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申请/专利权人:山东农业大学;四川农业大学
摘要:本发明公开了一种粗山羊草Yr4DS基因及其在麦族植物抗条锈病育种中的应用,本发明首次从粗山羊草中克隆得到了一个新型小麦抗条锈病基因Yr4DS,所述基因的序列如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10所示。该基因能够有效的调控小麦抗条锈病的能力,丰富了小麦抗条锈病基因资源。本发明发现Yr4DS的表达提供高水平抗条锈病的功能,预示着人为操作Yr4DS基因可以赋予小麦等麦族植物抗条锈病的特性。本发明可通过基于Yr4DS基因的分子标记辅助育种、遗传转化和基因编辑等技术,用于改良现有小麦等麦族植物的抗条锈病水平,培育和创制抗条锈病中间材料和生产品种。
主权项:1.一种抗条锈病基因,其特征在于,为以下a-c中任一项所述的核酸:a核酸,其碱基序列如SEQIDNO.1所示;b核酸,其碱基序列如SEQIDNO.10所示;c核酸,其碱基序列为编码SEQIDNO.2所示蛋白质的碱基序列。
全文数据:粗山羊草Yr4DS基因在麦族植物抗条锈病育种的应用技术领域本发明涉及分子遗传学技术领域,具体涉及一种粗山羊草Yr4DS基因及其在麦族植物抗条锈病育种的应用。背景技术小麦条锈病WheatStripeRust,orYellowRust是由小麦专化型条形柄锈菌Pucciniastriiformisf.sp.tritici.,Pst引起的真菌病害,在全球范围内危害小麦的生产。小麦条锈病通常可造成0.5%-5%的产量损失,严重发生时可导致5%-25%的产量损失,甚至绝产Chen.2005.CanadianJournalofPlantPathology27:314-337。中国是世界上小麦条锈病普遍发生的区域,曾发生4次小麦条锈病大流行1950、1964、1990和2002年,每次大流行均导致上亿公斤的产量损失,小麦产量损失总计约12亿公斤,其中1950年的产量损失约合全国小麦总产量的41.4%陈万权等,2013。中国农业科学46:4254-4262。利用抗病品种是防治病害最为经济、有效且环保的措施。小麦育种中普遍存在遗传基础狭窄的弊端,导致多数品种缺乏有效的抗条锈病基因或现有抗病基因的抗病性逐渐丧失等现象康振生等,2015。中国农业科学48:3439-3453。另外,小麦条锈菌存在有性生殖、毒性变异快,导致新型致病小种的不断进化,这也造成了很多小麦品种在推广3-5年后就“丧失”抗病性陈万权等,2013。中国农业科学46:4254-4262。当前,生产上可用的小麦抗病资源日渐匮乏、形势严峻。因此,分离新的抗病基因是当前小麦抗病育种中亟待解决的问题。通过抗病新基因的分离,可拓宽小麦抗源,促进抗病品种的培育;并可通过基因工程或分子标记辅助选择等方法与已知基因进行多基因聚合,培育具有持久抗性、广谱抗性的种质材料。利用抗病基因是控制条锈病的重要手段,全球范围内已经在小麦及其近缘植物基因组中定位了上百个抗条锈病基因,但由于小麦基因组巨大并复杂等因素,已知序列的抗条锈病基因的数量非常有限。迄今为止,只有Yr5、Yr7、Yr15、Yr18和Yr36五个基因得以克隆Fuetal.2009.Science323:1357-1360;Krattingeretal.2009.Science323:1360-1363;Marchaletal.2018.NaturePlants4:662-668;Klymiuketal.2018.NatureCommunications9:3735,大大限制了抗条锈病基因在小麦等作物育种中的有效利用。发明内容针对上述现有技术,为解决抗病基因资源缺乏的困难,本发明提供了一个从小麦祖先粗山羊草Ae.tauschiissp.strangulata中分离的新基因Yr4DS,并通过功能验证确认了该基因的表达可提高小麦和大麦的抗条锈病水平。利用本发明,可将Yr4DS抗条锈病基因应用于麦族植物抗病育种,促进抗条锈病小麦和大麦品种或种质材料的培育。为实现上述目的,本发明采用如下技术方案:本发明的第一方面,提供一种抗条锈病基因,命名为Yr4DS基因,所述Yr4DS基因为以下a-j中任一项所述的核酸:a核酸,其由SEQIDNO.1所示的碱基序列组成;b核酸,其由SEQIDNO.3所示的碱基序列组成;c核酸,其由SEQIDNO.5所示的碱基序列组成;d核酸,其由SEQIDNO.7所示的碱基序列组成;e核酸,其由SEQIDNO.9所示的碱基序列组成;f核酸,其由SEQIDNO.10所示的碱基序列组成;g核酸,其由编码SEQIDNO.2所示蛋白质的碱基序列组成;h核酸,其由编码SEQIDNO.4所示蛋白质的碱基序列组成;i核酸,其由编码SEQIDNO.6所示蛋白质的碱基序列组成;j核酸,其由编码SEQIDNO.8所示蛋白质的碱基序列组成。其中,Yr4DS基因全长cDNATV1序列如SEQIDNO.1所示;Yr4DS基因全长cDNATV2a序列如SEQIDNO.3所示;Yr4DS基因全长cDNATV3序列如SEQIDNO.5所示;Yr4DS基因全长cDNATV4序列如SEQIDNO.7所示;Yr4DS基因组全长表达框架,包括启动子、基因组编码区和终止子,其核甘酸序列如SEQIDNO.9或SEQIDNO.10所示。本发明的第二方面,提供一种由上述抗条锈基因编码的蛋白,所述蛋白的氨基酸序列如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示。其中,SEQIDNO.1所示的Yr4DS基因全长cDNATV1编码的蛋白Yr4DS蛋白TV1,其氨基酸序列如SEQIDNO.2所示;SEQIDNO.3所示的Yr4DS基因全长cDNATV2a编码的蛋白Yr4DS蛋白TV2a,其氨基酸序列如SEQIDNO.4所示;SEQIDNO.5所示的Yr4DS基因全长cDNATV3编码的蛋白Yr4DS蛋白TV3,其氨基酸序列如SEQIDNO.6所示;SEQIDNO.7所示的Yr4DS基因全长cDNATV4编码的蛋白Yr4DS蛋白TV4,其氨基酸序列如SEQIDNO.8所示;携带上述抗条锈病基因的重组表达载体、转基因细胞系或基因工程菌也是本发明的保护范围。本发明的第三方面,提供如下a-f中任一项所述的DNA片段作为抗条锈病基因在植物育种或小麦和大麦条锈病防治中的应用;aSEQIDNO.1、SEQIDNO.3、SEQIDNO.5或SEQIDNO.7所示的cDNA片段;b编码SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示氨基酸序列的cDNA片段;cSEQIDNO.9或SEQIDNO.10所示的DNA片段;d编码SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示氨基酸序列的DNA片段;ecDNA片段或DNA片段,其编码的蛋白在功能上与SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示的蛋白等价,但在氨基酸序列上存在一个、数个或数十个氨基酸的替换、删除或插入;fcDNA片段或DNA片段,其在严格条件下与a或c的DNA片段杂交,且编码SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示的蛋白。本发明的第四方面,提供如下1-4中任一项所述的DNA片段通过调控抗条锈病基因表达在植物育种或小麦和大麦条锈病防治中的应用;1DNA片段,其转录产物在植物细胞中上调SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的表达;2DNA片段,其翻译产物在植物细胞中上调SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的表达;3DNA片段,其转录产物在植物细胞中对SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的转录RNA具有上调作用;4DNA片段,其翻译产物在植物细胞中对SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因编码的蛋白具有上调作用。本发明的第五方面,提供含有SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的重组表达载体、转基因细胞系或基因工程菌或小麦抗条锈基因编码的蛋白在培育条锈病抗性提高或降低的麦族植物中的应用;优选的,所述麦族植物为小麦或大麦。本发明的第六方面,提供一种抗条锈病小麦的培育方法,所述培育方法包括:将SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因转入小麦或大麦中,获得抗条锈病小麦或大麦;或者上调小麦或大麦基因组中SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的表达,筛选得到条锈病抗性提高的小麦或大麦植株。上述培育方法中,将抗条锈病基因转入小麦或大麦中的方法包括:聚乙二醇法、农杆菌介导法或基因枪轰击法。上述培育方法中,上调小麦或大麦基因组中的抗条锈病基因的表达的方法包括:导入能够激活或提高小麦抗条锈病基因的转录水平或翻译水平或蛋白活性的DNA片段;或者控制特异小RNA分子的合成,上调小麦抗条锈病基因mRNA的积累。所述特异小RNA分子包括:微小RNA分子microRNA,miRNA、干扰小RNAsmallinterferingRNA,siRNA或人工miRNAartificialmicroRNA,amiRNA。本发明的第七方面,提供一种培育条锈病抗性降低的小麦或大麦的方法,所述方法包括:抑制小麦或大麦基因组中SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的表达,筛选得到条锈病抗性降低的小麦或大麦植株。上述方法中,抑制小麦或大麦基因组中的抗条锈病基因的表达的方法包括:突变或敲除小麦或大麦中SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示基因的全部或者部分序列;或者使用干扰RNA干扰SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示基因的表达;或者使用基因沉默系统使SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示基因沉默。本发明的第八方面,提供一种用于鉴定小麦抗条锈病基因的分子标记,所述分子标记有三个,分别命名为Yr4DS-PM、Yr4DS-GM和Yr4DS-TM。上述三个分子标记均可以区分抗条锈病和感条锈病的不同粗山羊草家系。其中,Yr4DS-PM是基于Yr4DS基因启动子区域设计的PCR标记,其核苷酸序列如SEQIDNO.61所示。用于扩增该Yr4DS-PM的引物为Yr4DS-FP1SEQIDNO.11和Yr4DS-RP1SEQIDNO.12。若扩增条带长度为727bp,则与粗山羊草高抗小麦条锈病的性状紧密连锁;若不扩增或扩增产物为575bp,则建议相应的粗山羊草不抗小麦条锈病。Yr4DS-GM是基于Yr4DS基因编码区区域设计的PCR标记,其核苷酸序列如SEQIDNO.62所示。用于扩增Yr4DS-GM的引物为Yr4DS-FP2SEQIDNO.13和Yr4DS-RP2SEQIDNO.14。将扩增产物用HaeIII酶酶切后,若出现一条361bp的条带,则与粗山羊草高抗小麦条锈病的性状紧密连锁;若出现两条240bp和121bp的条带,则建议相应的粗山羊草不抗小麦条锈病。Yr4DS-TM是基于Yr4DS基因终止子区域设计的PCR标记,其核苷酸序列如SEQIDNO.63所示。用于扩增Yr4DS-TM的引物为Yr4DS-FP3SEQIDNO.15和Yr4DS-RP3SEQIDNO.16。若扩增条带长度为858bp,则与粗山羊草高抗小麦条锈病的性状紧密连锁;若不扩增,则建议相应的粗山羊草不抗小麦条锈病。本发明的第九方面,提供一种获得携带所述抗条锈病基因的植物细胞的方法,通过转基因或基因组编辑手段而获得。本发明的第十方面,提供一种获得携带所述抗条锈病基因的植株的方法,由上述方法获得的植物细胞再生成苗。本发明的有益效果:本发明首次从粗山羊草中克隆得到了一个新型抗条锈病基因Yr4DS,该基因能够有效的调控麦族植物特别是小麦和大麦抗条锈病的能力,丰富了抗条锈病基因资源。本发明发现Yr4DS的表达提供高水平抗条锈病的功能,预示着人为操作Yr4DS基因可以赋予小麦等麦族植物抗条锈病的特性。本发明可通过基于Yr4DS基因的分子标记辅助育种、遗传转化和基因编辑等技术,用于改良现有小麦、大麦等麦族植物的抗条锈病水平,培育和创制抗条锈病中间材料和生产品种。附图说明图1:鉴定粗山羊草Yr4DS基因的三个有效标记Yr4DS-PM、Yr4DS-GM和Yr4DS-TM;其中,Yr4DS-PM和Yr4DS-TM为PCR的直接扩增产物;Yr4DS-GM为PCR扩增产物的HaeIII酶切结果。粗山羊草CIae9、PI511383高抗小麦条锈病;粗山羊草PI486274和PI560536高感小麦条锈病;SM1675和SW3为携带抗病型Yr4DS拷贝的六倍体小麦;中国春CS和六倍体小麦CB037不携带抗病型Yr4DS基因。品系后括弧内的字母代表该品系抗条锈病R或感条锈病S,星号*表示其抗病性受遗传背景或其它抗病基因的影响。M代表分子量标准样品。图2:Yr4DS基因在粗山羊草成株期叶片中的表达;其中,Rpool、Spool分别代表组成抗病池和感病池的F6代纯合株系;图中:上图应用RT-PCR检测了Yr4DScDNA的表达情况,样品包括抗条锈病粗山羊草亲本PI511383,感条锈病粗山羊草亲本PI486274以及二者来源的F6代纯合株系所组成的BSA抗病池Rpool,包括12个株系,每个株系都显示Yr4DScDNA阳性,这里只展示其中的4个株系和BSA感病池Spool,包括11个株系,每个株系都显示Yr4DScDNA阴性,这里只展示其中的4个株系。RT-PCR检测应用到PCR引物Yr4DS-FP4和Yr4DS-RP4表1。下图应用RT-PCR测试了对应样品中粗山羊草Actin基因内源参照的表达情况,应用到PCR引物Actin-FP和Actin-RP表1。图3:抗病型Yr4DS基因的突变导致人工合成小麦在成株期高感小麦条锈病。图中,L68和L91为人工合成小麦Syn-SAU-93,携带抗病型Yr4DS基因的M3代EMS突变体表2,高感小麦条锈病感染型介于8-9。相应的野生型对照WT表现中抗小麦条锈病感染型介于3-4。比例尺=1cm。人工合成小麦‘Syn-SAU-93AS2382AS2388’表现良好的成株抗条锈病水平,感染型介于3-4Infectiontype或IT;LineandQayoum.1992.USDATechnicalBulletin1788。图3为M3突变体L68和L91;表2及野生型对照WT=Syn-SAU-93在成株期感染小麦条锈菌后的发病情况,Yr4DS基因突变体高感小麦条锈病IT介于8-9,野生型对照表现中抗小麦条锈病IT介于3-4。图4:利用小麦‘CB037’和大麦‘GoldenPromise’开展Yr4DS等基因的遗传互补研究图;图中:A图片为携带Yr4DS等基因组全长表达框架SEQIDNO:5的质粒,该质粒骨架是pCC1FOS,红色线条代表载体区段;共转化载体为PC174,上面携带除草剂筛选标记BAR基因,该质粒骨架为pCAMBIA1300。B图片为转基因小麦植株接菌后的反应,并利用PCR方法验证了植株中三个基因的整合和表达情况。三个基因的排列为RLK1左、RLK2中和Yr4DS右。C图片为转基因大麦植株接菌后的反应,也检测了三个基因的整合和表达情况左边RLK1,中间RLK2,右边Yr4DS,“+”代表整合或表达,“-”代表未整合或未表达。小麦“CB037”和大麦“GoldenPromise”GDP为非转基因野生型对照。比例尺=1cm。图5:转基因小麦和大麦植株中RLK1、RLK2和Yr4DS基因的全长表达情况;图中展示了针对PC1104质粒不同类型转基因植株G1-G6中三个基因的表达,不同类型参照表3。使用双轮PCR检测目标基因的表达:第一轮使用长跨度PCR引物富集目标及其同源基因的全长转录本,第二轮使用特异PCR引物仅扩增三个目标基因的转录本。对于Yr4DS基因,同时检测了两类全长转录本,分别为Yr4DSTV1和Yr4DSTV4。另外使用肌动蛋白ACTIN基因作为内参对照,该对照只需要一轮PCR。虚线左边为小麦植株,虚线右边为大麦植株。PI511383为野生型阳性对照PC;CB037和GDP为野生型阴性对照NC。图中白色箭头指示基因组拷贝的扩增产物或其它非特异扩增产物。M代表分子量标准样品。比例尺=1cm。具体实施方式应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。