根癌农杆菌介导的柳树痂囊腔菌遗传转化探究

Study on Genetic Transformation of Elsino?murrayae Mediated by Agrobacterium tumefaciens

柳树痂囊腔菌是柳属植物重要的真菌病原,为了建立该真菌的遗传转化体系,本研究测试了柳树痂囊腔菌对潮霉素B、卡那霉素和草铵膦的敏感性,确定80μg/mL潮霉素B用于转化子筛选;构建了双元载体,使用构巢曲霉色氨酸合成基因启动子和柳树痂囊腔菌翻译延伸因子基因启动子分别驱动潮霉素抗性基因和GFP基因;在农杆菌介导的遗传转化中发现使用生长6 d菌落的分生孢子作为受体材料,农杆菌诱导培养基为pH=5.2,并添加乙酰丁香酮200μg/mL的条件下,柳树痂囊腔菌转化效率为0.62个转化子/10000个孢子;抗性菌落的PCR检测产生特异性条带,定量PCR结果显示外源基因高水平表达,并且在蓝色激发光下释放绿色荧光。使用反向PCR鉴定T-DNA插入位点,在2个转化子中发现插入突变的目标基因,进一步分析未在这两个突变体中发现生长发育和致病性的改变。本研究在柳树痂囊腔菌中建立了高效的根癌农杆菌介导遗传转化方法,有利于该菌的基因功能研究,并且促进痂囊腔属真菌共有致病机制的探索。

Elsinoë murrayae is an important fungal pathogen of Salix spp..In order to establish an genetic transformation method of E.murrayae.The study tested the susceptibility of E.murrayae to hygromycin B,kanamycin and glufosinate ammonium,and determined 80μg/mL hygromycin B was appropriate for screening transformants.A binary vector was constructed,in which the hygromycin resistance gene was driven by Aspergillus nidulans tryptophan C gene promoter,and the P gene was driven by translation elongation factor gene promoter of E.murrayae.In agrobacterium-mediated genetic transformation,it was found that the conidia of 6-day-old colony as recipient material and the A.tumefaciens induced in induction medium(pH=5.2)with 200μg/mL acetosyringone,the transformation efficiency of E.murrayae was 0.62 transformants/10000 conidia.PCR detection of resistant colonies produced specific band,and qPCR results revealed high expression level of exogenous genes,and hyphae of resistant colonies released green fluorescence under blue excitation light.The T-DNA insertion sites were identified by inverse PCR,and T-DNA insertion mutagenesis were revealed in two transformants.Further analysis found no changes in the growth,development and pathogenicity of the two mutants.In this study,an efficient method of genetic transformation mediated by A.tumefaciens was established in E.murrayae,which facilitates the study of the gene function of the fungus and promotes the exploration of the common pathogenic mechanism of Elsinoë spp..