绿色荧光蛋白基因标记棉花黄萎病菌

Verticillium dahliae labeled with green fluorescent protein gene

以携带有潮霉素抗性筛选标记的pCTHyg载体为骨架,构建了含有增强型绿色荧光蛋白基因sGFP的载体pCH-sGFP,并通过农杆菌介导的遗传转化法导入引起棉花黄萎病的高致病力大丽轮枝菌Vd991,获得了sGFP整合到大丽轮枝菌基因组的转化株。通过转化子荧光信号、生长表型和致病力筛选鉴定,获得了1株与Vd991生长和致病力无显著差异且荧光信号强烈的转化株Vd-gfp77。侵染棉花根部试验表明,Vd-gfp77侵染棉花后快速扩展繁殖,子代仍然能发出强烈的荧光信号。本试验绿色荧光蛋白标记大丽轮枝菌的成功构建,为后续大丽轮枝菌侵染棉花过程的组织学和致病机理研究提供了良好的研究材料。

The expression vector of pCH-Sgfp was constructed by integration of the enhanced green fluorescent protein gene（sGFP）into the pCTHyg vector with hygromycin-resisitant gene,and transformed into Verticillium dahliae strain Vd991 highly virulent to cotton by Agrobacterium tumefaciens-mediated transformation.A transformant with strong fluorescence intensity,namely strain Vd-gfp77,was obtained,which had the same growth phenotype and virulence with the wild type.Infection experiment of Vd-gfp77 to cotton demonstrated that the strain Vd-gfp77 can excite fluorescence after invading and spreading in cotton tissue.The successful construction of GFP-tagged isolate of V.dahliae is beneficial to the further research on pathogen infection process to cotton and its pathogenesis.