CN107354139B - 小麦als突变型蛋白、核酸及其应用 - Google Patents

小麦als突变型蛋白、核酸及其应用 Download PDF

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CN107354139B
CN107354139B CN201710842285.7A CN201710842285A CN107354139B CN 107354139 B CN107354139 B CN 107354139B CN 201710842285 A CN201710842285 A CN 201710842285A CN 107354139 B CN107354139 B CN 107354139B
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张保龙
王金彦
凌溪铁
陈天子
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Abstract

本发明公开了一种小麦ALS突变型蛋白，其在小麦A基因组6AL染色体的ALS基因、B基因组6BL染色体的ALS基因和小麦D基因组的6DL染色体的ALS基因序列发生突变。本发明还公开了编码该小麦ALS突变型蛋白的核酸。本发明还公开了上述小麦ALS突变型蛋白的应用。绿色植物表达该蛋白序列可以抗（耐）乙酰乳酸合成酶抑制剂类除草剂，特别是咪唑啉酮类除草剂。本发明的小麦ALS突变蛋白的小麦1叶1芯幼苗在施用1mL百垄通/L水（3倍推荐使用浓度）后，植株仍然正常生长发育和结实。

Description

小麦ALS突变型蛋白、核酸及其应用
技术领域
本发明属于植物蛋白和植物抗除草剂领域，涉及一种小麦ALS突变型蛋白及其核酸及其应用；具体而言，本发明涉及小麦的乙酰乳酸合成酶(ALS)突变蛋白，该蛋白能赋予植物尤其小麦抗乙酰乳酸合成酶抑制剂类除草剂的特性。本发明公开了该蛋白的序列，以及它们在植物抗除草剂领域中的应用。
背景技术
农田杂草是导致农作物减产的最主要原因之一。与传统依靠栽培措施、人工除草和机械除草等方法相比，化学除草剂的使用是人们公认的防除农田杂草最高效的、简便的和经济的治理方法。
植物和微生物支链氨基酸(缬氨酸、亮氨酸和异亮氨酸)的生物合成需要4种酶共同催化作用，乙酰乳酸合成酶(acetolactate synthase，ALS)、酮醇还原异构酶(ketol-acid reductoisomerase)、二羟基酸脱水酶(dihydroxyavid dehydratase)、支链氨基酸转氨酶(branched-chain amino acidtransaminase)。乙酰乳酸合成酶是生物合成过程中第一阶段的关键酶，在缬氨酸和亮氨酸的合成中催化2分子丙酮酸生成乙酰乳酸和二氧化碳，在异亮氨酸的合成中催化1分子丙酮酸与1分子α丁酮酸生成2-乙醛基-2-羟基丁酸和二氧化碳。ALS抑制剂类除草剂通过抑制植物体内的ALS酶活性，从而阻止支链氨基酸的合成，导致蛋白质的合成受到破坏，阻碍细胞分裂期的DNA合成，从而使植物细胞的有丝分裂停止在Gl阶段的S期(DNA合成期)和G2阶段的M期，干扰了DNA的合成，细胞因此不能完成有丝分裂，进而使植物停止生长，最终导致植物个体死亡。
乙酰乳酸合成酶(ALS)(也称乙酰羟酸合成酶，AHAS；EC 4.1.3.18)抑制剂类除草剂以ALS作为靶标而导致杂草死亡，主要包括磺酰脲类(Sulfonylureas,SU)、咪唑啉酮类(Imidazolinones,IMI)、三唑嘧啶类(Triazolopyrimidines,TP)、嘧啶氧(硫)苯甲酸类[Pyrimidinylthio(or oxy)–benzoates,PTB；pyrimidinyl-carboxyherbicides；PCs]和磺酰胺基羰基三唑啉酮类(Sulfonylamino-carbonyltriazolinones,SCT)等13类化合物。乙酰乳酸合成酶，存在于植物生长过程中，它能以高度专一性和极高的催化效率催化丙酮酸为乙酰乳酸，从而导致支链氨基酸的生物合成。
成熟的ALS蛋白大约由670个氨基酸组成，其序列在不同物种间高度保守。ALS蛋白在Gly 95、Ala 96、Ala 122、Pro 171、Pro 196、Pro 197、Ala 205、Asp 376、Trp 537、Trp548、Trp 552、Trp 557、Trp 563、Trp 574、Ser 621、Ser 627、Ser 638、Ser 653、Gly 654、Val 669等氨基酸位点(以模式植物拟南芥的ALS氨基酸位置计算)发生突变可以产生ALS抑制剂类除草剂抗性，这在多种农作物(包括玉米、小麦、小麦、油菜、向日葵等)、模式植物拟南芥和上百种杂草中均有报道。
生产上广泛种植的普通小麦是一种异源六倍体，含有A、B和D三个基因组。小麦已知的抗ALS抑制剂突变位点包括Ala 179、Ser 627(授权专利CN102559646B)。基因序列比对分析发现，该已知的Ala 179、Ser 627突变位于小麦D基因组的6DL染色体上。ALS突变体抗除草剂水平与ALS氨基酸突变的位置有关，还与突变后的氨基酸种类及突变氨基酸的数目有关。
目前，ALS抑制剂类除草剂的作用机理尚未确定，很难提前预测ALS蛋白其它氨基酸位点的突变是否会产生除草剂抗性，只能依赖于科研人员长期、艰苦的实践探索，并凭借一些运气才可能发现ALS蛋白新的除草剂抗性位点。
发明内容
发明目的：本发明所要解决的技术问题是提供了小麦ALS突变型蛋白。该小麦来源于徐麦33号。该徐麦33号的突变基因的核苷酸序列或者氨基酸序列在植物中表达时能赋予植物增加的对咪挫啉酮类除草剂的耐受性。
本发明还要解决的技术问题是提供了编码该蛋白的核酸以及表达盒、载体和细胞等。
本发明还要解决的技术问题是提供了获得具有除草剂抗性的植物的方法和应用。
本发明还要解决的技术问题是提供了判断植物是否采用本发明方法获得的鉴定方法。
技术方案：为解决上述技术问题，本发明采用的技术方案如下：小麦ALS突变型蛋白，其在小麦A基因组ALS基因上的第353位氨基酸由天冬氨酸突变为谷氨酸，和/或在小麦A基因组ALS基因上的第551位氨基酸由色氨酸突变为半胱氨酸，和/或在小麦A基因组ALS基因上的第631位氨基酸由甘氨酸变为天冬氨酸，和/或在小麦B基因组ALS基因上的第352位氨基酸由天冬氨酸突变为谷氨酸，和/或在小麦B基因组ALS基因上的第550位氨基酸由色氨酸变为半胱氨酸，和/或在小麦B基因组ALS基因上的第630位氨基酸由甘氨酸变为天冬氨酸，和/或在小麦D基因组ALS基因上的第350位氨基酸由天冬氨酸突变为谷氨酸，和/或在小麦D基因组ALS基因上的第548位氨基酸由色氨酸变为半胱氨酸，和/或在小麦D基因组ALS基因上的第628位氨基酸由甘氨酸变为天冬氨酸。
本发明的徐麦33号的A基因组ALS基因的野生型氨基酸序列参见SEQ ID No:2所示，徐麦33号的B基因组ALS基因的野生型氨基酸序列参见SEQ ID No:4所示，徐麦33号的D基因组ALS基因的野生型氨基酸序列参见SEQ ID No:6所示。
本发明的突变型蛋白能增强植物、植物组织、植物细胞对于至少一种除草剂的耐受性，所述除草剂干扰ALS酶的活性。本发明的突变型蛋白使植物具有除草剂抗性，该除草剂为咪唑啉酮类除草剂。
本发明所述的增强植物、植物组织或者植物细胞对除草剂的抗性方法可以通过转化或者杂交、自交和无性繁殖的方法实施，也可通过化学诱变或基因定点突变的方式获得，是改变的植株具有本发明的核苷酸序列。
