胆木异胡豆苷合成酶基因NoSTR1克隆及表达分析

Cloning and expression analysis of NoSTR1 gene of Nauclea officinalis

  • 摘要: 【目的】 克隆胆木(Nauclea officinalis)异胡豆苷合成酶(STR)基因NoSTR1,分析其在胆木不同组织中的表达差异,为解析胆木吲哚类生物碱合成途径和机制提供理论依据。【方法】 基于胆木转录组数据筛选出NoSTR1基因并进行克隆,对NoSTR1蛋白理化性质、结构及系统发育进行分析。以胆木根、茎和叶为试验材料,通过实时荧光定量PCR检测NoSTR1基因在胆木不同组织中的相对表达量,采用高效液相色谱(HPLC)检测胆木不同组织中吲哚类生物碱异长春花苷内酰胺含量。【结果】 克隆获得的NoSTR1基因编码区(CDS)长度为1032 bp,编码344个氨基酸残基。NoSTR1蛋白的分子式为C1720H2631N431O513S7,分子量为37779.81 Da,理论等电点为5.00,为稳定的亲水蛋白,具有信号肽,无跨膜结构域。NoSTR1蛋白二级结构中α-螺旋占18.37%,β-折叠占29.15%,无规则卷曲占52.48%,三级结构预测结果表明其为典型的6叶β-螺旋桨折叠结构蛋白。NoSTR1蛋白具有Str_synth superfamily保守结构域。系统发育分析结果表明,NoSTR1蛋白与常绿钩吻藤(Gelsemium sempervirens)、日本蛇根草(Ophiorrhiza japonica)和短小蛇根草(Ophiorrhiza pumila)的STR蛋白聚为一支,亲缘关系较近。NoSTR1基因在胆木的根、茎和叶中均有表达,其中茎的NoSTR1基因相对表达量最高,叶的NoSTR1基因相对表达量低。胆木茎的异长春花苷内酰胺含量最高,叶的异长春花苷内酰胺含量最低,与NoSTR1基因表达相对表达量的变化规律相似。【结论】 NoSTR1基因编码蛋白具有STR蛋白特有的Str_synth superfamily保守结构域。胆木不同组织NoSTR1基因相对表达量与异长春花苷内酰胺含量的变化规律相似,表明NoSTR1基因是胆木吲哚类生物碱合成途径中的关键候选基因。

     

    Abstract: 【Objective】 This study aimed to clone the strictosidine synthase (STR) gene of Nauclea officinalisNoSTR1) and analyze its tissue-specific expression difference, providing theoretical basis for elucidating pathways and mechanisms of indole alkaloid synthesis in Nauclea officinalis. 【Method】 The NoSTR1 gene was identified based on transcriptome data of Nauclea officinalis and cloned, and the physicochemical properties, structure, and phylogeny of NoSTR1 protein were analyzed. The roots, stems, and leaves of Nauclea officinalis were used as experimental materials. Real-time fluorescence quantitative PCR was employed to detect the relative expression of NoSTR1 gene in different tissues of Nauclea officinalis, and high performance liquid chromatography (HPLC) was used to detect the content of strictosamide, one of indole alkaloids, in different tissues of Nauclea officinalis. 【Result】 The cloned NoSTR1 gene contained a coding sequence (CDS) of 1032 bp, encoding 344 amino acid residues. The molecular formula of NoSTR1 protein was C1720H2631N431O513S7, with a molecular weight of 37779.81 Da, theoretical isoelectric point of 5.00, indicating that it was a stable hydrophilic protein with a signal peptide without transmembrane regions. In the secondary structure of NoSTR1 protein, α-helix accounted for 18.37%, β-sheet 29.15%, and random coils 52.48%; tertiary structure predictions indicated that it was a typical 6-bladed β-propeller fold protein. The NoSTR1 protein had a conserved domain of Str_synth superfamily. Phylogenetic tree analysis showed that the NoSTR1 protein was clustered in the same clade with STR proteins of Gelsemium sempervirensOphiorrhiza japonica, and Ophiorrhiza pumila, showing a close relationship. The NoSTR1 gene was expressed in roots, stems, and leaves of Nauclea officinalis, with the highest expression observed in stems, and the lowest in leaves. The strictosamide content of Nauclea officinalis was the highest in stems and the lowest in leaves, exhibiting a similar change pattern with the relative expression of NoSTR1 gene. 【Conclusion】 The encoded proteins of NoSTR1 gene contains a conserved domain, which is specifically for STR protein Str_synth superfamily. The relative expression of NoSTR1 gene in different tissues of Nauclea officinalis shows a similar change pattern with strictosa-mide content, indicating that the NoSTR1 gene is the key candidate gene of indole alkaloid synthesis pathways of Nauclea officinalis.

     

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