橡胶树胶孢炭疽菌T-DNA插入突变体库构建及其致病缺陷转化子筛选

Generation of a transformant library of Colletotrichum gloeosporioides by ATMT and screen of the mutants defective in pathogenicity

摘要:

【目的】进一步研究橡胶树胶孢炭疽菌致病分子机理。【方法】通过含ILV1基因(具氯嘧磺隆抗性)的pSULF·gfp双元载体农杆菌AGL-1介导进行橡胶树胶孢炭疽菌遗传转化, 利用氯嘧磺隆抗性标记筛选转化子, 对转化子PCR验证及荧光显微观察; 采用离体古铜期橡胶树叶无伤接种法进行致病性缺陷转化子筛选, 并对转化子进行遗传稳定性检测。【结果】获得含3 721个转化子的T-DNA插入突变体库, 转化效率为150?400个转化子/106孢子, 从3 721个转化子中筛选得到致病性缺陷转化子25个; 随机选取20个转化子进行遗传稳定性测定, 在不含氯嘧磺隆PDA平板上继代培养10次后仍保持氯嘧磺隆抗性, 且表型稳定, 表明插入外源基因能够稳定遗传。【结论】可以利用根癌农杆菌介导橡胶孢炭疽菌转化, 构建橡胶树胶孢炭疽菌T-DNA插入突变体库, 筛选致病缺陷突变菌, 为进一步研究该菌致病相关基因提供材料。

Abstract:

[Objective] Generation of C. gloeosporioides T-DNA insertion mutant library through Agrobacterium-mediated transformation was conducted in order to get more insight into the molecular mechanisms underlying the pathogenicity of C. gloeosporioides. [Methods] Agrobacterium containing the binary vector pSULF-gfp was used for transformation of C. gloeosporioides. Chlorimuron-ethyl resistant transformants were screened out and subjected to biological, morphological observation and PCR test. Detached copper-colour rubber leaves were used for pathogenicity assay. [Results] The transformation efficiency was up to 150?400 transformants per 106 conidia and 3721transtormants were generated, 25 transformants defective in pathogenicity were screened out from 3721 transformants. All of the transformants tested remained mitotically stable, maintaining their Chlorimuron-ethyl resistance after tenth generations of growth in the absence of Chlorimuron-ethyl. [Conclution] ATMT can be used to rapidly generate a large library of the fungal transformants, which offers highly efficient means for characterizing the genes that are important for the pathogenicity of C. gloeosporioides and should facilitate further molecular studies of this important plant pathogen.