本发明内容还包括核酸，其编码所述的突变型蛋白。本发明的核酸，其小麦A基因组ALS基因上的核苷酸序列在第1059位由T突变为A，和/或小麦A基因组ALS基因上的核苷酸序列在第1653位由G突变为T，和/或小麦A基因组ALS基因上的核苷酸序列在第1892位由G突变为A，和/或小麦B基因组ALS基因上的核苷酸序列在第1056位由T突变为A，和/或小麦B基因组ALS基因上的核苷酸序列在第1650位由G突变为T，和/或小麦B基因组ALS基因上的核苷酸序列在第1889位由G突变为A；和/或小麦D基因组ALS基因上的核苷酸序列在第1050位由T突变为A，和/或小麦D基因组ALS基因上的核苷酸序列在第1644位由G突变为T，和/或小麦D基因组ALS基因上的核苷酸序列在第1883位由G突变为A。
本发明的徐麦33号的A基因组ALS基因的野生型核苷酸序列参见SEQ ID No:1所示，徐麦33号的A基因组ALS基因野生型氨基酸序列如SEQ ID No:2所示，徐麦33号的B基因组ALS基因的野生型核苷酸序列参见SEQ ID No:3所示，徐麦33号的B基因组ALS基因的野生型氨基酸序列如SEQ ID No:4所示，徐麦33号的D基因组ALS基因的野生型核苷酸序列参见SEQ ID No:5所示，徐麦33号的D基因组ALS基因的野生型氨基酸序列如SEQ ID No:6所示。
本发明内容还包括表达盒、重组载体或细胞，其含有所述的核酸。
本发明内容还包括所述的蛋白、核酸、表达盒、重组载体或细胞在植物抗除草剂方面的应用。
其中，上述植物为小麦。
本发明内容还包括获得具有除草剂抗性的植物的方法，包括如下步骤：
1)使植物包含本发明所述的核酸；或
2)使植物表达所述的小麦ALS突变型蛋白。
本发明内容还包括鉴定植物的方法，其中植物是包含所述的核酸的植物、表达所述的蛋白的植物或所述的方法获得的植物，包括以下步骤：
1)测定所述植物是否包含所述的核酸；或，
2)测定所述植物是否表达所述的蛋白。
本发明内容还包括一种除草剂的防护剂，该防护剂由所述的小麦ALS突变型蛋白制成。
本发明人通过长期、艰苦的研究，对野生型小麦品种徐麦33进行EMS诱变处理，对这些的EMS突变植株(野生型徐麦33号为市场售小麦品种)进行长期、不断地筛选，发现了一系列ALS突变蛋白，包括前文所述的某些已知ALS突变蛋白和新ALS突变蛋白，其对ALS抑制剂类除草剂不敏感，从而使得植物具有ALS抑制剂类除草剂抗性。本发明在植物育种中的应用，可用于培育具有除草剂抗性的植物，尤其是农作物，还开发了这些蛋白及其编码基因在转基因或者非转基因如小麦等作物中的应用。
有益效果：田间喷施ALS抑制剂类除草剂“百垄通”的实验结果表明，含有本发明的小麦ALS突变蛋白的小麦1叶1芯幼苗在施用1mL百垄通/L水(3倍推荐使用浓度)后，植株仍然正常生长发育和结实，而野生型小麦幼苗在施用1mL百垄通/3L水(3倍推荐使用浓度)后，植株生长逐渐停止，叶片逐渐失绿、变浅黄色，植株不能拔节，最终整株死亡。
附图说明
图1百垄通除草剂筛选获得的抗性小麦突变体6AL-631；
图2百垄通除草剂筛选获得的抗性小麦突变体6BL-630；
图3百垄通除草剂筛选获得的抗性小麦突变体6DL-628；
图4PCR扩增9个小麦抗除草剂株系的ALS基因全长结果图；第1泳道为Marker；Marker分子量从上到下依次为5kb、3kb、2kb、1kb、750bp、500bp，第2泳道为抗除草剂突变植株(6AL-631)的DNA，第3泳道为抗除草剂突变植株(6AL-353)的DNA，第4泳道为抗除草剂突变植株(6AL-551)的DNA，第5泳道为抗除草剂突变植株(6BL-352)的DNA，第6泳道为抗除草剂突变植株(6BL-550)的DNA，第7泳道为抗除草剂突变植株(6BL-630)的DNA，第8泳道为抗除草剂突变植株(6DL-350)的DNA，第9泳道为抗除草剂突变植株(6DL-548)的DNA，第10泳道为抗除草剂突变植株(6DL-628)的DNA。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：小麦抗咪唑啉酮类除草剂突变体获取过程(百垄通)
将徐麦33号的种子(市场售小麦品种)(此为M0，用清水浸泡2小时)120kg分6次用0.4-0.6％(w/w)甲磺酸乙酯(EMS)室温下浸泡6-9小时，期间每1小时摇动一次种子；弃去EMS溶液，自来水翻动浸泡种子5次，每次5分钟，然后用自来水冲洗种子过夜，次日进行田间播种，并进行常规肥水管理(此为M1)。植株成熟后，种子混收、晾干，过冬保存。次年播种田间。待小麦(此为M2)幼苗长至1叶1芯期时，喷施为1mL百垄通/L水(“百垄通”是德国巴斯夫公司生产一种水剂型咪唑啉酮类除草剂，推荐最低使用浓度为1mL百垄通/1.5～3L水)，3个月后还呈正常绿色、且能正常拔节的植株即为抗咪唑啉酮类除草剂的小麦突变植株(图1)。共计获得抗除草剂的M2单株186株，这些抗性单株进行常规肥水管理，均可正常结实，种子成熟后，单株收种、晾干，过冬保存。我们对上述获得的186株M2进行单株收获，并进行种植M3种子。
实施例2：抗除草剂小麦突变体突变位点分析
小麦基因组中的ALS基因有三个拷贝，分别位于小麦A、B和D基因组中。上述实施例1获得的抗除草剂小麦突变植株中，选取突变植株的叶片和野生型植株的叶片，分别提取基因组DNA，并用对小麦ALS基因进行扩增并测序。测序结果发现：与野生型植株相比，上述抗除草剂的小麦突变体在3个基因组ALS基因上各有3个位点发生突变，分别命名为6AL-353突变体、6AL-551突变体、6AL-631突变体、6BL-352突变体、6BL-550突变体、6BL-630突变体、6DL-350突变体、6DL-548突变体、6DL-628突变体。其中，6AL-353突变体的小麦A基因组ALS基因核苷酸序列在SEQ ID NO:1的第1059位由T突变为A，导致其编码的氨基酸序列在SEQID NO:2的第353位氨基酸由天冬氨酸突变为谷氨酸；6AL-551突变体的小麦A基因组ALS基因核苷酸序列在SEQ ID NO:1的第1653位由G突变为T，导致其编码的氨基酸序列在SEQ IDNO:2的第551位氨基酸由色氨酸变为半胱氨酸；6AL-631突变体的小麦A基因组ALS基因核苷酸序列在SEQ ID NO:1的第1892位由G突变为A，导致其编码的氨基酸序列在SEQ ID NO:2的第631位氨基酸由甘氨酸变为天冬氨酸；6BL-352突变体的小麦B基因组ALS基因核苷酸序列在SEQ ID NO:3的第1056位由T突变为A，导致其编码的氨基酸序列在SEQ ID NO:4的第352位氨基酸由天冬氨酸突变为谷氨酸；6BL-550突变体的小麦B基因组ALS核苷酸序列在SEQID NO:3的第1650位由G突变为T，导致其编码的氨基酸序列在SEQ ID NO:4的第550位氨基酸由色氨酸变为半胱氨酸；6BL-630突变体的小麦B基因组ALS核苷酸序列在SEQ ID NO:3的第1889位由G突变为A，导致其编码的氨基酸序列在SEQ ID NO:4的第630氨基酸由甘氨酸变为天冬氨酸；6DL-350突变体的小麦B基因组ALS核苷酸序列在SEQ ID NO:3的第1050位由T突变为A，导致其编码的氨基酸序列在SEQ ID NO:4的第350位氨基酸由天冬氨酸突变为谷氨酸；6DL-548突变体的小麦D基因组ALS核苷酸序列第1644位由G突变为T，导致其编码的氨基酸序列在SEQ ID NO:4的第548位氨基酸由色氨酸变为半胱氨酸；6DL-628突变体的小麦D基因组ALS核苷酸序列第1883位由G突变为A，导致其编码的氨基酸序列在SEQ ID NO:4的第628位氨基酸由甘氨酸变为天冬氨酸。
实施例3抗咪唑啉酮类除草剂小麦突变体ALS基因克隆
分别取上述抗除草剂的6AL-353突变体、6AL-551突变体、6AL-631突变体、6BL-352突变体、6BL-550突变体、6BL-630突变体、6DL-350突变体、6DL-548突变体、6DL-628突变体的叶片，分别提取基因组DNA。根据测序序列设计扩增ALS基因全长基因的特异引物为：正向引物5’-CCCATCTGAACCACACGCTCACC-3’、反向引物5’-CTGATTGCGCATGTCACACTTG-3’。采用Takara PrimerSTAR Max DNA Polymerase聚合酶(10U/ul)(购自Takara公司)扩增ALS基因全长序列，其反应体系如下：
