CN116802304A - 用于转移对亚洲大豆锈病的抗性的多核苷酸和方法 - Google Patents
用于转移对亚洲大豆锈病的抗性的多核苷酸和方法 Download PDFInfo
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Abstract
本文公开了用于改善或增强豆类植物中的病原体抗性的组合物和方法。包含由本文公开的CcRpp2‑R1和CcRpp2‑R3多核苷酸编码的多肽的组合物可用于改善豆类对亚洲大豆锈病(ASR)的抗性。还公开了使用CcRpp2‑R1和CcRpp2‑R3基因制备转基因ASR抗性豆类植物的方法。
Description
相关申请的交叉引用
本申请要求2020年12月23日提交的美国临时申请号63/130,261的权益,该美国临时申请特此通过援引以其全文并入本文。
以电子方式递交的序列表的引用
该序列表的官方副本经由EFS-Web作为ASCII格式的序列表以电子方式提交,文件名为“RTS21584B-WO-PCT_SequenceListing_ST25”,创建于2021年12月3日,且具有163千字节大小,并与本说明书同时提交。包含在此ASCII格式的文件中的序列表是本说明书的一部分并且通过援引以其全文并入本文。
技术领域
本公开涉及可用于通过向植物提供与对亚洲大豆锈病(ASR)的致病原的抗性相关的一个或多个基因来增强豆类植物,并且更特别地大豆植物中的病原体抗性的组合物和方法。本公开进一步涉及能够增强豆类对ASR的抗性的多核苷酸以及使用这些多核苷酸序列制备对ASR具有抗性的转基因豆类植物的方法。
背景技术
大豆(Glycine max)是一种主要的工业用途作物,也是最重要的蛋白质来源作物之一,并且被许多公共卫生组织(包括美国糖尿病协会、美国心脏病协会和美国癌症协会)认为是用于预防疾病和优化健康的关键食物组。亚洲大豆锈病(ASR)是影响大豆的主要作物病害,并且可不利地影响生长和产量。它是由专性生物营养真菌豆薯层锈菌(Phakopsorapachyrhizi)和在较小程度上紧密相关的真菌山马蝗层锈菌(Phakopsora meibomiae)引起的。该病害可引起10%-90%的产量损失。
发明内容
本公开涉及用于鉴定来自豆类物种的ASR抗性基因并将那些基因转化到豆类作物或豆类作物物种诸如大豆(Glycine max)中以产生对ASR具有抗性的植物的组合物和方法。
本文公开了分离的多核苷酸,其包含编码与本文公开的豆类序列具有至少90%氨基酸序列同一性的豆类来源的二元CcRpp2-R1和CcRpp2-R3多肽中的一种或多种的核苷酸序列。在一个实施例中,该多核苷酸是包含异源启动子的重组序列,该异源启动子可操作地连接到编码豆类来源的二元CcRpp2-R1和CcRpp2-R3多肽中的一种或多种的核苷酸序列。已经证明,当与易感植物和/或非转化植物相比时,用表达此类二元多核苷酸的多核苷酸转化的大豆植物显示出增强的对亚洲大豆锈病的抗性。还公开了包含本文所述的多核苷酸的重组DNA构建体,其中CcRpp2-R1和CcRpp2-R3编码序列可操作地连接到异源调控元件,用于在植物细胞中表达CcRpp2-R1和CcRpp2-R3基因产物。
本文公开了有用的多核苷酸,其可包含SEQ ID NO:1或3的核酸序列及其变体,或替代性地由这些核酸序列及其变体组成或基本上由这些核酸序列及其变体组成。由此编码的多肽能够用作二元多肽,并且可用于在豆类作物中赋予对ASR的抗性的组合物和方法中。
本文公开了赋予豆类作物物种(例如,大豆)抗病性的方法,该方法包括用编码异源豆类来源的二元CcRpp2-R1和CcRpp2-R3多核苷酸的核酸序列转化豆类作物物种,这些多核苷酸赋予豆类作物物种病害(例如,ASR)抗病性。
根据一个实施例,提供了一种转基因植物细胞,其中该植物细胞包含编码赋予豆类作物物种病害(例如,ASR)抗病性的多肽的重组多核苷酸,其中所编码的多肽与选自由SEQ ID NO:2和21-36组成的组的氨基酸序列和/或选自由SEQ ID NO:4和48-58组成的组的氨基酸序列具有至少65%、75%、85%、90%、95%或99%序列同一性。
本文公开了一种用重组DNA构建体稳定转化的转基因豆类作物植物,该重组DNA构建体包含编码一种或多种豆类来源的CcRpp2-R1和CcRpp2-R3基因的多核苷酸。在一个方面,该多核苷酸包含一个或多个非豆类来源的CcRpp2-R1和CcRpp2-R3抗性基因和任选的赋予植物病害抗性的另外的非CcRpp2-R1和CcRpp2-R3抗性基因。本文所述的多核苷酸还可包含抗性基因的任何组合。转基因豆类作物植物可包含一种或多种输入性状和/或农艺性状。也考虑从此类转基因豆类作物植物获得种子。此外,本公开的特征还在于稳定转化的转基因豆类作物植物,其包含与本文所述的序列(包括例如SEQ ID NO 1、3、5-20和37-47)具有至少90%或95%序列同一性的豆类来源的二元CcRpp2-R1和CcRpp2-R3多核苷酸。
本文公开了鉴定赋予植物病害(例如,ASR)抗性的一个或多个抗性基因的方法。本文公开了用于检测生物样品中的CcRpp2-R1和CcRpp2-R3抗性基因的方法,其中所述方法包括使用对CcRpp2-R1和CcRpp2-R3抗性基因序列特异性的引物或探针筛选从生物样品回收的核酸序列,任选地其中这些引物和探针在严格洗涤条件下与选自SEQ ID NO 1、3、5-20和37-47的核酸序列杂交。
本文公开了通过将CcRpp2-R1和CcRpp2-R3抗性基因引入先前的ASR易感植物谱系中来产生ASR抗性植物(例如,豆类物种)的方法。在一个实施例中,该方法包括用豆类来源的二元CcRpp2-R1和CcRpp2-R3抗性基因转化植物细胞。在一个实施例中,该方法包括用核酸序列转化植物细胞,该核酸序列包括选自由SEQ ID NO:1、SEQ ID NO:5-12和14-20和SEQID NO:13组成的组的序列和选自由SEQ ID NO:3、SEQ ID NO:37-46和SEQ ID NO:47组成的组的序列。该方法可进一步包括从转化的植物细胞再生转化的植物。在一个方面,该方法包括使转化的植物生长,使得豆类来源的CcRpp2-R1和CcRpp2-R3抗性基因的表达产生显示出增强的对ASR病害抗性的转化的植物。
在一个实施例中,产生包含CcRpp2-R1和CcRpp2-R3抗性基因之一的转基因植物。在该实施例中,通过将包含CcRpp2-R1基因的第一植物与包含CcRpp2-R3基因的第二植物杂交,并选择包含CcRpp2-R1和CcRpp2-R3抗性基因两者的ASR抗性子代植物,来产生表现出增强的ASR抗性的植物。
本文公开了产生豆类植物的方法,该豆类植物是来自与包含本文所述的豆类来源的CcRpp2-R1和CcRpp2-R3二元抗性基因的豆类植物杂交的子代,其中选择保留CcRpp2-R1和CcRpp2-R3二元抗性基因的子代。
本文公开了测定豆类植物对植物病害(例如,ASR)的抗病性的方法。在一个方面,该方法包括将豆类植物的一部分暴露于植物病原体(例如,豆薯层锈菌);在暴露于该植物病原体的该豆类植物上测量植物病害症状;以及将这些植物病害症状与抗病性的参考标准进行比较。
本文公开了增强植物对ASR病害的抗性的方法。在一个方面,该方法包括通过在对ASR抗性的育种计划中将豆类来源的CcRpp2-R1和CcRpp2-R3二元抗性基因渗入种质(例如,豆类作物物种)中来赋予对ASR病原体(例如,豆薯层锈菌)的抗性。该方法的特征在于编码CcRpp2-R1和CcRpp2-R3多肽的豆类来源的CcRpp2-R1和CcRpp2-R3二元抗性基因。在一个方面,CcRpp2-R1和CcRpp2-R3多肽包含与本文公开的豆类来源的CcRpp2-R1和CcRpp2-R3多肽具有至少90%同源性的氨基酸序列。本文所述的方法的特征还在于用该多肽转化的植物,该植物当与易感植物相比时显示出增强的对ASR的抗性。
附图说明
图1是具有参考木豆(C.cajan)支来LGCc02的CcRpp2的精细作图的示意图。其中在远侧(左)有一个功能获得和丧失重组体,在近侧(右)有3个功能丧失重组体,将间隔界定为121,252bp。使用位于该间隔中的三个标志筛选的Rd BAC文库:Rdint_264620、dCAPS_393933和Rdint_385686。
具体实施方式
定义
在描述和要求保护本发明时,将根据以下陈述的定义使用以下术语。
如本文所使用的术语“约”意指大于或小于所陈述的值或值的范围的百分之十,但并非旨在仅针对此更宽泛的定义来指定任何值或值的范围。术语“约”之后的每个值或值的范围也旨在涵盖所陈述绝对值或值的范围的实施例。
如本文所使用的,术语“植物”包括整株植物以及任何后代,细胞、组织或植物的一部分。
术语“植物部位”包括植物的任何部位,包括例如且不限于:种子(包括成熟种子和未成熟种子);植物切段;植物细胞;植物细胞培养物;植物器官(例如花粉、胚、花朵、果实、枝、叶、根、茎和外植体)。植物组织或植物器官可以是种子、愈伤组织或组织成结构或功能单元的任何其他组的植物细胞。植物细胞或组织培养物可能能够再生具有从其获得细胞或组织的植物的生理和形态特征的植物,并且能够再生具有与该植物基本上相同的基因型的植物。相反,一些植物细胞不能再生以产生植物。植物细胞或组织培养物中的可再生细胞可以是胚胎、原生质体、分生组织细胞、愈伤组织、花粉、叶、花药、根、根尖、花丝、花、籽粒、穗、穗轴、苞叶或茎秆。植物的可收获部分可以是植物的任何有用的部分,包括例如但不限于:花、花粉、秧苗、块茎、叶、茎、果实、种子和根。
植物细胞是植物的结构和生理单位。如本文所用,植物细胞包括原生质体和具有细胞壁的原生质体。植物细胞可以呈分离的单个细胞或细胞聚集物(例如,易碎的愈伤组织和培养的细胞)的形式,并且可以是更高组织的单元(例如,植物组织、植物器官和植物)的部分。因此,植物细胞可以是原生质体、产生配子的细胞或可以再生为整株植物的细胞或细胞集合。这样,包含多个植物细胞并且能够再生为整株植物的种子在本文的实施例中被认为是“植物部分”。
如本文所用,术语“原生质体”是指其细胞壁被完全或部分去除而其脂双层膜裸露的植物细胞。通常,原生质体是无细胞壁的分离的植物细胞,其具有再生为细胞培养物或整株植物的潜能。
如本文所使用的,术语“天然的”或“自然的”定义了天然存在的状况。“天然DNA序列”是自然界中存在的DNA序列,其是通过自然手段或传统育种技术产生的,但不是通过基因工程(例如,使用分子生物学/转化技术)产生的。
如本文所用,“内源序列”定义了多核苷酸、基因或多肽在生物体或生物体基因组中的其天然位置的天然形式。
如本文所用,术语“外源序列”是已经导入细胞的任何核酸序列,其中导入的核酸序列的至少一部分对于该宿主细胞不是天然的。例如,外源DNA序列可包含来自另一物种的序列。
如本文所用,术语“异源序列”是已经从其天然位置去除并插入新位置从而改变已移动的核酸序列侧翼的序列的任何核酸序列。异源序列可以是源于外来物种的外源序列,或者如果源于相同物种,则是通过蓄意人为干预从其在组合物和/或基因组基因座中的天然形式进行实质性修饰得到的序列。例如,异源启动子是已与编码序列可操作地连接的启动子序列,从而形成重组核酸序列,该编码序列不与启动子天然连接。
如本文所用,术语“分离的”意指已从其天然环境中去除。
如本文所用,术语“纯化的”涉及以基本上不含通常与在天然或自然环境中的分子或化合物相关的污染物的形式分离分子或化合物,并且意指由于与原始组合物中的其他组分分离而纯度提高了。术语“纯化的核酸”在本文中用于描述已与包括但不限于多肽、脂质和碳水化合物的其他化合物分离的核酸序列。
术语“多肽”、“肽”以及“蛋白质”可互换使用,是指氨基酸残基的聚合物。该术语也适用于其中一个或多个氨基酸是对应天然存在的氨基酸的化学类似物或经修饰的衍生物的氨基酸聚合物。
“互补序列”在本文中用于指与给定核酸序列互补的核酸序列,使得其可以与该给定核酸序列杂交从而形成稳定的双链体。在一些实施例中,核酸序列是完全互补的,具有100%序列同一性。
“多核苷酸序列变体”在本文中用于指除遗传密码的简并性之外编码相同多肽的核酸序列。
本文在两个核酸或多肽序列的上下文中使用的术语“序列同一性”或“同一性”是指当在指定比较窗口上进行最大对应性比对时两个序列中相同的残基。
如本文所用,术语“序列同一性百分比”是指通过在比较窗口上比较两个最佳比对序列(例如,核酸序列和氨基酸序列)确定的值,其中用于两个序列的最佳比对,与参考序列(其不包含添加或缺失)相比,比较窗口中的序列部分可能包含添加或缺失(即,缺口)。通过如下方式来计算百分比:确定两个序列中出现相同核苷酸或氨基酸残基的位置数以产生匹配位置数,将匹配位置数除以比较窗口中的位置总数,并且将结果乘以100以得出序列同一性百分比。
用于比较的序列比对方法是本领域熟知的。各种程序和比对算法描述于例如:Smith和Waterman(1981)Adv.Appl.Math.[应用数学进展]2:482;Needleman和Wunsch(1970)J.Mol.Biol.[分子生物学杂志]48:443;Pearson和Lipman(1988)Proc.Natl.Acad.Sci.U.S.A.[美国科学院院报]85:2444;Higgins和Sharp(1988)Gene[基因]73:237-44;Higgins和Sharp(1989)CABIOS[计算机应用生物科学]5:151-3;Corpet等人(1988)Nucleic Acids Res.[核酸研究]16:10881-90;Huang等人(1992)Comp.Appl.Biosci.[生物科学中的计算机应用]8:155-65;Pearson等人(1994)MethodsMol.Biol.[分子生物学方法]24:307-31;Tatiana等人(1999)FEMS Microbiol.Lett.[FEMS微生物学快报]174:247-50。序列比对方法和同源性计算的详细考虑可见于例如Altschul等人(1990)J.Mol.Biol.[分子生物学杂志]215:403-10。国家生物技术信息中心(NCBI)基本局部比对搜索工具(BLASTTM;Altschul等人(1990))可从几个来源获得,包括国家生物技术信息中心(马里兰州贝塞斯达(Bethesda,MD)),以及在互联网上,用于与几个序列分析程序结合使用。如何使用此程序确定序列同一性的描述可在互联网上在BLASTTM的“帮助”部分下获得。对于核酸序列的比较,可以使用默认参数采用BLASTTM(Blastn)程序的“Blast 2序列”功能。当通过这种方法评估时,与参考序列具有甚至更大相似性的核酸序列将显示出增加的同一性百分比。本领域的技术人员可以确定用于比对序列的适当参数,包括在进行比较的序列的全长度上实现最大比对所需的任何算法。
导致特定严格性程度的杂交条件将根据选择的杂交方法的性质以及杂交核酸序列的组成和长度而变化。通常,杂交温度和杂交缓冲液的离子强度(尤其是Na+和/或Mg++浓度)将决定杂交的严格性,尽管洗涤时间也会影响严格性。关于获得特定严格性程度所需的杂交条件的计算是本领域普通技术人员已知的,并且在例如以下文献中讨论:Sambrook等人(编辑)Molecular Cloning:A Laboratory Manual[分子克隆:实验室手册],第2版,第1-3卷,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY[冷泉港实验室出版社,纽约州冷泉港市],1989,第9和11章;以及Hames和Higgins(编辑)Nucleic AcidHybridization[核酸杂交],IRL Press,Oxford[牛津IRL出版社],1985。关于核酸杂交的更详细的说明和指导可见于例如Tijssen,“Overview of principles of hybridizationand the strategy of nucleic acid probe assays[杂交原理和核酸探针检测策略概述],”Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes[生物化学和分子生物学实验室技术-核酸探针杂交],第I部分,第2章,Elsevier,NY[纽约爱思唯尔],1993;以及Ausubel等人编辑,Current Protocols in Molecular Biology[分子生物学当前方案],第2章,GreenePublishing and Wiley-Interscience,NY[格林出版与威利交叉科学出版社,纽约],1995。
如本文所用,“严格条件”涵盖仅在杂交分子与靶核酸分子内的序列之间的错配率小于20%时才发生杂交的条件。“严格条件”包括进一步的严格的特定水平。因此,如本文所用,“中严格性”条件是指在这些条件下序列错配率超过20%的分子不会杂交;“高严格性”条件是指在这些条件下错配率超过10%的序列不会杂交;以及“非常高严格性”条件是指在这些条件下错配率超过5%的序列不会杂交。以下是代表性的非限制性的杂交条件。
高严格性条件(检测共享至少90%序列同一性的序列):在5x SSC缓冲液(其中SSC缓冲液含有洗涤剂诸如SDS,和另外试剂如鲑精DNA、EDTA等)中在65℃下杂交16小时;在2xSSC缓冲液(其中SSC缓冲液含有洗涤剂诸如SDS,和另外试剂如鲑精DNA、EDTA等)中在室温下洗涤两次,每次15分钟;以及在0.5x SSC缓冲液(其中SSC缓冲液含有洗涤剂诸如SDS,和另外试剂如鲑精DNA、EDTA等)中在65℃下洗涤两次,每次20分钟。
中严格性条件(检测共享至少80%序列同一性的序列):在5x-6x SSC缓冲液(其中SSC缓冲液含有洗涤剂诸如SDS,和另外试剂如鲑精DNA、EDTA等)中在65℃-70℃下杂交16-20小时;在2x SSC缓冲液(其中SSC缓冲液含有洗涤剂诸如SDS,和另外试剂如鲑精DNA、EDTA等)中在室温下洗涤两次,每次5-20分钟;以及在1x SSC缓冲液(其中SSC缓冲液含有洗涤剂诸如SDS,和另外试剂如鲑精DNA、EDTA等)中在55℃-70℃下洗涤两次,每次30分钟。
非严格控制条件(共享至少50%序列同一性的序列将杂交):在6x SSC缓冲液(其中SSC缓冲液含有洗涤剂诸如SDS,和另外试剂如鲑精DNA、EDTA等)中在室温至55℃下杂交16-20小时;在2x-3x SSC缓冲液(其中SSC缓冲液含有洗涤剂诸如SDS,和另外试剂如鲑精DNA、EDTA等)中在室温至55℃下洗涤至少两次,每次20-30分钟。
可操作地连接:当第一核苷酸序列与第二核苷酸序列处于功能关系时,第一核苷酸序列与第二核苷酸序列“可操作地连接”。例如,如果启动子影响编码序列的转录或表达,则启动子可操作地连接到该编码序列。当重组产生时,可操作地连接的核苷酸序列通常是连续的,并且在需要连接两个蛋白质编码区的情况下,它们在同一阅读框中。然而,核苷酸序列不必连续地可操作地连接。
当关于调控序列和编码序列使用时,术语“可操作地连接”意指调控序列影响所连接的编码序列的表达。“调控序列”、“调控元件”或“控制元件”是指影响相关编码序列的转录、RNA加工或稳定性或翻译的时间和水平/量的核苷酸序列。调控序列可以包括启动子、翻译前导序列、内含子、增强子、茎环结构、阻遏物结合序列、终止序列、聚腺苷酸化识别序列等等。特定的调控序列可以位于与之可操作地连接的编码序列的上游和/或下游。同样,可操作地连接到编码序列的特定调控序列可以位于双链核酸分子的相关互补链上。
当用于提及两个或更多个氨基酸序列时,术语“可操作地连接”意指第一氨基酸序列与至少一个另外的氨基酸序列处于功能关系。
术语“抗性”在本文中用于意指植物中由植物病原体引起的一种或多种病害症状的缺乏或减少。抗性可意指当与对该病害易感的植物或不含减少一种或多种病害症状的有效抗性基因(诸如CcRpp2-R1和CcRpp2-R3基因)的植物相比,病害症状(诸如病变数、脱叶和相关产量损失)减少、最小化或减轻。此外,抗性可包括防止或延迟病原体(例如,真菌)的增殖。
“植物病原体”或“真菌病原体”在本文中可用于意指例如层锈菌属(Phakopsora)的真菌病原体,包括豆薯层锈菌和山马蝗层锈菌(Phakopsora meibomiae)物种。已知这些物种在植物中引起ASR。例如,植物病害或豆类作物物种病害可以是ASR。
术语“抗病性基因”或“抗性基因”在本文中用于意指编码能够增强或改善植物的防御或免疫系统应答的蛋白质或多肽的基因。
在本公开中,“核酸”是指单链或双链形式的脱氧核糖核苷酸或核糖核苷酸聚合物,并且除非另外限制,否则涵盖由于以与天然存在的核苷酸相似的方式杂交至单链核酸而具有天然核苷酸的基本性质的已知类似物(例如肽核酸)。
术语“编码”在本文中用于意指核酸包含所需的信息,其通过使用密码子来指导核苷酸序列(例如,豆类序列)翻译成指定的蛋白质而指定。编码蛋白质的核酸在该核酸的翻译区之内可以包含非翻译序列(例如,内含子)或可能缺乏此类插入的非翻译序列(例如,在cDNA中)。
本公开的各方面涵盖分离的或重组的多核苷酸或蛋白质组合物。“分离的”或“重组的”核酸分子(或DNA)在本文中用于指不再在其自然环境中的核酸序列(或DNA),例如在体外或异源重组细菌或植物宿主细胞中。当通过重组技术产生时,“分离的”或“重组的”核酸分子或其生物活性部分基本上不含其他细胞材料或培养基,或者当化学合成时基本上不含化学前体或其他化学品。
如本文所用的术语“抑制(inhibit、inhibition、inhibiting)”、“减少的”、“减少”等意指靶基因产物的表达或功能的任何降低,包括表达或功能的任何相对降低,直至并包括靶基因产物的表达或功能的完全消除。
术语“增加”、“增加的”、“增强”、“增强的”等在本文中用于意指与易感植物相比的靶基因(例如,TIR基因)产物的表达、功能或活性的任何加强或获得或升高,从而提供对一种或多种病原体(例如,层锈菌属物种)或对病害(例如,ASR)增加的抗性。此外,如本文所用的术语“诱导”或“增加”可意指靶基因产物的更高表达,使得水平相对于缺乏本公开的靶基因或蛋白质的细胞或植物增加10%或更多、50%或更多或100%。
如本文所用的术语“表达”是指例如产生多核苷酸的生物合成或过程,包括基因产物的转录和/或翻译。例如,本公开的多核苷酸可以从DNA模板转录(例如转录成mRNA或其他RNA转录物)和/或转录的mRNA随后被翻译成多肽或蛋白质的过程。
术语“基因产物”可指例如转录物和编码的多肽。基因产物(即,目的基因产物)的表达或功能的抑制(或增加)可以在任何两种植物之间的比较的背景下进行,例如基因产物在遗传改变的植物中的表达或功能相对于该基因产物在对应的但易感的野生型植物或其他易感植物中的表达或功能。野生型植物中基因产物的表达水平可能不存在。例如,“野生型”植物可以是不表达外源CcRpp2-R1和/或CcRpp2-R3核酸或外源CcRpp2-R1和/或CcRpp2-R3蛋白的植物、植物细胞或植物部分。
替代性地,靶基因产物的表达或功能的抑制(或增加)可在同一植物内或植物之间的植物细胞、细胞器、器官、组织或植物部分之间进行比较,并且包括同一植物内或植物之间的发育或时间阶段之间的比较。在转录或翻译水平下调靶基因产物表达或下调靶基因产物功能活性的任何方法或组合物可用于实现对靶基因产物表达或功能的抑制。类似地,在转录或翻译水平诱导或上调靶基因产物的表达,或增加或激活或上调靶基因产物的功能活性的任何方法或组合物可用于实现靶基因或蛋白质的增加的表达或功能。抑制或增强基因表达的方法是本领域熟知的。
如本文所用的术语“引入”定义了通过使用传统育种或重组转化技术改变细胞/植物内容物的方法。当使用重组转化技术时,核酸或蛋白质穿过植物细胞膜或细胞壁进入植物细胞内部。将多核苷酸引入植物的方法是本领域已知的,包括产生稳定转化方法或瞬时转化方法的程序。这些方法包括使用磷酸钙转染、聚凝胺、原生质体融合、PEG、电穿孔、超声方法(例如,声致穿孔)、脂质体、显微注射、裸DNA、质粒载体、病毒载体(游离型和整合型两者)以及将克隆的基因组DNA、cDNA、合成DNA或其他外源遗传材料引入宿主细胞的任何其他熟知的方法。
“稳定转化”或“稳定转化的”意指引入植物中的核苷酸构建体整合到该植物的基因组中,并且能够被其子代遗传。如本文所用,“瞬时转化”意指将多核苷酸引入该植物中并且不整合到该植物的基因组中,或者将多肽引入植物中。
术语“转化”在本文中用于意指将例如核酸片段转移到宿主生物体的基因组中,导致遗传稳定的遗传。含有转化的核酸片段的宿主生物体被称为“转基因的”生物体。术语“宿主细胞”是指其中发生重组DNA构建体转化的细胞,并且可包括酵母细胞、细菌细胞和/或植物细胞。植物转化方法的实例包括农杆菌属介导的转化和粒子轰击。然后可通过本领域技术人员已知的方法将转化的植物细胞用于再生转化的植物。
术语“转基因”在本文中用于指植物,包括源自植物的任何部分,诸如细胞、组织或器官,其中外源核酸(例如包含一种或多种核酸的重组构建体、载体或表达盒)通过遗传工程方法(诸如农杆菌属转化)整合到基因组中。通过实施基因技术方法,外源核酸被稳定地整合到染色体中,使得连续世代也可以是转基因的。如本文所用,“转基因”还涵盖包括植物杂交和/或天然重组的生物过程。
实施例
作物病害引起严重的作物管理问题,并且有时可能导致作物全面歉收。亚洲大豆锈病是对世界大豆生产的威胁,并且目前通过使用叶面杀真菌剂来解决。商业植物品系中的稳定和可靠的遗传抗性是与大豆作物产量相关的重要特征,并且目前,还没有获得对由豆薯层锈菌引起的亚洲大豆锈病具有完全抗性的商业培育的大豆栽培品种。ASR的致病原,即豆薯层锈菌和山马蝗层锈菌,感染来自广泛范围的豆类植物的叶组织(分别为,17个属中的至少31个物种;Slaminko等人(2008)Plant Dis.[植物病害],92:797-771;以及19个属中的至少42个物种;Frederick等人(2002)Mycology[真菌学],92:217-227)。总计,其他属中的另外152个物种已被描述为豆薯层锈菌的潜在宿主(Bonde等人(2008)Plant Dis.[植物病害],92:30-38;Goellner等人(2010)Molecular Plant Pathology[分子植物病理学],11:169-177;Ono等人(1992)Mycol.Res.[真菌学研究],96(10):825-850;以及Slaminko等人(2008)Plant Dis.[植物病害],92:797-771)。目前,杀真菌剂应用是唯一可商业获得的减轻ASR的方法。除了杀真菌剂之外,在南美国家如巴西可以使用另一种管理策略来减轻ASR。特别地,使用在生长季节开始时种植(允许作物避免病害有利条件)和无宿主期的短周期品种减少了初级接种物的量。
目前,还没有完全抵抗豆薯层锈菌的商业种植大豆栽培品种。大豆对豆薯层锈菌的抗性是罕见的;USDA评价了整个美国大豆种质资源,并且发现少于5%对豆薯层锈菌具有抗性或部分抗性。此外,这些大豆成员中可用的基因仅提供分离株特异性的抗性;因此,这些来源在诸如存在多个种族的田间条件下不能提供持久的抗性。
鉴于ASR是大豆生产的主要威胁,鉴定抗性基因的来源并将这些转基因掺入豆类种质诸如大豆中以增强保护是有益的。为了鉴定新的抗性基因,筛选几种非大豆豆类物种对豆薯层锈菌的抗性变异。鉴定了显性抗性基因并证实其为抗性(R)基因的TIR-TIR类的成员。当转移到大豆中时,本文公开的二元CcRpp2-R1和CcRpp2-R3抗性基因可通过异源表达提供对豆薯层锈菌的抗性。
植物可通过多种细胞机制防御自身。目前的理解是,植物免疫系统由细胞外部的受体(通常称为第一层免疫)和细胞内部的受体(通常称为第二层免疫)组成。这两组受体均可检测到病原体并对其做出响应。第一层响应于病原体的主要成分,导致激活病原体相关分子模式(PAMP)触发的免疫。成功的病原体通过分泌称为“效应蛋白”或“效应物”的分子克服PAMP触发的免疫,这些分子定位于植物质外体或被吸收到植物细胞中。效应物操纵宿主细胞功能以抑制宿主免疫应答,从而促进感染的建立或以其他方式增强病原体的生长条件,例如通过确保对营养物的可利用性。在一些情况下,植物进化出第二层免疫,其中R基因产物识别特定效应物的活性,导致效应物触发的免疫。R基因通常编码以C末端富含亮氨酸的重复序列(LRR)和核苷酸结合位点(NBS)结构域为特征的蛋白质。此类核酸结合LRR被称为核苷酸结合LRR(NLR)蛋白。NBS结构域根据所结合的核苷酸起分子开关的作用:ADP-在静止状态结合以及ATP-在活化状态结合。通常认为LRR结构域参与效应物识别和自身抑制(Ting等人,Immunity[免疫],28(2008),第285-287页)。典型的植物NLR几乎普遍地以另外的卷曲螺旋(CC)或Toll/白介素-1受体(TIR)N-末端结构域为特征。这些N-末端结构域用于将植物NLR分类为两个主要的组,称为CNL(CC-NLR)和TNL(TIR-NLR)。已经证明CC和TIR结构域在二聚体和寡聚体的形成中均起关键作用。
在植物中,TIR结构域出现在(TIR-NLR)家族抗性受体(R蛋白)的主要亚类的N末端,其在感知病原体效应物后触发防御反应(Dodds和Rathjen,Nat Rev Genet.[自然综述:遗传学]2010;11:539-548)。最近的发现表明,R蛋白中的TIR结构域起NAD+切割酶的作用以触发局部细胞死亡,称为过敏反应(HR)。参见:TIR-only protein regulates cell deathin plants[仅TIR的蛋白调节植物细胞死亡]Proceedings of the National Academy ofSciences[美国科学院院报]2017年3月,114(10)E2053-E2062;DOI:10.1073/pnas.16209731142)以及TIR domains of plant immune receptors are NAD+-cleavingenzymes that promote cell death.[植物免疫受体的TIR结构域是促进细胞死亡的NAD+切割酶]SCIENCE[科学]2019年8月23日:799-803。植物R蛋白的LRR结构域似乎是识别特异性的主要决定因素。NB结构域与哺乳动物核苷酸结合寡聚化结构域(NOD)样受体(NLR)共享,后者也充当先天免疫应答和凋亡的调节剂。证据表明NB结构域可以结合并水解核苷酸,并且结合的ATP或ADP的存在可以确定R蛋白是否处于活性或非活性信号传导状态。然而,在植物R蛋白中,对效应物识别与防御信号传导的激活相关的机制知之甚少。
作为宿主和病原体之间“武装竞赛”的结果,病原体效应物可以具有无毒或毒力效应。效应物的毒力活性与正常宿主细胞功能的操作或病原体对宿主免疫应答的抑制有关,以便建立成功的感染。在无毒的情况下,由对应植物R蛋白的识别激活宿主免疫或防御反应,导致程序性细胞死亡和对病原体的抗性。
本文公开的核酸和多肽可用于产生表现出真菌抗性的转基因植物以及用于赋予或增强或增加植物(例如豆类作物物种)的真菌抗性的方法中。本文公开的方法和组合物可包含以下多肽和多核苷酸序列:
SEQ ID NO:1来自木豆(Cajanus cajan)的CcRpp2-R1Aa编码序列(多核苷酸序列)。
SEQ ID NO:2:CcRpp2-R1Aa(多肽序列)。
SEQ ID NO:3:来自木豆的CcRpp2-R3Aa编码序列(多核苷酸序列)。
SEQ ID NO:4:CcRpp2-R3Aa(多肽序列)。
在另一个实施例中,本文公开的SEQ ID NO:5-20的CcRpp2-R1多核苷酸和SEQ IDNO:37-47的CcRpp2-R3多核苷酸以及SEQ ID NO:21-36的相应CcRpp2-R1多肽和SEQ ID NO:48-58的CcRpp2-R3多肽可用于产生表现出真菌抗性的转基因植物,以及用于赋予或增强或增加植物(例如豆类作物物种)真菌抗性的方法中。
在一些实施例中,提供了CcRpp2-R1多肽以及其氨基酸取代、缺失、插入、片段以及它们的组合,该多肽与SEQ ID NO:2和21-36中任一项相比具有至少约40%、45%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大序列同一性。
在一些实施例中,提供了CcRpp2-R3多肽以及其氨基酸取代、缺失、插入、片段以及它们的组合,该多肽与SEQ ID NO:4和48-58中任一项相比具有至少约40%、45%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大序列同一性。
本公开的多肽可以从本文公开的核酸或通过使用标准分子生物学技术来产生。例如,本公开的CcRpp2-RI和/或CcRpp2-R3蛋白可以通过在适当的宿主细胞中表达实施例的重组核酸或替代性地通过离体程序的组合来产生。
本文公开的组合物和方法可用于保护植物免受真菌病原体侵害。宿主和病原体之间的相互作用可用“免疫”到“易感性”的连续体来描述。术语“免疫性”或“免疫的”在本文中用于意指不存在任何肉眼可见的病害症状。术语“抗性”在本文中用于意指存在肉眼可见的病变,没有或有限的孢子形成,和/或任何病害症状的范围或程度的降低和/或进展的延迟,并且可以例如表现为病变的数量减少或具有孢子形成减少的病变。如本文所用,术语对ASR的“易感性”或短语“缺乏抗性”是指孢子形成水平等于或高于参考标准(诸如栽培品种Williams或Peking)中观察到的孢子形成水平的病变的发生。
可实施本公开的方法,例如,以提供大豆对专性生物营养真菌豆薯层锈菌(ASR的主要致病原)或山马蝗层锈菌的增强的抗性。例如,可将对真菌病原体的抗性增加或增强与易感植物(诸如Williams或Peking)的反应进行比较。抗性可以变化并且与在植物或植物部分(例如,叶)上观察到的病害症状(例如,病变)的比例(即,百分比)相关。可给出免疫力、抵抗力和易感性的数值分数或值。例如,抗性的数值分数表示植物表现出对植物病害(例如ASR)的抗性程度。数值分数还可用于比较例如目的植物(例如转基因豆类作物植物)与易感植物(例如Williams或Peking)或参考标准之间的抗性程度。
本文公开的用于抗性的方法和组合物涉及从豆类物种分离一种或多种抗性基因,并随后将这些抗性基因中的一种或多种抗性基因转移至另一植物,例如大豆,以经由同源或异源表达提供对层锈菌属物种的抗性。本公开的一个方面包括将功能性TIR基因转移至性相容或不相容的物种以产生抗病性。本文所述的多肽和TIR基因(例如,CcRpp2-R1和CcRpp2-R3多肽以及CcRpp2-R1和CcRpp2-R3基因)可单独使用或与其他抗性基因诸如R基因(包括NB-LRR抗性基因)堆叠使用或与非R基因(包括非NB-LRR抗性基因)堆叠使用以提供对ASR的抗性。
因此,本公开的转基因方法可单独使用或与其他策略组合使用以在植物中产生或赋予ASR抗性。其他有用的策略包括但不限于阻断效应物的功能活性、抑制病原体或病原体因子(例如,真菌)摄入宿主细胞(例如,植物细胞)和/或用于抗性的常规育种。
在一个实施例中,本公开的转基因方法可与CcRpp1多核苷酸(例如SEQ ID NO:59)和由其编码的多肽(SEQ ID NO:60)的转基因表达组合使用(还参见美国专利申请公布号US2018-0103600中公开的NB-LRR2多核苷酸和由其编码的多肽,其通过援引以其全文并入本文)。此类方法可以是通过含有CcRpp1基因和本文公开的CcRpp2-R1和CcRpp2-R3基因的二元物的育种堆叠物或分子堆叠物的方式。
本公开的方法可提供或增强植物的抗性,使得病害(诸如ASR)的致病原不再繁殖。术语“增强”意指改善、增加、放大、倍增、升高和/或提高,从而减少一种或多种病害症状。因此,植物(例如大豆)当与对层锈菌属物种敏感或耐受的植物相比时表现出增加的对病害(例如ASR)的抗性。在一个方面,本文所述的方法可以减少豆类植物病害(例如,ASR)的一种或多种症状(即,病害症状)。一种方法可包括将转基因豆类作物植物(例如大豆)暴露于豆类植物病害,导致转基因豆类作物植物对植物病害具有增强的抗性。在一些方面,转基因豆类作物植物包含CcRpp2-R1和CcRpp2-R3多核苷酸。一种或多种豆类来源的CcRpp2-R1和CcRpp2-R3多核苷酸可与本文公开的序列具有至少90%序列同一性。
在一个方面,植物、植物部分或植物细胞源自植物,包括但不限于苜蓿、三叶草、豌豆、菜豆、兵豆、羽扇豆、牧豆树(mesquite)、角豆树、大豆、花生和罗望子。本文公开的抗病植物的子代、变体和突变体在本公开的范围内,条件是这些子代、变体和突变体包含亲本植物的原始/修饰的多核苷酸。
在一个实施例中,植物是豆类。在另一个实施例中,CcRpp2-R1和CcRpp2-R3多肽、CcRpp2-R1和CcRpp2-R3多核苷酸和/或CcRpp2-Rl和CcRpp2-R3抗性基因源自豆类。豆类的实例包括但不限于菜豆属(Phaseolus)(例如,法国菜豆、矮秆豆、菜豆(Phaseolusvulgaris)、棉豆(Phaseolus lunatus)、宽叶菜豆(Phaseolus acutifolius)、红花菜豆(Phaseolus coccineus));大豆属(例如野大豆(Glycine soja)、大豆(Glycine max(L.)));豌豆(Pisum)(例如,脱荚豌豆(有时称为光皮或圆籽豌豆);Pisum sativum);皱粒豌豆(Pisum sativum)、糖豌豆(Pisum sativum),也称为糖荚豌豆、食荚豌豆或嫩豌豆(mangetout)(Pisum granda));花生(Arachis hypogaea)、三叶草(三叶草属(Trifolium)物种)、苜蓿属(Medicago)、葛根(Pueraria lobata)、普通苜蓿、苗蓿(紫花苜蓿(Medicagosativa))、鹰嘴豆(鹰嘴豆属(Cicer))、兵豆(Lens culinaris)、羽扇豆(Lupinus);野豌豆(野豌豆属(Vicia))、田豆、蚕豆(Vicia faba)、山黧豆(山黧豆属(Lathyrus))(例如,家山黧豆(Lathyrus sativus)、玫红山黧豆(Lathyrus tuberosus));豇豆属(例如,乌头叶菜豆(Vigna aconiti folia)、赤豆(Vigna angularis)、黑吉豆(Vigna mungo)、绿豆(Vignaradiata)、班巴拉花生(Vigna subterrane)、赤小豆(Vigna umbellata)、野豇豆(Vignavexillata)、豇豆(Vigna unguiculata)(也称为长豇豆、豇豆));木豆(Cajanus cajari;Cajanus cajan)、硬皮豆属(Macrotyloma)(例如,落花生(Macrotyloma geocarpum)、马蚕豆(Macrotyloma uniflorum);四棱豆(Psophocarpus tetragonolobus)、非洲豆薯(Sphenostylis stenocarpa)、埃及黑豆、鹊豆(Lablab purpureus)、豆薯(Pachyrhizuserosus)、瓜尔豆(Cyamopsis tetragonolobus);和/或刀豆属(例如,矮刀豆(Canavaliaensiformis))、红刀豆(Canavalia gladiata)。
本文所述的组合物和方法可产生农艺学上所希望的品系或品种。农学特性或性状包括但不限于除草剂耐受性、增加的产量、昆虫防治、杂草防治、有害生物防治、病原体抗病性(例如,真菌、病毒、细菌)、高蛋白质产量、萌发和幼苗生长控制、增强的营养、环境胁迫抗性、增加的可消化性、雄性不育、开花时间或转化技术性状诸如细胞周期调节和/或基因靶向。
本公开提供了用于筛选或测定豆类植物对植物病害的抗性、免疫性或易感性的方法。测定植物对特定病原体的抗性、免疫性或易感性的一般方法是本领域技术人员已知的。例如,筛选或测定豆类植物对植物病害的抗性、免疫性或易感性的方法可包括将植物细胞、组织或器官(例如叶)暴露于病原体(例如豆薯层锈菌),然后确定和/或测量暴露的植物对由病原体引起的对植物病害(例如ASR)的抗性、免疫性和/或易感性的程度。该方法可进一步包括测量暴露于植物病原体的植物上的任何可观察到的植物病害症状,然后将植物病害症状与参考标准比较以确定抗病性的程度或范围。
将植物细胞、组织或器官暴露于病原体的方法是本领域已知的。测量、比较和确定对由病原体引起的病害(例如ASR)的抗性、免疫性和/或易感性(例如植物病害症状)的水平的方法也是本领域已知的。可以进一步评估暴露的植物以分离与植物对特定病原体或病害的抗性、免疫性或易感性相关、相联系和/或赋予植物对特定病原体或病害的抗性、免疫性或易感性的多核苷酸、氨基酸序列和/或遗传标志。进一步的评估包括但不限于从暴露的植物分离多核苷酸、核酸或氨基酸序列,对分离的多核苷酸或核酸进行测定,例如,以检测与一种或多种农艺特征或性状(包括但不限于抗性、免疫性和/或易感性)相关的一种或多种生物标志或分子标志。从此类方法收集的信息可用于例如育种计划中。
在一个实施例中,提供了分离的或重组的核酸,该核酸不含在衍生该核酸的生物体基因组DNA中天然地位于该核酸侧翼的序列(即,位于该核酸的5′和3′端的序列)(最佳地编码蛋白质的序列)。例如,在本公开的一些实施例中,编码本文公开的抗性蛋白的分离的多核苷酸序列可含有小于约5kb、约4kb、约3kb、约2kb、约1kb、约0.5kb或约0.1kb的核苷酸序列,在衍生该多核苷酸的细胞的基因组DNA中,该核苷酸序列天然地位于该多核苷酸的侧翼。基本上不含细胞物质的蛋白质包括具有小于约30%、约20%、约10%、约5%、或约1%(以干重计)的污染蛋白质的蛋白质制剂。当重组生产实施例的蛋白质或其生物活性部分时,最佳地,培养基表示少于约30%、约20%、约10%、约5%或约1%(以干重计)的化学前体或非目的蛋白质化学品。
与核苷酸序列和编码的蛋白质相关的片段和变体在本公开的范围内。“片段”是指核苷酸序列的一部分或氨基酸序列的一部分以及由此编码的蛋白质。核苷酸序列的片段可以编码保留天然蛋白质的生物活性并具有赋予植物抗性(即真菌抗性)的能力的蛋白质片段。替代性地,用作杂交探针的核苷酸序列的片段不一定编码保留生物活性的片段蛋白。因此,核苷酸序列的片段范围可以是从至少约15个核苷酸、约50个核苷酸、约100个核苷酸并且高达编码本公开的多肽的全长核苷酸序列。“功能片段”、“在功能上等价的片段”和“功能等价片段”在本文中可互换地使用。这些术语是指本公开的多肽序列的一部分或子序列,其中保留了其天然能力。
编码本公开的多肽的生物活性部分的核苷酸序列片段可编码至少约15、约25、约30、约40或45至约50个连续氨基酸,或高达存在于实施例的全长多肽中的氨基酸总数个连续氨基酸(例如,由SEQ ID NO:2编码的肽的341个氨基酸)。
用作杂交探针或PCR引物的核苷酸序列片段通常不需要编码蛋白质的生物活性部分。
在一些实施例中,CcRpp2-R1多肽片段是来自SEQ ID NO:2和21-36的CcRpp2-R1多肽的N-末端的至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或更多个氨基酸的N-末端截短。
在一些实施例中,CcRpp2-R3多肽片段是来自SEQ ID NO:4和48-58的CcRpp2-R3多肽的N-末端的至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或更多个氨基酸的N-末端截短。
在一些实施例中,CcRpp2-R1多肽片段是来自相对于SEQ ID NO:2和21-36的CcRpp2-R1多肽的N-末端和/或C-末端的至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或更多个氨基酸的N-末端和/或C-末端截短。
在一些实施例中,CcRpp2-R3多肽片段是来自相对于SEQ ID NO:4和48-58的CcRpp2-R3多肽的N-末端和/或C-末端的至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或更多个氨基酸的N-末端和/或C-末端截短。
在一些实施例中,CcRpp2-R1多肽包含与SEQ ID NO:2和21-36的CcRpp2-R1多肽中任一项的氨基酸序列具有至少约40%、45%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大同一性的氨基酸序列,其中该CcRpp2-R1多肽具有抗真菌活性。
在一些实施例中,CcRpp2-R3多肽包含与SEQ ID NO:4和48-58的CcRpp2-R3多肽中任一项的氨基酸序列具有至少约40%、45%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大同一性的氮基酸序列,其中该CcRpp2-R3多肽具有抗真菌活性。
在一些实施例中,CcRpp2-R1多肽包含与跨SEQ ID NO:2和21-36的CcRpp2-R1多肽中任一项的氨基酸序列的整个长度具有至少约80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大同一性的氨基酸序列。
在一些实施例中,CcRpp2-R3多肽包含与跨SEQ ID NO:4和48-58的CcRpp2-R3多肽中任一项的氨基酸序列的整个长度具有至少约80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大同一性的氨基酸序列。
在一些实施例中,多肽片段是通过以下各项进行的来自N-末端和/或C-末端的至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31或更多个氨基酸的N-末端和/或C-末端截短:通过蛋白水解、通过插入起始密码子、通过缺失编码缺失的氨基酸的密码子并且同时插入起始密码子、和/或插入终止密码子。
当提及指定的多核苷酸时,术语“全长序列”意指具有天然序列的完整核酸序列。在一个实施例中,提供了本文公开的多核苷酸序列的片段,包括SEQ ID NO:1和3。此类片段可用作杂交探针或PCR引物,并且不一定编码保留生物活性的片段蛋白。因此,核苷酸序列的片段范围可以是从至少约15个核苷酸、约50个核苷酸、约100个核苷酸并且高达编码本公开的多肽的全长核苷酸序列。
根据一个实施例,提供了鉴定包含本公开的CcRpp2-R1和/或CcRpp2-R3基因的植物的方法。该方法包括从一种或多种植物获得核酸样品,并使所述核酸样品与特异性结合本公开的CcRpp2-R1和/或CcRpp2-R3基因的核酸序列接触,并且检测该核酸与其靶序列的特异性结合。例如,该方法可通过使用经标记的探针或通过用仅在靶序列的存在下产生扩增子的合适PCR引物进行PCR反应来检测靶序列。在一个实施例中,该方法包括从一种或多种植物获得核酸样品,并使该核酸样品与以下物质接触
i)多核苷酸,其包含与选自由SEQ ID NO:1、3、5-20和37-47组成的组的连续序列或其互补序列相同或具有至少90%-95%序列同一性的至少8个核苷酸的序列;其中所述方法进一步包括使所述样品和所述多核苷酸经受严格杂交条件:以及测定所述样品中所述多核苷酸与所述DNA的杂交;或者
ii)一对PCR引物,其中第一PCR引物和第二PCR引物各自特异性结合选自由SEQ IDNO:1、3、5-20和37-47组成的组的序列,其中所述第一PCR引物和第二PCR引物当与其靶互补序列结合并经受标准PCR反应条件时能够产生扩增子;使所述样品经受聚合酶链反应条件;以及测定在所述第一引物和第二引物之间产生的扩增子。
在一些实施例中,提供了包含本公开的CcRpp2-R1多肽和/或CcRpp2-R3多肽的融合蛋白,该融合蛋白由选自由以下组成的组的式表示:
R1-L-R2、R2-L-R1、R1-R2或R2-R1
其中R1是CcRpp2-R1多肽、本公开的嵌合CcRpp2-R1多肽或目的蛋白,并且R2是CcRpp2-R3多肽、本公开的嵌合CcRpp2-R3多肽或目的蛋白。该R1多肽直接或通过接头(L)区段融合至R2多肽。术语“直接”定义在没有肽接头的情况下连接多肽的融合。因此,“L”表示R1和R2与之框内融合的化学结合或多肽区段,最常见的是,L是R1和R2通过酰胺键将R1的羧基末端连接到L的氨基末端并且将L的羧基末端连接到R2的氨基末端的线性肽。所谓“框内融合”意指R1和R2的阅读框之间没有翻译终止或中断。连接基团(L)通常是长度在1至500个氨基酸之间的多肽。
本公开的核苷酸序列的片段可以编码多肽的生物活性部分,或者它可以是可以使用下文公开的方法用作杂交探针或PCR引物的片段。赋予抗性的多肽的生物活性部分可以通过以下来制备:分离实施例的核苷酸序列之一的一部分,表达蛋白质的编码部分,并评估蛋白质的编码部分赋予或增强植物中真菌抗性的能力。作为实施例的核苷酸序列的片段的核酸分子包含至少约15、约20、约50、约75、约100或约150个核苷酸,或高达比本文公开的全长核苷酸序列中存在的核苷酸总数少一个(例如,SEQ ID NO:8的5210个核苷酸)。
编码CcRpp2-R1和/或CcRpp2-R3多肽或相关蛋白质的多核苷酸的一个来源是选自但不限于以下的物种:落花生属、紫荆属、木豆属、大豆属、苜蓿属、菜豆属、豌豆属或豇豆属物种的物种,其含有同源的CcRpp2-R1多核苷酸或CcRpp2-R3多核苷酸。
SEQ ID NO:1和5-20以及3和37-47的多核苷酸可用于在豆类宿主植物中分别表达CcRpp2-R1和CcRpp2-R3多肽,该豆类宿主植物包括但不限于苜蓿、三叶草、豌豆、菜豆、兵豆、羽扇豆、牧豆树、角豆树、大豆、花生或罗望子。
这些多核苷酸还可用作用于分离编码CcRpp2-R1和CcRpp2-R3多肽或相关蛋白质的同源或基本上同源的多核苷酸的探针。此类探针可用于鉴定同源或基本上同源的多核苷酸,这些多核苷酸源自选自但不限于落花生属、紫荆属、木豆属、大豆属、苜蓿属、菜豆属、豌豆属或豇豆属的物种。
编码CcRpp2-R1和CcRpp2-R3多肽的多核苷酸也可以从CcRpp2-R1或CcRpp2-R3多肽序列从头合成。多核苷酸基因的序列可以通过使用遗传密码从CcRpp2-R1或CcRpp2-R3多肽序列推导出来。计算机程序如“BackTranslate”(GCGTM包,加利福尼亚州圣迭戈市的阿克莱瑞公司(Acclerys,Inc.San Diego,Calif.))可用于将肽序列转换成编码该肽的相应核苷酸序列。可用于获得对应核苷酸编码序列的CcRpp2-R1或CcRpp2-R3多肽序列的实例包括但不限于SEQ ID NO:2、4、21-36和48-58的CcRpp2-R1或CcRpp2-R3多肽。