正如背景技术部分所介绍的,由于小麦基因组巨大并且复杂,已知序列的抗条锈病基因的数量非常有限,大大限制了抗条锈病基因在小麦育种中的有效利用。基于此,本发明的目的是提供一种新的抗条锈病基因,并将其用于小麦、大麦等麦族植物的育种。本发明利用RNA测序RNA-seq和集团分离分析法BulkedSegregantAnalysis,BSA,比较了粗山羊草分离群体F6世代的抗条锈病BSA池和感条锈病BSA池在成株期的叶片转录组transcriptome,从中鉴定到只在抗条锈病亲本PI511383和抗条锈病BSA池中表达的基因,进一步筛选到一个在PI511383上特异表达、具有NBS-LRR结构域、位于粗山羊草4DS染色体的基因,故命名为Yr4DS基因。结果表明,人为操作Yr4DS基因可以提高小麦和大麦的抗条锈病水平。本发明可用于提高小麦等麦族植物的抗条锈病水平、培育高抗条锈病的中间材料和生产品种。Yr4DS基因的全长cDNA序列如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5或SEQIDNO.7所示;Yr4DS基因编码的蛋白即Yr4DS蛋白,其氨基酸序列如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示;Yr4DS基因组全长表达框架,包括启动子、基因组编码区和终止子,其核甘酸序列如SEQIDNO.10所示。本发明涉及编码粗山羊草Yr4DS蛋白及其同源蛋白的cDNA、合成DNA和基因组DNA的应用。本领域的技术人员利用常规技术即可获得Yr4DS基因相关的cDNA和基因组DNA。cDNA的制备涉及如下步骤:a从粗山羊草或其它物种中提取信使RNAmessageRNA,mRNA;b利用mRNA作为模板合成cDNA;c根据本发明Yr4DS基因全长cDNA序列SEQIDNO.1、SEQIDNO.3、SEQIDNO.5或SEQIDNO.7设计PCR引物,然后从cDNA模板中扩增Yr4DS基因或其同源基因;d将PCR产物克隆到质粒载体,分离出Yr4DS基因或其同源基因的cDNA;e利用Yr4DS基因cDNA序列为模板,委托商业服务人工合成DNA。同理,本领域的技术人员也可以从粗山羊草或其它物种中提取基因组DNA,用以创建基因组DNA文库如BAC、cosmid、fosmid等类型文库,再利用基于本发明Yr4DS基因组全长表达框架的核苷酸序列如SEQIDNO.10的DNA探针或PCR引物筛选DNA文库,获得携带Yr4DS基因的阳性质粒。也可采用长片段PCR方法,利用本发明Yr4DS基因组全长表达框架的核苷酸序列如SEQIDNO.10的特异PCR引物,从植物基因组DNA或质粒DNA扩增Yr4DS基因或其同源基因,再将PCR产物连接到克隆载体。本发明包括Yr4DS同源DNA片段,只要它们编码的蛋白与Yr4DS蛋白如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8功能等价。本文所指的“与Yr4DS蛋白功能等价”意味着目标DNA片段所编码的蛋白在生物学功能和生理生化特征等方面与本发明中Yr4DS蛋白如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8接近。Yr4DS蛋白典型的生物学功能是提供抗条锈病功能。为验证Yr4DS基因是否抗小麦条锈病,可使用甲基磺酸乙酯Ethylmethylsulfone,EMS创建‘携带Yr4DS基因的人工合成小麦’的突变群体实施例4和5,通过接种鉴定出高感小麦条锈病的突变个体,再分析Yr4DS基因的突变情况,分析Yr4DS基因的突变频率和感病表型的相关性。为明确Yr4DS基因的功能,也可采用遗传互补geneticcomplementation手段加以验证。本发明利用基因枪轰击biolisticsbombardment技术将Yr4DS基因组全长表达框架载体如SEQIDNO.10导入高感条锈病的小麦‘CB037’和大麦‘GoldenPromise’实施例6;利用所获得的转基因植株,分析了Yr4DS转基因的表达对小麦和大麦抗条锈病的贡献。如果DNA片段编码的蛋白功能等价于Yr4DS蛋白如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8,这些DNA片段的优选来源是单子叶植物monocotyledon,更优选来源是禾本科植物Gramineae,最优选来源是小麦族植物Triticeae。这些DNA片段包括本发明核苷酸序列如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5或SEQIDNO.7对应的等位基因、同源基因、突变基因和衍生基因;它们编码的蛋白类似于本发明Yr4DS蛋白氨基酸序列如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8,或存在一个、数个或数十个氨基酸的替换、删除或插入现象,都属于本发明内容。基因组编辑genomeediting技术,可以定向敲除目的基因,在动植物上均有应用ChengandAlper.2014.CurrentOpinioninBiotechnology30:87-94。部分基因组编辑技术,如规律性重复短回文序列簇clustered,regularlyinterspaced,shortpalindromicrepeats,CRISPR在小麦等作物上Shanetal.2014.NatureProtocols9:2395-2410;Wangetal.2014.NatureBiotechnology32:947-951;Zhangetal.2016.NatureCommunications7:12617都得到了成功应用。基因组编辑技术会造成靶标基因的特定区域发生一个、数个或数十个碱基的删除或插入、导致基因突变,位于转录区域的DNA变异则可能造成编码蛋白的变异或截短truncationWangetal.2014.NatureBiotechnology32:947-951。转录区域DNA的碱基突变也会引起编码蛋白的氨基酸改变。利用基因组编辑或碱基突变创建的DNA片段,其编码的蛋白与Yr4DS蛋白如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8相比,或存在一个、数个或数十个氨基酸的替换、删除或插入等现象,但只要该DNA片段编码的蛋白与Yr4DS蛋白如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8功能等价,该DNA片段即属于本发明的应用内容。本发明界定的DNA片段还包括那些发生了碱基突变,但不改变编码蛋白序列的突变,即保守突变conservativemutation。对于本领域的技术人员,可以采用基因组编辑技术改变本发明内容中Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5或SEQIDNO.7及其同源基因序列。另外,目标基因的变异可以通过突变诱导产生或通过种质筛选鉴定本已存在的自然变异。比如,Slade等人2005创建了小麦的EMS突变群体,进而采用定向诱导基因组局部突变TargetingInducedLocalLesionsINGenomes,TILLING技术鉴定目标基因的点突变Sladeetal.2005.NatureBiotechnology23:75-81。自然种质的长期进化积累了大量的变异,也可以采用Ecotilling手段从自然种质或育成品种中鉴定目标基因的变异Tilletal.2006.NatureProtocols1:2465-2477。对于本领域的技术人员,可以创建相关植物材料的突变群体,再从中筛选本发明内容中DNA片段如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10发生变异的个体。本领域的技术人员还可以从相关植物的自然种质或育成品种中鉴定本发明DNA片段如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10的自然变异。因此,本发明还涵盖:a所有通过基因组编辑、诱变或自然变异筛选而获得的携带本发明内容的DNA片段如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10发生变异的植物细胞;b携带a项植物细胞的植株;c来自b项植株的无性克隆或植株后代等,只要它们仍携带a项植物细胞;d来自b和c项内容的植物种子、植物组织或植物器官等,只要它们仍携带a项植物细胞。对于本领域的技术人员,获得DNA片段、使得它们编码的蛋白与Yr4DS蛋白如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8功能等价的方法很多,如PCR方法Saikietal.1985.Science230:1350-1354;Hemsleyetal.1989.NucleicAcidsResearch17:6545-6551;Landtetal.1990.Gene96:125-128、DNA重组技术和DNA人工合成技术KosuriandChurch.2014.NatureMethods11:499-507。可以说,对于本领域的技术人员,从小麦或其它植物中获得与Yr4DS基因高度同源的DNA片段为常规技术,可利用对应本发明核苷酸序列如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10的PCR引物,或使用对应本发明核酸序列如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10的DNA探针,通过筛选基因组DNA或cDNA文库,获得相应的DNA片段。对于DNA片段的获得,无论是通过PCR方法、DNA重组、DNA人工合成还是其它同类技术,只要这些DNA片段所编码的蛋白与Yr4DS蛋白如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8功能等价,这些DNA片段就属于本发明内容。这些DNA片段所编码的氨基酸序列应当与本发明Yr4DS蛋白氨基酸序列如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8高度同源;这里所指的高度同源,意味着两者之间在能够匹配的区段上,氨基酸序列的等同率sequenceidentity至少50%或更高,优选70%或更高,更优选90%或更高比如95%、96%、97%、98%和99%或更高。氨基酸或核苷酸序列的等同率可采用BLAST算法测定Altschuletal.1990.JournalofMolecularBiology215:403-410;KarlinandAltschul.1993.ProceedingsoftheNationalAcademyofSciences90:5873-5877。分子标记辅助选择育种利用有效分子标记加速育种进程和提高育种效果。当前,有效利用的标记包括:单核苷酸多态性Singlenucleotidepolymorphism、酶切扩增多态性序列Cleavedamplifiedpolymorphicsequence、衍生酶切扩增多态性序列Derivedcleavedamplifiedpolymorphicsequence和竞争性等位基因特异性PCRkompetitiveallelespecificPCR等。本领域的技术人员可以运用类似的标记创建方法,根据本发明Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10或其同源基因的DNA片段,设计可用于抗条锈病基因回交转育和抗病基因聚合的分子标记比如本发明设计的Yr4DS-PM标记;实施例3、图1;利用有关分子标记开展小麦等作物的抗条锈病分子育种属于本发明内容。从应用角度出发,本发明Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10或其同源基因的DNA片段,当导入高感条锈病的植物后,很可能创制出高抗条锈病的表型。换句话说,对于本发明Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10或其同源基因的DNA片段或该DNA片段的重组载体,将它们导入高感条锈病的植物后,转基因细胞则分化再生形成高抗条锈病的转基因植株,从而将感病植物转变为抗病植物。反过来,也可以创制调控Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10或其同源基因表达的转基因植物。这里所指的“调控表达”包括DNA转录水平、cDNA翻译水平和蛋白产物活性三个层面,包括上调和下调两种程度。比如说,根据本发明Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10或其同源基因挖掘到的能够激活或提高该基因的转录水平或翻译水平或蛋白活性等的DNA片段,或将它们插入合适质粒载体,将以上DNA片段或其承载质粒导入携带Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10或其同源基因的植株细胞,转基因细胞分化形成高抗条锈病的转基因植株。再比如,对于Yr4DS基因或其同源基因,或存在与之作用的微小RNA分子microRNA,miRNA、干扰小RNAsmallinterferingRNA,siRNA或与之作用的人工miRNAartificialmicroRNA,amiRNA;通过合理控制特异小RNA分子amiRNA、miRNA和siRNA的表达,从而调控Yr4DS基因或其同源基因mRNA的积累,提高植物的抗条锈病水平。本发明对用于开展植物细胞转化的质粒载体没有限制,只要它们在植物细胞中能够表达承载基因。比如,使用携带组成型启动子如玉米Ubi启动子;Christensenetal.1992.PlantMolecularBiology18:675-689或叶片特异启动子的载体。本发明所指的“植物细胞”形式多样,包括各类具备生命全能性的单细胞、多细胞、植物组织或器官,可以是悬浮培养细胞、原生质体细胞,也可以是植物切片组织、植物愈伤组织,它们统一的特点是通过分化再生或无性繁殖能形成植株或部分植株。本发明所指的“植物细胞”还涵盖各种植物来源的细胞,比如小麦、大麦和其他麦族植物,它们统一的特点是通过分化再生或无性繁殖能形成植株或部分植株。对于本领域的技术人员,可以使用各种手段将质粒载体导入植物细胞,比如聚乙二醇polyethyleneglycol,PEG法,电穿孔electroporation法,农杆菌介导法,基因枪轰击法等,并将转化细胞发展成为转基因植株。在植物领域,各种转基因技术趋向成熟,并得到广泛应用。以上这些方法和其他类似方法均适用于本发明领域。对于携带本发明DNA片段的转基因植株,进而通过有性或无性方式得到它们的有性后代如种子、无性克隆如不定枝、愈伤、原生质体等、分生组织如芽点、茎尖、根尖等,可以大量繁殖转基因植株。因此,本发明涵盖:a所有携带本发明内容DNA片段的转基因植物细胞;b携带a项转基因细胞的植株;c来自b项植株的无性克隆和植株后代等,只要它们仍携带a项转基因细胞;d来自b和c项内容的植物种子、植物组织或植物器官等,只要它们仍携带a项转基因细胞。利用以上方式获得的转基因植株或携带转基因细胞的植株,其抗条锈病能力在理论上将有别于野生型对照植株。比如,将粗山羊草Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10或其同源基因导入感条锈病植株,创制高抗条锈病的植株。综上所述,本发明集中在Yr4DS基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10在麦族植物抗条锈病领域的应用。利用感条锈病的植物作为受体,在叶片等组织中表达Yr4DS基因或其同源基因,可能将感条锈病植物转变为高抗条锈病的植物。由此推测,Yr4DS及其同源基因将在麦族植物的抗条锈病育种中发挥作用。为了使得本领域技术人员能够更加清楚地了解本申请的技术方案,以下将结合具体的实施例详细说明本申请的技术方案。本发明实施例和对比例中所用的试验材料均为本领域常规的试验材料,均可通过商业渠道购买得到。未注明详细条件的实验方法是按照常规试验方法或按照供应商所建议的操作说明书进行的。本发明利用粗山羊草PI511383和PI486274作为亲本,创制了杂交高代的重组自交系F6。为测试它们的抗条锈病水平,本发明利用小麦条锈菌Pst小种包括CYR29,CYR30,CYR31,CYR32,CYR33,CYR34,贵22-1,SY11-4,或HY46-8等接种鉴定。粗山羊草在温室条件下种植,温室配备长日照16h、105μmolm-2s-1,白天温度25-30℃,夜间温度15-20℃。粗山羊草的接种采用注射法或抖粉法,然后在10℃、黑暗和高湿100%条件下保持24h,随后转移到人工气候室:8h黑夜、12℃;16h光照、18℃。