
cagagattcg cttaatgatt cgagcaatcg tctaaaatgg acacgcgatt ttgtgagccc 300
ttgctatagt tggtcttatg ttacctgcag aggccagagt gttgtggctc taaatcttgc 360
ctcgagtgga ttcacaggaa cactctctcc agctattaca aaactgaagt tcttggttac 420
cttagagtta cagaacaata gtttatctgg tgccttacca gattctcttg ggaacatggt 480
taatctacag actttaaacc tatcagtgaa tagtttcagc ggatcgatac cagcgagctg 540
gagtcagctc tcgaatctaa agcacttgga tctctcatcc aataatttaa caggaagcat 600
cccaacacaa ttcttctcaa tcccaacatt cgatttttca ggaactcagc ttatatgcgg 660
taaaagtttg aatcagcctt gttcttcaag ttctcgtctt ccagtcacat cctccaagaa 720
aaagctgaga gacattactt tgactgcaag ttgtgttgct tctataatct tattccttgg 780
agcaatggtt atgtatcatc accatcgcgt ccgcagaacc aaatacgaca tcttttttga 840
tgtagctggg gaagatgaca ggaagatttc ctttggacaa ctaaaacgat tctctttacg 900
tgaaatccag ctcgcaacag atagtttcaa cgagagcaat ttgataggac aaggaggatt 960
tggtaaagta tacagaggtt tgcttccaga caaaacaaaa gttgcagtga aacgccttgc 1020
ggattacttc agtcctggag gagaagctgc tttccaaaga gagattcagc tcataagcgt 1080
tgcggttcat aaaaatctct tacgccttat tggcttctgc acaacttcct ctgagagaat 1140
ccttgtttat ccatacatgg aaaatcttag tgttgcatat cgactaagag atttgaaagc 1200
gggagaggaa ggattagact ggccaacaag gaagcgtgta gcttttggtt cagctcacgg 1260
tttagagtat ctacacgaac attgtaaccc gaagatcata caccgcgatc tcaaggctgc 1320
aaacatactt ttagacaaca attttgagcc agttcttgga gatttcggtt tagctaagct 1380
tgtggacaca tctctgactc atgtcacaac tcaagtccga ggcacaatgg gtcacattgc 1440
gccagagtat ctctgcacag gaaaatcatc tgaaaaaacc gatgtttttg gttacggtat 1500
aacgcttctt gagcttgtta ctggtcagcg cgcaatcgat ttttcacgct tggaagaaga 1560
ggaaaatatt ctcttgcttg atcatataaa gaagttgctt agagaacaga gacttagaga 1620
cattgttgat agcaatttga ctacatatga ctccaaagaa gttgaaacaa tcgttcaagt 1680
ggctcttctc tgcacacaag gctcaccaga agatagacca gcgatgtctg aagtggtcaa 1740
aatgcttcaa gggactggtg gtttggctga gaaatggact gaatgggaac aacttgaaga 1800
agttaggaac aaagaagcat tgttgcttcc gactttaccg gctacttggg atgaagaaga 1860
aaccaccgtt gatcaagaat ctatccgatt atcgacagca agatgaagaa gaaacagaga 1920
gagaaagata tctatgaaaa caaacttgca ttacagaaga taaacttaga aagtatttta 1980
agctgctaat tgtattgaac caggtgggga aaacgaagca aacacacaac gttgatttgt 2040
gtaatagatg atatgatata cataactagt tgtgttttgt atatatatga atttggttat 2100
tttcgt 2106
<210>41
<211>614
<212>PRT
<213>拟南芥
<400>41
Met Ala His Ser Gly Asn Gly Glu Ser Phe His Asp Pro Leu Arg Gly
1 5 10 15
Phe Ile Gln Arg Asn Cys Phe Arg Trp Asn Asn Gln Lys Leu Ile Leu
20 25 30
Gln Cys Phe Met Ala Leu Ala Phe Val Gly Ile Thr Ser Ser Thr Thr
35 40 45
Gln Pro Asp Ile Glu Gly Gly Ala Leu Leu Gln Leu Arg Asp Ser Leu
50 55 60
Asn Asp Ser Ser Asn Arg Leu Lys Trp Thr Arg Asp Phe Val Ser Pro
65 70 75 80
Cys Tyr Ser Trp Ser Tyr Val Thr Cys Arg Gly Gln Ser Val Val Ala
85 90 95
Leu Asn Leu Ala Ser Ser Gly Phe Thr Gly Thr Leu Ser Pro Ala Ile
100 105 110
Thr Lys Leu Lys Phe Leu Val Thr Leu Glu Leu Gln Asn Asn Ser Leu
115 120 125
Ser Gly Ala Leu Pro Asp Ser Leu Gly Asn Met Val Asn Leu Gln Thr
130 135 140
Leu Asn Leu Ser Val Asn Ser Phe Ser Gly Ser Ile Pro Ala Ser Trp
145 150 155 160