Figure BDA0001411053470000071
PCR扩增反应程序采用两步法，退火和延伸合为一起，采用68℃。
程序如下：预变性：98℃3min；35个循环；变性98℃10sec；延伸68℃3min；保温：72℃10min。
取2μlPCR产物经1％琼脂糖凝胶电泳检测，发现有预期大小的片段后(图4)，将上述的剩余的PCR产物经PCR清洁试剂盒(购自Axygen公司)清洁回收后，克隆至pMD19-T载体(购自Takara公司)，然后转化大肠杆菌。每个转化随机挑取36个大肠杆菌单克隆进行PCR检测，取PCR结果呈阳性的至少30个单克隆，送金斯瑞生物科技有限公司测序，获得突变ALS基因序列。
实施例4抗咪唑啉酮类除草剂小麦突变体后代咪挫啉酮类除草剂的抗性
将鉴定为纯合突变体的株系后代单株收种，并播种，待小麦长至三叶期时，喷施为1mL百垄通/L水(推荐浓度3倍)，喷施一个月后调查枯死情况，发现野生型对照在喷施百垄通后几乎停止生长，新长出来的叶片完全枯黄，并逐渐枯死。而我们筛选到的不同位点的抗除草剂阳性苗则全部对百垄通表现出抗的表型，其正常的生长发育几乎没有受到影响，最终正常拔节抽穗。这表明我们筛选到的抗除草剂突变体确实能够稳定遗传的获得抗咪挫啉酮类除草剂的抗性。
实施例5抗百垄通除草剂小麦突变体的ALS酶活测定
为了验证小麦突变体的除草剂抗性是否由ALS突变所致，本发明人进行了ALS酶活性测定。测定方法参照Singh等的方法(Singh B.K.,Stidham M.A.,Shaner D.L.Assay ofacetohydroxyacid synthase.Analytical Biochemistry,1988,171:173-179.)。具体的，分别取野生型小麦和突变体小麦的叶片0.2g，在研钵中用液氮研磨粉碎，加入2mL提取液(100mM K2HPO4，pH 7.5、10mM丙酮酸钠、5mM EDTA、1mM缬氨酸、1mM亮氨酸、10mM半胱氨酸、0.1mM黄素腺嘌呤二核苷酸、5mM氯化镁、10％(V/V)甘油、1％(w/v)聚乙烯吡咯烷酮)，待提取液解冻后继续研磨1min左右。12000rpm、4℃离心30min，吸取上清液，加入硫酸铵使之达到50％饱和度，于冰上放置半小时，12000rpm、4℃离心30min，弃上清，将沉淀溶解于0.2mL反应缓冲液(100mM K2HPO4,pH 7.0、1mM EDTA、10mM氯化镁、100mM丙酮酸钠、1mM焦磷酸硫胺素、0.1mM黄素腺嘌呤二核苷酸)，分别得各植株的ALS提取液。
在获得ALS提取液中分别加入10μL除草剂“百垄通”(水剂，有效成分240g/L)，混匀，37℃孵育1h，加0.1ml 3M硫酸终止反应，反应混合物在60℃反应孵育30分钟便于脱羧。然后加0.4mL显色液(0.09g/L 1-萘酚和0.009g/L肌酸，用2.5M NaOH溶解)。混合液在37℃孵育30分钟进行显色(ALS蛋白催化丙酮酸形成乙酰乳酸，乙酰乳酸脱羧形成3-羟基丁酮，再与肌酸和1-萘酚形成粉红色复合物，该复合物在530nm处有最大吸收值)，随后测定其530nm的吸光度，ALS蛋白活性用A530吸光值表示，A530吸光值的高低反映ALS蛋白活性的高低。实验以水为对照，设3次重复。
A530吸光值测定结果发现，当野生型和小麦突变体的ALS提取液中没有百垄通时，它们的A530吸光值均在1.3-1.5之间，表明野生型和突变体的ALS酶活性无显著性差异(表1)；而加入百垄通后，野生型植株的A530吸光值仅为0.21，突变体小麦植株的A530吸光值为0.98-1.12之间，即野生型植株的ALS酶活性仅为对照的16％，而突变体小麦植株的ALS酶活性66.2％-82.61％之间(表1)，表明突变体小麦的ALS基因突变后对百垄通不敏感，从而赋予了抗性。
表1
Figure BDA0001411053470000081
Figure BDA0001411053470000091
实施例6抗性材料的两两杂交
为了检测是否具有双重或双重以上突变位点的抗性植株会比具有单位点抗性植株的除草剂耐受能力更强，我们将实施例1和2中获得的具有不同突变位点的抗性植株进行两两杂交。选6AL-631突变体的株系命名为M121、6BL-630突变体的株系命名为M082和6DL-628突变体的株系命名为M167，将这三个株系在温室中种植，开花期将M121与M082、M082和M167、M121和M167进行杂交。具体方法是：将母本人工去雄并套袋，父本植株开花的时候，将父本花药中的花粉抖出，授粉到母本柱头上，重复两次，带种子成熟后收种，为F1带杂交种。收获的F1代杂交种繁殖一代，收获F2代群体，M121与M082杂交F2代群体中鉴定带有纯合的6AL-631突变体的突变和6BL-630突变体的突变的植株；M082和M167杂交的F2群体中鉴定带有纯合6BL-630突变体的突变和6DL-628突变体突变的植株；M121与M167杂交的F2群体中鉴定带有纯合6AL-631突变体的突变和6DL-628突变体突变的植株。用百垄通对这些含有双位点突变的纯合植株进行喷施实验，结果表明，同时突变两个位点的植株相对于只有一个突变位点的植株，其ALS的酶活性要高出15％(表2)。
表2
Figure BDA0001411053470000092
Figure BDA0001411053470000101
尽管本发明的具体实施方式已经得到详细描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些均在本发明的保护范围内。本发明的全部范围由所附专利要求极其任何等同物给出。
序列表
<110> 江苏省农业科学院
<120> 小麦ALS突变型蛋白、核酸及其应用
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Ala Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro
290 295 300
Val Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro
305 310 315 320
Leu Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr
325 330 335
Ala Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp
340 345 350
Asp Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile
355 360 365
Val His Ile Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro
370 375 380
His Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn
385 390 395 400
Ala Leu Leu Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro
405 410 415
Trp His Lys Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe
420 425 430
Lys Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu
435 440 445
