摘要
目的研究当归咖啡酸-O-甲基转移酶(AsCOMT)在大肠杆菌(E.coli)BL21(DE3)中的表达,以及AsCOMT基因在当归抽苔植株和紫外线(UV)-B辐射胁迫下不同组织中的表达。方法基于前期克隆的AsCOMT基因全长cDNA序列,利用双酶切技术构建pGEX-4T-3-AsCOMT原核表达载体,将其转化至E.coli BL21(DE3)中,优化重组蛋白诱导表达条件,通过聚丙烯酰胺凝胶制备试剂盒(SDS-PAGE)电泳检测;并利用实时荧光定量PCR(qRT-PCR)分析AsCOMT在当归抽苔植株和UV-B辐射胁迫下不同组织中的表达模式。结果异源表达得到重组蛋白分子量为40 KDa,大小与预测分子量一致,AsCOMT蛋白主要以包涵体形式存在。qRT-PCR分析显示,抽苔当归茎中COMT的表达量显著高于其他组织(P<0.05);UV-B辐射胁迫下,茎和叶中AsCOMT基因的表达水平差异有统计学意义(P<0.05),即随UV-B辐射增强,AsCOMT基因的表达量在茎和叶中呈先增加后减少的趋势,而在根中无明显变化。结论AsCOMT在不同组织和处理下表达水平存在差异,且在E.coli中成功表达,为后期深入研究当归阿魏酸生物合成调控研究奠定了基础。
Objective To study the expression of Angelica sinensis caffeic acid-O-methyltransferase(As-COMT)in E.coli BL21(DE3)and the expression of AsCOMT gene in different tissues of Angelica sinensis under the radiation stress of bolting plant and ultra violet(UV)-B.Methods Based on the full-length cDNA sequence of AsCOMT gene cloned previously,the prokaryotic expression vector pGEX-4T-3-AsCOMT was constructed by double enzymes digestion technique and transformed into E.coli BL21(DE3)to optimize the expression conditions of recombinant protein.Electrophoresis detection was performed with sodium dodecyl sulfate polyacrylamide gel preparation kit(SDS-PAGE).Real-time fluorescent quantitative PCR(qRT-PCR)was used to analyze the expression pattern of AsCOMT in bolting plants of Angelica sinensis and different tissues of UV-B stress.Results The heterologous expression gave the recombinant protein a molecular weight of 40 KDa,which was consistent with the predicted molecular weight.AsCOMT protein was mainly in the form of inclusion bodies.qRT-PCR analysis showed that the expression level of COMT in the stem of bolted Angelica sinensis was significantly higher than that of other tissues(P<0.05).Under UV-B radiation stress,there was significant difference in the expression level between stem and leaf(P<0.05),that was,AsCOMT gene expression increased first and then decreased in stem and leaf with UV-B radiation,but it did not change significantly in root.Conclusion AsCOMT was expressed at different levels in different tissues and treatments,and was successfully expressed in E.coli.It lays a foundation for further research on the biosynthetic regulation of Angelica sinensis ferulic acid.
作者
杨彩霞
王引权
雒军
张亚丽
YANG Caixia;WANG Yinquan;LUO Jun;ZHANG Yali(School of Pharmacy,Gansu University of Chinese Medicine,Lanzhou,Gansu,730000,China;College of Chinese and Western Integrated Medicine,Gansu University of Chinese Medicine,Lanzhou,Gansu,730000,China)
出处
《甘肃中医药大学学报》
2019年第6期1-6,共6页
Journal of Gansu University of Chinese Medicine
基金
国家自然科学基金地区基金项目(81660625)
国家重点研发计划项目(2017YFC700705)
甘肃省高校协同创新科技团队支持计划项目(2016C-05FF09)。
关键词
当归
咖啡酸-O-甲基转移酶
异源表达
组织表达
Angelica sinensis
caffeic acid-O-methyltransferase
heterologous expression
tissue expression
作者简介
杨彩霞(1991-),女,在读硕士研究生。研究方向:药用植物资源保护与开发利用研究;通信作者:王引权(1963-),男,教授,博士生导师,农学博士,主要从事中药质量综合评价研究。E-mail:kjkfpp@163.com。