在一些实施例中,编码CcRpp2-R1或CcRpp2-R3多肽的核酸分子是具有SEo ID NO:1、3、5-20和37-47中任一项所示的序列的多核苷酸、及其变体、片段和互补序列。还涵盖了与实施例的核酸序列互补或与实施例的序列杂交的核酸序列。这些核酸序列可以在DNA构建体或表达盒中使用,以用于在多种生物体(包括微生物和植物)中进行转化和表达。核苷酸或氨基酸序列可以是合成序列,该合成序列已经被设计用于在生物体中表达,该生物体包括但不限于植物。
在一些实施例中,编码CcRpp2-R1或CcRpp2-R3多肽的核酸分子是非基因组核酸序列。
在一些实施例中,编码CcRpp2-R1或CcRpp2-R3多肽的核酸分子是具有如下核苷酸序列的非基因组多核苷酸,该核苷酸序列与SEQ ID NO:1、3、5-20和37-47的核酸序列中任一项具有至少50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大同一性,其中所编码的CcRpp2-R1或CcRpp2-R3多肽具有真菌抗性活性。
在一些实施例中,CcRpp2-R1多核苷酸编码与SEQ ID NO:2和21-36中任一项相比具有至少约40%、45%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大序列同一性的CcRpp2-R1多肽,并且与天然序列相比具有至少一个氨基酸取代、缺失、插入或它们的组合。
在一些实施例中,CcRpp2-R3多核苷酸编码与SEQ ID NO:4和48-58中任一项相比具有至少约40%、45%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大序列同一性的CcRpp2-R3多肽,并且与天然序列相比具有至少一个氨基酸取代、缺失、插入或它们的组合。
在一些实施例中,该核酸分子编码包含与跨SEQ ID NO:2和21-36的氨基酸序列中任一项的全长具有至少约75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大同一性的氨基酸序列的CcRpp2-R1多肽。
在一些实施例中,该核酸分子编码包含与跨SEQ ID NO:4和48-58的氨基酸序列中任一项的全长具有至少约75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大同一性的氨基酸序列的CcRpp2-R3多肽。
在一些实施例中,核酸分子编码CcRpp2-R1多肽,该多肽包含SEQ ID NO:2和21-36中任一项的与在相应的SEQ ID NO:2和21-36的对应位置处的氨基酸相比具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35或更多个氨基酸取代、缺失和/或插入的氨基酸序列。
在一些实施例中,核酸分子编码CcRpp2-R3多肽,该多肽包含SEQ ID NO:4和48-58中任一项的与在相应的SEQ ID NO:4和48-58的对应位置处的氨基酸相比具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35或更多个氨基酸取代、缺失和/或插入的氨基酸序列。
可以修饰多核苷酸编码序列以在甲硫氨酸起始密码子后面的位置处添加密码子以产生用于重组克隆目的和/或用于表达目的的限制性酶切位点。在一些实施例中,CcRpp2-R1和/或CcRpp2-R3多肽在翻译起始因子甲硫氨酸之后的位置处进一步包含丙氨酸残基。
“变体”意指通过在天然蛋白质或多肽中的一个或多个内部位点处缺失或添加一个或多个氨基酸和/或在天然蛋白质或多肽中的一个或多个位点处取代一个或多个氨基酸而衍生自天然蛋白质或多肽的蛋白质或多肽。本公开所涵盖的变体表现出天然蛋白质或多肽序列的生物活性。对于多核苷酸,变体包含如下多核苷酸,该多核苷酸在该天然多核苷酸内的5′和/或3′末端具有缺失(即截短)和/或在一个或多个内部位点具有一个或多个核苷酸的缺失和/或添加,和/或在该天然多核苷酸的一个或多个位点具有一个或多个核苷酸的取代。如本文所用,“天然”多核苷酸或多肽分别包含天然存在的核苷酸序列或氨基酸序列。本领域技术人员可认识到将构建实施例的核酸变体以使得保持可读框。对于多核苷酸,保守变体包括由于遗传密码的简并性而编码实施例的多肽之一的氨基酸序列的那些序列。可以使用熟知的分子生物学技术来鉴定天然存在的等位基因变体如,例如使用下文概述的聚合酶链反应(PCR)和杂交技术鉴定。变体多核苷酸还包括合成地衍生的多核苷酸,例如那些例如通过使用定点诱变产生但仍编码实施例的蛋白质的多核苷酸。通常,本公开的特定多核苷酸的变体可与该特定多核苷酸具有至少约40%、约45%、约50%、约55%、约60%、约65%、约70%、约75%、约80%、约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%或更多序列同一性,如由本领域已知的序列比对程序确定的。
还可以通过比较由变体多核苷酸编码的多肽与由参考多核苷酸编码的多肽之间的序列同一性百分比来评估实施例的特定多核苷酸(即参考多核苷酸)的变体。可以使用本领域已知的序列比对程序来计算任何两个多肽之间的百分比序列同一性。在本公开的任何给定的多核苷酸对通过比较它们编码的两个多肽共有的序列同一性百分比来评估的情况下,其中所编码的两个多肽之间的序列同一性百分比是至少约40%、约45%、约50%、约55%、约60%、约65%、约70%、约75%、约80%、约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%或更多序列同一性。
“变体蛋白质”意指通过在天然蛋白质中的一个或多个位点处缺失或添加一个或多个氨基酸的和/或在天然蛋白质中的一个或多个位点处取代一个或多个氨基酸而衍生自天然蛋白质的蛋白质。本公开的一些方面所涵盖的变体蛋白质具有生物活性,即它们继续具有天然蛋白质的所希望的生物活性,即赋予或增强如本文所述的植物抗性(即植物真菌病原体抗性)的能力。此类变体可以例如由遗传多态性或人为操作产生。实施例的天然蛋白质的生物活性变体可与天然蛋白质的氨基酸序列具有至少约40%、约45%、约50%、约55%、约60%、约65%、约70%、约75%、约80%、约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%或更多序列同一性,如由本领域已知的序列比对程序确定的。本公开的蛋白质的生物活性变体可以与该蛋白质相差少至约1-15个氨基酸残基、少至约1-10个、例如约6-10个、少至约5个、少至4个、3个、2个、或甚至1个氨基酸残基。
可例如通过包括氨基酸取代、缺失、截短和插入来改变本文公开的蛋白质。用于此类操作的方法是本领域已知的。例如,可以通过DNA中的突变来制备抗性蛋白质的氨基酸序列变体和片段。用于诱变和多核苷酸改变的方法是本领域已知的。
变体多核苷酸和蛋白质还涵盖衍生自诱变或重组程序(包括但不限于诸如DNA改组的程序)的序列和蛋白质。可从相关序列多核苷酸的群体产生重组多核苷酸文库,该相关序列多核苷酸包含具有基本序列同一性并且能够在体外或体内同源重组的序列区域。例如,使用这种方法,编码目的结构域的序列基序可以在本公开的蛋白质基因和其他已知蛋白质基因之间改组以获得编码具有改善的目的性质的蛋白质的新基因,诸如增加的赋予或增强植物对真菌病原体的抗性的能力。这种DNA改组的策略在本领域中是已知的。
可以通过进行随机突变来制备变体,或者可以设计变体。在设计的突变体的情况下,当在考虑生物活性或参与确定最终负责生物活性的三维构型的多肽的关键区域中保持氨基酸同一性时,存在产生具有与天然多肽相似活性的变体的高可能性。如果取代是保守的,也将会高可能性地保留活性。氨基酸可分为以下类别:非极性、不带电的极性、碱性和酸性。其中一种类别的氨基酸被相同类别的另一种氨基酸取代的保守取代最不可能实质上改变变体的生物活性。表1提供了属于每个类别的氨基酸的实例的列表。
表1
氨基酸类别
本文所述的多核苷酸可用于从其他生物体,特别是其他植物中分离对应的序列。以这种方式,可使用诸如PCR或杂交的方法基于此类序列与本文所示序列的序列同一性来鉴定这些序列。本公开涵盖基于序列与本文所述的完整序列或其变体和片段的序列同一性而分离的序列。此类序列包括作为所公开序列的直向同源物的序列。术语“直向同源物”是指衍生自共同祖先基因并且由于物种形成而在不同物种中发现的基因。当在不同物种中发现的基因的核苷酸序列和/或其编码的蛋白质序列共享至少约60%、约70%、约75%、约80%、约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%或更大序列同一性时,这些基因被认为是直系同源物。直系同源物的功能通常在物种间是高度保守的。因此,本公开涵盖编码赋予或增强真菌植物病原体抗性的蛋白质并与本文公开的序列或其变体或片段杂交的分离的多核苷酸。
在PCR方法中,可以设计寡核苷酸引物用于PCR反应,以从由任何目的生物体提取的cDNA或基因组DNA扩增相应的DNA序列。用于设计PCR引物与PCR克隆的方法是本领域已知的并公开于Sambrook等人(1989)Molecular Cloning:A Laboratory Manual[分子克隆:实验室手册](第2版,Cold Spring Harbor Laboratory Press[冷泉港实验室出版社],Plainview,N.Y.[纽约州平景城])。已知的PCR方法包括但不限于:使用成对引物、巢式引物、单特异性引物、简并引物、基因特异性引物、载体特异性引物、部分错配引物等的方法。
在杂交技术中,利用已知多核苷酸的全部或部分作为探针,该探针选择性杂交来自所选生物体的一群克隆基因组DNA片段或cDNA片段(即基因组或cDNA文库)中存在的其他相应多核苷酸。这些杂交探针可以是基因组DNA片段、cDNA片段、RNA片段、或其他寡核苷酸,并且可以用可检测基团如32p或任何其他可检测标志进行标记。因此,例如,可通过基于实施例的多核苷酸标记合成的寡核苷酸来制得用于杂交的探针。制备杂交用探针和构建cDNA和基因组文库的方法是本领域已知的。
可以使用各种程序来检查是否存在特定的DNA、RNA或蛋白质序列。这些程序包括例如DNA印迹、RNA印迹、蛋白质印迹和ELISA分析。这些技术是本领域熟知的。
本公开的组合物和方法可用于调节植物中一种或多种蛋白质的水平。术语“调节”在本文中用于意指相对于来自对应未转化植物(即,未根据本公开的方法遗传改变的植物)的蛋白质水平,遗传改变的(即转化的)植物内的蛋白质水平的增加或降低。
本公开的基因和多核苷酸包括天然存在的序列以及突变体或改变形式。本文公开的蛋白质还涵盖天然存在的蛋白质及其变体、片段和修饰形式。此类变体和片段将继续具有赋予或增强植物真菌病原体抗性所希望的能力。在一个方面,在编码变体或其片段的DNA中进行的突变通常不将序列置于阅读框之外,并且最佳地不产生可能产生二级mRNA结构的互补区域。
本公开的一个或多个基因可作为转基因表达,以使植物对ASR具有抗性。使用本文所述或本领域技术人员已知的不同启动子的使用将允许在不同情况下调节基因的表达(即,可基于所希望的结果来选择启动子)。例如,可能需要在特定组织系统或器官(例如,叶)中更高水平的表达以增强抗性。可将整个基因作为转基因插入(例如,天然启动子和编码序列两者),从而允许与其他性状诸如昆虫或除草剂抗性快速组合。
根据一个实施例,提供了一种编码与选自由SEQ ID NO:2、4、21-36和48-58组成的组的序列具有至少85%、90%、95%或99%序列同一性的多肽的多核苷酸,其中该多肽当在植物细胞中表达时赋予所述植物对亚洲大豆锈病(ASR)病害的抗性。在另一个实施例中,该多核苷酸选自由SEQ ID NO:1、3、5-20和37-47或与SEQ ID NO:1、3、5-20和37-47具有至少85%、90%、95%或99%序列同一性的多核苷酸组成的组。在一个实施例中,这些多核苷酸序列可与在植物细胞中表达编码的CcRpp2-R1和CcRpp2-R3基因产物所必需的异源调控元件可操作地连接。例如,调控元件可包括启动子、翻译前导序列、增强子、终止序列和聚腺苷酸化识别序列。在一个实施例中,提供了一种重组多核苷酸,其中异源植物启动子与选自由SEQ ID NO:1、3、5-20和37-47或与SEQ ID NO:1、3、5-20和37-47具有至少85%、90%、95%或99%序列同一性的多核苷酸组成的组的CcRpp2-R1或CcRpp2-R3编码序列可操作地连接。在一个实施例中,提供了一种重组多核苷酸,其中异源植物启动子与选自由SEQ ID NO:1或3或与SEQ ID NO:1或3具有至少85%、90%、95%或99%序列同一性的多核苷酸组成的组的CcRpp2-R1或CcRpp2-R3编码序列可操作地连接。在一个实施例中,提供了一种重组多核苷酸,其中异源植物启动子与选自由SEQ ID NO:1或3组成的组的CcRpp2-R1或CcRpp2-R3编码序列可操作地连接。
在本公开的一些方面,核酸序列可与目的多核苷酸序列的任何组合堆叠以产生具有所希望的表型的植物。此类堆叠可通过DNA构建体中的基因组合,或通过将一种或多种具有转基因的植物与包含所希望的组合的另一种植物品系杂交来实现。例如,本公开的多核苷酸或其片段可与本公开的任何其他多核苷酸或与其他基因堆叠。产生的组合还可以包括目的多核苷酸中的任何一个的多个拷贝。本公开的多核苷酸还可与任何其他基因或基因组合堆叠以产生具有多种所希望的性状组合的植物,这些性状组合包括但不限于动物饲料所希望的性状(诸如高油基因、平衡的氨基酸、增加的可消化性、昆虫、病害或除草剂抗性、无毒性和抗病性基因)、农艺性状(例如雄性不育、开花时间)和/或转化技术性状(例如细胞周期调节或基因靶向)。
这些堆叠组合可以通过如下任何方法产生,该方法包括但不限于通过任何常规或已知的方法或遗传转化杂交育种植物。如果通过对植物进行遗传转化来堆叠这些性状,那么目的多核苷酸序列可以在任何时候并且以任何顺序组合。例如,可以使用包含一种或多种所需性状的转基因植物作为靶标,以通过随后的转化来引入另外的性状。可以用共转化方案将这些性状与转化盒的任何组合所提供的目的多核苷酸一起引入。例如,若引入两个序列,则这两个序列可包含在分开的转化盒(反式)或包含在同一个转化盒(顺式)中。这些序列的表达可以通过相同的启动子或通过不同的启动子驱动。在某些情况下,可能需要引入可抑制目的多核苷酸的表达的转化盒。这可以与其他抑制盒或过度表达盒的任何组合进行组合以在该植物中产生所需性状组合。
在一个实施例中,堆叠组合包括一种或多种编码杀有害生物蛋白的基因,包括但不限于:来自假单胞菌属物种的杀昆虫蛋白,如PSEEN3174(Monalysin;(2011)PLoSPathogens[PLoS病原体],7:1-13);来自假单胞菌蛋白菌(Pseudomonas protegens)菌株CHA0和Pf-5(之前为荧光假单胞菌(fluorescens))(Pechy-Tarr,(2008)EnvironmentalMicrobiology[环境微生物学]10:2368-2386;基因库登录号EU400157)的杀昆虫蛋白;来自台湾假单胞菌(Liu等人,(2010)J.Agric.Food Chem.[农业与食品化学杂志],58:12343-12349)和来自假产碱假单胞菌(Zhang等人,(2009)Annals of Microbiology[微生物学杂志]59:45-50和Li等人,(2007)Plant Cell Tiss.Organ Cult.[植物细胞,组织和器官培养杂志]89:159-168)的杀昆虫蛋白;来自发光杆菌属物种和致病杆菌属物种的杀昆虫蛋白(Hinchliffe等人,(2010)The Open Toxinology Joumal[开放的毒理学杂志]3:101-118和Morgan等人,(2001)Applied and Envir.Micro.[应用与环境微生物学]67:2062-2069);来自美国专利号6,048,838和美国专利号6,379,946的杀昆虫蛋白;美国专利号9,688,730的PIP-1多肽;美国专利号9,475,847的AfIP-1A和/或AfIP-1B多肽;美国公布号US20160186204的PIP-47多肽;国际专利申请公布号WO 2016/114973的IPD045多肽、IPD064多肽、IPD074多肽、IPD075多肽、和IPD077多肽;PCT序列号PCT/US 17/56517的IPD080多肽;序列号PCT/US 17/54160的IPD078多肽、IPD084多肽、IPD085多肽、IPD086多肽、IPD087多肽、IPD088多肽、和IPD089多肽;美国专利公布号US 20160366891的PIP-72多肽;美国公布号US20170166921的PtIP-50多肽和PtIP-65多肽;美国序列号62/521084的IPD098多肽、IPD059多肽、IPD108多肽、IPD109多肽;美国公布号US20160347799的PtIP-83多肽;美国公布号US20170233440的PtIP-96多肽;PCT公布号WO 2017/23486的IPD079多肽;PCT公布号WO 2017/105987的IPD082多肽、序列号PCT/US 17/30602的IPD090多肽、美国序列号62/434020的IPD093多肽;序列号PCT/US 17/39376的IPD103多肽;美国序列号62/438179的IPD101多肽;美国序列号US 62/508,514的IPD121多肽;和δ-内毒素,包括但不限于Cryl、Cry2、Cry3、Cry4、Cry5、Cry6、Cry7、Cry8、Cry9、Cry10、Cry11、Cry12、Cry13、Cry14、Cry15、Cry16、Cry17、Cry18、Cry19、Cry20、Cry21、Cry22、Cry23、Cry24、Cry25、Cry26、Cry27、Cry28、Cry29、Cry30、Cry31、Cry32、Cry33、Cry34、Cry35、Cry36、Cry37、Cry38、Cry39、Cry40、Cry41、Cry42、Cry43、Cry44、Cry45、Cry46、Cry47、Cry49、Cry50、Cry51、Cry52、Cry53、Cry54、Cry55、Cry56、Cry57、Cry58、Cry59、Cry60、Cry61、Cry62、Cry63、Cry64、Cry65、Cry66、Cry67、Cry68、Cry69、Cry70、Cry71、和Cry 72类的δ-内毒素多肽以及苏云金芽孢杆菌细胞溶解性cyt1和cyt2基因。这些类别的苏云金芽孢杆菌杀昆虫蛋白的成员可在以下中找到:Crickmore等人,“Bacillus thuringiensis toxin nomenclature[苏云金芽孢杆菌毒素命名法]”(2011),网址为lifesci.Sussex.ac.uk/home/Neil_Crickmore/Bt/,其可以使用“www”前缀在万维网上访问)。
在另一个实施例中,堆叠组合包括编码对抑制生长点或分生组织的除草剂(诸如咪唑啉酮或磺酰脲)的抗性的多核苷酸。这个类别的示例性基因编码突变体ALS和AHAS酶,例如分别如以下文献中所述:Lee等人,(1988)EMBO J.[欧洲分子生物学学会杂志]7:1241和Miki等人,(1990)Theor.Appl.Genet.[理论与应用遗传学]80:449。还参见,美国专利号5,605,011、5,013,659、5,141,870、5,767,361、5,731,180、5,304,732、4,761,373、5,331,107、5,928,937和5,378,824;美国专利申请序列号11/683,737和国际公布WO 1996/33270。
在另一个实施例中,堆叠组合包括编码对草甘膦(分别由突变体5-烯醇丙酮酰-3-磷酸莽草酸合酶(EPSP)和aroA基因赋予的抗性)和其他膦酰基化合物如草铵膦(草丁膦乙酰转移酶(PAT)和吸水链霉菌草丁膦乙酰转移酶(bar)基因)和吡啶氧基或苯氧基丙酸和环己酮(ACC酶抑制剂编码基因)具有抗性的蛋白质的多核苷酸。参见,例如Shah等人的美国专利号4,940,835,其公开了EPSPS形式的能赋予草甘磷抗性的核苷酸序列。Barry等人的美国专利号5,627,061也描述了编码EPSPS酶的基因。还参见,美国专利号6,566,587、6,338,961、6,248,876、6,040,497、5,804,425、5,633,435、5,145,783、4,971,908、5,312,910、5,188,642、5,094,945、4,940,835、5,866,775、6,225,114、6,130,366、5,310,667、4,535,060、4,769,061、5,633,448、5,510,471、Re.36,449、RE 37,287和5,491,288以及国际公布EP 1173580、WO 2001/66704、EP 1173581和EP 1173582,为此目的将以上专利通过援引并入本文。还向表达编码草甘磷氧化还原酶的基因的植物赋予草甘磷抗性,如在美国专利号5,776,760和5,463,175中进行了更全面地描述,为此目的将这两份专利通过援引并入本文。另外,可通过过表达编码草甘磷N-乙酰转移酶的基因,来赋予植物草甘磷抗性。参见,例如美国专利号7,462,481、7,405,074以及美国专利申请公布号2008/0234130。编码突变体aroA基因的DNA分子可以在登录号39256下获得,并且该突变体基因的核苷酸序列公开于Comai的美国专利号4,769,061中。Kumada等人的欧洲申请号0 333 033和Goodman等人的美国专利号4,975,374公开了赋予除草剂(如L-草铵膦)抗性的谷氨酰胺合成酶基因的核苷酸序列。Leemans等人的EP申请号0 242 246和0 242 236中提供了草丁膦乙酰基转移酶基因的核苷酸序列;De Greef等人,(1989)Bio/Technology[生物/技术]7:61,描述了表达编码草丁膦乙酰转移酶活性的嵌合bar基因的转基因植物的产生。还参见,美国专利号5,969,213、5,489,520、5,550,318、5,874,265、5,919,675、5,561,236、5,648,477、5,646,024、6,177,616和5,879,903,为此目的将以上专利通过援引并入本文。赋予苯氧基丙酸和环己酮(诸如稀禾啶和吡氟氯禾灵)抗性的示例性基因是由Marshall等人,(1992)Theor.Appl.Genet.[理论与应用遗传学]83:435描述的Acc1-S1、Acc1-S2和Acc1-S3基因。
在另一个实施例中,堆叠组合包括编码对抑制光合作用的除草剂(诸如三嗪(psbA和gs+基因)和苯甲氰(腈水解酶基因))具有抗性的蛋白质的多核苷酸。Przibilla等人(1991)Plant Cell[植物细胞]3:169,描述了用编码突变体psbA基因的质粒对衣藻进行转化。Stalker的美国专利号4,810,648中公开了腈水解酶基因的核苷酸序列,并且包含这些基因的DNA分子可在登录号53435、67441和67442下获得。编码谷胱甘肽S-转移酶的DNA的克隆和表达描述于以下文献中:Hayes等人,(1992)Biochem.J.[生物化学杂志]285:173。
在另一个实施例中,堆叠组合包括编码已经被引入到各种植物中对乙酰羟酸合酶具有抗性的蛋白的多核苷酸,已经发现该蛋白使表达该酶的植物对多种类型的除草剂具有抗性(参见,例如,Hattori等人,(1995)Mol Gen Genet.[分子和普通遗传学]246:419)。赋予除草剂抗性的其他基因包括:编码大鼠细胞色素P4507A1和酵母NADPH-细胞色素P450氧化还原酶的嵌合蛋白的基因(Shiota等人,(1994)Plant Physiol[植物生理学]106:17),针对谷胱甘肽还原酶和超氧化物歧化酶的基因(Aono等人,(1995)Plant Cell Physiol[植物细胞生理学]36:1687)和各种磷酸转移酶的基因(Datta等人,(1992)Plant Mol Biol[植物分子生理学]20:619)。
在另一个实施例中,堆叠组合包括编码对靶向原卟啉原氧化酶(protox)的除草剂的抗性的多核苷酸,该原卟啉原氧化酶是生产叶绿素所必需的。原卟啉原氧化酶用作多种除草剂化合物的靶标。这些除草剂还抑制存在的所有不同种类的植物的生长,导致其完全破坏。对这些除草剂具有抗性的、含有经改变的原卟啉原氧化酶活性的植物的开发描述于以下文献中:美国专利号6,288,306、6,282,83和5,767,373以及国际公布WO 2001/12825。
在另一个实施例中,堆叠组合包括编码芳氧基链烷酸酯双加氧酶(AAD-1)蛋白的aad-1基因(最初来自食除草剂鞘脂菌(Sphingobium herbicidovorans))。该性状赋予对2,4-二氯苯氧基乙酸和芳氧基苯氧基丙酸酯(通常称为“fop”除草剂,如喹禾灵)除草剂的耐受性。用于植物中除草剂耐受性的aad-1基因本身首先在WO 2005/107437中公开(还参见US2009/0093366)。来自食酸丛毛单胞菌(Delftia acidovorans)的aad-12基因,其编码芳氧基链烷酸酯双加氧酶(AAD-12)蛋白,该蛋白通过用芳氧基链烷酸酯部分(包括苯氧基生长素(例如2,4-D,MCPA)以及吡啶氧基生长素(例如氯氟吡氧乙酸,三氯吡氧乙酸))使几种除草剂失活来赋予对2,4-二氯苯氧基乙酸和吡啶氧基乙酸酯除草剂的耐受性。
在另一个实施例中,堆叠组合包括编码美国专利申请公布2003/0135879中公开的、用于赋予麦草畏耐受性的除草剂抗性麦草畏单加氧酶的多核苷酸。
在另一个实施例中,堆叠组合包编码美国专利号4,810,648中公开的用于赋予溴草腈耐受性的溴草腈腈水解酶(Bxn)的多核苷酸。
在另一个实施例中,堆叠组合包括描述于以下文献中的用于达草灭耐受性的编码八氢番茄红素(crtl)的多核苷酸:Misawa等人,(1993)Plant J.[植物杂志]4∶833-840和Misawa等人,(1994)Plant J.[植物杂志]6:481-489。
在另一个实施例中,堆叠组合包括编码蛋白质的多核苷酸,该蛋白质例如通过如下方式赋予或有助于改变的谷粒特性,诸如改变的脂肪酸:
(1)下调硬脂酰-ACP以增加植物的硬脂酸含量。参见,Knultzon等人,(1992)Proc.Natl.Acad.Sci.USA[美国科学院院报]89:2624和WO 1999/64579(Genes to AlterLipid Profiles in Com[改变玉米脂质谱的基因])。
(2)通过FAD-2基因修饰提高油酸和/或通过FAD-3基因修饰降低亚麻酸(参见,美国专利号6,063,947、6,323,392、6,372,965和WO 1993/11245)。
(3)改变共轭亚麻酸或亚油酸含量,如在WO 2001/12800中。
(4)改变LEC1,AGP,Dek1,Superal1,mi1 ps和各种Ipa基因如Ipa1、Ipa3、hpt或hggt。例如,参见WO 2002/42424、WO 1998/22604、WO 2003/011015、WO 2002/057439、WO2003/011015、美国专利号6,423,886、6,197,561、6,825,397、和美国专利申请公布号US2003/0079247、US 2003/0204870以及Rivera-Madrid等人,(1995)Proc.Natl.Acad.Sci.[美国科学院院报]92:5620-5624。
(5)基因编码用于制备长链多不饱和脂肪酸的δ-8去饱和酶(美国专利号8,058,571和8,338,152)、用于降低饱和脂肪的δ-9去饱和酶(美国专利号8,063,269)、用于改善ω-3脂肪酸谱的报春花属(Primula)Δ6-去饱和酶。
(6)与脂质和糖代谢调节相关的分离的核酸和蛋白质,特别是用于生产转基因植物和调节种子储存化合物(包括脂质、脂肪酸、淀粉或种子储存蛋白)的水平的方法中以及用于调节植物种子大小、种子数、种子重量、根长和叶子大小的方法中的脂质代谢蛋白(LMP)(EP 2404499)。
(7)改变植物中糖诱导型2(HSI2)蛋白的高水平表达,以增加或减少植物中HSI2的表达。增加HSI2的表达增加油含量,而降低HSI2的表达降低脱落酸敏感性和/或增加抗旱性(美国专利申请公布号2012/0066794)。
(8)细胞色素b5(Cb5)单独或与FAD2一起表达,以调节植物种子中的油含量,特别是增加ω-3脂肪酸的水平,并改善ω-6与ω-3脂肪酸的比例(美国专利申请公布号2011/0191904)。
(9)核酸分子编码wrinkled1样多肽,用于调节糖代谢(美国专利号8,217,223)。
本公开的特征是包括将多核苷酸引入植物的方法。在本公开的一些方面,多核苷酸可以序列获得进入植物细胞内部(包括其可能插入植物基因组中)的方式呈现。本公开的方法不取决于将序列引入植物中的特定方法,只要多核苷酸进入植物的至少一个细胞的内部即可。
可将多核苷酸瞬时或稳定地引入宿主细胞,并且可保持非整合状态,例如作为质粒。
转化方法以及将多核苷酸序列引入植物中的方法可取决于转化所针对的植物或植物细胞的类型,即单子叶植物或双子叶植物。将多肽和多核苷酸引入植物细胞中的合适方法包括但不限于显微注射、电穿孔、直接基因转移、Lecl转化和弹道粒子加速。随着更新的方法变得可用,它们也可应用于本公开,因为转化或转染的方法不是关键的。
可根据常规方式将已转化的细胞培育成植株。然后可以培育这些植株,并用相同的经转化的株系或者不同的株系进行授粉,并鉴定出具有所希望的表型特征的组成型表达的所得子代。可以培育两代或更多代,以确保所希望的表型特征的表达稳定地保持并遗传,并且然后收获种子以确保已经实现了所希望的表型特征的表达。在本公开的一些方面,将具有核苷酸构建体或表达盒的转化种子或转基因种子稳定掺入到它们的基因组中。
在一个方面,本公开涵盖包含本文公开的多核苷酸序列的种子,与例如野生型品种的植物种子相比,该种子可发育成或可用于发育对病原体(例如真菌)或由病原体引起的感染具有增加或增强的抗性的一种或多种植物。在一个方面,本公开的特征在于来自转基因豆类作物植物的种子,其中该种子包含本文公开的多核苷酸。
本公开可用于任何植物物种(包括但不限于单子叶植物和双子叶植物)的转化。目的植物的实例包括但不限于玉米(玉蜀黍),芸苔属物种(例如,甘蓝型油菜(Brassicanapus)、芜菁(Brassica rapa)、芥菜(Brassica juncea))(特别是可用作种子油来源的那些芸苔属物种),苜蓿(紫花苜蓿(Medicago sativa)),稻(rice,Oryza sativa),黑麦(rye,Secale cereale),高粱(甜高粱(Sorghum bicolor),高粱(Sorghum vulgare)),粟(例如,珍珠粟(御谷(Pennisetum glaucum)),黍(粟米(Panicum miliaceum)),粟(谷子(Setariaitalica)),穇子(龙爪稷(Eleusine coracana))),向日葵(sunflower,Helianthusannuus),红花(safflower,Carthamus tinctorius),小麦(wheat,Triticum aestivum),大豆(soybean,Glycine max),烟草(tobacco,Nicotiana tabacum),马铃薯(potato,Solanumtuberosum),花生(peanut,Arachis hypogaea),棉花(海岛棉(Gossypium barbadense)、陆地棉(Gossypium hirsutum)),甘薯(番薯(Ipomoea batatas)),木薯(cassava,Manihotesculenta),咖啡(咖啡属(Coffea)物种),椰子(coconut,Cocos nucifera),菠萝(pineapple,Ananas comosus),柑橘树(柑橘属(Citrus)物种),可可(cocoa,Theobromacacao),茶树(tea,Camellia sinensis),香蕉(芭蕉属(Musa)物种),鳄梨(avocado,Perseaamericana),无花果(fig或(Ficus casica)),番石榴(guava,Psidium guajava),芒果(mango,Mangifera indica),橄榄(olive,Olea europaea),木瓜(番木瓜(Caricapapaya)),腰果(cashew,Anacardium occidentale),澳洲坚果(macadamia,Macadamiaintegrifolia),巴旦杏(almond,Prunus amygdalus),甜菜(sugar beets,Betavulgaris),甘蔗(甘蔗属(Saccharum)物种),燕麦,大麦,蔬菜,观赏植物和针叶树。
在一个方面,目的植物包括豆类作物物种,包括但不限于苜蓿(紫花苜蓿);三叶草或车轴草(三叶草属物种);豌豆,包括大豌豆(Pisum satinum)、木豆(Cajanus cajan)、豇豆(Vigna unguiculata)和山黧豆属(Lathyrus)物种;菜豆(豆科(Fabaceae或Leguminosae));兵豆(Lens culinaris);羽扇豆(羽扇豆属(Lupinus)物种);牧豆树(牧豆树属(Prosopis)物种);角豆树(Ceratonia siliqua)、大豆(Glycine max)、花生(Arachishy pogaea)或罗望子(Tamarindus indica)。术语“豆类物种”和“豆类作物物种”在本文中用于指植物,并且可以是例如目的植物。在一些方面,豆类物种或豆类作物物种是植物、植物部分或植物细胞。
在一个方面,构建体或载体或表达盒不存在于原始植物的基因组中或存在于转基因植物的基因组中,但不存在于原始植物基因组的其天然基因座处。
本文公开的组合物可通过渗入过程产生或维持。当该过程重复两次或更多次时,渗入有时被称为“回交”。在渗入或回交中,“供体”亲本是指具有要渗入的所希望的基因或基因座的亲本植物。“受体”亲本(使用一次或多次)或“轮回”亲本(使用两次或更多次)是指基因或基因座被渗入到其中的亲本植物。初始杂交产生Fl代;然后术语“BC1”是指轮回亲本的第二次使用,并且“BC2”是指轮回亲本的第三次使用等等。
因此,本公开的一个方面是增强植物对植物病害诸如ASR的抗性的方法。该方法可以包括在育种程序(即,对ASR抗性的育种程序)中通过将豆类来源的CcRpp2-R1和CcRpp2-R3二元抗性基因或其同源物渗入种质中来赋予对病原体(例如,引起ASR的病原体)的抗性。
术语“种质”在本文中用于意指遗传物质,其属于或来自于个体(例如,植物)、个体的组(例如,植物品系、品种或家族)或衍生自品系、品种、物种或培养物的克隆。种质可以是生物体或细胞的一部分,或者可以与生物体或细胞分开。种质提供了具有特定分子组成的遗传物质,其为生物体或细胞培养物的一些或全部遗传特性提供了物理基础。在本公开上下文中的种质包括由此可以生长出新植物的细胞、种子或组织,或者可以培养成整株植物的植物部分,诸如叶、茎、花粉或细胞。
本公开的方面包括用于鉴定作为抗性来源的种质的方法,该种质包括但不限于以下属中的一种或多种的种质:大豆属、豇豆属和扁豆属。
在一个实施例中,豆类作物物种或豆类来源的基因源自大豆属。大豆属物种的实例包括但不限于沙生大豆(Glycine arenaria)、银毛大豆(Glycine argyrea)、弯裂片大豆(Glycine cyrtoloba)、灰毛大豆(Glycine canescens)、澎湖大豆(Glycineclandestine)、弯荚大豆(Glycine curvata)、镰荚大豆(Glycine falcata)、宽叶大豆(Glycine latifolia)、小叶大豆(Glycine microphylla)、Glycine pescadrensis、Glycine stenophita、Glycine syndetica、野大豆(Glycine soja)、烟豆(Glycinetabacina)和短绒野大豆(Glycine tomentella)。
在另一个实施例中,豆类作物物种或豆类来源的基因源自豇豆属。豇豆属是包括大约100个物种的泛嗜性属。其是细分成以下亚属的分类群:豇豆属、Haydonia、Plectotropis(非洲)、Ceratotropis(亚洲)、Sigmoidotropis和Lasiopron。该属包括经济上相关的物种,诸如Vigna unguiculata(L.)Walp(豇豆)、Vigna radiata(L.)Wilczek(绿豆)、Vigna angularis(赤豆)Ohwi和Ohashi(赤小豆)、Vigna mungo(L.)Hepper(黑吉豆)、和Vigna umbellata(赤小豆)Ohwi和Ohashi(饭豆)。在豇豆中识别出四种亚种:dekindtiana,栽培的亚种的野生近缘种;眉豆(cylindrica),栽培的乌豇豆;长豇豆(sesquipedalis),栽培的长豇豆;和豇豆(unguiculata),栽培的黑眼豆。豇豆属物种豇豆进一步分成以下栽培品种群:豇豆,其作为豆类种植;Biflora或Cilindrica(乌豇豆),主要用作饲料;长豇豆(yardlong或豇豆),其作为蔬菜种植;Textilis,其为长花序梗的纤维而栽培;以及Melanophthalmus(黑眼豆)。已经在田间和温室条件下记录了若干豇豆属物种,包括绿豆、黑吉豆和豇豆对豆薯层锈菌的易感性。
在另一个实施例中,豆类作物物种或豆类来源的基因源自扁豆属。扁豆(Lablabpurpureus(L.)Sweet)是原产于亚洲和非洲(Pengelly和Maass,(2001)Gen.resour.cropev.[遗传资源与作物进化]48:261-272)的豆类物种(Verdcourt(1971)Flora of TropicalEast Africa[热带东非植物志],第696-699页,Crown Agents,London,UK[皇家代理,英国伦敦];以及Duke等人(1981)Handbook of Legumes of World Economic Importance[世界经济重要性豆类手册],第102-106页,Plenum Press,New York,USA and London,UK[殷实出版社,美国纽约和英国伦敦])。其通常被称为鹊豆、扁豆、紫扁豆、田豆、埃及豆、穷人豆(poor man′s bean)、香翅豆(Tonga bean)(英国),以及至少20个另外的俗名。其在非洲、亚洲和加勒比地区作为豆类作物或作为绿色蔬菜种植(Duke等人(1981)Handbook ofLegumes of World Economic Importance[世界经济重要性豆类手册],第102-106页,Plenum Press,New York,USA and London,UK[殷实出版社,美国纽约和英国伦敦]);以及Pengelly和Maass,(2001)Gen.resour.crop ev.[遗传资源与作物进化]48:261-272)。
鹊豆(Lablab purpureas)已被报道为豆薯层锈菌的替代宿主(Perez-Hemandez,(2007)Alternative hosts of Phakopsora pachyrhizi in the Americas:An analysisof their role in the epidemiology of Asian soybean rust in the continentalU.S.M.Sc.thesis.[美国大陆理科硕士论文:豆薯层锈菌在美洲的替代宿主:分析它们在亚洲大豆锈病的流行病学中的作用]Iowa State University.[爱荷华州立大学]Ames,Iowa.U.S.A.[美国爱荷华州艾姆斯];Vakili(1981)Plant Dis.[植物病害]65:817-819;以及Poonpolgul和Surin,(1980)Soybean Rust[大豆锈病]Newsletter[时事通讯],3:30-31)。
在一个方面,豆类作物物种或豆类来源的基因源自鹰嘴豆属、木豆属、苜蓿属、菜豆属、豌豆属、葛属或三叶草属。鹰嘴豆属物种的实例包括但不限于鹰嘴豆、Cicerechinospermum、Cicer reticulatum和Cicer pinnatifldum。木豆属物种的实例包括但不限于木豆。苜蓿属物种的实例包括但不限藜苜蓿(Medicago truncatula)和紫花苜蓿。菜豆属物种的实例包括但不限于菜豆、棉豆、宽叶菜豆或红花菜豆。豌豆属物种的实例包括但不限于Pisum abyssinicum、豌豆、Pisum elatius、Pisum fulvum、Pisum transcaucasium和Pisum humile。葛属物种的实例包括但不限于葛根。三叶草属物种的实例包括但不限于黄车轴草(Trifolium aureum)和Trifolium occidentale。
本公开还包括可在表达盒或DNA构建体中提供用于在目的植物中表达的本文所述的序列。在一个方面,该盒可包括与本文公开的序列可操作连接的5’和3’异源调控序列。“可操作地连接”是指两个或更多个核酸片段在单个核酸片段上的缔合,这样使得一个核酸片段的功能受到另一个的影响。例如,当启动子能够影响编码序列的表达时,它与编码序列可操作地连接(即编码序列在启动子的转录控制下)。编码序列可以在正义或反义方向上可操作地连接到调控序列上。此类调控序列是本领域熟知的,并且包括指导核苷酸序列在许多类型的宿主细胞中组成型表达的那些和指导核苷酸序列在某些宿主细胞中或在某些条件下表达的那些。载体的设计可取决于例如待转化的宿主细胞的类型或所希望的核酸的表达水平。该盒可以含有一个或多个待共转化到植物中的另外的基因。并且,可以在多个表达盒上提供任何另外的基因。
本公开的表达盒可以包括许多限制性位点,用于插入受调控区域转录调控的核苷酸序列。表达盒还可含有选择性标志基因。
表达盒还可在转录的5’-3’方向上包括转录和翻译起始区、本公开的DNA序列以及在植物中起作用的转录和翻译终止区。转录起始区,即启动子,对于植物宿主可以是天然的或类似的,或外来的或异源的。此外,启动子可以是天然序列,或替代性地,是合成序列。术语“外来的”意指转录起始区未见于该转录起始区引入到其中的天然植物中。如本文所用,嵌合基因包含与转录起始区可操作地连接的编码序列,该转录起始区对于该编码序列是异源的。启动子的实例包括但不限于花椰菜花叶病毒35S和大豆泛素6。
虽然可优选使用异源启动子表达序列,但是可使用同源启动子或天然启动子序列。此类构建体将改变宿主细胞(即植物或植物细胞)中的表达水平。因此,宿主细胞(即植物或植物细胞)的表型被改变。
终止区可以与转录起始区一起是天然的,可以与可操作连接的目的DNA序列一起是天然的,或者衍生自另一个来源。方便的终止区可获自根癌农杆菌(Agrobacteriumtumefaciens)的Ti质粒,诸如章鱼碱合酶和胭脂碱合酶终止区。
在一个方面,内源或转基因抗性直向同源物可以通过同源或非同源重组方法改变,诸如通过基因组编辑。此类改变是指当与其未修饰的序列相比时具有至少一个修饰的核苷酸序列,并且包括例如:(i)至少一个核苷酸的替换,(ii)至少一个核苷酸的缺失,(iii)至少一个核苷酸的插入,或(iv)(i)-(iii)的任何组合。
在一些实施例中,可以使用基因组编辑技术将所公开的CcRpp2-R1和CcRpp2-R3多核苷酸组合物引入植物的基因组中,或者可以使用基因组编辑技术编辑植物基因组中先前引入的CcRpp2-R1和CcRpp2-R3多核苷酸。
例如,可以通过使用双链断裂技术(如TALEN、大范围核酸酶、锌指核酸酶、CRISPR-Cas等)将所公开的多核苷酸引入植物基因组中需要的位置上。例如,为了位点特异性插入的目的,可以使用CRISPR-Cas系统将所公开的多核苷酸引入基因组中需要的位置上。植物基因组中需要的位置可以是任何对于插入来说需要的靶位点,如适于育种的基因组区域,或者可以是位于具有现有的目的性状的基因组窗口中的靶位点。现有的目的性状可能是内源性状或先前引入的性状。
“靶位点”、“靶序列”、“靶DNA”、“靶基因座”、“基因组靶位点”、“基因组靶序列”以及“基因组靶基因座”在本文中可互换使用,并且是指例如在细胞的基因组(包括叶绿体DNA和线粒体DNA)中的多核苷酸序列,核酸内切酶被募集到该多核苷酸序列,并且任选地切开或切割靶位点的DNA。靶位点可以是植物基因组中的内源性位点,或者替代性地,靶位点可以与植物异源,从而不是基因组中天然存在的,或者与其在自然界中存在的地方相比,靶位点可以发现于异源基因组位置中。
在一些实施例中,当所公开的CcRpp2-R1和CcRpp2-R3多核苷酸先前已被引入基因组或为其他豆类物种中CcRpp2-R1和CcRpp2-R3的内源同源物时,可使用基因组编辑技术来改变或修饰所引入的多核苷酸序列或内源同源物。可以将位点特异性修饰引入所公开的CcRpp2-R1和CcRpp2-R3多核苷酸组合物中,这些位点特异性修饰包括使用用于引入位点特异性修饰的任何方法产生的修饰,该方法包括但不限于通过使用基因修复寡核苷酸(例如美国专利申请公布号2013/0019349),或通过使用双链断裂技术,如TALEN、大范围核酸酶、锌指核酸酶、CRISPR-Cas等。此类技术可用于通过在引入的多核苷酸内的核苷酸的插入、缺失或取代来修饰先前引入的多核苷酸。替代性地,可以使用双链断裂技术向引入的多核苷酸中添加另外的核苷酸序列。可以添加的另外的序列包括另外的表达元件(如增强子序列和启动子序列)。在另一个实施例中,可以使用基因组编辑技术在植物基因组内紧邻本文公开的所公开的CcRpp2-R1和CcRpp2-R3多核苷酸组合物定位另外的ASR抗性蛋白的编码序列,以产生ASR抗性蛋白的分子堆叠物。
“改变的靶位点”、“改变的靶序列”、“修饰的靶位点”和“修饰的靶序列”在本文中可互换地使用,并且是指如本文公开的靶序列,该靶序列当与未改变的靶序列相比时包含至少一个改变。此类“改变”包括例如:(i)至少一个核苷酸的替代、(ii)至少一个核苷酸的缺失、(iii)至少一个核苷酸的插入、或(iv)(i)-(iii)的任何组合。
可根据需要优化基因以增加转化的植物中的表达。换句话讲,可以使用植物偏好密码子合成基因用于改进表达。用于合成植物偏好基因的方法是本领域已知的。
已知有另外的序列修饰能增强细胞宿主中的基因表达。这些包括消除以下序列:编码假聚腺苷酸化信号、外显子-内含子剪接位点信号、转座子样重复的序列、及可能不利于基因表达的其他经充分表征的序列。可以将序列的G-C含量调整至通过参照宿主细胞中表达的已知基因而计算出的给定细胞宿主的平均水平。如果可能,修饰序列以避免出现预测的发夹二级mRNA结构。
表达盒可另外在表达盒构建体中包含5′前导序列。此类前导序列可以起到增强翻译的作用。翻译前导序列在本领域是已知的,并且包括:小核糖核酸病毒前导序列,例如,EMCV前导序列(脑心肌炎5′非编码区);马铃薯Y病毒前导序列,例如,TEV前导序列(烟草蚀纹病毒),和人免疫球蛋白重链结合蛋白(BiP);来自苜蓿花叶病毒的外壳蛋白mRNA的非翻译前导序列(AMV RNA 4);烟草花叶病毒前导序列(TMV);以及玉蜀黍褪绿斑驳病毒前导区(MCMV)(Lommel等人(1991)Virology[病毒学]81:382385)。还可以利用已知增强翻译的其他方法,诸如内含子。
可以在制备表达盒的同时操作各种DNA片段,以确保DNA序列处于正确的取向,并且如果合适,处于正确的阅读框中。为此,可以使用衔接子或接头来连接DNA片段。替代性地,可以使用其他操作来提供方便的限制性位点、去除多余的DNA或去除限制性位点。出于这个目的,可以涉及体外诱变、引物修复、限制性酶切(restriction)、退火、再取代(例如转换和颠换)。
通常,表达盒可包含选择性标志基因,用于选择转化细胞。利用选择性标志基因来选择转化的细胞或组织。标志基因包括编码抗生素抗性的基因,诸如编码新霉素磷酸转移酶II(NEO)和潮霉素磷酸转移酶(HPT)的基因,以及赋予除草化合物(诸如草甘膦、草铵膦、溴草腈、咪唑啉酮和2,4-二氯苯氧乙酸(2,4-D))抗性的基因。以上选择性标志基因的列表无意为限制性的。任何选择性标志基因都可用于本公开中。
为了在植物或植物细胞中表达本公开的靶基因和/或蛋白质(例如,一种或多种CcRpp2-R1和CcRpp2-R3基因和/或一种或多种CcRpp2-R1和CcRpp2-R3蛋白质),本文所述的方法包括用例如如本文所公开的编码该靶蛋白的多核苷酸转化植物或植物细胞。本文所述的多核苷酸可以可操作地连接到在植物细胞中驱动表达的启动子。本领域已知的任何启动子可用于本公开的方法中,包括但不限于组成型启动子、病原体诱导型启动子、伤口诱导型启动子、组织偏好性启动子和化学调节型启动子。启动子的选择可取决于转化的植物中所希望的表达时间和位置以及本领域技术人员已知的其他因素。可种植或培育转化的细胞或植物以产生包含被引入细胞或植物中的例如编码CcRpp2-R1和CcRpp2-R3蛋白的多核苷酸中的一种或多种的植物。
多种启动子可用于本公开的实践中。可基于所需结果,选择启动子。也就是说,核酸可与组成型、组织偏好性或其他启动子组合用于在目的宿主细胞中的表达。此类组成型启动子包括例如Rsyn7启动子的核心启动子和其他在WO 99/43838和美国专利号6,072,050中公开的组成型启动子、核心CaMV 35S启动子、稻肌动蛋白、泛素、pEMU、MAS、ALS等等。其他组成型启动子包括例如以下美国专利号中所公开的那些:5,608,149、5,608,144、5,604,121、5,569,597、5,466,785、5,399,680、5,268,463、5,608,142和6,177,611,这些启动子在本领域中是已知的,并且可考虑用于本公开。
通常,从诱导型启动子,特别是从病原体诱导型启动子表达基因是有益的。这些启动子包括来自在被病原体感染后被诱导的病程相关蛋白(PR蛋白)的那些启动子,例如PR蛋白、SAR蛋白、β-1,3-葡聚糖酶、几丁质酶等。
引人关注的是在病原体感染部位处或附近局部表达的启动子。此外,由于病原体通过伤口或昆虫损伤找到进入植物的入口,伤口诱导型启动子可以用于本公开的构建中。此类伤口诱导型启动子包括马铃薯蛋白酶抑制剂(pin II)基因、wun1和wun2、win1和win2、系统素(systemin)、WIP1、MPI基因等。
