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目的克隆绞股蓝Gynostemma pentaphyllum鲨烯合成酶基因(GpSS1),进行序列及表达分析,并研究茉莉酸甲酯(Me JA)对其调节的影响。方法基于Gen Bank提供的Gp SS基因序列设计GpSS1引物,利用RT-PCR方法获取GpSS1基因编码区序列;运用Protparam及Tmpred软件预测GpSS1蛋白的理化性质和跨膜结构域;采用Motif Scan及Bio Edit软件分析GpSS1酶催化活性保守区及蛋白多重序列比对;实时荧光定量PCR分析GpSS1基因的表达模式及Me JA对其调节的影响。结果 GpSS1(Gen Bank号为KU363976)编码区长1 254 bp,编码417个氨基酸;GpSS1基因具有3个与该酶催化活性相关的保守区、1个天冬氨酸富含区和1个碳端内质网锚定区。GpSS1基因在幼叶中表达最高,其次是老叶,在根状茎中的表达较低。不同浓度Me JA喷施绞股蓝植株后均引起了GpSS1基因的表达上调,其中50μmol/L的Me JA对GpSS1基因表达的影响最大。GpSS1基因在幼叶和老叶中的表达均呈现出先逐渐上升后有所下降的趋势,但在幼叶中的表达水平高于老叶。结论绞股蓝GpSS1基因的克隆及Me JA对其调控的分析,为进一步研究GpSS1基因的功能和Me JA对其调节的机制,以及为改善绞股蓝皂苷品质或提高其产量提供了科学理论依据。
Objective To clone Gynostemma pentaphyllum Squalene synthase gene (GpSS1), analyze its sequence and expression, and investigate the effect of methyl jasmonate (Me JA) on its regulation. Methods GpSS1 primers were designed based on the Gp SS gene sequence provided by Gen Bank and the coding region of GpSS1 gene was obtained by RT-PCR. The physical and chemical properties and transmembrane domain of GpSS1 protein were predicted by Protparam and Tmpred software. Motif Scan and Bio Edit software Analysis of GpSS1 enzymatic activity conserved regions and multiple sequence alignment of proteins; Real-time PCR analysis of GpSS1 gene expression patterns and Me JA regulation. Results The coding region of GpSS1 (GenBank accession number KU363976) was 1 254 bp, encoding 417 amino acids. The GpSS1 gene had 3 conserved regions, 1 aspartate-rich region and 1 carbon end Endoplasmic reticulum anchoring region. GpSS1 gene was the highest expression in young leaves, followed by old leaves, and lower expression in rhizomes. The GpSS1 gene expression was induced by different concentrations of Me JA sprayed on Gynostemma pentaphyllum, and the effect of Me JA at 50 μmol / L on GpSS1 gene expression was the highest. The expression of GpSS1 in young leaves and old leaves showed a trend of first decreasing and then decreasing, but the expression level of GpSS1 in young leaves was higher than that of old leaves. Conclusion Cloning of GpSS1 gene from Gynostemma pentaphyllum and analysis of its regulation by Me JA provide a scientific basis for further study on the function of GpSS1 gene and the mechanism by which Me JA regulate it, as well as to improve the quality of Gynostemma pentaphylla and increase its yield.