一个蛇苔氧甲基转移酶基因的克隆与功能鉴定  

Cloning and functional characterization of an O-methyltransferase gene from Conocephalum conicum

作　　者：李丹丹[1] 刘慧[1] 程爱霞[1] 

机构地区：[1]山东大学药学院天然产物化学生物学教育部重点实验室,山东济南250012

出　　处：《山东大学学报（医学版）》2016年第12期20-26,共7页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(31370330)

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c DNA全长。构建原核表达质粒p ET32a-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 k Da,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。Objective To isolate and characterize an O-methyltransferase（ OMT） gene from Conocephalum conicum.Methods The full length c DNA sequence of CcOMT1 was obtained by RT-PCR and inserted into the prokaryotic expression plasmid pET32 a,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.

关 键 词：蛇苔 氧甲基转移酶 黄酮 基因克隆 酶活分析 

分 类 号：Q789[生物学—分子生物学]