盐穗木醛脱氢酶基因HcALDH7A1原核表达载体的构建及蛋白诱导表达与优化

The Vector Construction and Prokaryotic Expression of Halostachys caspica Aldehyde Dehydrogenase Gene( Hc ALDH7A1) in E. coli

【目的】为构建盐穗木盐响应基因Hc ALDH7A1的原核表达载体并进行外源基因的诱导表达和优化。【方法】以实验室已构建好的p m D18-T Si mple-Hc ALDH7A1/DH5α(Pst I,Sac I)重组载体中盐穗木醛脱氢酶基因(Hc ALDH7A1)为模板,设计带有酶切位点(EcoR I,Xho I)的引物通过PCR扩增亚克隆到p m D18-T Si mple vector中,测序鉴定正确后,通过EcoR I和Xho I双酶切,构建重组的原核表达载体p ET28aHc ALDH7A1。将鉴定正确的重组质粒转化到大肠杆菌Transetta(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE检测目的蛋白表达,并对其表达体系进行优化。【结果】成功构建盐穗木醛脱氢酶基因Hc ALDH7A1的原核表达载体,诱导Hc ALDH7A1外源基因表达蛋白优化的最佳条件为IPTG终浓度为0.6 mmol/L,30℃诱导时间为4 h。融合蛋白分子量大小为61 k D。【结论】成功在大肠杆菌体内诱导表达盐穗木醛脱氢酶Hc ALDH7A1基因,并明确目的蛋白的最佳表达条件。为后续在原核表达系统大肠杆菌细胞中探索盐穗木Hc ALDH7A1的生物和非生物胁迫的功能奠定了基础。

【Objective】In order to construct the prokaryotic expression vector for Halostachys caspica aldehyde dehydrogenase gene（ Hc ALDH7A1） and carry out prokaryotic expression of Hc ALDH7A1 gene.【Method】Specific primers containing EcoR I and Xho I restriction sites were designed and Hc ALDH7A1 gene was amplified from the p MD18- T Simple- Hc ALDH7A1 plasmid with the two restriction sites of Pst I and Sac I obtained in this laboratory previously,then PCR product was subcloned into p MD18- T simple vector. After enzyme digestion and sequencing were made sure correct by the identifications of PCR,the p ET28 a and p MD18- T Simple- Hc ALDH7A1 plasmid were digested by EcoR I and Xho I,lineared p ET28 a and sticky-end target gene were ligated into the recombinant prokaryotic expression plasmid（ p ET28a- Hc ALDH7A1） and the plasmid of p ET28a- Hc ALDH7A1 was transformed into E. coli（ Transetta）. Transetta containing recombi-nant and empty plasmids was induced by IPTG and the fusion protein（ 61 k D） of Hc ALDH7A1 was detected by SDS- PAGE.【Result】The recombinant prokaryotic expression vector（ p ET28a- Hc ALDH7A1） was constructed; Optimized condition for inducing Hc ALDH7A1 prokaryotic expression was 0. 6 mmol / L IPTG at a final concentration and induction time of 4 hours under the temperature of 30 ℃.【Conclusion】The expression of exogenous gene（ Hc ALDH7A1） was successfully induced in E. coli and the optimal expression conditions gained. These results might be able to lay the solid foundation for further exploration of the function of Hc ALDH7A1 from Halostachys caspicthe under biotic and abiotic stress.