百蕊草苯丙氨酸解氨酶基因的克隆与表达分析  

Cloning and Expression Analysis of PAL Genes from Thesium chinense Turcz.

作　　者：张文香 杨再琴 潘柔柔 罗倩倩 吴超 曹剑锋 Zhang Wenxiang;Yang Zaiqin;Pan Rourou;Luo Qianqian;Wu Chao;Cao Jianfeng(Institute of Medicinal Plant Biotechnology,School of Biological Sciences,Guizhou Education University,Guiyang,550018)

机构地区：[1]贵州师范学院生物科学学院,药用植物生物技术研究所,贵阳550018

出　　处：《分子植物育种》2025年第4期1098-1104,共7页Molecular Plant Breeding

基　　金：贵州省科技厅自然科学研究基金项目(黔科合基础〔2016〕1415,黔科合基〔2018〕1117);贵州省科技厅平台人才项目(黔教合KY字〔2017〕5790-06);贵州师范学院大学生创新创业项目(202014223032);贵州省教育厅生物科学一流专业项目(〔2019〕46)共同资助。

摘　　要：苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)是百蕊草(Thesium chinense Turcz.)中黄酮类成分合成调节的关键基因,为了解百蕊草PAL(TcPAL)基因编码蛋白的理化性质和表达模式,以百蕊草茎和叶部位为主要材料,采用逆转录PCR(RT-PCR)法扩增其PAL基因,获得该基因的编码序列(CDS),并进行生物信息学分析;利用荧光定量(RT-qPCR)技术检测PAL基因在不同组织部位的表达模式。结果显示,扩增后的PAL的CDS全长为1674 bp,编码557个氨基酸,分子量为64.10 kD,等电点为6.06。其编码蛋白是一类稳定的亲水性蛋白,含有丝氨酸、苏氨酸和酪氨酸3个磷酸化位点,二级结构以α-螺旋和无规卷曲为主,具有典型的苯丙氨酸和组氨酸解氨酶保守结构域。空间结构模型显示其为同型四聚体,每个单体由3个结构域组成,其保守结构域含有PAL酶家族的保守序列和Ala-Ser-Gly三肽活性中心。荧光定量结果显示TcPAL基因在百蕊草叶和茎中表达较高,根中表达较少。本研究为深入研究TcPAL基因参与百蕊草黄酮类物质合成和积累的分子机制提供理论依据。Phenylalanine ammonia-lyase(PAL)gene is the key gene regulating synthesis of flavonoids in Thesium chinense Turcz.,to clarify the physicochemical properties of the proteins encoded by the PAL gene,and to explore their expression pattern.The major stem and leaves of Thesium chinense Turcz.as the experimental materials,the PAL gene was amplified by reverse transcription PCR(RT-PCR),the coding sequence(CDS)of the gene was obtained,and bioinformatics analysis was performed.The expression patterns of PAL gene in different tissues were detected by fluorescence quantification(RT-qPCR).The results showed that the gene contains a 1674 bp CDS encoding 557 amino acids,with a calculated molecular weight of 64.10 kD and a theoretical isoelectric point of 6.06.TcPAL proteins is a stable hydrophilic protein without signal peptide,including three phosphorylation sites(serine,threonine and tyrosine).The secondary structure is dominated by alpha helix and random coil,with typical conserved domains of phenylalanine and histidine ammonia-lyases.The tertiary structure of TcPAL indicated that TcPAL was a homotypic tetramer,and each monomer was consisted of three domains.The sconserved structure domains was containing a conserved sequence of the PAL family and Ala-Ser-Gly triad residues.The results of fluo rescence quantification showed that TcPAL gene was highly expressed in the leaves and stems,but less expressed in the roots.The results of this study could provide the theoretical basis for further understanding the molecular mechanism of flavonoid biosynthesis and accumulation of TcPAL gene in Thesium chinense Turcz.

关 键 词：百蕊草 苯丙氨酸氨解酶 基因克隆 表达分析 

分 类 号：Q943.2[生物学—植物学]