Ser Gln Leu Ser Asn Leu Lys His Leu Asp Leu Ser Ser Asn Asn Leu
165 170 175
Thr Gly Ser Ile Pro Thr Gln Phe Phe Ser Ile Pro Thr Phe Asp Phe
180 185 190
Ser Gly Thr Gln Leu Ile Cys Gly Lys Ser Leu Asn Gln Pro Cys Ser
195 200 205
Ser Ser Ser Arg Leu Pro Val Thr Ser Ser Lys Lys Lys Leu Arg Asp
210 215 220
Ile Thr Leu Thr Ala Ser Cys Val Ala Ser Ile Ile Leu Phe Leu Gly
225 230 235 240
Ala Met Val Met Tyr His His His Arg Val Arg Arg Thr Lys Tyr Asp
245 250 255
Ile Phe Phe Asp Val Ala Gly Glu Asp Asp Arg Lys Ile Ser Phe Gly
260 265 270
Gln Leu Lys Arg Phe Ser Leu Arg Glu Ile Gln Leu Ala Thr Asp Ser
275 280 285
Phe Asn Glu Ser Asn Leu Ile Gly Gln Gly Gly Phe Gly Lys Val Tyr
290 295 300
Arg Gly Leu Leu Pro Asp Lys Thr Lys Val Ala Val Lys Arg Leu Ala
305 310 315 320
Asp Tyr Phe Ser Pro Gly Gly Glu Ala Ala Phe Gln Arg Glu Ile Gln
325 330 335
Leu Ile Ser Val Ala Val His Lys Asn Leu Leu Arg Leu Ile Gly Phe
340 345 350
Cys Thr Thr Ser Ser Glu Arg Ile Leu Val Tyr Pro Tyr Met Glu Asn
355 360 365
Leu Ser Val Ala Tyr Arg Leu Arg Asp Leu Lys Ala Gly Glu Glu Gly
370 375 380
Leu Asp Trp Pro Thr Arg Lys Arg Val Ala Phe Gly Ser Ala His Gly
385 390 395 400
Leu Glu Tyr Leu His Glu His Cys Asn Pro Lys Ile Ile His Arg Asp
405 410 415
Leu Lys Ala Ala Asn Ile Leu Leu Asp Asn Asn Phe Glu Pro Val Leu
420 425 430
Gly Asp Phe Gly Leu Ala Lys Leu Val Asp Thr Ser Leu Thr His Val
435 440 445
Thr Thr Gln Val Arg Gly Thr Met Gly His Ile Ala Pro Glu Tyr Leu
450 455 460
Cys Thr Gly Lys Ser Ser Glu Lys Thr Asp Val Phe Gly Tyr Gly Ile
465 470 475 480
Thr Leu Leu Glu Leu Val Thr Gly Gln Arg Ala Ile Asp Phe Ser Arg
485 490 495
Leu Glu Glu Glu Glu Asn Ile Leu Leu Leu Asp His Ile Lys Lys Leu
500 505 510
Leu Arg Glu Gln Arg Leu Arg Asp Ile Val Asp Ser Asn Leu Thr Thr
515 520 525
Tyr Asp Ser Lys Glu Val Glu Thr Ile Val Gln Val Ala Leu Leu Cys
530 535 540
Thr Gln Gly Ser Pro Glu Asp Arg Pro Ala Met Ser Glu Val Val Lys
545 550 555 560
Met Leu Gln Gly Thr Gly Gly Leu Ala Glu Lys Trp Thr Glu Trp Glu
565 570 575
Gln Leu Glu Glu Val Arg Asn Lys Glu Ala Leu Leu Leu Pro Thr Leu
580 585 590
Pro Ala Thr Trp Asp Glu Glu Glu Thr Thr Val Asp Gln Glu Ser Ile
595 600 605
Arg Leu Ser Thr Ala Arg
610
<210>42
<211>1854
<212>DNA
<213>拟南芥
<400>42
atggctctgc ttattatcac tgccttagtt tttagtagtt tatggtcatc tgtgtcacca 60
gatgctcaag gggatgcatt atttgcgttg aggagctcgt tacgtgcatc tcctgaacag 120
cttagtgatt ggaaccagaa tcaagtcgat ccttgtactt ggtctcaagt tatttgtgat 180
gacaagaaac atgttacttc tgtaaccttg tcttacatga acttctcctc gggaacactg 240
tcttcaggaa taggaatctt gacaactctc aagactctta cattgaaggg aaatggaata 300
atgggtggaa taccagaatc cattggaaat ctgtctagct tgaccagctt agatttggag 360
gataatcact taactgatcg cattccatcc actctcggta atctcaagaa tctacagttc 420
ttgaccttga gtaggaataa ccttaatggt tctatcccgg attcacttac aggtctatca 480
aaactgataa atattctgct cgactcaaat aatctcagtg gtgagattcc tcagagttta 540
ttcaaaatcc caaaatacaa tttcacagca aacaacttga gctgtggtgg cactttcccg 600
caaccttgtg taaccgagtc cagtccttca ggtgattcaa gcagtagaaa aactggaatc 660
atcgctggag ttgttagcgg aatagcggtt attctactag gattcttctt ctttttcttc 720
