球毛壳菌荧光标记与重寄生现象研究

Study on GFP-labeling and mycoparasitism in Chaetomium globosum

球毛壳菌是一种非常重要的生防真菌.为了从分子生物学水平研究球毛壳菌与立枯丝核菌的拮抗机制,选用GFP(green fluorescent protein)作为报告基因对球毛壳菌进行标记.通过构建和转化EGFP(Enhanced GFP)表达载体,得到氯嘧磺隆抗性的转化子.经过几轮验证筛选,包括选择性平板筛选、PCR扩增目的片段验证、Southern杂交、荧光镜检等,确认SUR基因和EGFP基因已经插入球毛壳菌的基因组并在其中表达标记.取一个确认为荧光标记转化子的菌株,进行生防特性分析.主要通过与病原真菌平板共培养、载玻片对峙培养等方法初步研究了球毛壳菌与立枯丝核菌的拮抗机理.结果发现,在对立枯丝核菌的抑制过程中,球毛壳菌的拮抗机理主要是重寄生作用.

As an important biocontrol agent, the antagonistic mechanism of Chaetomium globosum to phytopathogens still leaves a lot to be studied. This paper aimed to investigate the antagonism between Chaetomium globosum and Rhizoctonia solani with GFP（green fluorescent protein） as reporter gene. Sulfonylrate-resistant transformants were obtained by construction and transformation of EGFP （Enhanced GFP）-expression vectors. After rounds of verification, including screening with selective plates, PCR amplification of targeted fragment, Southern blot and examination under fluoroscope, SUR and EGFP fragments were proved to be inserted and expressed in the genome of C. globosum. One fluorescing transformant was chosen to do biocontrol analysis. The mechanism of antagonism between C. globosum and R. solani was studied in the way of co-culture on plate and confrontation on slide. It is found that C. globosum has apparent process of mycoparasitism during the confrontation with R. solani.