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。

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.