生物技术通报2025，Vol.41Issue(2)：295-308,14.DOI:10.13560/j.cnki.biotech.bull.1985.2024-0760
当归肉桂醇脱氢酶AsCAD功能鉴定及表达分析
Functional Identification and Expression Analysis of Cinnamonyl Alcohol Dehydrogenase AsCAD in Angelica sinensis
摘要
Abstract
[Objective]The study is to explore the catalytic activity and expression characteristics of cinnamyl alcohol dehydrogenase,which is a key enzyme of lignin synthesis in Angelica sinensis,and to provide a new research idea and theoretical basis for solving the issue of lignification in the roots of A.sinensis.[Method]Unbolted roots(UBP)and bolted roots(BP)were used as experimental materials to identify candidate cinnamyl alcohol dehydrogenase genes based on transcriptome data.The full-length CDS of AsCADs gene was cloned,the prokaryotic expression vector pET-28a-AsCADs was constructed,transferred into E.coli BL21(DE3),and the recombinant protein expression was induced and enzyme activity was measured in vitro.The active CAD protein sequences were analyzed by bioinformatics.Real-time quantitative PCR was used to analyze the gene expression patterns of UBP and BP.[Result]A total of 30 AsCADs genes were identified,among which 3 cinnamyl alcohol dehydrogenase genes,AsCAD1,AsCAD4 and AsCAD24,were reported and cloned,all containing two Zn2+binding sites and 1 NAD(H)coenzyme binding site.In vitro enzymatic assay revealed that AsCAD1 catalyzed the reduction of coniferaldehyde and cafferaldehyde to the corresponding alcohols,and AsCAD4 and AsCAD24 catalyzed the reduction of p-coumaraldehyde,coniferaldehyde,cafferaldehyde and sinapaldehyde to the corresponding alcohols.AsCAD1,AsCAD4 and AsCAD24 contained an open reading frame of 1 083 bp,encoded 360 amino acids,and theoretical isoelectric points(pI)of 6.1 and 5.52,of which AsCAD1 was a hydrophobic protein,AsCAD4 and AsCAD24 were hydrophilic proteins,none of which contained a transmembrane structure and signal peptide,and the subcellular localization were in cytoplasm.RT-qPCR results showed that the expression of AsCAD1 was high in UBP,while AsCAD4 and AsCAD24 was high in BP.[Conclusion]The gene of AsCAD1,AsCAD4 and AsCAD24 are successfully cloned.The proteins all have catalytic activity after prokaryotic expression,and their relative expression levels are different in UBP and BP.关键词
当归/木质化/肉桂醇脱氢酶/原核表达/体外酶活/表达分析Key words
Angelica sinensis/lignification/cinnamyl alcohol dehydrogenase/prokaryotic expression/enzyme activity in vitro/expression analysis引用本文复制引用
向春繁,李勒松,王娟,梁艳丽,杨生超,栗孟飞,赵艳..当归肉桂醇脱氢酶AsCAD功能鉴定及表达分析[J].生物技术通报,2025,41(2):295-308,14.基金项目
云南特色植物提取实验室自主研究项目基金(2022YKZY001),云南省科技计划项目(202304B1090009),云南省兴滇英才支持计划"青年人才"项目(XDYC-QNRC-2022-0219),甘肃省科技厅重点研发计划(22YF7NA111) （2022YKZY001）
