普通野生稻抗细菌性条斑病基因BLS1的图位克隆和功能分析

Bacterial leaf streak (BLS) of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc), has become the fourth devastating disease in rice, and caused severe damage to rice product in China. Growing resistant varieties is the most effective way to manage this disease, and cloning of disease resistance gene is critical for applying molecular breeding techniques to make the breeding of rice with resistance to BLS more efficient. 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 BLS resistance gene BLS1, originated from common wild rice (Oryza rufipogon Griff.) in Guangxi will be cloned. Metabolomics and genes expression profiles and resistance spectrum analysis will be carried out to elucidate the mechanism of BLS resistance controlled by BLS1.Moreover, gene targeted markers available for marker assisted selection in BLS-resistant rice breeding will be developed, and breeding value of transgene of BLS1 will be evaluated. As results, a BLS resistance gene isolated by map-based cloning with own intellectual property right, breeding materials and gene information will be available for BLS resistant rice breeding and further research of BLS resistant genes.

水稻细菌性条斑病（简称细条病）是水稻第四大病害，对我国水稻生产造成严重危害。培育抗病品种是防治该病害最为经济有效的方法，而克隆抗性基因是利用分子育种技术高效率培育抗病品种的关键。本研究在基因精细定位的基础上,对目的基因所在区域进行生物信息学分析，完成各候选基因的预测和分离，进行抗、感病水稻间候选基因结构和表达差异分析，确认最合理的候选基因，利用RT-PCR技术获得其全长cDNA，然后通过转基因功能验证，克隆出源自广西普通野生稻的主效抗细条病基因BLS1；再分析基因抗谱，研究细条病侵染前后水稻的基因表达和代谢组成分变化情况，阐明该抗病基因的作用机理；此外，根据基因序列，开发出在育种中应用便利的目的基因标记，进行BLS1转基因育种价值验证。本研究通过图位克隆将获得具有自主知识产权的抗细条病基因，积累一批育种材料、基因资源和分子信息，为提高水稻抗细条病育种效率和深入研究抗细病基因提供关键基础。

水稻细菌性条斑病（简称细条病）是水稻第四大病害，对我国水稻生产造成严重危害。培育抗病品种是防治该病害最为经济有效的方法，而克隆抗性基因是利用分子育种技术高效率培育抗病品种的关键。本研究在基因精细定位的基础上,对目的基因所在区域的4个候选基因进行表达分析，结果表明OsMPK6（LOC_Os06g06090）是BLS1最理想的候选基因；利用Agilent捕获平台对目标区域进行测序，发现目目的基因在抗感亲本和抗感后代单株之间存在丰富的基因结构差异；反转录CDNA，设计引物，克隆候选基因OsMPK6全长CDNA，利用CRISPR/Cas9 技术研究基因功能，结果表明BLS1负调控稻株对细菌性条斑病的抗性。根据基因序列，开发出在育种中应用便利的目的基因标记M5860和M6200，获得25份农艺性状优良抗细条病基因敲除材料。本研究所获得基因、育种材料基因资源和分子信息，为提深入研究抗细病基因提供关键基础。