稻曲病菌T-DNA插入突变体B-766丧失厚垣孢子形成能力的分子机制

Rice false smut, which is caused by Ustilaginoidea virens(Cke.)Tak., has been becoming an important rice disease in China in the past few years. It was not only cause yield loss, but also polluted rice with ustiloxins. Chlamydospores, known as the important primary- and secondary-infection resources of rice false smut, are copiously produced on the surface of smut balls. To better understand molecular mechanism of chlamydospores production in U.virens, a chlamydospores formation defective mutant B-766, which derived from wild-type strain 70-22, was screened out from a T-DNA insertional mutant library as the candidate. In the preliminary study, a single copy of T-DNA in B-766 was confirmed by southern blot assay, and the T-DNA flanking sequence was cloned as well. In this project, T-DNA insertional mutated gene in B-766 will be cloned, and its role in this particular process will be uncovered. The project will be executed as follow: (1) genes, which predicted to be disrupted by T-DNA insertion, will be firstly colned by 5'-RACE and 3'-RACE; (2)Agrobacterium tumefaciens mediated transformation (ATMT) based gene deletion,complementation and over-expression methods have been established and will be used to validate the function of the putative key chlamydospore-formation gene; (3)Function of the key chlamydospore-formation gene(designated as chspo1) will be further uncovered via GFP-fusion expession based protein subcellular location,function domain prediction and validation, and three-dimension structure prediction; (4)Downstream relative genes of chspo1 gene were screened out by gene expression profile comparision between trophic growth mycelia and initial chlamydospore formation structure. Finally,the key chlamydospore-formation gene will be cloned and well characterized, the prospective results will help us taking insight into molecular mechanism of chlamydospores formation in Ustilaginoidea virens, and will be benefit for generating control strategy for rice false smut.The key chlamydospore-formation protein could also be recruited as a referenced target for fungicide molecular design.

稻曲病严重影响稻米产量和品质，已成为我国水稻上重要病害之一。厚垣孢子是该病害的初侵染来源和二次侵染源，然而决定稻曲病菌厚垣孢子形成的分子机制还不清楚。本项目前期获得了丧失厚垣孢子形成能力的T-DNA插入突变体B-766，利用southern杂交和TAIL-PCR克隆到单拷贝插入T-DNA的侧翼序列。为此，本项目将通过RACE法克隆B-766中突变基因编码区；借助基因敲除、回复和过量表达突变明确该基因在厚垣孢子形成中的关键作用；通过GFP融合表达蛋白的亚细胞定位、功能结构域分析验证和蛋白三维结构模拟明确其生物学功能；借助RNA-seq技术比较野生型菌株与基因敲除突变体在营养生长和厚垣孢子形成初期的转录表达谱，寻找下游相关基因，解析该基因在厚垣孢子形成中的作用机制。预期结果将从分子水平揭示稻曲病菌厚垣孢子形成机制，为稻曲病防治策略制定提供理论依据，为防控药剂的分子设计研发提供候选作用靶标。