本发明中分子生物学实验的用水、常规药品以及植物激素购自美国飞世尔科技公司FisherScientific,Pittsburgh,PA,USA和美国西格玛奥德里奇公司Sigma-Aldrich,St.Louis,MO,USA,植物组织培养基购自美国植物技术公司PhytoTechnologyLaboratories,OverlandPark,KS,USA,微生物培养基购自美国BD公司Becton,DickinsonandCompany,FranklinLakes,NJ,USA,抗生素和双丙氨膦bialaphos购自美国GoldBio公司GoldBiotechnology,St.Louis,MO,USA。本发明所涉及的PCR引物及序列见表1。表1:本发明中应用到的PCR引物备注:RT-PCR中内参引物Actin-FP和Actin-RP在小麦大麦和粗山羊草上都工作。表2:高感条锈病人工合成小麦突变体中候选基因的突变情况11本表格描述了Yr4DS区域的三个基因的删除和点突变情况。肌动蛋白ACTIN基因用作内参评估DNA样品的质量。扩增引物包括RLK1RLK1-FP1和RLK1-RP1,RLK2RLK2-FP1和RLK2-RP1,Yr4DSYR4DS-FP5和YR4DS-RP5和ACTINActin-FP和Actin-RP。“+”代表PCR扩增阳性;“-”代表PCR扩增阴性,反映目标基因的整体或部分删除。目标基因扩增阳性的植株,对于所携带的RLK2和Yr4DS基因进行测序,所有个体的RLK2基因未出现编码序列的碱基突变,但Yr4DS基因的编码序列存在碱基突变。2左侧字母为抗病型Yr4DS基因的碱基,中间数字代表cDNA水平相对于起始密码子ATG的碱基位置,右侧字母为突变后的碱基。3左侧字母为抗病型Yr4DS蛋白中对应的氨基酸,中间数字代表相对于首位氨基酸的位置,右侧字母为突变后的氨基酸。表3:不同目标基因的表达对转基因植株抗条锈病水平的影响1首先酶切处理质粒PC1104,再用于小麦和大麦的转化。质粒的酶切处理包括:未酶切Intact=I,BsrGI酶切B1,NotI酶切N1,XbaI酶切X1,和XbaI+KpnI双酶切XK1。通过检测Yr4DS区域的三个基因的表达及其转基因植株的抗条锈病水平,判断各个基因在抗条锈病中的作用。扩增引物包括RLK1一轮引物RPK1-FP1和RLK1-RP2,二轮引物RPK1-FP3和RLK1-RP3,RLK2一轮引物RLK2-FP2和RLK2-RP2,二轮引物RLK2-FP3和RLK2-RP3,Yr4DSTV1一轮引物YR4DS-FP6和YR4DS-RP6,TV1二轮引物YR4DS-FP7和YR4DS-RP7;TV4一轮引物YR4DS-FP8和YR4DS-RP3,TV4二轮引物YR4DS-FP9和YR4DS-RP9和ACTIN单轮引物Actin-FP和Actin-RP。“+”代表表达;“-”代表未表达。2对于双数字,加号前的数字代表小麦的独立转基因株系的数目,加号后的数字代表大麦的独立转基因株系的数目。对于单个数字,只代表小麦的独立转基因株系的数目。实施例1:利用转录组学鉴定抗条锈病亲本和抗病BSA池中特异表达的基因本发明利用RNA测序RNA-seq和集团分离分析法BulkedSegregantAnalysis,BSA,比较了粗山羊草分离群体F6世代的抗条锈病BSA池和感条锈病BSA池在成株期的叶片转录组transcriptome。RNA测序利用高通量测序技术直接测定样本cDNA分子。本发明通过RNA测序对比了抗条锈病粗山羊草PI511383,感条锈病粗山羊草PI486274以及二者来源的F6代纯合株系中12个抗条锈病株系组成的BSA抗病池Rpool和由11个感条锈病株系组成的BSA感病池Spool的成株期叶片的转录组。不同样本分别测定了一个生物学重复。总RNA的提取采用TRIzol试剂及相关方法LifeTechnologies,GrandIsland,NY,USA。RNA测序涉及的文库构建优选500bp左右的mRNA碎片和高通量双端测序HiSeq2500,Illumina,SanDiego,CA,USA;paired-end,PE125由北京贝瑞和康生物技术有限公司承担BerryGenomicsCompany,Beijing,China。对于RNA-seq原始数据,首先剔除接头信息、低质量碱基Q值≤3的碱基,占整个read的50%以上和未测出碱基N比例大于3%,获得有效数据;利用Trinity软件Haasetal.2013.NatureProtocols8:1494-1512开展有效数据的从头拼装。通过比较PI511383、PI486274、抗病池Rpool和感病池Spool各自的叶片转录组数据,发现个别基因只在抗条锈病亲本PI511383和抗病池Rpool中表达。本发明鉴定到一个未知基因,其序列如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10所示;用引物组合Yr4DS-FP4Yr4DS-RP4检测到该基因在抗病亲本PI511383和抗病池中表达,但在感病亲本PI486274及感病池中不表达图2。发明人预测该基因如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10可能影响粗山羊草抵抗小麦条锈病的水平。利用粗山羊草的基因组序列比对,发现该基因位于4DS染色体http:aegilops.wheat.ucdavis.eduATGSPblast.php,本发明暂将该基因命名为Yellowrustresistancegene4DSYr4DS基因,并围绕该基因开展了功能研究。实施例2:粗山羊草Yr4DS基因全长cDNA的验证为验证Yr4DS基因的全长full-lengthcDNA,使用TRIzol试剂提取了粗山羊草PI511383接条锈菌10天后的叶片总RNA,然后利用RevertAidFristStrandcDNASynthesis试剂盒ThermoScientific,Waltham,MA,USA制备了cDNA模板。采用cDNA末端快速扩增技术rapidamplificationofcDNAends,RACE分离Yr4DS基因SEQIDNO.1和3全长cDNA的5'和3'末端,使用SMARTerRACEcDNAAmplification试剂盒ClontechLaboratories,MountainView,CA,USA,操作方法参照试剂盒说明书。5'端RACEPCR的嵌套引物为Yr4DS-RP10和Yr4DS-RP11,其中Yr4DS-RP11为Yr4DS-RP10的巢式引物nestedprimer。本基因存在两个3'端,第一个3'端RACEPCR的嵌套引物为Yr4DS-FP10和Yr4DS-FP11,其中Yr4DS-FP11为Yr4DS-FP10的巢式引物;第二个3'端RACEPCR的嵌套引物为Yr4DS-FP12和Yr4DS-FP13,其中Yr4DS-FP13为Yr4DS-FP12的巢式引物。对RACEPCR产物进行测序,证实了Yr4DS基因全长cDNA序列如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5或SEQIDNO.7两端的完整性,说明RNA测序及序列拼装高度可靠。Yr4DS基因全长cDNATV1SEQIDNO.1共4,266bp,包含一个3,207bp的开放阅读框openreadingframe,ORF;在起始密码子的上游30bp位置,有1个框内终止密码子in-framestopcodon,表明当前预测的ORF可靠、代表Yr4DS蛋白的一个异构体isoform。Yr4DS基因全长cDNATV2aSEQIDNO.3共3,477bp,包含一个1,227bp的ORF,代表Yr4DS蛋白的另外一个异构体。Yr4DS基因全长cDNATV3SEQIDNO.5共2,853bp,包含一个1,488bp的ORF,代表Yr4DS蛋白的第三个异构体。Yr4DS基因全长cDNATV4SEQIDNO.7共2,609bp,包含一个1,416bp的ORF,代表Yr4DS蛋白的第四个异构体。实施例3:‘PI511383’基因组Fosmid文库的构建和筛选为方便基因组DNA的克隆,创建了PI511383基因组DNAFosmid文库,构建方法参照已发表的文献Jetty.2005.TheorApplGenet111:1596-1607。首先从PI511383叶片中提取大分子量基因组DNAhigh-molecularweight,HMW,利用重复冻融液氮45℃,20-30次将大分子量基因组DNA随机“切割”成不同大小的片段;利用1%的琼脂糖凝胶及脉冲电泳pulsed-fieldgelelectrophoresis,PFGE游离DNA片段产物,从中纯化介于36-60kb的DNA片段;用末端修复酶补平DNA片段,重复脉冲电泳和片段纯化步骤,将经过两次纯化获得的DNA片段克隆到Fosmid载体pCC1FOS;经过噬菌体提取物包装后侵染宿主菌EPI300-T1R菌株细胞,将感染了噬菌体的宿主菌涂布到含有12.5ugml-1氯霉素的LB平板上,37℃过夜培养,收集平板上的Fosmid克隆。PI511383基因组Fosmid文库包含大约1百万个克隆、保存在622个超级池中。根据随机挑选的120个Fosmid克隆检测文库质量,文库空载率为0,单克隆的平均插入片段35kb,覆盖粗山羊草全基因组按4.3Gb计算约8.2倍。为获得携带Yr4DS基因的Fosmid克隆,根据Yr4DS基因全长cDNATV1SEQIDNO.1和粗山羊草基因组参考序列设计了多套PCR引物,测试不同引物组合在粗山羊草基因组DNA上的扩增效果,确定了两对用于文库筛选的引物组合:Yr4DS-FP1Yr4DS-RP1和Yr4DS-FP14Yr4DS-RP14。Yr4DS-FP1Yr4DS-RP1在抗条锈病山羊草CIae9、PI511383中扩增出727bp的条带图1,在感条锈病山羊草PI486274和PI560536中不扩增特异条带,但能从感条锈病山羊草AL878扩增出575bp的条带。为验证该标记和山羊草抗条锈病的关系,本发明检测了来自粗山羊草组合PI486274PI511383,AS2388AS87的6,000多个分离个体,该标记727bp条带与抗条锈病表型完全连锁。为了进一步确定该标记的有效性,我们筛选了175份粗山羊草种质材料,其中69份抗性山羊草上扩增出727bp的条带,35份感条锈病山羊草上扩增出575bp的条带,另外71份感病粗山羊草上没有特异的扩增条带,该标记的基因型与现有粗山羊草种质的抗条锈病性状完全吻合。因此,Yr4DS-FP1Yr4DS-RP1可以用作诊断粗山羊草抗条锈病表型的可靠标记,命名为Yr4DS-PMpromter-derivedmarker。另外,我们也针对Yr4DS基因区和终止区设计了特异分子标记,命名为Yr4DS-GMgene-derivedmarker和Yr4DS-TMterminator-derivedmarker图1,都能用作诊断粗山羊草抗条锈病表型的可靠标记。利用Yr4DS-PM标记,通过菌液PCR筛选了PI511383Fosmid文库的622个超级池,首先确认了阳性Fosmid单克隆所在的超级池,再通过逐级稀释筛选,获得阳性Fosmid单克隆,共筛选到10独立单克隆。然后利用Yr4DS基因引物Yr4DS-FP15Yr4DS-RP15进一步筛选,得到8独立单克隆;克隆F2-1即质粒PC1104携带39,535bp的插入片段SEQIDNO.10;图4A,该序列有3个表达基因,分别为类受体激酶蛋白1receptor-likekinasegene1,RLK1、类受体激酶蛋白2receptor-likekinasegene2,RLK2和Yr4DS。实施例4:‘人工合成小麦’EMS突变群体的创建为确认抗病型Yr4DS基因的功能,本发明利用化学诱变剂甲基磺酸乙酯Ethylmethanesulfonate,EMS水溶液处理人工合成小麦Syn-SAU-93AS2382AS2388Zhangetal.2010.Euphytica172:285-294。前期结果表明AS2388和PI511383携带相同的抗病基因Liuetal.2013.CropScience53:2014-2020;实际上,二者的Yr4DS基因序列如SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10一致。突变群体的创建如下:共处理1,850粒种子,每100毫升EMS水溶液78mM处理200粒种子,放置于水平摇床震荡12h150rpm,25℃,然后在室温下用自来水冲洗12h;将冲洗后的种子简单晾干,然后播种到四川农业大学小麦所试验田,共获得了613个M2株系。实施例5:筛选人工合成小麦突变群体、确认Yr4DS等基因的突变及功能将所有M2株系衍生得到的M3或M3种子混合种植在四川农业大学小麦所实验田,在苗期使用当前流行的条锈菌混合生理小种接菌,包括CYR30,CYR31,CYR32,CYR33,CYR34,贵22-1,SY11-4和HY46-8;待感病对照充分发病后进行人工合成小麦感条锈病突变体的鉴定。采用十二烷基肌氨酸钠Sarkosyl方法Yuanetal.2012.JournalofGeneticsandGenomics39:587-592提取感病植株的基因组DNA,并利用NanoDropTMOne超微量分光光度计ThermoFisherScientific,Madison,WI,USA测定DNA浓度,统一调整为100ngul-1。利用PCR扩增技术,以高感条锈病的突变体DNA为模板克隆Yr4DS基因。针对Yr4DS基因中3,956bp的区域分三段扩增:第一段使用PCR引物Yr4DS-FP16和Yr4DS-RP16,第二段使用PCR引物Yr4DS-FP17和Yr4DS-RP17,第三段使用PCR引物Yr4DS-FP5和Yr4DS-RP5;PCR反应条件为,初始变性94℃、3min,10个循环变性94℃、30sec;退火65℃每个循环降0.5℃、30sec;延伸72℃、105sec;28个循环变性94℃、30sec;退火60℃、30sec;延伸72℃、105sec,终延伸72℃、6min。另外,我们还针对紧靠Yr4DS的RLK1和RLK2基因,检测它们在高感条锈病的突变体中的变异情况,使用到PCR扩增引物RLK1-FP1和RLK1-RP1、RLK1-FP4和RLK1-RP4、RLK2-FP4和RLK2-RP1表1。PCR扩增产物委托生工生物工程上海股份有限公司测序完成SangonBiotech,Chengdu,China。在M3或M4混合世代中,共检测到60株高感小麦条锈病的人工合成小麦植株表2,图3。通过检测Yr4DS区域的3个基因,发现22个突变体发生了Yr4DS基因的删除,其中的21个突变体其删除伸展到RLK2基因;同时呈现RLK2和Yr4DS删除的植株中又有9个突变体其删除伸展到RLK1基因。但是,在所有60株高感小麦条锈病的突变体中,没有发现只有RLK1或RLK2基因删除的现象。Yr4DS基因删除占所有植株的36%,与Ni等人揭示约29%的目标基因删除相似Nietal.2017.NatureCommunications8:15121。对比RLK1、RLK2和Yr4DS的删除情况,Yr4DS基因的删除可能造成合成小麦抗条锈病能力的丧失。对于未出现Yr4DS基因删除的38株高感条锈病的突变植株,10株出现Yr4DS基因的点突变,造成单个氨基酸的改变或蛋白翻译的提前终止。根据他人的研究,使用0.8%EMS或78mM处理普通小麦,每个突变体中点突变的平均步长约30kbKrasilevaetal.2017.PNAS114:E913–921。假设Yr4DS基因和小麦抗条锈病性状无关H0假设,在其余38株高感条锈病的个体中,理论上会发现5=38×3.956÷30;Yr4DS基因的检测区域为3,956bp株存在Yr4DS基因的突变,另外33株不存在Yr4DS基因被检测区段上的突变。实际上,在38株高感条锈病的个体中,28个植株未发现Yr4DS基因的有效点突变,但另外10株存在Yr4DS基因的有效点突变。据此开展卡方拟合优度检验χ2=5.8,df=1,P=0.016,在α=0.05的显著水平下推翻H0假设。因此,Yr4DS基因影响粗山羊草的抗条锈病水平。另外,我们也测定了高感条锈病突变体中的RLK2基因,该基因位于Yr4DS基因远着丝粒端小于3kb的距离,但未发现任何有效点突变。与此相比,在携带Yr4DS基因但高感条锈病突变体中,Yr4DS基因发生点突变的频率为26.3%。可见,Yr4DS基因影响粗山羊草的抗条锈病水平。实施例6:转基因小麦和大麦的获得、表型分析及分子验证为开展小麦遗传互补实验,我们开展了携带Yr4DS等基因的FosmidF2-1或质粒PC1104,其插入序列为SEQIDNO.10;图4A的遗传转化,并利用到共转化载体PC174图4A。共转化载体携带Bar筛选标记Blocketal.1987.TheEMBOJournal6:2513-2518。