Asp Glu Leu Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln
450 455 460
His Gln Met Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln
465 470 475 480
Trp Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala
485 490 495
Ala Ala Gly Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile
500 505 510
Asp Gly Asp Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile
515 520 525
Arg Ile Glu Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His
530 535 540
Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg
545 550 555 560
Ala His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro
565 570 575
Asp Phe Val Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val
580 585 590
Thr Lys Lys Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr
595 600 605
Pro Gly Pro Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val
610 615 620
Leu Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu
625 630 635 640
Gly Asp Gly Arg Thr Ser Tyr
645
<210> 3
<211> 1938
<212> DNA
<213> 徐麦33号的B基因组的野生型序列(Xu mai 33)
<220>
<221> CDS
<222> (1)..(1938)
<400> 3
atg gcc gca gcc acc tcc ccc gcc gtc gca ttc tcg ggc gcc gcc gcc 48
Met Ala Ala Ala Thr Ser Pro Ala Val Ala Phe Ser Gly Ala Ala Ala
1 5 10 15
gcc gcc gcc gcc ata ccc aaa ccc gcc cgc cag cct ctc ccg cgc cac 96
Ala Ala Ala Ala Ile Pro Lys Pro Ala Arg Gln Pro Leu Pro Arg His
20 25 30
cag ccc gcc tcg cgc cgc gcg ctc ccc gcc cgc atc gtc agg tgc tgc 144
Gln Pro Ala Ser Arg Arg Ala Leu Pro Ala Arg Ile Val Arg Cys Cys
35 40 45
gcc gcg tcc ccc gcc gcc acc tcc gtc gcg cct ccc gcc acc gcg ctc 192
Ala Ala Ser Pro Ala Ala Thr Ser Val Ala Pro Pro Ala Thr Ala Leu
50 55 60
cgg ccg tgg ggc ccc tcc gag ccc cgc aag ggc gcc gac atc ctc gtc 240
Arg Pro Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val
65 70 75 80
gag gcg ctg gag cgc tgc ggc atc gtc gac gtc ttc gcc tac cct ggc 288
Glu Ala Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly
85 90 95
ggc gcg tcc atg gag atc cac cag gcg ctg acg cgc tcg cca gtc atc 336
Gly Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile
100 105 110
acc aac cac ctc ttc cgc cac gag cag ggg gag gcg ttc gcg gcg tcc 384
Thr Asn His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser
115 120 125
ggg tac gcc cgc gcg tcc ggc cgc gtc ggc gtc tgc gtc gcc acc tcc 432
Gly Tyr Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser
130 135 140
ggc ccg ggg gcc acc aac ctc gtc tcc gcg ctc gcc gac gct ctc ctc 480
Gly Pro Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu
145 150 155 160
gac tcc atc ccc atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg 528
Asp Ser Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met
165 170 175
atc ggc acg gat gcg ttc cag gag acg ccc atc gtg gag gtc acg cgc 576
Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg
180 185 190
tcc atc acc aag cac aac tac ctg gtc ctt gac gtg gag gat atc ccc 624
Ser Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro
195 200 205
cgc gtc atc cag gaa gcc ttc ttc ctc gca tcc tct ggc cgc ccg ggg 672
Arg Val Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly
210 215 220
ccg gtg ctg gtt gat atc ccc aag gac atc cag cag cag atg gct gtg 720
Pro Val Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val
225 230 235 240
cct gtc tgg gac acg ccg atg agt ttg cca ggg tac atc gcc cgc ctg 768
Pro Val Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu
245 250 255
ccc aag cca cca tct act gaa tcg ctt gag cag gtc ctg cgt ctg gtt 816
