【作者】 徐艳；

【导师】 李茵；

【作者基本信息】 东华大学 ， 环境工程， 2011， 硕士

【摘要】 Pantoea ananatis (P. ananatis)是一种对许多重要经济农作物造成广泛危害的革兰氏阴性菌,尤其在麦穗成熟期侵染水稻而造成白叶枯病和不实,侵染洋葱的叶和茎,造成洋葱“中心腐烂”疾病和洋葱叶弯落。然而,P. ananatis的主要致病因子尚未完全明确。已有研究证实该植物病原菌毒性与其生产胞外多糖(EPS)相关。胞外多糖在植物病原菌入侵植物体,生物膜形成和枯萎病诱导方面起着至关重要的作用,被认为是许多植物病原菌的致病性决定性因素。研究表明,Pantoea stewartii,一种引起玉米病变的病原体,其致病性随着胞外多糖合成基因群的突变而丧失。Erwinia amylovora,其变异体由于缺乏合成胞外多糖基因,致病性也随之减小。因此可以推断胞外多糖的合成与P. ananatis的病原性相关。本课题的研究目的就是确认并分离能大量生产胞外多糖的P. ananatis野生株的变异体,提取变异体的质粒DNA,鉴定突变基因,从而分析其与过量生产胞外多糖的关系。为了鉴定能大量生产胞外多糖的变异体的基因,用电击转化法将转座子Tn5-OT182导入P. ananatis野生株。利用含四环素的LB培养基培养得到野生株变异体的克隆体,克隆体于含四环素的TSB培养基上培养,一共得到大约3000个生产胞外多糖变异体的克隆体,并成功分离出其中21个能大量生产胞外多糖的变异体。为了确认变异基因,抽出变异体的染色体DNA,用四种限制性内切酶EcoRI, SadI XhoI或HindⅢ进行酶切,染色体片段自我连结后导入大肠杆菌,用LB培养基培养后得到形质转换体。采用改良的碱裂解法提取质粒,通过BigDye Terminator ver.3.1和ABI Prism 3100基因分析仪进行DNA测序。特异性的引物与其互补的序列杂交,将获得的DNA序列与DDBJ/EMBL/GenBank数据库比较,推断突变基因的表达功能。突变体33,91,40,11,46,81,29和98的突变基因分别与已知基因高度相似。能大量生产胞外多糖的变异体33,91和40的变异基因假定功能表达分别为黏性蛋白质,细胞外膜蛋白和脂蛋白,通过分析这些蛋白质功能,细胞表面结构的突变可能导致大量的胞外多糖的产生。变异体11的变异基因在从活性糖配体到单糖或非单糖受体的糖移动过程中起催化作用。变异体46和81变异基因的功能表达分别是糖基转移酶和铁离子运输脂蛋白,可能影响胞外多糖的合成。变异体29和98功能表达为感应蛋白质或核糖核苷酸酶R,可能与胞外多糖的成分之一核酸的合成有关。更多还原

【Abstract】 Pantoea ananatis(P. ananatis) is one of the gram-negative bacteria and encountered worldwide. P. ananatis was reported as a common colonist of wheat heads at ripening and caused disease symptoms in a wide range of economically important agricultural crops. Especially, "center rot" disease of onion caused by P. ananatis induced that the infected leaves were usually collapsed and hang down beside the neck. However, the major virulent factors of P. ananatis had not been elucidated yet.It was reported that virulence of the plant pathogens were correlated with the ability to produce extracellular polysaccharide (EPS). EPS was considered a virulence determinant in many bacterial plant pathogens because of its central role in wilt induction, host colonization, biofilm formation. It was thus likely to be important in physical interactions with the host. In Pantoea stewartii, which was a vascular pathogen of corn maize, mutations at the EPS biosynthetic gene cluster generally lead to loss of pathogenicity. In Erwinia amylovora, which was the cause of fire blight disease, mutants that lacked the synthesis of EPS were reduced in virulence. From these reports, it was assumed that EPS biosynthesis correlated with the pathogenicity of P. ananatis. The purpose of this study was to isolate and characterize the transposon mutants which caused hyper production of EPS, to extract the plasmid DNA of the mutants and identify the mutated genes, and to investigate the relationship between the mutated genes and excessive production of EPS.In order to identify specific genes which caused hyper production of EPS, P. ananatis strain SK-1 was mutagenized with the transposon Tn5-OT182. Transposon carrier plasmid pOT182 was transformed into SK-1 by electropolation. Transposon mutants of SK-1 were selected by plating on LB agar plates containing tetracycline. Transposon mutants were replica-plated onto TSB agar plates containing tetracycline, a total of 3,000 colonies were screened. As the results,21 mutants, that showed hyper production of EPS, were successfully isolated. In order to identify the mutated gene, chromosomal DNA of mutants was extracted, digested with following restriction enzymes: EcoRⅠ, SacⅠ, XhoⅠor HindⅢThe chromosomal fragments were self-ligated and transformed into Escherichia coli DH5α. Transformants were selected by plating on LB agar plates containing tetracycline. Plasmids were extracted by improved alkali lysis method from the transformants. DNA sequencing was performed by using BigDye Terminator ver. 3.1 and ABI Prism 3100 Genetic Analyzer (Applied Biosystems). The specific primers were used to initiate DNA sequence reactions with plasmids obtained by self-cloning. DNA sequences were compared with the sequences in the DDBJ/EMBL/GenBank databases, whose predicted functions were deduced.As the results, the transposon integration of the mutant 33,91,40,11,46,81,29 and 98 was observed to be located in a gene with strong homology to known proteins. The mutation sites of the EPS hyper-producing mutant 33,91, and 40 were genes that encoded an adhesion-like protein, outer membrane protein and lipoprotein, respectively. Mutation of cell surface structure might cause excessive EPS in SK-1. The mutation site of the EPS hyper-producing mutant 11 was a gene that catalyzed the transfer of a sugar moiety from an activated sugar donor to saccharide or non-saccharide acceptors. Mutation of glycosyltransferase or iron transport lipoprotein in mutant 46 and 81 might affect metabolisms of EPS biosynthesis. Mutation of putative sensor protein or ribonuclease R in mutant 29 and 98 were related to biosynthesizing of nucleic acid, one composition of EPS.更多还原