紫金龙异紫堇定生物合成相关6-OMT基因克隆与功能表征  

Cloning and Functional Characterization of 6-OMT Gene Related to Isocorydine Biosynthesis in Dactylicapnos scandens

作　　者：李明 刘祥宇[1,2,3] 王益娜 和四梅 沙本才 LI Ming;LIU Xiang-yu;WANG Yi-na;HE Si-mei;SHA Ben-cai(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201;National Local Joint Engineering Research Center for Germplasm Innovation and Utilization of Chinese Medicinal Materials in Southwest,Yunnan Agricultural University,Kunming 650201;Yunnan Key Laboratory of Medicinal Plant Biology,Yunnan Agricultural University,Kunming 650201;Yunnan Characteristic Plant Extraction Laboratory,Kunming 650106)

机构地区：[1]云南农业大学农学与生物技术学院,昆明650201 [2]云南农业大学西南中药材种质创新与利用国家地方联合工程研究中心,昆明650201 [3]云南农业大学云南省药用植物生物学重点实验室,昆明650201 [4]云南特色植物提取实验室,昆明650106

出　　处：《生物技术通报》2025年第2期309-320,共12页Biotechnology Bulletin

基　　金：国家自然科学基金项目(82160727);云南特色植物提取实验室自主研究项目基金(2022YKZY001)。

摘　　要：【目的】(S)-去甲乌药碱O-甲基转移酶(6-OMT)是异紫堇定生物合成的关键限速酶,通过克隆与体外酶活验证紫金龙6-OMT基因功能,为解析紫金龙异紫堇定生物合成途径奠定基础。【方法】从紫金龙转录组数据中挖掘DsOMT基因,通过PCR扩增获得全长cDNA序列,并通过生物信息学分析DsOMT蛋白结构;分析DsOMT基因在不同组织中的表达水平;构建pET-28a-DsOMT原核表达载体,将其转入大肠杆菌BL21(DE3)进行诱导表达,纯化蛋白后进行体外酶反应,以表征其功能。【结果】从转录组数据中挖掘到4个DsOMT候选基因,分别命名为DsOMT07、DsOMT08、DsOMT010、DsOMT012,并成功扩增得到全长cDNA序列;4个DsOMT蛋白都不存在跨膜结构域和信号肽,属于膜外蛋白。系统发育显示4个DsOMT与6-OMT亚家族亲缘关系较近。对4个DsOMT的氨基酸序列进行分析,显示可能具有上游途径(S)-去甲乌药碱6-OH位点的催化活性。表达谱分析发现4个DsOMT基因在根中高表达。SDS-PACE结果表明DsOMT蛋白可溶性高,并在大肠杆菌中高效表达,体外酶反应后发现DsOMT010能够催化(S)-去甲乌药碱的C6的O-甲基化形成(S)-衡州乌药碱。【结论】成功克隆了4个DsOMT基因,属于膜外蛋白,与6-OMT亚家族亲缘关系较近,4个DsOMT基因在根中高表达;同时在大肠杆菌中实现了DsOMT的异源表达,并纯化蛋白进行了体外酶功能表征,鉴定到1个(S)-去甲乌药碱C6位O-甲基转移酶DsOMT010。【Objective】(S)-norcoclaurine O-methyltransferase(6-OMT)is the key rate-limiting enzyme in the biosynthesis of isocorydine.This study is aimed to verify the function of the 6-OMT gene from Dactylicapnos scandens through cloning and in vitro enzyme activity assays,thereby laying the foundation for elucidating the isocorydine biosynthetic pathway in D.scandens.【Method】The DsOMT gene was mined from the transcriptome data of D.scandens.The full-length cDNA sequence was obtained via PCR amplification,and the protein structure of DsOMT was analyzed using bioinformatics.The expressions of the DsOMT gene in different tissues were examined.A pET-28a-DsOMT prokaryotic expression vector was constructed and transferred into Escherichia coli BL21(DE3)for induced expression.The protein was then purified and subjected to in vitro enzyme assays to characterize its function.【Result】Four DsOMT candidate genes,named DsOMT07,DsOMT08,DsOMT010,and DsOMT012,were identified from the transcriptome data,and their full-length cDNA sequences were successfully amplified.All four DsOMT proteins lacked transmembrane domains and signal peptides,classifying them as extramembrane proteins.Phylogenetic analysis indicated that these genes were closely related to the 6-OMT subfamily.Amino acid sequence analysis suggested potential catalytic activity at the 6-OH site of the upstream pathway for(S)-norcoclaurine.Expression profiling revealed that these four DsOMT genes were highly expressed in the roots.SDS-PAGE results showed that DsOMT proteins were highly soluble and efficiently expressed in Escherichia coli.In vitro enzyme assays demonstrated that DsOMT010 catalyzed the O-methylation of the C6 position of(S)-norcoclaurine,forming(S)-coclaurine.【Conclusion】Four DsOMT genes are successfully cloned and identified as extramembrane proteins closely related to the 6-OMT subfamily.These genes show high expressions in the roots.Additionally,heterologous expression of DsOMT is achieved in E.coli,and the purified proteins are characterized fo

关 键 词：紫金龙 O-甲基转移酶 生物信息学分析 原核表达 功能表征 

分 类 号：Q943.2[生物学—植物学] S567.2[农业科学—中草药栽培]