Heterogeneous expression of DnaK gene from Alicyclobacillus acidoterrestris improves the resistance of Escherichia coli against heat and acid stress.
Heterogeneous expression of DnaK gene from Alicyclobacillus acidoterrestris improves the resistance of Escherichia coli against heat and acid stress.
复制标题
酸土脂环酸芽孢杆菌DnaK基因的异质表达提高了大肠杆菌对热和酸胁迫的抵抗力
DOI：
10.1186/s13568-017-0337-x
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发表时间：
2017-12
期刊：
影响因子：
3.7
通讯作者：
Zhao R
中科院分区：
文献类型：
--
作者：
Xu X;Jiao L;Feng X;Ran J;Liang X;Zhao R
酸土脂环酸芽孢杆菌是一种嗜酸嗜热菌，是筛选抗逆基因的重要微生物资源。本研究将A.将Acidoterrestris基因进行亚克隆，构建重组质粒pET 28 a-DnaK。经双酶切和测序分析证实质粒构建成功。将重组质粒转化大肠杆菌BL 21，用异丙基-β-d-硫代半乳糖苷（isopropy-β-d-thiogalactoside，IPTG）诱导表达重组E. coli中表达DnaK基因。经SDS-PAGE鉴定，融合蛋白分子量约为70 kD，表明A. acidoterrestris成功表达。在pH5.0 -7.0的条件下，分别在54、56和58 °C的高温下对重组菌和野生菌进行处理，以比较异源表达A.对植物抗逆性的影响。实验结果表明，重组菌BL 21-DnaK在高温和酸胁迫下的存活率比野生菌BL 21有明显提高，D值比野生菌提高了14.7-72%，表明A.酸土芽孢杆菌能显著增强宿主菌E.大肠杆菌抵抗热和酸胁迫。
Alicyclobacillus acidoterrestris, an acidophilic and thermophilic bacteria, is an important microbial resource for stress resistance genes screening. In this study, DnaK gene from A. acidoterrestris was subcloned to construct the recombinant plasmid pET28a–DnaK. The successful construction of the plasmid was verified by double-enzyme digestion and sequencing analysis. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-β-d-thiogalactoside (IPTG) was used to induce recombinant E. coli to express DnaK gene. A 70 kD fusion protein was identified by SDS-PAGE, which suggested that DnaK gene from A. acidoterrestris was successfully expressed. The recombinant and wild BL21 were treated with high temperatures of 54, 56 and 58 °C at pH values of 5.0–7.0 to compare the effects of heterogeneous expression of the DnaK gene from A. acidoterrestris on the stress resistance. The experimental results showed that survival rate of recombinant BL21–DnaK has been improved considerably under heat and acid stresses in contrast with the wild BL21, and D-values of recombinant BL21 were 14.7–72% higher than that of wild BL21, which demonstrated that heterogeneous expression of DnaK gene from A. acidoterrestris could significantly enhance the resistance of host bacteria E. coli against heat and acid stresses.