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辣椒胶孢炭疽菌遗传转化体系的建立

作  者：
曾泉;满益龙;陈岳;张鑫;张卓;李成刚;杜娇;张德咏;刘勇;谭新球
单  位：
湖南大学研究生院隆平分院;湖南省植物保护研究所;湖南省农业科学院农业生物技术研究所
关键词：
辣椒炭疽病菌;遗传转化;GFP;生长;致病性
摘  要：
由辣椒胶孢炭疽菌Colletotrichum gloeosporioides导致的辣椒炭疽病是辣椒生产上最为严重的真菌病害之一.本文以辣椒胶孢炭疽菌CSLL11为供试菌株,采用PEG-CaCl2介导的原生质体转化法,将含有潮霉素B抗性基因和eGFP表达基因的DNA片段成功转入辣椒胶孢炭疽菌的原生质体中,获得了稳定表达绿色荧光的转化子,从而成功建立了辣椒胶孢炭疽菌的遗传转化体系.试验结果表明,可有效筛选阳性转化子的潮霉素B浓度为500 mg/L;PCR及Southern blot结果显示,eGFP表达基因已单拷贝整合至辣椒胶孢炭疽菌转化子的基因组中;使用荧光显微镜观察第一代及继代培养后的转化子,菌丝与分生孢子均表现出强烈的绿色荧光信号,说明GFP能在转化子中稳定遗传;将转化子与野生型菌株相比,菌落形态、生长速率及致病力水平无明显差异.本研究建立了辣椒胶孢炭疽菌遗传转化体系并获得了稳定表达绿色荧光蛋白的转化子,对辣椒炭疽菌与寄主互作的研究及病害防治具有重要意义.
作  者：
ZENG Quan;MAN Yilong;CHEN Yue;ZHANG Xin;ZHANG Zhuo;LI Chenggang;DU Jiao;ZHANG Deyong;LIU Yong;TAN Xinqiu;Long Ping Branch, Graduate School of Hunan University;Plant Protection Institute, Hunan Academy of Agricultural Sciences;Hunan Key Laboratory of Integrated Pest Management in Horticultural Crops;Agricultural Biotechnology Institute, Hunan Academy of Agricultural Sciences;
单  位：
ZENG Quan%MAN Yilong%CHEN Yue%ZHANG Xin%ZHANG Zhuo%LI Chenggang%DU Jiao%ZHANG Deyong%LIU Yong%TAN Xinqiu%Long Ping Branch, Graduate School of Hunan University%Plant Protection Institute, Hunan Academy of Agricultural Sciences%Hunan Key Laboratory of Integrated Pest Management in Horticultural Crops%Agricultural Biotechnology Institute, Hunan Academy of Agricultural Sciences
关键词：
Colletotrichum gloeosporioides;;genetic transformation;;GFP;;growth;;pathogenicity
摘  要：
Capsicum anthracnose, caused by Colletotrichum gloeosporioides, is one of the most serious fungal diseases in pepper production. In this paper, we used the CSLL11 as the tested strain, and the DNA fragment containing hygromycin B resistance gene and eGFP expression gene was successfully transferred into chili Colletotrichum gloeosporioidess by using PEG-CaCl2-mediated protoplast transformation. A transformant stably expressing green fluorescence was obtained, and the genetic transformation system of chili Colletotrichum gloeosporioidess was successfully established. The results showed that the concentration of hygromycin B for effective screening of the positive transformants was 500 mg/L. The results of PCR and Southern blot showed that the eGFP expression gene was integrated into the genome of the transformant. The hyphae and the conidia of the transformants after the first generation and subculture showed strong green fluorescence signals, indicating that GFP could be stably inherited in the transformants. Moreover, there was no significant difference in colony morphology, growth rate and pathogenicity between the transformants and the wild-type strains. In this study, the genetic transformation system of chili Colletotrichum gloeosporioidess and the stable expression of green fluorescent protein were obtained, which is of great significance for the study of the interaction between anthracnose and host and the disease control.

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