薇甘菊萎蔫病毒遗传多样性分析及致病机理的研究

Mikania micrantha H. B. K. is an important invasive alien plant in South China. A new virus named Mikania micrantha wilt virus (MMWV) isolated by our team can strongly inhibit the growth of M. micrantha. To determine molecular variations and evolution mechanisms of MMWV, the genetic diversity of the virus population will be analyzed using RT-PCR, single-strand conformation polymorphism (SSCP), DNA sequencing and phylogenetic analysis tools. Comparative transcriptome analysis of differentially expressed genes in MMWV-infected and non-infected M. micrantha, and subsequent gene ontology and pathway analyses will be conducted to identify genes associated with MMWV pathogenesis. To gain further understanding concerning the pathogenesis and photosynthetic efficiency in different host plants, changes in cell ultrastructure will be observed by using a transmission electron microscope and photosynthetic activity will be evaluated in leaf tissues following MMWV infection. Co-expression studies with green fluorescence protein (GFP) and MMWV proteins genes will reveal the subcellular localization and expression of MMWV proteins in host plants during pathogenesis.Moreover, contents of endogenous auxin in infected and non-infected leaves will be determined by high performance liquid chromatography (HPLC) analysis. The results of the proposed work will provide detail information on the molecular mechanisms of growth inhibition and wilting of MMWV-infected M. micrantha plants, as well as useful epidemiological and molecular information on MMWV, which will lay a good foundation for use of this virus as biological control agent for invasive M. micrantha.

薇甘菊是华南地区重要的外来有害入侵植物，本课题组新发现的薇甘菊萎蔫病毒(Mikania micrantha wilt virus, MMWV)可强烈抑制薇甘菊生长。本项目拟应用RT-PCR和单链构象多态性(SSCP)研究MMWV在华南地区的分布及群体遗传多样性组成，探讨MMWV分子变异与演化机制；分析比较感染MMWV的薇甘菊转录组差异表达基因，阐明薇甘菊对MMWV侵染的分子响应机制；利用透射电镜观察不同寄主感染MMWV后细胞超微结构变化，测定MMWV侵染对不同寄主叶片光合效率的影响；利用GFP标记MMWV的功能蛋白，确定致病相关蛋白表达和亚细胞定位情况；利用HPLC分析MMWV侵染对薇甘菊关键内源激素含量的影响，揭示MMWV引起薇甘菊叶片萎蔫和皱缩及抑制薇甘菊生长的机制。研究从分子水平阐明MMWV的遗传多样性及致病机理,为薇甘菊等外来入侵植物的生物防治提供理论依据。

本项目应用单链构象多态性(single-strand conformation polymorphism, SSCP)方法分析在广东、广西和海南等地73个种群中291个有代表性的MMWV田间分离物的遗传多样性，探讨MMWV在华南地区的发生情况及群体遗传多样性组成；应用Illumina Hiseq 4000测序平台比较分析健康薇甘菊和MMWV侵染薇甘菊叶片15天和30后转录组差异表达基因；利用投射电镜观察MMWV侵染寄主植物的细胞病理特征；通过构建pCHF3-GFP-MP表达载体，确定MMWV致病基因的亚细胞定位和表达分析；分析MMWV侵染对寄主内源激素生长素(IAA)，赤霉素(GA3)，脱落酸(ABA)，细胞分裂素(CTK)等含量的影响，阐明MMWV 引起薇甘菊叶片萎蔫和皱缩及抑制薇甘菊生长的机制。研究从分子、亚细胞和细胞水平解释MMWV遗传多样性及致病机理，为薇甘菊等外来入侵植物的生物防治提供理论依据。