西瓜食酸菌抗铜性相关基因cusB的功能分析

发布时间：2018-06-15 03:52

本文选题：西瓜食酸菌 + RND家族外排转运体　；参考：《新疆农业大学》2015年硕士论文

【摘要】：西瓜食酸菌(Acidovorax citrulli)侵染葫芦科植物会使其产生一种破坏力极大的瓜类细菌性果斑病(bacterial fruit blotch,BFB),该病害已在世界各国迅速传播,对当地的西甜瓜产业造成严重的经济损失。目前铜素类杀菌剂广泛应用于防治瓜类细菌性果斑病,但过度使用,使得西瓜食酸菌抗铜能力逐渐增强,因此,研究该病菌的抗铜机理显得尤为突出。本研究首先对西瓜食酸菌RND(耐药结节细胞分化)家族外排转运体的膜融合蛋白基因cusB进行生物信息学分析,然后利用实验室保存的cusB基因缺失的突变体△cusB,通过比较野生型和突变体以及功能互补菌株在抗铜性、胞外分泌物检测、致病性及过敏性坏死反应等相关的生物学性状之间的差异分析,研究cusB基因的功能,主要获得以下结论:首先,cusB基因的生物信息学分析,cusB基因大小为1185bp,编码具有394个氨基酸残基的CusB蛋白亚基,主要作为一个转运体参与RND外排转运体的正常工作,西瓜食酸菌的CusB蛋白与大肠杆菌的CusB蛋白在理化性质以及亲缘关系上较近,预测该蛋白在1-30氨基酸残基处疏水性较高,且存在一个跨膜结构域,预测发现存在多个磷酸化位点,分泌蛋白的亚细胞定位于周质空间,其二级结构存在α-螺旋、β-折叠、无规则卷曲。其次,cusB基因缺失突变体与野生型和功能互补菌株表型测定比较本研究利用双亲杂交的方法构建功能互补菌株,与野生型相比,突变体在抗铜性,胞外多糖分泌,生物膜形成中显著降低,而在功能互补菌株中这些差异明显减小,表明西瓜食酸菌的cusB基因与这3种表型联系紧密,但cusB基因的缺失没有影响致病性及过敏性坏死反应、胞外纤维素酶的分泌、胞外蛋白酶的分泌,表明该基因与这3种表型关系不密切。
[Abstract]:The infection of Acidovorax citrulliae with Cucurbitaceae will produce a kind of bacterial fruit blotchchus bFGF, which has been spreading rapidly in many countries all over the world, and has caused serious economic losses to the local muskmelon industry. At present, copper fungicides are widely used to control bacterial fruit spot disease of melon, but the copper resistance ability of watermelon acid-eating bacteria is gradually enhanced by excessive use. Therefore, the study of copper resistance mechanism of this pathogen is particularly prominent. In this study, the membrane fusion protein gene cusB of the efflux transporter of RND (resistant nodular cell differentiation) family in watermelon was analyzed by bioinformatics. Then we compared the copper resistance and extracellular secretion of wild-type mutants and functional complementary strains by using the mutant cusB gene deleted in laboratory. To study the function of cusB gene by analyzing the differences of biological characters such as pathogenicity and anaphylactic necrosis reaction. The main conclusions are as follows: firstly, the bioinformatics analysis of CusB gene is 1185 BP, encoding 394 amino acid residues of CusB protein subunit, mainly as a transporter to participate in the normal work of RND efflux transporter. The relationship between CusB protein and Escherichia coli CusB protein was close in physicochemical properties and phylogenetic relationship. It was predicted that the protein was hydrophobic at 1-30 amino acid residues and had a transmembrane domain. It was predicted that there were many phosphorylation sites. The subcell of secretory protein is located in the periplasmic space, and its secondary structure is 伪 -helix, 尾 -fold and irregular curl. Secondly, the phenotypic determination of CusB gene deletion mutants was compared with wild type and functional complementary strains. In this study, we used the method of parental hybridization to construct functional complementary strains. Compared with wild type, the mutants were copper resistant and exopolysaccharide secreted. These differences were significantly decreased in biofilm formation and decreased in functional complementary strains, indicating that the cusB gene was closely related to the three phenotypes, but the absence of cusB gene had no effect on pathogenicity and anaphylactic necrosis. The secretion of extracellular cellulase and extracellular protease showed that the gene was not closely related to the three phenotypes.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.4

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