稻曲病菌有性繁殖过程中交配型基因座MAT1-1作用机制及其互作因子的功能研究

Rice false smut is one of the major diseases, caused by Villosiclava virens, which heavily affected on the production safety and quality of rice; sexual reproduction process of V. virens played an important role in infection cycle, however, the molecular mechanism ofsexual reproduction is still unconfirmed. We cloned the mating type locies of V. virens and analysis theirs sequence characteristics. Based on these results, we want carry out some study works as follows: 1) by using RFP and GFP as nuclear localization tag to reveal nuclear phase distribution and clarify the function of mating type locus MAT1-1 during V. virens sexual reproduction; 2)furthermore, MAT1-1 locus key factor MAT1-1-3 will be employed as bait to screen 2-3 MAT1-1-3 strong interaction factors by using the yeast two-hybrid technology; 3)among these factors, 1-2 factors will be focused to study their function. The knockout and complementary mutants will be constructed to analyze their biological characteristics, and formation of sclerotia and sexual reproduction after combination with MAT1-2 strain after inoculated on rice; 4)at last，Real-time PCR and GFP fusion expression technology will be used to explain the expression characteristics and subcellular localization of MAT1-1-3 strong interaction factors. According these research works, it is expected to reveal the sexual cycle and molecular mechanism of sclerotia formation,sexual reproduction process in V.virens, alsoit is exhibited significant value in understanding the V. virens pathogenic mechanisms and disease epidemics rules; the results also provided reference for the development of the disease control strategy basis.

稻曲病是严重影响我国水稻安全生产和稻米品质的重大病害；申请者克隆了稻曲病菌交配型基因座并分析了序列特征，初步确定了其有性繁殖方式。为了进一步阐明该病原菌有性繁殖过程中交配型基因座MAT1-1的作用机制及其调控网络，本项目拟在前期研究基础上，利用GFP和RFP核定位稻曲病菌（分别含有基因座MAT1-1、MAT1-2），研究稻曲病菌在有性繁殖过程中的核相分布；采用酵母双杂交技术，以MAT1-1基因座中重要因子MAT1-1-3为诱饵，筛选2-3个与MAT1-1-3强互作因子,并以其中1-2因子为重点，构建敲除和回复突变体，明确其生物学特性及与MAT1-2型菌株组合接种后的有性繁殖特性；采用RT- PCR、GFP融合表达技术，揭示MAT1-1-3强互作因子实时表达特征和亚细胞定位。预期研究结果对揭示稻曲病菌的有性繁殖的分子机制、致病机理与病害流行规律有重要价值，并为制定稻曲病的控制策略提供依据。

水稻稻曲病是由稻曲病菌侵染引起的水稻主要病害之一，该病害的发生会严重影响水稻产量和品质。稻曲病菌有性繁殖产生的子囊孢子是水稻稻曲病可能的初侵染源之一，交配型基因座对真菌有性繁殖中的性别控制起着决定性作用。本项目通过自然田间稻曲球及菌核萌发、交配型基因座检测等试验，初步阐明了该病原菌交配型基因座 MAT1-1 在有性繁殖过程中具有重要作用。自然田间无菌核稻曲球中，存在MAT1-1和MAT1-2两个基因座的稻曲球占总数的13.3%，而且这种稻曲球的地区分布与采集到的菌核的地区分布一致。用砂培法萌发稻曲球显示所有供试无菌核稻曲球均不能萌发产生子囊孢子，而带菌核稻曲球有71.42%可已萌发。进一步用分子生物学检测菌核中的交配型基因座发现，含MAT1-1和MAT1-2两个基因座的菌核可以正常萌发，而仅含MAT1-1基因座的菌核不能正常萌发。上述研究说明菌核应该是两种交配型细胞发生质配核配等有性行为的重要场所，而当同时含有MAT1-1和MAT1-2两个基因座时，才具有潜在的可育性。两个基因座包含的五个基因的表达特点分析显示，五个基因均能在菌核中表达，而在菌丝中除MAT1-1-2外也均能表达，结果表明MAT1-1-2可能特异性参与有性繁殖过程。本项目研究结果从多角度分析稻曲病菌交配型基因座MAT1-1与稻曲病菌有性繁殖循环过程、稻曲病菌菌核形成的关系，可为揭示稻曲病菌菌核形成与稻曲病菌交配型关系，阐明稻曲病菌有性繁殖分子基础，制定稻曲病防控策略提供依据。