农杆菌介导的绿木霉遗传转化及重寄生缺陷突变体的筛选  

Agrobacterium tumefaciens-mediated Transformation of Trichoderma virens and Screening for Defective Mutants in Hyperparasitism

作　　者：刘连盟[1] 王玲[1] 黄世文[1,2] 侯恩庆[1,2] 肖丹凤[1] 

机构地区：[1]中国水稻研究所,杭州310006 [2]广西大学农学院,南宁530004

出　　处：《基因组学与应用生物学》2013年第5期562-568,共7页Genomics and Applied Biology

基　　金：中央级公益性科研院所基本科研专项资助项目(2011RG009和2012RG003-4);国家转基因重大专项"转基因植物新材料育种价值评估"(2011ZX08010-005);水稻重大病虫害防控技术研究与集成示范(2012BAD19B03)共同资助

摘　　要：绿木霉（Trichodermavirens）是一种重要菌寄生真菌，该菌己广泛应用于多种病害的生物防治上。本研究构建了双元载体pCATPH—I（GenBank登录号为：KC252999），并利用该载体成功实现了农杆菌介导的绿木霉遗传转化，转化效率可达385～460个转化子／10s绿木霉分生孢子。转化子的PCR和遗传稳定性分析表明，T—DNA己整合进该菌的基因组中且在无选择压力的条件下能够稳定遗传。利用该转化体系成功建立了超过2000个转化子的转化子库，通过转化子与病原真菌立枯丝核菌限hizoctoninsolani）对峙培养在该转化子库中成功筛选到两个重寄生缺陷突变体，D441和A281。本研究的完成可为绿木霉的功能基因组学及重寄生分子机制的研究打下基础。Trichoderma virens, which has been extensively used as a biocontrol agent against lots of plant diseases for many years, was an important mycoparasite. In this research, a binary vector, pCATPH- I, was constructed and submitted in GenBank under accession number KC252999. Trichoderma virens was successfully transformed for random integration of transforming DNA （T-DNA） with a high transformation efficiency of385-460 transformants /10^6 conidia by co-culturing with Agrobacterium tumefaciens AGL-1 which carries pCATPH-1. PCR and genetic stability analysis of the transformants showed that T-DNA integrated into the genome and can be stably inherited without selective pressure. A T-DNA insertion library of more than 2 000 transformants was constructed by this transformation system. By confronting cultivation with Rhizoctonia solani, two mutants defective in hyperpara- sitism, designated as D441 and A281, were screened out from that T-DNA insertion library. This work can lay a foundation for researching on the functional genome and hyperparasitism mechanism of Trichoderma virens.

关 键 词：绿木霉 农杆菌介导的转化 pCATPH—I 重寄生 重寄生缺陷突变体 

分 类 号：S476[农业科学—农业昆虫与害虫防治]