油菜BnNRsPGIP2基因在菌核病抗性调控中的功能及机理研究

Sclerotinia stem rot (SSR) caused by the necrotrophic fungus Sclerotinia sclerotiorum is a major disease in rapeseed (Brassica napus) worldwide. There is no genetic source of complete plant resistance to this broad host range pathogen known to date. Cultispecies “Ning RS-1” resistant to SSR was screened from thousands of Rapeseed varieties. Studies have shown that BnNRsPGIP2 is associated with the disease resistance of “Ning RS-1”. This suggests that studying the functional of BnNRsPGIP2 gene is helpful for SSR resistance breeding of rapeseed. This study intend to analys the resistance to PG protein and the interaction between BnNRsPGIP2 and PG protein through express BnNRsPGIP2 gene in vitro. Then, confirm the SSR resistance in transgenic Arabidopsis and Brassica napus when overexpressing BnNRsPGIP2 gene. Thirdly, find out the regulatory sequences, target genes and interacting proteins relate to hormone signal pathway from “Ning RS-1” or S. sclerotiorum through mRNA-seq, sRNA-seq and yeast-two-hybrid. The results of this study will not only lay the foundation of building a signal transduction network of disease resistance, provide the basis for explaining the molecular mechanism of S. sclerotiorum regulating phytohormone pathways and plant defence response, but also leads to a new approach to improve the resistant ability of rapeseed and promote the SSR resistance breeding.

菌核病是油菜的主要病害，到目前为止尚未在十字花科及其野生同源植物中找到抗源。甘蓝型油菜宁RS-1，是我们经过多年筛选获得的抗菌核病材料。研究表明BnNRsPGIP2基因可能与宁RS-1的抗病性有关。研究BnNRsPGIP2基因的功能，有利于更好的利用宁RS-1进行油菜抗菌核病育种。本项目拟通过体外表达BnNRsPGIP2蛋白，研究其对PG蛋白的抗性及相互作用；通过转基因技术导入拟南芥和油菜感病品种进行异源表达，验证其抗性功能；以转BnNRsPGIP2基因油菜为材料，利用mRNA-seq和sRNA-seq联合分析，结合酵母双杂交筛库，挖掘宁RS-1中与信号通路相关调控序列、靶标基因和互作蛋白以探究其抗性机理。研究结果可为构建抗病信号调控网络奠定基础，为解析核盘菌调控宁RS-1信号通路和防御反应的分子机理提供依据，为应用油菜内源基因提高油菜的抗菌核病能力，进而为油菜抗菌核病育种开辟新的途径。

菌核病（Sclerotinia stem rot, SSR）是油菜的主要病害，常导致油菜严重减产和品质下降。长期以来，筛选抗、耐菌核病材料一直是油菜抗病育种的重要研究内容之一。经过数十年努力，已经得到了数份高耐菌核病的甘蓝型油菜种质，但真正的抗源至今未见报道。因此，挖掘现有高耐菌核病材料中的相关抗病基因或标记，是开展油菜抗病育种的首要选择。本研究，以抗菌核病种质宁Rs-1为材料，在前期研究的基础上，主要开展了三方面的研究：. A、将BnNRsPGIP2和BnJAR1基因分别导入到甘蓝型油菜Westar中，转基因油菜的菌核病抗性与对照相比明显提高；分别把BnNRsPGIP2和BnPGIP2蛋白与pGADT7-SsPG1载体做酵母双杂交，通过滴度试验发现BnNRsPGIP2与SsPG1的互作比BnPGIP2更强，说明单碱基突变提高了BnPGIP2基因与SsPG1基因的互作能力。. B、针对BnNRsPGIP2与BnPGIP2之间的SNP位点，设计了3组PARMS基因分型引物，经PCR鉴定，获得一组标记可用于区分NRsBnPGIP2和BnPGIP2基因；将这组标记用于检测中油821/宁RS-1和宁RS-1/Westar这两个F2群体，分型成功率达到95%以上；此外，这组标记还可以应用于转NRsBnPGIP2基因油菜后代的分型与阳性植株鉴定。. C、以宁RS-1为材料，分别测定了核盘菌接种前、接种后6h、24h和48h的mRNA和sRNA转录组并获得了一些靶标候选基因；此外，以BnJar1基因（XM_013856053）构建了诱饵载体pGBKT7-BnJar1，并在油菜-核盘菌互作文库中进行筛选，获得了7个互作蛋白，注释到的蛋白质中有5个来自油菜，分别是BnAOC2、BnCOP9、BnDcoH、BnNIT2和BnTSJT1蛋白。GO富集分析表明，5个蛋白主要富集在激素合成、泛素化降解、低温和盐胁迫等生物学过程。还有2个蛋白来自核盘菌，分别是SsMFS和SsRho3蛋白，GO富集在跨膜运输活性和调控信号转导，推测与核盘菌的致病过程密切相关。. 研究结果为进一步解析宁Rs-1抗菌核病的机理，挖掘相关致病和抗病基因应用甘蓝型油菜抗菌核病分子标记选育奠定了坚实基础。