青海湖盐单胞菌QHL1 ectABC基因簇克隆鉴定及二氨基丁酸转氨酶基因ectB的异源表达

Characterization of the ectABC gene cluster and heterologous expression of the ectB gene from Halomonas sp. QHL1 in Qinghai Lake

从青海湖20份水样筛选得到一株优势中度嗜盐菌QHL1,经初步鉴定为盐单胞菌属。设计保守基因ectB的引物,以基因组DNA为模板,PCR扩增获得ectB基因(1269 bp)。利用染色体步移技术,克隆获得四氢嘧啶合成基因簇ectABC及其上下游调控序列。DNAStar软件分析表明ectA、ectB和ectC位于同一个操纵子上,大小分别为579 bp、1269 bp和390 bp,预测分别编码192、422和129个氨基酸的肽链。同源性分析表明:Halomonas sp.QHL1ectABC基因簇所编码的二氨基丁酸转乙酰基酶(EctA)、二氨基丁酸转氨酶(EctB)和四氢嘧啶合成酶(EctC)与Halomonas sp.Nj223 ectABC基因簇所编码的酶蛋白相似性分别达57%、96%和85%。利用分子克隆技术构建二氨基丁酸转氨酶基因ectB的重组表达载体pET-28-ectB,通过限制性内切酶酶切和测序分析,结果表明其目的基因的插入位置、大小和读码框均正确。诱导表达重组菌,SDS-PAGE分析,目的蛋白条带约46 kDa。

The moderate halophile QHL1 isolated from 20 water samples of Qinghai Lake was preliminarily identified as a member of the genus Holomonas. The conserved ectB gene of 1269bp from this strain was amplified by PCR. The ectABC gene cluster for biosyn- thesis of ectoine was further cloned by genome walking methods. Analysis of the DNAStar software showed that ectA, ectB and ectC genes organized as an operon and the size were 579bp, 1269bp and 390bp predicting to encode peptides of 192, 422 and 129 amino acids, respectively. The deduced amino acid sequences of EctA, EctB and EctC shared 57% , 96% and 85% identity to 2, 4-diami- nobutyric acid acetyltransferase, 2, 4-diaminobutyric acid transaminase and ectoine synthase of Halomonas sp. Nj223 respectively ac- cording to homology analysis. The recombinant expression vector pET-28-ectB was constructed by molecular cloning. Indueion and ex- pression of the recombinant strain by IPTG and SDS-PAGE showed that the relative molecular mass of the expression product was 46kDa as predicted.