【作者】 刘小红；

【导师】 林福呈；

【作者基本信息】 浙江大学 ， 植物病理， 2004， 硕士

【摘要】 稻瘟病是水稻上重要的病害之一，其病原为子囊菌Magnaporthe grisea。了解M.grisea的致病机理不仅有利于稻瘟病的防治，而且作为研究植物病原真菌与寄主互作的理想模式系统，对于了解其它真菌的致病机理也有重要意义。致病相关基因的克隆和分析是达到这一目标的有效途径。突变体的获得及表型分析是鉴定致病相关基因的有效策略。 获得突变体的方法很多，如限制性内切酶介导的转化，农杆菌介导的转化，转座子标签技术等。然而，这些方法不能研究以下基因：在实验室条件下功能上多余的基因；在多个水平上都有作用的基因；低水平表达的重要基因。启动子陷阱的方法，并不需要产生可见的功能缺陷，只需根据报告基因的表达就可将转化子筛选出来，然后通过启动子陷阱载体上的已知序列克隆目的基因。本研究，将启动子陷阱的技术应用于稻瘟病菌，通过构建启动子陷阱载体，利用限制性内切酶介导转化，建立的突变体库。研究结果如下： 1．构建了启动子陷阱载体pEGFP-HPH和pCB1003-EGFP，均具有具有潮霉素抗性基因和无启动子的eGFP基因； 2．建立了稻瘟病菌遗传转化体系，转化效率达到40-60个突变体／微克DNA； 3．获得突变体1077株，通过荧光显微镜检测，489株突变体荧光表达，eGFP基因表达率45.4％；生长速度明显比野生型减慢的突变体3株；菌落白色的突变体9株；产孢减少的有35株；致病性有所降低的菌株18株；随机挑取8株突变体进行Southern杂交分析，可以杂交到强弱不同的信号。 实验数据表明，启动子陷阱技术具有良好的前景，通过这种方法，我们可以得到许多在形态、代谢、致病性等各个方面有变化的突变体。更多还原

【Abstract】 Magnaporthe grisea is a causal agent of rice blast and diseases of many other different grasses. Understanding of the molecular basis of this disease is not only benificial for rice blast control, but also can serve as a model for revealing of other fungal pathogen-plant interactions. Obviously, identification of genes required for pathogenicity is a key step toward achieving this goal. The parastic interaction between the pathogenic fungus and its plant host is a complex biological process. Mutation analysis of pathogen is a powerful strategy in study of this process. To elucidate the mechanisms of the pathogenesis, we clone genes by using of insertional mutagenesis. As we know, strategies, such as REMI, ATMT and trasnposon tagging have cloned many interesting genes in many plant pathogenic fungi so far. However, those methods will not permit isolation of genes without clear mutant phenotypes during development either because of gene redundancy or due to the gene product not being essential for development under the conditions in the laboratory. One way to gain access to such genes is to use a method called promoter trapping, in which expression of a reporter gene can be initiated only from an endogenous promoter because the reporter gene lacks its own promoter. In this method, it is not necessary to obtain the obvious phenotype mutants. Here we discribe this promoter trapping method for M. grisea that depends upon restriction enzyme-mediated insertion of a vector carrying a promoterless eGFP (green fluorescent protein) gene. New promoter trapping vectors pEGFP-HPH and pCB 1003-EGFP have been developed to trap genes in M. grisea. The redults are shown as follows:1. New promoter trapping vectors pEGFP-HPH and pCB1003-EGFP have been constructed which possess the marker hygromycin gene and a promoterless eGFP gene;2. Transformation system is stabilized. The frequency of the transformation is 40-60mutants/ug DNA;3. We constructed a library in Magnaporthe grisea which consisted of 1077mutants. eGFP expression was detected by fluoroscopy. eGFP expression in 489 mutants. The frequency of the eGFP expression is 45.4%. By analysing of the 489 mutants, 3 mutants grow slowly, 9 mutants’clony are white, the spore production of 35 mutants are decreased and 18 mutants deduced in pathogenecity. We use Southern blot analysis to check the library and get the different signals. These data indicated that the promoter trapping technique has a good future. Byanalyzing the mutants, we can get the useful mutants which have defects inpathogenicity, metabolism, and alterations in morphology.更多还原