肇东盐单胞菌中胞苷酸激酶基因cmk的克隆与功能分析

为探究一株中度嗜盐菌-肇东盐单胞菌(Halomonas zhaodongensis)NEAU-ST10-25T的耐盐碱机制,采用基因文库筛选方法从该菌株获得一个重组质粒p UC-LYS1。该质粒可恢复大肠杆菌(Escherichia coli)Na+/H+逆向转运蛋白缺陷株KNabc在含有0.2 mol·L-1Na Cl的LBK培养基上生长。序列分析显示,重组质粒p UC-LYS1中外源DNA片段由1个N端截短的ORF1、三个完整的ORF(ORF2-4)以及1个C端截短的ORF5组成。其中,ORF4与伸长盐单胞菌(Halomonas enlongata)中1个推测的胞苷酸激酶(Cytidylate kinase,CMK)同源性最高(86%)。为便于区分与其同源物,编码ORF4的基因cmk来自肇东盐单胞菌(Halomonas zhaodongensis)被定名为Hz_cmk。Hz_CMK也与包括已被鉴定来自大肠杆菌(E.coli)的Ec_CMK等其他同源物具有较高同源性。为进一步鉴定Hz_cmk是否编码胞苷酸激酶,通过PCR扩增分别将Hz_cmk和大肠杆菌(E.coli)的同源基因Ec_cmk(作为正对照)构建至原核表达载体p ET19,转化到大肠杆菌(E.coli)C41(DE3)感受态细胞中诱导表达。SDS-PAGE表明其以可溶形式存在。酶学分析表明Hz_CMK与Ec_CMK均具有较高胞苷酸激酶活性。为国内外首次中度嗜盐菌的胞苷酸激酶基因功能鉴定。

Cloning and functional analysis of cmk encoding cytidylate kinase in Halomonas zhaodongensis

In order to explore the halo-alkaline-tolerant mechanism of the moderate halophile Halomonas zhaodongensis NEAU-ST10-25T,genomic DNA was screened from this strain by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters.One recombinant plasmid designated pUCLYS1 enabled E.coli KNabc to grow in the presence of 0.2 mol· L-1 NaCl.Sequence analysis showed that one N-terminal ORF1,three intact ORF (ORF2-4) and one C-terminal ORF5 are included in the DNA sequence inserted in the recombinant plasmid pUC-LYS1.Of three intact ORFs,ORF4 had the highest identity of 86％ with a putative cytidylate kinase (CMK) from Halomonas enlongata.For the convenience of differentiation of its homologs,the gene cmk encoding ORF4 from H.zhaodongensis was designated Hz_cmk.Hz_CMK has also the higher identity with its homologs including the identified Ec_CMK from E.coll In order to determine whether it encodes a cytidylate kinase,Hz_cmk,as well as Ec_cmk from E.coli as a positive control,was respectively constructed into a prokaryotic expression vector,pET19,and then transformed into the competent cells of E.coli C41 (DE3),followed by the induction by with isopropyl-D-1thiogalactopyranoside (IPTG).SDS-PAGE showed that Hz_CMK or Ec_CMK was overexpressed in E.coli C41 (DE3) and soluble analysis also showed that either of them is present mostly in a soluble form,revealing that Hz_CMK or Ec_CMK should be functional in E.coli.Finally,enzymatic assay showed that Hz_CMK,as well as Ec_CMK,displayed the cytidylate kinase activity.To the best of our knowledge,this is the first report on the cloning and functional analysis of the cytidylate kinase gene from the moderate halophile.