阴沟肠杆菌的Hy3AL和hycA部分基因克隆及序列分析

Cloning and Sequence Analysis of Large Subunit of Hydrogenase 3 and Formate Hydrogen Lyase Repressor of Enterobacter cloacae

为了克隆氢化酶3大亚基(HyA3L)和甲酸氢酶抑制因子(hycA),研究其在阴沟肠杆菌产氢代谢中的作用和机制,笔者利用CODEHOP设计阴沟肠杆菌NRRL B-414的HyA3L和hycA简并引物,选用引物HyA3J和HycAJ扩增基因组DNA,分别得到约1500、150bp的PCR产物,该产物连接pMD 18-T载体,并克隆转化至E.coli DH5α感受态细胞中,经筛选后测序。推导的HyA3J扩增产物氨基酸序列与Enterobacter cancerogenus ATCC 35316的HyA3L蛋白一致性最高达到99%,仅有3个氨基酸的差异,表明其为阴沟肠杆菌的HyA3L;推导的HycAJ扩增产物氨基酸序列与Enterobacter cloacae subsp.cloacae ATCC 13047的HycA蛋白一致性最高达到92%,仅有4个氨基酸的差异,表明其为阴沟肠杆菌的hycA。通过以上分析可得出,采用CODEHOP程序设计的简并引物可信性强,阳性率高。阴沟肠杆菌NRRL B-414的氢化酶3大亚基和甲酸氢酶抑制因子部分基因的成功克隆,为获得氢化酶3大亚基和甲酸氢酶抑制因子的基因全长克隆奠定基础,不但可以增加这2个基因的资源,也可为其基因敲除等代谢工程研究提供科学依据和工作基础。

In order to study the function of formate hydrogen lyase repressor （hycA） and large subunit of hydrogenase 3 （HyA3） in the producing-hydrogen metabolism,the authors used CODEHOP to design the degenerate primers of hycA and HyA3L,and chose two pairs of degenerate primers named HyA3J and HycAJ respectively,then used Enterobacter cloacae NRRL B-414 genome DNA as template to make degenerate PCR,got about 1500 bp and 150 bp PCR product respectively,they were transformed into the E.coli DH5α through being linked with pMD18-T vector and sequenced after filtration.Similarity alignment showed that the 1500 bp products were very similar to the large subunit of hydrogenase 3 genes,and shared 99% identity to the large subunit of hydrogenase 3 from Enterobacter cancerogenus ATCC 35316,and only had the difference of three amino acids;the 150 bp products were very similar to the hycA genes,and shared 92% identity to the hycA from Enterobacter cloacae subsp.cloacae ATCC 13047,and only had the difference of four amino acids.Cloning these two fragments would not only enrich the gene resources of hycA and large subunit of hydrogenase 3 genes,but also give the scientific warrant for the metabolic engineering research.