tgcaaggata aacataaagg atataaacga gacgtatttg tggatgttgc aggaacgaac 780
tttaaaaaag gtttgatttc aggtgaagtg gacagaagga ttgcttttgg acagttgaga 840
agatttgcat ggagagagct tcagttggct acagatgagt tcagtgaaaa gaatgttctc 900
ggacaaggag gctttgggaa agtttacaaa ggattgcttt cggatggcac caaagtcgct 960
gtaaaaagat tgactgattt tgaacgtcca ggaggagatg aagctttcca gagagaagtt 1020
gagatgataa gtgtagctgt tcataggaat ctgcttcgcc ttatcggctt ttgtacaaca 1080
caaactgaac gacttttggt gtatcctttc atgcagaatc taagtgttgc atattgctta 1140
agagagatta aacccgggga tccagttctg gattggttca ggaggaaaca gattgcgtta 1200
ggtgcagcac gaggactcga atatcttcat gaacattgca acccgaagat catacacaga 1260
gatgtgaaag ctgcaaatgt gttactagat gaagactttg aagcagtggt tggtgatttt 1320
ggtttagcca agttggtaga tgttagaagg actaatgtaa ccactcaggt ccgaggaaca 1380
atgggtcata ttgcaccaga atgtatatcc acagggaaat cgtcagagaa aaccgatgtt 1440
ttcgggtacg gaattatgct tctggagctt gtaactggac aaagagcaat tgatttctcg 1500
cggttagagg aagaagatga tgtcttattg ctagaccatg tgaagaaact ggaaagagag 1560
aagagattag aagacatagt agataagaag cttgatgagg attatataaa ggaagaagtt 1620
gaaatgatga tacaagtagc tctgctatgc acacaagcag caccggaaga acgaccagcg 1680
atgtcggaag tagtaagaat gctagaagga gaagggcttg cagagagatg ggaagagtgg 1740
cagaatcttg aagtgacgag acaagaagag tttcagaggt tgcagaggag atttgattgg 1800
ggtgaagatt ccattaataa tcaagatgct attgaattat ctggtggaag atag 1854
<210>43
<211>617
<212>PRT
<213>拟南芥
<400>43
Met Ala Leu Leu Ile Ile Thr Ala Leu Val Phe Ser Ser Leu Trp Ser
1 5 10 15
Ser Val Ser Pro Asp Ala Gln Gly Asp Ala Leu Phe Ala Leu Arg Ser
20 25 30
Ser Leu Arg Ala Ser Pro Glu Gln Leu Ser Asp Trp Asn Gln Asn Gln
35 40 45
Val Asp Pro Cys Thr Trp Ser Gln Val Ile Cys Asp Asp Lys Lys His
50 55 60
Val Thr Ser Val Thr Leu Ser Tyr Met Asn Phe Ser Ser Gly Thr Leu
65 70 75 80
Ser Ser Gly Ile Gly Ile Leu Thr Thr Leu Lys Thr Leu Thr Leu Lys
85 90 95
Gly Asn Gly Ile Met Gly Gly Ile Pro Glu Ser Ile Gly Asn Leu Ser
100 105 110
Ser Leu Thr Ser Leu Asp Leu Glu Asp Asn His Leu Thr Asp Arg Ile
115 120 125
Pro Ser Thr Leu Gly Asn Leu Lys Asn Leu Gln Phe Leu Thr Leu Ser
130 135 140
Arg Asn Asn Leu Asn Gly Ser Ile Pro Asp Ser Leu Thr Gly Leu Ser
145 150 155 160
Lys Leu Ile Asn Ile Leu Leu Asp Ser Asn Asn Leu Ser Gly Glu Ile
165 170 175
Pro Gln Ser Leu Phe Lys Ile Pro Lys Tyr Asn Phe Thr Ala Asn Asn
180 185 190
Leu Ser Cys Gly Gly Thr Phe Pro Gln Pro Cys Val Thr Glu Ser Ser
195 200 205
Pro Ser Gly Asp Ser Ser Ser Arg Lys Thr Gly Ile Ile Ala Gly Val
210 215 220
Val Ser Gly Ile Ala Val Ile Leu Leu Gly Phe Phe Phe Phe Phe Phe
225 230 235 240
Cys Lys Asp Lys His Lys Gly Tyr Lys Arg Asp Val Phe Val Asp Val
245 250 255
Ala Gly Thr Asn Phe Lys Lys Gly Leu Ile Ser Gly Glu Val Asp Arg
260 265 270
Arg Ile Ala Phe Gly Gln Leu Arg Arg Phe Ala Trp Arg Glu Leu Gln
275 280 285
Leu Ala Thr Asp Glu Phe Ser Glu Lys Asn Val Leu Gly Gln Gly Gly
290 295 300
Phe Gly Lys Val Tyr Lys Gly Leu Leu Ser Asp Gly Thr Lys Val Ala
305 310 315 320
Val Lys Arg Leu Thr Asp Phe Glu Arg Pro Gly Gly Asp Glu Ala Phe
325 330 335
Gln Arg Glu Val Glu Met Ile Ser Val Ala Val His Arg Asn Leu Leu