可以使用化学调节型启动子以通过应用外源化学调节剂来调节植物中的基因表达。取决于目标,在应用化学品诱导基因表达的情况下启动子可以是化学诱导型启动子,或者在应用化学品阻抑基因表达的情况下启动子可以是化学阻抑型启动子。化学诱导型启动子是本领域已知的,并且包括但不限于由苯磺酰胺除草剂安全剂激活的玉蜀黍In2-2启动子、由用作萌前除草剂的疏水亲电子化合物激活的玉蜀黍GST启动子、以及由水杨酸激活的烟草PR-1a启动子。其他目的化学调节型启动子包括类固醇响应性启动子(例如糖皮质激素诱导型启动子以及四环素诱导型和四环素阻遏型启动子)。
可利用组织偏好性启动子来靶向特定植物组织中靶基因或蛋白(例如,编码豆类来源的CcRpp2-R1和CcRpp2-R3多肽的多核苷酸序列)的增强表达。此类组织偏好性启动子包括但不限于叶偏好性启动子、根偏好性启动子、种子偏好性启动子和茎偏好性启动子。组织偏好性启动子包括Yamamoto等人(1997)Plant J.[植物杂志]12(2):255-265;Kawamata等人(1997)Plant Cell Physiol.[植物细胞生理学]38(7):792-803;Hansen等人(1997)Mol.Gen Genet.[分子和普通遗传学]254(3):337-343;Russell等人(1997)TransgenicRes.[转基因研究]6(2):157-168;Rinehart等人(1996)Plant Physiol[植物生理学]112(3):1331-1341;Van Camp等人(1996)Plant Physiol.[植物生理学]112(2):525-535;Canevascini等人(1996)Plant Physiol.[植物生理学]112(2):513-524;Yamamoto等人(1994)Plant Cell Physiol[植物细胞生理学]35(5):773-778;Lam(1994)ResultsProbl.Cell Differ.[细胞分化的结果和问题]20:181-196;Orozco等人(1993)Plant MolBiol.[植物分子生物学]23(6):1129-1138;Matsuoka等人(1993)Proc Natl.Acad.Sci.USA[美国科学院院报]90(20):9586-9590;以及Guevara-Garcia等人(1993)Plant J.[植物学]4(3):495-505。此类启动子可被修饰。
叶特异性启动子是本领域已知的。参见,例如,Yamamoto等人(1997)Plant J.[植物杂志]12(2):255-265;Kwon等人(1994)Plant Physiol.[植物生理学]105:357-67;Yamamoto等人(1994)Plant Cell Physiol[植物细胞生理学]35(5):773-778;Gotor等人(1993)Plant J.[植物杂志]3∶509-18;Orozco等人(1993)Plant Mol.Biol.[植物分子生物学]23(6):1129-1138;以及Masuoka等人(1993)Proc.Natl.Acad.Sci.USA[美国科学院院报]90(20):9586-9590。
“种子偏好性”启动子包括“种子特异性”启动子(在种子发育期间有活性的那些启动子如种子贮藏蛋白的启动子)以及“种子发芽性”启动子(在种子发芽期间有活性的那些启动子)。此类种子偏好性启动子包括但不限于Ciml(细胞分裂素诱导的信号)、cZ19Bl(玉蜀黍19kDa玉米醇溶蛋白)、milps(肌醇-1-磷酸盐合酶)和celA(纤维素合酶)(参见WO 00/11177,其通过援引并入本文)。γ-玉蜀黍醇溶蛋白是优选的胚乳特异性启动子。Glob-1是优选的胚胎特异性启动子。对于双子叶植物,种子特异性启动子包括但不限于:菜豆β-菜豆素、油菜籽蛋白、β-伴大豆球蛋白、大豆凝集素、十字花科蛋白等。对于单子叶植物,种子特异性启动子包括但不限于玉蜀黍15kDa玉米醇溶蛋白、22kDa玉米醇溶蛋白、27kDa玉米醇溶蛋白、g-玉米醇溶蛋白、蜡质、收缩素(shrunken)1、收缩素2、球蛋白1等。还参见WO 00/12733,其中公开了来自end1和end2基因的种子偏好性启动子;该文献通过援引并入本文。
本公开的多核苷酸的表达可以涉及使用完整的、天然的CcRpp2-R1和CcRpp2-R3基因,其中该表达是由同源5′上游启动子序列驱动的。替代性地,可使用由在宿主豆类中表达的异源CcRpp2-R1和CcRpp2-R3抗病性基因提供的5’转录控制序列组装的构建体来产生表达。根据本申请的传授内容,本领域技术人员将能够鉴定编码CcRpp2-R1和CcRpp2-R3蛋白的基因,以评价它们的表达水平,并选择可用于表达目的CcRpp2-R1和/或CcRpp2-R3基因的优选启动子序列。使用同源或异源CcRpp2-R1和CcRpp2-R3启动子序列提供了调节蛋白质表达的选择,以避免或最小化与不适当或不想要的表达和植物防御激活相关的任何潜在的不希望的结果。
特定的大豆启动子包括但不限于用于组成型表达的大豆泛素(subi-1)、延伸因子1A和S-腺苷甲硫氨酸合酶,以及Rpp4、RPG1-B和基因模型中包含的启动子,诸如Glyma启动子。
在另一个实施例中,表达本文公开的多核苷酸和多肽(即CcRpp2-R1和CcRpp2-R3抗性基因和多肽序列)的转基因植物还可以具有一种或多种杀真菌剂,其作为进一步防止对豆类作物物种的ASR相关损害的方法施用于转基因植物。这些杀真菌化合物还可用于补充包含CcRpp2-R1和CcRpp2-R3抗性基因序列的转基因豆类作物物种对多种不希望的病害的保护。这些杀真菌剂可与本文公开的其他杀真菌剂配制或桶混,或与其他杀真菌剂顺序施用。此类杀真菌剂可以包括2-(硫氰酰甲硫基)-苯并噻唑、2-苯基苯酚、8-羟基喹啉硫酸盐、唑嘧菌胺、aminopyrifen、安美速、抗霉素、白粉寄生孢(Ampelomyces quisqualis)、阿扎康唑、嘧菌酯、枯草芽孢杆菌(Bacillus subtilis)、枯草芽孢杆菌菌株QST713、苯霜灵、苯菌灵、苯噻菌胺、苯并烯氟菌唑、苄基苯胺-磺酸盐(BABS)、碳酸氢盐、联苯、噻枯唑、联苯三唑醇、联苯吡菌胺、灭瘟素、硼砂、波尔多混合剂、啶酰菌胺、溴菌唑、乙嘧酚磺酸酯、多硫化钙、敌菌丹、克菌丹、多菌灵、萎锈灵、环丙酰菌胺、香芹酮、氯芬同(chlazafenone)、氯英康唑(chloroinconazide)、地茂散、百菌清、乙菌利、盾壳霉(Coniothyrium minitans)、氢氧化铜、辛酸铜、氯氧化铜、硫酸铜、硫酸铜(三元)、氧化亚铜、氰霜唑、环氟菌胺、霜脲氰、环唑醇、嘧菌环胺、棉隆、咪菌威、联胺乙烯双-(二硫代氨基甲酸酯)、抑菌灵、二氯芬、双氯氰菌胺、哒菌酮、氯硝胺、乙霉威、苯醚甲环唑、野燕枯(difenzoquat ion)、二氟林、烯酰吗啉、醚菌胺、烯唑醇、烯唑醇-M、消螨通、敌螨普、二苯胺、二噻农、吗菌灵醋酸盐、吗菌灵醋酸盐、多果定、多果定游离碱、克瘟散、enestrobin、烯肟菌酯、氟环唑、噻唑菌胺、乙氧喹、土菌灵、噁唑菌酮、咪唑菌酮、氯苯嘧啶醇、腈苯唑、甲呋酰胺、环酰菌胺、氰菌胺、拌种咯、苯锈啶、丁苯吗啉、胺苯吡菌酮、三苯锡、薯瘟锡、毒菌锡、福美铁、嘧菌腙、氟啶胺、咯菌腈、氟茚唑菌胺、氟吗啉、氟吡菌胺、氟吡菌酰胺、唑呋草、fluoxapiprolin、氟嘧菌酯、氟喹唑、氟硅唑、磺菌胺、flutianil、氟酰胺、粉唑醇、氟唑菌酰胺、灭菌丹、甲醛、乙膦酸、三乙膦酸铝、麦穗宁、呋霜灵、呋吡菌胺、双胍盐、双胍盐醋酸盐、GY-81、六氯苯、己唑醇、恶霉灵、抑霉唑、抑霉唑硫酸盐、亚胺唑、双胍辛胺、双胍辛胺三乙酸盐、双胍三辛烷苯基磺酸盐、inpyrfluxam、iodocarb、种菌唑、ipfenpyrazolone、异稻瘟净、异菌脲、丙森锌、isofetamide、isoflucypram、稻瘟灵、吡唑萘菌胺、异噻菌胺、春日霉素、春日霉素盐酸盐水合物、kresoxium-methyl、海带多糖、代森锰铜、代森锰锌、双炔酰菌胺、代森锰、精甲霜灵、嘧菌胺、灭锈胺、敌螨普、氯化汞、氧化汞、甘汞、甲霜灵、精甲霜灵、威百亩、安百亩、metam-potassium、威百亩、叶菌唑、磺菌威、甲基碘、敌线酯、代森联、苯氧菌胺、苯菌酮、米多霉素、腈菌唑、代森钠、酞菌酯、氟苯嘧啶醇、辛噻酮、甲呋酰胺、油酸(脂肪酸)、肟醚菌胺、恶霜灵、氟噻唑吡乙酮、喹啉铜、咪唑富马酸盐、氧化萎锈灵、稻瘟酯、戊菌唑、戊菌隆、氟唑菌苯胺、五氯苯酚、月桂酸五氯苯酯、吡噻菌胺、醋酸苯汞、膦酸、四氯苯酞、啶氧菌酯、多氧菌素B、多抗霉素、保粒霉素、碳酸氢钾、羟基喹啉硫酸氢钾盐、噻菌灵、咪鲜胺、腐霉利、霜霉威、霜霉威盐酸盐、丙环唑、甲基代森锌、丙氧喹啉、丙硫菌唑、氟唑菌酰羟胺、唑胺菌酯、唑菌酯、唑菌胺酯、pyraziflumid、定菌磷、吡菌苯威、稗草畏、啶斑肟、嘧霉胺、甲氧苯啶、咯喹酮、灭藻醌、喹氧灵、五氯硝基苯、大虎杖(Reynoutria sachalinensis)提取物、氟唑环菌胺、硫硅菌胺、硅氟唑、2-苯基苯酚钠、碳酸氢钠、五氯酚酸钠、螺环菌胺、硫磺、SYP-Z048、煤焦油、戊唑醇、特弗喹啉(tebufloquin)、四氧硝基苯、氟醚唑、噻苯咪唑、噻呋酰胺、甲基硫菌灵、福美双、噻酰菌胺、甲基立枯磷、对甲抑菌灵、三唑酮、唑菌醇、咪唑嗪、三环唑、克啉菌、肟菌酯、氟菌唑、嗪氨灵、灭菌唑、有效霉素、精高效氯氟氰菊酯、霜霉灭、农利灵、代森锌、福美锌、苯酰菌胺、嗜油假丝酵母(Candida oleophila)、尖孢镰刀菌(Fusarium oxysporum)、粘帚霉属(Gliocladium)物种、大伏革菌(Phlebiopsis gigantea)、灰绿链霉菌(Streptomycesgriseoviridis)、木霉属(Trichoderma)物种、(RS)-N-(3,5-二氯苯基)-2-(甲氧基甲基)-琥珀酰亚胺、1,2-二氯丙烷、1,3-二氯-1,1,3,3-四氟丙酮水合物、1-氯-2,4-二硝基萘、1-氯-2-硝基丙烷、2-(2-十七烷基-2-咪唑啉-1-基)乙醇、2,3-二氢-5-苯基-1,4-二噻1,1,4,4-四氧化物、2-醋酸甲氧基乙基汞、2-甲氧乙氯汞、2-灭菌硅、3-(4-氯苯基)-5-甲基罗丹宁、4-(2-硝基丙-1-烯基)苯基硫氰酸酯、氨基丙基磷酸、防霉灵、氧化福美双、多硫化钡、Bayer 32394、麦锈灵、敌菌腙、bentaluron、benzamacril;benzamacril-isobutyl、抑菌啉(benzamorf)、乐杀螨、双(甲基汞)硫酸盐、双(三丁基锡)氧化物、丁硫啶、镉钙铜锌的硫酸铬酸盐、吗菌威、CECA、灭瘟唑、双胺灵、chlorfenazole、四氯喹恶啉、氯咪巴唑、双(3-苯基水杨酸)铜、铬酸铜锌、丁香菌酯、硫杂灵、硫酸肼铜、福美铜氯、环菌胺、青菌灵(cypendazole)、酯菌胺、癸磷锡、dichlobentiazox、二氯萘醌、菌核利、苄氯三唑醇、二甲嘧酚、邻敌螨消、硝辛酯、硝丁酯、dipymetitrone、双硫氧吡啶、灭菌磷、多地辛、敌菌酮、EBP、烯肟菌酯(enoxastrobin)、ESBP、乙环唑、代森硫、乙嘧酚(ethirim)、烯肟菌胺、敌磺钠、咪菌腈、种衣酯、fenpicoxamid、florylpicoxamid、氟苯醚酰胺(flubeneteram)、氟菌螨酯、氟醚菌酰胺、三氟苯唑、二甲呋酰胺、呋菌唑、呋醚唑、拌种胺、呋菌隆、果绿啶、灰黄霉素、丙烯酸喹啉酯、Hercules3944、环己硫磷、ICIA0858、ipfentrifluconazole、ipflufenoquin、异潘松(isopamphos)、异瓦二酮(isovaledione)、mandestrobin、邻酰胺、甲威苯咪、氯氟醚菌唑、肼菌酮(metazoxolon)、呋菌胺、氰胍甲汞、噻菌胺、metyltetraprole、代森环、糠氯酸酐、甲菌利、N-3,5-二氯苯基-琥珀酰亚胺、N-3-硝基苯基衣康酰亚胺、纳他霉素、N-乙基汞-4-甲苯磺酰苯胺、双(二甲基二硫氨基甲酸酯)镍、OCH、苯基汞二甲基二硫代氨基甲酸酯、苯硝酸汞、氯瘟磷、硫菌威;扑菌硫、比锈灵、吡丙炔(pyrapropoyne)、吡啶氯甲基(pyridachlometyl)、啶菌腈、啶菌唑、吡氯灵、氯吡呋醚、喹烯酮;喹烯酮硫酸盐、酯菌腙、喹唑菌酮、奎诺夫林(quinofumelin)、吡咪唑、水杨酰苯胺、SSF-109、戊苯砜、福代硫(tecoram)、噻二氟、噻菌腈、硫氯苯亚胺、硫菌灵、克杀螨、硫氰苯甲酰胺(tioxymid)、三唑磷胺、嘧菌醇、叶锈特、水杨菌胺、氯啶菌酯、三氟苯嘧啶、福美甲胂、氰菌胺、(2S,3S)-3-(邻甲苯基)丁-2-基(4-甲氧基-3-(丙酰氧基)吡啶甲酰基)-L-丙氨酸酯以及它们的任何组合。
本公开还包括用于本文所述的测定的试剂盒。多肽序列和多核苷酸可以作为试剂盒的组分与用于完成本文公开的测定的说明书一起包装。本公开的试剂盒可包括本文所述的多肽和/或多核苷酸以及合适的说明书(书面和/或作为音频、视频或视听材料提供)的任何组合。在一个实施例中,该试剂盒涉及用于鉴定针对ASR的TIR基因(例如,CcRpp2-R1和CcRpp2-R3基因)或CcRpp2-RI和CcRpp2-R3蛋白的DNA检测试剂盒。提供了利用本文公开的任何序列来鉴定植物中针对ASR的功效的转基因事件(例如,CcRpp2-R1和CcRpp2-R3)的试剂盒。例如,该试剂盒可包含特异性探针,其序列对应于或互补于与转基因事件的特定区域具有80%至100%序列同一性的序列。该试剂盒可包括进行测定或检测方法所需的任何试剂和材料。
根据实施例1,提供了一种选自以下的ASR抗性多肽:
a)CcRpp2-R1多肽,其包含与SEQ ID NO:2和21-36中任一项的氨基酸序列相比具有大于60%序列同一性的氨基酸序列;或者
b)CcRpp2-R3多肽,其包含与SEQ ID NO:4和48-58中任一项的氨基酸序列相比具有大于60%序列同一性的氨基酸序列。
根据实施例2,提供了一种ASR抗性组合物,其包含如实施例1所述的CcRpp2-R1多肽和如实施例1所述的CcRpp2-R3多肽。
根据实施例3,提供了一种多核苷酸,其编码ASR抗性多肽,其中所编码的多肽选自:
a)CcRpp2-R1多肽,其包含与SEQ ID NO:2和21-36中任一项的氨基酸序列相比具有大于60%序列同一性的氨基酸序列,任选地其中编码ASR抗性多肽的该多核苷酸与异源调控元件诸如异源植物启动子可操作地连接;或者
b)CcRpp2-R3多肽,其包含与SEQ ID NO:4和48-58中任一项的氨基酸序列相比具有大于60%序列同一性的氨基酸序列,任选地其中编码ASR抗性多肽的该多核苷酸与异源调控元件诸如异源植物启动子可操作地连接。
根据实施例4,提供了如实施例3所述的重组多核苷酸,其中该重组多核苷酸选自:
a)与SEQ ID NO:1和5-20中任一项的多核苷酸具有至少70%序列同一性的多核苷酸;以及
b)与SEQ ID NO:3和37-47中任一项的多核苷酸具有至少70%序列同一性的多核苷酸。
根据实施例5,提供了一种DNA构建体,其包含如实施例3或4所述的重组多核苷酸和与该重组多核苷酸可操作地连接的异源调控序列。
根据实施例6,提供了一种转基因植物或植物细胞,其包含如实施例1-5任一项所述的DNA构建体。
根据实施例7,提供了如权利要求6所述的转基因植物,其中该植物是豆类作物植物。
根据实施例8,提供了如实施例7所述的转基因豆类作物植物,其中转基因豆类作物植物是大豆。
根据实施例9,提供了一种赋予豆类作物物种抗病性的方法,其中该方法包括用赋予对豆类作物物种病害的抗病性的异源豆类来源的CcRpp2-R1基因和异源豆类来源的CcRpp2-R3基因转化豆类作物物种。
根据实施例10,提供了如实施例9所述的方法,其中该豆类作物物种病害由植物病原体引起。
根据实施例11,提供了如实施例9或10所述的方法,其中该植物病原体是豆薯层锈菌或山马蝗层锈菌。
根据实施例12,提供了如实施例9-11中任一项所述的方法,其中该豆类作物物种病害是亚洲大豆锈病。
根据实施例13,提供了如实施例9、10、11或12中任一项所述的方法,其中该豆类作物物种是苜蓿、三叶草、豌豆、菜豆、兵豆、羽扇豆、牧豆树、角豆树、大豆、花生或罗望子。
根据实施例14,提供了如实施例9、10、11、12或13中任一项所述的方法,其中该豆类作物物种是大豆。
根据实施例15,提供了如实施例9、10、11、12、13或14中任一项所述的方法,其中该豆类来源的CcRpp2-R1或CcRpp2-R3基因源自落花生属、紫荆属、木豆属、大豆属、苜蓿属、菜豆属、豌豆属或豇豆属。
根据实施例16,提供了如实施例6-8中任一项所述的转基因豆类作物植物,其进一步包含一个或多个另外的抗性基因,任选地其中该另外的抗性基因是CcRpp1基因。
根据实施例17,提供了如实施例6-8或16中任一项所述的转基因豆类作物植物,其进一步包含改善的农艺性状。
根据实施例18,一种种子,其来自如实施例16或17所述的转基因豆类作物植物,其中该种子具有如实施例1-5中任一项所述的DNA构建体。
根据实施例19,提供了一种减少豆类植物病害的一种或多种症状的方法,其中该方法包括将如权利要求6-8中任一项所述的转基因豆类作物植物暴露于该豆类植物病害,其中该转基因豆类作物植物具有增强的对该植物病害的抗性。
根据实施例20,提供了如实施例19所述的方法,其中该植物病害是亚洲大豆锈病。
根据实施例21,提供了一种产生亚洲大豆锈病抗性植物的方法,其中该方法包括用豆类来源的CcRpp2-R1基因和豆类来源的CcRpp2-R3基因转化植物细胞。
根据实施例22,提供了如实施例21所述的方法,其进一步包括从该转化的植物细胞再生该转化的植物。
根据实施例22,提供了如实施例22所述的方法,其进一步包括使该转化的植物生长的步骤,其中该豆类来源的CcRpp2-R1基因和该豆类来源的CcRpp2-R3基因的该表达导致该转化的植物中增强的对亚洲大豆锈病的抗性。
根据实施例23,提供了如实施例21-23中任一项所述的方法,其中该亚洲大豆锈病抗性植物是豆类物种。
根据实施例24,提供了一种豆类植物,其是来自包含本文公开的豆类来源的CcRpp2-R1基因和豆类来源的CcRpp2-R3基因的转基因豆类植物与未用该豆类来源的CcRpp2-R1基因和该豆类来源的CcRpp2-R3基因转化的类似豆类植物之间的杂交的子代。
根据实施例25,提供了如实施例24所述的植物,其中该豆类植物是苜蓿、三叶草、豌豆、菜豆、兵豆、羽扇豆、牧豆树、角豆树、大豆、花生或罗望子物种。
根据实施例26,提供了一种测定豆类植物对植物病害的抗病性的方法,其中该方法包括将包含豆类来源的CcRpp2-R1基因和豆类来源的CcRpp2-R3基因的该豆类植物的一部分暴露于植物病原体;在暴露于该植物病原体的该豆类植物上测量植物病害症状;以及将这些植物病害症状与抗病性的参考标准进行比较,任选地其中该植物病害由植物病原体引起,任选地其中该植物病原体由豆薯层锈菌或山马蝗层锈菌引起,任选地其中该植物病害是亚洲大豆锈病。
根据实施例27,提供了一种增强植物对亚洲大豆锈病(ASR)病害的抗性的方法,其中该方法包括在针对ASR的抗性的育种程序中通过将豆类来源的CcRpp2-R1基因和豆类来源的CcRpp2-R3基因渗入种质中来赋予对ASR病原体的抗性。
根据实施例28,提供了如实施例27所述的方法,其中该豆类来源的CcRpp2-R1基因编码与SEQ ID NO:2的多肽具有至少90%序列同一性的多肽,并且该豆类来源的CcRpp2-R3基因编码与SEQ ID NO:4的多肽具有至少90%序列同一性的多肽。
根据实施例29,提供了如实施例27或28中任一项所述的方法,其中该CcRpp2-R1基因编码SEQ ID NO:2的多肽并且该CcRpp2-R3基因编码SEQ ID NO:4的多肽,任选地其中该种质是豆类作物物种,任选地其中该豆类作物物种是苜蓿、三叶草、豌豆、菜豆、兵豆、羽扇豆、牧豆树、角豆树、大豆、花生或罗望子物种,任选地其中该豆类作物物种是大豆。
根据实施例30,提供了如实施例29所述的方法,其中当与易感植物相比,用该多肽转化的植物显示增强的对ASR的抗性。
根据实施例31,提供了如实施例5所述的重组DNA构建体,其进一步包含一种或多种NB-LRR多核苷酸或其片段。
根据实施例32,提供了如实施例5或31所述的重组DNA构建体,如权利要求5所述的重组DNA构建体,其进一步包含一种或多种抗性基因。
根据实施例32,提供了一种种子,其包含如实施例5、31或32中任一项所述的重组DNA构建体。
根据实施例33,提供了一种植物,其包含如实施例5、31或32中任一项所述的重组DNA构建体。
根据实施例34,提供了如实施例32所述的种子或如实施例33所述的植物,其中所述种子或所述植物包含编码选自以下的多肽的核酸序列:
a)CcRpp2-R1多肽,其包含与SEQ ID NO:2和21-36中任一项的氨基酸序列相比具有大于90%序列同一性的氨基酸序列;或者
b)CcRpp2-R3多肽,其包含与SEQ ID NO:4和48-58中任一项的氨基酸序列相比具有大于90%序列同一性的氨基酸序列。
根据实施例34,提供了如实施例33所述的种子或植物,其中该CcRpp2-R1多肽是SEQ ID NO:2并且该CcRpp2-R3多肽是SEQ ID NO:4,任选地其中该CcRpp2-R1多核苷酸是SEQ ID NO:1并且该CcRpp2-R3多核苷酸是SEQ ID NO:3。
以下实例是通过说明的方式但不是通过限制的方式来提供的。
实例1:CcRpp2基因的作图和克隆
木豆(Cajanus cajan)是二倍体豆类,其基因组大小为约830Mbp(Varshney等人(2012)Nat.Biotechnol.[自然-生物技术],30:83-89)。该植物是自花授粉的,并且具有2-3个月的种子间生命周期。木豆(登录号G108-99)先前被表征为表现出对亚洲大豆锈病(ASR)病害的抗性。如本文所公开的,进一步筛选该品种以研究除了已知的CcRpp1基因座之外,在植物的基因组中是否存在对亚洲大豆锈病(ASR)病害的另外的未表征的抗性基因。更特别地,通过将木豆(登录号G108-99)与显示完全易感性的成员(包括Ra、Rb、Rc、Rd、Re和Rf)杂交产生了几个分离的群体。分离分析表明,除了显示出15:1分离模式的Rd群体外,单个主要抗性基因在这些群体中赋予抗性。进一步的分析显示,除了在Rd群体中的潜在的第二基因座外,在这些成员中观察到的抗性映射到相同的基因座。
具体地,进行了分离和标志分析,并证明了登录号G108-99(Rd)中存在第二抗性基因座。用ASR分离株PPUFV-02筛选来自群体CG8-1(G48-95x G108-99)的292株F2植物。将266株植物分类为抗性的,并且将24株植物分类为易感的。观察到的数值符合两个独立基因预期的15:1分离比。使用在含有先前描述的CcRpp1的间隔侧翼的标志SSR10581和dCAPS239615筛选相同的群体。基于该筛选,选择对该间隔纯合敏感的56株植物,即在CcRpp1基因座不含抗性等位基因。在该间隔易感等位基因纯合的至少十二株植物显示出免疫表型(0类),证实了在登录号G108-99中存在新抗性基因座的假设。将这56株F2植物自交以获得F3种子。用分离株PPUFV-02接种F3家族,并使用标志SSR10581和dCAPS239615进行基因分型。这些F3家族中的一些的抗性显示3:1的分离,并且标志分析证实了在CcRpp1基因座存在G48-95DNA,证实了木豆属基因组中其他地方单个抗性基因的分离。
选择两个分离的F3家族用于新抗性基因座的作图。将DNA分离并送到爱荷华州立大学(Iowa State University)的DNA设施中,以使用Sequenom MassARRAY iPLEX平台进行SNP基因分型。待筛选的SNP以10-20cM的间隔跨越木豆属基因组。在比较G48-95和G108-99基因组Illumina数据后对它们进行选择。
精细作图和BAC文库筛选用于鉴定本文鉴定为CcRpp2的新抗性基因的物理间隔(参见图1)。从Rd文库中鉴定了三个BAC克隆:BACP6(116,744bp)、BACJ17(139,830bp)和xcajanus-2(161,443bp)。所有BAC包括标志Rdint_264620和dCAPS_393933,但它们不包含标志Rdint_385686。BACJ17包括完整的BACP6和第三个BAC,并且没有一个BAC完全覆盖121kb的间隔(图1)。使用合适的标志,鉴定了在标志位置Rdint_385686上的间隔的远侧(标志位置Rdint_264620)上的功能一失一得重组体以及在近侧上的三个功能丧失重组体,将间隔缩小至121,252bp。使用位于该间隔中的三个标志构建并筛选Rd BAC文库:Rdint_264620;dCAPS 393933和Rdint_385686。
contig_153610和contig_135277之间的区域含有类似TIR-NB-LRR基因(Glyma14g024500的同源物)的序列。然而,通过在SMART(简单模块化架构研究工具;提供蛋白质结构域的简单鉴定和广泛注释以及蛋白质结构域架构的探索的网络资源(http://smart.embl.de/))上检查木豆TIR-NB-LRR序列进行更详细的观察后,该序列不是完整的NB-LRR基因,而是TIR-TIR结构域。TIR结构域的四个拷贝在121,252bp的间隔中存在两次,并且并似乎是重复的。
为了显现表达的基因,使用程序Geneious和TopHat(用于RNAseq读段的快速剪接接合映射器),分析作图结果以鉴定外显子之间的剪接接合。BAC上的第一TIR-TIR结构域组在抗性和易感转录组读段之间没有显示任何多态性。有趣的是,在易感亲本的TIR-TIR序列中观察到由两个核苷酸缺失引起的明显移码。此外,TopHat分析显示在抗性亲本的转录组数据集中有轻微的诱导。因此,该TIR-TIR序列是赋予CcRpp2抗性的主要候选者。第二TIR-TIR结构域在抗性转录组读段中未显示出诱导,并且在所表达的重叠群中的抗性读段与易感读段之间没有检测到多态性。然而,在易感亲本中存在的基因变体中的移码也是令人感兴趣的。
在BACJ17(BACJ17位置:63,676至66,081)中发现的TIR-TIR基因在抗性亲本(G108-99)的转录组中相对于易感转录组亲本(G48-95)上调。由该基因编码的TIR-TIR结构域属于罕见的TIR-2超家族,其中双子叶植物家族成员的数量限于每个物种2-5个基因(Sarris等人,2016)。第二外显子上的CT缺失仅存在于易感等位基因(BACJ17上的位置:64,691)中,引起基因上的移码,这导致早期终止密码子,从而产生很可能无功能且仅含有一个TIR结构域的短多肽序列。在易感等位基因S48中发现的这种CT缺失在其他四种不在CcRpp2基因座传递抗性的木豆成员中是固定的,表明该基因可能是CcRpp2抗性基因候选者。将G108-99的CcRpp2基因座处存在的TIR-TIR基因与木豆的六个其他成员进行比较,揭示仅有G108-99经由CcRppl和CcRpp2基因座传递抗性。G48-95是用于杂交的易感亲本植物。G59-95、G119-99、G127-97和G146-97在CcRpp1基因座含有抗性,但不经由CcRpp2基因座传递抗性。
RNA连接酶介导cDNA末端的快速扩增。
(RLM-RACE)在抗性成员中存在的第一TIR-TIR基因上进行,以获得包括5′和3′UTR的完整TIR-TIR基因序列。用于RACE实验的RNA从未感染的组织和未黄化的叶材料中分离。有趣的是,观察到两种不同的全长转录物,其中一种全长转录物显示在第二外显子末端缺失51个核苷酸。在第一TIR-TIR抗性转录物中观察到的两个剪接变体是由导致肽序列的两个变异的替代剪接事件引起的,其中一个肽序列从其序列丢失17个aa,但是该事件不改变这两个变体的功能。
为了确定完整TIR-TIR转录物相对于剪接变体RACE产物的相对丰度,使用程序TopHat将两个亲本的转录物组与BACJ17的更新版本比对。完整TIR-TIR抗性转录物的相对丰度的覆盖度为220x读段数,而携带51nt缺失的变体为9x读段数。
为了鉴定TIR-TIR转录物的最小有效启动子,假定不含来自上游RNA解旋酶基因的读段的较短启动子长度具有驱动大豆转化体中两种TIR基因表达的所有转录元件。
实例2:用木豆基因CcRpp2-R1Aa(SEQ ID NO:1)和CcRpp2-R3Aa(SEQ ID NO:3)转化大豆
设计植物转化构建体以在大豆中提供CcRpp2-R1Aa(SEQ ID NO:1)的高水平组成型表达和CcRpp2-R3Aa(SEQ ID NO:3)的中等水平组成型表达。用含有CcRpp2-R1Aa编码区的1026bp SfiI片段产生狭槽载体(slot vector),该片段在5′端与1948bp大豆泛素启动子+IntronI片段连接,并且在3′端与1163bp菜豆素终止子片段连接。完整的启动子-编码区-终止子盒侧翼为attR1和attL4重组位点。用含有CcRpp2-R3Aa编码区的1035bpBamHI+SnaBI片段产生第二狭槽载体,该片段在5′端与2,576bp玉蜀黍组蛋白2B启动子+IntronI片段连接,并且在3′端与902bp大豆泛素14(UBQ14)终止子片段连接。完整的启动子-编码区-终止子盒位于Gateway attL3和attR2重组位点之间。通过将由1,531bp attL2和attL1侧翼缓冲片段分开的两个启动子-编码区-终止子盒重组在基于Gateway的植物表达载体中的相容attR4和attR3重组位点之间,产生最终的堆叠基因构建体。除了上述元件之外,该载体还含有用于细菌选择的壮观霉素抗性基因和作为植物选择性标志的除草剂抗性大豆ALS基因。
将最终的含有CcRpp2Aa二元物的植物表达载体电穿孔到大肠杆菌(Escherichiacoli)中。然后选择转化体并通过标准小量制备方法分离pDNA。通过对小量制备的DNA进行诊断性限制酶消化来表征转化体。选择含有预期消化带模式的阳性克隆并随后转化到根癌农杆菌中。选择转化体,验证序列含有二元CcRpp2Aa植物转化构建体,并提交用于农杆菌属介导的转化。
农杆菌属介导的大豆转化。按照农杆菌属介导的转化方案从未成熟种子培养物产生转基因大豆品系(Finer和McMullen 1991;Stewart等人1996;Cho等人2015)。简而言之,在标准条件下从温室中生长的植物的大豆荚中收获未成熟的种子。将种子表面灭菌,无菌切下未成熟子叶,并将培养物保持在250ml烧瓶中,该烧瓶含有50ml液体培养基、在26℃下在光周期为16/8h白天/夜晚的冷白色荧光下的旋转振荡器上(Samoylov等人1998;Cho等人2011)。将携带具有目的基因的质粒的根癌农杆菌用于转促未成熟子叶。选择转基因事件并再生至成熟。使这些植物在与野生型植物相同的条件下生长,但是在分开的生长室中。
组装另外的分子堆叠物构建体以分别在大豆泛素启动子或玉蜀黍组蛋白2B启动子后表达CcRpp2-R1Aa和CcRpp2-R3Aa的基因组和预测的cDNA,用于高水平或中等水平表达。从每个另外的构建体产生事件并证实其表达CcRpp2转基因。当用豆薯层锈菌攻击时,纯合和半合子植物显示红棕色(RB)表型;然而,与事件1-2(参见下文)相反,另外的构建体设计导致抗性谱降低,而没有检测到孢子形成的显著减少。
实例3:测试转基因植物针对ASR的功效
通过将植物表达构建体转化到大豆中,随后用豆薯层锈菌接种转基因植物并对植物病害症状评分来测试CcRpp2-R1Aa(SEQ ID NO:1)和CcRpp2-R3Aa(SEQ ID NO:3)基因的分子堆叠物针对ASR的功效。
从大豆转化实验中回收一个转基因事件,即1-2,并通过qPCR证实其含有CcRpp2-R1Aa和CcRpp2-R3Aa基因。该事件另外通过qRT-PCR显示表达CcRpp2-R1Aa转录物的197bp诊断性片段和CcRpp2-R3Aa转录物的202bp片段。
二元CcRpp2Aa针对豆薯层锈菌的功效的T1转基因测试。种植来自一个T1事件的种子并在生长室条件下生长15天直到Vc。在V1对植物取样用于qPCR以确定转基因拷贝数,并用豆薯层锈菌孢子的悬浮液接种。用收集自易感品种的夏孢子进行接种。将新鲜收获的孢子悬浮于0.01%吐温20的水溶液中并充分混合;然后用血细胞计数器将孢子浓度调节至4x103sp/ml。将植物用夏孢子悬浮液喷雾接种,在100%相对湿度下在黑暗中温育22小时,然后转移至针对病害发展优化的生长室(23℃,70%RH,16小时光周期),在那里使它们生长并发展症状15天。定期切除新的生长物,以便在实验期间保持单叶。
为了评估二元CcRpp2-R1Aa和CcRpp2-R3Aa的效果,将植物定性地评分为抗性(R;红棕色(RB),低或无孢子形成的病变)和易感(S;褐色,高度孢子形成的病变)和然后通过切除和扫描叶子定量地确定病变计数。接种后15天对无效、杂合和纯合植株进行评分。为了确定二元基因的效果,将转基因植物与来自相同事件的无效植物进行比较。
ASR感染测定结果总结于表2中。这些结果显示纯合样品中的二元CcRpp2Aa(CcRpp2-R1Aa和CcRpp2-R3Aa)提供对ASR的红棕色型抗性,具有很少至没有孢子形成。目测评估植物叶子的病变的存在,并用显微镜评价以检测夏孢子的存在。在95.45%的证实表达CcRpp2-R1Aa和CcRpp2-R3Aa的代表事件1-2的纯合植物上观察到低至无孢子形成,其中一个植物显示中等水平的孢子形成。杂合植物也显示抗性,红棕色病变,在89.47%植物上观察到低至无孢子形成;然而,在三株植物上观察到中等水平的孢子形成,并且一株杂合植物显示高水平的孢子形成。无效植物含有褐色的、高度孢子形成的病变,这是对病原体的典型易感反应。
这些ASR感染测定结果显示,当从宿主豆类木豆转基因转移到大豆植物时,二元CcRpp2基因CcRpp2-R1Aa和CcRpp2-R3Aa能够提供对豆薯层锈菌的抗性。
表2.携带二元CcRpp2Aa的事件1-2的测得性状。将合子型用作转基因拷贝数(无效=0,半合子=1,纯合=2);R=抗性,S=易感性;平均LC/cm2=每单位面积(em2)的平均病变计数;表型pct=具有观察到的表型的植物百分比
实例4-同源蛋白质的鉴定。
可以通过在默认参数下进行BLAST(基本局部比对20搜索工具;Altschul等人,(1993)J.Mol.Biol.[分子生物学杂志]215:403-410;还参见ncbi.nlm.nih.gov/BLAST/,可以通过使用www前缀登录该网址)搜索来确定基因同一性以获得与包含于公开可用的BLAST“nr”数据库(包括源自3维结构布鲁克海文蛋白质数据库(25SWISS-PROT蛋白质序列数据库的最后一个主要版本)、EMBL和DDBJ数据库的所有非冗余基因库CDS翻译、序列)中的序列的相似性。除了公共数据库之外,还搜索了专有内部的数据库。分析了某些多核苷酸序列。CcRpp2-R1Aa(SEQ ID NO:1)与某些同源蛋白质的所得百分比同一性值呈现于表3中。CcRpp2-R3Aa(SEQ ID NO:3)与某些同源蛋白质的所得百分比同一性值呈现于表4中。
表3:CcRpp2-R1Aa同源蛋白质及其来源
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*还可充当CcRpp2-R3多肽
表4:CcRpp2-R3Aa同源蛋白质及其来源
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*还可充当CcRpp2-R1多肽
对本公开的各种所说明的实施例的上述描述并不旨在是详尽的或者将范围限制于所公开的精确形式。虽然为了说明目的而在本文描述了具体实施例和实例,但是如相关领域的技术人员将认识到的,在本公开范围内的各种等效修改是可能的。本文提供的传授内容可以应用于除了上述实例之外的其他目的。根据上述传授内容,许多修改和变化是可能的,并且因此在所附权利要求书的范围内。
背景技术、具体实施方式和实例中引用的各文献(包括专利、专利申请、杂志文章、摘要、手册、书籍或其他公开内容)的全部公开内容通过援引以其全文并入本文。
就使用的数字(例如量、温度、浓度等)而言,已努力确保其准确性,但仍应允许有一些实验误差和偏差。除非另有说明,否则份为重量份,分子量为平均分子量:温度以摄氏度计;并且压力为大气压或接近大气压。
序列表
<110> 先锋良种国际有限公司(Pioneer Hi-Bred International, Inc.)
双刀片基金会(Two Blades Foundation)
维索萨联邦大学(Universidade Federal de Vicosa)
<120> 用于转移对亚洲大豆锈病的抗性的多核苷酸和方法
<130> 76751-327599 (RTS21584B-US-PSP)
<150> 63/130,261
<151> 2020-12-23
<160> 60
<170> PatentIn 3.5版
<210> 1
<211> 1026
<212> DNA
<213> 木豆(Cajanus cajan)
<400> 1
atgaagatgg gggatgaaaa agcgaagtgg tgctacgatg tttttctggc tttcagaggg 60
caagacacgc gctacaactt caccggtaat ctctacgatg ctttgcggcg cgctagaatc 120
agtactttca tggatgacag tgccttgaag ggtggtgacc aaatctcagc ctctcttctc 180
agagcgcttg aagcgtccag gatttcaatc gttgttctct ccgaaaacta tgcgttttcc 240
acgtggtgcc ttgaagaact tgtgaacatc gtccggtgta gaaggaccta tgcccaagtc 300
gttttgccaa tcttttacga cgtggatccc tccgatgtaa ggaagcagac aggtaggttt 360
gatgaagcca tggtccgaca tatacgtcgc ttcggagata aatcggagaa gataaaagag 420
tggaggtcag ctttgactga aatcgccaac ttgcgtggat gggatttccg atatggggac 480
cgatatgaat ataaactcat ccaagacatt gttagatggg tcaccaacac tgtgtcccgc 540
tatagcattt ttctgagttt ttgtggaaaa gatacccgct actcttttac aggttttctc 600
tacaatgctt tgtctcggag tggattccaa accttcatga acgatgggga ccacatttca 660
caatctaata ttgaagttga agcgattgaa aattcaaggc tttccatcat tgtattttct 720
gaaaactatg cacgttcctc ctcgtgtctt gatgagcttc tgacgatcct cgagtgtatg 780
aagctcaaaa accaactagt ttggcctgtc ttttacaaag tggaaccatc ggatttaagg 840
catcaaagaa atagttgggg tgaagccatg attgaacatg aaaacatgtt gggcaacgac 900
tctcagaagg tgcagaaatg gaggttagct ttacttgaag tcgccaactt gaaagggtgg 960
caccacaaaa ccgggtacga atatgaatta attgaaaaaa ttgtggaaat ggccactaaa 1020
atttaa 1026
<210> 2
<211> 341
<212> PRT
<213> 木豆(Cajanus cajan)
<400> 2
Met Lys Met Gly Asp Glu Lys Ala Lys Trp Cys Tyr Asp Val Phe Leu
1 5 10 15
Ala Phe Arg Gly Gln Asp Thr Arg Tyr Asn Phe Thr Gly Asn Leu Tyr
20 25 30
Asp Ala Leu Arg Arg Ala Arg Ile Ser Thr Phe Met Asp Asp Ser Ala
35 40 45
Leu Lys Gly Gly Asp Gln Ile Ser Ala Ser Leu Leu Arg Ala Leu Glu
50 55 60
Ala Ser Arg Ile Ser Ile Val Val Leu Ser Glu Asn Tyr Ala Phe Ser
65 70 75 80
Thr Trp Cys Leu Glu Glu Leu Val Asn Ile Val Arg Cys Arg Arg Thr
85 90 95
Tyr Ala Gln Val Val Leu Pro Ile Phe Tyr Asp Val Asp Pro Ser Asp
100 105 110
Val Arg Lys Gln Thr Gly Arg Phe Asp Glu Ala Met Val Arg His Ile
115 120 125
Arg Arg Phe Gly Asp Lys Ser Glu Lys Ile Lys Glu Trp Arg Ser Ala
130 135 140
Leu Thr Glu Ile Ala Asn Leu Arg Gly Trp Asp Phe Arg Tyr Gly Asp
145 150 155 160
Arg Tyr Glu Tyr Lys Leu Ile Gln Asp Ile Val Arg Trp Val Thr Asn
165 170 175
Thr Val Ser Arg Tyr Ser Ile Phe Leu Ser Phe Cys Gly Lys Asp Thr
180 185 190
Arg Tyr Ser Phe Thr Gly Phe Leu Tyr Asn Ala Leu Ser Arg Ser Gly
195 200 205
Phe Gln Thr Phe Met Asn Asp Gly Asp His Ile Ser Gln Ser Asn Ile
210 215 220
Glu Val Glu Ala Ile Glu Asn Ser Arg Leu Ser Ile Ile Val Phe Ser
225 230 235 240
Glu Asn Tyr Ala Arg Ser Ser Ser Cys Leu Asp Glu Leu Leu Thr Ile
245 250 255
Leu Glu Cys Met Lys Leu Lys Asn Gln Leu Val Trp Pro Val Phe Tyr
260 265 270
Lys Val Glu Pro Ser Asp Leu Arg His Gln Arg Asn Ser Trp Gly Glu
275 280 285
Ala Met Ile Glu His Glu Asn Met Leu Gly Asn Asp Ser Gln Lys Val
290 295 300
Gln Lys Trp Arg Leu Ala Leu Leu Glu Val Ala Asn Leu Lys Gly Trp
305 310 315 320
His His Lys Thr Gly Tyr Glu Tyr Glu Leu Ile Glu Lys Ile Val Glu
325 330 335
Met Ala Thr Lys Ile
340
<210> 3
<211> 1035
<212> DNA
<213> 木豆(Cajanus cajan)
<400> 3
atgttgatgg cgaatggtgc caccgaggaa caaaggagca gcaacaagta cgatgttttt 60
ctcagttttg caggtataga caaccgctac accttcaccg gtaatctcta caacgccttg 120
cgcagcaaga gaatcaacac ctttttcaat gaaaatggtg ataccaaagc tcttgaagca 180
attaaagaat cgaggatttc gatcgttgtg ctctccccaa actatgcatc ctcctcatgg 240
aggcttgatg aacttgtgac catccttgag tgtatgaaaa ccaagggcca actggtgtgg 300
cccatctatt acgaagtgga accgtcggaa gtaaggcgtc agagcggtat ctatggcgaa 360
gccatgtctg aatttgaaca aaaatttgga catgagagtg agatggtgtg gaaatggagg 420
agtgctttga ctgaagtcag cagcttgagt ggatgggttt acgaaactgg gtacgaatat 480
aaattcatcc gaaagattgt gcgattagca gtggaatctt tgccccgata tgatgttttc 540
ctgagtttta gcggagaaga tactcgctac tctttcacag gttttctcta taatgccttt 600
aggcgggagg ggttcaatat cttcatggat gatgagggat tggagggtgg caaccaaatt 660
tcagaaactc tcatgcgagc aattgaaatg tcaagacttt caattgtcgt cttctctgaa 720
aactatgcat attccacgtg gtgtcttgat gaacttgcca agatcatcga gtgcaagaag 780
accaagaatc agatggtttg gccaatattt cactatgtgg aaaagtctga tgtgtgcaat 840
caaacaaaaa gttatggtga agccatggct gcacatgaag aaagatttgg aaaggactct 900
gagaaggtgc aaaactggag gtctgctttg tctgaaattg ccaacttgga tggacaccat 960
ttcagagaaa atgagtacca atatgaattt atcgaaaggg ttgtggatct ggccattgct 1020
atcgggaatc agtag 1035
<210> 4
<211> 344
<212> PRT
<213> 木豆(Cajanus cajan)
<400> 4
Met Leu Met Ala Asn Gly Ala Thr Glu Glu Gln Arg Ser Ser Asn Lys
1 5 10 15
Tyr Asp Val Phe Leu Ser Phe Ala Gly Ile Asp Asn Arg Tyr Thr Phe
20 25 30
Thr Gly Asn Leu Tyr Asn Ala Leu Arg Ser Lys Arg Ile Asn Thr Phe
35 40 45
Phe Asn Glu Asn Gly Asp Thr Lys Ala Leu Glu Ala Ile Lys Glu Ser
50 55 60
Arg Ile Ser Ile Val Val Leu Ser Pro Asn Tyr Ala Ser Ser Ser Trp
65 70 75 80
Arg Leu Asp Glu Leu Val Thr Ile Leu Glu Cys Met Lys Thr Lys Gly
85 90 95
Gln Leu Val Trp Pro Ile Tyr Tyr Glu Val Glu Pro Ser Glu Val Arg
100 105 110
Arg Gln Ser Gly Ile Tyr Gly Glu Ala Met Ser Glu Phe Glu Gln Lys
115 120 125
Phe Gly His Glu Ser Glu Met Val Trp Lys Trp Arg Ser Ala Leu Thr
130 135 140
Glu Val Ser Ser Leu Ser Gly Trp Val Tyr Glu Thr Gly Tyr Glu Tyr
145 150 155 160
Lys Phe Ile Arg Lys Ile Val Arg Leu Ala Val Glu Ser Leu Pro Arg
165 170 175
Tyr Asp Val Phe Leu Ser Phe Ser Gly Glu Asp Thr Arg Tyr Ser Phe
180 185 190
Thr Gly Phe Leu Tyr Asn Ala Phe Arg Arg Glu Gly Phe Asn Ile Phe
195 200 205
Met Asp Asp Glu Gly Leu Glu Gly Gly Asn Gln Ile Ser Glu Thr Leu
210 215 220
Met Arg Ala Ile Glu Met Ser Arg Leu Ser Ile Val Val Phe Ser Glu
225 230 235 240
Asn Tyr Ala Tyr Ser Thr Trp Cys Leu Asp Glu Leu Ala Lys Ile Ile
245 250 255
Glu Cys Lys Lys Thr Lys Asn Gln Met Val Trp Pro Ile Phe His Tyr
260 265 270
Val Glu Lys Ser Asp Val Cys Asn Gln Thr Lys Ser Tyr Gly Glu Ala
275 280 285
Met Ala Ala His Glu Glu Arg Phe Gly Lys Asp Ser Glu Lys Val Gln
290 295 300
Asn Trp Arg Ser Ala Leu Ser Glu Ile Ala Asn Leu Asp Gly His His
305 310 315 320
Phe Arg Glu Asn Glu Tyr Gln Tyr Glu Phe Ile Glu Arg Val Val Asp
325 330 335
Leu Ala Ile Ala Ile Gly Asn Gln
340
<210> 5
<211> 1125
<212> DNA
<213> 蔓花生(Arachis duranensis)
<400> 5
atgggtctgc aactgcaacc atcccactcg tcttcttcct cttcctggca ttgggaatac 60