PC1104携带RLK1、RLK2和Yr4DS三个基因,为获得个别基因表达的转基因植株,我们还利用限制性内切酶,包括BsrGI、NotI、XbaI和KpnI+XbaINewEnglandBiolabs,美国分别处理PC1104,然后开展酶切产物的遗传共转化。小麦幼胚培养和基因枪轰击转化参照Lv等人的流程Lvetal.2014.PLoSONE9:e94171。其中,Yr4DS基因载体PC1104和共转化载体PC174按3:1的摩尔比例混合。本发明选择可感染条锈病的小麦‘CB037’和大麦‘GoldenPromise’作为转化受体。对于小麦转化,取开花后7-14d左右的幼胚,进行幼胚表面灭菌,首先70%酒精含0.05%吐温-20处理5min,然后20%高乐氏漂白液RegularBleach,Oakland,CA,USA;额外添加了0.05%吐温-20处理15min,最后用灭菌水冲洗3-5遍。在超净台上剥取幼胚幼胚长度介于1-1.5mm,按盾片朝上摆放到诱愈培养基MS基本培养基4.3gL-1,麦芽糖40gL-1,维生素B10.5mgL-1,天冬氨酸0.15gL-1,2,4-D2mgL-1,硫酸铜0.78mgL-1,植物凝胶2.5gL-1,pH5.8,于22-23℃条件下暗培养4-6d。将幼胚转移到高渗培养基即诱愈培养基+蔗糖171.15gL-1,pH5.8处理4h,随后开展基因枪轰击。轰击处理20h后,将幼胚转移到恢复培养基等同于诱愈培养基,22-23℃条件下暗培养2wk。将幼胚衍生的胚性愈伤转移到分化培养基即诱愈培养基+6-苄氨基嘌呤0.1mgL-1+双丙氨膦3mgL-1,pH5.8,22-23℃和16h光照25μmolm-2s-1条件下培养2wk。将分化出的再生苗高度2-3cm转移至生根培养基MS基本培养基2.15gL-1,麦芽糖20gL-1,维生素B10.25mgL-1,天冬氨酸0.075gL-1,2,4-D1mgL-1,硫酸铜0.39mgL-1,植物凝胶2.5gL-1,双丙氨膦3mgL-1,pH5.8,并在同样环境条件下培养。待再生苗根系发育完全后,转为盆栽,在温室条件下种植。轰击处理采用PDS-1000He台式基因枪Bio-RadLaboratories,Hercules,CA,USA。轰击微粒混合物的准备步骤如下:在1.5ml的硅化离心管中加入2mg金粉直径0.6μm,再加入35μl无水乙醇,震荡混匀,离心收集12,000rpm,5sec,弃上清;加入200μl冰预冷的灭菌水,震荡混匀,离心收集12,000rpm,5sec,弃上清;加20μg质粒DNA浓度约1μgμl-1,再加入冰预冷的灭菌水至245μl,震荡混匀;然后加入250μl冰预冷的氯化钙2.5M,震荡混匀;最后加入50μl亚精胺1.45%,vv,4℃条件下震荡15-20min,离心收集12,000rpm,10sec,弃上清;再加入36μl冰预冷的无水乙醇,震荡混匀。吸取10μl金粉和DNA悬浊液至载体膜中央,无菌风干后,将载体膜金粉面朝下置入微粒发射器microcarrierlaunchassembly,该发射器位于可裂膜1,100psi下方3cm处。接受轰击的愈伤摆放到高渗培养基直径约3.5cm的区域,然后放置于载体膜下方6cm处。PDS-1000He基因枪的使用参照仪器说明书,轰击参数为1,300psi轰击压力和25mmHg真空度。本发明共轰击了8,380个小麦幼胚包括未经酶切的PC1104质粒处理的1,590个幼胚和经过酶切的PC1104质粒处理的6,790个幼胚,经过组培、筛选和移栽,成功获得来自170个幼胚的222棵株系。利用PCR引物,检测了RLK1RLK1-FP5和RLK1-RP5、RLK2RLK2-FP3和RLK2-RP4和Yr4DSYR4DS-FP1和YR4DS-RP1、YR4DS-FP3和YR4DS-RP3在222棵株系中的整合情况。进而使用RT-PCR引物,RLK1首轮引物RLK1-FP1和RLK1-RP2;二轮引物RLK1-FP3和RLK1-RP3、RLK2首轮引物RLK2-FP2和RLK2-RP2;二轮引物RLK2-FP3和RLK2-RP3和Yr4DSTV1首轮引物YR4DS-FP6和YR4DS-RP6,二轮引物YR4DS-FP7和YR4DS-RP7;TV4首轮引物YR4DS-FP8和YR4DS-RP3,二轮引物YR4DS-FP9和YR4DS-RP9确认各个目标基因的表达表3,图5。还利用RT-PCR引物Actin-FP和Actin-RP确认了内源参照ACTIN基因的表达。通过小麦条锈病测试,所有222棵株系后代中,只有表现Yr4DS转基因表达的2个株系高抗小麦条锈病表3,图4和5。综上所述,Yr4DS基因组全长表达框架SEQIDNO.10的表达提供小麦高抗条锈病的能力,将感病小麦转变为抗条锈病小麦。大麦上只开展了未经酶切的PC1104质粒的共转化,其转化步骤类似小麦,但组培、再生和筛选采用Hao等人的方法Haoetal.2018.MolecularPlantPathology19:1995-2010。基因枪轰击后,将处理后的幼胚转移至诱愈筛选培养基中,24℃黑暗培养14d;将分化出愈伤的幼胚转移至诱愈筛选培养基中,进行继代筛选培养,24℃黑暗培养14d;挑取明黄色的胚性愈伤继续转移至诱愈筛选培养基中,进行继代筛选培养,24℃黑暗培养14d;将生长旺盛的愈伤转移至过渡培养基中,诱导愈伤分化再生,培养5-10d即可再生出绿芽,培养条件为24℃,弱光,光照强度为2μmolm-2s-1;14d后将愈伤转移至再生培养基中,培养条件为24℃,16h光照8h黑暗,光照强度为35μmolm-2s-1;大约14d后,将生长健壮的再生苗转移至生根培养基,培养条件与上一步一致;待再生苗根系发育良好后,将其移栽到温室种植。大麦组培所使用的各种培养基如下:a诱愈培养基包括MS盐4.3gL-1,麦芽糖30gL-1,酶水解酪蛋白1gL-1,A溶液10mLL-1,植物凝胶3.5gL-1,pH=5.8、高压灭菌121℃,15min后加入潮霉素25mgL-;b过渡培养基包括MS盐2.7gL-1,麦芽糖20gL-1,谷氨酸0.75gL-1,B溶液5mLL-1,植物凝胶3.5gL-1,pH=5.8、高压灭菌后加入潮霉素25mgL-1,2,4-D2.5mgL-1和6BA0.1mgL-1;c再生培养基包括MS盐2.7gL-1,麦芽糖20gL-1,谷氨酸0.75gL-1,B溶液5mLL-1,植物凝胶3.5gL-1,pH=5.8、高压灭菌后加入潮霉素25mgL-1;d生根培养基包括MS盐4.3gL-1,麦芽糖30gL-1,酶水解酪蛋白1gL-1,A溶液10mLL-1,植物凝胶3.5gL-1,pH=5.8、高压灭菌后加入潮霉素25mgL-1。A溶液为肌醇35gL-1,脯氨酸69gL-1,硫酸铜0.12gL-1,VB10.1gL-1,pH=5.8。B溶液为硝酸铵33gL-1,肌醇20gL-1,VB10.08gL-1,pH=5.8。本发明共轰击了2,200个大麦幼胚,经过组培、筛选和移栽,成功获得来自300个幼胚的540棵株系。利用小麦上介绍的PCR引物,检测了RLK1、RLK2和Yr4DS在540棵株系中的整合和表达情况表3,图5;另外利用到Actin-FP和Actin-RP确认了内源参照ACTIN基因的表达。通过对大麦条锈菌株PSH-72的测试,在5棵阳性转基因株系后代中,所有表现Yr4DS转基因表达的3个株系高抗大麦条锈病表3,图4和5。综上所述,Yr4DS基因组全长表达框架SEQIDNO.10的表达提供小麦和大麦高抗条锈病的能力,将感病小麦和大麦分别转变为抗条锈病小麦和大麦。由此可见,将Yr4DS基因组全长表达框架SEQIDNO.10导入感条锈病小麦等麦族植物,可以创建高抗条锈病的植物,这将在小麦等麦族植物的抗条锈病育种中发挥作用。以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。SEQUENCELISTING110山东农业大学,四川农业大学120粗山羊草Yr4DS基因在麦族植物抗条锈病育种的应用130201816063170PatentInversion3.521012114266212DNA213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4001gcagctccacctaccgcagctggccggtcgtcggtccttgcatcgcatccaaggcgacgc60cggctccaccactgccgccaccactgattggtaccttaagctcccgtgaagctacaaggg120aggaggccccctcgcctcccatccattgttcctcaacagcttgggtgccgagctccccca180ccaccacactagacgcgctgggattcattggatccgctccagcgagcgagccggattcag240ttcaccgagattatattcacccttcccgtctgccaactgattacctcccttttcccattc300taccctgggtgaccactctgctcactctcactctttctttctcctctacgccggccgtgc360acagcgcgacgccgacgaccgcatccccccgacgttgccgcagccgccatacaagtagaa420ggaccgagatggcaacctctcccagctttgtgctacaccgcaagtactaggagcattgct480gaatgccggatatagatccccagatctaggccaagggtctcgctttctgctcactgttac540ttctatcatctctccaatcgatccaagttccagctcaagccactggaccctgcaactact600tgcgagacagcacgggatatctatctctgcaacaagatccgtctctcatctctctagtcg660attcaagtactagttcaagcgtgagcaaaccctgcaaccaatggcgggggttctggatgc720tttggcatcctacgtgaccaacatgctcaccgagatggctaaagaggaggtggccatgct780aatcggcgtgtctgacgggatcaaagacctaagcatcaagcttggggacctcaagaattt840ccttgctgatgctgataggaggaacattaccgatgatagtgtgcgggggtgggtgggcga900actgaagcgtgccatgtacttggccactgacatcgtcgacctatgtcagctcaaggccat960ggagcaaggtcaaacaaaggacagggcgtgccggtgccttaaccctctgctcttttgcat1020gcggaatcccctccacgcccacgacatcggcacccgcatcaagctgctcaaccaaaattt1080ggatgatatttgcaagaggggcagcagtttcaatttcatcaagctagaagcctaccaaga1140ccaaaagaccactcggtctcccgccactgaccggaaaacaaattcactgattgagcggtc1200cggtgtggttggagagaagatcgaggaggacacgagggcacttgtggaggtgcttacaag1260ggaggcggtaggtgacaagagtggtcgcctcattgtggtcgccattgttggtatcggagg1320gattggtaagactaccctcggcaagaaggtcttcaatgatgaggccatcgaaggcaagtt1380tactaagaagatatggcttagcatcacacaagatttcaccgatgttgagttgttaagtac1440gaccatcactgccattggggcagatcttcctggagggggtggggctccagacaaggccct1500acttgtcgatgctctcaagaacgccatcaaggacaagaagttctttcttgtactagatga1560cctgtgggatgtcgatgcatggaacaaacatctaatgactccctttagctatggtggccc1620cggtagtagagtcctcatcaccaccagacatgacactgtagcccgaagcatgaaagcctt1680tcatccctaccatcatgttgacaaattagctccacaagatgcttggtcgttgctcaagaa1740gcaggtagtcacaggagaggaaaatgaaccagaagttgatatgctagaagatattgggct1800gcagattatagcaaaatgtgatggcttaccacttgctgtaaaagttatgggtggactcct1860atgcaagaaggagaaaacacgacgtgattggcaagacgtcttgaatgatgatatgtggtc1920agtatctcaaatgtcaaaggaactaaattatgccatatatcttagctatgaggatttgtc1980cccttacttaaaacagtgcttcttgcacttctccctcaaaccaaaaaagacagtgataac2040tgatactgaaatggtgtccatgtgggttggtgaaggattggttgaaggagacacatatac2100tcgtagtttggaagaagggaataagtactataaggagctaatagtaaggaaccttataga2160ggtagatacaaagtaccctagtcaacttatttgcaacatgcatgatgttattcgctcatt2220tgctcaatttgtggctagggatgaaacactagtaggtcacaatggagatactatcaaaac2280aaatcttagatcaccgaattatcttagattatccatagaaacgaaaggagtgggatccga2340tgaatttgagtggagatatttaagagagcaaaaattgcttaggtctctaatattaactgg2400aaacctcaaaagtcagcctggggattcgttgactatcttcccaagtctacgtcttttgca2460tatagaatcagcaaatattgctgcactagttgaatctatgtaccaactcaagcatttgag2520atatttggcattaaagaggactgatatgtgtagactaccagagaacatccatgagatgaa2580attcctacagcatattagccttgaaggttgtgaaagtttcatgaaacttcctgatagtat2640tatcaagctgcaagggttgagatatcttgatatgggtgacacacgtgtaagtagtattcc2700taggggtttccgagctcttacaaatttgacttcactatttgggtttccagcctatattga2760tggtgactggtgtagtctggaagagttggggtctctttcccagctcaatgaactttcact2820acagagcctagaaaatgtatctagtgccttgttggctgaaaaggcaagggtaaatgcaaa2880gaaacaacttaccgtacttgctttaaaatgtggtggtagagtgggacatgggttggtcca2940aggagaggtctctgagtctaaggaggaggagcaaataattgaggcggtgtttgatgtgct3000ctgtcctcagccttgcatagaacacatcagaatagaaagatattttggtcgtcggctccc3060aggatggatggcgtccacagctatggtgcccctcgagagcttgaagattctatgcctcga3120acacctgccctgctgcacccaactcccagatggcttgtgcaggctcccgtatttggagtg3180gataaaagtgatgaatgctccagtaatcaagtgtattggtcctgaattcgttcaacagta3240caatcagctgcaccgtccttcatctcagttggctgctacgtttcccaaactccagatgtt3300ggaatttcacggaatggaggaatgggaggagtgggtttgggagacggaagtgaaagctat3360gcccttattggaggaacttcgtatcacttcttgcagactgagccgtatgcctccaggact3420tatgtctcatgcaatggctttgaagaagctaacaatatggagcgtccaatgtctccactc3480tctagagaactttgtttctgtagttgaactcgaattgggaaacatacctgaactggccat3540gatctccaatcttccaaaattacaaaaacttacaatcgagtgctgcccaaagctcgagat3600gctgcaggagatggctgcactccggagactcgagctgaccattttcaacagcgaaaatca3660acttccggtctacctgcagactgtgaagcctagtaatttgctgctgacctgcaacctagc3720ggtactcacttccatggctgagggtgaatctagctccgagtgggacaagttcagtcatat3780caaccacgttgaggcttatgcagaggatggagaagatgagaagaaatggcacgtgttcta3840cacatctgaatcctgcaacatagagacaaatattcatcaggatcgattggtcgaagaaga3900ggagtaggctgaaactccagccgaacgaggaaggaccatatgatgcaaggatatataagt3960atgtctgctgttacaacttcaactagttttgagatcgaatccatgaagggatacggggaa4020aagcatcagccataatgccatatacacttacctgcagagattcacacatcatactaattt4080cttgtgaagtgtgacatatacagttacgaatttagagatgtaaacttcgtggcggactat4140ccagctggtgaagaaacatggtgaccttggcaaggctctcctttgtttagtgcaagcaac4200taatggagtttagactactctgctttgctctgctctctgaaagcagctcaggttgcaggt4260cctctg426621022111068212PRT213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4002MetAlaGlyValLeuAspAlaLeuAlaSerTyrValThrAsnMetLeu151015ThrGluMetAlaLysGluGluValAlaMetLeuIleGlyValSerAsp202530GlyIleLysAspLeuSerIleLysLeuGlyAspLeuLysAsnPheLeu354045AlaAspAlaAspArgArgAsnIleThrAspAspSerValArgGlyTrp505560ValGlyGluLeuLysArgAlaMetTyrLeuAlaThrAspIleValAsp65707580LeuCysGlnLeuLysAlaMetGluGlnGlyGlnThrLysAspArgAla859095CysArgCysLeuAsnProLeuLeuPheCysMetArgAsnProLeuHis100105110AlaHisAspIleGlyThrArgIleLysLeuLeuAsnGlnAsnLeuAsp115120125AspIleCysLysArgGlySerSerPheAsnPheIleLysLeuGluAla130135140TyrGlnAspGlnLysThrThrArgSerProAlaThrAspArgLysThr145150155160AsnSerLeuIleGluArgSerGlyValValGlyGluLysIleGluGlu165170175AspThrArgAlaLeuValGluValLeuThrArgGluAlaValGlyAsp180185190LysSerGlyArgLeuIleValValAlaIleValGlyIleGlyGlyIle195200205GlyLysThrThrLeuGlyLysLysValPheAsnAspGluAlaIleGlu210215220GlyLysPheThrLysLysIleTrpLeuSerIleThrGlnAspPheThr225230235240AspValGluLeuLeuSerThrThrIleThrAlaIleGlyAlaAspLeu245250255ProGlyGlyGlyGlyAlaProAspLysAlaLeuLeuValAspAlaLeu260265270LysAsnAlaIleLysAspLysLysPhePheLeuValLeuAspAspLeu275280285TrpAspValAspAlaTrpAsnLysHisLeuMetThrProPheSerTyr290295300GlyGlyProGlySerArgValLeuIleThrThrArgHisAspThrVal305310315320AlaArgSerMetLysAlaPheHisProTyrHisHisValAspLysLeu325330335AlaProGlnAspAlaTrpSerLeuLeuLysLysGlnValValThrGly340345350GluGluAsnGluProGluValAspMetLeuGluAspIleGlyLeuGln355360365IleIleAlaLysCysAspGlyLeuProLeuAlaValLysValMetGly370375380GlyLeuLeuCysLysLysGluLysThrArgArgAspTrpGlnAspVal385390395400LeuAsnAspAspMetTrpSerValSerGlnMetSerLysGluLeuAsn405410415TyrAlaIleTyrLeuSerTyrGluAspLeuSerProTyrLeuLysGln420425430CysPheLeuHisPheSerLeuLysProLysLysThrValIleThrAsp435440445ThrGluMetValSerMetTrpValGlyGluGlyLeuValGluGlyAsp450455460ThrTyrThrArgSerLeuGluGluGlyAsnLysTyrTyrLysGluLeu465470475480IleValArgAsnLeuIleGluValAspThrLysTyrProSerGlnLeu485490495IleCysAsnMetHisAspValIleArgSerPheAlaGlnPheValAla500505510ArgAspGluThrLeuValGlyHisAsnGlyAspThrIleLysThrAsn515520525LeuArgSerProAsnTyrLeuArgLeuSerIleGluThrLysGlyVal530535540GlySerAspGluPheGluTrpArgTyrLeuArgGluGlnLysLeuLeu545550555560ArgSerLeuIleLeuThrGlyAsnLeuLysSerGlnProGlyAspSer565570575LeuThrIlePheProSerLeuArgLeuLeuHisIleGluSerAlaAsn580585590IleAlaAlaLeuValGluSerMetTyrGlnLeuLysHisLeuArgTyr595600605LeuAlaLeuLysArgThrAspMetCysArgLeuProGluAsnIleHis610615620GluMetLysPheLeuGlnHisIleSerLeuGluGlyCysGluSerPhe625630635640MetLysLeuProAspSerIleIleLysLeuGlnGlyLeuArgTyrLeu645650655AspMetGlyAspThrArgValSerSerIleProArgGlyPheArgAla660665670LeuThrAsnLeuThrSerLeuPheGlyPheProAlaTyrIleAspGly675680685AspTrpCysSerLeuGluGluLeuGlySerLeuSerGlnLeuAsnGlu690695700LeuSerLeuGlnSerLeuGluAsnValSerSerAlaLeuLeuAlaGlu705710715720LysAlaArgValAsnAlaLysLysGlnLeuThrValLeuAlaLeuLys725730735CysGlyGlyArgValGlyHisGlyLeuValGlnGlyGluValSerGlu740745750SerLysGluGluGluGlnIleIleGluAlaValPheAspValLeuCys755760765ProGlnProCysIleGluHisIleArgIleGluArgTyrPheGlyArg770775780ArgLeuProGlyTrpMetAlaSerThrAlaMetValProLeuGluSer785790795800LeuLysIleLeuCysLeuGluHisLeuProCysCysThrGlnLeuPro805810815AspGlyLeuCysArgLeuProTyrLeuGluTrpIleLysValMetAsn820825830AlaProValIleLysCysIleGlyProGluPheValGlnGlnTyrAsn835840845GlnLeuHisArgProSerSerGlnLeuAlaAlaThrPheProLysLeu850855860GlnMetLeuGluPheHisGlyMetGluGluTrpGluGluTrpValTrp865870875880GluThrGluValLysAlaMetProLeuLeuGluGluLeuArgIleThr885890895SerCysArgLeuSerArgMetProProGlyLeuMetSerHisAlaMet900905910AlaLeuLysLysLeuThrIleTrpSerValGlnCysLeuHisSerLeu915920925GluAsnPheValSerValValGluLeuGluLeuGlyAsnIleProGlu930935940LeuAlaMetIleSerAsnLeuProLysLeuGlnLysLeuThrIleGlu945950955960CysCysProLysLeuGluMetLeuGlnGluMetAlaAlaLeuArgArg965970975LeuGluLeuThrIlePheAsnSerGluAsnGlnLeuProValTyrLeu980985990GlnThrValLysProSerAsnLeuLeuLeuThrCysAsnLeuAlaVal99510001005LeuThrSerMetAlaGluGlyGluSerSerSerGluTrpAspLys101010151020PheSerHisIleAsnHisValGluAlaTyrAlaGluAspGlyGlu102510301035AspGluLysLysTrpHisValPheTyrThrSerGluSerCysAsn104010451050IleGluThrAsnIleHisGlnAspArgLeuValGluGluGluGlu10551060106521032113477212DNA213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4003gcagctccacctaccgcagctggccggtcgtcggtccttgcatcgcatccaaggcgacgc60cggctccaccactgccgccaccactgattggtaccttaagctcccgtgaagctacaaggg120aggaggccccctcgcctcccatccattgttcctcaacagcttgggtgccgagctccccca180ccaccacactagacgcgctgggattcattggatccgctccagcgagcgagccggattcag240ttcaccgagattatattcacccttcccgtctgccaactgattacctcccttttcccattc300taccctgggtgaccactctgctcactctcactctttctttctcctctacgccggccgtgc360acagcgcgacgccgacgaccgcatccccccgacgttgccgcagccgccatacaagtagaa420ggaccgagatggcaacctctcccagctttgtgctacaccgcaagtactaggagcattgct480gaatgccggatatagatccccagatctaggccaagggtctcgctttctgctcactgttac540ttctatcatctctccaatcgatccaagttccagctcaagccactggaccctgcaactact600tgcgagacagcacgggatatctatctctgcaacaagatccgtctctcatctctctagtcg660attcaagtactagttcaagcgtgagcaaaccctgcaaccaatggcgggggttctggatgc720tttggcatcctacgtgaccaacatgctcaccgagatggctaaagaggaggtggccatgct780aatcggcgtgtctgacgggatcaaagacctaagcatcaagcttggggacctcaagaattt840ccttgctgatgctgataggaggaacattaccgatgatagtgtgcgggggtgggtgggcga900actgaagcgtgccatgtacttggccactgacatcgtcgacctatgtcagctcaaggccat960ggagcaaggtcaaacaaaggacagggcgtgccggtgccttaaccctctgctcttttgcat1020gcggaatcccctccacgcccacgacatcggcacccgcatcaagctgctcaaccaaaattt1080ggatgatatttgcaagaggggcagcagtttcaatttcatcaagctagaagcctaccaaga1140ccaaaagaccactcggtctcccgccactgaccggaaaacaaattcactgattgagcggtc1200cggtgtggttggagagaagatcgaggaggacacgagggcacttgtggaggtgcttacaag1260ggaggcggtaggtgacaagagtggtcgcctcattgtggtcgccattgttggtatcggagg1320gattggtaagactaccctcggcaagaaggtcttcaatgatgaggccatcgaaggcaagtt1380tactaagaagatatggcttagcatcacacaagatttcaccgatgttgagttgttaagtac1440gaccatcactgccattggggcagatcttcctggagggggtggggctccagacaaggccct1500acttgtcgatgctctcaagaacgccatcaaggacaagaagttctttcttgtactagatga1560cctgtgggatgtcgatgcatggaacaaacatctaatgactccctttagctatggtggccc1620cggtagtagagtcctcatcaccaccagacatgacactgtagcccgaagcatgaaagcctt1680tcatccctaccatcatgttgacaaattagctccacaagatgcttggtcgttgctcaagaa1740gcaggtagtcacaggagaggaaaatgaaccagaagttgatatgctagaagatattgggct1800gcagattatagcaaaatgtgatggcttaccacttgctgtaaaagttatgggtggactcct1860atgcaagaaggagaaaacacgacgtgattggcaagacgtcttgaatgatgatatgtgcct1920atattgatggtgactggtgtagtctggaagagttggggtctctttcccagctcaatgaac1980tttcactacagagcctagaaaatgtatctagtgccttgttggctgaaaaggcaagggtaa2040atgcaaagaaacaacttaccgtacttgctttaaaatgtggtggtagagtgggacatgggt2100tggtccaaggagaggtctctgagtctaaggaggaggagcaaataattgaggcggtgtttg2160atgtgctctgtcctcagccttgcatagaacacatcagaatagaaagatattttggtcgtc2220ggctcccaggatggatggcgtccacagctatggtgcccctcgagagcttgaagattctat2280gcctcgaacacctgccctgctgcacccaactcccagatggcttgtgcaggctcccgtatt2340tggagtggataaaagtgatgaatgctccagtaatcaagtgtattggtcctgaattcgttc2400aacagtacaatcagctgcaccgtccttcatctcagttggctgctacgtttcccaaactcc2460agatgttggaatttcacggaatggaggaatgggaggagtgggtttgggagacggaagtga2520aagctatgcccttattggaggaacttcgtatcacttcttgcagactgagccgtatgcctc2580caggacttatgtctcatgcaatggctttgaagaagctaacaatatggagcgtccaatgtc2640tccactctctagagaactttgtttctgtagttgaactcgaattgggaaacatacctgaac2700tggccatgatctccaatcttccaaaattacaaaaacttacaatcgagtgctgcccaaagc2760tcgagatgctgcaggagatggctgcactccggagactcgagctgaccattttcaacagcg2820aaaatcaacttccggtctacctgcagactgtgaagcctagtaatttgctgctgacctgca2880acctagcggtactcacttccatggctgagggtgaatctagctccgagtgggacaagttca2940gtcatatcaaccacgttgaggcttatgcagaggatggagaagatgagaagaaatggcacg3000tgttctacacatctgaatcctgcaacatagagacaaatattcatcaggatcgattggtcg3060aagaagaggagtaggctgaaactccagccgaacgaggaaggaccatatgatgcaaggata3120tataaagattcacacatcatactaatttcttgtgaagtgtgacatatacagttacgaatt3180tagagatgtaaacttcgtggcggactatccagctggtgaagaaacatggtgaccttggca3240aggctctcctttgtttagtgcaagcaactaatggagtttagactactctgctttgctctg3300ctctctgaaagcagctcaggttgcaggtcctctgaagcagctgcgtttttctttttcttt3360tttcgcgggtgtctttcattcgttctctggaaaactatgtttatttctgatagatcaatc3420aatagtatactggaacactaaatactgttagaacaaacccaatcaaaatgtactaat34772104211408212PRT213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4004MetAlaGlyValLeuAspAlaLeuAlaSerTyrValThrAsnMetLeu151015ThrGluMetAlaLysGluGluValAlaMetLeuIleGlyValSerAsp202530GlyIleLysAspLeuSerIleLysLeuGlyAspLeuLysAsnPheLeu354045AlaAspAlaAspArgArgAsnIleThrAspAspSerValArgGlyTrp505560ValGlyGluLeuLysArgAlaMetTyrLeuAlaThrAspIleValAsp65707580LeuCysGlnLeuLysAlaMetGluGlnGlyGlnThrLysAspArgAla859095CysArgCysLeuAsnProLeuLeuPheCysMetArgAsnProLeuHis100105110AlaHisAspIleGlyThrArgIleLysLeuLeuAsnGlnAsnLeuAsp115120125AspIleCysLysArgGlySerSerPheAsnPheIleLysLeuGluAla130135140TyrGlnAspGlnLysThrThrArgSerProAlaThrAspArgLysThr145150155160AsnSerLeuIleGluArgSerGlyValValGlyGluLysIleGluGlu165170175AspThrArgAlaLeuValGluValLeuThrArgGluAlaValGlyAsp180185190LysSerGlyArgLeuIleValValAlaIleValGlyIleGlyGlyIle195200205GlyLysThrThrLeuGlyLysLysValPheAsnAspGluAlaIleGlu210215220GlyLysPheThrLysLysIleTrpLeuSerIleThrGlnAspPheThr225230235240AspValGluLeuLeuSerThrThrIleThrAlaIleGlyAlaAspLeu245250255ProGlyGlyGlyGlyAlaProAspLysAlaLeuLeuValAspAlaLeu260265270LysAsnAlaIleLysAspLysLysPhePheLeuValLeuAspAspLeu275280285TrpAspValAspAlaTrpAsnLysHisLeuMetThrProPheSerTyr290295300GlyGlyProGlySerArgValLeuIleThrThrArgHisAspThrVal305310315320AlaArgSerMetLysAlaPheHisProTyrHisHisValAspLysLeu325330335AlaProGlnAspAlaTrpSerLeuLeuLysLysGlnValValThrGly340345350GluGluAsnGluProGluValAspMetLeuGluAspIleGlyLeuGln355360365IleIleAlaLysCysAspGlyLeuProLeuAlaValLysValMetGly370375380GlyLeuLeuCysLysLysGluLysThrArgArgAspTrpGlnAspVal385390395400LeuAsnAspAspMetCysLeuTyr40521052112853212DNA213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4005gcagctccacctaccgcagctggccggtcgtcggtccttgcatcgcatccaaggcgacgc60cggctccaccactgccgccaccactgattggtaccttaagctcccgtgaagctacaaggg120aggaggccccctcgcctcccatccattgttcctcaacagcttgggtgccgagctccccca180ccaccacactagacgcgctgggattcattggatccgctccagcgagcgagccggattcag240ttcaccgagattatattcacccttcccgtctgccaactgattacctcccttttcccattc300taccctgggtgaccactctgctcactctcactctttctttctcctctacgccggccgtgc360acagcgcgacgccgacgaccgcatccccccgacgttgccgcagccgccatacaagtagaa420ggaccgagatggcaacctctcccagctttgtgctacaccgcaagtactaggagcattgct480gaatgccggatatagatccccagatctaggccaagggtctcgctttctgctcactgttac540ttctatcatctctccaatcgatccaagttccagctcaagccactggaccctgcaactact600tgcgagacagcacgggatatctatctctgcaacaagatccgtctctcatctctctagtcg660attcaagtactagttcaagcgtgagcaaaccctgcaaccaatggcgggggttctggatgc720tttggcatcctacgtgaccaacatgctcaccgagatggctaaagaggaggtggccatgct780aatcggcgtgtctgacgggatcaaagacctaagcatcaagcttggggacctcaagaattt840ccttgctgatgctgataggaggaacattaccgatgatagtgtgcgggggtgggtgggcga900actgaagcgtgccatgtacttggccactgacatcgtcgacctatgtcagctcaaggccat960ggagcaaggtcaaacaaaggacagggcgtgccggtgccttaaccctctgctcttttgcat1020gcggaatcccctccacgcccacgacatcggcacccgcatcaagctgctcaaccaaaattt1080ggatgatatttgcaagaggggcagcagtttcaatttcatcaagctagaagcctaccaaga1140ccaaaagaccactcggtctcccgccactgaccggaaaacaaattcactgattgagcggtc1200cggtgtggttggagagaagatcgaggaggacacgagggcacttgtggaggtgcttacaag1260ggaggcggtaggtgacaagagtggtcgcctcattgtggtcgccattgttggtatcggagg1320gattggtaagactaccctcggcaagaaggtcttcaatgatgaggccatcgaaggcaagtt1380tactaagaagatatggcttagcatcacacaagatttcaccgatgttgagttgttaagtac1440gaccatcactgccattggggcagatcttcctggagggggtggggctccagacaaggccct1500acttgtcgatgctctcaagaacgccatcaaggacaagaagttctttcttgtactagatga1560cctgtgggatgtcgatgcatggaacaaacatctaatgactccctttagctatggtggccc1620cggtagtagagtcctcatcaccaccagacatgacactgtagcccgaagcatgaaagcctt1680tcatccctaccatcatgttgacaaattagctccacaagatgcttggtcgttgctcaagaa1740gcaggtagtcacaggagaggaaaatgaaccagaagttgatatgctagaagatattgggct1800gcagattatagcaaaatgtgatggcttaccacttgctgtaaaagttatgggtggactcct1860atgcaagaaggagaaaacacgacgtgattggcaagacgtcttgaatgatgatatgtggtc1920agtatctcaaatgtcaaaggaactaaattatgccatatatcttagctatgaggatttgtc1980cccttacttaaaacagtgcttcttgcacttctccctcaaaccaaaaaagacagtgataac2040tgatactgaaatggtgtccatgtgggttggtgaaggattggttgaaggagacacatatac2100tcgtagtttggaagaagggaataagtactataaggagctaatagtaaggaaccttataga2160ggatcgattggtggaagaagaggactagaccgaaactcctacggaacgagtttaggcgaa2220aggaccatatgatgcaaggatatacggatacctggaaagcatcagccataatgccatata2280cacttaacctgcagagattcggacaccacactaatttcttgcgaagtgtgacatatacac2340ttactaattttgagatgtaaacttcttggcgtactatctagctggtgaagaaacatggtg2400accttggcaaggctgtcctttgtgcagtgcaagcaactaatggagtttatgctactctgc2460tctgctctgctctgttctctgaaagcagctcaggaattccgaatcagagtgatgtatact2520gggaccaaacaaatgttggtgagatcctggtccacgaagatcacgatgatgacctgctga2580ctggatgttctagctatctttagtttcttcaattagtgaaatcaaattagatcctggcta2640atatggtcaaatccgtggcacgtgggtccacgaagaacacgtgggtctagctatctgttg2700tttcttcaatcagtgcagtcaaattaagttcaggttttccccgcaaaaaaaaaaatttaa2760gttcaggtttgttctggggctcaggacatatgttgtggatggaagcaactgtatcaggac2820atattggatcaggacatgaaactgtaattgcag28532106211495212PRT213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4006MetAlaGlyValLeuAspAlaLeuAlaSerTyrValThrAsnMetLeu151015ThrGluMetAlaLysGluGluValAlaMetLeuIleGlyValSerAsp202530GlyIleLysAspLeuSerIleLysLeuGlyAspLeuLysAsnPheLeu354045AlaAspAlaAspArgArgAsnIleThrAspAspSerValArgGlyTrp505560ValGlyGluLeuLysArgAlaMetTyrLeuAlaThrAspIleValAsp65707580LeuCysGlnLeuLysAlaMetGluGlnGlyGlnThrLysAspArgAla859095CysArgCysLeuAsnProLeuLeuPheCysMetArgAsnProLeuHis100105110AlaHisAspIleGlyThrArgIleLysLeuLeuAsnGlnAsnLeuAsp115120125AspIleCysLysArgGlySerSerPheAsnPheIleLysLeuGluAla130135140TyrGlnAspGlnLysThrThrArgSerProAlaThrAspArgLysThr145150155160AsnSerLeuIleGluArgSerGlyValValGlyGluLysIleGluGlu165170175AspThrArgAlaLeuValGluValLeuThrArgGluAlaValGlyAsp180185190LysSerGlyArgLeuIleValValAlaIleValGlyIleGlyGlyIle195200205GlyLysThrThrLeuGlyLysLysValPheAsnAspGluAlaIleGlu210215220GlyLysPheThrLysLysIleTrpLeuSerIleThrGlnAspPheThr225230235240AspValGluLeuLeuSerThrThrIleThrAlaIleGlyAlaAspLeu245250255ProGlyGlyGlyGlyAlaProAspLysAlaLeuLeuValAspAlaLeu260265270LysAsnAlaIleLysAspLysLysPhePheLeuValLeuAspAspLeu275280285TrpAspValAspAlaTrpAsnLysHisLeuMetThrProPheSerTyr290295300GlyGlyProGlySerArgValLeuIleThrThrArgHisAspThrVal305310315320AlaArgSerMetLysAlaPheHisProTyrHisHisValAspLysLeu325330335AlaProGlnAspAlaTrpSerLeuLeuLysLysGlnValValThrGly340345350GluGluAsnGluProGluValAspMetLeuGluAspIleGlyLeuGln355360365IleIleAlaLysCysAspGlyLeuProLeuAlaValLysValMetGly370375380GlyLeuLeuCysLysLysGluLysThrArgArgAspTrpGlnAspVal385390395400LeuAsnAspAspMetTrpSerValSerGlnMetSerLysGluLeuAsn405410415TyrAlaIleTyrLeuSerTyrGluAspLeuSerProTyrLeuLysGln420425430CysPheLeuHisPheSerLeuLysProLysLysThrValIleThrAsp435440445ThrGluMetValSerMetTrpValGlyGluGlyLeuValGluGlyAsp450455460ThrTyrThrArgSerLeuGluGluGlyAsnLysTyrTyrLysGluLeu465470475480IleValArgAsnLeuIleGluAspArgLeuValGluGluGluAsp48549049521072112609212DNA213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4007gcagctccacctaccgcagctggccggtcgtcggtccttgcatcgcatccaaggcgacgc60cggctccaccactgccgccaccactgattggtaccttaagctcccgtgaagctacaaggg120aggaggccccctcgcctcccatccattgttcctcaacagcttgggtgccgagctccccca180ccaccacactagacgcgctgggattcattggatccgctccagcgagcgagccggattcag240ttcaccgagattatattcacccttcccgtctgccaactgattacctcccttttcccattc300taccctgggtgaccactctgctcactctcactctttctttctcctctacgccggccgtgc360acagcgcgacgccgacgaccgcatccccccgacgttgccgcagccgccatacaagtagaa420ggaccgagatggcaacctctcccagctttgtgctacaccgcaagtactaggagcattgct480gaatgccggatatagatccccagatctaggccaagggtctcgctttctgctcactgttac540ttctatcatctctccaatcgatccaagttccagctcaagccactggaccctgcaactact600tgcgagacagcacgggatatctatctctgcaacaagatccgtctctcatctctctagtcg660attcaagtactagttcaagcgtgagcaaaccctgcaaccaatggcgggggttctggatgc720tttggcatcctacgtgaccaacatgctcaccgagatggctaaagaggaggtggccatgct780aatcggcgtgtctgacgggatcaaagacctaagcatcaagcttggggacctcaagaattt840ccttgctgatgctgataggaggaacattaccgatgatagtgtgcgggggtgggtgggcga900actgaagcgtgccatgtacttggccactgacatcgtcgacctatgtcagctcaaggccat960ggagcaaggtcaaacaaaggacagggcgtgccggtgccttaaccctctgctcttttgcat1020gcggaatcccctccacgcccacgacatcggcacccgcatcaagctgctcaaccaaaattt1080ggatgatatttgcaagaggggcagcagtttcaatttcatcaagctagaagcctaccaaga1140ccaaaagaccactcggtctcccgccactgaccggaaaacaaattcactgattgagcggtc1200cggtgtggttggagagaagatcgaggaggacacgagggcacttgtggaggtgcttacaag1260ggaggcggtaggtgacaagagtggtcgcctcattgtggtcgccattgttggtatcggagg1320gattggtaagactaccctcggcaagaaggtcttcaatgatgaggccatcgaaggcaagtt1380tactaagaagatatggcttagcatcacacaagatttcaccgatgttgagttgttaagtac1440gaccatcactgccattggggcagatcttcctggagggggtggggctccagacaaggccct1500acttgtcgatgctctcaagaacgccatcaaggacaagaagttctttcttgtactagatga1560cctgtgggatgtcgatgcatggaacaaacatctaatgactccctttagctatggtggccc1620cggtagtagagtcctcatcaccaccagacatgacactgtagcccgaagcatgaaagcctt1680tcatccctaccatcatgttgacaaattagctccacaagatgcttggtcgttgctcaagaa1740gcaggtagtcacaggagaggaaaatgaaccagaagttgatatgctagaagatattgggct1800gcagattatagcaaaatgtgatggcttaccacttgctgtaaaagttatgggtggactcct1860atgcaagaaggagaaaacacgacgtgattggcaagacgtcttgaatgatgatatgtggat1920cgattggtggaagaagaggactagaccgaaactcctacggaacgagtttaggcgaaagga1980ccatatgatgcaaggatatacggatacctggaaagcatcagccataatgccatatacact2040taacctgcagagattcggacaccacactaatttcttgcgaagtgtgacatatacacttac2100taattttgagatgtaaacttcttggcgtactatctagctggtgaagaaacatggtgacct2160tggcaaggctgtcctttgtgcagtgcaagcaactaatggagtttatgctactctgctctg2220ctctgctctgttctctgaaagcagctcaggaattccgaatcagagtgatgtatactggga2280ccaaacaaatgttggtgagatcctggtccacgaagatcacgatgatgacctgctgactgg2340atgttctagctatctttagtttcttcaattagtgaaatcaaattagatcctggctaatat2400ggtcaaatccgtggcacgtgggtccacgaagaacacgtgggtctagctatctgttgtttc2460ttcaatcagtgcagtcaaattaagttcaggttttccccgcaaaaaaaaaaatttaagttc2520aggtttgttctggggctcaggacatatgttgtggatggaagcaactgtatcaggacatat2580tggatcaggacatgaaactgtaattgcag26092108211471212PRT213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4008MetAlaGlyValLeuAspAlaLeuAlaSerTyrValThrAsnMetLeu151015ThrGluMetAlaLysGluGluValAlaMetLeuIleGlyValSerAsp202530GlyIleLysAspLeuSerIleLysLeuGlyAspLeuLysAsnPheLeu354045AlaAspAlaAspArgArgAsnIleThrAspAspSerValArgGlyTrp505560ValGlyGluLeuLysArgAlaMetTyrLeuAlaThrAspIleValAsp65707580LeuCysGlnLeuLysAlaMetGluGlnGlyGlnThrLysAspArgAla859095CysArgCysLeuAsnProLeuLeuPheCysMetArgAsnProLeuHis100105110AlaHisAspIleGlyThrArgIleLysLeuLeuAsnGlnAsnLeuAsp115120125AspIleCysLysArgGlySerSerPheAsnPheIleLysLeuGluAla130135140TyrGlnAspGlnLysThrThrArgSerProAlaThrAspArgLysThr145150155160AsnSerLeuIleGluArgSerGlyValValGlyGluLysIleGluGlu165170175AspThrArgAlaLeuValGluValLeuThrArgGluAlaValGlyAsp180185190LysSerGlyArgLeuIleValValAlaIleValGlyIleGlyGlyIle195200205GlyLysThrThrLeuGlyLysLysValPheAsnAspGluAlaIleGlu210215220GlyLysPheThrLysLysIleTrpLeuSerIleThrGlnAspPheThr225230235240AspValGluLeuLeuSerThrThrIleThrAlaIleGlyAlaAspLeu245250255ProGlyGlyGlyGlyAlaProAspLysAlaLeuLeuValAspAlaLeu260265270LysAsnAlaIleLysAspLysLysPhePheLeuValLeuAspAspLeu275280285TrpAspValAspAlaTrpAsnLysHisLeuMetThrProPheSerTyr290295300GlyGlyProGlySerArgValLeuIleThrThrArgHisAspThrVal305310315320AlaArgSerMetLysAlaPheHisProTyrHisHisValAspLysLeu325330335AlaProGlnAspAlaTrpSerLeuLeuLysLysGlnValValThrGly340345350GluGluAsnGluProGluValAspMetLeuGluAspIleGlyLeuGln355360365IleIleAlaLysCysAspGlyLeuProLeuAlaValLysValMetGly370375380GlyLeuLeuCysLysLysGluLysThrArgArgAspTrpGlnAspVal385390395400LeuAsnAspAspMetTrpIleAspTrpTrpLysLysArgThrArgPro405410415LysLeuLeuArgAsnGluPheArgArgLysAspHisMetMetGlnGly420425430TyrThrAspThrTrpLysAlaSerAlaIleMetProTyrThrLeuAsn435440445LeuGlnArgPheGlyHisHisThrAsnPheLeuArgSerValThrTyr450455460ThrLeuThrAsnPheGluMet46547021092118844212DNA213粗山羊草'PI511383'(Aegilopstauschii'PI511383')4009tgatcatcttcacacggaagaaccaagatcgttaacttggaaagataagttaaggatcac60aaccgaaacatgcaaagctcttgcataccttcactctgctgtttcagtccctgtaataca120tagagatatcaagccttccaacatacttcttgatgatgccttgacagcaaaagtgtcaga180ctttggagcttcaaggtacattgccatgaatcaaacaggaacaacaactgcagtgcaagg240aactataggttacttggatcctacatattattatagcagacgcctcacagaaagtagcga300tgtttatagctttggagttcttcttattgaattgcttactaggaggaagccatctttgta360tagatccaatgaaggcgttgggcttgtcatggagttcgttgcactacttgcagaaggcaa420tctatccgaaatactagacccaccagttgtagaagagggaggtagcaaagtcgaagacgt480agctaatctagctgtgtcgtgcgtgaaactgagagcagaggagcgaccaactatgaggca540agtggagatggcgctggaagctctccaagcacgcaaggagcatgtcatgggtgatttgat600agaagaaacaaacgggaagaaacatgcagcaccaaattgtccatcgactagcaaacgacc660aaagggaagtggagcaagcagatgctatagccaagaagaagaggtggtgttatccgcaac720attccctcgatagttttgcttgcccggaagtgatttttagtgatgttgtattctttctac780tatttggaatgagcgtgtgctattgtatgaatatatgagttacacataactctagtgtat840tatttaaaataaatttgaatgcacccaactctgttgtgctaattttgcatgttgtcgcgc900aatcagcatttggtcttgatgatcgacagttcatcactagtggaaaaagtgttagtcaca960atttttgttggtggcgcaccgactcctaacaacggtagtactattttttccaaataatgg1020tggcgtcggacaatttggtacgacattgatttgagcataccgctggcatagaatttttct1080cgtaccacataggcattttctgagtttttttccgtgtgctttcgttttctatagcgtggc1140ccattaggcatacgacagcggtatccattaggcccacgcgagcaaaataaaactacccta1200tccagtaaagatcagtgtaggtccgattcactaacctatcattttcgcctcttcttcatc1260ccccttctcgaccgccccgccaccgccactgcccgatgacgccgctcttcgccgccggaa1320ctgcgcggccgccgccaactccggcaaggtgctacactgtgtcatgtttggtcgataggt1380gctacgcacgacaaatcctttgcgtctggatggacgagcgcaggacgatagcgtcgtcgg1440gcgtcgtggtggcatcgacggaccggtaaggatgatgcggatctatctcctgaagatggg1500tcagcggtccgatgatgatggcggcttctaaaacgtgtgcatgtgttgtacgcattttgt1560ctgctgcgccggtgttagttctgatacattatgtggatggatcggcgacgacactggttt1620tagatatgggagtgagagccctccacattatcgagttttttagatgtgagtagtggtttc1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cgaaaaaagaatagtgcttgcagtgttgcggaggagct39240agatgtgtcatcccaatagtgggagaagggttaaggagggtatcgagctagggagagatg39300gtgggtctgtgtggtgggtgtgtgaaccgctgcagccttagtgaggagcgtgtggttaca39360agggaccatctagcttttttccgtttggatgagtgcacgtggttacagaagccctagcat39420tagtggggagacagccaggcctaggacacagcagaattatgtgatttagcagcacaaacc39480aagtgtctgttggcatgcttgttcgaaggccaaggttcaatcactatggaatttt395352101121122212DNA213人工序列40011tgtgtcatgtttggtcgatagg222101221120212DNA213人工序列40012tcctcccttgtagcttcacg202101321124212DNA213人工序列40013gcttccttgacttaaatttcaccg242101421122212DNA213人工序列40014ccacatatcatcattcaagacg