杜仲EuGT2基因的原核表达及蛋白纯化  

Prokaryotic Expression and Protein Purification of EuGT2 Gene from Eucommia ulmoides

作　　者：冷阳 刘世会[1,3] 赵懿琛 赵德刚[3,4] Leng Yang;Liu Shihui;Zhao Yichen;Zhao Degang(School of Pharmaceutical Sciences,Guizhou University,Guiyang,550025;School of Tea Sciences,Guizhou University,Guiyang,550025;The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Guizhou University,Guiyang,550025;Guiyang Station for DUS Testing Center of New Plant Varieties of MOA.P.R.China,Guizhou Academy of Agricultural Sciences,Guiyang,550006)

机构地区：[1]贵州大学药学院,贵阳550025 [2]贵州大学茶学院,贵阳550025 [3]贵州大学,山地植物资源保护与种质创新省部共建教育部重点实验室,贵阳550025 [4]贵州省农业科学研究院,国家农业农村部DUS中心贵阳分中心,贵阳550006

出　　处：《分子植物育种》2020年第10期3240-3245,共6页Molecular Plant Breeding

基　　金：国家自然科学基金(31660076);国家转基因生物新品种培育科技重大专项(2016ZX08010003-009);贵州省科学技术基金(黔科合J字[2015]2042)共同资助。

摘　　要：本研究以克隆所得杜仲(Eucommia ulmoides)EuGT2基因为基础,利用基因重组技术构建原核表达载体pMCSG19-EuGT2。重组载体转化到E.coli Rosetta(DE3)中,用0.05 mmol/L异丙基β-D-硫代半乳糖苷(IPTG)在37℃下诱导6 h。使用镍-亚氨基二乙酸(Ni-IDA)亲和层析柱进行纯化,并用15%十二烷基硫酸钠-聚丙烯酰胺(SDS-PAGE)凝胶电泳进行检测。研究结果表明,杜仲EuGT2基因在E.coli Rosetta(DE3)中得到成功表达,重组蛋白表达形式为部分包涵体、部分可溶性蛋白。经SDS-PAGE凝胶电泳检测,在相对分子质量约56 kD处有1条特异性蛋白条带,经质谱鉴定该特异性蛋白条带确为杜仲EuGT2蛋白。以上研究结果为后续研究杜仲糖基转移酶的功能提供了科学依据。In this study,the prokaryotic expression vector pMCSG19-EuGT2 was constructed by gene recombination based on EuGT2 gene from Eucommia ulmoides.The recombinant vector was transformed into E.coli Rosetta(DE3)and induced with 0.05 mmol/L isopropylβ-D-thiogalactoside(IPTG)for 6 hours under 37℃.Purification was carried out using a nickel-iminodiacetic acid(Ni-IDA)affinity chromatography column and detection by 15%sodium dodecyl sulfate-polyacrylamide(SDS-PAGE)gel electrophoresis.The results showed that the EuGT2 gene was successfully expressed in E.coli Rosetta(DE3),and the recombinant protein was expressed as partial inclusion bodies and partially soluble proteins.SDS-PAGE gel electrophoresis showed that there was a specific protein band at a relative molecular mass of about 56 kD.The specific protein band was identified as EuGT2 protein by mass spectrometry.The above results provide scientific basis for the subsequent study of the function of Eucommia ulmoides glycosyltransferase.

关 键 词：杜仲(Eucommia ulmoides) 糖基转移酶 原核表达 蛋白纯化 

分 类 号：S567.19[农业科学—中草药栽培]