周麦22中抗叶锈病基因LrZH22的精细定位和图位克隆

Leaf rust is an important wheat disease and can cause significant yield losses in China. During the last few years, leaf rust has become more serious in the major wheat production regions with increasing temperatures. Isolating wheat leaf rust resistance genes will provide effective genes for controlling leaf rust with genetic manipulation and establish a foundation for researching molecular mechanism of leaf rust resistance and obtaining durable resistance based on biotechnology. Results from previous research showed that a new leaf rust resistance gene LrZH22 was identified in Chinese wheat cultivar Zhoumai 22 and mapped on chromosome bin 2BS1-0.53-0.75. The two closest flanking SSR loci were Xbarc55 and Xgwm374 with genetic distances of 2.5 cM and 4.7 cM from LrZH22, respectively. In the present study, firstly high throughput SNP chips, transcriptome sequencing and comparative genomics will be used for fine mapping of LrZH22, and then construction of wheat BAC genomic library, chromosome walking technique and wheat genome sequence will be used for physical mapping of LrZH22, confirming physical interval containing LrZH22 and obtaining candidate gene for LrZH22, lastly mutant, transgenic technology and RNA silencing will be used for verifying the function of candidate gene for LrZH22.

小麦叶锈病是影响我国小麦生产的重要病害之一，随着气候变暖近年来有加重的趋势。克隆小麦抗叶锈病基因可为通过基因操作控制我国小麦叶锈病提供抗病基因，同时为研究小麦叶锈病的抗病机理、利用生物技术手段获得小麦对叶锈病的持久抗性奠定基础。前期研究结果表明小麦品种周麦22携带一个新的抗叶锈病基因LrZH22，位于2BS bin 2BS1-0.53-0.75染色体区段，与SSR位点Xbarc55、Xgwm374连锁距离分别为2.5、4.7cM。本研究拟利用630K的SNP芯片、转录组测序和比较基因组学技术对LrZH22进行精细定位，同时通过小麦基因组BAC文库构建和染色体步移技术，并结合小麦基因组序列信息构建抗病基因LrZH22的物理图谱，确定目标基因所在的物理区间、克隆LrZH22的候选基因，最后利用突变体、转基因技术及RNA沉默技术对候选基因进行功能验证。

小麦叶锈病是由小麦叶锈菌（Puccinia triticina）引起，严重威胁着我国小麦粮食安全，定位和克隆小麦抗叶锈病基因，可为培育抗病品种及研究抗病分子机理奠定基础。本课题组先期在周麦22中定位了一个苗期温敏抗叶锈病新基因LrZH22，距离两侧最近的SSR标记为Xbarc55 and Xgwm374，距离LrZH22分别为2.4cM和4.8cM。进一步利用标记检测7213个周麦22/郑州5389的F2:3家系，获得332个重组家系。结合重组体的表型数据并利用中国春参考序列设计特异SSR引物对LrZH22进行精细定位，构建了LrZH22所在区域的精细遗传连锁图谱，将LrZH22定位到70kb的物理区段。该区间有3个注释基因，其中1个是典型的与抗病相关的NBS-LRR基因作为LrZH22的候选基因。课题组同时选取抗病亲本周麦22和8个纯感EMS突变家系进行MutRenSeq，结果将抗病基因LrZH22成功定位在2BS的特定物理区间，该结果与精细定位的NBS-LRR类抗病基因结果一致。MutRenSeq测序后发现5个感病突变体在LrZH22的候选基因上均发生了G/A的SNP突变导致抗病功能丧失。LrZH22全长23.154kb，存在5种剪切方式，ORF共含有3219个碱基，编码1073个氨基酸。基因LrZH22在抗病亲本周麦22中受叶锈菌强烈的诱导表达，而在感病亲本中诱导表达量低。与此同时，我们为了更加准确的定位该基因蛋白的作用位置，进行了亚细胞定位试验，结果LrZH22-gfp融合蛋白在细胞核中表达。此外，开发出一个LrZH22的功能SNP标记，可以有效地检测出供试小麦品种是否携带该抗病基因。利用农杆菌介导技术成功将基因LrZH22遗传转化至感病小麦品种Fielder，获得了T0和T1转基因小麦植株，且对其进行抗性鉴定和功能验证。所克隆的抗病基因及其分子标记可用于小麦抗叶锈病育种。迄今为止，本项目在国内外核心期刊发表研究论文全文12篇，其中SCI 收录8篇；核心期刊4篇；参加国际学术会议专题2次，国内学术会议8次。 培养了博士生1名，硕士生7名。