农杆菌介导的哈茨木霉T-DNA转化系统优化及拮抗能力突变子的性质分析  

The Optimization of Agrobacterium tumefaciens Mediated Trichoderma harzianum Transformation System and Chariaterization Analysis of Antagonistic Mutants

作　　者：黄亚丽[1,2] 蒋细良[3] 田云龙[2] 叶小波[3] 郭萍[2] 朱昌雄[2] 

机构地区：[1]河北省生物研究所,石家庄050081 [2]中国农业科学院农业环境与可持续发展研究所,北京100081 [3]中国农科院植物保护研究所/植物病虫害生物学国家重点实验室,北京100081

出　　处：《中国生物防治》2009年第3期233-238,共6页Chinese Journal of Biological Control

基　　金：国家自然科学基金项目(30671400);国家"十一五"支撑项目(2006BAD08A02);国家"十一五"863项目(2006AA10A211)

摘　　要：采用单因子试验方法研究了农杆菌EHA105介导的哈茨木霉Th-33转化过程中,各主要因素对转化效率的影响,建立了高效的转化系统,使农杆菌转化哈茨木霉的效率达到60～150个转化子/10^6个木霉孢子,利用该转化系统构建了含有8000多个转化子的T-DNA插入突变库。通过转化子与立枯丝核菌的对峙试验,从1260株转化子中筛选到23株拮抗能力发生变化的突变子。随机挑选5株突变子对其遗传稳定性进行分析,表明5株突变子都具有稳定性,聚合酶链式反应（PCR）表明上述突变子均有T-DNA片段的插入。Factors influencing the transformation efficiency of Agrobacterium tumefaciens mediated transformation of Trichoderma harzianum were studied.Through the study,a high efficient transformation system was established.Co-cultivation of T.harzianum Th-33 conidia with A.tumefaciens EHA105 in the presence of acetosyringone resulted in the formation of hygromycin B resistant fungal colonies with high transformation efficient（about 60-150 transformants per 10 conidia）.T-DNA insertional library of T. harzianum Th-33 mediated by A.tumefaciens was constructed,which was consisted of more than 8000 transformants.We determined the antagonistic ability of 1260 transformants by antagonistic experiments against Rhizoctonia solani on PDA plate.Twenty-three mutants whose antagonistic ability changed were selected. Five randomly selected antagonistic mutants were proved to be mitotic cell division stable through five generations on PDA plate without hygromycin B.PCR analysis showed that T-DNA was inserted into all transformants genome.These suggest that A.tumefaciens mediated transformation is a potentially powerful tool towards tagged mutagenesis and functional gene clone for T.harzianum.

关 键 词：哈茨木霉 农杆菌 T-DNA 遗传转化 拮抗突变子 

分 类 号：Q319.2[生物学—遗传学] Q785