胶孢炭疽菌T-DNA插入突变体库的构建及其分子分析

Construction of a T-DNA Insertional Mutant Library of Colletotrichum gloeosporioides of Mango and Its Molecular Analysis

利用根癌农杆菌介导的T-DNA插入遗传转化体系转化杧果炭疽菌SC2菌株,共获得抗潮霉素B突变体4680个,平均每转化1.0×106个炭疽菌分生孢子可得到300～980个突变体,且潮霉素抗性在多次传代实验中得到稳定遗传。随机挑取38个突变体进行PCR检测,均可扩增出1条约1.2kb的目标谱带,说明突变体为T-DNA插入引起;进一步的Southern blot杂交分析结果表明,在随机挑选的20个突变体中,有1个(5%)为三拷贝T-DNA插入,4个(20%)为双拷贝插入,剩余的15个(75%)为单拷贝插入。

Mangifera anthracnose, caused by Colletotrichum gloeosporioides,is one of the most damaging phytopathogens of mango（Mangifera indica L） in the world.A highly efficient Agrobacterium tumefaciens-mediated transformation of C, gloeosporioides was established. Strain SC2 of C. gloeosporioides was mutated through T-DNA insertion mediated by A. tumefaciens strain Agl-1. The transformation efficiency ranged from a low of 300 to a high of 980 transformants/10^6 conidia, and 4 680 transformants were obtained. The PCR amplification found that 38 tested transformants randomly selected were all resistant to hygromycin B and contained hph gene, indicating that the transformants were T-DNA inserted mutants. Southern blotting analysis showed the 20 T-DNA inserted