根癌农杆菌介导的胶孢炭疽菌遗传转化体系的建立

Establishment of Agrobacterium-mediated transformation systems for Colletotrichum gloeosporioides

本研究基于农杆菌介导的遗传转化方法,建立了芒果胶孢炭疽菌高效的遗传转化体系,获得一批炭疽菌的T-DNA插入突变体,其目的是为炭疽菌的功能基因组学研究和致病相关基因的克隆奠定基础。结果如下:通过摸索并优化了体系的各项因子,在潮霉素筛选浓度为200μg/mL,菌液浓度为OD660=0.15条件下,AS为200μmol/L,选择pH5.5的IM共培养基中转化效果最好;进一步通过对转化子的继代稳定性和PCR检测,结果发现潮霉素抗性稳定遗传和假阳性率低;通过菌落形态观察和产孢能力的测定获得3个菌落形态异常突变体,6个产孢能力下降突变体。

Based on Agrobacterium-mediated T-DNA insertion, an efficient transformation system of Colletotrichum gloeosporioides （CG） from mango was established and some T-DNA insertion mutants of CG were obtained by this system in this study. The key conditions and parameters for co-cultivation of LCG5 and Agrobacterium were tested and optimized. The results showed that the optimal conditions were： OD660 of 0. 15 for Agrobacterium density, 200μg/mL of hygromycin B and 200μmol/L of AS. The clones maintained their hygromycin B resistance after 5 continuous transfers and incubation on PDA media without hygromycin B, suggesting that the resistance of the mutants to hygromycin B was stable. With primers designed based on hygromycin phosphotransferase gene in the plasmid used in LCG5 transformation, a fragment completely homologous to this gene was amplified by PCR from each clones randomly selected from the LCG5 mutants, indicating that the hygromycin gene had inserted into the LCG5 genome and there was a low rate of false positive clones from the LCG5 transformation system. Many LCG5 mutants with T- DNA insertion were obtained by using this transformation system. The colony morphology and sporulation capability of the mutants and their wild type （LCG5） were compared and 3 mutants were found to be significantly different from the wild type in colony appearance and color, while 6 mutants produced significantly less conidia.