生物技术通报2025，Vol.41Issue(2)：309-320,12.DOI:10.13560/j.cnki.biotech.bull.1985.2024-0544
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
摘要
Abstract
[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 for their in vitro enzyme functions.Among them,DsOMT010 is identified as a(S)-norcoclaurine C-6 O-methyltransferase.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Key words
Dactylicapnos scandens/O-methyltransferase/bioinformatics analysis/prokaryotic expression/functional characterization引用本文复制引用
李明,刘祥宇,王益娜,和四梅,沙本才..紫金龙异紫堇定生物合成相关6-OMT基因克隆与功能表征[J].生物技术通报,2025,41(2):309-320,12.基金项目
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) （82160727）