gatgtgttcc tcaacttcag aggccctgac actcgctacg gtttcacagg ctatctctac 120
aaagctctct gtgacaaggg aattcatgcc ttcatggatt ttgacgatat tcacagaggg 180
aatgaaatct ccgcatcact tatgaaggca attgaagcgt ccaggattgc gattcttgtg 240
ttctctaaga actatgctga atcttcttat tgcctaaacg aactggtgaa gatcatggag 300
tgctctcagc gtcatggcca gtttgttttg ccagtatttt acagcgtcga tcctagcgtc 360
gtgcggcatc agaaaggtat ttatgaggag gcattggcta agactggaag gacgtttgag 420
catgccatgg acagagtgca aagatggagg actgccatgg ctgacgcagc taacttgtct 480
ggcttgcatt ttaaagggga tggatacgga tatgagtttg ttgagaaaat tgttgaacaa 540
gtgtcaaggg tgattaaacg tgttggtgat tatccagttg aactagagtc ccaggaacaa 600
gcttcctact cgtctttggc tccttgcttg agcaatggat ggaaatacga tgtgttcctc 660
agcttcagag gcactgatac tcgatttggt ttcactggca atctctacaa tgttctttgt 720
ggcaagggaa tccacacctt catggatgat gaggctcttc atagagggaa tgaaatctca 780
ggaacacttg acaaggcaat tgaagggtcc aagattgcta tacttgtgtt ctcgaagaac 840
tatgcttatt cttcttattg cttagatgaa cttgttaaga tcatgaagtg ctctcaatct 900
aattcccagt gtgttttgcc ggttttttat aacgtggatc ctcctcacgt tcgacatcag 960
cgtggtagtt atgaggaagc actggctaag catgaggaga ggttcaagaa tgatgtagac 1020
agactgcgag attggagggc tgctctgcat caggcagcta acttaacagg cttccatttc 1080
aaaggacaac caacgcaaca cttcaaaagt caaagcagaa actga 1125
<210> 6
<211> 1455
<212> DNA
<213> 花生(Arachis hypogaea)
<400> 6
atgggtctgc aaccatccca ctcttcttct tccttttcct ggcattggga atacgatgtg 60
ttcctcaact tcagaggccc tgacactcgc tacggtttca caggctatct ctacaaagct 120
ctctgtgaca agggaattcg tgccttcatg gattttggcg atattcacag agggaatgaa 180
atctccgcat ctcttatgaa ggcaattgaa gcgtccagga ttgcgattct tgtgttctct 240
aagaactatg ctgaatcttc ttattgccta aacgaactag tgaagatcat ggagtgctct 300
cagcgtcatg gccagtttgt tttgccagtg ttttacggca tcgatcctag cgtcgtgcgg 360
catcagaaag gtatttacga ggaggcactg gctaagactg gaaggacgtt tgagcatgcc 420
atggacagag tgcaaagatg gaggactgcc atggctgacg cagctaactt gtctggcttg 480
cattttaaag gggatggata cggatatgaa tttgttgaga aaattgttga acaagtgtca 540
agggtgatta aacgtgttgg tgattatcca attgaactgg agtcccaaga acaagcttcc 600
tactcctctt tggctccttc ctcgagcaat ggatggaaat acgatgtgtt cctcagcttc 660
agaggtactg atactcgatt tggtttcact ggcaatctct acaatgttct ttgtggcaag 720
ggaatccaca ccttcatgga tgatgaggct cttcatagag ggaatgaaat ctcaggaaca 780
cttgacaagg caattgaagg gtccaagatt gctatacttg tgttctcgaa gaactatgct 840
tattcttctt attgcttaga tgaacttgtt aagatcatga agtgctctca atctcattcc 900
cagtgtgttt tgccggtttt ttataacgtg gatcctcctc acgttcgaca tcagcatggt 960
agttatgagg aagcactggc taagcatgag gagaggttca gtgatgtaga gagactgcga 1020
gattggaggg ctgctctgca tcaggcagct aacttaacag gcttccattt caaagggaat 1080
gagtatgaac atgagtttat tggaaagatc gttcgagtgg tgtcgcggaa tattagaaat 1140
attgcttctc ctgtggtagg aatccaagaa aatggtggta cagaagcaga gtctcttctc 1200
caagacaacc aacgcaacaa ttcaaaagtc aaagcagaaa ctgaagctaa tgttgaaaac 1260
ttcatggaag tcgatgtgtt tgagaagtct gtaggtgttc agttggaatc gttgaagcga 1320
aagaagcgag aacttgaagg gcaaatccgt gccattaatg accagataac agaattccaa 1380
agaaagacag ttgcaaagag aaaaaaagcg tttgacagtg gaaaaaaatg ccaagttgaa 1440
agcatagtta catga 1455
<210> 7
<211> 771
<212> DNA
<213> 木豆(Cajanus cajan)
<400> 7
atgaaaacca agggccaact ggtgtggccc atctattacg aagtggaacc gtcggaagta 60
aggcgtcaga gcggtatcta tggcgaagcc atgtctgaat ttgaacaaaa atttggacat 120
gagagtgaga tggtgtggaa atggaggagt gctttgactg aagtcagcag cttgagtgga 180
tgggtttacg aaactgggta tcattcgtac gaatataaat tcatccgaaa gattgtgcga 240
ttagcagtgg aatctttgcc ccgatatgat gttttcctga gttttagcgg agaagatact 300
cgctactctt tcacaggttt tctctataat gcctttaggc gggaggggtt caatatcttc 360
atggatgatg agggattgga gggtggcaac caaatttcag aaactctcat gcgagcaatt 420
gaaatgtcaa gactttcaat tgtcgtcttc tctgaaaact atgcatattc cacgtggtgt 480
cttgatgaac ttgccaagat catcgagtgc aagaagacca agaatcagat ggtttggcca 540
atatttcact atgtggaaaa gtctgatgtg tgcaatcaaa caaaaagtta tggtgaagcc 600
atggctgcac atgaagaaag atttggaaag gactctgaga aggtgcaaaa ctggaggtct 660
gctttgtctg aaattgccaa cttggatgga caccatttca gagaaaatga gtaccaatat 720
gaatttatcg aaagggttgt ggatctggcc attgctatcg ggaatcagta g 771
<210> 8
<211> 5211
<212> DNA
<213> 加拿大紫荆(Cercis canadensis)
<400> 8
atggcaaatt ttggagaagc atcttctagt tcctcctcca aacccaggta cacctatgac 60
gttttcctga gttttagagg tgaagatacc cgcaacacat ttaccggtaa tctctatgat 120
gctctgtgcc aaaggggttt caacaccttc atagatgatg acgggttgga gagaggccaa 180
caaatttcat atgctcttat caatgcaatt gaagaatcaa aggtttccat tgttgtgttc 240
tccgaaaact atgcatcttc cccgtggtgc cttgatgaac ttgtcaagat ccttgaatgc 300
aagaaggaaa agggtcaaat agtttggcca attttttaca aggttgaacc tgccgatgta 360
agagatcaga agaacagcta cgaagcagca atggcaaaac atgaaagtag gttcagcaat 420
gacaaagtga caaaatggag gtcagctttg aaagaagcag ctaacttgtc aggatcggaa 480
tacaaaactg ggtacgaata caaatttatc accaacatta tagaagtcgc ctccactaaa 540
ttacatgata agcatttata tattggtgag cacattgttg gactcaagtc tcgtttaaaa 600
gaagtaaagt caattttaga tattggatct cacaatacta ttggcatggt gggaattcat 660
ggaactggtg gaataggaaa aaccacactt gccaaggttt tgtataactt gattgttgat 720
caatttcact gtgcatgttt tcttgaatct gtcagagaag gttcaaaatg ttccatggac 780
cttgtaagtc tgcaaaagaa acttctatcc cagatattta ggaaggaaaa tttcaacttg 840
ggcaatgttg atgaaggtgc aaatataata aaacataggc ttcgcaatag aagagttctc 900
cttgtgcttg atgatgttgt taagggggag caattaaaaa aattggctgg gggatgtgat 960
tggtttggtc ctggtagtag gatcatcata acaacaaggg ataaacattt gctaattgct 1020
catggagtag aaaaaatata tgaaatgaac gagctagatg atgataaagc tcttgaactc 1080
ttttgttgga aggccttcaa aatgagtgaa cctacagaag gctttgtgga tatatcctat 1140
aagataataa aatatgccaa gggccttcca ttggctttaa atgtaatagg ttccaatttg 1200
tttggtagga gtttaaaggc atgggaatct gcatcggata aatataagag gattctggac 1260
aaaggtattc atgacatact tagagtaagc tatgatagct tggaggatga tcagaaaagc 1320
atttttctcg atattgcttg tttcttcaaa ggggagagat tggaagatgt tgaaaagata 1380
ctagatgctt gtgatctctc tccgcaatat aatattgaag tacttgttga taaatctctc 1440
ataactattg ggcttggcaa tttgtggatg catgatcttg ttcaagacat gggaaaagaa 1500
gttcttaagc aagatgcgtc atcaaaactt ggtgactaca gtagattatg gaatcatgag 1560
gctattttcg acggaagtga tagcattcaa ggaataatgt ttgatccacc tcaactagaa 1620
atggtagaat ggagtggtac tgccttcaag aagatgaaca atttaagaat tctcattgtc 1680
cgaaatgctg acttttcaac aggccctaaa aatttgccaa ataatttaag ggtgcttgaa 1740
tgggagagat atccttcaga gtctttacca caaggatttt atcctagaaa aattgttgtt 1800
ttaaagttac ggagtagctg tcttatctcc ttaaagccat tgcagaaatt tgaaaagctt 1860
acttgcattg atttctcgtg ctgtcaacta ttaactcaaa tacctgatat gtctagagcc 1920
ccaaatatag taactttgaa gcttaaccga tgcaccagtc taaaagaggt tcatgattct 1980
gttggagttc ttactaagct tcttggttta agccttgagg gatgcaccaa gctcaaaata 2040
tttccatatg gaattcagat gacatctctt atacatctta atctcaatga ttgcagaagt 2100
cttcaacact tcccagatat attgggacag atggatgaac tgaaacgtat tgatgcggaa 2160
aggaccggta ttaagcagat tccacattcc atttgttacc tccgggggct tgaattttta 2220
tgtatatcaa ataattacga tttgatatcc ctcccggaaa gcattaacca attagataga 2280
ttgatacacc ttaatattta taattgcaag aagcttcgac aaatttcagg aattccttca 2340
aatttggaac aaatacgtgc agaagaagca tcttctagtt cctcctttga accaaagttc 2400
tcctatgaca ttttgctgag ttttggaggt gaagatacct gcaacacctt tactgattat 2460
ctctacaatg ctctgtgcca aaggggtttc aacaccttca tagatgatgg cgggttagag 2520
agaggggaac aaatttcacc tgctcttctc aatgcaattg aagaatctag ggcttccatt 2580
gttgtgttgt ctgaaaactt cccatttttc acgtggtgcc ttgatgtagt tgtcaagatc 2640
cttgagtgca agaaggaaaa aggccaaatg gtttggccaa ttttctacca agtggaaccg 2700
tcctatgtaa gacttaagaa gaagagctac agagaagcaa tggctaaaca tgaaaatagg 2760
ttcagaaatg atatggacaa agtgagaaaa tggaggtcag ctttgaaaga agcagctgac 2820
ttgtatgaat ataaatttat ctcaaatatt atagaagagg tctctgtcac attacataat 2880
aagcatttat atgttggtga gcatatagtt ggactcaagt cttgcattga agaaatgaag 2940
tcaattttag atgttggatc taataatgat gttggcatgg tgggaattca tggaattggt 3000
gggatagaag catcttctag ttcctcctcc aaacccaggt acacctatga cgttttcctg 3060
agttttagag gtgaagatac ccgcaacaca tttaccggta atctctatga tgctctgtgc 3120
caaaggggtt tcaacacctt catagatgat gacgggttgg agagagggca acaaatttca 3180
tatgctctta tcaatgcaat tgaagaatca aaggtttcca ttgttgtgtt ctccgaaaac 3240
tatgcatctt ccccgtggtg ccttgatgaa cttgtcaaga tccttgaatg caagaaggaa 3300
aagggtcaaa tagtttggcc aattttttac aaggttgaac cggccgatgt aagacatctg 3360
aagaacagct acgaagcagc aatggcaaaa catgaaagta ggttcagcaa tgacaaagtg 3420
acaaaatgga ggtcagcttt gaaagaagca gctaacttgt caggatcgga atacaaaact 3480
gggtacgaat acaaatttat caccaacatt atagaagtgg cctccgctaa attacatgat 3540
aagcatttat atattggtga gcacattgtt ggactcaggt ctcgtttaaa agaagtaaag 3600
tcaattttag atattggatc tcacaatact attggcatgg tgggaattca tggaactggt 3660
ggtataggaa aaaccacact tgtcaaggtt ttgtataact tgattgttga tcaatttcac 3720
tgtgcatgtt ttcttgaatc tgccagagaa ggttcaaaat gttccatgga ccttgtaagt 3780
ctacaaaaga aacttctatc ccagatattt aggaaggaaa atttcaactt gggcaatgtt 3840
gatgaaggtg caaatataat aaaacacagg cttcgcaata ggagtgttct tcttgtgctt 3900
gatgatgttg ataaggggga gcaattaaaa aaattggctg ggggatgtga ttggtttggt 3960
cctggtagta ggatcatcat aacaacaagg gataaacatc tgctaattgc tcatggagta 4020
gaaaaaatat atgaaatgaa agagctagat gatgataaag ctcttgaact cttttgttgg 4080
aaggccttca aaacgagtga acctgcagaa agctttgtgg atatatccta taagataata 4140
aaatatgcca agggccttcc attggcttta aatgtaatag gttccaattt gtttggtagg 4200
agtttaaagg catgggaatc tgcattggat aaatataaga ggattctgga caaaggtatt 4260
catgacatac ttagagtaag ctatgatagc ttggaggatg atcagaaaag catttttctc 4320
gatattgctt gtttcttcaa aggggataga ttggaagatg ttgaaaagat actagaagct 4380
tgtgatctct ctccacaata taatattgaa ggaagtgata gcattcaagg aatcatgttt 4440
catccacctc aactagaaat ggtagaatgg agtggtactg ccttcgagaa gatgaacaat 4500
ttaagaattc tcattgtccg aaatgctaac ttttcaacaa gccctaaaaa tttgccaaat 4560
aatttaaggg tgcttgaatg ggaaagatat ccttcagact ctttaccaca aggattttat 4620
cctagaaaaa ttgttgtatt aaagttacga agtagctgtc ttaactcctt aaagccattg 4680
cagaaatttg aaaagcttac ttgcattgat ttctcgtgct gtcaactatt aactcaaata 4740
cctgatatgt ctaatgcccc aaatatagta actttgaagc ttgaccgatg caccagtctg 4800
aaagaggttc atgattctgt tggagttctt actaagcttc ttgatttaag ccttgaggga 4860
tgcaccaagc tcaaaatatt cccacatgga attcagatga catctcttag agatcttaat 4920
ctcaatgatt gcagaagtct tcaacacttc ccagatatat tgggacagat ggatgaacta 4980
agacgtattg atgcgaaaag gaccggtatt aagcagattc cacattccat ttgttacctc 5040
acaaggcttg gatttttact tatgtcagat aattatgatt tgatatccct cccggaaagc 5100
attagccaat tagatagatt gacagacctt aatatggata attgcaagaa gcttcgacaa 5160
atttcaggaa ttccttcaaa tttgaaacaa atacgtgcag atgttgcata a 5211
<210> 9
<211> 1023
<212> DNA
<213> 野大豆(Glycine soja)
<400> 9
atggcggatg aagggggaat tgagaagcgg cgctacgatg tttttctgtg ttttagagcg 60
caagacacgc gctacacctt cacaggtaat ctctatgctg ctttgcggca ggccagattg 120
aggaccttct tcgctggtgg gttgaagggt ggcgaccaaa tcttagacgc cattcttcaa 180
gcaattcagg aatcaaggat ttcaatcgtt gttctctccc aaagcttcgg gtcttccttg 240
tggtgccttg aagaacttgt gaagatcctt gagtgcaaga aaacaaagaa gcaactcctt 300
attccaatct tttaccgcgt ggatccctcc gacataagaa gacagactgg tagttttaag 360
gaggagctgc ttaaacatga aagtcgcttc ggaaaggact ccgagaaagt acgcaagtgg 420
aagtcagctt tgactcatgt cgccaccttg cccggatggt gtttcggaga tggaagctgc 480
aggtaccaat atgaatatga attcatcgaa aacattgtgc gagaagtcat cgccattgta 540
ccccgctata gcatttttct gagttttagt ggaaacgata cccgctcctt tacaggtttt 600
ctcaacaatg cattgtgcag gagtaggtac caaaccttca tgaatgatgg ggaccaaatt 660
tcacaatcta ctaatggagt tattgaagaa tcaaggcttt cgatcattgt attttctgaa 720
aactatgcac gttcctcgtc ctgtcttgat tttcttttga ccatccttga gtgtatgaag 780
acgaaaaacc aactggtttg ccccatcttt tacaaagtgt taccgtcgga tttaaggcat 840
caaagaaata gctatggtga agccatgact gagcatgaaa atatgttggg taaggactct 900
gagatggtga agaaatggag gtcagctttg tttgatgtcg ccaacttgaa aggattttac 960
ttgaaaaccg ggtacgaata tgaatttatt gacaaaatcg tggaaatggc cagtaaaatt 1020
taa 1023
<210> 10
<211> 1071
<212> DNA
<213> 藜苜蓿(Medicago truncatula)
<400> 10
atggccatga tgaatgaaga ggttgatggc agcagctaca agtacgatgt ttttctaagt 60
tttagagggg aagatactta ttgtaccttc gcaggtaatc tctatcatgc tttacgcaac 120
aagaaaatca agaccttctt cccacatgat caaattcaaa atgatgatga ggaacttcaa 180
ctttcacctt ctattctcaa ggcaattcag gaatcaagga tttcaatcgt cgttctctca 240
aaaaactatg caacctccac acgttgcctc aacgaacttg tgatcatcct tcagtgtatg 300
aagatgaaaa accagcttgt ttggccaatc ttttacgaag tacattcctc ggatgtaaag 360
cttcagagat gtaagtatgg tagtagcagc aaagccatat tgaagttccg agaaaggttt 420
aaagactacc ctcgcaggat gtgggaatgg cagcaagctt tgtctcaagt cacctctatt 480
gctggatgga attatggaat taaattcgaa tatgaactca tccaaaagat tgtggagtta 540
acagtacaat cgctaccccg ctacgatgtt tttctaagtt tttgcgggga agatactcgc 600
tactccttta cgggattcct ctatcatgct ttgcgtctag agggattcaa gatcttcatg 660
gatgatgagg gattggaggg tggaaaccaa atttcacaaa ctcttttaaa agcaattgaa 720
aaatcaaggc tctcaattgt tgttttgtcc gaaaattatg gatattcgac atggtgtctt 780
gatgaacttg tgaagattat ggagtgcaag aagaccaata ataaattggt ttggccacta 840
ttttacaaaa tagaacagtc agatttaagc tataaaaaaa gtagttatgg taaagccatg 900
gcagcacatg aggatagatt tggaaaggaa tctgagaatg tgcaaaaatg gagatcagct 960
ttgtcagaag ttgccttatt gaaagcagac catatcaaag aaaatgagca cgaatatgaa 1020
ttcataaaga agatcgtgga aagggccaat gaagctgaaa atcacatgtg a 1071
<210> 11
<211> 1005
<212> DNA
<213> 菜豆(Phaseolus vulgaris)
<400> 11
atggcgcgtt atgatgtttt tctgtgcttt agaggggaag acacgcgcct caccttcacg 60
ggtaacctct atgctgcttt gcagcaggca agattgagaa ctttcaggga tgagggagtg 120
ttgaagggtg gcgaccttgt atattccatc attgaagctt tggaagcatc cagggttgcc 180
atcgttgttc tctccgaaaa ctttgcgttt tctaggtggt gccttgacga acttgtcaag 240
atcctagact gcatgaagac aaagaaccaa atcgttattc caatctttta caacgtcgat 300
ccgtccgatg taaggaacct gagaggtagt tttgccgatg ccatggttga ccatgaacat 360
aggttcggaa agaactttga caaaatacga aactggaggt cagctttgac tgaagtggcc 420
aatttgtcag gatggtgttt gggaagagga agcaggttcg gatatgaata tgagtacatt 480
gaaaggattg tgagagactt gaccttgagg ctaccccgct atactatttt tctgagtttt 540
agtggaaaag atacacgctc cttctcgggt tttctctaca atgctctgag cagaagtgga 600
taccatacca tcctcaatga tggggaccag agttcacaat ctactgttgg ggttattgaa 660
aaatcaaaac tttcaatcat tgtcttttct gaaaactatg cacgttctcc ctcctgtctt 720
gatgagcttt tgaggatcct tgagtgcaag gagatgaaaa aacaactggt ttgccccatc 780
ttttacaaat tgttaccgtc tgatttaagg catcaaagaa atagttatgg tgaagccatg 840
agtgaacatg aaactatgat gggtaaggac tctgagaagg tgaagaaatg gaggtcagct 900
ttgtttgaag tcgccaactt gaaaggatgg tacatgaaaa cagggtacga atacgaattt 960
attgaaaaaa tcgtggaatt ggccaataaa atttctcggg tgtga 1005
<210> 12
<211> 531
<212> DNA
<213> 豌豆(Pisum sativum)
<400> 12
atggctaaat ggcaaatttt tcttagtttt agaggagaag atacgcgtta cgcgtttaca 60
ggttctcttt atcaatcttt atgtcaaggg ggattcaaga cctttatgga tgatagagga 120
ttgcagatgg gagaccaaat ttcaccatct cttttaaatg ccattgaagc atcgaggctt 180
tcgattattg ttttatctga aaactatgca aattcgacgt ggtgtctgga tgaacttgtc 240
aagattctcg agtgtatgaa attgaagaat caattggttt ggccaatctt ttataaagtg 300
gagccatctg atatcagaca tatgaaaaaa tcttatggaa aagacatggc tcgacatgaa 360
aatatatttg gaaatgattc tgagagagta caaaaatgga agtcagcttt agttgaagtg 420
tgtaatttgg ccaaaatggc ttattcaatc gggtatgaat atgaatttat tcagaagatt 480
gtggaagatg ccaatctcat tagaagacgt ttgcaaataa gaaacatata g 531
<210> 13
<211> 1014
<212> DNA
<213> 大豆(Glycine max)
<400> 13
atggcggatg aagggggaat tgagaagcgg cgctacgatg tttttctgtg ttttagaggg 60
gaagacacgc gctacacctt cacaggtaat ctctatgctg ctttgcggca ggcgagattg 120
aggaccttct tcgatgatgg gttcaagagt ggcgaccaaa tctttgacgt tgttcttcaa 180
gcaattcagg aatcaaggat ttcaatcgtt gttctctccg aaaacttcgc ttcttcctcg 240
tggtgccttg aagaacttgt gaagatcctt gagtgcaggg aaacaaagaa gcaactcgtt 300
attccaatct tttaccgcat ggatccctcc gacgtaagga gacagactgg ttgttatggg 360
gagagcctgg ctcaacatca atatgagttc agaagcgact ccgagaaagt acgcaattgg 420
caggaagctt tgactcatgt cgccaacttg cccggatgga ggttcagccg gtaccaatat 480
gaatatgaat tcatcgaaga cattgtgcga caagccatcg tcgccattgt accccgctat 540
agcatttttc tgagttttag tggaaacgat acccgctcct ttacaggttt tctcaacaat 600
gcattgtgca ggagtaggta ccaaaccttc atgaatgatg gggaccaaat ttcacaatct 660
actaatggag ttattgaaga atcaaggctt tcgatcattg tattttctga aaactatgca 720
cgttcctcgt cctgtcttga ttttcttttg accatccttg agtgtatgaa gacgaaaaac 780
caactggttt gccccatctt ttacaaagtg ttaccgtcgg atttaaggca tcaaagaaat 840
agctatggtg aagccatgac tgagcatgaa aatatgttgg gtaaggactc tgagatggtg 900
aagaaatgga ggtcagcttt gtttgatgtc gccaacttga aaggatttta cttgaaaacc 960
gggtacgaat atgaatttat tgacaaaatc gtggaaatgg ccagtaaaat ttaa 1014
<210> 14
<211> 1530
<212> DNA
<213> 红车轴草(Trifolium pratense)
<400> 14
atgtctggca gctgcaacaa tacggttgat ttcacaaaag aaaagaaccg attggttaca 60
catcgagatt ttgtgcgtga caaaattgaa agaactcata ataaaactca aaaggttagc 120
gatgttgtgt ttgagtggct aaatgaaaca gatatactca tacgtcaatt ggagaatctg 180
tcggcacaag caataacaag aaaacaattt aaaaaattgt tgaaaagagt aatggaacag 240
aacacgaaag taccttcagg aaattacatt caggaatctt caactccaat tccaagttta 300
gaacacttct cttcaggaaa tttaatgtgt tttaattcta gagagaagat ctccgatcaa 360
cttttcgtgg cattgaatga tgatagttgt tctatgattg gattgtatgg tagccagggc 420
tcgggtaaaa caacattggt tgaagcaatg gccaaaaaag taaagtattt agagattttt 480
catgagattt tatttgtcaa ggtaacccaa aattcaaata ttagaacaat gcaagatgaa 540
attgttgact cattaaatat gaaatttgat aaaaaaaaca gtgaatctgg aagagccaga 600
gaaatattct caacaataga aagtatgaat tgtccaattc tagtcatttt tgataatgtt 660
ccagcaaaat tcgacccaga agatataggc attccttgta atagtaatcg ctgcaagatt 720
cttttgacca catgttgcca aaaagattgt gacttgttgt ccggtcaaag gaagattcaa 780
cttgatccct tatctagaca ggaagcttgg attttgtttc aaaaacattc aggtatttat 840
gatgatgaga aatactcgag atttgactta ttgaatgtag catatgaagt tgctttagaa 900
tgtgaagggt tacctagaac aattaaagat gtgggacctt tcttaaaaag taaaccaatt 960
gaagaatgga agacaacact agatagtctg aaacattcaa tggccaaatg gcaaattttt 1020
cttagtttta gaggagaaga tacacgcaac tcttttacag gttatcttta tcaaacttta 1080
tctcaagcgg gattcaagac cttcatggat gatggaggac tgcatactgg agaccaaatt 1140
tcaccctctc ttgtaaatgc aattgaagca tcgaggcttt cgattattgt tttatctgaa 1200
aactatgcaa attcctcatg gtgtcttgaa gaacttgtca agattcttga gtgtatgaag 1260
ttgaagaatc aattagtttg gccaatcttt tacaaagtgt acccatcaga cataaggcat 1320
ctgagaaaat gttatggcaa agacatggat cgacatgaaa ataattttgg aattaactca 1380
gaaagagtgc agaaatggag gtcagctttg tttgaagtgt ctaatttatc cggaaaagct 1440
tatacaaccg ggtacgaata tgaatttatt caaaagatcg tggaagatgc caatcgtatt 1500
aaaagtcgtt tgcaaatacg aagagcataa 1530
<210> 15
<211> 1848
<212> DNA
<213> 赤豆(Vigna angularis)
<400> 15
atggaggcag aaagttacaa tagactgagt caacaagacc cttctggtgg tgccactgtt 60
tctcatccat ccagtgaacg aaaaaacttt gatgttttcc tcagtcttgg tggaaaagat 120
gttcgctaca ccttcactgg taatctcttt aatgctttgc gcagcaagag aattaagacc 180
cttttcagag aacatgaata tgaacctgat gatgacacca atatttcacc ctctgctctt 240
aaagcaatac aaacgtcaaa gatttccatc gttgttttct ccccagaata tgcatcctcc 300
tcaaaacgtc tcgatgaact tgtggccatc cttgagtgta ggatgaggac caaccaactc 360
gtatggccaa tcttttacgg agtggaaccg actgacgtaa gatttcagag aggtagatat 420
gaacaagcca ttaatagatt tgaagaaaga tattattccc cagagaggat gaataaatgg 480
agagcagctt tggctgaagt cagcaacttg agtggatggt tttaccaaaa ggggcacaaa 540
tacgaatata aattcatccg aaagattgtg gaagcagccg tgcaatgcct gtcaagatat 600
gatgtttttc tgagtttttg tggagaggat acccgctaca ctctcacagg ttttctatac 660
aatgcccttc gccgagaggg attcaaaatc ttcatggacg atgaagaatt ggagggtggg 720
aaccaaattt ctcaaaagct aatgggagca atagaaagtt caagggtttc aattgttgtg 780
ttctctgaaa actatggata ttccacctgg tgtgctttgt ctcaaatcac caacttggaa 840
ggagagcatc tcagtgaaaa tgagtctgta atgttaaatt ttattactgt atttataatt 900
ggtgtttgtt ggaatgtttt agggaaggac acgcgccaca ccctcacagg taacctctat 960
gctgctttgc agcaggctag attcagaact ttcatggatg atgacgagtt gaagggtgcc 1020
gaccaaattg catatacgat cgttttggaa gcatccagga tttcgatcgt tgttctctcc 1080
gaacactttg cgttttccag ttggtgcctt gatgaacttg ctaacatcct agactgcatg 1140
aacacaaaga accaagccgt ttttccaatc ttttacgaag tcgatccgtt ctatgtaagg 1200
cacctgaaag gtagttttgg cgaggccatg gttgcccatg aagctagatt cggaaaggac 1260
tctgagaaag tagaaaaatg gaggtcagct ttggctcaag tggccaactt gtcgggatgg 1320
tgttttggaa gaggaaggtg gtgccaatat gaatatgaat atgaattcat tgaaaggatt 1380
gtgcaacatg tgaccaagtt gttaccccgc tacagcattt ttctgagttt tagtggaaaa 1440
gatacacgct ccttcacggg ttttctctac aatgctttga gccgaagtgg ataccatact 1500
ttcatcagtg atggggaaca tagttcacaa tctattgttg gggttattga aaaatcaagg 1560
ctttcaatca ttgtcttttc tgaaaactat gcacgttccc cttcctctct tgatgagctt 1620
ttgaggatcc ttgagtgcat ggatatgaaa aaccacctgg tttgccccat cttttacaaa 1680
gtgttaccgt cggatataag gcatcagaga aaaagttatt gtgaagccat gattgaacat 1740
gaaaatgtga tgggtgagga ctctgagaag gtcaagaaat ggaggtcagc tttgtttcaa 1800
gtcgccaact tgaaaggatg gtgcatgaaa acggggtaca gtaaatag 1848
<210> 16
<211> 1065
<212> DNA
<213> 绿豆(Vigna radiata)
<400> 16
atgtatttca tttcagcatc agagagaagg aatgcagcaa cctgcaatga acatctaagt 60
aatcgcaata acactcagtt tgagttcttg attcatttca ttatcgatgc tatggcgcgt 120
tatgacgttt ttctgtgctt tagagggagg gacacgcgcc acaccttcac aggtaacctc 180
tatgctgctt tgcagcaggc tagattcaga actttcatgg atgatgacga gttgaagggt 240
gccgaccaaa ttgcatatac tatcgttttg gaagcatcca ggatttcgat cgttgttctc 300
tccgaacact ttgcattttc cagttggtgc cttgatgaac tagctaaaat cgtagactgc 360
atgaacacaa agaaccaagc cgtttttcca atcttttacg aagttgatcc gttctatgta 420
aggcacctga aaggtagttt tggcgaggcc atggttgccc atgaagctag gttcggaaag 480
gactctgaga gagtagagaa atggaggtca gctttgattc aagtgaccaa cttgtcagga 540
tggtgttttg caagaggaag gtggtgcgaa tacgaatatg aattcattga aaggattgtg 600
caacatgtga ccaagttggt accccgctac agaatttttg tgagttttag tggaaaagat 660
acacgctcct tcactggttt tctctgcaat gctttgagcc gaagtggata caatactttc 720
atcagtgatg gggaacagag ttcacaatct actgttgggg ttattgaaaa atcaaggctt 780
tcaatcattg tcttttctga aaactatgca cgttcccctt cctgtcttga tgagcttttg 840
agggtccttg agtgcatgga gatgaaaaac cagctggttt gccccatctt ttacaaagtg 900
ttaccatcgg atttaaggca tcagagaaga agttatggtg aagccatgat tgaacatgaa 960
aatgtgatgg gtgaaaactc tgagaaggtc aagaaatgga ggtcagcttt gtttcatgtc 1020
gccaacttga aaggatggtg catgaaaaca gggtacagta aatag 1065
<210> 17
<211> 2664
<212> DNA
<213> 豌豆(Pisum sativum)
<400> 17
atggcattgg cttgcaatag cctccaaagc agctcttctt caccaaagat gagatggaaa 60
tatgatgtgt ttgtgagctt tagaggagaa gacactcgca acaatttcac cgatcacctc 120
tttggcgctc ttcataagaa agctattatt accttcaggg atgatacaaa gctcaagaaa 180
ggggaagata tatcacttga gcttctacaa gctattgaag gatcccaaat tttgattgtt 240
attttctcaa caaactatgc ttcttccaca tggtgcttgc aagaagtagc aaaaattgct 300
