棉花GhVHA A基因的原核表达及其重组大肠杆菌的抗逆性分析

为进一步验证棉花GhVHA-A基因的功能,该研究将棉花GhVHA-A基因构建到原核表达载体pET28a上,利用IPTG诱导其在大肠杆菌BL21(DE3)中高效表达,同时对重组大肠杆菌BL21(pET28a-GhVHA-A)进行抗逆性分析。结果表明:(1)半定量RT-PCR分析发现,棉花幼苗液泡膜H+-ATPase基因(GhVHA-A)表达水平受脱水和高盐胁迫诱导。(2)将1 872bp长的编码区序列连接至原核表达载体pET28a上,成功构建了原核表达载体pET28a-GhVHA-A;SDS-PAGE电泳检测结果表明,在70kD左右处有1条特异表达的蛋白质条带,与预期的目的产物大小一致。(3)重组菌BL21(pET28a-GhVHA-A)的抗逆性分析发现,重组菌对PEG6000(20%)和NaCl(0.5mol/L)的抗性明显高于对照菌株BL21(pET28a),表明GhVHA-A基因在大肠杆菌中表达后能够增强菌株的抗性。本研究结果为GhVHA-A基因在植物抗逆基因工程中的应用提供了理论依据。

Prokaryotic Expression and Stress Tolerance of E.coli Expressing GhVHA A Gene from Cotton

In order to further verify the function of cotton GhVHA A gene, we constructed the GhVHA A gene into the prokaryotic expression vector pET28a and induced by IPTG in E. coli BL21 (DE3) for resistance analysis. The results indicated that: (1) semi quantitative RT PCR analysis showed that the expression level of GhVHA A gene in cotton seedlings was induced by dehydration and salt stress. (2) A 1 872 bp coding region was ligated into the prokaryotic expression vector pET28a. The prokaryotic expression vector pET28a GhVHA A was successfully constructed. The SDS PAGE electrophoresis results show that there was a specific protein band at about 70 kD, which was identical with the expected molecular weight of the recombinant protein. (3) The resistance of recombinant bacterium BL21 (pET28a GhVHA A) to PEG6000 (20%) and NaCl (0.5 mol/L) was significantly higher than that of the control strain BL21 (pET28a), and indicating that the expression of GhVHA A gene in E.coli can enhance the resistance of the strain. The results provided a theoretical basis for the application of GhVHA A gene in plant stress resistant genetic engineering.