一个蛇苔氧甲基转移酶基因的克隆与功能鉴定

doi:10.6040/j.issn.1671-7554.0.2016.1137

摘要/Abstract

摘要： 目的克隆蛇苔氧甲基转移酶(CcOMT1)基因并对其进行功能鉴定。方法通过RT-PCR技术获得CcOMT1基因的cDNA全长。构建原核表达质粒pET32a-CcOMT1,在大肠杆菌BL21(DE3)中表达后,利用Ni-NTA吸附柱对表达的重组蛋白进行纯化,并通过体外酶活分析其活性。采用DNAMAN 7.0和MEGA 5.1软件分别进行氨基酸多序列比对和进化关系分析。结果克隆得到的CcOMT1开放读码框为837 bp,编码278个氨基酸,预测分子量为30.95 kDa,等电点为5.59。与来自钝鳞紫背苔、紫花苜蓿和冰叶日中花的氧甲基转移酶的同源性分别为73.18%、53.60%和44.60%。对纯化的重组蛋白进行酶活检测,结果表明融合蛋白可以催化具有邻二酚羟基的黄酮和苯丙类化合物,产生相应的氧甲基化产物,其最适底物为槲皮素。结论克隆并鉴定了一个CcOMT1基因,为通过酶法实现化合物的氧甲基化修饰提供了一个候选基因。

关键词: 酶活分析, 氧甲基转移酶, 黄酮, 基因克隆, 蛇苔

Abstract: Objective To isolate and characterize an O-methyltransferase(OMT)gene from Conocephalum conicum. Methods The full length cDNA sequence of CcOMT1 was obtained by RT-PCR and inserted into the prokaryotic expression plasmid pET32a, and then was transformed into Escherichia coli BL21(DE3)for expression. The recombinant protein was collected by Ni-NTA affinity column and purified for enzyme characterization. The analyses of multiple alignment and phylogenetic tree were performed using DNAMAN 7.0 and MEGA 5.1 softwares. Results Sequence analysis showed that the full length cDNA of CcOMT1 was 837 bp and encoded a 278-aa protein with a calculated molecular weight of 30.95 kDa and an isoelectric point of 5.59. The amino acid sequence of CcOMT1 showed 73.18%, 53.60%, and 44.60% identity with O-methyltransferase(OMT)from Plagiochasma appendiculatum, Medicago sativa and Mesembryanthemum crystallinum, respectively. The purified recombinant protein was able to methylate a variety of flavonoids and phenylpropanoids with aromatic vicinal dihydroxyl groups, and the preferred substrate was quercetin. Conclusion An OMT gene from Conocephalum conicum is cloned and characterized. The present investigation would provide a candidate gene for synthesis of methylated compounds at specific position through enzymatic method.

Key words: Conocephalum conicum, O-methyltransferase, Flavonoids, Enzyme activity assay, Gene cloning

中图分类号:

Q789

李丹丹,刘慧,程爱霞. 一个蛇苔氧甲基转移酶基因的克隆与功能鉴定[J]. 山东大学学报（医学版）, 2016, 54(12): 20-26.

LI Dandan, LIU Hui, CHENG Aixia. Cloning and functional characterization of an O-methyltransferase gene from Conocephalum conicum[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2016, 54(12): 20-26.

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