340 345 350
Arg Leu Ile Gly Phe Cys Thr Thr Gln Thr Glu Arg Leu Leu Val Tyr
355 360 365
Pro Phe Met Gln Asn Leu Ser Val Ala Tyr Cys Leu Arg Glu Ile Lys
370 375 380
Pro Gly Asp Pro Val Leu Asp Trp Phe Arg Arg Lys Gln Ile Ala Leu
385 390 395 400
Gly Ala Ala Arg Gly Leu Glu Tyr Leu His Glu His Cys Asn Pro Lys
405 410 415
Ile Ile His Arg Asp Val Lys Ala Ala Asn Val Leu Leu Asp Glu Asp
420 425 430
Phe Glu Ala Val Val Gly Asp Phe Gly Leu Ala Lys Leu Val Asp Val
435 440 445
Arg Arg Thr Asn Val Thr Thr Gln Val Arg Gly Thr Met Gly His Ile
450 455 460
Ala Pro Glu Cys Ile Ser Thr Gly Lys Ser Ser Glu Lys Thr Asp Val
465 470 475 480
Phe Gly Tyr Gly Ile Met Leu Leu Glu Leu Val Thr Gly Gln Arg Ala
485 490 495
Ile Asp Phe Ser Arg Leu Glu Glu Glu Asp Asp Val Leu Leu Leu Asp
500 505 510
His Val Lys Lys Leu Glu Arg Glu Lys Arg Leu Glu Asp Ile Val Asp
515 520 525
Lys Lys Leu Asp Glu Asp Tyr Ile Lys Glu Glu Val Glu Met Met Ile
530 535 540
Gln Val Ala Leu Leu Cys Thr Gln Ala Ala Pro Glu Glu Arg Pro Ala
545 550 555 560
Met Ser Glu Val Val Arg Met Leu Glu Gly Glu Gly Leu Ala Glu Arg
565 570 575
Trp Glu Glu Trp Gln Asn Leu Glu Val Thr Arg Gln Glu Glu Phe Gln
580 585 590
Arg Leu Gln Arg Arg Phe Asp Trp Gly Glu Asp Ser Ile Asn Asn Gln
595 600 605
Asp Ala Ile Glu Leu Ser Gly Gly Arg
610 615
<210>44
<211>844
<212>DNA
<213>拟南芥
<400>44
ggcgaaaacc atggtggcgc aaaacagtcg gcgggagctt ctagcagctt ccctgatcct 60
aactttagct ctaattcgtc taacggaagc aaactccgaa ggggacgctc ttcacgcgct 120
tcgccggagc ttatcagatc cagacaatgt tgttcagagt tgggatccaa ctcttgttaa 180
tccttgtact tggtttcatg tcacttgtaa tcaacaccat caagtcactc gtctggattt 240
ggggaattca aacttatctg gacatctagt acctgaactt gggaagcttg aacatttaca 300
atatcttgaa ctctacaaaa acgagattca aggaactata ccttctgagc ttggaaatct 360
gaagagtcta atcagtttgg atctgtacaa caacaatctc accgggaaaa tcccatcttc 420
tttgggaaaa ttgaagtcac ttgttttttt gcggcttaac gaaaaccgat tgaccggtcc 480
tattcctaga gaactcacag ttatttcaag ccttaaagtt gttgatgtct cagggaatga 540
tttgtgtgga acaattccag tagaaggacc ttttgaacac attcctatgc aaaactttga 600
gaacaacctg agattggagg gaccagaact actaggtctt gcgagctatg acaccaattg 660
cacttaaaaa gaagttgaag aacctataaa gaagaatgtt aggtgacctt gtaagaactc 720
tgtaccaagt gtttgtaaat ctatatagag ccttgtttca tgttatatat gaaagctttg 780
agagacagta acttgcaatg tattggtatt ggtagaaaaa gttgaaatga gaattgcttt 840
gtaa 844
<210>45
<211>218
<212>PRT
<213>拟南芥
<400>45
Met Val Ala Gln Asn Ser Arg Arg Glu Leu Leu Ala Ala Ser Leu Ile
1 5 10 15
Leu Thr Leu Ala Leu Ile Arg Leu Thr Glu Ala Asn Ser Glu Gly Asp
20 25 30
Ala Leu His Ala Leu Arg Arg Ser Leu Ser Asp Pro Asp Asn Val Val
35 40 45
Gln Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val
50 55 60
Thr Cys Asn Gln His His Gln Val Thr Arg Leu Asp Leu Gly Asn Ser
65 70 75 80
Asn Leu Ser Gly His Leu Val Pro Glu Leu Gly Lys Leu Glu His Leu
85 90 95
Gln Tyr Leu Glu Leu Tyr Lys Asn Glu Ile Gln Gly Thr Ile Pro Ser
100 105 110
Glu Leu Gly Asn Leu Lys Ser Leu Ile Ser Leu Asp Leu Tyr Asn Asn
115 120 125
Asn Leu Thr Gly Lys Ile Pro Ser Ser Leu Gly Lys Leu Lys Ser Leu
