管花肉苁蓉花中二氢黄酮醇4-还原酶的克隆、表达分析和酶活性鉴定  

Cloning,expression analysis and enzyme activity verification of dihydroflavonol 4-reductase from Cistanche tubulosa(Schenk)Wight flower

作　　者：邱海玲 王方明 高博闻[4] 米芯雨 张泽坤[1,2] 杜宇 史社坡 屠鹏飞[3] 王晓晖 QIU Hai-ling;WANG Fang-ming;GAO Bo-wen;MI Xin-yu;ZHANG Ze-kun;DU Yu;SHI She-po;TU Peng-fei;WANG Xiao-hui(School of Chinese Pharmacy,Beijing University of Chinese Medicine,Beijing 102488,China;Modern Research Center for Traditional Chinese Medicine,School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China;State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Science,Peking University,Beijing 100191,China;Baotou Medical College,Baotou 014060,China)

机构地区：[1]北京中医药大学中药学院,北京102488 [2]北京中医药大学中药学院中药现代研究中心,北京100029 [3]北京大学药学院,天然药物及仿生药物国家重点实验室,北京100191 [4]包头医学院,内蒙古包头014060

出　　处：《药学学报》2023年第4期1079-1089,共11页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81773832)。

摘　　要：二氢黄酮醇4-还原酶(dihydroflavonol 4-reductase,DFR)是花色素苷生物合成途径的关键酶,在植物花色调控中发挥重要作用。本研究通过管花肉苁蓉花转录组数据分析,利用RT-PCR技术克隆出一条DFR基因并命名为CtDFR,其开放阅读框(ORF)长1263 bp,编码420个氨基酸,蛋白分子质量为47.5 kDa。序列分析表明CtDFR具有NADPH结合域和底物特异性结合域。实时荧光定量PCR表明CtDFR在管花肉苁蓉的红、紫色花中高表达,相对表达量分别是白色花的4.04、19.37倍。构建原核表达载体pET-28a-CtDFR,转入大肠杆菌BL21(DE3)菌株中,成功表达CtDFR蛋白,纯化得到可溶性重组蛋白。体外酶活性分析表明,重组蛋白CtDFR能催化二氢山柰酚、二氢槲皮素、二氢杨梅素生成无色天竺葵素、无色矢车菊素、无色飞燕草素。亚细胞定位实验结果表明CtDFR主要定位于细胞质中。本研究结果表明CtDFR在管花肉苁蓉花色调控中起到重要作用,为进一步探讨管花肉苁蓉花色形成及调控机制研究奠定了基础。Dihydroflavonol 4-reductase(DFR)plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color.Based on the transcriptome data of Cistanche tubulosa(Schenk)Wight,a fulllength cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction(RT-PCR).CtDFR contains an open reading frame(ORF)of 1263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa.The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate(NADPH)binding domain and a specific substrate binding domain.The expression analysis indicated that CtDFR was highly expressed in red and purple flowers,and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers,respectively.The recombinant CtDFR protein was expressed in E.coli BL21(DE3)using vector pET-28a-CtDFR and was purified.In vitro enzyme activity analysis,CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol,dihydroquercetin,and dihydromyricetin to leucopelargonidin,leucocyanidin and leucodelphinidin.Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm.These results demonstrate that CtDFR plays an important role in regulation of flower color in C.tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C.tubulosa(Schenk)Wight flower color.

关 键 词：管花肉苁蓉 花色 二氢黄酮醇4-还原酶 表达分析 酶活性分析 亚细胞定位 

分 类 号：R931[医药卫生—生药学]