Pro Lys Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val
260 265 270
ggc gag tca cgg cgc cca att ctg tat gtt ggt ggt ggc tgc gct gca 864
Gly Glu Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala
275 280 285
tct ggt gag gag ttg cgc cgc ttt gtt gag ctc act ggg att cca gtt 912
Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val
290 295 300
aca act act ctt atg ggc ctt ggc aac ttc ccc agt gac gac cca ctg 960
Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu
305 310 315 320
tct ctg cgc atg ctg ggg atg cat ggc act gtg tat gca aat tat gca 1008
Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala
325 330 335
gta gat aag gct gac ctg ttg ctt gca ttt ggt gtg cgg ttt gat gat 1056
Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp
340 345 350
cgt gtg acc ggg aaa atc gag gct ttt gca agc agg tcc aag att gtg 1104
Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val
355 360 365
cac att gac att gac cca gct gag att ggc aag aac aag cag cca cat 1152
His Ile Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His
370 375 380
gtc tcc att tgt gca gat gtt aag ctt gct tta cag ggg ttg aat gct 1200
Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala
385 390 395 400
cta tta aat ggg agc aaa gca caa cag ggt ctg gat ttt ggt cca tgg 1248
Leu Leu Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp
405 410 415
cac aag gag ttg gat cag cag aag agg gag ttt cct cta gga ttc aag 1296
His Lys Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys
420 425 430
act ttt ggt gag gcc atc ccg ccg caa tat gct atc cag gta ctg gat 1344
Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp
435 440 445
gag ctg aca aaa ggg gag gcg atc att gcc acc ggt gtt ggg cag cat 1392
Glu Leu Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His
450 455 460
cag atg tgg gcg gct cag tat tac act tac aag cgg cca cgg cag tgg 1440
Gln Met Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp
465 470 475 480
ctg tct tca tcc ggt ttg ggt gca atg gga ttt ggg ttg cca gct gca 1488
Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala
485 490 495
gct ggc gct gct gtg gcc aac cca ggt gtt aca gtt gtt gac att gat 1536
Ala Gly Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp
500 505 510
ggg gat ggt agt ttc ctc atg aac att cag gag ttg gcg ttg atc cgt 1584
Gly Asp Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg
515 520 525
att gag aac ctc cca gtg aag gtg atg ata ttg aac aac cag cat ctg 1632
Ile Glu Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu
530 535 540
gga atg gtg gtg cag tgg gag gat agg ttt tac aag gcc aac cgg gcg 1680
Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala
545 550 555 560
cac aca tac ctt ggc aac cca gaa aat gag agt gag ata tat cca gat 1728
His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp
565 570 575
ttt gtg acg att gct aaa gga ttc aac gtt ccg gca gtt cgt gtg acg 1776
Phe Val Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr
580 585 590
aag aag agc gaa gtc act gca gca atc aag aag atg ctt gag acc cca 1824
Lys Lys Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro
595 600 605
ggg cca tac ttg ttg gat atc att gtc ccg cat cag gag cac gtg ctg 1872
Gly Pro Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu
610 615 620
cct atg atc cca agc ggt ggt gct ttt aag gac atg atc atg gag ggt 1920
Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly
625 630 635 640