gcatgcattg aagttccagg acaatctgtt ctacctattt tctttgatgt cagtccttct 360
gaggtacgaa agcaatgcgg agattatgaa aaagcctttc aagggcatga agaaagattc 420
aaagcaactt tagagaaagt gcaaagatgg agaggagctc taacacaagt agccaatctc 480
tctggttggg acgttaggga taagccacaa tatgcagaga ttggagaaat tactaaaaag 540
gtaacatgct tattgggaaa taaatcttcg actttaccta gggatatagt tgggatgccc 600
tccagagtgg aagaattaga aaaccatctg aatttggact caaatgatga tgatgttcga 660
gtaataggga tttgtggaat gggtggaata ggaaagacga ctcttgctac tgctttgtat 720
gctagaatct ctaatcaatt tgatgcttgc tgctttattg atgatgtaag taaaatttat 780
ggagatcatg gcccaattgg agtacaaaag caacttctgc gtcaaactct aaatgaagaa 840
aatctccaaa tatgcaatct tcctatggct tctaatttga ttcgaactag gctatctcga 900
ttaaaatccc ttgtttttct tgataatgtg gatgaagttg aacaactgga taaattggat 960
atgagactag aatggctagg cagagggagt agaataatca taatttctag aaatgggcac 1020
atcttgacag agtatggagt agatgaagta tacagagttc gactcttgga tcgtaaatgt 1080
gcccttcaat tgttttgcca gaaagctttc aaaagtgatg acatcatgag tggttacata 1140
tacttgacaa aagaagtact agcatatgcc aatggccttc cactcgcaat taaagtgttg 1200
ggctcgtatc tgtatggtcg agatgtgtct gagtggagaa gtgcattgtc aagattaaga 1260
gagaacccaa tgacagatat tatgaatgta ctccgaatta gttttgatgg actagaggat 1320
acagaaaagg acatatttct tgatattgct tgtttctttc atggataccc gaagggatac 1380
ctgaagaaaa ttttagattt tcgtggattt catcctgaaa ttggtttaag agttctcgtt 1440
gataaatcat tcataactta caagaaacag ataatttgca tgcatgattt gtttagagaa 1500
ctgggaaaga gtatcgttag agaaaaatca cccaaagaac caagaaagtg gaacagggtg 1560
tgggactaca aagacgtcca caatgttatc tcagaaaaca tggcaacaga aaaccttgaa 1620
gccatgatga tggagtatga ttcagaacat gatatagaaa tacaacagat gacaacattg 1680
agggccgaag ctttagcaca aatgagtcgc cttaaattgc tcaggttgtt gacgtttaat 1740
ttctcaggaa gtctcaattt tctttcaagt gaattggggt atctacgttg ggataaatat 1800
cctttcacta gtttgccatc aagttttcag gcgtataaac ttgttgaact gatcctacgt 1860
catagcaaca ttaggaaact atgggaaggc acaaagtctc tacctaattt gacacgtatt 1920
gatctcagtt actcaaagaa tcttaatatg atgccaaatt ttgaggagac tccaaatctt 1980
gagagcctat gtcttgtagg atgtataaaa ctcgtgaaga ttgatccatc cattgctggg 2040
ttatatatat ttgactgccc cagtttagtt gagatggaaa gctattttgg aattgctttt 2100
tcttggatga tacaacttct tcaggttcac atgcaatccg agatcccaag gacagacatt 2160
acaattgtta ttcccaaaac tcaaattcca aagtggttca ccaaacaaca tgtaggcagt 2220
tcaataagca tcgatccatc atccattatg catgacaaga atttgatagg catcgcttgc 2280
tgcttaacat ttgtagcgca agataatcct actaatttaa gggaagaact gtcatcttat 2340
attgcgtttg gttttaaatg tacacagtgt ggggtctatt caattattcc catacttctt 2400
ggaaaagatc tggtcacagt tgatttagat cacctgttgc tagttttttt cagtaggaag 2460
aaatttattg atttgataag tgatgcaaca gatggatggg atgatattag tggtattgaa 2520
ttgagtgcta cagttaggca gccctttggt ttgcacttgg aagtaaaaaa ttgtggttat 2580
aattggatat ttaaggagga tctggaacaa ttaaatccac aaaagatgta caagggaaat 2640
tcgtcagttc agccatatta ttga 2664
<210> 18
<211> 978
<212> DNA
<213> 菜豆(Phaseolus vulgaris)
<400> 18
atggcgcgtt atgatgtttt tctgtgcttt agagggcgtg acacgcgcca caccttcatg 60
ggtaacctct atgctgcttt gcggcaggcc agattgagaa ctttcatgga tgagggcgtg 120
cttaaaggtg gcgacgttgc agatactatc attcaagctt tggaagcatc cagggtttcc 180
atcgttattc tctccgaaac ctttgcgtct tctaggtggt gccttgacga acttgtcaac 240
atcctagact gcatgaagac aaagaaccaa accgttattc caatctttta caacgtcgat 300
ccctccgatg taaggaacct gaaaggtagt tttggcgctg ccatggttgc ccatgaagat 360
gggttcggaa aggacaatga gagattacaa aagtggagat cagcattgac tcaagtggcc 420
aacttgtcag gatgctgttt gggtacagga agcaggttcg gatatgaata tgagtacatt 480
gaaaggattg tgagaagcgt gaccttggtg ataccccgct ataatatttt tgtgagtttc 540
agtggaaaag atacacgctc cttctcgggt tttctctaca atgctctgag cagaagggga 600
taccatacca tcctcaatga tggggaccag agttcacaat ctactactgt tggggttatt 660
gaaaaatcaa aactttcaat cattgtgttt tctgaaaact atgcacgttc tccctcatgt 720
cttgatgagc ttttgaggat ccttgagtgc aaggagatga aaaaccaact ggtttgcccc 780
atcttttaca aagtgttacc gtctgattta aggcatcaaa gaaatagtta tggtgaagcc 840
atgagtgaac atgaaaatat gatgggtaag gactctgaga aggtgaagat atggaggtca 900
gctttgtttg aagtcgccaa cttgaaagga tgtcctagtt ttgttgtgtg gcaattactt 960
tgtgcagata tactttga 978
<210> 19
<211> 984
<212> DNA
<213> 大豆(Glycine max)
<400> 19
atggcggatg aagggggaat tgagaagcgg cgctacgatg tttttctgtg ttttagaggg 60
gaagacacgc gcttcacctt cacaggtaat ctctgtgctg ctttgcggca ggccagattg 120
aggaccttct tcgatgatgg gttgaagggt ggcgaccaaa tcttagacgc cagtcttcaa 180
gcaattcagg aatcaaggat ttcaatcgtt gttctctccc acaacttcgc ttcttcctcg 240
tggtgccttg aagaacttgt gaagatcctt gagtgcaggg aaacaaagaa gcaactcgtt 300
attccaatct tttgccgggt ggatccctcc gacgtaagga gacagactgg tcgttttaag 360
gaggatctgc ttaaacatga aagtcgcttc gaaaaggact ccgagaaagt acgcaagtgg 420
aagtcagctt tgactcaagt cgccaccttg cccggatttt gtttcggaga tggaagctgc 480
agcgaccaat atgaatatga attcatccaa aacattgtgc gagaagccat cgccattgta 540
ccccgctata gcatttttct gagttttagt ggaaacgata cccgctcctt tacaggtttt 600
ctcaacaatg cattgtgcag gagtaggtac caaaccttca tgaatgatgg ggaccaaatt 660
tcacaatcta ctaatggagt tattgaagaa tcaaggcttt cgatcattgt attttctgaa 720
aactatgcac gttcctcgtc ctgtcttgat ttgcttttga ctatccttga gtgtatgaag 780
acgaaaaacc aactggtttg ccccatcttt tacaaagtgt taccgtcgga tttaaggcat 840
caaagaaata gctatggtga agccatgact gaacatgaaa atatgttggg taaggactct 900
gagagggtga agaaatggag gtcagctttg tttgatgtcg ccaacttgaa aggattttac 960
ttgaaaaccg ggtacaacac atag 984
<210> 20
<211> 1035
<212> DNA
<213> 大豆(Glycine max)
<400> 20
atggcggatg aagggggaat taagaagcgg cgctacgatg tttttctgtg ttttagaggg 60
gaagacacgc gctacacctt caccggtaat ctctatgctg ctttgcggca ggcgagattg 120
aggaccttct tcgatggtgg cattggtggg ttgaagggtg gcgaccaaat ctttgacgtt 180
cttcttcaag caattcagga atcaaggatt tcaatcgttg ttctctccca aaacttcggg 240
tcttccttgt ggtgccttga agaacttgtg aagatccttg agtgcaagaa aacaaagaag 300
caactcgtta ttccaatctt ttaccgcgtg gatcccgccg acgtaaggag acagactggt 360
agttttaagg agcagctgct taaacatgaa agtcgcttcg gaaaggactc cgagaaagta 420
cgccagtgga agtcagcttt gactcaagtc gccaccttgc ccggatggtg tttcggacat 480
ggaagctgca ggtaccaata tgaatatgaa ttcatcgaag acattgtgcg acaagccatc 540
gtcgccattg taccccgcta tagcattttt ctgagtttta gtggaaacga tacccgctcc 600
tttacaggtt ttctcaacaa tgcattgtgc aggagtaggt accaaacctt catgaatgat 660
ggggaccaaa tttcacaatc tactaatgga gttattgaag aatcaaggct ttcgatcatt 720
gtattttctg aaaactatgc acgttcctcg tcctgtcttg attttctttt gaccatcctt 780
gagtgtatga agacgaaaaa ccaactggtt tgccccatct tttacaaagt gttaccgtcg 840
gatttaaggc atcaaagaaa tagctatggt gaagccatga ctgagcatga aaatatgttg 900
ggtaaggact ctgagatggt gaagaaatgg aggtcagctt tgtttgatgt cgccaacttg 960
aaaggatttt acttgaaaac cgggtacgaa tatgaattta ttgacaaaat cgtggaaatg 1020
gccagtaaaa tttaa 1035
<210> 21
<211> 374
<212> PRT
<213> 蔓花生(Arachis duranensis)
<400> 21
Met Gly Leu Gln Leu Gln Pro Ser His Ser Ser Ser Ser Ser Ser Trp
1 5 10 15
His Trp Glu Tyr Asp Val Phe Leu Asn Phe Arg Gly Pro Asp Thr Arg
20 25 30
Tyr Gly Phe Thr Gly Tyr Leu Tyr Lys Ala Leu Cys Asp Lys Gly Ile
35 40 45
His Ala Phe Met Asp Phe Asp Asp Ile His Arg Gly Asn Glu Ile Ser
50 55 60
Ala Ser Leu Met Lys Ala Ile Glu Ala Ser Arg Ile Ala Ile Leu Val
65 70 75 80
Phe Ser Lys Asn Tyr Ala Glu Ser Ser Tyr Cys Leu Asn Glu Leu Val
85 90 95
Lys Ile Met Glu Cys Ser Gln Arg His Gly Gln Phe Val Leu Pro Val
100 105 110
Phe Tyr Ser Val Asp Pro Ser Val Val Arg His Gln Lys Gly Ile Tyr
115 120 125
Glu Glu Ala Leu Ala Lys Thr Gly Arg Thr Phe Glu His Ala Met Asp
130 135 140
Arg Val Gln Arg Trp Arg Thr Ala Met Ala Asp Ala Ala Asn Leu Ser
145 150 155 160
Gly Leu His Phe Lys Gly Asp Gly Tyr Gly Tyr Glu Phe Val Glu Lys
165 170 175
Ile Val Glu Gln Val Ser Arg Val Ile Lys Arg Val Gly Asp Tyr Pro
180 185 190
Val Glu Leu Glu Ser Gln Glu Gln Ala Ser Tyr Ser Ser Leu Ala Pro
195 200 205
Cys Leu Ser Asn Gly Trp Lys Tyr Asp Val Phe Leu Ser Phe Arg Gly
210 215 220
Thr Asp Thr Arg Phe Gly Phe Thr Gly Asn Leu Tyr Asn Val Leu Cys
225 230 235 240
Gly Lys Gly Ile His Thr Phe Met Asp Asp Glu Ala Leu His Arg Gly
245 250 255
Asn Glu Ile Ser Gly Thr Leu Asp Lys Ala Ile Glu Gly Ser Lys Ile
260 265 270
Ala Ile Leu Val Phe Ser Lys Asn Tyr Ala Tyr Ser Ser Tyr Cys Leu
275 280 285
Asp Glu Leu Val Lys Ile Met Lys Cys Ser Gln Ser Asn Ser Gln Cys
290 295 300
Val Leu Pro Val Phe Tyr Asn Val Asp Pro Pro His Val Arg His Gln
305 310 315 320
Arg Gly Ser Tyr Glu Glu Ala Leu Ala Lys His Glu Glu Arg Phe Lys
325 330 335
Asn Asp Val Asp Arg Leu Arg Asp Trp Arg Ala Ala Leu His Gln Ala
340 345 350
Ala Asn Leu Thr Gly Phe His Phe Lys Gly Gln Pro Thr Gln His Phe
355 360 365
Lys Ser Gln Ser Arg Asn
370
<210> 22
<211> 484
<212> PRT
<213> 花生(Arachis hypogaea)
<400> 22
Met Gly Leu Gln Pro Ser His Ser Ser Ser Ser Phe Ser Trp His Trp
1 5 10 15
Glu Tyr Asp Val Phe Leu Asn Phe Arg Gly Pro Asp Thr Arg Tyr Gly
20 25 30
Phe Thr Gly Tyr Leu Tyr Lys Ala Leu Cys Asp Lys Gly Ile Arg Ala
35 40 45
Phe Met Asp Phe Gly Asp Ile His Arg Gly Asn Glu Ile Ser Ala Ser
50 55 60
Leu Met Lys Ala Ile Glu Ala Ser Arg Ile Ala Ile Leu Val Phe Ser
65 70 75 80
Lys Asn Tyr Ala Glu Ser Ser Tyr Cys Leu Asn Glu Leu Val Lys Ile
85 90 95
Met Glu Cys Ser Gln Arg His Gly Gln Phe Val Leu Pro Val Phe Tyr
100 105 110
Gly Ile Asp Pro Ser Val Val Arg His Gln Lys Gly Ile Tyr Glu Glu
115 120 125
Ala Leu Ala Lys Thr Gly Arg Thr Phe Glu His Ala Met Asp Arg Val
130 135 140
Gln Arg Trp Arg Thr Ala Met Ala Asp Ala Ala Asn Leu Ser Gly Leu
145 150 155 160
His Phe Lys Gly Asp Gly Tyr Gly Tyr Glu Phe Val Glu Lys Ile Val
165 170 175
Glu Gln Val Ser Arg Val Ile Lys Arg Val Gly Asp Tyr Pro Ile Glu
180 185 190
Leu Glu Ser Gln Glu Gln Ala Ser Tyr Ser Ser Leu Ala Pro Ser Ser
195 200 205
Ser Asn Gly Trp Lys Tyr Asp Val Phe Leu Ser Phe Arg Gly Thr Asp
210 215 220
Thr Arg Phe Gly Phe Thr Gly Asn Leu Tyr Asn Val Leu Cys Gly Lys
225 230 235 240
Gly Ile His Thr Phe Met Asp Asp Glu Ala Leu His Arg Gly Asn Glu
245 250 255
Ile Ser Gly Thr Leu Asp Lys Ala Ile Glu Gly Ser Lys Ile Ala Ile
260 265 270
Leu Val Phe Ser Lys Asn Tyr Ala Tyr Ser Ser Tyr Cys Leu Asp Glu
275 280 285
Leu Val Lys Ile Met Lys Cys Ser Gln Ser His Ser Gln Cys Val Leu
290 295 300
Pro Val Phe Tyr Asn Val Asp Pro Pro His Val Arg His Gln His Gly
305 310 315 320
Ser Tyr Glu Glu Ala Leu Ala Lys His Glu Glu Arg Phe Ser Asp Val
325 330 335
Glu Arg Leu Arg Asp Trp Arg Ala Ala Leu His Gln Ala Ala Asn Leu
340 345 350
Thr Gly Phe His Phe Lys Gly Asn Glu Tyr Glu His Glu Phe Ile Gly
355 360 365
Lys Ile Val Arg Val Val Ser Arg Asn Ile Arg Asn Ile Ala Ser Pro
370 375 380
Val Val Gly Ile Gln Glu Asn Gly Gly Thr Glu Ala Glu Ser Leu Leu
385 390 395 400
Gln Asp Asn Gln Arg Asn Asn Ser Lys Val Lys Ala Glu Thr Glu Ala
405 410 415
Asn Val Glu Asn Phe Met Glu Val Asp Val Phe Glu Lys Ser Val Gly
420 425 430
Val Gln Leu Glu Ser Leu Lys Arg Lys Lys Arg Glu Leu Glu Gly Gln
435 440 445
Ile Arg Ala Ile Asn Asp Gln Ile Thr Glu Phe Gln Arg Lys Thr Val
450 455 460
Ala Lys Arg Lys Lys Ala Phe Asp Ser Gly Lys Lys Cys Gln Val Glu
465 470 475 480
Ser Ile Val Thr
<210> 23
<211> 256
<212> PRT
<213> 木豆(Cajanus cajan)
<400> 23
Met Lys Thr Lys Gly Gln Leu Val Trp Pro Ile Tyr Tyr Glu Val Glu
1 5 10 15
Pro Ser Glu Val Arg Arg Gln Ser Gly Ile Tyr Gly Glu Ala Met Ser
20 25 30
Glu Phe Glu Gln Lys Phe Gly His Glu Ser Glu Met Val Trp Lys Trp
35 40 45
Arg Ser Ala Leu Thr Glu Val Ser Ser Leu Ser Gly Trp Val Tyr Glu
50 55 60
Thr Gly Tyr His Ser Tyr Glu Tyr Lys Phe Ile Arg Lys Ile Val Arg
65 70 75 80
Leu Ala Val Glu Ser Leu Pro Arg Tyr Asp Val Phe Leu Ser Phe Ser
85 90 95
Gly Glu Asp Thr Arg Tyr Ser Phe Thr Gly Phe Leu Tyr Asn Ala Phe
100 105 110
Arg Arg Glu Gly Phe Asn Ile Phe Met Asp Asp Glu Gly Leu Glu Gly
115 120 125
Gly Asn Gln Ile Ser Glu Thr Leu Met Arg Ala Ile Glu Met Ser Arg
130 135 140
Leu Ser Ile Val Val Phe Ser Glu Asn Tyr Ala Tyr Ser Thr Trp Cys
145 150 155 160
Leu Asp Glu Leu Ala Lys Ile Ile Glu Cys Lys Lys Thr Lys Asn Gln
165 170 175
Met Val Trp Pro Ile Phe His Tyr Val Glu Lys Ser Asp Val Cys Asn
180 185 190
Gln Thr Lys Ser Tyr Gly Glu Ala Met Ala Ala His Glu Glu Arg Phe
195 200 205
Gly Lys Asp Ser Glu Lys Val Gln Asn Trp Arg Ser Ala Leu Ser Glu
210 215 220
Ile Ala Asn Leu Asp Gly His His Phe Arg Glu Asn Glu Tyr Gln Tyr
225 230 235 240
Glu Phe Ile Glu Arg Val Val Asp Leu Ala Ile Ala Ile Gly Asn Gln
245 250 255
<210> 24
<211> 1736
<212> PRT
<213> 加拿大紫荆(Cercis canadensis)
<400> 24
Met Ala Asn Phe Gly Glu Ala Ser Ser Ser Ser Ser Ser Lys Pro Arg
1 5 10 15
Tyr Thr Tyr Asp Val Phe Leu Ser Phe Arg Gly Glu Asp Thr Arg Asn
20 25 30
Thr Phe Thr Gly Asn Leu Tyr Asp Ala Leu Cys Gln Arg Gly Phe Asn
35 40 45
Thr Phe Ile Asp Asp Asp Gly Leu Glu Arg Gly Gln Gln Ile Ser Tyr
50 55 60
Ala Leu Ile Asn Ala Ile Glu Glu Ser Lys Val Ser Ile Val Val Phe
65 70 75 80
Ser Glu Asn Tyr Ala Ser Ser Pro Trp Cys Leu Asp Glu Leu Val Lys
85 90 95
Ile Leu Glu Cys Lys Lys Glu Lys Gly Gln Ile Val Trp Pro Ile Phe
100 105 110
Tyr Lys Val Glu Pro Ala Asp Val Arg Asp Gln Lys Asn Ser Tyr Glu
115 120 125
Ala Ala Met Ala Lys His Glu Ser Arg Phe Ser Asn Asp Lys Val Thr
130 135 140
Lys Trp Arg Ser Ala Leu Lys Glu Ala Ala Asn Leu Ser Gly Ser Glu
145 150 155 160
Tyr Lys Thr Gly Tyr Glu Tyr Lys Phe Ile Thr Asn Ile Ile Glu Val
165 170 175
Ala Ser Thr Lys Leu His Asp Lys His Leu Tyr Ile Gly Glu His Ile
180 185 190
Val Gly Leu Lys Ser Arg Leu Lys Glu Val Lys Ser Ile Leu Asp Ile
195 200 205
Gly Ser His Asn Thr Ile Gly Met Val Gly Ile His Gly Thr Gly Gly
210 215 220
Ile Gly Lys Thr Thr Leu Ala Lys Val Leu Tyr Asn Leu Ile Val Asp
225 230 235 240
Gln Phe His Cys Ala Cys Phe Leu Glu Ser Val Arg Glu Gly Ser Lys
245 250 255
Cys Ser Met Asp Leu Val Ser Leu Gln Lys Lys Leu Leu Ser Gln Ile
260 265 270
Phe Arg Lys Glu Asn Phe Asn Leu Gly Asn Val Asp Glu Gly Ala Asn
275 280 285
Ile Ile Lys His Arg Leu Arg Asn Arg Arg Val Leu Leu Val Leu Asp
290 295 300
Asp Val Val Lys Gly Glu Gln Leu Lys Lys Leu Ala Gly Gly Cys Asp
305 310 315 320
Trp Phe Gly Pro Gly Ser Arg Ile Ile Ile Thr Thr Arg Asp Lys His
325 330 335
Leu Leu Ile Ala His Gly Val Glu Lys Ile Tyr Glu Met Asn Glu Leu
340 345 350
Asp Asp Asp Lys Ala Leu Glu Leu Phe Cys Trp Lys Ala Phe Lys Met
355 360 365
Ser Glu Pro Thr Glu Gly Phe Val Asp Ile Ser Tyr Lys Ile Ile Lys
370 375 380
Tyr Ala Lys Gly Leu Pro Leu Ala Leu Asn Val Ile Gly Ser Asn Leu
385 390 395 400
Phe Gly Arg Ser Leu Lys Ala Trp Glu Ser Ala Ser Asp Lys Tyr Lys
405 410 415
Arg Ile Leu Asp Lys Gly Ile His Asp Ile Leu Arg Val Ser Tyr Asp
420 425 430
Ser Leu Glu Asp Asp Gln Lys Ser Ile Phe Leu Asp Ile Ala Cys Phe
435 440 445
Phe Lys Gly Glu Arg Leu Glu Asp Val Glu Lys Ile Leu Asp Ala Cys
450 455 460
Asp Leu Ser Pro Gln Tyr Asn Ile Glu Val Leu Val Asp Lys Ser Leu
465 470 475 480
Ile Thr Ile Gly Leu Gly Asn Leu Trp Met His Asp Leu Val Gln Asp
485 490 495
Met Gly Lys Glu Val Leu Lys Gln Asp Ala Ser Ser Lys Leu Gly Asp
500 505 510
Tyr Ser Arg Leu Trp Asn His Glu Ala Ile Phe Asp Gly Ser Asp Ser
515 520 525
Ile Gln Gly Ile Met Phe Asp Pro Pro Gln Leu Glu Met Val Glu Trp
530 535 540
Ser Gly Thr Ala Phe Lys Lys Met Asn Asn Leu Arg Ile Leu Ile Val
545 550 555 560
Arg Asn Ala Asp Phe Ser Thr Gly Pro Lys Asn Leu Pro Asn Asn Leu
565 570 575
Arg Val Leu Glu Trp Glu Arg Tyr Pro Ser Glu Ser Leu Pro Gln Gly
580 585 590
Phe Tyr Pro Arg Lys Ile Val Val Leu Lys Leu Arg Ser Ser Cys Leu
595 600 605
Ile Ser Leu Lys Pro Leu Gln Lys Phe Glu Lys Leu Thr Cys Ile Asp
610 615 620
Phe Ser Cys Cys Gln Leu Leu Thr Gln Ile Pro Asp Met Ser Arg Ala
625 630 635 640
Pro Asn Ile Val Thr Leu Lys Leu Asn Arg Cys Thr Ser Leu Lys Glu
645 650 655
Val His Asp Ser Val Gly Val Leu Thr Lys Leu Leu Gly Leu Ser Leu
660 665 670
Glu Gly Cys Thr Lys Leu Lys Ile Phe Pro Tyr Gly Ile Gln Met Thr
675 680 685
Ser Leu Ile His Leu Asn Leu Asn Asp Cys Arg Ser Leu Gln His Phe
690 695 700
Pro Asp Ile Leu Gly Gln Met Asp Glu Leu Lys Arg Ile Asp Ala Glu
705 710 715 720
Arg Thr Gly Ile Lys Gln Ile Pro His Ser Ile Cys Tyr Leu Arg Gly
725 730 735
Leu Glu Phe Leu Cys Ile Ser Asn Asn Tyr Asp Leu Ile Ser Leu Pro
740 745 750
Glu Ser Ile Asn Gln Leu Asp Arg Leu Ile His Leu Asn Ile Tyr Asn
755 760 765
Cys Lys Lys Leu Arg Gln Ile Ser Gly Ile Pro Ser Asn Leu Glu Gln
770 775 780
Ile Arg Ala Glu Glu Ala Ser Ser Ser Ser Ser Phe Glu Pro Lys Phe
785 790 795 800
Ser Tyr Asp Ile Leu Leu Ser Phe Gly Gly Glu Asp Thr Cys Asn Thr
805 810 815
Phe Thr Asp Tyr Leu Tyr Asn Ala Leu Cys Gln Arg Gly Phe Asn Thr
820 825 830
Phe Ile Asp Asp Gly Gly Leu Glu Arg Gly Glu Gln Ile Ser Pro Ala
835 840 845
Leu Leu Asn Ala Ile Glu Glu Ser Arg Ala Ser Ile Val Val Leu Ser
850 855 860
Glu Asn Phe Pro Phe Phe Thr Trp Cys Leu Asp Val Val Val Lys Ile
865 870 875 880
Leu Glu Cys Lys Lys Glu Lys Gly Gln Met Val Trp Pro Ile Phe Tyr
885 890 895
Gln Val Glu Pro Ser Tyr Val Arg Leu Lys Lys Lys Ser Tyr Arg Glu
900 905 910
Ala Met Ala Lys His Glu Asn Arg Phe Arg Asn Asp Met Asp Lys Val
915 920 925
Arg Lys Trp Arg Ser Ala Leu Lys Glu Ala Ala Asp Leu Tyr Glu Tyr
930 935 940
Lys Phe Ile Ser Asn Ile Ile Glu Glu Val Ser Val Thr Leu His Asn
945 950 955 960
Lys His Leu Tyr Val Gly Glu His Ile Val Gly Leu Lys Ser Cys Ile
965 970 975
Glu Glu Met Lys Ser Ile Leu Asp Val Gly Ser Asn Asn Asp Val Gly
980 985 990
Met Val Gly Ile His Gly Ile Gly Gly Ile Glu Ala Ser Ser Ser Ser
995 1000 1005
Ser Ser Lys Pro Arg Tyr Thr Tyr Asp Val Phe Leu Ser Phe Arg
1010 1015 1020
Gly Glu Asp Thr Arg Asn Thr Phe Thr Gly Asn Leu Tyr Asp Ala
1025 1030 1035
Leu Cys Gln Arg Gly Phe Asn Thr Phe Ile Asp Asp Asp Gly Leu
1040 1045 1050
Glu Arg Gly Gln Gln Ile Ser Tyr Ala Leu Ile Asn Ala Ile Glu
1055 1060 1065
Glu Ser Lys Val Ser Ile Val Val Phe Ser Glu Asn Tyr Ala Ser
1070 1075 1080
Ser Pro Trp Cys Leu Asp Glu Leu Val Lys Ile Leu Glu Cys Lys
1085 1090 1095
Lys Glu Lys Gly Gln Ile Val Trp Pro Ile Phe Tyr Lys Val Glu
1100 1105 1110
Pro Ala Asp Val Arg His Leu Lys Asn Ser Tyr Glu Ala Ala Met
1115 1120 1125
Ala Lys His Glu Ser Arg Phe Ser Asn Asp Lys Val Thr Lys Trp
1130 1135 1140
Arg Ser Ala Leu Lys Glu Ala Ala Asn Leu Ser Gly Ser Glu Tyr
1145 1150 1155
Lys Thr Gly Tyr Glu Tyr Lys Phe Ile Thr Asn Ile Ile Glu Val
1160 1165 1170
Ala Ser Ala Lys Leu His Asp Lys His Leu Tyr Ile Gly Glu His
1175 1180 1185
Ile Val Gly Leu Arg Ser Arg Leu Lys Glu Val Lys Ser Ile Leu
1190 1195 1200
Asp Ile Gly Ser His Asn Thr Ile Gly Met Val Gly Ile His Gly
1205 1210 1215
Thr Gly Gly Ile Gly Lys Thr Thr Leu Val Lys Val Leu Tyr Asn
1220 1225 1230
Leu Ile Val Asp Gln Phe His Cys Ala Cys Phe Leu Glu Ser Ala
1235 1240 1245
Arg Glu Gly Ser Lys Cys Ser Met Asp Leu Val Ser Leu Gln Lys
1250 1255 1260
Lys Leu Leu Ser Gln Ile Phe Arg Lys Glu Asn Phe Asn Leu Gly
1265 1270 1275
Asn Val Asp Glu Gly Ala Asn Ile Ile Lys His Arg Leu Arg Asn
1280 1285 1290
Arg Ser Val Leu Leu Val Leu Asp Asp Val Asp Lys Gly Glu Gln
1295 1300 1305
Leu Lys Lys Leu Ala Gly Gly Cys Asp Trp Phe Gly Pro Gly Ser
1310 1315 1320
Arg Ile Ile Ile Thr Thr Arg Asp Lys His Leu Leu Ile Ala His
1325 1330 1335
Gly Val Glu Lys Ile Tyr Glu Met Lys Glu Leu Asp Asp Asp Lys
1340 1345 1350
Ala Leu Glu Leu Phe Cys Trp Lys Ala Phe Lys Thr Ser Glu Pro
1355 1360 1365
Ala Glu Ser Phe Val Asp Ile Ser Tyr Lys Ile Ile Lys Tyr Ala
1370 1375 1380
Lys Gly Leu Pro Leu Ala Leu Asn Val Ile Gly Ser Asn Leu Phe
1385 1390 1395
Gly Arg Ser Leu Lys Ala Trp Glu Ser Ala Leu Asp Lys Tyr Lys
1400 1405 1410
Arg Ile Leu Asp Lys Gly Ile His Asp Ile Leu Arg Val Ser Tyr
1415 1420 1425
Asp Ser Leu Glu Asp Asp Gln Lys Ser Ile Phe Leu Asp Ile Ala
1430 1435 1440
Cys Phe Phe Lys Gly Asp Arg Leu Glu Asp Val Glu Lys Ile Leu
1445 1450 1455
Glu Ala Cys Asp Leu Ser Pro Gln Tyr Asn Ile Glu Gly Ser Asp
1460 1465 1470
Ser Ile Gln Gly Ile Met Phe His Pro Pro Gln Leu Glu Met Val
1475 1480 1485
Glu Trp Ser Gly Thr Ala Phe Glu Lys Met Asn Asn Leu Arg Ile
1490 1495 1500
Leu Ile Val Arg Asn Ala Asn Phe Ser Thr Ser Pro Lys Asn Leu
1505 1510 1515
Pro Asn Asn Leu Arg Val Leu Glu Trp Glu Arg Tyr Pro Ser Asp
1520 1525 1530
Ser Leu Pro Gln Gly Phe Tyr Pro Arg Lys Ile Val Val Leu Lys
1535 1540 1545
Leu Arg Ser Ser Cys Leu Asn Ser Leu Lys Pro Leu Gln Lys Phe
1550 1555 1560
Glu Lys Leu Thr Cys Ile Asp Phe Ser Cys Cys Gln Leu Leu Thr
1565 1570 1575
Gln Ile Pro Asp Met Ser Asn Ala Pro Asn Ile Val Thr Leu Lys
1580 1585 1590
Leu Asp Arg Cys Thr Ser Leu Lys Glu Val His Asp Ser Val Gly
1595 1600 1605
Val Leu Thr Lys Leu Leu Asp Leu Ser Leu Glu Gly Cys Thr Lys
1610 1615 1620
Leu Lys Ile Phe Pro His Gly Ile Gln Met Thr Ser Leu Arg Asp
1625 1630 1635
Leu Asn Leu Asn Asp Cys Arg Ser Leu Gln His Phe Pro Asp Ile
1640 1645 1650
Leu Gly Gln Met Asp Glu Leu Arg Arg Ile Asp Ala Lys Arg Thr
1655 1660 1665
Gly Ile Lys Gln Ile Pro His Ser Ile Cys Tyr Leu Thr Arg Leu
1670 1675 1680
Gly Phe Leu Leu Met Ser Asp Asn Tyr Asp Leu Ile Ser Leu Pro
1685 1690 1695
Glu Ser Ile Ser Gln Leu Asp Arg Leu Thr Asp Leu Asn Met Asp
1700 1705 1710
Asn Cys Lys Lys Leu Arg Gln Ile Ser Gly Ile Pro Ser Asn Leu
1715 1720 1725
Lys Gln Ile Arg Ala Asp Val Ala
1730 1735
<210> 25
<211> 340
<212> PRT
<213> 野大豆(Glycine soja)
<400> 25
Met Ala Asp Glu Gly Gly Ile Glu Lys Arg Arg Tyr Asp Val Phe Leu
1 5 10 15
Cys Phe Arg Ala Gln Asp Thr Arg Tyr Thr Phe Thr Gly Asn Leu Tyr
20 25 30
Ala Ala Leu Arg Gln Ala Arg Leu Arg Thr Phe Phe Ala Gly Gly Leu
35 40 45
Lys Gly Gly Asp Gln Ile Leu Asp Ala Ile Leu Gln Ala Ile Gln Glu
50 55 60
Ser Arg Ile Ser Ile Val Val Leu Ser Gln Ser Phe Gly Ser Ser Leu
65 70 75 80
Trp Cys Leu Glu Glu Leu Val Lys Ile Leu Glu Cys Lys Lys Thr Lys
85 90 95
Lys Gln Leu Leu Ile Pro Ile Phe Tyr Arg Val Asp Pro Ser Asp Ile
100 105 110
Arg Arg Gln Thr Gly Ser Phe Lys Glu Glu Leu Leu Lys His Glu Ser
115 120 125
Arg Phe Gly Lys Asp Ser Glu Lys Val Arg Lys Trp Lys Ser Ala Leu
130 135 140
Thr His Val Ala Thr Leu Pro Gly Trp Cys Phe Gly Asp Gly Ser Cys
145 150 155 160
Arg Tyr Gln Tyr Glu Tyr Glu Phe Ile Glu Asn Ile Val Arg Glu Val
165 170 175
Ile Ala Ile Val Pro Arg Tyr Ser Ile Phe Leu Ser Phe Ser Gly Asn
180 185 190
Asp Thr Arg Ser Phe Thr Gly Phe Leu Asn Asn Ala Leu Cys Arg Ser
195 200 205
Arg Tyr Gln Thr Phe Met Asn Asp Gly Asp Gln Ile Ser Gln Ser Thr
210 215 220
Asn Gly Val Ile Glu Glu Ser Arg Leu Ser Ile Ile Val Phe Ser Glu
225 230 235 240
Asn Tyr Ala Arg Ser Ser Ser Cys Leu Asp Phe Leu Leu Thr Ile Leu
