尼泊尔黄堇G6PDH蛋白基因的异源表达及蛋白纯化  

Prokaryotic Expression and Protein Purification of G6PDH Protein Gene in Corydalis hendersonii Hemsl.

作　　者：任玉玲 孙胜男 王伊慧 赵成周 李萍 Ren Yuling;Sun Shengnan;Wang Yihui;Zhao Chengzhou;Li Ping(College of Eco-environmental Engineering,Qinghai University,Xining,810016;College of Tibetan Medicine,Qinghai University,Xining,810016)

机构地区：[1]青海大学生态环境工程学院,西宁810016 [2]青海大学藏医学院,西宁810016

出　　处：《分子植物育种》2021年第20期6708-6715,共8页Molecular Plant Breeding

基　　金：国家自然科学基金地区项目(31660063);青海科技厅基础研究项目(2017-ZJ-950Q);教育部春晖计划项目(Z2017056);海南藏族自治州科技计划项目“海南州野生甘蒙锦鸡儿驯化研究”共同资助。

摘　　要：大量研究表明葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PDH)在响应生物和非生物胁迫方面具有重要作用,为进一步研究珍贵濒危藏药材尼泊尔黄堇对极端环境的适应性,本研究根据尼泊尔黄堇转录组信息设计ChG6PDH扩增引物,利用PCR克隆获得ChG6PDH基因,构建原核表达载体ChG6PDH-pMAL-C2x,将其导入大肠杆菌BL21菌株诱导表达,利用亲和层析法对重组蛋白进行纯化,并用6-磷酸葡萄糖为底物在37℃对其进行体外酶活力检测。结果表明,重组可溶性蛋白最佳诱导条件为0.5 mmol/L的异丙基β-D-硫代半乳糖苷(IPTG)在37℃诱导5 h,SDS-PAGE和Western Blot检测纯化的酶蛋白,发现ChG6PDH蛋白分子量为60 kD。体外活性测定表明MPB-ChG6PDH蛋白具有G6PDH酶催化能力,酶活力是3228 nmol·min^(-1)·mg^(-1)prot。该研究结果为尼泊尔黄堇G6PDH酶蛋白的功能研究提供基础。A large number of studies have shown that glucose-6-phosphate dehydrogenase(glucose-6-phosphate dehydrogenase,G6 PDH)plays an important role in response to biotic and abiotic stress.Corydalis hendersonii Hemsl is a kind of precious and endangered Tibetan medicine berth.For further study the mechanism of Corydalis hendersonii Hemsl adapting to extreme environment,according to the transcriptome information of Corydalis hendersonii Hemsl,the amplification primers were designed.The Ch G6 PDH was cloned,prokaryotic expression vector ChG6 PDH-pMAL-C2 x was constructed,and was transformed into E.coli BL21 cells for culture to obtain genetically engineered bacteria with stable expression in this research.The recombinant protein was purified by affinity chromatography and its enzyme activity was detected at 37℃using glucose 6-phosphate as substrate.The results showed that the optimal induction condition of the recombinant soluble protein was 37℃,and the isopropyl-D-thiogalactoside(IPTG)with the concentration of 0.5 mmol/L was induced at 37℃for 5 h.The purified protein was identified by SDS-PAGE and Western Blot,and the purified enzyme protein was detected,and the molecular weight of ChG6 PDH protein was found to be 60 k D.In vitro activity assay showed that MPB-ChG6 PDH protein had the catalytic ability of G6 PDH enzyme,and the enzyme activity was 3228 nmol·min^(-1)·mg^(-1)prot.The results will provide a theoretical basis for further research on the function of G6 PDH in Corydalis hendersonii Hemsl.

关 键 词：尼泊尔黄堇(Corydalis hendersonii Hemsl.) G6PDH蛋白 原核表达 蛋白纯化 蛋白质印迹法 

分 类 号：S567.239[农业科学—中草药栽培]