大丽轮枝菌ADP-ATP载体蛋白及其在能量代谢途径的调控机制

Verticillium dahliae causes a serious wilt disease of important crops and is difficult to control. The key genes involved in energy metabolism were interferenced led to the reduced pathogenicity in V. dahliae. In our previously study, transgenic tobacco producing dsRNA against ADP-ATP carrier protein (AAC) of Verticillium dahliae significantly inhibited AAC expression level, leading to reduced fungal biomass and disease index. In this project, dsAAC transgenic tobacco plants have been obtained. DsAAC expression were detected in the transgenic and wild-type (Wt) plants. At post-inoculation with V. dahliae, fungal biomass and disease index of plants were recorded. The expression profile of genes involved in energy metabolism would be determined in the strains isolated from plants. Meanwhile, ADP/ATP ratio will be detected by HPLC and secreted protein will be analyzed with MALDI-TOF. Furthermore, the transgenic and Wt plants will be inoculated with Vd-GFP, Vd-RFP and Vd-YFP strain. The fluorescence will be observed with confocal microscopy to analyze the fungal invasion, colonization and production. The main focus of this project is to analyze the interaction of dsAAC expressed in the transgenic plants with the fungi which will provide a theoretical basis for energy metabolism and the output would also be useful in protecting the crops against V. dahliae.

大丽轮枝菌可危害多种农作物，防治工作非常困难，干扰其能量代谢关键基因可有效降低病原菌致病能力。我们已有的研究表明，dsAAC转基因烟草可减少大丽轮枝菌ADP-ATP载体蛋白（AAC）转录水平，从而有效降低烟草体内病原菌生物量和病情指数。本项目拟以dsAAC转基因烟草入手，在大丽轮枝菌侵染后，分析植株病情指数及真菌生物量；分离病原菌并结合dsAAC表达情况，分析病原菌VdAAC及能量代谢相关基因间的调控关系；借助HPLC分析ADP/ATP比例变化以及MALDI-TOF分析多肽和分泌蛋白差异。同时，利用Vd-GFP、Vd-RFP和Vd-YFP接种野生型以及转基因烟草，通过激光共聚焦显微镜来实时观察病原菌侵入、定殖和繁殖过程，明确转基因烟草对病原菌的防御作用。本研究拟探讨转基因烟草体内的dsRNA与病原菌互作机制，来揭示AAC在能量代谢途径的调控机制，为大丽轮枝菌防治工作提供一定的理论基础。

棉花黄萎病是一种土传、维管束系统性病害，被称为“棉花癌症”，其致病菌为大丽轮枝菌。项目前期发现，ADP-ATP载体蛋白（AAC）基因与病原菌的致病力密切相关，dsAAC转基因烟草可显著提高植物抗病能力以及降低植物病情指数。因此，本项目以dsAAC转基因烟草为研究对象，首先将病原菌再次从转基因植物中进行分离，借助MALDI-TOF发现了病原菌体内非核体糖小肽发生了明显的变化；通过测序技术，发现了siRNA可通过植物进入病原菌体内。同时，利用将mcherry转化至大丽轮枝菌体内，揭示了病原菌在植物体内的侵染、定殖和增殖过程。通过表型测定和致病力测定，并结合代谢通路中相关基因的转录差异，鉴定了VdOGDH基因在生长发育及致病过程中的生物学功能。此外，通过HIGS技术获得了病原菌VdEG基因的最佳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，并开展了相关基因的功能分析，为后续研究的开展提供了坚实的工作基础。