水稻条纹叶枯病抗病基因的精细定位、克隆及功能分析

Rice stripe disease is a viral disease caused by Laodelphax Striatellus carrying RSV，which can seriously affect rice production, especially in japonica rice. The fundamental way to control the disease should be cultivating new resistance varieties. Discovering and utilizing of the resistance genes are essential for resistance varieties breeding. At present, information of gene mapping have been accumulated in quantity , and some important gene locus have been found, but the resistance genes have not been cloned yet. The applicant of this study found a major QTL in chromosome 11 as having the preliminary work on elemantary mapping with DH cross C161/99B. Recently, by transcriptome sequencing technique, she have studied the genes which played key roles in the interaction of RSV and rice， and molecular mechanism of virus resistance were revealed preliminarily. Based on previous studies, this research try to carry out the followed studies in depth: The first, to construct DH backcross ,screen high-density polymorphic locus by whole genome sequencing technique, and map the resistance genes site-specificly. The second, to identify candidate genes according differently express and functional annotation of genes. The third, to clone the resistance genes , verify their functions by genetic transformation and explore the molecular mechanism of resistance to virus.This study is important for resistance varieties breeding and control of rice virus disease.

水稻条纹叶枯病是灰飞虱携带水稻条纹病毒（RSV）引起的病毒病害，该病大发生可严重影响水稻（粳稻）的生产。从根本上防控该病的为害必须培育抗病品种，而抗病基因的发掘和利用是抗病品种选育的关键。目前已具抗病基因定位研究的积累信息，且已界定多个主效抗性位点，但抗病基因一直未被克隆，限制了其利用效率。本申请于前期选用C161/99B DH群体进行基因初定位时于第11染色体上已找到了主效抗病位点，并利用转录组测序技术初步界定了RSV与水稻互作的关键调控基因，初步明确了抗性分子机理。本申请拟在前期研究基础上，系统开展：1）构建回交DH群体，结合全基因组重测序技术筛选高密度多态性标记，精细定位抗病基因；2）根据基因功能注释和诱导差异表达确定候选基因；3）通过遗传转化验证候选基因功能，探讨水稻抗病毒的分子机理。预期研究对水稻条纹叶枯病抗病品种选育及有效地控制水稻病毒病具有重要学术价值和实践意义。

水稻条纹叶枯病是对我国水稻生产具严重威胁的病毒病害，该病由介体灰飞虱携带的水稻条纹病毒引起（RSV），由于灰飞虱为迁飞性害虫，导致该病难于防控，选育并推广抗病品种是解决该病为害的有效途径。本研究主要围绕水稻对RSV及灰飞虱的抗性分子机理、抗病基因的精细定位及功能分析进行研究，结果表明：①茉莉酸合成在品种对灰飞虱抗性中可以起到关键的作用；而由过氧化物酶、细胞壁结构蛋白及纤维素合成酶等介导的物理屏障强化可能是水稻抵抗条纹病毒侵染的一种重要机制，过敏性坏死反应与水稻对条纹叶枯病的抗性无关。②利用所开发的多态性标记，在回交群体中筛选到5个重组子，结合其表型将恢复系C161抗条纹叶枯病的基因精细定位于Chr11：17.35-18.22Mb范围内，该区域内包含5个与植物抗病性相关的基因，将其分别构建CRISPR基因敲除载体并转化抗病品种C161，结果表明只有当Loc_Os11g30110被敲除时转基因后代对条纹叶枯病表现感病（心叶枯死），进而确认该基因为控制水稻条纹叶枯病抗性的关键基因。③根据定位区域在抗感亲本间的插入缺失差异开发了一对InDel标记（InDel-R），并利用其辅助育成了一个杂交粳稻新组合辽73优62，该品种已审定并在辽宁地区大面积推广。④利用简化基因组测序技术研究导致辽宁地区条纹叶枯病大发生的灰飞虱介体来源，研究结果表明致病群体系由外地迁飞而来，且很可能由几个地域的灰飞虱混合组成，最大的可能性是来自于江苏省和山东省。