哈氏弧菌dam基因的克隆与分析

Cloning and analysis of the Vibrio harveyi dam gene

摘要:

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。

Abstract:

The DNA adenine methylase (dam) gene was cloned by degenerate PCR from Vibrio harveyi strain T4. The gene was 840bp in length and encoded a putative protein of 279 amino acids that shared relatively high homology with the Dam of other Vibrios, especially with that of V.parahaemolyticus (96% in identity). The V.harveyi dam gene was subcloned into plasmid pBR322 and the resulting plasmid pBD was introduced into the E.coli strain ER2925 in which the dam gene had been knocked out. DpnⅠ, DpnⅡ, and Sau3AⅠ restriction enzyme analysis of the genomic DNA of ER2925 transformed with pBD indicated that the cloned V.harveyi dam gene could functionally complement the E. coli dam mutant and methylate E.coli chromosome at the GATC sites. The 3251 bp upstream region of V.harveyi dam was obtained by genome walking and analyzed at the sequence level. It was found that this 3251 bp region contained two complete open reading frames (ORF): one was of 1101 bp in length and the other was of 1503 bp in length. The predicted amino acid sequence of ORF1101 shared 91% identity with the 3-dehydroquinate synthase of V. parahaemolyticus. The amino acid sequence of ORF1503 shared 80% identity with V. parahaemolyticus DamX. A truncated ORF was found at the upstream of ORF1101, encoding 169 amino acids that shared 94% identity with the shikimate kinase of V. parahaemolyticus. These three genes, together with dam, were arranged in the order of shikimate kinase-3-dehydroquinate synthase-damX-dam. The region immediate upstream of the V.harveyi dam structural gene was cloned in three fragments of different length: 78bp, 112 bp and 477bp (named P78, P112，and P477, respectively) and tested for promoter activity. The results showed that，while all the three fragments had detectable promoter activities, the activity of P78 appeared to be higher than that of P₁₁₂ and P₄₇₇.