gat ggc agg acc tcg tac 1938
Asp Gly Arg Thr Ser Tyr
645
<210> 4
<211> 646
<212> PRT
<213> 徐麦33号的B基因组的野生型序列(Xu mai 33)
<400> 4
Met Ala Ala Ala Thr Ser Pro Ala Val Ala Phe Ser Gly Ala Ala Ala
1 5 10 15
Ala Ala Ala Ala Ile Pro Lys Pro Ala Arg Gln Pro Leu Pro Arg His
20 25 30
Gln Pro Ala Ser Arg Arg Ala Leu Pro Ala Arg Ile Val Arg Cys Cys
35 40 45
Ala Ala Ser Pro Ala Ala Thr Ser Val Ala Pro Pro Ala Thr Ala Leu
50 55 60
Arg Pro Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val
65 70 75 80
Glu Ala Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly
85 90 95
Gly Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile
100 105 110
Thr Asn His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser
115 120 125
Gly Tyr Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser
130 135 140
Gly Pro Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu
145 150 155 160
Asp Ser Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met
165 170 175
Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg
180 185 190
Ser Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro
195 200 205
Arg Val Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly
210 215 220
Pro Val Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val
225 230 235 240
Pro Val Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu
245 250 255
Pro Lys Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val
260 265 270
Gly Glu Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala
275 280 285
Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val
290 295 300
Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu
305 310 315 320
Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala
325 330 335
Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp
340 345 350
Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val
355 360 365
His Ile Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His
370 375 380
Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala
385 390 395 400
Leu Leu Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp
405 410 415
His Lys Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys
420 425 430
Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp
435 440 445
Glu Leu Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His
450 455 460
Gln Met Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp
465 470 475 480
Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala
485 490 495
Ala Gly Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp
500 505 510
Gly Asp Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg
515 520 525
Ile Glu Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu
530 535 540
Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala
545 550 555 560
His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp
565 570 575
Phe Val Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr
580 585 590
Lys Lys Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro
595 600 605
Gly Pro Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu
610 615 620
Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly
625 630 635 640
Asp Gly Arg Thr Ser Tyr
645
<210> 5
<211> 1935
<212> DNA
<213> 徐麦33号的D基因组的野生型序列(Xu mai 33)
<220>
<221> CDS
<222> (1)..(1935)
<400> 5
atg gcc gcc gcc acc tcc ccc gcc gtc gca ttc tcg ggc gcc acc gcc 48
Met Ala Ala Ala Thr Ser Pro Ala Val Ala Phe Ser Gly Ala Thr Ala
1 5 10 15
gcc gcc atg ccc aaa ccc gcc cgc cat cct ctc ccg cgc cac cag ccc 96
