斑地锦查尔酮合酶基因及启动子的克隆与分析  

Cloning and Analysis of Chalcone Synthase Gene and Its Promoter from Euphorbia maculata

作　　者：郭三保[1] 宋美玲 李灵心 尧子钊 桂明明 黄胜和 GUO San-bao;SONG Mei-ling;LI Ling-xin;YAO Zi-zhao;GUI Ming-ming;HUANG Sheng-he(Department of Pharmacy,Jiangxi College of Traditional Chinese Medicine,Fuzhou 344000;Department of Basic Medicine,Jiangxi College of Traditional Chinese Medicine,Fuzhou 344000;Fuzhou Medical College,Nanchang University,Fuzhou 344000)

机构地区：[1]江西中医药高等专科学校药学系,抚州344000 [2]江西中医药高等专科学校医学基础部,抚州344000 [3]南昌大学抚州医学院,抚州344000

出　　处：《生物技术通报》2023年第4期148-156,共9页Biotechnology Bulletin

基　　金：江西省中医药管理局科技计划项目(2022B1000);江西省教育厅科学技术研究重点项目(GJJ203301);江西省抚州市科技计划项目重点研发计划(青年科技领军人才计划)(2020ED66)。

摘　　要：黄酮类化合物槲皮素是斑地锦主要药效成分之一,而查尔酮合酶(CHS)是槲皮素生物合成过程中的关键酶。为阐明斑地锦中槲皮素生物合成机制,本研究以斑地锦为材料,根据转录组测序数据结合3'RACE、Tail-PCR技术,克隆了CHS基因及其启动子,将该基因命名为EmCHS(GenBank登录号为ON652865)。对EmCHS蛋白进行氨基酸序列比对、理化性质、跨膜结构域、亚细胞定位和系统进化树等分析,并采用实时荧光定量PCR技术检测EmCHS在不同生长期不同组织中的表达情况。结果显示,EmCHS开放阅读框(ORF)全长为1194 bp,编码397个氨基酸,EmCHS蛋白定位于细胞质,理论等电点为5.96,相对分子质量为43.48 kD,不含跨膜区域,为结构稳定的亲水性蛋白。氨基酸序列比对和系统进化树分析结果显示,EmCHS与同为大戟科的木薯氨基酸序列相似性最高(91.92%),符合植物分类学的特点。RT-qPCR实验表明,EmCHS基因在斑地锦不同生长期不同组织中均有表达,且存在明显差异,在生殖生长期叶内表达水平最低,生殖生长期根内表达水平最高。克隆到的EmCHS启动子(GenBank登录号为OP626754),长度为971 bp,生物信息学分析结果显示,其既含TATA-box、CAAT-box等基本序列,也含有MYB、MYC等转录因子结合位点、多个光反应元件(G-box等)和激素反应元件(ABRE等)等顺式作用元件。实验结果为进一步研究斑地锦CHS基因功能及表达调控奠定基础。Quercetin is one of the main medicinal ingredients of Euphorbia maculata,and chalcone synthase(CHS)is one of key enzymes in the biosynthesis of quercetin.To explore the mechanism of quercetin biosynthesis in E.maculata,a CHS gene named EmCHS(GenBank access number:ON652865)was cloned from E.maculata based on transcriptome sequencing results,combined with technologies of 3'RACE and Tail-PCR.The amino acid sequence was aligned,and physicochemical properties,transmembrane region,subcellular localization and phylogenetic relationships of EmCHS protein were analyzed.And the expressions of EmCHS in different tissues during different growth stages were detected with RT-qPCR.The results showed that the ORF of EmCHS gene was 1194 bp,encoding 397 amino acids.The EmCHS protein was located in the cytoplasm.The theoretical isoelectric point of the protein was 5.96 and the theoretical molecular weight was 43.48 kD,and it did not contain a transmembrane region and was a hydrophilic protein with stable structure.The results of amino acid sequence alignment and phylogenetic relationships analysis revealed that EmCHS had the highest similarity(91.92%)with the amino acid sequence of Manihot esculenta in Euphorbiaceae family,which was consistent with the characteristics of plant taxonomy.The analysis of RT-qPCR confirmed that EmCHS gene was expressed in different tissues during different growth stages,and there were significant differences.The expression of EmCHS gene was the lowest in the leaf at the reproduction stage,while the highest in the root at the reproduction stage.Besides,the promoter(GenBank access number:OP626754)length of EmCHS gene cloned was 971 bp,including TATA-box,CAAT-box,MYB and MYC transcription factor binding sites,multiple light response and hormone response cis-acting elements,and so on.These results will provide a foundation for further research on gene function and expression regulation of EmCHS gene.

关 键 词：斑地锦 CHS 启动子 克隆 热不对称交错PCR 生物信息学 基因表达 

分 类 号：S567.219[农业科学—中草药栽培]