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

作　　者：向春繁 李勒松 王娟 梁艳丽 杨生超 栗孟飞 赵艳 XIANG Chun-fan;LI Le-song;WANG Juan;LIANG Yan-li;YANG Sheng-chao;LI Meng-fei;ZHAO Yan(Yunnan Agricultural University,National-Local Joint Engineering Research Center on Gemplasm Innovation&Utilization of Chinese Medici-nal Materials in Southwest China,Key Laboratory of Medicinal Plant Biology,College of Agronomy and Biotechnology,Kunming 650201;Yunnan Characteristic Plant Extraction Laboratory,Kunming 650106;Agronomy College,Gansu Agricultural University,Lanzhou 730070)

机构地区：[1]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云南省药用植物生物学重点实验室农学与生物技术学院,昆明650201 [2]云南特色植物提取实验室,昆明650106 [3]甘肃农业大学农学院,兰州730070

出　　处：《生物技术通报》2025年第2期295-308,共14页Biotechnology Bulletin

基　　金：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)。

摘　　要：【目的】探究当归中木质素合成关键肉桂醇脱氢酶的催化活性及表达特性,为解决当归抽薹后根部木质化问题提供新的研究思路和理论基础。【方法】以非木质化根(UBP)和木质化根(BP)为实验材料,基于转录组数据挖掘候选肉桂醇脱氢酶基因;克隆AsCADs基因的全长CDS,构建pET-28a-AsCADs原核表达载体,转入大肠杆菌(E.coli)BL21(DE3),并诱导重组蛋白表达及体外酶活测定;生物信息学分析具有活性的CAD蛋白序列;利用实时荧光定量PCR对木质化和非木质化根部进行基因表达模式分析。【结果】共鉴定出30个AsCADs基因,其中挖掘并克隆到3个肉桂醇脱氢酶基因AsCAD1、AsCAD4和AsCAD24,均包含2个Zn^(2+)结合位点和1个NAD(H)辅酶结合位点。体外酶活测定发现AsCAD1能催化松柏醛和咖啡醛还原为相应的醇,AsCAD4和AsCAD24能催化对羟基肉桂醛、松柏醛、咖啡醛和芥子醛还原为相应的醇。AsCAD1、AsCAD4和AsCAD24开放阅读框为1083 bp,编码360个氨基酸,理论等电点(p I)为6.1和5.52,AsCAD1为疏水性蛋白,AsCAD4和AsCAD24为亲水性蛋白,均不含跨膜结构和信号肽,亚细胞定位均定位在细胞质中。RT-q PCR结果显示AsCAD1在非木质化根(UBP)中的表达量高,而AsCAD4和AsCAD24在木质化根(BP)中的表达量高。【结论】成功克隆了AsCAD1、AsCAD4和AsCAD24基因,原核表达后蛋白均具有催化活性,其相对表达水平在木质化与非木质化根中存在差异。【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 Zn^(2+)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 1083 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 d

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 

分 类 号：S567.239[农业科学—中草药栽培]