棉花黄萎病菌T-DNA插入突变体库的构建及变异性状的分析

Construction of T-DNA Inserted Transformation Library of Verticillium dahliae on Cotton and Mutative Traits of the Mutants

利用农杆菌介导遗传转化(ATMT)的方法,成功构建含2 000个转化子的棉花黄萎病菌强毒菌株Vd080的突变体库。200μmol/L乙酰丁香酮(AS)作诱导剂,25℃共培养48h,棉花黄萎病菌孢子量为106 mL-1,其转化效率达150～540个转化子。进一步研究发现,供试突变体的T-DNA成功插入Vd080基因组,且均为单拷贝插入,转化子的潮霉素B基因能够稳定遗传。309个突变体中,53.7%的突变体菌落形态与初始菌株Vd080相同,均产生黑色的微菌核,不产生黑色素微菌核的菌丝型仅占17.5%。与初始菌株相比,随机选取的85个突变体,产孢量、生长速率、粗毒素分泌量及致病力发生显著变异的菌株分别占36.4%、12.9%、50.6%和29.4%,四者之间不存在明显的相关性。

A mutant library, which intiated from a high virulent Verticillium dahliae strain Vd080 and contained 2 000 mutants, was constructed by Agrobacterium-mediated transformation. 200 μmol/L Acetosyringone and 48 hours of co-cultivation period at 25 ℃ were optimal for the trans- formation and reached 150-540 transformants per 106 spores. The results of further study indicated that T-DNA of tested mutants inserted into the genome of Vd080 successfully, and T-DNAs in 6 mutants selected randomly were all single copy. Besides, all of the tested transformants maintained their resistance to hygromycin B. Like wild strain Vd080, 53.7%mutants produced black microsclerotia in their colonies. 17.5% mutants produced white or yellow mycelia without black microsclerotia. Mutants which exhibited significantly different from the wild strain Vd080 on spore yield, growth rate,crude toxin and pathogenicity were 36.4%, 12.9%, 50.6% and 29.4%, respectively. There was no correlation among the four parameters.