盐芥谷胱甘肽过氧化物酶基因(ThGPX8)的克隆与原核表达

Cloning and prokaryotic expression of ThGPX8from Thellungiella salsuginea

【目的】克隆盐芥谷胱甘肽过氧化物酶(GPXs)基因ThGPX8,对其进行序列分析,构建原核表达载体,为进一步研究ThGPX8的结构和功能奠定基础。【方法】以盐芥为材料,利用RT-PCR技术获得其ThGPX8基因的全长cDNA序列;应用生物信息学手段对该基因编码的蛋白结构和系统进化关系进行分析;构建原核表达载体pET-ThGPX8,转化E.coli BL21(DE3),进行IPTG诱导表达、SDS-PAGE检测和Western blotting鉴定。【结果】获得的ThGPX8基因序列的开放阅读框(ORF)为504bp,编码167个氨基酸,具有GPXs的特征结构域,不是跨膜蛋白;其二级结构主要由无规则卷曲组成,三级结构为二聚体。进化树分析表明,盐芥ThGPX8与拟南芥AlyGPX8、AtGPX8和油菜BnGPX8具有较高的同源性。盐芥ThGPX8与ThGPX6的理化性质存在差异,但具有相似的二级结构和三级结构。将ThGPX8转入E.coli BL21(DE3)中,经IPTG诱导表达得到了分子质量约23ku的蛋白,该蛋白在37℃下诱导5h可获得较高的表达量。【结论】获得的ThGPX8基因cDNA序列所编码的蛋白属于植物GPXs家族成员,利用构建的原核表达载体pET-ThGPX8转化E.coli BL21(DE3)获得了融合表达蛋白。

【Objective】In the study,ThGPX8 （Thellungiella salsuginea GPX8） was isolated and analyzed using bioinformatics tools to improve the structural and functional analysis of ThGPX8.【Method】ThGPX8 was cloned from T.salsuginea by RT-PCR.The structure and phylogenetic tree of ThGPX8 were analyzed by bioinformatics software.ThGPX8 was ligated into pET28a vector,and transferred into E.coli strain BL21（DE3） for heterologous expression.【Result】Nucleotide analysis showed that ThGPX8 sequence contained a 504 bp open reading frame （ORF）,which encoded 167 amino acid residues.Amino sequence analysis demonstrated that the deduced amino sequences of ThGPX8 had characteristic domain of GPXs.ThGPX8 was not a trans-membrane protein.The secondary structure of ThGPX8 mainly included random coils,and its tertiary structure was a dimer.The phylogenetic analysis of ThGPX8 revealed that ThGPX8 shared higher homology with AlyGPX8,AtGPX8 and BnGPX8.There were differences in physicochemical properties between ThGPX8 and ThGPX6,but they were similar in secondary and tertiary structure.After being induced by IPTG,a 23 ku recombinant protein was highly expressed in 37 ℃ for 5 h.【Conclusion】ThGPX8 was cloned from T.salsuginea,and the deduced amino sequence of ThGPX8 belonged to glutathione peroxidases in plant.pET-ThGPX8 was constructed and transferred into E.coli strain BL21（DE3）,and a 23 ku recombinant protein was expressed.