中药材2026，Vol.49Issue(3)：558-566,9.DOI:10.13863/j.issn1001-4454.2026.03.002
当归苯丙氨酸解氨酶AsPAL基因的克隆与功能表征
Cloning and Functional Characterization of the AsPAL Gene of Angelica sinensis Phenylalanine Ammonia-Lyase
摘要
Abstract
Objective:To explore the catalytic activity and expression characteristics of phenylalanine ammonia-lyase,a key enzyme for lignin synthesis in Angelica sinensis,so as to provide a basis for analyzing the early bolting problem of Angelica sinensis.Methods:The PAL gene family of Angelica sinensis was explored through bioinformatics methods.Protein physicochemical properties,secondary struc-ture prediction,tertiary structure prediction,amino acid homology sequence alignment and phylogenetic analysis were carried out.Gene expression patterns were analyzed by heterologous expression in Escherichia coli and in vitro enzyme activity assay,and real-time fluores-cence quantitative PCR method was used.Results:A total of 4 AsPALs genes were identified,among which 3 phenylalanine ammoniase genes were successfully cloned(named AsPAL1,AsPAL2 and AsPAL3,respectively),encoding 708 to 718 amino acids,with relative mo-lecular masses ranging from 77 034.19 to 78 324.77 Da.The theoretical isoelectric point(PI)range was 6.00 to 6.52,and the second-ary structure was mainly α-helix and random curl.In vitro enzyme activity assay revealed that AsPAL1～3 could respectively catalyze phenylalanine and tyrosine to generate cinnamic acid and p-coumaric acid.The RT-qPCR results showed that the expression levels of AsPAL1 and AsPAL2 were high in the roots of Angelica sinensis without bolting,while the expression level of AsPAL3 in the roots of An-gelica sinensis with bolting was high.Conclusion:The AsPAL1,AsPAL2 and AsPAL3 genes are successfully cloned.After prokaryotic ex-pression,all proteins have catalytic activity,and their relative expression levels are different in the roots of Angelica sinensis that bolted and those that did not.关键词
当归/抽薹/苯丙氨酸解氨酶/原核表达/体外酶活/功能表征Key words
Angelica sinensis(Oliv.)Diels/Bolting/Phenylalanine ammonia-lyase/Prokaryotic expression/In vitro enzyme activi-ty/Functional characterization分类
医药卫生引用本文复制引用
张金金,李勒松,贺佳蝶,梁艳丽,杨生超,栗孟飞,赵艳..当归苯丙氨酸解氨酶AsPAL基因的克隆与功能表征[J].中药材,2026,49(3):558-566,9.基金项目
国家自然科学基金项目(82460743) （82460743）
云南省科技计划项目(202502AS100012) （202502AS100012）
云南特色植物提取实验室自主研究项目基金(2022YKZY001) （2022YKZY001）
云南省兴滇英才支持计划"青年人才"项目(XDYC-QNRC-2022-0219) （XDYC-QNRC-2022-0219）
