水稻纹枯病菌致病相关基因Rstpp和Rsgst的寄主诱导转基因沉默研究

To date, limited studies have examined the function of the genes associated with growth, development, pathogenicity and virulence of the fungal pathogen Rhizoctonia solani AG1-IA, and the mechanism of the interaction between the pathogen and the host plant rice cultivars is not well understood, thereby the rice cultivars are facing impediment of developing stable disease resistance with a high efficiency. Host Induced Gene Silencing (HIGS) is based on the silencing of genes in plant pathogens by expressing an RNAi construct against specific genes endogenous to the pathogen in the host plant. It has been previously employed to enhance the disease resistance of the crops and can be further used in the development of disease resistant crop breeding. In our previous study, we have successfully silenced the genes of Rstpp and Rsgst in R. solani using the virus induced gene silencing based on HIGS strategy. The infection incidence of the rice cultivars by R. solani was shown to be inhibited by the silenced expression of the two genes. In this project, we will study the functions of the two genes Rstp and Rsgst in R. solani based on the HIGS strategy. We will investigate the sheath blight disease index on the susceptible rice varieties infected by the virulent strain GD118 of R. solani. The efficacy of gene silencing and its impact on the rice cultivar disease resistance will also be further evaluated by estimating the efficiency of gene silencing, biomass statistics and histopathology. We expect to understand the relationship between the rice sheath blight disease resistance and the pathogenic genes of Rstpp and Rsgst and develop the new rice cultivar resistant to rice sheath blight. Our study will not only provide a theoretical basis for the study of rice resistance to R. solani, but also provide germplasm resources for the development of new resistant rice varieties to R. solani in practice, which will guide the the prevention and control of rice sheath blight most economically and efficiently.

目前有关水稻纹枯病菌生长发育和致病相关基因的功能及与水稻的互作机理尚不清楚，直接影响水稻高效稳定抗性品种的培育。寄主诱导基因沉默（HIGS）技术是通过沉默病原菌的靶标基因来提高作物的抗病性，在抗病育种中极具应用前景。本实验室前期通过病毒诱导基因沉默筛选出了水稻纹枯病菌Rstpp和Rsgst基因，它们的沉默抑制了病原菌的侵染。本项目将以水稻纹枯病菌强致病力菌株GD118和水稻纹枯病感病品种为材料，用HIGS技术验证Rstpp和Rsgst基因的功能，通过病情指数调查、沉默效率评估、生物量统计、组织学观察等一系列方法，评估基因沉默效果和水稻对纹枯病的抗性。预期结果将明确Rstpp和Rsgst基因与水稻抗纹枯病的关系，并可望获得抗纹枯病的水稻新品种。本研究不仅在理论上对水稻抗纹枯病的研究提供了基础，而且在实践上还将为水稻抗纹枯病新品种的培育提供抗病种质资源，为水稻纹枯病的防治提供最经济有效的措施。

水稻纹枯病是由立枯丝核菌（Rhizoctonia solani AG1-IA）引起的重要水稻真菌病害之一，该病每年在我国的发病面积超过2亿亩，对水稻生产造成严重的影响。目前仍没有高抗纹枯病的水稻品种。由于纹枯病菌遗传操作等的技术难题，有关该菌致病相关基因的功能也很少被报道。寄主诱导基因沉默技术是一种基于RNA干扰的技术，通过在寄主植物中表达病原物的重要基因的RNAi载体，寄主产生的小干扰RNA可以被病原菌吸收，从而沉默掉病原菌的致病基因，从而导致病菌致病力的减弱，从而提高了寄主的抗病性。因此本研究旨在建立一套适宜于筛选水稻纹枯病菌致病相关基因的技术体系，可以快速、批量筛选可能与致病相关的基因。在通过构建水稻表达载体，通过HIGS技术创制转基因水稻品种，旨在获得可以抗纹枯病的株系。. 本研究以R. solani AG1-IA编码蛋白序列进行预测，通过PHI等数据库，筛选到33个致病力有关的基因。根据这33基因的cDNA序列设计特异引物，并分别构建到TRV2载体中，获得病毒诱导基因沉默载体。通过转化根癌脓杆菌后用本氏烟草进行微注射，并优化其接种方法。通过统计病情指数，结果发现，瞬时转化Rsgst、Rsatg22、Rsmapk、Rsgsh和Rstps2五个基因的本氏烟草显著提高了对R. solani AG1-IA的抗性。选择这5个基因进行构建表达载体，用于转化水稻。通过生物量、基因表达水平和病情指数等指标发现，转化了Rstps2的转基因水稻也显著提高了对R. solani AG1-IA。小RNA测序结果也表明，所构建的转基因水稻比野生型菌株产生了更多的小片段干扰RNA序列。这些结果表明Rstps2基因与R. solani AG1-IA的致病有关。但转化Rsgst的水稻植株对R.solani AG1-IA的抗性不显著。.本研究通过优化接种体系，建立了一套R. solani AG1-IA与本氏烟草互作的体系，该体系可以用快速筛选R. solani的致病关键基因，可以用于其它融合群基因的筛选，因此也可以用R. solani与其它寄主的基因功能研究。本研究揭示Rstps2基因与R.solani AG1-IA的致病有关，说明海藻糖代谢途径在R. solani与水稻互作中发挥了重要作用。本研究也获得了可以高抗纹枯病的转基因水稻植株，对该病的抗病育种具有应用意义。