棉花黄萎病菌中硫氨素转运蛋白及其在硫氨素代谢途径的调控机制

Verticillium dahliae, the pathogen of Verticillium Wilt of Cotton, has a very complex pathogenic mechanism. Our previous study indicated a close association between Verticillium dahilae thiamine transporter (VdThit) and the fungal pathogenicity. Transgenic Nicotiana benthamiana expressing dsRNA against VdThit (dsVdThit) were significantly resistant to V. dahliae. VdThit also plays an important role in the regulation of thiamine metabolism pathway along with VdSti35 and Vdthi11 genes. In this project, the VdThit gene deletion mutant and complementary strains have been obtained. Furthermore, gene disruption mutants and complementary strains for VdSti35 and Vdthi11 will be constructed further analyse the expression profiles to investigate the regulation among these genes. The pyruvate level and polypeptides differences will be detected using GC-MS and MALDI-TOF analysis, respectively. DsVdThit transgenic cotton would be generated using Agrobacterium mediated transformation (AMT) and inoculated with Vd-GFP, Vd-RFP, Vd-YFP and wild-type (Wt) strains. The related genes’ expression level, pyruvate level and polypeptides differences of strains, isolated fungi from transgenic and Wt plants, would be analyzed. Meanwhile, the florescence would be observed during different stages of fungal infection, such as invasion, colonization and spore production using confocal microscope. The main goal of this project is to investigate the molecular mechanism of VdThit in regulation of thiamine metabolism pathway and the interaction between transgenic cotton and fungi. The outcome of this project will provide a theoretical basis for thiamine metabolism and cultivation of transgenic cotton against V. dahliae.

棉花黄萎病的致病菌是大丽轮枝菌，致病机理非常复杂。我们已有的研究表明，其硫胺素转运蛋白（VdThit）与致病性密切相关；dsVdThit转基因烟草抗病性提高；与VdSti35和Vdthi11共同调控硫氨素代谢通路。本项目以VdThit突变体和回补体入手，构建VdSti35和Vdthi11突变体和回补体，进行表达谱分析基因间调控关系；利用GC-MS分析丙酮酸代谢变化及MALDI-TOF分析多肽和分泌蛋白差异。获得dsVdThit转基因棉花，分析与野生型植株抗病性差异，分离病原菌进行生理检测、表达谱分析、GC-MS分析以及MALDI-TOF分析。在已获得转YFP、GFP和RFP菌株的基础上，观察其在野生型及转基因棉花中侵染、定殖和繁殖过程。旨在探讨VdThit在硫氨素代谢通路中调控机制及转基因棉花与病原菌互作的分子机理，为棉花黄萎病中硫氨素代谢途径以及培育抗黄萎病棉花新种质材料提供理论基础。

棉花黄萎病是由大丽轮枝菌引起的一种土传维管束真菌病害，侵染范围非常广泛。鉴于其严重的危害性，探究大丽轮枝菌致病机理和寄主与病原菌之间的相互作用机制一直是研究的热点，从而提高寄主植物体对大丽轮枝菌的抗病性是亟待解决的关键问题。.项目前期发现，大丽轮枝菌硫胺素转运蛋白（VdThit）与病原菌生长发育、产孢量及致病性密切相关；与VdSti35和Vdthi11共同调控硫氨素代谢通路，并且dsVdThit转基因烟草抗病性显著提高。因此，本项目以大丽轮枝菌硫胺素代谢关键基因VdSti35和Vdthi11为研究对象，构建突变体和回补体，深入其在菌落直径、产孢量以及致病力中的作用，并通过表达谱分析基因间调控关系；利用GC-MS分析丙酮酸代谢变化及MALDI-TOF分析多肽和分泌蛋白差异，揭示该基因在病原菌次级代谢中的功能。同时，构建含有mcherry标签的大丽轮枝菌，通过分子手段以及荧光显微镜技术实时监测病原菌在植物体内的侵染、定殖和增殖过程。构建了病原菌致病关键基因VdFreB、VdAK、VdPLP和VdOGDH的突变体和回补体，并通过一系列分子生物学手段揭示了其在生长发育及致病过程中的生物学功能。此外，利用分别构建病原菌VdEG、VdPLP和VdPRMT1基因的HIGS质粒，并转化烟草，通过病情指数及分子生物学检测，获得了最佳RNAi区段。总之，本项目阐释了转基因烟草体内的dsRNA与病原菌互作机制，并开展了病原菌关键致病基因的生物学功能研究，为后续科研工作的开展提供了坚实的理论基础和数据支撑。