野生甘蓝抗菌核病关键蛋白鉴定及抗病候选基因筛选

Uncovering the mechanism involved in the resistance against Sclerotinia sclerotiorum rot (infected by Sclerotinia sclerotiorum) in oilseed rape is very important to stimulate breeding focusing on resistance improvement. However, the lack of resistant resource in cultivated species has dramatically impeded the related research and the corresponding application. Fortunately, our group have discovered Brassica incana has more excellent resistance compared with ZhongYou 821, commonly recognized as tolerance.We mapped QTL for Sclerotinia resistance in it. We created BC3F2 population using the suceptible one as recurrent parent with the assistance of MAS. To more deeply understand the mechanism of resistance, we will construct resistant pools and susceptible ones using the most resistant individuals and the most susceptible ones in BC3F2 population; investigate the proteome change resulting from S. sclerotiorum challenge; locate the corresponding coding genes on the reference genome of B. oleracea; determine the resistance-related genes based on the QTL analysis results in our group; and screen out the candidated resistance genes based on the verfication of genome and transcriptome level. Finally, we will duscuss the resistance mechanism of B. incana. The implement of the project will be beneficial to deeply understand the resistance mechanism in B. incana and enrich the resistace theory, laying the foundation for the utilization of the resistance genes in B.oleracea to breed B. napus with higher level resistance against S. sclerotiorum.

深入了解油菜菌核病的抗病机制对有效促进油菜抗菌核病育种有重要意义。现有油菜中缺乏有效的菌核病抗源，限制了对其抗病机制的研究与应用。本课题组前期研究发现一份野生甘蓝（Brassica incana）对核盘菌有突出抗性，并对其进行了抗病QTL定位。我们以感病亲本为轮回亲本，结合分子标记辅助回交，构建了抗、感分离的BC3F2群体。为研究该高抗菌核病野生甘蓝的抗病机理，本项目申请拟在BC3F2中构建抗、感病混合池，运用蛋白质组技术鉴定接种前后抗、感混合池的差异蛋白，并将其编码基因锚定到甘蓝参考基因组上，结合前期QTL定位结果获取抗性相关基因，并在BC3F2群体中进行DNA和转录水平上的验证，筛选候选基因；同时我们还将通过生物信息学分析解析该野生甘蓝的抗病机理。该项目的实施将有助于深入研究野生甘蓝抗菌核病机理，丰富菌核病抗性理论，为利用高抗病野生甘蓝改良油菜菌核病抗性奠定理论基础。

（１）项目背景.油菜的近缘物种中的抗性资源具有改良甘蓝型油菜菌核病抗性的潜力。甘蓝型油菜为异源四倍体（AACC），由甘蓝（CC）和白菜（AA）杂交并经自然加倍而成。野生甘蓝（Brassica incana）对菌核病有突出的抗性，其抗性受以加性效应为主的多基因控制，最重要的抗病QTL位于C9染色体上。从蛋白质组层面深入挖掘其抗病机理具有重要意义。.（２）研究内容及重要结果.以高抗菌核病野生甘蓝（B. incana）与感病甘蓝杂交，以感病甘蓝为轮回亲本发展回交群体，进行前景选择和背景选择、抗性鉴定，发展BC3F1群体及BC3F2群体。 分别以BC3F2群体的叶片和茎杆材料，活体接种核盘菌，以LC-MS/MS技术，分析核盘菌胁迫下甘蓝的蛋白质组响应，对抗病相关蛋白进行定性与定量分析，并结合抗病QTL定位及转录组表达分析，筛选抗病候选基因 .在BC3F2群体中，挑选极抗材料、极感材料及亲本，分别以叶片、茎杆活体接种，构建叶片组抗、感池和茎杆组抗、感病池，分析核盘菌胁迫下叶片、茎杆蛋白质组响应。将差异蛋白编码基因映射到拟南芥基因组，进行GO和KEGG pathway分析。将叶片差异蛋白编码基因与课题组抗病QTL结果进行比较，有6个基因落在抗病QTL区间。通过与RNA-seq差异表达基因进行比较，这6个基因中有2个RNA-seq结果一致。将茎杆差异蛋白编码基因映射到拟南芥基因组，并GO和KEGG pathway分析。与课题组的QTL结果进行比较，18个基因落在QTL置信区间。其中有11个基因在scaffold0000132区间，有7个基因落在scaffold000030_P2区间。这18个基因同为RNA-seq结果产生的差异基因。项目组将这些候选基因开展功能研究。.（３）科学意义.项目组对核盘菌胁迫下甘蓝叶片、茎杆的蛋白质组响应进行了定性与定量分析，对产生的差异蛋白编码基因进行了GO及KEGG分析。将蛋白质组、转录组及QTL定位结合起来，综合多组学、多层次研究结果，初步筛选出甘蓝叶片抗菌核病基因2个、茎杆抗菌核病基因18个，为进一步进行甘蓝抗菌核病的分子机制研究奠定了基础。