130 135 140
Val Phe Leu Arg Leu Asn Glu Asn Arg Leu Thr Gly Pro Ile Pro Arg
145 150 155 160
Glu Leu Thr Val Ile Ser Ser Leu Lys Val Val Asp Val Ser Gly Asn
165 170 175
Asp Leu Cys Gly Thr Ile Pro Val Glu Gly Pro Phe Glu His Ile Pro
180 185 190
Met Gln Asn Phe Glu Asn Asn Leu Arg Leu Glu Gly Pro Glu Leu Leu
195 200 205
Gly Leu Ala Ser Tyr Asp Thr Asn Cys Thr
210 215
<210>46
<211>1154
<212>DNA
<213>拟南芥
<400>46
accaatcgca taatcgattt cttccaactt caataaaggg gaaccaacgt aaccctaatt 60
ttgctttctc ctctttgttc agaaaatttt ccctttactc tcaaattcct tttcgatttc 120
cctctcttaa acctccgaaa gctcacatgg cgtctcgaaa ctatcggtgg gagctcttcg 180
cagcttcgtt aaccctaacc ttagctttga ttcacctggt cgaagcaaac tccgaaggag 240
atgctctcta cgctcttcgc cggagtttga cagatccaga ccatgtcctc cagagctggg 300
atccaactct tgttaatcct tgtacctggt tccatgtcac ctgtaaccaa gacaaccgcg 360
tcactcgtgt ggatttggga aattcaaacc tctctggaca tcttgcgcct gagcttggga 420
agcttgaaca tttacagtat ctagagctct acaaaaacaa catccaagga actatacctt 480
ccgaacttgg aaatctgaag aatctcatca gcttggatct gtacaacaac aatcttacag 540
ggatagttcc cacttctttg ggaaaattga agtctctggt ctttttacgg cttaatgaca 600
accgattgac cggtccaatc cctagagcac tcacggcaat cccaagcctt aaagttgttg 660
acgtctcaag caatgatttg tgtggaacaa tcccaacaaa cggacccttt gctcacattc 720
ctttacagaa ctttgagaac aacccgagat tggagggacc ggaattactc ggtcttgcaa 780
gctacgacac taactgcacc tgaaacaact ggcaaaacct gaaaatgaag aattgggggg 840
tgaccttgta agaacacttc accactttat caaatatcac atctattatg taataagtat 900
atatatgtag taaaaacaaa aaaaatgaag aatcgaatcg gtaatatcat ctggtctcaa 960
ttgagaactt cgaggtctgt atgtaaaatt tctaaatgcg attttcgctt actgtaatgt 1020
tcggttgtgg gattctgaga agtaacattt gtattggtat ggtatcaagt tgttctgcct 1080
tgtctgcatt taacacttgt gttttagatc tgttatataa agccaaaaaa ggttttgtgt 1140
gatttggtac tatc 1154
<210>47
<211>218
<212>PRT
<213>拟南芥
<400>47
Met Ala Ser Arg Asn Tyr Arg Trp Glu Leu Phe Ala Ala Ser Leu Thr
1 5 10 15
Leu Thr Leu Ala Leu Ile His Leu Val Glu Ala Asn Ser Glu Gly Asp
20 25 30
Ala Leu Tyr Ala Leu Arg Arg Ser Leu Thr Asp Pro Asp His Val Leu
35 40 45
Gln Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val
50 55 60
Thr Cys Asn Gln Asp Asn Arg Val Thr Arg Val Asp Leu Gly Asn Ser
65 70 75 80
Asn Leu Ser Gly His Leu Ala Pro Glu Leu Gly Lys Leu Glu His Leu
85 90 95
Gln Tyr Leu Glu Leu Tyr Lys Asn Asn Ile Gln Gly Thr Ile Pro Ser
100 105 110
Glu Leu Gly Asn Leu Lys Asn Leu Ile Ser Leu Asp Leu Tyr Asn Asn
115 120 125
Asn Leu Thr Gly Ile Val Pro Thr Ser Leu Gly Lys Leu Lys Ser Leu
130 135 140
Val Phe Leu Arg Leu Asn Asp Asn Arg Leu Thr Gly Pro Ile Pro Arg
145 150 155 160
Ala Leu Thr Ala Ile Pro Ser Leu Lys Val Val Asp Val Ser Ser Asn
165 170 175
Asp Leu Cys Gly Thr Ile Pro Thr Asn Gly Pro Phe Ala His Ile Pro
180 185 190
Leu Gln Asn Phe Glu Asn Asn Pro Arg Leu Glu Gly Pro Glu Leu Leu
195 200 205
Gly Leu Ala Ser Tyr Asp Thr Asn Cys Thr
210 215
<210>48
<211>1722
<212>DNA
<213>拟南芥
<400>48
atgaagattc aaattcatct cctttactcg ttcttgttcc tctgtttctc tactctcact 60
ctatcttctg agcccagaaa ccctgaagtt gaggcgttga taagtataag gaacaatttg 120
