西瓜嗜酸菌趋化性测定及趋化基因cheA和cheY功能研究

发布时间：2018-03-14 10:48

本文选题：西瓜嗜酸菌　切入点：趋化性　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：瓜类细菌性果斑病(Bacterial Fruit Blotch,简称BFB)是世界范围的检疫性细菌病害,主要为害西瓜、甜瓜等葫芦科作物,造成重大的经济损失。西瓜嗜酸菌(Acidovorax citrulli,Schaad et al,2008)是其病原菌,有研究表明病原细菌在寄主表面的定殖能力与其致病能力关系密切,而病原细菌的趋化能力是决定定殖能力的关键因素之一。本研究主要围绕西瓜嗜酸菌的趋化性测定及其趋化基因的功能展开,主要结果如下:1、初步测定西瓜嗜酸菌Ac-5菌株的趋化性利用毛细管法测定碳源、氨基酸、有机酸等对西瓜嗜酸菌趋化性的影响。结果发现:麦芽糖、葡萄糖、乳糖、蔗糖和半乳糖显著促进西瓜嗜酸菌的趋化性;L-亮氨酸、L-异亮氨酸、L-色氨酸、L-缬氨酸、L-脯氨酸、L-谷氨酸和L-谷氨酰胺显著促进西瓜嗜酸菌的趋化性,而L-蛋氨酸显著抑制其趋化性;柠檬酸和苹果酸显著促进西瓜嗜酸菌的趋化性;氯化钠、硫酸镁等对西瓜嗜酸菌的趋化性无显著影响。2、Ac-5菌株趋化基因che A和che Y的生物信息学分析登录NCBI数据库西瓜嗜酸菌全基因序列查找che A和che Y基因的相关信息。che A和che Y基因分别编码Che A和Che Y蛋白。Che A和Che Y蛋白均存在多个疏水性氨基酸区域;Che A蛋白是一种跨膜蛋白,在600-700氨基酸处(即Che W结合区域)有跨膜结构,Che Y蛋白不存在跨膜结构,是一种膜内蛋白,用于接收Che A蛋白的信号;西瓜嗜酸菌Che A蛋白与青枯菌的Che A蛋白亲缘关系最近,和荧光假单胞菌的亲缘关系最远。3、Ac-5菌株趋化基因che A和che Y3的功能研究构建Δche A和Δche Y3基因缺失突变株以及Cche A互补菌株,并进行表型分析。结果发现:Δche A和Δche Y3基因缺失突变株与野生型菌Ac-5相比,趋化及运动能力下降;在种子表面的定殖能力下降;病株病情指数先下降后恢复野生型菌株Ac-5水平;生物膜形成能力增强;引起非寄主烟草的过敏性反应。4、che A基因缺失对其他基因表达量的影响利用荧光定量PCR分析che A基因缺失对其他基因表达量的影响。结果显示che A基因在突变株中不表达,T3SS中hrc N和hrp E基因,毒性蛋白trb C和vir B基因,鞭毛合成基因mot A和动力蛋白基因fli M以及信号接收蛋白基因che Y1均上调表达。
[Abstract]:Bacterial Fruit blotch (BFB) is a quarantine bacterial disease in the world, which mainly damages watermelon, muskmelon and other cucurbitaceae crops and causes great economic losses. Acidovorax citrullirii Schaad et Alba 2008 is the pathogen of this disease. Some studies have shown that the colonization ability of pathogenic bacteria on the host surface is closely related to its pathogenicity. The chemotactic ability of pathogenic bacteria is one of the key factors to determine colonization ability. This study focused on the chemotaxis of eosinophilic bacteria in watermelon and the function of chemoattractant genes. The main results were as follows: 1. The chemotaxis of watermelon acidophilic strain Ac-5 was determined by capillary method. The effects of carbon sources, amino acids and organic acids on the chemotaxis of watermelon acidophilic bacteria were determined by capillary method, and the results showed that: maltose, glucose, lactose, Sucrose and galactose promoted the chemotaxis of watermelon acidophilic bacteria significantly. L- leucine, L-leucine, L-tryptophan, L-valine, L-proline, L-glutamine, and L-glutamine significantly promoted the chemotaxis of watermelon acidophilic bacteria. L-methionine significantly inhibited its chemotaxis; citric acid and malic acid significantly promoted the chemotaxis of acidophilic bacteria in watermelon; Bioinformatics analysis of chemotaxis genes che A and che Y of Ac-5 strain in watermelon; bioinformatics analysis of the chemoattractant genes che A and che Y of watermelon strain Ac-5; access to the whole gene sequence of eosinophilic bacteria in watermelon in NCBI database to find the relevant information of che A and che Y genes. The A and che Y genes encode Che A and Che Y proteins. CHE A and Che Y proteins have many hydrophobic amino acid regions. CHE A protein is a transmembrane protein. At 600-700 amino acids (i.e., Che W binding region), the transmembrane structure of Che Y protein is not transmembrane structure, it is a kind of intramembrane protein, which is used to receive the signal of Che A protein, and the relationship between Che A protein of watermelon acidophilic bacteria and Che A protein of bacterial wilt is very close. The function of chemoattractant genes che A and che Y3 of Ac-5 strain was the furthest related to Pseudomonas fluorescein. The deletion mutants of 螖 che A and 螖 che Y3 genes and the complementary strain Cche A were constructed. The results of phenotypic analysis showed that the chemotaxis and motor ability of 螖 che A and 螖 che Y3 mutant were lower than those of wild type strain Ac-5, the colonization ability on seed surface was decreased, the disease index of disease plant was decreased first, and then the Ac-5 level of wild type strain was restored. The ability of biofilm formation was enhanced; Effects of allergy. 4che A gene deletion on the expression of other genes in non-host tobacco the effect of che A gene deletion on the expression of other genes was analyzed by fluorescence quantitative PCR. The results showed that the che A gene was expressed in mutant strains. Hrc N and hrp E genes were not expressed in T3SS. The expression of trb C and vir B genes, flagellum synthase gene mot A, dynamic protein gene fli M and signal receptor-protein gene che Y1 were up-regulated.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.4

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