海南本地稻来源的广谱抗稻瘟病新基因图位克隆与功能分析

Blast disease caused by the fungal pathogen Magnaporthe oryzae is the most severe diseases of rice. The use of resistant rice varieties continues to be the most economical and effective way to combat this disease. Exploitation, cloning and utilization of major resistance genes is the basis of breeding for resistance. One rice landrace, 63xuan, from Hainan Island, exhibits broad-spectrum resistance to rice blast. A novel dominant resistance gene, designated Xuan63, in 63xuan against 40 blast isolates collected from 6 provinces controls partial resistance. It was roughly mapped to a 183-kb region on the long arm of chromosome 11, flanked by markers RM27306 and RM11-12S. In the present study, based on fine mapping, bioinformatics analysis of the target mapping region will be carried out, and the DNA sequences of all candidate genes will be isolated by PCR. By comparison the structures and expression profiles of candidate genes, the most reasonable candidate gene will be screened out, and its full-length cDNA will be isolated by RT-PCR. Then according to the result of transgene function analysis of the most reasonable candidate gene, the broad-spectrum blast resistance gene Xuan63 will be cloned with own intellectual property right. Fuctional marker will be developed based on the genetic sequence, which is precise, easier and highly ef?cient in molecular breeding. We are going to develop new varieties through molecular marker-assisted selection and provide the new gene source for the rice disease-resistant breeding. The rice materials containing resitance gene Xuan63 were inoculated with more blast isolates from different parts of the world to better understand the broad-spectrum resistance to rice blast, which is a breeding guide in utilization of genetic resources. It was the gene expression analyses of gene Xuan63 and relative genes in plant disease resistance pathways, which were to elucidate gene function, resistance signal transduction pathway and the mechanism of rice blast resistance controlled by Xuan63 in real-time quantitative PCR and northern blotting analyses.

稻瘟病是水稻最主要病害，培育抗病品种是防治病害最经济有效的手段，挖掘、克隆和利用新基因是抗病育种的基础。我们从海南本地水稻资源中，挖掘了1个广谱抗稻瘟病新基因，对国内6个省份的40个稻瘟病菌生理小种表现部分抗性，已将其定位在水稻第11号染色体长臂上，位于标记RM27306和RM11-12S之间183kb区域内，暂命名Xuan63。本研究对基因精细定位区域进行生物信息学分析，预测和确认最合理的候选基因，利用RT-PCR技术获得基因全长cDNA，进行转基因功能验证，获得具有自主知识产权的广谱抗稻瘟病新基因；开发基因功能标记，培育一批育种新材料，准确、方便地利用新基因提高分子育种效率；深入鉴定基因抗谱，为育种利用提供指导；利用定量PCR及Northern blot技术，分析稻瘟病菌侵染前后，基因Xuan63及其抗病途径中相关基因表达变化，研究基因功能和抗病信号传导途径，揭示广谱抗稻瘟病机制。

发掘广谱抗稻瘟病新基因是抗稻瘟病育种的基础。本研究鉴定了广谱抗稻瘟病新基因Xuan63的抗谱；在基因精细定位的基础上，进行生物信息学分析，结合抗病和感病水稻材料间候选基因表达差异分析，确认了4个候选基因；设计候选基因引物并扩增基因组DNA，对候选基因进行序列比对；并构建4个候选基因的crispr/cas9双靶点基因敲除突变体及转基因互补突变体用于进一步的基因功能分析。根据精细定位结果，确定了抗性基因紧密连锁的标记RM27306和RM11-12S为抗性基因分子标记辅助选择的有效标记，用常用的恢复系昌恢T1211、昌恢851、R800及优良常规稻品种黄华占、美香占与抗源Xuan63杂交、回交，并进行分子标记辅助选择，已获得具有渗入基因区段及表型的BC4F3近等基因系22份。本研究对于利用新基因提高育种效率，创制自主知识产权的新基因具有重要意义。同时为揭示广谱稻瘟病抗性机制奠定了基础。