catgatcctc atggagcttt gaacaattgg gacgagtttt cagttgatcc ttgtagctgg 180
gctatgatca cttgctctcc cgacaacctc gtcattggac taggagcgcc gagccagtct 240
ctctcgggag gtttatctga gtctatcgga aatctcacaa atctccgaca agtgtcattg 300
caaaataaca acatctccgg caaaattcca ccggagctcg gttttctacc caaattacaa 360
accttggatc tttccaacaa ccgattctcc ggtgacatcc ctgtttccat cgaccagcta 420
agcagccttc aatatctgga cttgtcttac aacaatctca gtggccctgt tcctaaattc 480
ccagcaagga ctttcaacgt tgctggtaat cctttgattt gtagaagcaa cccacctgag 540
atttgttctg gatcaatcaa tgcaagtcca ctttctgttt ctttgagctc ttcatcagca 600
gataaacaag aggaagggct tcaaggactt gggaatctaa gaagcttcac attcagagaa 660
ctccatgttt atacagatgg tttcagttcc aagaacattc tcggcgctgg tggattcggt 720
aatgtgtaca gaggcaagct tggagatggg acaatggtgg cagtgaaacg gttgaaggat 780
attaatggaa cctcagggga ttcacagttt cgtatggagc tagagatgat tagcttagct 840
gttcataaga atctgcttcg gttaattggt tattgcgcaa cttctggtga aaggcttctt 900
gtttaccctt acatgcctaa tggaagcgtc gcctctaagc ttaaatctaa accggcattg 960
gactggaaca tgaggaagag gatagcaatt ggtgcagcga gaggtttgtt gtatctacat 1020
gagcaatgtg atcccaagat cattcataga gatgtaaagg cagctaatat tctcttagac 1080
gagtgctttg aagctgttgt tggtgacttt ggactcgcaa agctccttaa ccatgcggat 1140
tctcatgtca caactgcggt ccgtggtacg gttggccaca ttgcacctga atatctctcc 1200
actggtcagt cttctgagaa aaccgatgtg tttgggttcg gtatactatt gctcgagctc 1260
ataaccggac tgagagctct tgagtttggt aaaaccgtta gccagaaagg agctatgctt 1320
gaatgggtga ggaaattaca tgaagagatg aaagtagagg aactattgga tcgagaactc 1380
ggaactaact acgataagat tgaagttgga gagatgttgc aagtggcttt gctatgcaca 1440
caatatctgc cagctcatcg tcctaaaatg tctgaagttg ttttgatgct tgaaggcgat 1500
ggattagccg agagatgggc tgcttcgcat aaccattcac atttctacca tgccaatatc 1560
tctttcaaga caatctcttc tctgtctact acttctgtct caaggcttga cgcacattgc 1620
aatgatccaa cttatcaaat gtttggatct tcggctttcg atgatgacga tgatcatcag 1680
cctttagatt cctttgccat ggaactatcc ggtccaagat aa 1722
<210>49
<211>573
<212>PRT
<213>拟南芥
<400>49
Met Lys Ile Gln Ile His Leu Leu Tyr Ser Phe Leu Phe Leu Cys Phe
1 5 10 15
Ser Thr Leu Thr Leu Ser Ser Glu Pro Arg Asn Pro Glu Val Glu Ala
20 25 30
Leu Ile Ser Ile Arg Asn Asn Leu His Asp Pro His Gly Ala Leu Asn
35 40 45
Asn Trp Asp Glu Phe Ser Val Asp Pro Cys Ser Trp Ala Met Ile Thr
50 55 60
Cys Ser Pro Asp Asn Leu Val Ile Gly Leu Gly Ala Pro Ser Gln Ser
65 70 75 80
Leu Ser Gly Gly Leu Ser Glu Ser Ile Gly Asn Leu Thr Asn Leu Arg
85 90 95
Gln Val Ser Leu Gln Asn Asn Asn Ile Ser Gly Lys Ile Pro Pro Glu
100 105 110
Leu Gly Phe Leu Pro Lys Leu Gln Thr Leu Asp Leu Ser Asn Asn Arg
115 120 125
Phe Ser Gly Asp Ile Pro Val Ser Ile Asp Gln Leu Ser Ser Leu Gln
130 135 140
Tyr Leu Asp Leu Ser Tyr Asn Asn Leu Ser Gly Pro Val Pro Lys Phe
145 150 155 160
Pro Ala Arg Thr Phe Asn Val Ala Gly Asn Pro Leu Ile Cys Arg Ser
165 170 175
Asn Pro Pro Glu Ile Cys Ser Gly Ser Ile Asn Ala Ser Pro Leu Ser
180 185 190
Val Ser Leu Ser Ser Ser Ser Ala Asp Lys Gln Glu Glu Gly Leu Gln
195 200 205
Gly Leu Gly Asn Leu Arg Ser Phe Thr Phe Arg Glu Leu His Val Tyr
210 215 220
