稻曲病菌交配型基因座MAT1-2位点调控其有性生殖的分子机制研究

Rice false smut, caused by Ustilaginoidea virens, is an important disease of rice in the worldwide, also heavily reduced the quantity and quality of rice. The sexual reproduction process of U. virens played an important role in the infection cycle. However, the molecular mechanism of sexual reproduction is still uncovered. We have cloned the mating-type idiomorphs of U. virens (MAT1-1 and MAT1-2), and proved this pathogen as a heterothallic ascomycetous fungus. To further elucidate the mechanisms of mating-type idiomorph MAT1-2 involving in sexual reproduction, we will carry out studies in this project as follows: 1) Construct single-gene knockout, double-gene knockout and complementary mutants of MAT1-2-1 and MAT1-2-8 mating-type genes in MAT1-2 idiomorph, respectively. Study the biological characteristics, sclerotia formation and sexual reproduction processes after inoculating these mutants in combination with MAT1-1 mating-type strains on rice; 2) Explain the temporal and spatial expression patterns of the mating-type genes in MAT1-2 idiomorph and subcellular localization of their protein, by using the real-time PCR and GFP fusion expression technologies respectively; 3) Analysis the transcriptomes of gene-deletion mutants and wild-type strain mentioned in “1)” to carry out the differentially expressed genes associated with the MAT1-2 idiomorph; The MAT1-2-1 and MAT1-2-8 will be employed as bait to screen 2-4 interaction factors by using yeast two-hybrid technology; 4) Study will be further focused on the interaction factors mentioned in“3)”, to elucidate their function in the sexual reproduction of U. virens. Generally, this research project will be therefore of great significance to reveal the sexual life cycle and molecular mechanism of sexual reproduction process of U. virens, and also will be valuable for understanding the pathogenic mechanisms of U. virens and disease epidemic rules and development of the disease control strategy.

稻曲病严重影响我国水稻安全生产和稻米品质。有性生殖是稻曲病菌重要的繁殖方式，申请者克隆了稻曲病菌交配型基因座；本项目拟在前期研究基础上，阐明交配型基因座MAT1-2位点调控稻曲病菌有性生殖过程；构建MAT1-2位点基因MAT1-2-1和MAT1-2-8的敲除突变体及回复突变体，分析其在有性生殖过程中生物学特性；采用qRT-PCR和荧光蛋白标记技术明确MAT1-2-1 和MAT1-2-8的表达规律及表达产物亚细胞定位；通过转录组测序比较基因敲除突变体与野生型菌株差异，分析参与MAT1-2位点调控的基因，同时采用酵母双杂交、基因功能互补等方法筛选和验证MAT1-2-1、MAT1-2-8互作因子，解析相关因子参与MAT1-2位点调控稻曲病菌有性生殖的信号网络。预期结果对在分子水平认识稻曲病菌有性生殖和侵染循环过程、制定稻曲病防控策略有重要价值。

稻曲病是威胁我国水稻安全生产的重要病害；但对稻曲病侵染循环的研究并不是很清楚，特别是该病害的有性生殖过程及其调控机制并不清楚。因此，本项目主要是在研究交配型基因座MAT1-1位点功能的基础上，进一步对MAT1-2位点参与调控的分子机制和信号网络加以解析，更完整揭示稻曲病菌有性生殖过程。.本项目主要结果：1）阐明了MAT1-1-1对稻曲病菌的菌丝生长、分生孢子形态、有性生殖的起始是必须的；同时对盐和渗透胁迫耐受性增加，蛋白亚细胞定位在细胞核中。MAT1-1-2能够影响稻曲病菌的分生孢子产量、菌核形成和致病性；对盐及氧胁迫的耐受性增加，亚细胞定位显示蛋白信号主要在细胞质中。MAT1-1-3敲除后菌落生长减慢，无法形成菌核，致病性降低；对盐、氧胁迫和渗透胁迫耐受性增加，亚细胞定位显示蛋白信号主要在细胞质和细胞核中。.2）研究发现MAT1-1-3可能通过负调控假基因MAT1-2-1的表达来抑制稻曲病菌有性生殖方式从异宗配合向同宗配合的转换。酵母双杂交实验显示MAT1-1-1与MAT1-1-2、MAT1-1-3及MAT1-2-1进行互作，MAT1-1-2和MAT1-2-1与MAT1-2-8互作，说明交配型蛋白在有性生殖过程中可能形成复合体发挥作用。亚细胞定位显示MAT1-2-1定位在细胞核中，MAT1-2-8主要定位在细胞质中。.3）检测了MAT1-2-1和MAT1-2-8在稻曲病菌不同生长时期的表达量，结果表明在有性生殖过程中的核萌发形成菌丝时期表达量最高。亚细胞定位了MAT1-2-1和MAT1-2-8，并利用酵母双杂交系统检测了交配型基因的互作。我们鉴定到一些与MAT1-2-1可能相互作用的蛋白。对MAT1-2-1和MAT1-2-8的表达量、蛋白亚细胞定位和互作蛋白的研究，这些为了更好地研究MAT1-2位点的功能提供了基础。相关研究结果对解析稻曲病菌的遗传、变异和病害的流行预警都有重要的价值。.依托本项目撰写论文9篇。已经发表论文5篇，其中SCI论文2篇，中文核心期刊1篇，会议论文两篇；另有3篇SCI论文两篇返修，1篇在审稿中，1篇中文核心在审稿中。