盐单胞菌属BYS1四氢嘧啶合成基因ectABC克隆及其盐激表达

利用SEFA-PCR技术从中度嗜盐菌Halomonassp.BYS-1总DNA中克隆了四氢嘧啶合成基因ectABC及其上游序列(GenBank accession number DQ017757);OMIGA软件分析结果显示ectA、ectB、ectC位于同一个操纵子上,大小分别为573bp1、251bp和387bp,预测编码的DAT(L-二氨基丁酸转氨酶)、DAA(L-二氨基丁酸乙酰转移酶)和ES(四氢嘧啶合酶)大小分别为21.1kDa(191 amino acid)、45.7kDa(417 amino acid)和14.5kDa(129 amino acid);将包含ectABC基因及其上游1000bp序列的片段克隆到pUC19中并转化E.coliDH5α,转化子E.coli(pUC19ECT)能够在盐激条件下合成四氢嘧啶,但其耐盐能力没有得到显著改善。

Cloning of the ectoine biosynthesis gene ectABC from Halomonas sp.BYS-1 and salt stressed expression in Escherichia coli

Ectoine was the main compatible solute of moderately halophilic bacteria. In order to clone the ectABC gene which involved in the ectoine biosynthesis pathway from total DNA of moderately halophilic bacteria Halomonas sp. BYS-1, firstly a 750bp fragment of ectABC gene was amplified by PCR using combinations of forward primers and reverse primers designed according to the ectABC genes of Halomonas elongata 2851T and Halomonas elongata DSM3043. Then the upstream and downstream sequences of the 750bp fragment were amplified by SEFA PCR (SElf-Formed Adaptor PCR), a new PCR method amplified relatively long flanking sequences from tagged sequences in a simple way without enzyme excision and ligation. The 3532bp fragment include 2423bp ectABC, 980bp upstream sequences and 129bp downstream sequences were cloned from Halomonas sp. BYS-1 using a pair of conserved primers designed according to acquired sequences by SEFA PCR. The GenBank accession number of the 3532bp fragment is DQ017757. ORF analysis revealed that ectA, ectB, ectC cluster to an operon, the size of ectA, ectB, and ectC were 573bp, 1251bp and 387bp respectively. The predicted molecular masses of the encoded proteins were 21.1kDa (191 amino acids, EctA), 45.7 kDa (417 amino acids, EctB), and 14.5 kDa (129 amino acids, EctC) respectively. The 3532bp fragment was ligated to the MCS site of vector pUC19 and transformed E. coli DH5alpha to construct E. coli (pUC19ECT). Transformant E. coli (pUC19ECT) could synthesis ectoine under salt stress, the intracellular ectoine level were 7.1, 19.4 and 32.3 micromol/(g x dry x wt) when the salinities of the mediums were 0, 0.4 and 0.8mol/L sodium chloride respectively. But the accumulation of ectoine could not promote the growth of E. coli (pUC19ECT)under high salinity.