Cloning and tissue expression specificity analysis of UrTSA gene of Uncaria rhynchophylla
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摘要: 【目的】克隆钩藤碱的合成关键酶即色氨酸合成酶α链基因(UrTSA),并分析其组织表达特异性,为探究该基因的钩藤碱生物合成机制提供理论参考。【方法】从生物项目数据库(BioProject)筛选得到钩藤碱的生物合成相关酶基因UrTSA,以钩藤的根、叶、茎钩和蒴果的cDNA为模板,PCR克隆UrTSA基因的开放阅读框(ORF),并进行生物信息学分析,结合实时荧光定量PCR检测UrTSA基因在钩藤不同组织中的表达模式。【结果】UrTSA基因的ORF为963bp,编码320个氨基酸残基,相对分子量为33924.49Da,理论等电点(pI)为9.11,不稳定指数Ⅰ为33.50,脂溶指数为102.12,总平均亲水性指数为0.117,说明UrTSA蛋白为稳定、脂溶性好的疏水蛋白。UrTSA蛋白亚细胞定位于叶绿体,不含信号肽和跨膜区,为非分泌型蛋白;不具有潜在的糖基化位点,有30个潜在的磷酸化位点,包括16个丝氨酸(Ser)、13个苏氨酸(Thr)和1个酪氨酸(Tyr);二级结构由α-螺旋(43.44%)、无规则卷曲(31.87%)、延伸链(15.94%)和β-折叠(8.75%)组成。UrTSA蛋白与其他11个植物物种的TSA蛋白具有较高的氨基酸序列相似性(69.78%~92.77%),其中与阿拉比卡咖啡(Coffee arabica)和欧基尼奥伊德斯种咖啡(Coffea eugenioides)TSA蛋白的亲缘关系较近。UrTSA基因在钩藤的根、叶、茎钩和蒴果中均有表达,其中在蒴果、根和叶中相对表达量均极显著高于茎钩中的相对表达量(P<0.01),分别是茎钩的2.17、1.58和1.20倍。【结论】UrTSA基因具有组织表达特异性,主要在蒴果钩藤碱合成中发挥调控作用,为后续研究钩藤碱在钩藤中不同部位的累积及其生物合成途径提供思路和线索。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 realtime fluorescence quantitative PCR to analyze the expression pattern of UrTSA gene in different tissues of U. rhynchophylla. 【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 hydrophobic protein. The UrTSA protein was subcellularly localized to chloroplasts without signal peptide and transmembrane region, and was non-secretory protein. Without potential glycosylation sites, it had 30 potential phosphorylation sites, including 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 alkaloid 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.
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