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围小丛壳菌,一种子囊菌真菌,能引起苹果炭疽叶枯病。本研究的目的是建立和优化农杆菌介导的苹果炭疽叶枯病菌遗传转化技术体系,获得产孢与致病性变异的突变体,为开展该菌的分生孢子形成机制、致病机理研究奠定基础。对影响转化效率的主要因子进行单因子条件测验,对转化子进行PCR鉴定、绿色荧光观察和产孢及致病性分析。优化的遗传转化体系:预诱导4 h,培养介质滤纸,分生孢子浓度1伊106个/m L,农杆菌浓度OD600=0.3,乙酰丁香酮浓度50滋mol/m L,21℃,共培养时间24 h。利用这一体系,每106个分生孢子能产生350~400个转化子。通过转化子潮霉素抗性基因的PCR检测和荧光观察,证实潮霉素抗性基因和gfp基因已成功整合到该病菌基因组中。产孢和致病性检测结果显示,转化子M421、M755和M903致病力明显下降,转化子M285和M744丧失了致病能力,转化子M598和M856丧失了产孢能力和致病能力。本研究建立和优化了农杆菌介导的苹果炭疽叶枯病菌遗传转化技术体系,构建了该病菌的绿色荧光蛋白标记的突变体库,获得了产孢与致病性变异的转化子。
Around the small cluster of bacteria, an ascomycete fungus, can cause apple anthracnose leaf blight. The purpose of this study is to establish and optimize Agrobacterium-mediated genetic transformation system of apple anthracnose, and obtain the mutants of sporulation and pathogenicity. To study the mechanism of conidial formation and pathogenicity Lay the foundation. The main factors affecting the transformation efficiency of single factor test conditions, the transformants were identified by PCR, green fluorescence and sporulation and pathogenicity analysis. The optimized genetic transformation system was as follows: pre-induction for 4 h, filter media for culture medium, conidia concentration of 1 106 cells / ml, Agrobacterium concentration of OD600 = 0.3, acetosyringone concentration of 50 mM mol / ml, Time 24 h. With this system, 350-400 transformants are produced per 106 conidia. By PCR detection and fluorescence observation of the hygromycin resistance gene, it was confirmed that the hygromycin resistance gene and the gfp gene have been successfully integrated into the genome of the bacterium. The results of sporulation and pathogenicity test showed that the pathogenicity of transformants M421, M755 and M903 was significantly decreased, and the transformants M285 and M744 lost their pathogenicity. The transformants M598 and M856 lost their sporulation and pathogenicity. In this study, we established and optimized Agrobacterium-mediated genetic transformation system of apple anthracnose, constructed green fluorescent protein labeled mutants of this bacterium, and obtained transformants with sporulation and pathogenicity.