中国水产科学2011，Vol.18Issue(5)：1100-1107,8.DOI:10.3724/SP.J.1118.2011.01100
柱状黄杆菌乙酰辅酶A合成酶基因及其上游调控序列分析
Acetyl-coenzyme A synthetase gene and its upstream promoter in the bacterial pathogen Flavobacterium columnare
摘要
Abstract
Flavobacterium columnare, the etiological agent of columnaris disease, is one of the most important bacterial pathogens of freshwater fish in the world. However, suitable genetic manipulation of this bacterium which has been challenging and producing genetic mutations in this bacterium has not been reported. Therefore, isolation of an effective promoter in F. Columnare may provide tools for the development of a genetic manipulation system in the bacterium. In this study, the acetyl-coenzyme A synthetase gene (acs) and flanking sequences were analyzed to determine promoter function. The acs gene is 2 323 bp long, encoding a protein of 635 amino acids. A TAAAA motif was identified as the conserved sequence for ribosome binding site (RBS) and TATTTTCG and TTG were determined to be -7 and -33 promoter motifs, upstream of the acs start codon. When a plasmid was constructed containing the acs upstream regulation sequence (Pacs) fused upstream of the chloramphenicol ace-tyltransferase (cat) gene and was introduced into F. Columnare G4 strain, the host cells gained chloramphenicol (Cm) resistance. The transcriptional start position (TSP) of acs and cat was both determined to be T locating 46 bp upstream of the start codon. Deletion analysis of the promoter showed that at least 164 bp nucleotides upstream of the start codon were required for promoter activity and for the expression of CAT to sufficiently confer the resistance in F. Columnare. Nucleotide analysis and alignment of the putative RBS for 32 protein-coding genes in F. Columnare G4 revealed the conserved RBS consensus, TAAAA, in Flavobacterium located 10 bp upstream of start codon of acs. The current study described the first successful construction of a plasmid that was able to express cloned genes in F. Columnare, which will allow further studies of the important columnaris disease in fish.关键词
柱状黄杆菌/乙酰辅酶A合成酶基因/启动子/核糖体结合位点Key words
Flavobacterium columnare/ acetyl-coenzyme A synthetase gene/ promoter/ ribosome binding site分类
农业科技引用本文复制引用
张金,邹红,姚卫建,聂品..柱状黄杆菌乙酰辅酶A合成酶基因及其上游调控序列分析[J].中国水产科学,2011,18(5):1100-1107,8.基金项目
国家重点基础研究发展计划项目(2009CB11 8703). （2009CB11 8703）
