北细辛AhOMT1基因的克隆及原核表达分析

Cloning and Prokaryotic Expression Analysis of AhOMT1 Gene from Asarum heterotropoides

为进一步挖掘甲基丁香酚合成相关酶基因,解析北细辛次生代谢途径,根据北细辛转录组数据库信息,筛选和克隆了AhOMT1基因,并进行了相应的生物信息学分析及原核表达分析。结果表明,AhOMT1基因包含的开放阅读框为1089 bp,编码362个氨基酸,理论分子质量为40.23 ku,等电点为5.34,AhOMT1蛋白为亲水性蛋白,无跨膜结构,无信号肽序列;AhOMT1基因具有二聚化结构域与甲基转移酶结构域,AhOMT1与催化类黄酮、丁香酚类化合物的O-甲基转移酶的同源性较高;构建pET-32b-AhOMT1原核表达载体,成功诱导表达约60 ku的重组蛋白;确定了最佳诱导条件是0.2 mmol/L IPTG,16℃诱导16 h,该诱导条件下的重组蛋白多以可溶性蛋白形式存在;使用Ni^(2+)树脂分离纯化AhOMT1蛋白,以200 mmol/L咪唑洗脱可得到最大量的蛋白。该研究首次克隆了北细辛AhOMT1基因,构建原核表达载体,筛选出AhOMT1蛋白最佳诱导条件。

In order to further mine the enzyme gene related with methyl-eugenol synthesis and to elucidate the secondary metabolism pathway of Asarum heterotropoides.According to the transcriptome database information of Asarum heterotropoides,AhOMT1 gene was screened and cloned,and the corresponding bioinformatics analysis and prokaryotic expression analysis were carried out.The results showed that AhOMT1 contained an open reading frame of 1089 bp,encoding 362 amino acids,with a theoretical molecular weight of 40.23 ku and an isoelectric point of 5.34.AhOMT1 protein was a hydrophilic protein without transmembrane structure and signal peptide sequence.AhOMT1 gene had dimerization domain and methyltransferase domain.AhOMT1 was close with the O-methyltransferase catalyzing flavonoids and eugenols.The prokaryotic expression vector pET-32b-AhOMT1 was successfully constructed and the recombinant protein of about 60 ku was successfully induced.The optimal induction condition was 0.2 mmol/L IPTG and induced at 16℃for 16 h.Most of the recombinant proteins existed in the form of soluble proteins.AhOMT1 protein was separated and purified with Ni^(2+)resin,and the maximum amount of protein was obtained by eluting with 200 mmol/L imidazole.AhOMT1 gene of Asarum heterotropoides was cloned for the first time,prokaryotic expression vector was constructed,and the best induction conditions of the protein were screened.