【作者】 方丽； 宋凤鸣；

【Author】 FANG Li, SONG Fengming, ZHENG Zhong (Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China)

【机构】 浙江大学生物技术研究所；

【摘要】 建立并优化了农杆菌介导转化瓜类炭疽菌获得T-DNA插入突变体的体系, 包括农杆菌使用浓度、农杆菌与炭疽菌孢子共培养时间、抗生素配合使用抑制农杆菌污染等。所用的转化载体pBIG2RHPH2-GUS-GFP除了抗生素选择标记外还携带有融合GFP-GUS报告基因。转化106个孢予平均可获得约150-200个潮霉素抗性转化子。PCR检测表明,所有潮霉素抗性转化子菌株都能从其基因组DNA中扩增到转化载体中的GUS基因片段,而且都能产生GFP荧光或GUS染色阳性。转化子菌株经过连续继代培养后仍然稳定。随机对36转化子菌株进行致病性测定,发现1个转化子菌株对黄瓜致病性下降,1个丧失致病性,而大多数表现为野生型致病性。农杆菌介导转化瓜类炭疽菌体系的建立,为进一步构建大库容量的T-DNA 插入突变体库、致病性突变体筛选以及致病性相关基因的克隆鉴定奠定了基础。更多还原

【Abstract】 We report the establishment and optimization of the conditions for T-DNA insertional mutagenesis of Colletotrichum lagenarium, the casual agent of cucumber anthracnose , using Agrobacterium-mediated transformation approach. Binary vector BIG2RHPH2-GUS-GFP was used in our study, which carries the bacterial hygromycin B phosphotransferase gene cassette under the control of Aspergillus nidulans trpC promoter as a selection marker and a GFP-GUS fusion cassette driven by the A. nidulans trpC promoter enabling detection of GFP microscopically and GUS stain in the resultant .transformants. In average, 150-200 hygromycin-resistant transformants could be obtained when transforming 106 spores of C. lagenarium under the optimized conditions. All hygromycin-resistant transformants contain T-DNA insertions in their genomes as detected by PCR amplification of the GUS gene in the T-DNA cassette with genomic DNA as templates. These hygromycin-resistant transformants also showed GFP fluorescence as visualized under microscope and GUS activity as detected by staining. The T-DNA inserted transformants appear to be stable as they showed hrgromycin resistance, GFP fluorescence and GUS activity after 4 generations. Preliminary results from pathogenicity test of randomly selected 36 hygromycin-resistant transformants on leaves of two-week-old cucumber seedlings identified two transformants with reduced pathogenicity or loss-of-pathogenicity, respectively, except that the majority of the transformants showed wild-type pathogenicity. The establishment and optimization of the conditions for Agrobacterium-mediated transformation will facilitate the construction of the T-DNA inserted mutant population, screening of mutants with altered pathogenicity and identification of pathogenicity-related genes in C lagenarium.更多还原