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目的:克隆霍乱弧菌色素产生相关基因,阐明霍乱弧菌色素产生的机制。方法:利用转座子插入失活产色素霍乱弧菌的色素产生相关基因,获取不产生色素的突变株。采用质粒拯救法,将相关片段克隆于测序载体。测序后,序列在NCBI网站上进行同源性比较。结果:利用转座子插入失活法筛选到4株突变菌株,基因同源性分析的结果显示克隆到的色素产生基因为对羟基丙酮酸氧化物酶(HPD),该序列和标准菌株N16961的序列差异主要集中在启动子区;对羟基丙酮酸氧化物酶基因的启动子区显示了一定的菌群特异性。结论:霍乱弧菌产生的色素属于黑素类色素,对羟基丙酮酸氧化物酶是色素产生过程中的关键酶,但霍乱弧菌色素产生与否不是羟基丙酮酸过氧化物酶基因序列差异的直接结果。
OBJECTIVE: To clone the genes related to Vibrio cholerae pigment production and elucidate the mechanism of Vibrio cholerae pigment production. Methods: Transposons were used to insert the genes related to the pigment production of Vibrio cholerae inactivation, and the mutants without pigment production were obtained. Using plasmid rescue method, the relevant fragments were cloned into sequencing vector. After sequencing, sequences were compared for homology on the NCBI website. Results: Four mutant strains were screened by transposon insertion inactivation method. The results of gene homology analysis showed that the cloned gene was HPD, which was identical to the standard strain N16961 The sequence differences mainly concentrated in the promoter region; the promoter region of the pyruvate oxidase gene showed a certain flora specificity. CONCLUSION: The pigment produced by Vibrio cholerae belongs to the melanoid pigment and the p-hydroxy pyruvate oxidase is the key enzyme in the pigment production. However, the cholera Vibrio pigment production is not different from that of the hydroxypyruvate peroxidase gene sequence Direct result.