Thr Asp Gly Phe Ser Ser Lys Asn Ile Leu Gly Ala Gly Gly Phe Gly
225 230 235 240
Asn Val Tyr Arg Gly Lys Leu Gly Asp Gly Thr Met Val Ala Val Lys
245 250 255
Arg Leu Lys Asp Ile Asn Gly Thr Ser Gly Asp Ser Gln Phe Arg Met
260 265 270
Glu Leu Glu Met Ile Ser Leu Ala Val His Lys Asn Leu Leu Arg Leu
275 280 285
Ile Gly Tyr Cys Ala Thr Ser Gly Glu Arg Leu Leu Val Tyr Pro Tyr
290 295 300
Met Pro Asn Gly Ser Val Ala Ser Lys Leu Lys Ser Lys Pro Ala Leu
305 310 315 320
Asp Trp Asn Met Arg Lys Arg Ile Ala Ile Gly Ala Ala Arg Gly Leu
325 330 335
Leu Tyr Leu His Glu Gln Cys Asp Pro Lys Ile Ile His Arg Asp Val
340 345 350
Lys Ala Ala Asn Ile Leu Leu Asp Glu Cys Phe Glu Ala Val Val Gly
355 360 365
Asp Phe Gly Leu Ala Lys Leu Leu Asn His Ala Asp Ser His Val Thr
370 375 380
Thr Ala Val Arg Gly Thr Val Gly His Ile Ala Pro Glu Tyr Leu Ser
385 390 395 400
Thr Gly Gln Ser Ser Glu Lys Thr Asp Val Phe Gly Phe Gly Ile Leu
405 410 415
Leu Leu Glu Leu Ile Thr Gly Leu Arg Ala Leu Glu Phe Gly Lys Thr
420 425 430
Val Ser Gln Lys Gly Ala Met Leu Glu Trp Val Arg Lys Leu His Glu
435 440 445
Glu Met Lys Val Glu Glu Leu Leu Asp Arg Glu Leu Gly Thr Asn Tyr
450 455 460
Asp Lys Ile Glu Val Gly Glu Met Leu Gln Val Ala Leu Leu Cys Thr
465 470 475 480
Gln Tyr Leu Pro Ala His Arg Pro Lys Met Ser Glu Val Val Leu Met
485 490 495
Leu Glu Gly Asp Gly Leu Ala Glu Arg Trp Ala Ala Ser His Asn His
500 505 510
Ser His Phe Tyr His Ala Asn Ile Ser Phe Lys Thr Ile Ser Ser Leu
515 520 525
Ser Thr Thr Ser Val Ser Arg Leu Asp Ala His Cys Asn Asp Pro Thr
530 535 540
Tyr Gln Met Phe Gly Ser Ser Ala Phe Asp Asp Asp Asp Asp His Gln
545 550 555 560
Pro Leu Asp Ser Phe Ala Met Glu Leu Ser Gly Pro Arg
565 570<210>50<211>1301<212>DNA<213>稻<400>50
tctcttctga agctgaagcc ctgcgaaata ggcctttaaa cgctttaagg ttactggatg 60
atcatatcgg cgtaagaccg gtttaaacat ggtttcgctt tgtgaatcca atgtgagtca 120
cgacgtgaca catggcacgt ccttggagct ttagacatat cgaatctgag cactggagtg 180
gccgagtggg tgagcggcca aatccgtttt agacagatcg cactgacacg atgttgatca 240
ttgatactaa taccatttta tcaagcagta gtgttgaaaa aaaaacttat gttctcttca 300
actgtgagat ttcatcccgt ttcaagatga acaagccatg catgtgagat gtgaacagaa 360
ggcagaagac agtggaaaga caggacaaat aagtgaagag ggatcaaatc aatgggcctg 420
acggtttctg aaagttgaca tggaaatcgc cggtgatcac cggtttatac gttatttaaa 480
tctgcgattt ccactttcgt ttgctttcgg ggttccaatt tgagtcacgc acatattctt 540
catcgtgctt tggatctcag caccgtagta acttttggac aaattgcatt cgccgacact 600
aataacatgt tctttttatg ctgctttaca tatactgctt atccacaccc aatcccatgt 660
tcatatatta tgagatggag ggagtaaact ttgttaacag caacattttt tatattaaag 720
catcaactaa ttaaagcaca agatacgcat gttatctcaa taaatcttcc agtgcatgta 780
taaagaagat gtcgccgcta acttagataa tttttgtgac ttttatcctg gccggcataa 840
ttaattcttc cggaaattaa aagctagttt ttccatattc atcagtacag acaagacagc 900
atagtaagcg aagcatacct gacgtgttag ctcattgtaa ctcgatctgg aacactcgat 960
gctagataca gacagacact cctcgtgatg aacgttagca tttagcaaca tacggtgata 1020
aagcagctgg ggatcgatcc atccatccat cgtctttaca cgtacttacc ttgctaaccg 1080
cactgtcgac tcttgcatgt ttgcatgtaa tccaaatgga ccccacgtgg aacatgctca 1140
