根癌农杆菌介导荸荠秆枯病菌转化体系构建及突变体筛选

Development of Agrobacterium tumefaciens Mediated Transformation of Cylindrosporium eleocharidis and Analysis of T-DNA Insertional Mutants

【目的】建立农杆菌介导荸荠秆枯病菌(Cylindrosporium eleocharidis)的遗传转化体系,构建该病菌的突变体库并进行突变体筛选,为荸荠秆枯病病原菌的分子机理研究提供参考依据。【方法】运用农杆菌介导的真菌转化方法,将含有绿色荧光蛋白(GFP)的双元载体pEX4转化至荸荠秆枯病菌野生型菌株Ceh中,利用PCR、Southern blotting杂交和荧光显微镜检测等技术验证转化子。通过单因子变量[共培养基中的乙酰丁香酮(AS)、农杆菌诱导时间、病原菌孢子浓度、IM诱导培养基pH、共培养时间和共培养介质]试验,优化农杆菌遗传转化体系,并建立该病菌突变体库,分析突变体的形态变异和致病性。【结果】获得的最佳遗传转化条件为:共培养基中AS浓度为300μmol/L,农杆菌诱导时间6 h,真菌孢子浓度为107个/mL,诱导培养基pH 5.6,共培养时间72 h,共培养介质为硝酸纤维素膜。在该优化条件下,农杆菌遗传转化体系转化效率达600~700个转化子/107个孢子。随机挑取转化子进行PCR检测均为阳性,T-DNA单拷贝插入率达77.8%,荧光显微镜均可检测到荧光信号。【结论】成功构建了农杆菌介导的荸荠秆枯病病原菌遗传转化体系和T-DNA插入突变体库,并筛选获得表型和致病性缺陷突变体,可用于指导荸荠秆枯病菌基因功能和病菌与寄主互作研究。

【Objective】Genetic transformation method mediated by Agrobacterium tumefaciens was developed for generation of a T-DNA insertional mutant library of Cylindrosporium eleocharidis,in order to provide reference basis for study of function of the pathogen on molecular level.【Method】C.eleocharidis wild type isolate Ceh was transformed by A.tumefaciens containing green fluorescent protein(GFP)with bianry vector pEX4.Transformants were randomly selected and verified by PCR amplification and southern blotting assay as well as fluorescence microscopy detection.Single factors including acetosyringone(AS)in co-cultivation medium,inducing time of A.tumefaciens,spore concentration of C.eleocharidis,pH of induction medium(IM),co-culture time and substrates were examined respectively.Furthermore,mutants library was built and mutants were analyzed by describing morphological character and pathogenicity tests.【Result】The optimized conditions included using AS in co-cultivation medium,300μmol/L;6 hours induction of A.tumefaciens with AS;107CFU/mL of the spore concentration for C.eleocharidis;pH 5.6 of induction medium(IM),72 hours co-culture of C.eleocharidis spore with A.tumefaciens,and using nitrocellulose membrane as co-culture medium.The transformation efficiency of genetic transformation system for C.eleocharidis was in the range of 600-700 transformants per 107 conidia through the improved conditions.All the randomly selected transformants were positive by PCR examination,whose single copy insertion rate of T-DNA reached 77.8%and fluorescence signals could be detected by fluorescence microscopy.【Conclusion】ATMT and TDNA insertion mutant library for C.eleocharidis genetic tranfoemation system induced by A.tumefaciens were successfully established,pathogenicity and phenotype changing mutants were screened,which could be used for the study of genes functional analysis and interaction between pathogen and host.