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；稻曲病菌有性繁殖是其侵染循环的重要环节，但分子机制并不明确。申请者克隆了其交配型基因座并分析了序列特征。本项目拟在此基础上开展以下研究工作：1）采用酵母双杂交技术，以MAT1-1基因座中重要基因MAT1-1-3为诱饵，筛选2-3个与MAT1-1-3强互作因子；2）并以其中1-2个因子为重点，构建基因敲除和回复突变体，观察突变体与MAT1-2型菌株组合接种后的有性繁殖，明确其生物学功能；3）最后，采用Realtime PCR、GFP融合表达技术，明确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有性繁殖分子机制、致病机理与病害流行规律具有重要价值，并可为制定稻曲病控制策略提供依据。

Rice false smut, caused by Villosiclava virens, is one of the major rice diseases in China, and heavily affected on the rice production safety and quality; sexual reproduction process of V. virens played an important role in the infection cycle, however, the molecular mechanism of sexual reproduction is still uncovered. We cloned the mating type loci of V. virens and analyzed their DNA sequence features. Based on these results, we want carry out some work as follows: 1)MAT1-1 locus key factor MAT1-1-3 will be employed as bait to screen 2-3 MAT1-1-3 strong interaction factor using the yeast two-hybrid technology; 2)among these interabe focused on to study their function. To reveal the biological function of the chosen MAT1-1-3 interaction factors, The gene knockout and complementary mutants will be constructed to observe the processes of sclerotia formation and sexual reproduction after combined inoculation ction factors, 1-2 factor will on rice with MAT1-2 strain; 3)at last, Realtime PCR and GFP fusion expression technology will be used to reveal the expression patterns and subcellular localization of MAT1-1-3 interaction factors. These research works are expected to reveal the molecular mechanism of sclerotia formation and sexual reproduction of V. virens, and will exhibited important value in understanding its pathogenic mechanisms and disease epidemics of rice false smut; the results will also provide theoretical basis for the development of the disease control strategy.

提取稻曲病菌P1的总RNA，经电泳检测后，28s rRNA和18s rRNA条带清晰，浓度和纯度都较好。采用SMART反转录技术和LD-PCR将总RNA反转录为双链cDNA，cDNA片段范围是250-5000 bp，将cDNA与载体pGADT7-Rec转化进感受态酵母菌株Y187中，获得合格的稻曲病菌酵母双杂交AD文库。以不具有毒性和自我激活性的pGBKT7-MAT1-1-3为诱饵在稻曲病菌P1的cDNA文库中筛选互作因子，在四缺培养基上共挑取纯化了61个阳性克隆，成功测序46个，最终确定了7个与MAT1-1-3互作的候选蛋白，包括Putative O-methyltransferase，Putative SUMO ligase SizA，Type II restriction m6 adenine DNA methyltransferase或TATA box-binding protein-associated factor，Eukaryotic translation initiation factor 3 subunit 6，60S acidic ribosomal protein P2，Pre-mRNA Splicing Factor和Ankyrin repeat protein。收集菌核，萌发70 d时分为四个关键阶段：菌核萌发初期，子座顶端膨大期，子囊壳形成初期和子囊孢子成熟期。以α-tubulin-2为内参基因，以相应基因在无性营养体内的表达量为对照，利用qRT-PCR测定交配型基因MAT1-1-1、MAT1-1-2、MAT1-1-3和MAT1-2-8在菌核萌发四个关键阶段的表达水平，采用2？ ΔΔCT分析方法处理数据。结果显示各交配型基因在菌核萌发四个关键阶段的表达量明显低于对照组，这些基因可能在稻曲病菌的有性生殖过程中发挥着负调控的作用。另外MAT1-1-1、MAT1-1-3和MAT1-2-8在稻曲病菌菌核萌发四个阶段的表达量有明显的差异，表明其在有性生殖中发挥着相对重要的调控作用。这些结果为阐明稻曲病菌有性生殖的信号通路和分子机制具有重要的理论价值。