南方农业学报2024，Vol.55Issue(4)：1079-1088,10.DOI:10.3969/j.issn.2095-1191.2024.04.016
钩藤UrTSA基因克隆及组织表达特异性分析
Cloning and tissue expression specificity analysis of UrTSA gene of Uncaria rhynchophylla
摘要
Abstract
[Objective]The purpose of the study was to clone the key enzyme for the alkaloid synthesis of Uncaria rhynchophylla,the tryptophan synthase α-chain gene(UrTSA),and to organize the expression specificity analysis,so as to provide theoretical references for exploring the mechanism of alkaloid biosynthesis in U.rhynchophylla by this gene.[Method]The alkaloid biosynthesis-related enzyme gene UrTSA of U.rhynchophylla was screened and obtained from the BioProject database.The opening reading frame(ORF)of UrTSA gene was cloned by PCR using cDNAs of root,leaf,stem hook and capsule of U.rhynchophylla as templates,and bioinformatic analysis was performed,combined with real-time fluorescence quantitative PCR to analyze the expression pattern of UrTSA gene in different tissues of U.rhyncho-phylla.[Result]The ORF of UrTSA gene was 963 bp,encoding 320 amino acid residues,with relative molecular weight of 33924.49 Da,theoretical isoelectric point(pI)of 9.11,instability index Ⅰ of 33.50,lipid solubility index of 102.12,and total average hydrophilicity index of 0.117.It indicated that the UrTSA protein was a stable,lipid-soluble hydropho-bic protein.The UrTSA protein was subcellularly localized to chloroplasts without signal peptide and transmembrane re-gion,and was non-secretory protein.Without potential glycosylation sites,it had 30 potential phosphorylation sites,in-cluding 16 serine(Ser),13 threonine(Thr)and 1 tyrosine(Tyr).The secondary structure consisted of α-helix(43.44% ),random coil(31.87% ),extended strand(15.94% ),and β-fold(8.75% ).The UrTSA protein showed relatively high amino acid sequence similarity(69.78% to 92.77% )with TSA proteins from 11 other plants,with closer affinities to the TSA proteins of Coffee arabica and Coffea eugenioides.The UrTSA gene was expressed in roots,leaves,stem hooks and capsule of U.rhynchophylla,with the relative expression in capsules,roots and leaves being extremely significantly higher than that in stem hooks(P<0.01),which was as 2.17,1.58 and 1.20 times as that in stem hooks,respectively.[Conclusion]UrTSA gene has tissue expression specificity and mainly plays a regulatory role in the synthesis of capsule al-kaloid in U.rhynchophylla,which provides ideas and clues for the subsequent study on the accumulation of alkaloids in different tissues of U.rhynchophylla and its biosynthetic pathway.关键词
钩藤/UrTSA基因/基因克隆/生物信息学分析/实时荧光定量PCRKey words
Uncaria rhynchophylla/UrTSA gene/gene cloning/bioinformatics analysis/real-time fluorescence quantitative PCR分类
农业科技引用本文复制引用
章瑶,穆德添,王丽娅,陆英,唐其,朱丽娜..钩藤UrTSA基因克隆及组织表达特异性分析[J].南方农业学报,2024,55(4):1079-1088,10.基金项目
湖南省自然科学基金项目(2022JJ30305) （2022JJ30305）
湖南省教育厅重点项目(21A0138) （21A0138）
湖南省研究生科研创新项目(CX20 220689) （CX20 220689）
湖南农业大学研究生科研创新项目(2022XC062) Hunan Natural Science Foundation(2022JJ30305) （2022XC062）
Key Project of Hunan Department of Educa-tion(21A0138) （21A0138）
Hunan Graduate Research Innovation Project(CX20220689) （CX20220689）
Hunan Agricultural University Graduate Research Innovation Project(2022XC062) （2022XC062）