245 250 255
Glu Cys Met Lys Thr Lys Asn Gln Leu Val Cys Pro Ile Phe Tyr Lys
260 265 270
Val Leu Pro Ser Asp Leu Arg His Gln Arg Asn Ser Tyr Gly Glu Ala
275 280 285
Met Thr Glu His Glu Asn Met Leu Gly Lys Asp Ser Glu Met Val Lys
290 295 300
Lys Trp Arg Ser Ala Leu Phe Asp Val Ala Asn Leu Lys Gly Phe Tyr
305 310 315 320
Leu Lys Thr Gly Tyr Glu Tyr Glu Phe Ile Asp Lys Ile Val Glu Met
325 330 335
Ala Ser Lys Ile
340
<210> 26
<211> 356
<212> PRT
<213> 藜苜蓿(Medicago truncatula)
<400> 26
Met Ala Met Met Asn Glu Glu Val Asp Gly Ser Ser Tyr Lys Tyr Asp
1 5 10 15
Val Phe Leu Ser Phe Arg Gly Glu Asp Thr Tyr Cys Thr Phe Ala Gly
20 25 30
Asn Leu Tyr His Ala Leu Arg Asn Lys Lys Ile Lys Thr Phe Phe Pro
35 40 45
His Asp Gln Ile Gln Asn Asp Asp Glu Glu Leu Gln Leu Ser Pro Ser
50 55 60
Ile Leu Lys Ala Ile Gln Glu Ser Arg Ile Ser Ile Val Val Leu Ser
65 70 75 80
Lys Asn Tyr Ala Thr Ser Thr Arg Cys Leu Asn Glu Leu Val Ile Ile
85 90 95
Leu Gln Cys Met Lys Met Lys Asn Gln Leu Val Trp Pro Ile Phe Tyr
100 105 110
Glu Val His Ser Ser Asp Val Lys Leu Gln Arg Cys Lys Tyr Gly Ser
115 120 125
Ser Ser Lys Ala Ile Leu Lys Phe Arg Glu Arg Phe Lys Asp Tyr Pro
130 135 140
Arg Arg Met Trp Glu Trp Gln Gln Ala Leu Ser Gln Val Thr Ser Ile
145 150 155 160
Ala Gly Trp Asn Tyr Gly Ile Lys Phe Glu Tyr Glu Leu Ile Gln Lys
165 170 175
Ile Val Glu Leu Thr Val Gln Ser Leu Pro Arg Tyr Asp Val Phe Leu
180 185 190
Ser Phe Cys Gly Glu Asp Thr Arg Tyr Ser Phe Thr Gly Phe Leu Tyr
195 200 205
His Ala Leu Arg Leu Glu Gly Phe Lys Ile Phe Met Asp Asp Glu Gly
210 215 220
Leu Glu Gly Gly Asn Gln Ile Ser Gln Thr Leu Leu Lys Ala Ile Glu
225 230 235 240
Lys Ser Arg Leu Ser Ile Val Val Leu Ser Glu Asn Tyr Gly Tyr Ser
245 250 255
Thr Trp Cys Leu Asp Glu Leu Val Lys Ile Met Glu Cys Lys Lys Thr
260 265 270
Asn Asn Lys Leu Val Trp Pro Leu Phe Tyr Lys Ile Glu Gln Ser Asp
275 280 285
Leu Ser Tyr Lys Lys Ser Ser Tyr Gly Lys Ala Met Ala Ala His Glu
290 295 300
Asp Arg Phe Gly Lys Glu Ser Glu Asn Val Gln Lys Trp Arg Ser Ala
305 310 315 320
Leu Ser Glu Val Ala Leu Leu Lys Ala Asp His Ile Lys Glu Asn Glu
325 330 335
His Glu Tyr Glu Phe Ile Lys Lys Ile Val Glu Arg Ala Asn Glu Ala
340 345 350
Glu Asn His Met
355
<210> 27
<211> 334
<212> PRT
<213> 菜豆(Phaseolus vulgaris)
<400> 27
Met Ala Arg Tyr Asp Val Phe Leu Cys Phe Arg Gly Glu Asp Thr Arg
1 5 10 15
Leu Thr Phe Thr Gly Asn Leu Tyr Ala Ala Leu Gln Gln Ala Arg Leu
20 25 30
Arg Thr Phe Arg Asp Glu Gly Val Leu Lys Gly Gly Asp Leu Val Tyr
35 40 45
Ser Ile Ile Glu Ala Leu Glu Ala Ser Arg Val Ala Ile Val Val Leu
50 55 60
Ser Glu Asn Phe Ala Phe Ser Arg Trp Cys Leu Asp Glu Leu Val Lys
65 70 75 80
Ile Leu Asp Cys Met Lys Thr Lys Asn Gln Ile Val Ile Pro Ile Phe
85 90 95
Tyr Asn Val Asp Pro Ser Asp Val Arg Asn Leu Arg Gly Ser Phe Ala
100 105 110
Asp Ala Met Val Asp His Glu His Arg Phe Gly Lys Asn Phe Asp Lys
115 120 125
Ile Arg Asn Trp Arg Ser Ala Leu Thr Glu Val Ala Asn Leu Ser Gly
130 135 140
Trp Cys Leu Gly Arg Gly Ser Arg Phe Gly Tyr Glu Tyr Glu Tyr Ile
145 150 155 160
Glu Arg Ile Val Arg Asp Leu Thr Leu Arg Leu Pro Arg Tyr Thr Ile
165 170 175
Phe Leu Ser Phe Ser Gly Lys Asp Thr Arg Ser Phe Ser Gly Phe Leu
180 185 190
Tyr Asn Ala Leu Ser Arg Ser Gly Tyr His Thr Ile Leu Asn Asp Gly
195 200 205
Asp Gln Ser Ser Gln Ser Thr Val Gly Val Ile Glu Lys Ser Lys Leu
210 215 220
Ser Ile Ile Val Phe Ser Glu Asn Tyr Ala Arg Ser Pro Ser Cys Leu
225 230 235 240
Asp Glu Leu Leu Arg Ile Leu Glu Cys Lys Glu Met Lys Lys Gln Leu
245 250 255
Val Cys Pro Ile Phe Tyr Lys Leu Leu Pro Ser Asp Leu Arg His Gln
260 265 270
Arg Asn Ser Tyr Gly Glu Ala Met Ser Glu His Glu Thr Met Met Gly
275 280 285
Lys Asp Ser Glu Lys Val Lys Lys Trp Arg Ser Ala Leu Phe Glu Val
290 295 300
Ala Asn Leu Lys Gly Trp Tyr Met Lys Thr Gly Tyr Glu Tyr Glu Phe
305 310 315 320
Ile Glu Lys Ile Val Glu Leu Ala Asn Lys Ile Ser Arg Val
325 330
<210> 28
<211> 176
<212> PRT
<213> 豌豆(Pisum sativum)
<400> 28
Met Ala Lys Trp Gln Ile Phe Leu Ser Phe Arg Gly Glu Asp Thr Arg
1 5 10 15
Tyr Ala Phe Thr Gly Ser Leu Tyr Gln Ser Leu Cys Gln Gly Gly Phe
20 25 30
Lys Thr Phe Met Asp Asp Arg Gly Leu Gln Met Gly Asp Gln Ile Ser
35 40 45
Pro Ser Leu Leu Asn Ala Ile Glu Ala Ser Arg Leu Ser Ile Ile Val
50 55 60
Leu Ser Glu Asn Tyr Ala Asn Ser Thr Trp Cys Leu Asp Glu Leu Val
65 70 75 80
Lys Ile Leu Glu Cys Met Lys Leu Lys Asn Gln Leu Val Trp Pro Ile
85 90 95
Phe Tyr Lys Val Glu Pro Ser Asp Ile Arg His Met Lys Lys Ser Tyr
100 105 110
Gly Lys Asp Met Ala Arg His Glu Asn Ile Phe Gly Asn Asp Ser Glu
115 120 125
Arg Val Gln Lys Trp Lys Ser Ala Leu Val Glu Val Cys Asn Leu Ala
130 135 140
Lys Met Ala Tyr Ser Ile Gly Tyr Glu Tyr Glu Phe Ile Gln Lys Ile
145 150 155 160
Val Glu Asp Ala Asn Leu Ile Arg Arg Arg Leu Gln Ile Arg Asn Ile
165 170 175
<210> 29
<211> 337
<212> PRT
<213> 大豆(Glycine max)
<400> 29
Met Ala Asp Glu Gly Gly Ile Glu Lys Arg Arg Tyr Asp Val Phe Leu
1 5 10 15
Cys Phe Arg Gly Glu Asp Thr Arg Tyr Thr Phe Thr Gly Asn Leu Tyr
20 25 30
Ala Ala Leu Arg Gln Ala Arg Leu Arg Thr Phe Phe Asp Asp Gly Phe
35 40 45
Lys Ser Gly Asp Gln Ile Phe Asp Val Val Leu Gln Ala Ile Gln Glu
50 55 60
Ser Arg Ile Ser Ile Val Val Leu Ser Glu Asn Phe Ala Ser Ser Ser
65 70 75 80
Trp Cys Leu Glu Glu Leu Val Lys Ile Leu Glu Cys Arg Glu Thr Lys
85 90 95
Lys Gln Leu Val Ile Pro Ile Phe Tyr Arg Met Asp Pro Ser Asp Val
100 105 110
Arg Arg Gln Thr Gly Cys Tyr Gly Glu Ser Leu Ala Gln His Gln Tyr
115 120 125
Glu Phe Arg Ser Asp Ser Glu Lys Val Arg Asn Trp Gln Glu Ala Leu
130 135 140
Thr His Val Ala Asn Leu Pro Gly Trp Arg Phe Ser Arg Tyr Gln Tyr
145 150 155 160
Glu Tyr Glu Phe Ile Glu Asp Ile Val Arg Gln Ala Ile Val Ala Ile
165 170 175
Val Pro Arg Tyr Ser Ile Phe Leu Ser Phe Ser Gly Asn Asp Thr Arg
180 185 190
Ser Phe Thr Gly Phe Leu Asn Asn Ala Leu Cys Arg Ser Arg Tyr Gln
195 200 205
Thr Phe Met Asn Asp Gly Asp Gln Ile Ser Gln Ser Thr Asn Gly Val
210 215 220
Ile Glu Glu Ser Arg Leu Ser Ile Ile Val Phe Ser Glu Asn Tyr Ala
225 230 235 240
Arg Ser Ser Ser Cys Leu Asp Phe Leu Leu Thr Ile Leu Glu Cys Met
245 250 255
Lys Thr Lys Asn Gln Leu Val Cys Pro Ile Phe Tyr Lys Val Leu Pro
260 265 270
Ser Asp Leu Arg His Gln Arg Asn Ser Tyr Gly Glu Ala Met Thr Glu
275 280 285
His Glu Asn Met Leu Gly Lys Asp Ser Glu Met Val Lys Lys Trp Arg
290 295 300
Ser Ala Leu Phe Asp Val Ala Asn Leu Lys Gly Phe Tyr Leu Lys Thr
305 310 315 320
Gly Tyr Glu Tyr Glu Phe Ile Asp Lys Ile Val Glu Met Ala Ser Lys
325 330 335
Ile
<210> 30
<211> 509
<212> PRT
<213> 红车轴草(Trifolium pratense)
<400> 30
Met Ser Gly Ser Cys Asn Asn Thr Val Asp Phe Thr Lys Glu Lys Asn
1 5 10 15
Arg Leu Val Thr His Arg Asp Phe Val Arg Asp Lys Ile Glu Arg Thr
20 25 30
His Asn Lys Thr Gln Lys Val Ser Asp Val Val Phe Glu Trp Leu Asn
35 40 45
Glu Thr Asp Ile Leu Ile Arg Gln Leu Glu Asn Leu Ser Ala Gln Ala
50 55 60
Ile Thr Arg Lys Gln Phe Lys Lys Leu Leu Lys Arg Val Met Glu Gln
65 70 75 80
Asn Thr Lys Val Pro Ser Gly Asn Tyr Ile Gln Glu Ser Ser Thr Pro
85 90 95
Ile Pro Ser Leu Glu His Phe Ser Ser Gly Asn Leu Met Cys Phe Asn
100 105 110
Ser Arg Glu Lys Ile Ser Asp Gln Leu Phe Val Ala Leu Asn Asp Asp
115 120 125
Ser Cys Ser Met Ile Gly Leu Tyr Gly Ser Gln Gly Ser Gly Lys Thr
130 135 140
Thr Leu Val Glu Ala Met Ala Lys Lys Val Lys Tyr Leu Glu Ile Phe
145 150 155 160
His Glu Ile Leu Phe Val Lys Val Thr Gln Asn Ser Asn Ile Arg Thr
165 170 175
Met Gln Asp Glu Ile Val Asp Ser Leu Asn Met Lys Phe Asp Lys Lys
180 185 190
Asn Ser Glu Ser Gly Arg Ala Arg Glu Ile Phe Ser Thr Ile Glu Ser
195 200 205
Met Asn Cys Pro Ile Leu Val Ile Phe Asp Asn Val Pro Ala Lys Phe
210 215 220
Asp Pro Glu Asp Ile Gly Ile Pro Cys Asn Ser Asn Arg Cys Lys Ile
225 230 235 240
Leu Leu Thr Thr Cys Cys Gln Lys Asp Cys Asp Leu Leu Ser Gly Gln
245 250 255
Arg Lys Ile Gln Leu Asp Pro Leu Ser Arg Gln Glu Ala Trp Ile Leu
260 265 270
Phe Gln Lys His Ser Gly Ile Tyr Asp Asp Glu Lys Tyr Ser Arg Phe
275 280 285
Asp Leu Leu Asn Val Ala Tyr Glu Val Ala Leu Glu Cys Glu Gly Leu
290 295 300
Pro Arg Thr Ile Lys Asp Val Gly Pro Phe Leu Lys Ser Lys Pro Ile
305 310 315 320
Glu Glu Trp Lys Thr Thr Leu Asp Ser Leu Lys His Ser Met Ala Lys
325 330 335
Trp Gln Ile Phe Leu Ser Phe Arg Gly Glu Asp Thr Arg Asn Ser Phe
340 345 350
Thr Gly Tyr Leu Tyr Gln Thr Leu Ser Gln Ala Gly Phe Lys Thr Phe
355 360 365
Met Asp Asp Gly Gly Leu His Thr Gly Asp Gln Ile Ser Pro Ser Leu
370 375 380
Val Asn Ala Ile Glu Ala Ser Arg Leu Ser Ile Ile Val Leu Ser Glu
385 390 395 400
Asn Tyr Ala Asn Ser Ser Trp Cys Leu Glu Glu Leu Val Lys Ile Leu
405 410 415
Glu Cys Met Lys Leu Lys Asn Gln Leu Val Trp Pro Ile Phe Tyr Lys
420 425 430
Val Tyr Pro Ser Asp Ile Arg His Leu Arg Lys Cys Tyr Gly Lys Asp
435 440 445
Met Asp Arg His Glu Asn Asn Phe Gly Ile Asn Ser Glu Arg Val Gln
450 455 460
Lys Trp Arg Ser Ala Leu Phe Glu Val Ser Asn Leu Ser Gly Lys Ala
465 470 475 480
Tyr Thr Thr Gly Tyr Glu Tyr Glu Phe Ile Gln Lys Ile Val Glu Asp
485 490 495
Ala Asn Arg Ile Lys Ser Arg Leu Gln Ile Arg Arg Ala
500 505
<210> 31
<211> 615
<212> PRT
<213> 赤豆(Vigna angularis)
<400> 31
Met Glu Ala Glu Ser Tyr Asn Arg Leu Ser Gln Gln Asp Pro Ser Gly
1 5 10 15
Gly Ala Thr Val Ser His Pro Ser Ser Glu Arg Lys Asn Phe Asp Val
20 25 30
Phe Leu Ser Leu Gly Gly Lys Asp Val Arg Tyr Thr Phe Thr Gly Asn
35 40 45
Leu Phe Asn Ala Leu Arg Ser Lys Arg Ile Lys Thr Leu Phe Arg Glu
50 55 60
His Glu Tyr Glu Pro Asp Asp Asp Thr Asn Ile Ser Pro Ser Ala Leu
65 70 75 80
Lys Ala Ile Gln Thr Ser Lys Ile Ser Ile Val Val Phe Ser Pro Glu
85 90 95
Tyr Ala Ser Ser Ser Lys Arg Leu Asp Glu Leu Val Ala Ile Leu Glu
100 105 110
Cys Arg Met Arg Thr Asn Gln Leu Val Trp Pro Ile Phe Tyr Gly Val
115 120 125
Glu Pro Thr Asp Val Arg Phe Gln Arg Gly Arg Tyr Glu Gln Ala Ile
130 135 140
Asn Arg Phe Glu Glu Arg Tyr Tyr Ser Pro Glu Arg Met Asn Lys Trp
145 150 155 160
Arg Ala Ala Leu Ala Glu Val Ser Asn Leu Ser Gly Trp Phe Tyr Gln
165 170 175
Lys Gly His Lys Tyr Glu Tyr Lys Phe Ile Arg Lys Ile Val Glu Ala
180 185 190
Ala Val Gln Cys Leu Ser Arg Tyr Asp Val Phe Leu Ser Phe Cys Gly
195 200 205
Glu Asp Thr Arg Tyr Thr Leu Thr Gly Phe Leu Tyr Asn Ala Leu Arg
210 215 220
Arg Glu Gly Phe Lys Ile Phe Met Asp Asp Glu Glu Leu Glu Gly Gly
225 230 235 240
Asn Gln Ile Ser Gln Lys Leu Met Gly Ala Ile Glu Ser Ser Arg Val
245 250 255
Ser Ile Val Val Phe Ser Glu Asn Tyr Gly Tyr Ser Thr Trp Cys Ala
260 265 270
Leu Ser Gln Ile Thr Asn Leu Glu Gly Glu His Leu Ser Glu Asn Glu
275 280 285
Ser Val Met Leu Asn Phe Ile Thr Val Phe Ile Ile Gly Val Cys Trp
290 295 300
Asn Val Leu Gly Lys Asp Thr Arg His Thr Leu Thr Gly Asn Leu Tyr
305 310 315 320
Ala Ala Leu Gln Gln Ala Arg Phe Arg Thr Phe Met Asp Asp Asp Glu
325 330 335
Leu Lys Gly Ala Asp Gln Ile Ala Tyr Thr Ile Val Leu Glu Ala Ser
340 345 350
Arg Ile Ser Ile Val Val Leu Ser Glu His Phe Ala Phe Ser Ser Trp
355 360 365
Cys Leu Asp Glu Leu Ala Asn Ile Leu Asp Cys Met Asn Thr Lys Asn
370 375 380
Gln Ala Val Phe Pro Ile Phe Tyr Glu Val Asp Pro Phe Tyr Val Arg
385 390 395 400
His Leu Lys Gly Ser Phe Gly Glu Ala Met Val Ala His Glu Ala Arg
405 410 415
Phe Gly Lys Asp Ser Glu Lys Val Glu Lys Trp Arg Ser Ala Leu Ala
420 425 430
Gln Val Ala Asn Leu Ser Gly Trp Cys Phe Gly Arg Gly Arg Trp Cys
435 440 445
Gln Tyr Glu Tyr Glu Tyr Glu Phe Ile Glu Arg Ile Val Gln His Val
450 455 460
Thr Lys Leu Leu Pro Arg Tyr Ser Ile Phe Leu Ser Phe Ser Gly Lys
465 470 475 480
Asp Thr Arg Ser Phe Thr Gly Phe Leu Tyr Asn Ala Leu Ser Arg Ser
485 490 495
Gly Tyr His Thr Phe Ile Ser Asp Gly Glu His Ser Ser Gln Ser Ile
500 505 510
Val Gly Val Ile Glu Lys Ser Arg Leu Ser Ile Ile Val Phe Ser Glu
515 520 525
Asn Tyr Ala Arg Ser Pro Ser Ser Leu Asp Glu Leu Leu Arg Ile Leu
530 535 540
Glu Cys Met Asp Met Lys Asn His Leu Val Cys Pro Ile Phe Tyr Lys
545 550 555 560
Val Leu Pro Ser Asp Ile Arg His Gln Arg Lys Ser Tyr Cys Glu Ala
565 570 575
Met Ile Glu His Glu Asn Val Met Gly Glu Asp Ser Glu Lys Val Lys
580 585 590
Lys Trp Arg Ser Ala Leu Phe Gln Val Ala Asn Leu Lys Gly Trp Cys
595 600 605
Met Lys Thr Gly Tyr Ser Lys
610 615
<210> 32
<211> 354
<212> PRT
<213> 绿豆(Vigna radiata)
<400> 32
Met Tyr Phe Ile Ser Ala Ser Glu Arg Arg Asn Ala Ala Thr Cys Asn
1 5 10 15
Glu His Leu Ser Asn Arg Asn Asn Thr Gln Phe Glu Phe Leu Ile His
20 25 30
Phe Ile Ile Asp Ala Met Ala Arg Tyr Asp Val Phe Leu Cys Phe Arg
35 40 45
Gly Arg Asp Thr Arg His Thr Phe Thr Gly Asn Leu Tyr Ala Ala Leu
50 55 60
Gln Gln Ala Arg Phe Arg Thr Phe Met Asp Asp Asp Glu Leu Lys Gly
65 70 75 80
Ala Asp Gln Ile Ala Tyr Thr Ile Val Leu Glu Ala Ser Arg Ile Ser
85 90 95
Ile Val Val Leu Ser Glu His Phe Ala Phe Ser Ser Trp Cys Leu Asp
100 105 110
Glu Leu Ala Lys Ile Val Asp Cys Met Asn Thr Lys Asn Gln Ala Val
115 120 125
Phe Pro Ile Phe Tyr Glu Val Asp Pro Phe Tyr Val Arg His Leu Lys
130 135 140
Gly Ser Phe Gly Glu Ala Met Val Ala His Glu Ala Arg Phe Gly Lys
145 150 155 160
Asp Ser Glu Arg Val Glu Lys Trp Arg Ser Ala Leu Ile Gln Val Thr
165 170 175
Asn Leu Ser Gly Trp Cys Phe Ala Arg Gly Arg Trp Cys Glu Tyr Glu
180 185 190
Tyr Glu Phe Ile Glu Arg Ile Val Gln His Val Thr Lys Leu Val Pro
195 200 205
Arg Tyr Arg Ile Phe Val Ser Phe Ser Gly Lys Asp Thr Arg Ser Phe
210 215 220
Thr Gly Phe Leu Cys Asn Ala Leu Ser Arg Ser Gly Tyr Asn Thr Phe
225 230 235 240
Ile Ser Asp Gly Glu Gln Ser Ser Gln Ser Thr Val Gly Val Ile Glu
245 250 255
Lys Ser Arg Leu Ser Ile Ile Val Phe Ser Glu Asn Tyr Ala Arg Ser
260 265 270
Pro Ser Cys Leu Asp Glu Leu Leu Arg Val Leu Glu Cys Met Glu Met
275 280 285
Lys Asn Gln Leu Val Cys Pro Ile Phe Tyr Lys Val Leu Pro Ser Asp
290 295 300
Leu Arg His Gln Arg Arg Ser Tyr Gly Glu Ala Met Ile Glu His Glu
305 310 315 320
Asn Val Met Gly Glu Asn Ser Glu Lys Val Lys Lys Trp Arg Ser Ala
325 330 335
Leu Phe His Val Ala Asn Leu Lys Gly Trp Cys Met Lys Thr Gly Tyr
340 345 350
Ser Lys
<210> 33
<211> 887
<212> PRT
<213> 豌豆(Pisum sativum)
<400> 33
Met Ala Leu Ala Cys Asn Ser Leu Gln Ser Ser Ser Ser Ser Pro Lys
1 5 10 15
Met Arg Trp Lys Tyr Asp Val Phe Val Ser Phe Arg Gly Glu Asp Thr
20 25 30
Arg Asn Asn Phe Thr Asp His Leu Phe Gly Ala Leu His Lys Lys Ala
35 40 45
Ile Ile Thr Phe Arg Asp Asp Thr Lys Leu Lys Lys Gly Glu Asp Ile
50 55 60
Ser Leu Glu Leu Leu Gln Ala Ile Glu Gly Ser Gln Ile Leu Ile Val
65 70 75 80
Ile Phe Ser Thr Asn Tyr Ala Ser Ser Thr Trp Cys Leu Gln Glu Val
85 90 95
Ala Lys Ile Ala Ala Cys Ile Glu Val Pro Gly Gln Ser Val Leu Pro
100 105 110
Ile Phe Phe Asp Val Ser Pro Ser Glu Val Arg Lys Gln Cys Gly Asp
115 120 125
Tyr Glu Lys Ala Phe Gln Gly His Glu Glu Arg Phe Lys Ala Thr Leu
130 135 140
Glu Lys Val Gln Arg Trp Arg Gly Ala Leu Thr Gln Val Ala Asn Leu
145 150 155 160
Ser Gly Trp Asp Val Arg Asp Lys Pro Gln Tyr Ala Glu Ile Gly Glu
165 170 175
Ile Thr Lys Lys Val Thr Cys Leu Leu Gly Asn Lys Ser Ser Thr Leu
180 185 190
Pro Arg Asp Ile Val Gly Met Pro Ser Arg Val Glu Glu Leu Glu Asn
195 200 205
His Leu Asn Leu Asp Ser Asn Asp Asp Asp Val Arg Val Ile Gly Ile
210 215 220
Cys Gly Met Gly Gly Ile Gly Lys Thr Thr Leu Ala Thr Ala Leu Tyr
225 230 235 240
Ala Arg Ile Ser Asn Gln Phe Asp Ala Cys Cys Phe Ile Asp Asp Val
245 250 255
Ser Lys Ile Tyr Gly Asp His Gly Pro Ile Gly Val Gln Lys Gln Leu
260 265 270
Leu Arg Gln Thr Leu Asn Glu Glu Asn Leu Gln Ile Cys Asn Leu Pro
275 280 285
Met Ala Ser Asn Leu Ile Arg Thr Arg Leu Ser Arg Leu Lys Ser Leu
290 295 300
Val Phe Leu Asp Asn Val Asp Glu Val Glu Gln Leu Asp Lys Leu Asp
305 310 315 320
Met Arg Leu Glu Trp Leu Gly Arg Gly Ser Arg Ile Ile Ile Ile Ser
325 330 335
Arg Asn Gly His Ile Leu Thr Glu Tyr Gly Val Asp Glu Val Tyr Arg
340 345 350
Val Arg Leu Leu Asp Arg Lys Cys Ala Leu Gln Leu Phe Cys Gln Lys
355 360 365
Ala Phe Lys Ser Asp Asp Ile Met Ser Gly Tyr Ile Tyr Leu Thr Lys
370 375 380
Glu Val Leu Ala Tyr Ala Asn Gly Leu Pro Leu Ala Ile Lys Val Leu
385 390 395 400
Gly Ser Tyr Leu Tyr Gly Arg Asp Val Ser Glu Trp Arg Ser Ala Leu
405 410 415
Ser Arg Leu Arg Glu Asn Pro Met Thr Asp Ile Met Asn Val Leu Arg
420 425 430
Ile Ser Phe Asp Gly Leu Glu Asp Thr Glu Lys Asp Ile Phe Leu Asp
435 440 445
Ile Ala Cys Phe Phe His Gly Tyr Pro Lys Gly Tyr Leu Lys Lys Ile
450 455 460
Leu Asp Phe Arg Gly Phe His Pro Glu Ile Gly Leu Arg Val Leu Val
465 470 475 480
Asp Lys Ser Phe Ile Thr Tyr Lys Lys Gln Ile Ile Cys Met His Asp
485 490 495
Leu Phe Arg Glu Leu Gly Lys Ser Ile Val Arg Glu Lys Ser Pro Lys
500 505 510
Glu Pro Arg Lys Trp Asn Arg Val Trp Asp Tyr Lys Asp Val His Asn
515 520 525
Val Ile Ser Glu Asn Met Ala Thr Glu Asn Leu Glu Ala Met Met Met
530 535 540
Glu Tyr Asp Ser Glu His Asp Ile Glu Ile Gln Gln Met Thr Thr Leu
545 550 555 560
Arg Ala Glu Ala Leu Ala Gln Met Ser Arg Leu Lys Leu Leu Arg Leu
565 570 575
Leu Thr Phe Asn Phe Ser Gly Ser Leu Asn Phe Leu Ser Ser Glu Leu
580 585 590
Gly Tyr Leu Arg Trp Asp Lys Tyr Pro Phe Thr Ser Leu Pro Ser Ser
595 600 605
Phe Gln Ala Tyr Lys Leu Val Glu Leu Ile Leu Arg His Ser Asn Ile
610 615 620
Arg Lys Leu Trp Glu Gly Thr Lys Ser Leu Pro Asn Leu Thr Arg Ile
625 630 635 640
Asp Leu Ser Tyr Ser Lys Asn Leu Asn Met Met Pro Asn Phe Glu Glu
645 650 655
Thr Pro Asn Leu Glu Ser Leu Cys Leu Val Gly Cys Ile Lys Leu Val
660 665 670
Lys Ile Asp Pro Ser Ile Ala Gly Leu Tyr Ile Phe Asp Cys Pro Ser
675 680 685
Leu Val Glu Met Glu Ser Tyr Phe Gly Ile Ala Phe Ser Trp Met Ile
690 695 700
Gln Leu Leu Gln Val His Met Gln Ser Glu Ile Pro Arg Thr Asp Ile
705 710 715 720
Thr Ile Val Ile Pro Lys Thr Gln Ile Pro Lys Trp Phe Thr Lys Gln
725 730 735
His Val Gly Ser Ser Ile Ser Ile Asp Pro Ser Ser Ile Met His Asp
740 745 750
Lys Asn Leu Ile Gly Ile Ala Cys Cys Leu Thr Phe Val Ala Gln Asp
755 760 765
Asn Pro Thr Asn Leu Arg Glu Glu Leu Ser Ser Tyr Ile Ala Phe Gly
770 775 780
Phe Lys Cys Thr Gln Cys Gly Val Tyr Ser Ile Ile Pro Ile Leu Leu
785 790 795 800
Gly Lys Asp Leu Val Thr Val Asp Leu Asp His Leu Leu Leu Val Phe
805 810 815
Phe Ser Arg Lys Lys Phe Ile Asp Leu Ile Ser Asp Ala Thr Asp Gly
820 825 830
Trp Asp Asp Ile Ser Gly Ile Glu Leu Ser Ala Thr Val Arg Gln Pro
835 840 845
Phe Gly Leu His Leu Glu Val Lys Asn Cys Gly Tyr Asn Trp Ile Phe
850 855 860
Lys Glu Asp Leu Glu Gln Leu Asn Pro Gln Lys Met Tyr Lys Gly Asn
865 870 875 880
Ser Ser Val Gln Pro Tyr Tyr
885
<210> 34
<211> 202
<212> PRT
<213> 菜豆(Phaseolus vulgaris)
<400> 34
Lys Asp Asn Glu Arg Leu Gln Lys Trp Arg Ser Ala Leu Thr Gln Val
1 5 10 15
Ala Asn Leu Ser Gly Cys Cys Leu Gly Thr Gly Ser Arg Phe Gly Tyr
20 25 30
Glu Tyr Glu Tyr Ile Glu Arg Ile Val Arg Ser Val Thr Leu Val Ile
35 40 45
Pro Arg Tyr Asn Ile Phe Val Ser Phe Ser Gly Lys Asp Thr Arg Ser
50 55 60
Phe Ser Gly Phe Leu Tyr Asn Ala Leu Ser Arg Arg Gly Tyr His Thr
65 70 75 80
Ile Leu Asn Asp Gly Asp Gln Ser Ser Gln Ser Thr Thr Val Gly Val
85 90 95
Ile Glu Lys Ser Lys Leu Ser Ile Ile Val Phe Ser Glu Asn Tyr Ala
100 105 110
Arg Ser Pro Ser Cys Leu Asp Glu Leu Leu Arg Ile Leu Glu Cys Lys
115 120 125
Glu Met Lys Asn Gln Leu Val Cys Pro Ile Phe Tyr Lys Val Leu Pro
130 135 140
Ser Asp Leu Arg His Gln Arg Asn Ser Tyr Gly Glu Ala Met Ser Glu
145 150 155 160
His Glu Asn Met Met Gly Lys Asp Ser Glu Lys Val Lys Ile Trp Arg
165 170 175
Ser Ala Leu Phe Glu Val Ala Asn Leu Lys Gly Cys Pro Ser Phe Val
180 185 190
Val Trp Gln Leu Leu Cys Ala Asp Ile Leu
195 200
<210> 35
<211> 327
<212> PRT
<213> 大豆(Glycine max)
<400> 35
Met Ala Asp Glu Gly Gly Ile Glu Lys Arg Arg Tyr Asp Val Phe Leu
1 5 10 15
Cys Phe Arg Gly Glu Asp Thr Arg Phe Thr Phe Thr Gly Asn Leu Cys
20 25 30
Ala Ala Leu Arg Gln Ala Arg Leu Arg Thr Phe Phe Asp Asp Gly Leu
35 40 45
Lys Gly Gly Asp Gln Ile Leu Asp Ala Ser Leu Gln Ala Ile Gln Glu
50 55 60
Ser Arg Ile Ser Ile Val Val Leu Ser His Asn Phe Ala Ser Ser Ser
65 70 75 80
Trp Cys Leu Glu Glu Leu Val Lys Ile Leu Glu Cys Arg Glu Thr Lys
85 90 95
Lys Gln Leu Val Ile Pro Ile Phe Cys Arg Val Asp Pro Ser Asp Val
100 105 110
Arg Arg Gln Thr Gly Arg Phe Lys Glu Asp Leu Leu Lys His Glu Ser
115 120 125
Arg Phe Glu Lys Asp Ser Glu Lys Val Arg Lys Trp Lys Ser Ala Leu
130 135 140
Thr Gln Val Ala Thr Leu Pro Gly Phe Cys Phe Gly Asp Gly Ser Cys
145 150 155 160
Ser Asp Gln Tyr Glu Tyr Glu Phe Ile Gln Asn Ile Val Arg Glu Ala
165 170 175
Ile Ala Ile Val Pro Arg Tyr Ser Ile Phe Leu Ser Phe Ser Gly Asn
180 185 190
Asp Thr Arg Ser Phe Thr Gly Phe Leu Asn Asn Ala Leu Cys Arg Ser
195 200 205
Arg Tyr Gln Thr Phe Met Asn Asp Gly Asp Gln Ile Ser Gln Ser Thr
210 215 220
Asn Gly Val Ile Glu Glu Ser Arg Leu Ser Ile Ile Val Phe Ser Glu
225 230 235 240
Asn Tyr Ala Arg Ser Ser Ser Cys Leu Asp Leu Leu Leu Thr Ile Leu
245 250 255
Glu Cys Met Lys Thr Lys Asn Gln Leu Val Cys Pro Ile Phe Tyr Lys
260 265 270
Val Leu Pro Ser Asp Leu Arg His Gln Arg Asn Ser Tyr Gly Glu Ala
275 280 285
Met Thr Glu His Glu Asn Met Leu Gly Lys Asp Ser Glu Arg Val Lys
290 295 300
Lys Trp Arg Ser Ala Leu Phe Asp Val Ala Asn Leu Lys Gly Phe Tyr
305 310 315 320
Leu Lys Thr Gly Tyr Asn Thr
325
<210> 36
<211> 344
<212> PRT
<213> 大豆(Glycine max)
<400> 36
Met Ala Asp Glu Gly Gly Ile Lys Lys Arg Arg Tyr Asp Val Phe Leu
1 5 10 15
Cys Phe Arg Gly Glu Asp Thr Arg Tyr Thr Phe Thr Gly Asn Leu Tyr
20 25 30
Ala Ala Leu Arg Gln Ala Arg Leu Arg Thr Phe Phe Asp Gly Gly Ile
35 40 45
Gly Gly Leu Lys Gly Gly Asp Gln Ile Phe Asp Val Leu Leu Gln Ala
50 55 60
Ile Gln Glu Ser Arg Ile Ser Ile Val Val Leu Ser Gln Asn Phe Gly
65 70 75 80
Ser Ser Leu Trp Cys Leu Glu Glu Leu Val Lys Ile Leu Glu Cys Lys
85 90 95
Lys Thr Lys Lys Gln Leu Val Ile Pro Ile Phe Tyr Arg Val Asp Pro
100 105 110
Ala Asp Val Arg Arg Gln Thr Gly Ser Phe Lys Glu Gln Leu Leu Lys
115 120 125
His Glu Ser Arg Phe Gly Lys Asp Ser Glu Lys Val Arg Gln Trp Lys
130 135 140
Ser Ala Leu Thr Gln Val Ala Thr Leu Pro Gly Trp Cys Phe Gly His
145 150 155 160
Gly Ser Cys Arg Tyr Gln Tyr Glu Tyr Glu Phe Ile Glu Asp Ile Val
165 170 175
Arg Gln Ala Ile Val Ala Ile Val Pro Arg Tyr Ser Ile Phe Leu Ser
180 185 190
Phe Ser Gly Asn Asp Thr Arg Ser Phe Thr Gly Phe Leu Asn Asn Ala
195 200 205
Leu Cys Arg Ser Arg Tyr Gln Thr Phe Met Asn Asp Gly Asp Gln Ile
210 215 220
Ser Gln Ser Thr Asn Gly Val Ile Glu Glu Ser Arg Leu Ser Ile Ile
225 230 235 240
