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Alternative TitleThe Screening of Flavobacterium columnare Virulent Strain G4 Specific DNA Sequences and Characterization of Possible Virulence Factors
Thesis Advisor聂品
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword柱状黄杆菌 抑制性差减杂交 毒力因子 I型限制修饰系统 电转化效率
Abstract柱状黄杆菌是鱼类柱形病的病原,分为三种基因组型,不同基因组型的菌株致病力不同。为探明强、弱毒菌株基因组水平上的差异,以本实验室保存的分属两个不同基因组型的柱状黄杆菌G4和G18菌株为研究对象,开展毒力基因的筛选和鉴定方面的工作。分别以G4为检测子、G18为驱动子,采用抑制性差减杂交的方法,筛选到46个G4特异基因。其中,35个与已知蛋白具有同源性;分为12类,与DNA重组修复、无机离子转运代谢、外膜蛋白、肠毒素、转座酶和金属蛋白酶等功能相关。 通过扩增和分析柱状黄杆菌G4 I型限制修饰系统基因序列的全长,发现该系统含有四个基因(fclIM、fclIS、fclIR和一个功能未知的基因);fclIM和fclIS的启动子序列和核糖体结合位点与肝素黄杆菌相似。RT-PCR证明fclIM和fclIS在G4中有明显转录。Western blotting表明,FclIM亚基分子量约为90 kD。克隆fclIM和fclIS基因全长并转化E. coli TOP10,RT-PCR证明这两个基因能正常表达。将pGL006转化含有fclIM和fclIS基因的E. coli使pGL006甲基化。经甲基化的pGL006转化G4,该G4的电转化效率提高3.6倍,从而建立了通过E. coli体内甲基化质粒以提高柱状黄杆菌电转化效率的方法。 扩增和分析TonB依赖的受体、转座酶和ABC转运超家族等基因的全长,并证明这些基因仅在柱状黄杆菌部分菌株中存在,为认识其与疾病发生的关系奠定了基础。
Other AbstractColumnris disease occurs in freshwater fish throughout the world. Its causative agent has been identified as the bacterium Flavobacterium columnare. Recent investigations on genetic diversity of this bacterium have revealed the occurrence of three genomovars of F. columnare. In order to understand the possible difference in virulence among different strains of the bacterium, two strains of F. columnare, i.e. G4 and G18, which were shown to belong to different genomovars in a previous study in our laboratory, were employed in the present study. F. columnare G4 strain, which has been recognized as a strong virulence strain, was examined for its specific genes when compared with a low virulence strain, G18, by Suppression Subtractive Hybridization (SSH). A total of 50 clones which were specifically detected in F. columnare G4 were sequenced, and 46 sequences of different genes obtained. 35 of the 46 genes showed some degree of homology with known proteins and can be classified into following categories, related with DNA replication, or recombination and repairing, inorganic ion transport or metabolism, outer-membrane proteins, enterotoxin, transposase, and metabolism etc. The Type I restriction modification system (R-M system) in F. columnare G4 strain was described in this study. The complete sequence of Type I R-M system FclI was cloned by genomic DNA walking. Through analyzing the open reading frames (ORFs) of FclI, sequences encoding three subunits, i.e.m HsdM, HsdS and HsdR of Type I R-M system are identified, which were then named as fclIM, fclIS and fclIR in F. columnare G4, respectively. The ribose-binding site (RBS) and the putative promoter of fclIM and fclIS were analyzed, which are similar to those in F. hibernum. The transcription of fclIM, fclIS and fclIR in F. columnare G4 was determined by RT-PCR. fclIM and fclIS responsible for DNA methylation were transcribed, but fclIR associated with restriction was not. In addition, the size FclIM subunit was identified as about 90 kD by Western blotting. To identify the function of fclIM and fclIS in F. columnare G4, these genes were cloned into plasmid pACYC184 and transformed into E. coli TOP10. The expression of fclIM and fclIS in E. coli was identified by RT-PCR. The integrative plasmid pGL006 was transformed into E. coli containing fclIM and fclIS and was methylated. Using the methylated pGL006, the electroporation efficiency of F. columnare G4 increased 3.6 times under the voltage of 2.0 kV/cm. Several genes encoding putative virulence factors in F. columanre strain G4, such as TonB-dependent receptor, transposases, as well as ABC transporter permease protein were cloned by genome walking. he flanking sequence of a putative transposase was cloned. A putative Rhs element was located at the downstream of the putative transposase.
Document Type学位论文
Recommended Citation
GB/T 7714
李楠. 柱状黄杆菌强毒株特异基因的筛选及相关毒力因子的研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2009.
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