利用启动子捕获技术构建橡胶炭疽病菌突变体库

Mutant Library Constructed by Using Promoter trapping in Colletotrichum gloeosporioides

通过不同限制性内切酶分别处理载体pCAMBIA1300、pCSN43和pBluescriptSK+后,构建启动子捕获载体pCAHPH,该载体经农杆菌介导转化橡胶炭疽病菌,建立起橡胶炭疽病菌T-DNA插入突变体库。经鉴定结果表明,突变体库的转化子数量为1105个,均抗潮霉素;随机测定248个转化子,有19个转化子致病力明显降低;随机挑取7个转化子做PCR检测,均可扩增出一条约800bp的目标条带,说明突变体为启动子捕获载体插入所引起;致病力显著减弱的转化子经southern检测,均为单拷贝插入。该结果旨在为开展橡胶炭疽病菌致病遗传分析和后续致病基因研究奠定基础。

Promoter-trapping vectors, designated as pCAHPH, were constructed and transformed into Collectotrichurn gloeosporioides via ATMT （Agrobacterium tumefaciens-mediated transformation）. A library of 1 105 transformants resistant to hygromycin B was generated. Of all the transformants, 248 transformants were randomly selected for detection, of which 19 transformants were reduced greatly in pathogenicity. PCR amplification of randomly selected 7 transformants showed that they were all resistant to hygromycin B and contained hph gene, indicating that the transformants were T-DNA inserted mutants. Southern blotting analysis showed the T-DNA inserted mutants with single copy insertion were reduced significantly in pathogenicity. The promoter trapping technique has been applied successfully in C. gloeosporioides and can be used as a tool for functional genomic analysis.