农杆菌介导的黄瓜炭疽菌遗传转化

建立并优化了农杆菌介导转化瓜类炭疽菌(Colletotrichum lagenarium)获得T-DNA插入突变体体系,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个孢子平均可获得约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保持稳定.随机对36个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.

Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose

To elucidate the molecular mechanism of pathogenicity in Colletotrichum lagenarium,the casual agent of cucumber anthracnose,conditions for T-DNA insertional mutagenesis using Agrobacterium-mediated transformation approach were established and optimized.Binary vector BIG2RHPH2-GUS-GFP was used,which carried 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, a total of 150-200 hygromycin-resistant transformants could be obtained for transforming 10~6 spores of C.lagenarium under the optimized conditions.All hygromycin-resistant transformants contained T-DNA insertions in their genomes which were 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 visualized under microscope and GUS activity detected by staining.The T-DNA inserted transformants appeared to be stable as they showed hygromycin resistance,GFP fluorescence and GUS activity after 4 generations.Preliminary results from pathogenicity test of randomly selecting 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 system facilitate the construction of the TDNA inserted mutant population,screening of mutants with altered pathogenicity and identification of pathogenicity-related genes in C.lagenarium.