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
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转基因生物新品种培育重大专项(2017ZX08005004);


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

    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1 872 bp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70 kD 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(0.5 mol/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。

    Abstract:

    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.

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刘 娜,倪志勇,芮 存,等.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[J].西北植物学报,2018,38(1):41-47

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  • 在线发布日期: 2018-02-05
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