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《Journal of Northeast Agricultural University》 2017-07
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Cloning and functional analysis of cmk encoding cytidylate kinase in Halomonas zhaodongensis

JIANG Juquan;YANG Lina;LIU Yanshuang;SUN Kaifu;School of Life Sciences, Northeast Agricultural University;  
In order to explore the halo-alkaline-tolerant mechanism of the moderate halophile Halomonas zhaodongensis NEAU-ST10-25 T, genomic DNA was screened from this strain by selection in Escherichia coli KNabc lacking three major Na+/H+antiporters. One recombinant plasmid designated pU CLYS1 enabled E. coli KNabc to grow in the presence of 0.2 mol · L-1NaC l. 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 pU C-LYS1. Of three intact ORFs, ORF4 had the highest identity of 86% with a putative cytidylate kinase(CMK) from Halomonas enlongata. For the convenience ofdifferentiation 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.coli. 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, pE T19, and then transformed into the competent cells of E. coli C41(DE3), followed by the induction by with isopropyl-D-1-thiogalactopyranoside(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.
【Fund】: 国家自然科学基金面上项目(31570045);; 黑龙江省自然科学基金面上项目(C201417);; 黑龙江省博士后科研启动金(LBH-Q14022)
【CateGory Index】: Q78;Q936
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