Ala Ala Met Pro Lys Pro Ala Arg His Pro Leu Pro Arg His Gln Pro
20 25 30
gtc tcg cgc cgc gcg ctc ccc gcc cgc gtc gtc agg tgt tgc gcc gcg 144
Val Ser Arg Arg Ala Leu Pro Ala Arg Val Val Arg Cys Cys Ala Ala
35 40 45
tcc ccc gcc gcc acc tcc gcc gcg cct ccc gca acc gcg ctc cgg ccc 192
Ser Pro Ala Ala Thr Ser Ala Ala Pro Pro Ala Thr Ala Leu Arg Pro
50 55 60
tgg ggc ccg tcc gag ccc cgc aag ggc gcc gac atc ctc gtc gag gcg 240
Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
ctc gag cgc tgc ggc atc gtc gac gtc ttc gcc tac ccc ggc ggc gcc 288
Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcg ccc gtc atc acc aac 336
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
cac ctc ttc cgc cac gag cag ggg gag gcg ttc gcg gcg tcc ggc tac 384
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
gcc cgc gcg tcc ggc cgc gtc ggc gtc tgc gtc gcc acc tcc ggc ccg 432
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
ggg gcc acc aac ctc gtc tcc gcg ctc gcc gac gcc ctc ctc gac tcc 480
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
atc ccc atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg atc ggc 528
Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
acg gac gcg ttc cag gag acg ccc ata gtg gag gtc acg cgc tcc atc 576
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
acc aag cac aac tac ctg gtc ctt gac gtg gag gat atc ccc cgc gtc 624
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
atc cag gaa gcc ttc ttc ctt gca tcc tct ggc cgc ccg ggg ccg gtg 672
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
cta gtt gat atc ccc aag gac atc cag cag cag atg gct gtg ccc gtc 720
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
tgg gac act cca atg agt ttg cca ggg tac atc gcc cgc ctg ccc aag 768
Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
cca cca tct act gaa tcg ctt gag cag gtc ctg cgt ctg gtt ggc gag 816
Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
tca cgg cgc cca att ctg tat gtt ggt ggt ggc tgc gct gcg tct ggc 864
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala Ser Gly
275 280 285
gag gag ttg cgc cgc ttt gtt gag ctt act ggg att cca gtt aca act 912
Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
act ctg atg ggc ctt ggc aac ttc ccc agc gac gac cca ctg tct ctg 960
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
cgc atg ctt ggg atg cat ggc act gtg tat gca aat tat gca gta gat 1008
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
aag gct gac ctg ttg ctc gca ttt ggt gtg cgg ttt gat gat cgt gtg 1056
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
act ggg aaa atc gag gct ttt gca agc agg tcc aag att gtg cac att 1104
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val His Ile
355 360 365
gac att gac cca gct gag att ggc aag aac aag cag cca cat gtc tcc 1152
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
att tgt gca gat gtt aag ctt gct tta cag ggg ttg aat gat cta tta 1200
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Asp Leu Leu
385 390 395 400
aat ggg agc aaa gca caa cag ggt ctg gat ttt ggt cca tgg cac aag 1248
Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp His Lys
405 410 415
gag ttg gat cag cag aag agg gag ttt cct cta gga ttc aag act ttt 1296
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys Thr Phe
420 425 430
ggc gag gcc atc ccg ccg caa tat gct atc cag gta ctg gat gag ctg 1344
Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