Val Phe Ser Glu Asn Tyr Ala Arg Ser Ser Ser Cys Leu Asp Phe Leu
245 250 255
Leu Thr Ile Leu Glu Cys Met Lys Thr Lys Asn Gln Leu Val Cys Pro
260 265 270
Ile Phe Tyr Lys Val Leu Pro Ser Asp Leu Arg His Gln Arg Asn Ser
275 280 285
Tyr Gly Glu Ala Met Thr Glu His Glu Asn Met Leu Gly Lys Asp Ser
290 295 300
Glu Met Val Lys Lys Trp Arg Ser Ala Leu Phe Asp Val Ala Asn Leu
305 310 315 320
Lys Gly Phe Tyr Leu Lys Thr Gly Tyr Glu Tyr Glu Phe Ile Asp Lys
325 330 335
Ile Val Glu Met Ala Ser Lys Ile
340
<210> 37
<211> 1104
<212> DNA
<213> 野大豆(Glycine soja)
<400> 37
atgtcgaatg aactagaaaa ctacgatgtt tttctcagtt ttcatggcca agattcccgt 60
tacaccttca ctggtactct ctataatgct ttgcgcagca agagaatcaa gacctttttc 120
acagaacatg aatatgatcg taaactacac actgatgaca gccaaattcc accctctact 180
cttaaagcaa ttaaggaatc aaggatttcg gtggttgttt tgtcggaaaa ctatgcatcc 240
tcctcaagat gtcttgatga acttgtggcc atccttgagt gtaacaggac gataaaccaa 300
ctggtgtggc ccatctttta cgaagtgtat ccgtggcacg taaggcagca gagaggtagc 360
tatgaagcac ccatgtctaa atttgaagaa atatttggag attccaatga gagggtgaag 420
caatggagag cagctttatt tgaagtcacc aacttgaatg gatggtgtta caaaactggg 480
acgtcccagt acgaatatga attgatcgaa aagatcgtgg aatcaaccgt gcaagccttg 540
cccggatatg atgtttttct gagttttacc ggagaggata cccgctacac tttcacaggt 600
tttctctata atgcctttcg ccgagaggga ttcaaaatct tcatggatga tgaggaattg 660
gagagtggga accaaatttc acagaagctt atgggagcaa ttgagagttc aaagatttca 720
attgttgtgc tctctgaaaa ctatgcatat tccacctggt gtcttgatga acttgccaag 780
atcattgagt gtatgaagac caacaatcaa atggtttggc caatatttta caatgtgcaa 840
aagtcggatg tatgcaatca aacaaaaagt tatggtgaag ccatgactga acatgaaaaa 900
agatttggaa aggactctga gaaagtgcag aaatggaggt ctgctttgtc tgaaatcaac 960
aacttggaag gagaccatgt caaacaaaat gagtaccaac atgaattaat cgaaaggatt 1020
gtggaaaagg tcattaaaat tgaaggtagc aagcatacag caaatccttt ccttttatcc 1080
catgacagct acgaggaaga atga 1104
<210> 38
<211> 1092
<212> DNA
<213> 菜豆(Phaseolus vulgaris)
<400> 38
atgtccaatg aaccaaaaaa ctatgatgtt tttctgagtc ttggtggtga agatgttcgc 60
tacaccttca ctggtagtct cttcagtgct ttgtgcagca agagaatcaa gacgtttttc 120
agagaacatg aagaagatcc tgagccatac actaatgaca ccaatatttc accctcttct 180
cttaaagcaa tacaagagtc aaagatttcc atcgttgttt tctcccaagg atatgcatcc 240
tcctcaagat gtctcgatga acttgtggcc atccttgact gttggatgaa gagcgaccaa 300
cttgtctggc caatctatta cggagtggat ccgagtgagg taagaactca gaaaggtgga 360
tttggacaag ccatgttcag agttagagaa aggtattcca cagagaggat gaacaaatgg 420
agagaagctt tggttgaggt tagcagattc agtggatggg tttaccaaat ggggtccaag 480
tacgaataca aattcatccg aaagattgtg gaagcagccc tgcaatccct gccaagatat 540
gatgtttttc tgagtttttg tggagaggat acgcgccata ctctcactgg ttttctcttt 600
gatgccattc gccgagaggg attcaaaatc ttcatggacg atgaagaatt ggagggtggg 660
aaccaaattt ctgaaacgct catgggagca attcaaagtt caaggatttc cattgttgtg 720
ttctctgaaa actatggata ttccacttgg tgtcttgatg aacttgccaa gatcactgag 780
tgtatgaaca ccaagaatca gaaggtttgg ccaatatttt acaatgtgga gaagttggat 840
gtgtgcaatc agacaaaaag ttatggtgaa gccatgactg cacatgaaaa aagatttgga 900
aaggactctg agaaggtgct gaaatggagg tctgctttgt ctcaaatcac caacttggat 960
ggagagcatc tcagtgaaaa tgagttccaa catgaatcta tcgaaaggat tgtggaacgg 1020
ctcattaata ttgaagatgg gaagcatata gcaagtcctt tccttattca agacaacaat 1080
ggagaagaat ga 1092
<210> 39
<211> 819
<212> DNA
<213> 豌豆(Pisum sativum)
<400> 39
atgcagatcc ttgtgaccgc acgttgccaa caagaatgtg atttgatgta ctgtcaaagg 60
gatgttcaac ttgatccctt atctaaagag gaggcttgga ctttgtttga aaaacattcg 120
ggcattcatg acgaggactg ctcctcctcg cccgacttat cgaatgtcgc acgtgaagtt 180
gcttttgaat gtgaaggggt gcctagatta attaaagatg tggcgtcttc cttaagaaat 240
aaaccaattg atgaatggaa agcatcacta gatagtctca aacattcaat ggctaaatgg 300
caaatttttc ttagttttcg aggagaagat acgcgttacg cgtttacagg ttctctttat 360
caatctttac gtcaaggggg attcaagacc tttatggatg atggaggatt ggagacggga 420
gaccaaattt caccatctct tctaaatgcc attgaagcgt cgaggctttc gattattgtt 480
ttatctgaaa actatgcaaa ttcgacgtgg tgtctggatg agcttgtcaa gattctcgag 540
tgtaagaaat tgaagaatca attggtttgg ccaatctttt ataaagtgga tccatctgat 600
atcagacata tgagaaaatg ttatggaaaa gacatggctc gacatgaaaa tagatttgga 660
attgattctg agagagtaca aaaatggaag tcagctttag atgaagtgtg taatttgtcc 720
ggaaaggctt attcaatcgg gtatgaatat gaatttattc agaagattgt ggaacatgcc 780
aatctcatta gaagtcgttt gcaaataaga aacatatag 819
<210> 40
<211> 1068
<212> DNA
<213> 大豆(Glycine max)
<400> 40
atgtcgaatg aactaaaaaa ctatgatgtt tttctcaatt ttcatggcaa agattccggt 60
tacaccttca ctggtactct ctataatgct ttgcgcagca agagaatcaa gacctttttc 120
acaaaacatg aatatggtcg taaactacac actgatgaca gccacattcc accctttact 180
cttaaagcaa ttaaggaatc aaggatttcg gtggttgttt tgtcggaaaa ctatgcatcc 240
tcctcaagat gtcttgatga acttgtggcc atccttgagt gtaaaaggac gataaaccaa 300
ctggtgtggc ccatctttta caaagtggat ccgtcgcaag taaggcacca gaaaggtagc 360
tatggagaac acatttgtaa ttttaaaaaa atttttagag attacaacga ttccaatgag 420
agggtgaagc aatggagagc agctttatct gaagtcagca aattgagtgg atggctttac 480
aatgatcgga ggtcccagta cgaatatgaa ttcatcgaaa ggatcgtgga atcaaccgtg 540
caagccttgc ccggatatga tgtttttctg agttttaccg gagaggatac ccgctacact 600
ttcacaggtt ttctctataa tgcctttcgc cgagagggat tcaaaatctt catggatgat 660
gaggaattgg agagtgggaa ccaaatttca cagaagctta tgagagcaat tgagagttca 720
aagatttcaa ttgttgtgct ctctgaaaac tatgcatatt ccacctggtg tcttgatgaa 780
cttgccaaga tcattgagtg tatgaagacc aacaatcaaa tggtttggcc aatattttac 840
aatgtgcaaa agtcggatgt atgcaatcaa acaaaaagtt atggtgaagc catgactgaa 900
catgaaaaaa gatttggaaa ggactctgag aaggtgcaga aatggaggtc tgctttgtct 960
gaaatcaaaa acttggaagg agaccatgtc aaacaaaatg agtacgtaat gcttttaatt 1020
aattacttaa tatatgcttc cctcttaagt gtttttgacc atctttga 1068
<210> 41
<211> 1113
<212> DNA
<213> 大豆(Glycine max)
<400> 41
atgtcgaatg aactaaaaaa ctatgatgtt tttctcaatt ttcatggcaa agattccggt 60
tacaccttca ctggtactct ctataatgct ttgcgcagca agagaatcaa gacctttttc 120
acaaaacatg aatatggtcg taaactacac actgatgaca gccacattcc accctttact 180
cttaaagcaa ttaaggaatc aaggatttcg gtggttgttt tgtcggaaaa ctatgcatcc 240
tcctcaagat gtcttgatga acttgtggcc atccttgagt gtaaaaggac gataaaccaa 300
ctggtgtggc ccatctttta caaagtggat ccgtcgcaag taaggcacca gaaaggtagc 360
tatggagaac acatttgtaa ttttaaaaaa atttttagag attacaacga ttccaatgag 420
agggtgaagc aatggagagc agctttatct gaagtcagca aattgagtgg atggctttac 480
aatgatcgga ggtcccagta cgaatatgaa ttcatcgaaa ggatcgtgga atcaaccgtg 540
caagccttgc ccggatatga tgtttttctg agttttaccg gagaggatac ccgctacact 600
ttcacaggtt ttctctataa tgcctttcgc cgagagggat tcaaaatctt catggatgat 660
gaggaattgg agagtgggaa ccaaatttca cagaagctta tgagagcaat tgagagttca 720
aagatttcaa ttgttgtgct ctctgaaaac tatgcatatt ccacctggtg tcttgatgaa 780
cttgccaaga tcattgagtg tatgaagacc aacaatcaaa tggtttggcc aatattttac 840
aatgtgcaaa agtcggatgt atgcaatcaa acaaaaagtt atggtgaagc catgactgaa 900
catgaaaaaa gatttggaaa ggactctgag aaggtgcaga aatggaggtc tgctttgtct 960
gaaatcaaaa acttggaagg agaccatgtc aaacaaaatg agtaccaaca tgaattaatc 1020
gaaaggattg tggaaaaggt cattaaaatt gaaggtagca agcatacagc aaatcctttc 1080
cttttatcca atgacagcta cgaggaagaa tga 1113
<210> 42
<211> 1098
<212> DNA
<213> 绿豆(Vigna radiata)
<400> 42
atgtcttgtg aaccaaaaag ctatgatgtt tttctcagtc ttggtggaaa agatgttcgt 60
tacaccttca ctggtaatct ctttaatgct ttgcgcagca agagaattaa gacccttttc 120
agagaacatg aatatgaacc tgagctacac actcatcaaa ctaatatttc accctctgtt 180
cttaaagcac tacaaacgtc aaagatttcc atcgttgttt tctccccaga atatgcatcc 240
tcctcaagac gtctcgatga acttgtggcc atccttgagt gtaggatgag gaccaaccaa 300
ctcgtatggc caatctttta cggagtggaa cccactgacg taagatttca gagaggtaga 360
tatgaacaag ccatgaatac atttgaagaa agatattccc cagagaggat gaataaatgg 420
agatcagctt tggctgaagt cagcaacttg agtggatggt tttaccaaaa ggagcacaaa 480
tacgaatata aattcatcag aaagattgtg gaagcagccg tgcaatccct gtcaagatat 540
gatgtttttt tgagtttttg tggagaggat acccgctaca ctctcacagg ttttctatac 600
aatgccctta gccgagaggg attcaaaatc ttcatggacg atgaagaatt ggagggtggg 660
aaccaaattt ctcaaaagct aatgggagca atagaaagtt caagggtttc aattgttgtg 720
ttctctgaaa actatggata ttccacctgg tgtcttgatg aactggccaa gatcactgag 780
tgtatgaaga ccaagaatca gatggtttgg ccaatatttt acaatgtgga aaagtcggat 840
gtatgcaatc aaacaaaaag ttatggtgag gccatgactg cacatgaaaa aaggtttggg 900
aaggactccg agaaggtgca aaaatggagg tctgctttgt ctcaaatcac caacttggaa 960
ggagagcatc tcagtgaaaa tgagttccaa catgaatcta tcgaaaagat tgtggaacgg 1020
ctcattaata ttgaagatgg gaagcatata gcaagtcctt tccttgttcc aatgacagct 1080
acggagaaca atgaatga 1098
<210> 43
<211> 513
<212> DNA
<213> 豌豆(Pisum sativum)
<400> 43
atgtctcttc caaagtatga cgtcttcatc agctttagag gagacgacac tcgtgacaac 60
ttcacgagtc atctttatgc agaattgcgt aggaaaaata ttgaaacatt catagattat 120
agacttggca gaggggaaga gattttccca actctgtgca aagcaataga agaatcagct 180
atttatgtgg ttattttgtc agaacactat gcttcttcca cttggtgttt ggaagaactc 240
acaaagatac tcgaatgtaa ggagagacat ggaaggaaag tgattcctgt cttctacaag 300
gtggatccgt caactgttag acatcagaca cagagttatg cagatgattt tgttaaacat 360
caacaacgat ttggcgacaa agtggatgca tggaaggctg ctctaaccca aatagctaat 420
ctttctggca tggattcaca caaaatcagg gatgacctaa acggagctgg aatgcctgtt 480
ttccacccta attctagttg tattaagttg tga 513
<210> 44
<211> 1098
<212> DNA
<213> 菜豆(Phaseolus vulgaris)
<400> 44
atgtccaatg aaccaaaaaa ctatgatgtt tttctgagtc ttggtggtga agatgttcgc 60
tacaccttca ctggtaatct cttttatgct ttgtgcagca agagaatcaa gacgtttttc 120
agagaacatg aacatgatcc tgagctatac actaatgaca ccaatatttc accctctgct 180
cttaaagcaa tacaagagtc aaagatttcc atggttgttt tctcccaaca atatgcatcc 240
tcctcaagat gtctcgatga acttgtggcc atccttgact gtaggatgaa gagcgaccaa 300
cttgtctggc caatctatta tggagtggat ccgagtgacc tagtaactca cgaaggtaga 360
ttcggacagg ccatgtgcag agttgaagaa aaatattcca cagagaggat aaacaaatgg 420
agagaagctt tggttcaagt tggccaatta agtggatggg tttatcaaag gaggttcaag 480
tacgaatata aattcatccg aaagatcgtg aaagcagctg tgcaatccct gccaagatat 540
gatgtttttc tgagcttttg tggagaggat acccgctaca ctcttgcagg ttttctctat 600
aatgccatta gccgagaggg attcaaaatc ttcatggacg atgaagaatt ggagggtggg 660
aaccaaattt ctgaaacgct catgggagca attgaaagtt caaggatttc aattgttgtg 720
ttctctgaaa actatggata ttccacttgg tgtcttgatg aacttgccaa gatcactgag 780
tgtatgaaca ccaagaatca gaaggtttgg ccaatatttt acaatgtgga gaagatggat 840
gtgtgcaatc aaacaaaaag ttatggtgag gccatgactg cacatggaaa aatgtttgga 900
aaggagtctg agaaggtgca gaaatggaag tctgctttgt ctcagatcac caacttggat 960
ggagagcatc tcattgaaaa tgagttccaa catgaatcta tcgaaaggat tgtggaacag 1020
ctcattaata ttgaagatgg gaagcatata gcaagtcctt tccttgttcc aacgacagct 1080
gctgagagga atgaatga 1098
<210> 45
<211> 1059
<212> DNA
<213> 大豆(Glycine max)
<400> 45
atgtcgaatg aactagaaaa ctacgatgtt tttctcagtt ttcatggcca agattcccgt 60
tacaccttca ctggtactct ctataatgct ttgcgcagca agagaatcaa gacctttttc 120
acagaacatg aatatgatcg taaactacac actgatgaca gccaaattcc accctctact 180
cttaaagcaa ttaaggaatc aaggatttcg gtggttgttt tctcggaaaa ctatgcatcc 240
tcctcaagat gtcttgatga acttgtggcc atccttgagt gtaacaggac gataaaccaa 300
ctggtgtggc ccatctttta cgaagtgtat ccgtggcacg taaggcagca gagaggtagc 360
tatgaagcac ccatgtctaa atttgaagaa atatttggag attccaatga gagggtgaag 420
caatggagag cagctttatt tgaagtcacc aacttgaatg gatggtgtta caaaactggg 480
acgtcccagt acgaatatga attcatcgaa aagatcgtgg aatcaaccgt gcaagccttg 540
cccggatatg atgtttttct gagttttacc ggagaggata cccgctacac tttcacaggt 600
tttctctatg atgcctttcg ccgagaggga ttcaaaatct tcatggatga tgaggaattg 660
gagagtggga accaaatttc acagaagctt atgggagcaa ttgagagttc aaagatttca 720
attgttgtgc tctctgaaaa ctatgcatat tccacctggt gtcttgatga acttgccaag 780
atcattgagt gtatgaagac caacaatcaa atggtttggc caatatttta caatgtgcaa 840
aagtcggatg tatgcaatca aacaaaaagt tatggtgaag ccatgactga acatgaaaaa 900
agatttggaa aggactctga gaaggtgcag aaatggaggt ctgctttgtc tgaaatcaac 960
aacttggaag gagaccatgt caaacaaaat gagtacgtaa tgcttttaat taattactta 1020
atatatgctt ccctcttaag tgtttttgaa catctgtga 1059
<210> 46
<211> 1113
<212> DNA
<213> 大豆(Glycine max)
<400> 46
atgtcgaatg aactaaaaaa ctatgatgtt tttctcaatt ttcatggcaa agattccggt 60
tacaccttca ctggtactct ctataatgct ttgcgcagca agagaatcaa gacctttttc 120
acaaaacatg aatatggtcg taaactacac actgatgaca gccacattcc accctttact 180
cttaaagcaa ttaaggaatc aaggatttcg gtggttgttt tgtcggaaaa ctatgcatcc 240
tcctcaagat gtcttgatga acttgtggcc atccttgagt gtaaaaggac gataaaccaa 300
ctggtgtggc ccatctttta caaagtggat ccgtcgcaag taaggcacca gaaaggtagc 360
tatggagaac acatttgtaa tttaaaaaaa aattttagag attacaacga ttccaatgag 420
agggtgaagc aatggagagc agctttatct gaagtcagca aattgagtgg atggctttac 480
aatgatcgga ggtcccagta cgaatatgaa ttcatcgaaa ggatcgtgga atcaaccgtg 540
caagccttgc ccggatatga tgtttttctg agttttaccg gagaggatac ccgctacact 600
ttcacaggtt ttctctataa tgcctttcgc cgagagggat tcaaaatctt catggatgat 660
gaggaattgg agagtgggaa ccaaatttca cagaagctta tgagagcaat tgagagttca 720
aagatttcaa ttgttgtgct ctctgaaaac tatgcatatt ccacctggtg tcttgatgaa 780
cttgccaaga tcattgagtg tatgaagacc aacaatcaaa tggtttggcc aatattttac 840
aatgtgcaaa agtcggatgt atgcaatcaa acaaaaagtt atggtgaagc catgactgaa 900
catgaaaaaa gatttggaaa ggactctgag aaggtgcaga aatggaggtc tgctttgtct 960
gaaatcaaaa acttggaagg agaccatgtc aaacaaaatg agtaccaaca tgaattaatc 1020
gaaaggattg tggaaaaggt catcaaaatt gaaggtagca agcatacagc aaatcctttc 1080
cttttatcca atgacagcta cgaggaagaa tga 1113
<210> 47
<211> 1113
<212> DNA
<213> 野大豆(Glycine soja)
<400> 47
atgtcgaatg aactaaaaaa ctatgatgtt tttctcaatt ttcatggcaa agattccggt 60
tacaccttca ctggtactct ctataatgct ttgcgcagca agagaatcaa gacctttttc 120
acaaaacatg aatatggtcg taaactacac actgatgaca gccacattcc accctttact 180
cttaaagcaa ttaaggaatc aaggatttcg gtggttgttt tgtcggaaaa ctatgcatcc 240
tcctcaagat gtcttgatga acttgtggcc atccttgagt gtaaaaggac gataaaccaa 300
ctggtgtggc ccatctttta caaagtggat ccgtcgcaag taaggcacca gaaaggtagc 360
tatggagaac acatttgtaa ttttaaaaat ttttttagag attacaacga ttccaatgag 420
agggtgaagc aatggagagc agctttatct gaagtcagca aattgagtgg atggctttac 480
aatgatcgga ggtcccagta cgaatatgaa ttcatcgaaa ggatcgtgga atcaaccgtg 540
caagccttgc ccggatatga tgtttttctg agttttaccg gagaggatac ccgctacact 600
ttcacaggtt ttctctataa tgcctttcgc cgagagggat tcaaaatctt catggatgat 660
gaggaattgg agagtgggaa ccaaatttca cagaagctta tgagagcaat tgagagttca 720
aagatttcaa ttgttgtgct ctctgaaaac tatgcatatt ccacctggtg tcttgatgaa 780
cttgccaaga tcattgagtg tatgaagacc aacaatcaaa tggtttggcc aatattttac 840
aatgtgcaaa agtcggatgt atgcaatcaa acaaaaagtt atggtgaagc catgactgaa 900
catgaaaaaa gatttggaaa ggactctgag aaggtgcaga aatggaggtc tgctttgtct 960
gaaatcaaaa acttggaagg agaccatgtc aaacaaaatg agtaccaaca tgaattaatc 1020
gaaaggattg tggaaaaggt cattaaaatt gaaggtagca agcatacagc aaatcctttc 1080
cttttatcca atgacagcta cgaggaagaa tga 1113
<210> 48
<211> 367
<212> PRT
<213> 野大豆(Glycine soja)
<400> 48
Met Ser Asn Glu Leu Glu Asn Tyr Asp Val Phe Leu Ser Phe His Gly
1 5 10 15
Gln Asp Ser Arg Tyr Thr Phe Thr Gly Thr Leu Tyr Asn Ala Leu Arg
20 25 30
Ser Lys Arg Ile Lys Thr Phe Phe Thr Glu His Glu Tyr Asp Arg Lys
35 40 45
Leu His Thr Asp Asp Ser Gln Ile Pro Pro Ser Thr Leu Lys Ala Ile
50 55 60
Lys Glu Ser Arg Ile Ser Val Val Val Leu Ser Glu Asn Tyr Ala Ser
65 70 75 80
Ser Ser Arg Cys Leu Asp Glu Leu Val Ala Ile Leu Glu Cys Asn Arg
85 90 95
Thr Ile Asn Gln Leu Val Trp Pro Ile Phe Tyr Glu Val Tyr Pro Trp
100 105 110
His Val Arg Gln Gln Arg Gly Ser Tyr Glu Ala Pro Met Ser Lys Phe
115 120 125
Glu Glu Ile Phe Gly Asp Ser Asn Glu Arg Val Lys Gln Trp Arg Ala
130 135 140
Ala Leu Phe Glu Val Thr Asn Leu Asn Gly Trp Cys Tyr Lys Thr Gly
145 150 155 160
Thr Ser Gln Tyr Glu Tyr Glu Leu Ile Glu Lys Ile Val Glu Ser Thr
165 170 175
Val Gln Ala Leu Pro Gly Tyr Asp Val Phe Leu Ser Phe Thr Gly Glu
180 185 190
Asp Thr Arg Tyr Thr Phe Thr Gly Phe Leu Tyr Asn Ala Phe Arg Arg
195 200 205
Glu Gly Phe Lys Ile Phe Met Asp Asp Glu Glu Leu Glu Ser Gly Asn
210 215 220
Gln Ile Ser Gln Lys Leu Met Gly Ala Ile Glu Ser Ser Lys Ile Ser
225 230 235 240
Ile Val Val Leu Ser Glu Asn Tyr Ala Tyr Ser Thr Trp Cys Leu Asp
245 250 255
Glu Leu Ala Lys Ile Ile Glu Cys Met Lys Thr Asn Asn Gln Met Val
260 265 270
Trp Pro Ile Phe Tyr Asn Val Gln Lys Ser Asp Val Cys Asn Gln Thr
275 280 285
Lys Ser Tyr Gly Glu Ala Met Thr Glu His Glu Lys Arg Phe Gly Lys
290 295 300
Asp Ser Glu Lys Val Gln Lys Trp Arg Ser Ala Leu Ser Glu Ile Asn
305 310 315 320
Asn Leu Glu Gly Asp His Val Lys Gln Asn Glu Tyr Gln His Glu Leu
325 330 335
Ile Glu Arg Ile Val Glu Lys Val Ile Lys Ile Glu Gly Ser Lys His
340 345 350
Thr Ala Asn Pro Phe Leu Leu Ser His Asp Ser Tyr Glu Glu Glu
355 360 365
<210> 49
<211> 363
<212> PRT
<213> 菜豆(Phaseolus vulgaris)
<400> 49
Met Ser Asn Glu Pro Lys Asn Tyr Asp Val Phe Leu Ser Leu Gly Gly
1 5 10 15
Glu Asp Val Arg Tyr Thr Phe Thr Gly Ser Leu Phe Ser Ala Leu Cys
20 25 30
Ser Lys Arg Ile Lys Thr Phe Phe Arg Glu His Glu Glu Asp Pro Glu
35 40 45
Pro Tyr Thr Asn Asp Thr Asn Ile Ser Pro Ser Ser Leu Lys Ala Ile
50 55 60
Gln Glu Ser Lys Ile Ser Ile Val Val Phe Ser Gln Gly Tyr Ala Ser
65 70 75 80
Ser Ser Arg Cys Leu Asp Glu Leu Val Ala Ile Leu Asp Cys Trp Met
85 90 95
Lys Ser Asp Gln Leu Val Trp Pro Ile Tyr Tyr Gly Val Asp Pro Ser
100 105 110
Glu Val Arg Thr Gln Lys Gly Gly Phe Gly Gln Ala Met Phe Arg Val
115 120 125
Arg Glu Arg Tyr Ser Thr Glu Arg Met Asn Lys Trp Arg Glu Ala Leu
130 135 140
Val Glu Val Ser Arg Phe Ser Gly Trp Val Tyr Gln Met Gly Ser Lys
145 150 155 160
Tyr Glu Tyr Lys Phe Ile Arg Lys Ile Val Glu Ala Ala Leu Gln Ser
165 170 175
Leu Pro Arg Tyr Asp Val Phe Leu Ser Phe Cys Gly Glu Asp Thr Arg
180 185 190
His Thr Leu Thr Gly Phe Leu Phe Asp Ala Ile Arg Arg Glu Gly Phe
195 200 205
Lys Ile Phe Met Asp Asp Glu Glu Leu Glu Gly Gly Asn Gln Ile Ser
210 215 220
Glu Thr Leu Met Gly Ala Ile Gln Ser Ser Arg Ile Ser Ile Val Val
225 230 235 240
Phe Ser Glu Asn Tyr Gly Tyr Ser Thr Trp Cys Leu Asp Glu Leu Ala
245 250 255
Lys Ile Thr Glu Cys Met Asn Thr Lys Asn Gln Lys Val Trp Pro Ile
260 265 270
Phe Tyr Asn Val Glu Lys Leu Asp Val Cys Asn Gln Thr Lys Ser Tyr
275 280 285
Gly Glu Ala Met Thr Ala His Glu Lys Arg Phe Gly Lys Asp Ser Glu
290 295 300
Lys Val Leu Lys Trp Arg Ser Ala Leu Ser Gln Ile Thr Asn Leu Asp
305 310 315 320
Gly Glu His Leu Ser Glu Asn Glu Phe Gln His Glu Ser Ile Glu Arg
325 330 335
Ile Val Glu Arg Leu Ile Asn Ile Glu Asp Gly Lys His Ile Ala Ser
340 345 350
Pro Phe Leu Ile Gln Asp Asn Asn Gly Glu Glu
355 360
<210> 50
<211> 272
<212> PRT
<213> 豌豆(Pisum sativum)
<400> 50
Met Gln Ile Leu Val Thr Ala Arg Cys Gln Gln Glu Cys Asp Leu Met
1 5 10 15
Tyr Cys Gln Arg Asp Val Gln Leu Asp Pro Leu Ser Lys Glu Glu Ala
20 25 30
Trp Thr Leu Phe Glu Lys His Ser Gly Ile His Asp Glu Asp Cys Ser
35 40 45
Ser Ser Pro Asp Leu Ser Asn Val Ala Arg Glu Val Ala Phe Glu Cys
50 55 60
Glu Gly Val Pro Arg Leu Ile Lys Asp Val Ala Ser Ser Leu Arg Asn
65 70 75 80
Lys Pro Ile Asp Glu Trp Lys Ala Ser Leu Asp Ser Leu Lys His Ser
85 90 95
Met Ala Lys Trp Gln Ile Phe Leu Ser Phe Arg Gly Glu Asp Thr Arg
100 105 110
Tyr Ala Phe Thr Gly Ser Leu Tyr Gln Ser Leu Arg Gln Gly Gly Phe
115 120 125
Lys Thr Phe Met Asp Asp Gly Gly Leu Glu Thr Gly Asp Gln Ile Ser
130 135 140
Pro Ser Leu Leu Asn Ala Ile Glu Ala Ser Arg Leu Ser Ile Ile Val
145 150 155 160
Leu Ser Glu Asn Tyr Ala Asn Ser Thr Trp Cys Leu Asp Glu Leu Val
165 170 175
Lys Ile Leu Glu Cys Lys Lys Leu Lys Asn Gln Leu Val Trp Pro Ile
180 185 190
Phe Tyr Lys Val Asp Pro Ser Asp Ile Arg His Met Arg Lys Cys Tyr
195 200 205
Gly Lys Asp Met Ala Arg His Glu Asn Arg Phe Gly Ile Asp Ser Glu
210 215 220
Arg Val Gln Lys Trp Lys Ser Ala Leu Asp Glu Val Cys Asn Leu Ser
225 230 235 240
Gly Lys Ala Tyr Ser Ile Gly Tyr Glu Tyr Glu Phe Ile Gln Lys Ile
245 250 255
Val Glu His Ala Asn Leu Ile Arg Ser Arg Leu Gln Ile Arg Asn Ile
260 265 270
<210> 51
<211> 355
<212> PRT
<213> 大豆(Glycine max)
<400> 51
Met Ser Asn Glu Leu Lys Asn Tyr Asp Val Phe Leu Asn Phe His Gly
1 5 10 15
Lys Asp Ser Gly Tyr Thr Phe Thr Gly Thr Leu Tyr Asn Ala Leu Arg
20 25 30
Ser Lys Arg Ile Lys Thr Phe Phe Thr Lys His Glu Tyr Gly Arg Lys
35 40 45
Leu His Thr Asp Asp Ser His Ile Pro Pro Phe Thr Leu Lys Ala Ile
50 55 60
Lys Glu Ser Arg Ile Ser Val Val Val Leu Ser Glu Asn Tyr Ala Ser
65 70 75 80
Ser Ser Arg Cys Leu Asp Glu Leu Val Ala Ile Leu Glu Cys Lys Arg
85 90 95
Thr Ile Asn Gln Leu Val Trp Pro Ile Phe Tyr Lys Val Asp Pro Ser
100 105 110
Gln Val Arg His Gln Lys Gly Ser Tyr Gly Glu His Ile Cys Asn Phe
115 120 125
Lys Lys Ile Phe Arg Asp Tyr Asn Asp Ser Asn Glu Arg Val Lys Gln
130 135 140
Trp Arg Ala Ala Leu Ser Glu Val Ser Lys Leu Ser Gly Trp Leu Tyr
145 150 155 160
Asn Asp Arg Arg Ser Gln Tyr Glu Tyr Glu Phe Ile Glu Arg Ile Val
165 170 175
Glu Ser Thr Val Gln Ala Leu Pro Gly Tyr Asp Val Phe Leu Ser Phe
180 185 190
Thr Gly Glu Asp Thr Arg Tyr Thr Phe Thr Gly Phe Leu Tyr Asn Ala
195 200 205
Phe Arg Arg Glu Gly Phe Lys Ile Phe Met Asp Asp Glu Glu Leu Glu
210 215 220
Ser Gly Asn Gln Ile Ser Gln Lys Leu Met Arg Ala Ile Glu Ser Ser
225 230 235 240
Lys Ile Ser Ile Val Val Leu Ser Glu Asn Tyr Ala Tyr Ser Thr Trp
245 250 255
Cys Leu Asp Glu Leu Ala Lys Ile Ile Glu Cys Met Lys Thr Asn Asn
260 265 270
Gln Met Val Trp Pro Ile Phe Tyr Asn Val Gln Lys Ser Asp Val Cys
275 280 285
Asn Gln Thr Lys Ser Tyr Gly Glu Ala Met Thr Glu His Glu Lys Arg
290 295 300
Phe Gly Lys Asp Ser Glu Lys Val Gln Lys Trp Arg Ser Ala Leu Ser
305 310 315 320
Glu Ile Lys Asn Leu Glu Gly Asp His Val Lys Gln Asn Glu Tyr Val
325 330 335
Met Leu Leu Ile Asn Tyr Leu Ile Tyr Ala Ser Leu Leu Ser Val Phe
340 345 350
Asp His Leu
355
<210> 52
<211> 370
<212> PRT
<213> 大豆(Glycine max)
<400> 52
Met Ser Asn Glu Leu Lys Asn Tyr Asp Val Phe Leu Asn Phe His Gly
1 5 10 15
Lys Asp Ser Gly Tyr Thr Phe Thr Gly Thr Leu Tyr Asn Ala Leu Arg
20 25 30
Ser Lys Arg Ile Lys Thr Phe Phe Thr Lys His Glu Tyr Gly Arg Lys
35 40 45
Leu His Thr Asp Asp Ser His Ile Pro Pro Phe Thr Leu Lys Ala Ile
50 55 60
Lys Glu Ser Arg Ile Ser Val Val Val Leu Ser Glu Asn Tyr Ala Ser
65 70 75 80
Ser Ser Arg Cys Leu Asp Glu Leu Val Ala Ile Leu Glu Cys Lys Arg
85 90 95
Thr Ile Asn Gln Leu Val Trp Pro Ile Phe Tyr Lys Val Asp Pro Ser
100 105 110
Gln Val Arg His Gln Lys Gly Ser Tyr Gly Glu His Ile Cys Asn Phe
115 120 125
Lys Lys Ile Phe Arg Asp Tyr Asn Asp Ser Asn Glu Arg Val Lys Gln
130 135 140
Trp Arg Ala Ala Leu Ser Glu Val Ser Lys Leu Ser Gly Trp Leu Tyr