cagtgctttg cagctgcttt ccaaaatgct ttctttcact tcttccattc ctctgtccac 1200
aaaaaaagta gtgtgttctt gagcctatat aagagagggt cacacgctcc agtcgactca 1260
ccatcgatcc atctgacggt tagttccaag ggaaagaaga a 1301
Claims (26)
1.改良植物生长特性的方法,包括增加植物中亚家族II富含亮氨酸重复受体样蛋白(LRR-II-RLP)编码核酸的表达,和任选地选择具有改良的生长特性的植物,其中所述改良的植物生长特性是相对于相应的野生型植物而言增加的产率,优选增加的种子产率。
2.根据权利要求1的方法,其中通过引入遗传修饰来实现所述增加的表达,优选在编码RKS11的基因座或编码RKS4的基因座或编码LRR-II-RLP蛋白的基因座引入遗传修饰。
3.根据权利要求2的方法,其中通过如下一种或多种方法实现所述遗传修饰:定点诱变、转座子诱变、定向进化、同源重组、TILLING和T-DNA激活。
4.改良植物生长特性的方法,包括在植物中引入和表达分离的LRR-II-RLP编码核酸。
5.根据权利要求4的方法,其中所述改良的植物生长特性是相对于相应的野生型植物而言增加的产率,优选增加的种子产率。
6.根据权利要求4或5的方法,其中所述LRR-II-RLP编码核酸或其变体在植物中过表达。
7.根据权利要求4至6中任一项的方法,其中所述LRR-II-RLP编码核酸是植物来源的,优选来自双子叶植物,优选来自十字花科,更加优选来自拟南芥。
8.根据权利要求4至7中任一项的方法,其中所述LRR-II-RLP编码核酸编码如SEQ ID NO:10或SEQ ID NO:14所示的多肽。
9.根据权利要求4至8中任一项的方法,其中所述LRR-II-RLP编码核酸有效连接于种子特异性启动子。
10.根据权利要求9的方法,其中所述种子特异性启动子如SEQ IDNO:19所示。
11.根据权利要求1至10中任一项的方法,其中所述增加的种子产率选自以下任何一项或多项:(i)增加的种子重量;(ii)增加的(饱满)种子数;(ii)增加的收获指数和(iv)改进的代谢物组成。
12.可根据权利要求1至11中任一项的方法获得的植物。
13.构建体,其包含:
i.LRR-II-RLP编码核酸;
ii.能够驱动(i)中核酸序列表达的一个或多个控制序列;和任选的
iii.转录终止序列。
14.根据权利要求13的构建体,其中所述启动子为种子特异性启动子。
15.根据权利要求14的构建体,其中所述启动子如SEQ ID NO:19所示。
16.用根据权利要求13至15中任一项的构建体转化的植物。
17.产生相对于相应的野生型植物具有改良的生长特性、优选增加的产率、更优选增加的种子产率的转基因植物的方法,该方法包括:
i.在植物细胞中引入和表达LRR-II-RLP编码核酸或其变体;和
ii.在促进植物生长和发育的条件下培养植物细胞。
18.相对于相应的野生型植物具有改良的生长特性、优选增加的产率的转基因植物,其通过向所述植物中引入LRR-II-RLP编码核酸或其变体产生。
19.根据权利要求12、16或18的转基因植物,其中所述植物是诸如大豆、向日葵、芸苔、苜蓿、油菜籽或棉花的作物植物;或者其中所述植物是诸如甘蔗的单子叶植物;或者其中所述植物是诸如稻、玉米、小麦、大麦、粟、黑麦、燕麦或高粱的谷类。
20.根据权利要求12、16、18或19中任一项的植物的可收获部分,以及由它们直接衍生的产品。
21.根据权利要求20的可收获部分,其中所述可收获部分是种子。
22.LRR-II-RLP编码核酸/基因或LRR-II-RLP多肽在改良植物生长特性、特别是提高产率、尤其是种子产率中的用途。
23.根据权利要求22的用途,其中所述增加的种子产率包括以下一项或多项:增加的(饱满)种子数;增加的种子重量;增加的收获指数和改进的代谢物组成。
24.LRR-II-RLP编码核酸/基因或LRR-II-RLP多肽作为分子标记的用途。
25.选自下组的分离的LRR-II-RLP蛋白:
a)无激酶活性的多肽,其包括(i)信号序列,(ii)亮氨酸拉链基序,具有被6个其他氨基酸分隔开的2个或3个Leu残基,(iii)具有2个保守半胱氨酸残基的基序,(iv)4个富含亮氨酸重复单位,各约23个氨基酸残基,(v)富含丝氨酸和脯氨酸残基的结构域,(vi)单个跨膜结构域,和(vii)部分或完整的RELH结构域;
b)基本上缺乏整个激酶结构域的亚家族II富含亮氨酸重复受体样激酶;
c)如SEQ ID NO:10所示的多肽;
d)具有与SEQ ID NO:10所示的一个或多个氨基酸序列具有至少90%序列同一性、优选95%、96%、97%、98%或99%序列同一性的氨基酸序列的多肽,
前提是所述LRR-II-RLP蛋白不是SEQ ID NO:14所示的蛋白。
26.选自下组的分离的核酸:
i)如SEQ ID NO:9所示的核酸序列或其互补链;
ii)编码SEQ ID NO:10所示氨基酸序列的核酸序列;
iii)能够在严谨条件下与上述(i)或(ii)中核酸序列杂交的核酸序列,所述杂交序列编码LRR-II-RLP蛋白;
iv)编码权利要求25中所述蛋白的核酸;
v)上述(i)至(iii)中任一核酸序列的一部分,所述部分编码LRR-II-RLP蛋白,
前提是所述LRR-II-RLP编码核酸并非如SEQ ID NO:13所示或者并不编码SEQ ID NO:14的蛋白。
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PCT/EP2006/063017 WO2006131547A1 (en) | 2005-06-08 | 2006-06-08 | Plants having improved growth characteristics and method for making the same |
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AR053893A1 (es) | 2007-05-23 |
CN101189342B (zh) | 2013-04-03 |
BRPI0611801A2 (pt) | 2008-12-09 |
CA2611253A1 (en) | 2006-12-14 |
US7956240B2 (en) | 2011-06-07 |
AU2006256760B2 (en) | 2011-04-21 |
MX2007015417A (es) | 2008-02-19 |
AU2006256760A1 (en) | 2006-12-14 |
US20090138991A1 (en) | 2009-05-28 |
ZA200710640B (en) | 2009-07-29 |
EP1893760A1 (en) | 2008-03-05 |
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