aca aaa ggg gag gcg atc att gcc act ggt gtt ggg cag cac cag atg 1392
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
tgg gcg gct cag tat tac act tac aag cgg cca cgg cag tgg ctg tct 1440
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
tcg tct ggt ttg ggg gca atg gga ttt ggg tta cca gct gca gct ggc 1488
Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
gct gct gtg gcc aac cca ggt gtt aca gtt gtt gac att gat ggt gat 1536
Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
ggt agt ttc ctc atg aac att cag gag ttg gcg ttg atc cgc att gag 1584
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
aac ctc cca gtg aag gtg atg ata ttg aac aac cag cat ctg gga atg 1632
Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu Gly Met
530 535 540
gtg gtg cag tgg gag gat agg ttt tac aag gcc aat cgg gcg cac aca 1680
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
tac ctt ggc aac cca gaa aat gag agt gag ata tat cca gat ttt gtg 1728
Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
acg att gct aaa gga ttc aac gtt cca gca gtt cga gtg acg aag aag 1776
Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr Lys Lys
580 585 590
agc gaa gtc act gca gca atc aag aag atg ctt gag acc cca ggg cca 1824
Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
tac ttg ttg gat atc ata gtc ccg cat cag gag cac gtg ctg cct atg 1872
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
atc cca agc ggt ggt gct ttc aag gac atg atc atg gag ggt gat ggc 1920
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly Asp Gly
625 630 635 640
agg acc tcg tac tga 1935
Arg Thr Ser Tyr
<210> 6
<211> 644
<212> PRT
<213> 徐麦33号的D基因组的野生型序列(Xu mai 33)
<400> 6
Met Ala Ala Ala Thr Ser Pro Ala Val Ala Phe Ser Gly Ala Thr Ala
1 5 10 15
Ala Ala Met Pro Lys Pro Ala Arg His Pro Leu Pro Arg His Gln Pro
20 25 30
Val Ser Arg Arg Ala Leu Pro Ala Arg Val Val Arg Cys Cys Ala Ala
35 40 45
Ser Pro Ala Ala Thr Ser Ala Ala Pro Pro Ala Thr Ala Leu Arg Pro
50 55 60
Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala Ser Gly
275 280 285
Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Asp Leu Leu
385 390 395 400
Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp His Lys
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys Thr Phe
420 425 430
Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly Asp Gly
625 630 635 640
Arg Thr Ser Tyr
<210> 7
<211> 30
<212> DNA
<213> ALS正向引物(人工序列)
<400> 7
cgcggatccc catcacccct ccccaattcc 30
<210> 8
<211> 30
<212> DNA
<213> ALS反向引物(人工序列)
<400> 8
tccccgcggc acttgtaggt cttgtaggtc 30

Claims (11)

1.小麦ALS突变型蛋白，其在小麦A基因组ALS基因的第631位氨基酸由甘氨酸变为天冬氨酸。
2.根据权利要求1所述的突变型蛋白，其还包括在小麦B基因组ALS基因的第630位氨基酸由甘氨酸变为天冬氨酸或其在小麦D基因组ALS基因的第628位氨基酸由甘氨酸变为天冬氨酸。
3.根据权利要求1所述的小麦ALS突变型蛋白，其特征在于，该突变型蛋白使小麦具有除草剂抗性，该除草剂为咪唑啉酮类除草剂。
4.核酸，其编码权利要求1~3任一项所述的突变型蛋白。
5.根据权利要求4所述的核酸，其小麦A基因组ALS基因的核苷酸序列在第1892位由G突变为A。
6.根据权利要求5所述的核酸，其还包括在小麦B基因组ALS基因的核苷酸序列在第1889位由G突变为A或在小麦D基因组ALS基因的核苷酸序列在第1883位由G突变为A。
7.表达盒、重组载体或细胞，其含有权利要求4或5或6所述的核酸。
8.权利要求1~3任一项所述的蛋白，权利要求4或5或6所述的核酸，权利要求7所述的表达盒、重组载体或细胞在小麦抗除草剂方面的应用。
9.获得具有除草剂抗性的植物的方法，其特征在于，包括如下步骤：
1）使植物包含权利要求4或5或6所述的核酸；或
2）使植物表达权利要求1~3之任一所述的小麦ALS突变型蛋白，所述植物为小麦。
10.鉴定植物的方法，其中植物是包含权利要求4或5所述的核酸的植物、表达权利要求1~2之任一所述的蛋白的植物或权利要求9所述的方法获得的植物，其特征在于，包括以下步骤：
1) 测定所述植物是否包含权利要求4或5所述的核酸；或，
2) 测定所述植物是否表达权利要求1~3之任一所述的蛋白，所述植物为小麦。
11.一种除草剂的防护剂，其特征在于，该防护剂由权利要求1~3之任一所述的小麦ALS突变型蛋白制成。
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