145 150 155 160
Asn Asp Arg Arg Ser Gln Tyr Glu Tyr Glu Phe Ile Glu Arg Ile Val
165 170 175
Glu Ser Thr Val Gln Ala Leu Pro Gly Tyr Asp Val Phe Leu Ser Phe
180 185 190
Thr Gly Glu Asp Thr Arg Tyr Thr Phe Thr Gly Phe Leu Tyr Asn Ala
195 200 205
Phe Arg Arg Glu Gly Phe Lys Ile Phe Met Asp Asp Glu Glu Leu Glu
210 215 220
Ser Gly Asn Gln Ile Ser Gln Lys Leu Met Arg Ala Ile Glu Ser Ser
225 230 235 240
Lys Ile Ser Ile Val Val Leu Ser Glu Asn Tyr Ala Tyr Ser Thr Trp
245 250 255
Cys Leu Asp Glu Leu Ala Lys Ile Ile Glu Cys Met Lys Thr Asn Asn
260 265 270
Gln Met Val Trp Pro Ile Phe Tyr Asn Val Gln Lys Ser Asp Val Cys
275 280 285
Asn Gln Thr Lys Ser Tyr Gly Glu Ala Met Thr Glu His Glu Lys Arg
290 295 300
Phe Gly Lys Asp Ser Glu Lys Val Gln Lys Trp Arg Ser Ala Leu Ser
305 310 315 320
Glu Ile Lys Asn Leu Glu Gly Asp His Val Lys Gln Asn Glu Tyr Gln
325 330 335
His Glu Leu Ile Glu Arg Ile Val Glu Lys Val Ile Lys Ile Glu Gly
340 345 350
Ser Lys His Thr Ala Asn Pro Phe Leu Leu Ser Asn Asp Ser Tyr Glu
355 360 365
Glu Glu
370
<210> 53
<211> 365
<212> PRT
<213> 绿豆(Vigna radiata)
<400> 53
Met Ser Cys Glu Pro Lys Ser Tyr Asp Val Phe Leu Ser Leu Gly Gly
1 5 10 15
Lys Asp Val Arg Tyr Thr Phe Thr Gly Asn Leu Phe Asn Ala Leu Arg
20 25 30
Ser Lys Arg Ile Lys Thr Leu Phe Arg Glu His Glu Tyr Glu Pro Glu
35 40 45
Leu His Thr His Gln Thr Asn Ile Ser Pro Ser Val Leu Lys Ala Leu
50 55 60
Gln Thr Ser Lys Ile Ser Ile Val Val Phe Ser Pro Glu Tyr Ala Ser
65 70 75 80
Ser Ser Arg Arg Leu Asp Glu Leu Val Ala Ile Leu Glu Cys Arg Met
85 90 95
Arg Thr Asn Gln Leu Val Trp Pro Ile Phe Tyr Gly Val Glu Pro Thr
100 105 110
Asp Val Arg Phe Gln Arg Gly Arg Tyr Glu Gln Ala Met Asn Thr Phe
115 120 125
Glu Glu Arg Tyr Ser Pro Glu Arg Met Asn Lys Trp Arg Ser Ala Leu
130 135 140
Ala Glu Val Ser Asn Leu Ser Gly Trp Phe Tyr Gln Lys Glu His Lys
145 150 155 160
Tyr Glu Tyr Lys Phe Ile Arg Lys Ile Val Glu Ala Ala Val Gln Ser
165 170 175
Leu Ser Arg Tyr Asp Val Phe Leu Ser Phe Cys Gly Glu Asp Thr Arg
180 185 190
Tyr Thr Leu Thr Gly Phe Leu Tyr Asn Ala Leu Ser Arg Glu Gly Phe
195 200 205
Lys Ile Phe Met Asp Asp Glu Glu Leu Glu Gly Gly Asn Gln Ile Ser
210 215 220
Gln Lys Leu Met Gly Ala Ile Glu Ser Ser Arg Val Ser Ile Val Val
225 230 235 240
Phe Ser Glu Asn Tyr Gly Tyr Ser Thr Trp Cys Leu Asp Glu Leu Ala
245 250 255
Lys Ile Thr Glu Cys Met Lys Thr Lys Asn Gln Met Val Trp Pro Ile
260 265 270
Phe Tyr Asn Val Glu Lys Ser Asp Val Cys Asn Gln Thr Lys Ser Tyr
275 280 285
Gly Glu Ala Met Thr Ala His Glu Lys Arg Phe Gly Lys Asp Ser Glu
290 295 300
Lys Val Gln Lys Trp Arg Ser Ala Leu Ser Gln Ile Thr Asn Leu Glu
305 310 315 320
Gly Glu His Leu Ser Glu Asn Glu Phe Gln His Glu Ser Ile Glu Lys
325 330 335
Ile Val Glu Arg Leu Ile Asn Ile Glu Asp Gly Lys His Ile Ala Ser
340 345 350
Pro Phe Leu Val Pro Met Thr Ala Thr Glu Asn Asn Glu
355 360 365
<210> 54
<211> 170
<212> PRT
<213> 豌豆(Pisum sativum)
<400> 54
Met Ser Leu Pro Lys Tyr Asp Val Phe Ile Ser Phe Arg Gly Asp Asp
1 5 10 15
Thr Arg Asp Asn Phe Thr Ser His Leu Tyr Ala Glu Leu Arg Arg Lys
20 25 30
Asn Ile Glu Thr Phe Ile Asp Tyr Arg Leu Gly Arg Gly Glu Glu Ile
35 40 45
Phe Pro Thr Leu Cys Lys Ala Ile Glu Glu Ser Ala Ile Tyr Val Val
50 55 60
Ile Leu Ser Glu His Tyr Ala Ser Ser Thr Trp Cys Leu Glu Glu Leu
65 70 75 80
Thr Lys Ile Leu Glu Cys Lys Glu Arg His Gly Arg Lys Val Ile Pro
85 90 95
Val Phe Tyr Lys Val Asp Pro Ser Thr Val Arg His Gln Thr Gln Ser
100 105 110
Tyr Ala Asp Asp Phe Val Lys His Gln Gln Arg Phe Gly Asp Lys Val
115 120 125
Asp Ala Trp Lys Ala Ala Leu Thr Gln Ile Ala Asn Leu Ser Gly Met
130 135 140
Asp Ser His Lys Ile Arg Asp Asp Leu Asn Gly Ala Gly Met Pro Val
145 150 155 160
Phe His Pro Asn Ser Ser Cys Ile Lys Leu
165 170
<210> 55
<211> 365
<212> PRT
<213> 菜豆(Phaseolus vulgaris)
<400> 55
Met Ser Asn Glu Pro Lys Asn Tyr Asp Val Phe Leu Ser Leu Gly Gly
1 5 10 15
Glu Asp Val Arg Tyr Thr Phe Thr Gly Asn Leu Phe Tyr Ala Leu Cys
20 25 30
Ser Lys Arg Ile Lys Thr Phe Phe Arg Glu His Glu His Asp Pro Glu
35 40 45
Leu Tyr Thr Asn Asp Thr Asn Ile Ser Pro Ser Ala Leu Lys Ala Ile
50 55 60
Gln Glu Ser Lys Ile Ser Met Val Val Phe Ser Gln Gln Tyr Ala Ser
65 70 75 80
Ser Ser Arg Cys Leu Asp Glu Leu Val Ala Ile Leu Asp Cys Arg Met
85 90 95
Lys Ser Asp Gln Leu Val Trp Pro Ile Tyr Tyr Gly Val Asp Pro Ser
100 105 110
Asp Leu Val Thr His Glu Gly Arg Phe Gly Gln Ala Met Cys Arg Val
115 120 125
Glu Glu Lys Tyr Ser Thr Glu Arg Ile Asn Lys Trp Arg Glu Ala Leu
130 135 140
Val Gln Val Gly Gln Leu Ser Gly Trp Val Tyr Gln Arg Arg Phe Lys
145 150 155 160
Tyr Glu Tyr Lys Phe Ile Arg Lys Ile Val Lys Ala Ala Val Gln Ser
165 170 175
Leu Pro Arg Tyr Asp Val Phe Leu Ser Phe Cys Gly Glu Asp Thr Arg
180 185 190
Tyr Thr Leu Ala Gly Phe Leu Tyr Asn Ala Ile Ser Arg Glu Gly Phe
195 200 205
Lys Ile Phe Met Asp Asp Glu Glu Leu Glu Gly Gly Asn Gln Ile Ser
210 215 220
Glu Thr Leu Met Gly Ala Ile Glu Ser Ser Arg Ile Ser Ile Val Val
225 230 235 240
Phe Ser Glu Asn Tyr Gly Tyr Ser Thr Trp Cys Leu Asp Glu Leu Ala
245 250 255
Lys Ile Thr Glu Cys Met Asn Thr Lys Asn Gln Lys Val Trp Pro Ile
260 265 270
Phe Tyr Asn Val Glu Lys Met Asp Val Cys Asn Gln Thr Lys Ser Tyr
275 280 285
Gly Glu Ala Met Thr Ala His Gly Lys Met Phe Gly Lys Glu Ser Glu
290 295 300
Lys Val Gln Lys Trp Lys Ser Ala Leu Ser Gln Ile Thr Asn Leu Asp
305 310 315 320
Gly Glu His Leu Ile Glu Asn Glu Phe Gln His Glu Ser Ile Glu Arg
325 330 335
Ile Val Glu Gln Leu Ile Asn Ile Glu Asp Gly Lys His Ile Ala Ser
340 345 350
Pro Phe Leu Val Pro Thr Thr Ala Ala Glu Arg Asn Glu
355 360 365
<210> 56
<211> 352
<212> PRT
<213> 大豆(Glycine max)
<400> 56
Met Ser Asn Glu Leu Glu Asn Tyr Asp Val Phe Leu Ser Phe His Gly
1 5 10 15
Gln Asp Ser Arg Tyr Thr Phe Thr Gly Thr Leu Tyr Asn Ala Leu Arg
20 25 30
Ser Lys Arg Ile Lys Thr Phe Phe Thr Glu His Glu Tyr Asp Arg Lys
35 40 45
Leu His Thr Asp Asp Ser Gln Ile Pro Pro Ser Thr Leu Lys Ala Ile
50 55 60
Lys Glu Ser Arg Ile Ser Val Val Val Phe Ser Glu Asn Tyr Ala Ser
65 70 75 80
Ser Ser Arg Cys Leu Asp Glu Leu Val Ala Ile Leu Glu Cys Asn Arg
85 90 95
Thr Ile Asn Gln Leu Val Trp Pro Ile Phe Tyr Glu Val Tyr Pro Trp
100 105 110
His Val Arg Gln Gln Arg Gly Ser Tyr Glu Ala Pro Met Ser Lys Phe
115 120 125
Glu Glu Ile Phe Gly Asp Ser Asn Glu Arg Val Lys Gln Trp Arg Ala
130 135 140
Ala Leu Phe Glu Val Thr Asn Leu Asn Gly Trp Cys Tyr Lys Thr Gly
145 150 155 160
Thr Ser Gln Tyr Glu Tyr Glu Phe Ile Glu Lys Ile Val Glu Ser Thr
165 170 175
Val Gln Ala Leu Pro Gly Tyr Asp Val Phe Leu Ser Phe Thr Gly Glu
180 185 190
Asp Thr Arg Tyr Thr Phe Thr Gly Phe Leu Tyr Asp Ala Phe Arg Arg
195 200 205
Glu Gly Phe Lys Ile Phe Met Asp Asp Glu Glu Leu Glu Ser Gly Asn
210 215 220
Gln Ile Ser Gln Lys Leu Met Gly Ala Ile Glu Ser Ser Lys Ile Ser
225 230 235 240
Ile Val Val Leu Ser Glu Asn Tyr Ala Tyr Ser Thr Trp Cys Leu Asp
245 250 255
Glu Leu Ala Lys Ile Ile Glu Cys Met Lys Thr Asn Asn Gln Met Val
260 265 270
Trp Pro Ile Phe Tyr Asn Val Gln Lys Ser Asp Val Cys Asn Gln Thr
275 280 285
Lys Ser Tyr Gly Glu Ala Met Thr Glu His Glu Lys Arg Phe Gly Lys
290 295 300
Asp Ser Glu Lys Val Gln Lys Trp Arg Ser Ala Leu Ser Glu Ile Asn
305 310 315 320
Asn Leu Glu Gly Asp His Val Lys Gln Asn Glu Tyr Val Met Leu Leu
325 330 335
Ile Asn Tyr Leu Ile Tyr Ala Ser Leu Leu Ser Val Phe Glu His Leu
340 345 350
<210> 57
<211> 370
<212> PRT
<213> 大豆(Glycine max)
<400> 57
Met Ser Asn Glu Leu Lys Asn Tyr Asp Val Phe Leu Asn Phe His Gly
1 5 10 15
Lys Asp Ser Gly Tyr Thr Phe Thr Gly Thr Leu Tyr Asn Ala Leu Arg
20 25 30
Ser Lys Arg Ile Lys Thr Phe Phe Thr Lys His Glu Tyr Gly Arg Lys
35 40 45
Leu His Thr Asp Asp Ser His Ile Pro Pro Phe Thr Leu Lys Ala Ile
50 55 60
Lys Glu Ser Arg Ile Ser Val Val Val Leu Ser Glu Asn Tyr Ala Ser
65 70 75 80
Ser Ser Arg Cys Leu Asp Glu Leu Val Ala Ile Leu Glu Cys Lys Arg
85 90 95
Thr Ile Asn Gln Leu Val Trp Pro Ile Phe Tyr Lys Val Asp Pro Ser
100 105 110
Gln Val Arg His Gln Lys Gly Ser Tyr Gly Glu His Ile Cys Asn Leu
115 120 125
Lys Lys Asn Phe Arg Asp Tyr Asn Asp Ser Asn Glu Arg Val Lys Gln
130 135 140
Trp Arg Ala Ala Leu Ser Glu Val Ser Lys Leu Ser Gly Trp Leu Tyr
145 150 155 160
Asn Asp Arg Arg Ser Gln Tyr Glu Tyr Glu Phe Ile Glu Arg Ile Val
165 170 175
Glu Ser Thr Val Gln Ala Leu Pro Gly Tyr Asp Val Phe Leu Ser Phe
180 185 190
Thr Gly Glu Asp Thr Arg Tyr Thr Phe Thr Gly Phe Leu Tyr Asn Ala
195 200 205
Phe Arg Arg Glu Gly Phe Lys Ile Phe Met Asp Asp Glu Glu Leu Glu
210 215 220
Ser Gly Asn Gln Ile Ser Gln Lys Leu Met Arg Ala Ile Glu Ser Ser
225 230 235 240
Lys Ile Ser Ile Val Val Leu Ser Glu Asn Tyr Ala Tyr Ser Thr Trp
245 250 255
Cys Leu Asp Glu Leu Ala Lys Ile Ile Glu Cys Met Lys Thr Asn Asn
260 265 270
Gln Met Val Trp Pro Ile Phe Tyr Asn Val Gln Lys Ser Asp Val Cys
275 280 285
Asn Gln Thr Lys Ser Tyr Gly Glu Ala Met Thr Glu His Glu Lys Arg
290 295 300
Phe Gly Lys Asp Ser Glu Lys Val Gln Lys Trp Arg Ser Ala Leu Ser
305 310 315 320
Glu Ile Lys Asn Leu Glu Gly Asp His Val Lys Gln Asn Glu Tyr Gln
325 330 335
His Glu Leu Ile Glu Arg Ile Val Glu Lys Val Ile Lys Ile Glu Gly
340 345 350
Ser Lys His Thr Ala Asn Pro Phe Leu Leu Ser Asn Asp Ser Tyr Glu
355 360 365
Glu Glu
370
<210> 58
<211> 370
<212> PRT
<213> 野大豆(Glycine soja)
<400> 58
Met Ser Asn Glu Leu Lys Asn Tyr Asp Val Phe Leu Asn Phe His Gly
1 5 10 15
Lys Asp Ser Gly Tyr Thr Phe Thr Gly Thr Leu Tyr Asn Ala Leu Arg
20 25 30
Ser Lys Arg Ile Lys Thr Phe Phe Thr Lys His Glu Tyr Gly Arg Lys
35 40 45
Leu His Thr Asp Asp Ser His Ile Pro Pro Phe Thr Leu Lys Ala Ile
50 55 60
Lys Glu Ser Arg Ile Ser Val Val Val Leu Ser Glu Asn Tyr Ala Ser
65 70 75 80
Ser Ser Arg Cys Leu Asp Glu Leu Val Ala Ile Leu Glu Cys Lys Arg
85 90 95
Thr Ile Asn Gln Leu Val Trp Pro Ile Phe Tyr Lys Val Asp Pro Ser
100 105 110
Gln Val Arg His Gln Lys Gly Ser Tyr Gly Glu His Ile Cys Asn Phe
115 120 125
Lys Asn Phe Phe Arg Asp Tyr Asn Asp Ser Asn Glu Arg Val Lys Gln
130 135 140
Trp Arg Ala Ala Leu Ser Glu Val Ser Lys Leu Ser Gly Trp Leu Tyr
145 150 155 160
Asn Asp Arg Arg Ser Gln Tyr Glu Tyr Glu Phe Ile Glu Arg Ile Val
165 170 175
Glu Ser Thr Val Gln Ala Leu Pro Gly Tyr Asp Val Phe Leu Ser Phe
180 185 190
Thr Gly Glu Asp Thr Arg Tyr Thr Phe Thr Gly Phe Leu Tyr Asn Ala
195 200 205
Phe Arg Arg Glu Gly Phe Lys Ile Phe Met Asp Asp Glu Glu Leu Glu
210 215 220
Ser Gly Asn Gln Ile Ser Gln Lys Leu Met Arg Ala Ile Glu Ser Ser
225 230 235 240
Lys Ile Ser Ile Val Val Leu Ser Glu Asn Tyr Ala Tyr Ser Thr Trp
245 250 255
Cys Leu Asp Glu Leu Ala Lys Ile Ile Glu Cys Met Lys Thr Asn Asn
260 265 270
Gln Met Val Trp Pro Ile Phe Tyr Asn Val Gln Lys Ser Asp Val Cys
275 280 285
Asn Gln Thr Lys Ser Tyr Gly Glu Ala Met Thr Glu His Glu Lys Arg
290 295 300
Phe Gly Lys Asp Ser Glu Lys Val Gln Lys Trp Arg Ser Ala Leu Ser
305 310 315 320
Glu Ile Lys Asn Leu Glu Gly Asp His Val Lys Gln Asn Glu Tyr Gln
325 330 335
His Glu Leu Ile Glu Arg Ile Val Glu Lys Val Ile Lys Ile Glu Gly
340 345 350
Ser Lys His Thr Ala Asn Pro Phe Leu Leu Ser Asn Asp Ser Tyr Glu
355 360 365
Glu Glu
370
<210> 59
<211> 2778
<212> DNA
<213> 木豆(Cajanus cajan)
<400> 59
atggcagaca gtgtggtttc ctttgtttta gatcacttgt cccaactggt ggaacatgaa 60
gcaaggttgc taagtggcgt ggaagacaag gtgaagtccc tcgagaggga gcttcagatg 120
atcaacgtca tcctaagaac cacaaacagc aacaacgaca ttcagaaaac agtggtgagc 180
caaatcagag atgtggccca tgaagctgag gatgtcattg acacgtacgt tgccaaagtg 240
gccctgcaca acaggagaac catgctgggg aggctactcc atggagttga ccaaacaaag 300
ttgctccatg acgtatccga gaaaatagac gagatcataa caactctcaa ccagatacgt 360
gaaaacaaga tcaaatacag cgagttccaa gaaagaaatc atcaatccat agcagaagag 420
gaggaggagg agaaggagag ggagaggtta cttcacaagc taagaagaaa tgtagaggag 480
gaacatgtag tgggttttat ccgtggatct caagcaatca tcaagctact caaggaaggt 540
ggctcacggc gtaatgtggt ctccatcatc ggcatggggg ggttgggcaa gaccaccctt 600
gcccgaaaag tttataatga tagcaaggtg aaacaaggct ttagttgttg tgtttgggtg 660
tacgtgtcaa acgagtgtag agctaaggag cttttgctta gtcttcttaa gcatttgagg 720
ccaaacctcg aaactgaact tcaagaagaa aacaacaaag gaaaaaaatt cactgaagaa 780
caagacattt ttaacttgag tgtggaggag ctgaagaaac tggtgcggca atacttggag 840
aggaaaacaa ggtatctggt ggtcctcgat gacttgtgga aaacacaaga ttgggacgag 900
gtgcaagatg cttttcccga caacaacaga ggcaacagaa tattgatcac tagtcgtttg 960
aaagaggtgg ccttgcatac tagtcttcat cctccatact accttcaatt tctcagccaa 1020
gaagaaagct gggagctctt tcgtaggaaa gtgtttagag gggaagaatg cccttttgaa 1080
ctagagcctc taggcaaaca aatagtggca agttgtcgcg gtttgccact ctctattgtt 1140
gtattagcag gattgctagc caacaaggaa aagtcacaca gggaatggtc caaagtggtg 1200
ggtcacgtca actggtatct tactcaagac gagactcaag tgaaggatat agttctgaag 1260
ctcagttatg ataacttgcc aagaagattg aaaccatgct ttctatattt tgggatattc 1320
cctgaagact ttgaaatccc tgttaggcca ttactacaac aatgggttgc agaagggttt 1380
atacaagaaa caagaaatag agacccagat gatgtggcag aagactactt gtacgagctc 1440
attgatcgta gtttggtcca agtagcagca ataaagacta gtggaggtgt gaaaacttgt 1500
cacatccatg atcttctccg agatctttgt gtatcgcaga gcaaagggga caagattttt 1560
gaagtctgct cagataatga cattcaaatt ctaacaaaac ctcgcaggtt gtccttccat 1620
tgtgacatgg gccactacat ttcttcaagc aacaaagacc attcatgtat ccgttctttg 1680
ttcttctttg gaatatattc caattttact gggaacgagt gggaatggct tttcaaaggc 1740
ttcaaattgg ttcgagtgtt agagcttgga aaaaaccatt gcgcaggaaa gatcccatct 1800
aatttggggg actttatcca cttaaggtat ttgagaattg actcgaattt tggtataatt 1860
attccaactt ccatacttac ccttcagaat ttacaaacag tagatttagg taattggttt 1920
agggaaatcc caatttgttt ccctgctcaa atgtggaagc tcaaacattt aaggcacctg 1980
tatgggcaag gacctgtgaa gcttcaaggc cactattcag gatcaaatga ggttatgtgg 2040
aatctccgaa ccatcttccc cattgatatt gatacacaaa cattgtctct gatgaagaaa 2100
ggaagcttcc ccaatcttgt gaaattgggg ttgtcaatca attcggaccg ccaaggtaag 2160
tggccaaagt tgttgcagag cttacaagaa ttaagtcatt tgaatatctt aaagatttgc 2220
ctccgagggg attttgatgc ttcaataggc acagtgtcaa gcatatggcg gtttggttgt 2280
gagccacagg agctattaca aagcctaggg ttgttgactc atataactac gttgaaaatc 2340
accaatatct gcagccttat gataacggtt cctccaaatg tcaccaagtt aacattgcgt 2400
ggtattagta gcatcactag ggaggggctg aatgcgttga gaaatcacac caaactccaa 2460
attttgagtc tatatggaga ctatggctct aacattaacc tcaattgtgt tgtaggcggc 2520
tttccacaac tgcaagtatt gcaattgaaa aagttcacct ctgtaaattg gaaattaggc 2580
aatggtgcaa tgccacgtct tcacactcta gtcatcatca actgtcaaag tttagatgat 2640
cttccaaatg aattgtggtc tctcactgcc ttcagaaaac tgcatgtaaa acaaccctca 2700
caaccaatgc ttcgtatgct acgggatttg aaaataaagg atagggttca agtcatagtc 2760
gatgatcatg acaactag 2778
<210> 60
<211> 925
<212> PRT
<213> 木豆(Cajanus cajan)
<400> 60
Met Ala Asp Ser Val Val Ser Phe Val Leu Asp His Leu Ser Gln Leu
1 5 10 15
Val Glu His Glu Ala Arg Leu Leu Ser Gly Val Glu Asp Lys Val Lys
20 25 30
Ser Leu Glu Arg Glu Leu Gln Met Ile Asn Val Ile Leu Arg Thr Thr
35 40 45
Asn Ser Asn Asn Asp Ile Gln Lys Thr Val Val Ser Gln Ile Arg Asp
50 55 60
Val Ala His Glu Ala Glu Asp Val Ile Asp Thr Tyr Val Ala Lys Val
65 70 75 80
Ala Leu His Asn Arg Arg Thr Met Leu Gly Arg Leu Leu His Gly Val
85 90 95
Asp Gln Thr Lys Leu Leu His Asp Val Ser Glu Lys Ile Asp Glu Ile
100 105 110
Ile Thr Thr Leu Asn Gln Ile Arg Glu Asn Lys Ile Lys Tyr Ser Glu
115 120 125
Phe Gln Glu Arg Asn His Gln Ser Ile Ala Glu Glu Glu Glu Glu Glu
130 135 140
Lys Glu Arg Glu Arg Leu Leu His Lys Leu Arg Arg Asn Val Glu Glu
145 150 155 160
Glu His Val Val Gly Phe Ile Arg Gly Ser Gln Ala Ile Ile Lys Leu
165 170 175
Leu Lys Glu Gly Gly Ser Arg Arg Asn Val Val Ser Ile Ile Gly Met
180 185 190
Gly Gly Leu Gly Lys Thr Thr Leu Ala Arg Lys Val Tyr Asn Asp Ser
195 200 205
Lys Val Lys Gln Gly Phe Ser Cys Cys Val Trp Val Tyr Val Ser Asn
210 215 220
Glu Cys Arg Ala Lys Glu Leu Leu Leu Ser Leu Leu Lys His Leu Arg
225 230 235 240
Pro Asn Leu Glu Thr Glu Leu Gln Glu Glu Asn Asn Lys Gly Lys Lys
245 250 255
Phe Thr Glu Glu Gln Asp Ile Phe Asn Leu Ser Val Glu Glu Leu Lys
260 265 270
Lys Leu Val Arg Gln Tyr Leu Glu Arg Lys Thr Arg Tyr Leu Val Val
275 280 285
Leu Asp Asp Leu Trp Lys Thr Gln Asp Trp Asp Glu Val Gln Asp Ala
290 295 300
Phe Pro Asp Asn Asn Arg Gly Asn Arg Ile Leu Ile Thr Ser Arg Leu
305 310 315 320
Lys Glu Val Ala Leu His Thr Ser Leu His Pro Pro Tyr Tyr Leu Gln
325 330 335
Phe Leu Ser Gln Glu Glu Ser Trp Glu Leu Phe Arg Arg Lys Val Phe
340 345 350
Arg Gly Glu Glu Cys Pro Phe Glu Leu Glu Pro Leu Gly Lys Gln Ile
355 360 365
Val Ala Ser Cys Arg Gly Leu Pro Leu Ser Ile Val Val Leu Ala Gly
370 375 380
Leu Leu Ala Asn Lys Glu Lys Ser His Arg Glu Trp Ser Lys Val Val
385 390 395 400
Gly His Val Asn Trp Tyr Leu Thr Gln Asp Glu Thr Gln Val Lys Asp
405 410 415
Ile Val Leu Lys Leu Ser Tyr Asp Asn Leu Pro Arg Arg Leu Lys Pro
420 425 430
Cys Phe Leu Tyr Phe Gly Ile Phe Pro Glu Asp Phe Glu Ile Pro Val
435 440 445
Arg Pro Leu Leu Gln Gln Trp Val Ala Glu Gly Phe Ile Gln Glu Thr
450 455 460
Arg Asn Arg Asp Pro Asp Asp Val Ala Glu Asp Tyr Leu Tyr Glu Leu
465 470 475 480
Ile Asp Arg Ser Leu Val Gln Val Ala Ala Ile Lys Thr Ser Gly Gly
485 490 495
Val Lys Thr Cys His Ile His Asp Leu Leu Arg Asp Leu Cys Val Ser
500 505 510
Gln Ser Lys Gly Asp Lys Ile Phe Glu Val Cys Ser Asp Asn Asp Ile
515 520 525
Gln Ile Leu Thr Lys Pro Arg Arg Leu Ser Phe His Cys Asp Met Gly
530 535 540
His Tyr Ile Ser Ser Ser Asn Lys Asp His Ser Cys Ile Arg Ser Leu
545 550 555 560
Phe Phe Phe Gly Ile Tyr Ser Asn Phe Thr Gly Asn Glu Trp Glu Trp
565 570 575
Leu Phe Lys Gly Phe Lys Leu Val Arg Val Leu Glu Leu Gly Lys Asn
580 585 590
His Cys Ala Gly Lys Ile Pro Ser Asn Leu Gly Asp Phe Ile His Leu
595 600 605
Arg Tyr Leu Arg Ile Asp Ser Asn Phe Gly Ile Ile Ile Pro Thr Ser
610 615 620
Ile Leu Thr Leu Gln Asn Leu Gln Thr Val Asp Leu Gly Asn Trp Phe
625 630 635 640
Arg Glu Ile Pro Ile Cys Phe Pro Ala Gln Met Trp Lys Leu Lys His
645 650 655
Leu Arg His Leu Tyr Gly Gln Gly Pro Val Lys Leu Gln Gly His Tyr
660 665 670
Ser Gly Ser Asn Glu Val Met Trp Asn Leu Arg Thr Ile Phe Pro Ile
675 680 685
Asp Ile Asp Thr Gln Thr Leu Ser Leu Met Lys Lys Gly Ser Phe Pro
690 695 700
Asn Leu Val Lys Leu Gly Leu Ser Ile Asn Ser Asp Arg Gln Gly Lys
705 710 715 720
Trp Pro Lys Leu Leu Gln Ser Leu Gln Glu Leu Ser His Leu Asn Ile
725 730 735
Leu Lys Ile Cys Leu Arg Gly Asp Phe Asp Ala Ser Ile Gly Thr Val
740 745 750
Ser Ser Ile Trp Arg Phe Gly Cys Glu Pro Gln Glu Leu Leu Gln Ser
755 760 765
Leu Gly Leu Leu Thr His Ile Thr Thr Leu Lys Ile Thr Asn Ile Cys
770 775 780
Ser Leu Met Ile Thr Val Pro Pro Asn Val Thr Lys Leu Thr Leu Arg
785 790 795 800
Gly Ile Ser Ser Ile Thr Arg Glu Gly Leu Asn Ala Leu Arg Asn His
805 810 815
Thr Lys Leu Gln Ile Leu Ser Leu Tyr Gly Asp Tyr Gly Ser Asn Ile
820 825 830
Asn Leu Asn Cys Val Val Gly Gly Phe Pro Gln Leu Gln Val Leu Gln
835 840 845
Leu Lys Lys Phe Thr Ser Val Asn Trp Lys Leu Gly Asn Gly Ala Met
850 855 860
Pro Arg Leu His Thr Leu Val Ile Ile Asn Cys Gln Ser Leu Asp Asp
865 870 875 880
Leu Pro Asn Glu Leu Trp Ser Leu Thr Ala Phe Arg Lys Leu His Val
885 890 895
Lys Gln Pro Ser Gln Pro Met Leu Arg Met Leu Arg Asp Leu Lys Ile
900 905 910
Lys Asp Arg Val Gln Val Ile Val Asp Asp His Asp Asn
915 920 925
Claims (36)
1.一种多肽,其包含与选自由SEQ ID NO:2和21-36组成的组的序列具有至少85%序列同一性的氨基酸序列,其中所述多肽当在植物细胞中表达时赋予所述植物对亚洲大豆锈病(ASR)病害的抗性。
2.如权利要求1所述的多肽,其中所述氨基酸序列与选自由SEQ ID NO:2和21-36组成的组的全长序列具有至少95%序列同一性。
3.如权利要求1所述的多肽,其中所述氨基酸序列与SEQ ID NO:2的序列具有至少95%序列同一性。
4.如权利要求1-3中任一项所述的多肽,其中将所述多肽用可检测标志进行标记。
5.一种组合物,其包含如权利要求1-4中任一项所述的多肽、和第二多肽,所述第二多肽包含与选自由SEQ ID NO:4和48-58组成的组的序列具有至少95%序列同一性的氨基酸序列。
6.如权利要求5所述的组合物,其中将所述第二多肽用可检测标志进行标记。
7.一种多核苷酸,其编码如权利要求1-4中任一项所述的多肽。
8.如权利要求7所述的多核苷酸,其中所述多核苷酸选自由SEQ ID NO:1和5-20或与SEQ ID NO:1和5-20具有至少95%序列同一性的多核苷酸组成的组。
9.如权利要求7或8所述的多核苷酸,其进一步包含与重组多核苷酸可操作地连接的异源调控序列。
10.如权利要求9所述的多核苷酸,其中所述异源调控序列是植物可操作启动子。
11.一种多核苷酸,其选自由SEQ ID NO:3和37-47或与选自由SEQ ID NO:3和37-47组成的组的序列具有至少95%序列同一性的多核苷酸组成的组,其中所述多核苷酸进一步包含与重组多核苷酸可操作地连接的异源调控序列。
12.如权利要求11所述的多核苷酸,其中所述异源调控序列是植物可操作启动子。
13.一种植物转化构建体,其包含如权利要求8-12所述的多核苷酸中的一种或多种。
14.一种植物转化构建体,其包含与SEQ ID NO:1具有至少95%序列同一性的第一多核苷酸和与SEQ ID NO:3具有至少95%序列同一性的第二多核苷酸,其中所述第一多核苷酸和所述第二多核苷酸与在植物细胞中可操作的异源调控序列可操作地连接。
15.一种转基因植物细胞,其包含如权利要求7-14中任一项所述的多核苷酸。
16.一种植物或植物部分,其包含多个如权利要求15所述的植物细胞。
17.如权利要求16所述的植物或植物部分,其中所述植物是豆类作物植物。
18.如权利要求17所述的豆类作物植物,其中转基因豆类作物植物是大豆。
19.一种转基因植物细胞,其包含编码赋予豆类作物物种病害抗性的多肽的重组多核苷酸,其中所编码的多肽与选自由SEQ ID NO:2和21-36组成的组的氨基酸序列或选自由SEQ ID NO:4和48-58组成的组的氨基酸序列具有至少85%序列同一性。
20.如权利要求19所述的转基因植物细胞,其包含编码与选自由SEQ ID NO:2和21-36组成的组的氨基酸序列具有至少95%序列同一性的多肽的第一多核苷酸,以及编码与选自由SEQ ID NO:4和48-58组成的组的氨基酸序列具有至少95%序列同一性的多肽的第二多核苷酸。
21.如权利要求19或20所述的转基因植物细胞,其进一步包含编码具有SEQ ID NO:60的序列或与SEQ ID NO:60具有95%序列同一性的序列的多肽的CcRpp1基因。
22.一种植物或植物部分,其包含多个如权利要求19、20或21中任一项所述的植物细胞。
23.如权利要求22所述的植物部分,其中所述植物部分是种子。
24.如权利要求22或23所述的植物或植物部分,其中所述植物是豆类作物植物,任选地其中所述植物是选自由苜蓿、三叶草、豌豆、菜豆、兵豆、羽扇豆、牧豆树、角豆树、大豆、木豆、花生和罗望子组成的组的豆类,任选地其中所述植物是大豆植物。
25.一种检测植物组织中存在的编码CcRpp2-R1或CcRpp2-R3的核酸的方法,所述方法包括
从所述植物组织获得核酸样品;以及
i)使所述核酸样品与多核苷酸接触,所述多核苷酸包含与选自由SEQ ID NO:1、3、5-20和37-47组成的组的连续序列或其互补序列相同的至少8个核苷酸的序列;
使所述样品和所述多核苷酸经受严格杂交条件;以及
测定所述样品中所述多核苷酸与所述DNA的杂交;或者
ii)使所述核酸样品与第一PCR引物和第二PCR引物接触,其中所述第一PCR引物和所述第二PCR引物各自特异性结合选自由SEQ ID NO:1、3、5-20和37-47组成的组的序列;
使所述样品经受聚合酶链反应;以及
测定在所述第一引物和所述第二引物之间产生的扩增子。
26.一种赋予豆类作物物种抗病性的方法,所述方法包括向豆类作物物种中引入赋予对豆类作物物种病害的抗病性的重组CcRpp2-R1基因和重组CcRpp2-R3基因,其中所述衍生的CcRpp2-R1基因包含与选自由SEQ ID NO:1和5-20组成的组的序列具有至少95%序列同一性的序列,并且所述CcRpp2-R3基因包含与选自由SEQ ID NO:3和37-47组成的组的序列具有至少95%序列同一性的序列。
27.如权利要求26所述的方法,其中通过用与选自由SEQ ID NO:1和5-20组成的组的序列具有至少95%序列同一性的序列和与选自由SEQ ID NO:3和37-47组成的组的序列具有至少95%序列同一性的序列转化所述豆类作物物种的细胞来将所述CcRpp2-R1基因和CcRpp2-R3基因引入所述豆类作物物种中。
28.如权利要求27所述的方法,其中将转化的细胞再生为植物。
29.如权利要求27或28所述的方法,其中将所述细胞用表达构建体转化,所述表达构建体包含与SEQ ID NO:1具有至少95%序列同一性的第一多核苷酸和与SEQ ID NO:3具有至少95%序列同一性的第二多核苷酸,其中所述第一多核苷酸和所述第二多核苷酸与在植物细胞中可操作的异源调控序列可操作地连接。
30.如权利要求26所述的方法,其中通过将包含所述CcRpp2-R1基因的第一豆类作物植物与包含所述CcRpp2-R3基因的第二豆类作物植物杂交并选择表达所述CcRpp2-R1基因和所述CcRpp2-R3基因的子代来将所述CcRpp2-R1基因和所述CcRpp2-R3基因引入所述豆类作物物种中。
31.如权利要求26所述的方法,其进一步包括将编码具有SEQ ID NO:60的序列或与SEQID NO:60具有95%序列同一性的序列的多肽的CcRpp1基因引入包含重组CcRpp2-R1基因和重组CcRpp2-R3基因的豆类作物物种中的步骤。
32.如权利要求26-31中任一项所述的方法,其中所述豆类作物物种病害由选自由豆薯层锈菌(Phakopsora pachyrhizi)或山马蝗层锈菌(Phakopsora meibomiae)或它们的组合组成的组的植物病原体引起。
33.如权利要求26-32中任一项所述的方法,其中所述豆类作物物种病害是亚洲大豆锈病。
34.如权利要求26-33中任一项所述的方法,其中所述豆类作物物种是大豆。
35.一种防止对豆类作物物种的ASR相关损害的方法,所述方法包括用如权利要求23所述的种子种植田地。
36.如权利要求35所述的方法,其中将所述田地用杀真菌剂处理。
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