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一肇东盐单胞菌（Halomonaszhaodongemis）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pUC—LYSl。该质粒可恢复大肠杆菌（Eschenchiacoli）Na＊／H’逆向转运蛋白缺陷株KNabc在含有0．2mol·L-1 NaCl的LBK培养基上生长。序列分析显示，重组质粒pUC—LYSl中外源DNA片段由1个N端截短的ORFl、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（Halomonasenlongata）中1个推测的胞苷酸激酶（Cytidylatekinase...

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Bibliographic Details
Published in东北农业大学学报 Vol. 48; no. 7; pp. 33 - 40
Main Author 姜巨全 杨立娜 刘艳双 孙开福
Format Journal Article
LanguageChinese
Published 东北农业大学生命科学学院,哈尔滨,150030 2017
Subjects
原核表达
肇东盐单胞菌
胞苷酸激酶（cMK）
酶活性
胞苷酸激酶(CMK)
肇东盐单胞菌
原核表达
enzymatic activity
cytidylate kinase (CMK)
prokaryotic expression
酶活性
Halomonas zhaodongensis
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Summary:为探究一株中度嗜盐菌一肇东盐单胞菌（Halomonaszhaodongemis）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pUC—LYSl。该质粒可恢复大肠杆菌（Eschenchiacoli）Na＊／H’逆向转运蛋白缺陷株KNabc在含有0．2mol·L-1 NaCl的LBK培养基上生长。序列分析显示，重组质粒pUC—LYSl中外源DNA片段由1个N端截短的ORFl、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（Halomonasenlongata）中1个推测的胞苷酸激酶（Cytidylatekinase，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（Halomonaszhaodongensis）被定名为Hz_cmk。Hz—CMK也与包括已被鉴定来自大肠杆菌（Ecoli）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（Ecoli）的同源基因Ec_cmk（作为正对照）构建至原核表达载体pETl9，转化到大肠杆菌（Ecoli）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HzCMK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。
Bibliography:JIANG Juquan, YANG Lina, LIU Yanshuang, SUN Kaifu （School of Life Sciences, Northeast Agricultural University, Harbin 150030, China）
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 pUC- LYS1 enabled E. coli KNabc to grow in the presence of 0.2 mol. L-1 NaCI. 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-LYSI. 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. coli. In
ISSN:1005-9369