222101521122212DNA213人工序列40015agatgagaagaaatggcacgtg222101621123212DNA213人工序列40016ccagtatacatcactctgattcg232101721120212DNA213人工序列40017atattcacccttcccgtctg202101821120212DNA213人工序列40018cttgccaatcacgtcgtgtt202101921120212DNA213人工序列40019gcaccgtccttcatctcagt202102021120212DNA213人工序列40020tgcttttccccgtatccctt202102121121212DNA213人工序列40021tagttcaagcgtgagcaaacc212102221120212DNA213人工序列40022ccatgtttcttcaccagctg202102321122212DNA213人工序列40023ctgtagttgaactcgaattggg222102421120212DNA213人工序列40024atggctgatgcttttccccg202102521120212DNA213人工序列40025actacttgcgagacagcacg202102621122212DNA213人工序列40026gaagcatgaaagcctttcatcc222102721121212DNA213人工序列40027tccattagttgcttgcactgc212102821120212DNA213人工序列40028tgagagacggatcttgttgc202102921120212DNA213人工序列40029agtagttgcagggtccagtg202103021120212DNA213人工序列40030aaagctcgagatgctgcagg202103121120212DNA213人工序列40031tccgagtgggacaagttcag202103221122212DNA213人工序列40032cacaggagaggaaaatgaacca222103321121212DNA213人工序列40033caagaaggagaaaacacgacg212103421120212DNA213人工序列40034tgtggctagggatgaaacac202103521120212DNA213人工序列40035catcatatggtccttcctcg202103621122212DNA213人工序列40036gcaaaatgtgatggcttaccac222103721120212DNA213人工序列40037actagtgtttcatccctagc202103821120212DNA213人工序列40038tgtgcactgtctttgcaagc202103921120212DNA213人工序列40039gtgtagtcccaaacgacgtg202104021120212DNA213人工序列40040gcatgatgtacggcttctca202104121120212DNA213人工序列40041gagtggagacattggacgct202104221120212DNA213人工序列40042gatgaagatagggatgccgg202104321120212DNA213人工序列40043agaacttctgtctcagcgcc202104421121212DNA213人工序列40044tagaacaacatagttgggtgc212104521120212DNA213人工序列40045gtgtcggagactttcaagtc202104621119212DNA213人工序列40046gatgtcggccctgtgagaa192104721120212DNA213人工序列40047tttctgcttcggggactgtg202104821122212DNA213人工序列40048aacagaaacaattcaccatggc222104921120212DNA213人工序列40049agcgagtgatatagatgcgc202105021120212DNA213人工序列40050tgcaaatggccagagttcac202105121120212DNA213人工序列40051cttcacatgtgcacatgtcc202105221123212DNA213人工序列40052tattcatacaatagcacacgctc232105321120212DNA213人工序列40053tctgcaagagcacccatagc202105421120212DNA213人工序列40054aaaatcacttccgggcaagc202105521121212DNA213人工序列40055gtcaaataatacagtcggggc212105621123212DNA213人工序列40056tgaaggtatgcaagagctttgca232105721120212DNA213人工序列40057acacaggtatgacacgcacc202105821120212DNA213人工序列40058caagcctgcgagcttgattg202105921120212DNA213人工序列40059tatgccagcggtcgaacaac202106021120212DNA213人工序列40060ggaacagcacctcagggcac2021061211727212DNA213粗山羊草'PI511383'(Aegilopstauschii'PI511383')40061tgtgtcatgtttggtcgataggtgctacgcacgacaaatcctttgcgtctggatggacga60gcgcaggacgatagcgtcgtcgggcgtcgtggtggcatcgacggaccggtaaggatgatg120cggatctatctcctgaagatgggtcagcggtccgatgatgatggcggcttctaaaacgtg180tgcatgtgttgtacgcattttgtctgctgcgccggtgttagttctgatacattatgtgga240tggatcggcgacgacactggttttagatatgggagtgagagccctccacattatcgagtt300ttttagatgtgagtagtggtttcgggtggcttgatgtatattcctgttcgacctttgttg360aataataaataaagatgaccgtatgcatcgattgatgcagaggccggtgttttaacctcc420tttttgaaaggaaaagtactatgttagtagagtgcttagctgattaaggatgaaaactgc480aatagctccttactttccctggtaaaactacaaatatcacaaatgcaaacccacccatcc540cggtagcctcgtcaaattaaaataaaaaaaaatataagaaaaggaaaatgcgaccggcgg600ctagcagctccacctaccgcagctggccggtcgtcggtccttgcatcgcatccaaggcga660cgccggctccaccactgccgccaccactgattggtaccttaagctcccgtgaagctacaa720gggagga72721062211361212DNA213粗山羊草'PI511383'(Aegilopstauschii'PI511383')40062gcttccttgacttaaatttcaccgtaacacgagttcatcaagtttgtatatgaaattagg60aagatattaaacaatattttcatttgtatgcatgatgtacggcttctcaatttgtggatt120attccggacttaagtttagcaattatcagggattgcatccagcttctcaattagtttcta180cttgcaggtagtcacaggagaggaaaatgaaccagaagttgatatgctagaagatattgg240gctgcagattatagcaaaatgtgatggcttaccacttgctgtaaaagttatgggtggact300cctatgcaagaaggagaaaacacgacgtgattggcaagacgtcttgaatgatgatatgtg360g36121063211858212DNA213粗山羊草'PI511383'(Aegilopstauschii'PI511383')40063agatgagaagaaatggcacgtgttctacacatctgaatcctgcaacatactttggtttcc60ctctctttcaatctactagctaataatattcttccttaatgtaatctttgttggttccat120gcatattgttatcatcagtattatataggatagctgaatgtatctcccactattttgttt180tgcttgccaggatcgattggtggaagaagaggactagaccgaaactcctacggaacgagt240ttaggcgaaaggaccatatgatgcaaggatatacgtatgtctactgttatagcttctact300agttttgggtattcttacctagtgccatgaccattggaatctcatgtttaatattatgat360cgaatccatgcagggatacctggaaagcatcagccataatgccatatacacttaacctgc420agagattcggacaccacactaatttcttgcgaagtgtgacatatacacttactaattttg480agatgtaaacttcttggcgtactatctagctggtgaagaaacatggtgaccttggcaagg540ctgtcctttgtgcagtgcaagcaactaatggagtttatgctactctgctctgctctgctc600tgttctctgaaagcagctcaggttgcagcttctccattgtggcgtttttctttttctttt660tcacgggtgttttgcgttggttctctgaaaaactatgttttttttgatagaccagtcaat720agtgtactggtatttcagcttttaatttgtactgcaaccacacaaatgactgtcagagag780tgtagtttattcaagcttccggctgctgatttgtaactgtatgtcataggaattccgaat840cagagtgatgtatactgg858
权利要求:1.一种抗条锈病基因,其特征在于,为以下a-j中任一项所述的核酸:a核酸,其由SEQIDNO.1所示的碱基序列组成;b核酸,其由SEQIDNO.3所示的碱基序列组成;c核酸,其由SEQIDNO.5所示的碱基序列组成;d核酸,其由SEQIDNO.7所示的碱基序列组成;e核酸,其由SEQIDNO.9所示的碱基序列组成;f核酸,其由SEQIDNO.10所示的碱基序列组成;g核酸,其由编码SEQIDNO.2所示蛋白质的碱基序列组成;h核酸,其由编码SEQIDNO.4所示蛋白质的碱基序列组成;i核酸,其由编码SEQIDNO.6所示蛋白质的碱基序列组成;j核酸,其由编码SEQIDNO.8所示蛋白质的碱基序列组成。2.一种由权利要求1所述的抗条锈基因编码的蛋白,其特征在于,所述蛋白的氨基酸序列如SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示。3.携带权利要求1所述的抗条锈病基因的重组表达载体、转基因细胞系或基因工程菌。4.如下a-f中任一项所述的DNA片段作为抗条锈病基因在植物育种或小麦条锈病防治中的应用;aSEQIDNO.1、SEQIDNO.3、SEQIDNO.5或SEQIDNO.7所示的cDNA片段;b编码SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示氨基酸序列的cDNA片段;cSEQIDNO.9或SEQIDNO.10所示的DNA片段;d编码SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示氨基酸序列的DNA片段;ecDNA片段或DNA片段,其编码的蛋白在功能上与SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示的蛋白等价,但在氨基酸序列上存在一个、数个或数十个氨基酸的替换、删除或插入;fcDNA片段或DNA片段,其在严格条件下与a或c的DNA片段杂交,且编码SEQIDNO.2、SEQIDNO.4、SEQIDNO.6或SEQIDNO.8所示的蛋白。5.如下1-4中任一项所述的DNA片段通过调控抗条锈病基因表达在植物育种或麦族植物条锈病防治中的应用;1DNA片段,其转录产物在植物细胞中上调SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的表达;2DNA片段,其翻译产物在植物细胞中上调SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的表达;3DNA片段,其转录产物在植物细胞中对SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的转录RNA具有上调作用;4DNA片段,其翻译产物在植物细胞中对SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因编码的蛋白具有上调作用。6.含有SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的重组表达载体、转基因细胞系或基因工程菌或权利要求2所述的蛋白在培育条锈病抗性提高或降低的麦族植物中的应用;优选的,所述麦族植物为小麦或大麦。7.一种抗条锈病小麦或大麦的培育方法,其特征在于,所述培育方法包括:将SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因转入小麦或大麦中,获得抗条锈病小麦或大麦;或者上调小麦或大麦基因组中SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的表达,筛选得到条锈病抗性提高的小麦或大麦植株;优选的,将抗条锈病基因转入小麦或大麦中的方法包括:聚乙二醇法、农杆菌介导法或基因枪轰击法;优选的,上调小麦或大麦基因组中的抗条锈病基因的表达的方法包括:导入能够激活或提高抗条锈病基因的转录水平或翻译水平或蛋白活性的DNA片段;或者控制特异小RNA分子的合成,上调抗条锈病基因mRNA的积累。8.一种培育条锈病抗性降低的小麦或大麦的方法,其特征在于,所述方法包括:抑制小麦或大麦基因组中SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示的抗条锈病基因的表达,筛选得到条锈病抗性降低的小麦或大麦植株;优选的,抑制小麦或大麦基因组中的抗条锈病基因的表达的方法包括:突变或敲除小麦或大麦中SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示基因的全部或者部分序列;或者使用干扰RNA干扰SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示基因的表达;或者使用基因沉默系统使SEQIDNO.1、SEQIDNO.3、SEQIDNO.5、SEQIDNO.7、SEQIDNO.9或SEQIDNO.10中至少一项所示基因沉默。9.一种用于鉴定权利要求1所述小麦抗条锈病基因的PCR标记,其特征在于,所述分子标记有三个,分别命名命名为Yr4DS-PM、Yr4DS-GM和Yr4DS-TM,其核苷酸序列分别如SEQIDNO.61、SEQIDNO.62和SEQIDNO.63所示。10.用于扩增权利要求8所述PCR标记的引物,其特征在于,扩增Yr4DS-PM的引物的序列如SEQIDNO.11和SEQIDNO.12所示;扩增Yr4DS-GM的引物的序列如SEQIDNO.13和SEQIDNO.14所示;扩增Yr4DS-TM的引物的序列如SEQIDNO.15和SEQIDNO.16所示。11.权利要求9所述的PCR标记或权利要求10所述的引物在如下任一项中的应用:1小麦或大麦抗条锈病基因的鉴定;2植物育种;3小麦或大麦条锈病防治。12.一种获得携带权利要求1所述抗条锈病基因的植物细胞的方法,其特征在于,通过转基因或基因组编辑手段而获得。13.一种获得携带权利要求1所述抗条锈病基因的植株的方法,其特征在于,由权利要求12所述的方法获得的植物细胞再生成苗。
百度查询: 山东农业大学 四川农业大学 粗山羊草Yr4DS基因在麦族植物抗条锈病育种的应用
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