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Alternative TitleConstruction Deletion Mutant Strain of Flavobacterium colunmare
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 自杀质粒 滑动基因 蔗糖筛选
Abstract柱状黄杆菌(Flavobacterium columnare)是鱼类柱形病(Columnaris disease)的病原,该菌在淡水中分布广泛,可感染野生、养殖及观赏的各种冷水和热带鱼类。柱状黄杆菌主要感染鱼类的鳃组织,导致大面积的鳃部坏死;也会感染体表组织导致引起体表溃烂。 为鉴定可能毒力因子的功能,本研究就构建滑动基因GldH(AY781295)的缺失突变子的可行性进行了研究。本研究依据overlap PCR、启动子的鉴定、自杀质粒的构建、结合转移、同源重组及蔗糖负选择的方法来构建滑动基因GldH的缺失突变子。通过overlap PCR获得了大小为1658bp的滑动基因GldH的的缺失片段ΔMH,该片段为GldH内部缺失了一小段,但是却不发生移码的序列,这样也就保证了突变为非极性突变。通过氯霉素抗性筛选,鉴定了一段327bp的含胶原蛋白酶启动子的序列,该序列位于柱状黄杆菌胶原蛋白酶基因(EF501979)起始密码子前,可启动氯霉素基因的表达并赋予大肠杆菌17μg/ml浓度的氯霉素抗性。依据上述胶原蛋白酶基因启动子,本研究构建了该启动子与氯霉素基因ORF的融合片段,并将此融合片段替换了自杀质粒pRE112的氯霉素基因片段,在连接入滑动基因GldH的的缺失片段ΔMH后得到了新的自杀质粒pREFC-ΔMH。将自杀质粒pREFC-ΔMH转化到大肠杆菌S17-1(λpir),然后与对数生长期的柱状黄杆菌进行结合转移。24小时后通过双抗平板(cm 3.4μg/ml, To 1μg/ml)来筛选基因组整合有质粒pREFC-ΔMH的柱状黄杆菌,结果表明质粒pREFC-ΔMH成功整合到了柱状黄杆菌基因组中。将上一步得到的柱状黄杆菌进行多次传代,然后涂含10%蔗糖的Shieh平板以筛选发生了第二次同源重组的突变子,结果未筛选到发生了第二次同源重组的突变子。 另外,本研究分析了柱状黄杆菌细胞和细胞裂解物对FALPGPA(胶原蛋白酶特异性底物)的降解活性,两者结果都表明柱状黄杆菌具有水解胶原的能力。
Other AbstractFlavobacterium columnare is the causative agent of Columnris disease. It infects wild, farmed and ornamental fish species in freshwater worldwide. F. columnare causes erosion and necrosis of external tissues, with gills often being a major site of damage. In order to identify the function of possible virulent factors, this study tried to construct the deletion mutation of GldH (AY781295) gene. The technology of overlapping PCR, promoter identification, suicide plasmid construction, trans-conjugation, homologous recombination and sucrose counter-selection were employed. A deletion fragment of GldH was constructed and this 1658 bp fragment was named ΔMH, in which a small fragment of GldH was deleted and no frame-shift mutation occurred, so this kind of mutation was non-polar mutation. By the selection of Chloramphenicol, a 327 bp length sequence was identified with promoter function. It is the leading sequence of the initiation codon of collagenase gene (EF501979). This sequence promoted the expression of Chloramphenicol gene, and enabled E. coli to duplicate in the LB broth with 17 μg/ml Chloramphenicol. A fusion fragment was constructed with promoter sequence and Chloramphenicol gene ORF. The Chloramphenicol gene of suicide plasmid pRE112 was replaced with the fusion fragment, further more ΔMH was cloned into this plasmid, then a new plasmid pREFCΔMH was constructed. Suicide plasmid pREFCΔMH was transformed into E. coli S17-1(λpir). Bacterial mating was done when both donor and recipient cells were grown to mid-log phase. 24 h later, double antibiotic resistance (cm 3.4 µg/ml, To 1 µg/ml) plate was used to select trans-conjugation, the result demonstrated that plasmid pREFCΔMH was integrated into the genome of F. columnare. The positive trans-conjugation was sub-cultured in Shieh broth (To+) for several generations, then the Shieh plate with 10% sucrose was used to select the mutant with the occurrence of second cross-over. In the present study, the ability of a high virulence strain (G4) and a low virulence strain (G18) of F. columnare to hydrolyze the synthetic peptide FALGPA, which is similar in structure to collagen, was investigated by the frozen-thawing procedure method. This suggests a cell-associated collagenolytic activity in F. columnare.
Document Type学位论文
Recommended Citation
GB/T 7714
张立强. 柱状黄杆菌缺失突变株的构建[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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