柱花草炭疽病菌T-DNA 插入突变体库的构建与分析

胶孢炭疽菌(Colletotrichum gloeosporioides)引起的柱花草炭疽病是热带牧草柱花草的主要病害,利用根癌农杆菌介导转化(Agrobacterium tumefaciens mediated transformation,ATMT)的T-DNA插入突变技术,可使炭疽病菌基因组上被插入部位的基因丧失功能,是研究柱花草炭疽病菌致病机理的重要方法.在先前优化根癌农杆菌介导柱花草炭疽病菌遗传转化体系的基础上,构建了4 616个转化子的突变体库,从中随机选取部分突变体,对其T-DNA插入拷贝数、遗传稳定性、生长速度、产孢量、分生孢子萌发等进行分析.结果表明,所得转化子多为单拷贝,能稳定遗传.一些转化子的生长速度、产孢量、分生孢子萌发率与野生型相比有明显差异.

Construction and analysis of a T-DNA insertional mutant library for Colletotrichum gloeosporioides

The anthracnose caused by Colletotrichum gloeosporioides is a serious threat to Stylosanthes guianensis, an important tropical forage. Agrobacterium tumefaciens -mediated transformation as a tool for insertional mutagenesis, is the important way to study pathogenesis of C. gloeosporioides. Based on the previously optimized Agrobacterium -mediated genetic transformation system, a mutant library containing 4 616 putative mutants was constructed, from which some mutants were randomly selected for analysis of the copy number of T-DNA insertion, genetic stability, growth rate, spore amount, spore germination and appressorium formation. The results showed that most of the mutants had single T-DNA insertion and could be stably inherited. The growth rate, spore amount, spore germination rate, appressorium formation rate of some mutants were significantly different from the wild type.