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[摘要]
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(MeJA)对其调节的影响。方法 基于GenBank提供的GpSS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MotifScan及BioEdit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MeJA对其调节的影响。结果 GpSS1(GenBank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MeJA喷施绞股蓝植株后均引起了GpSS1基因的表达上调,其中50 μmol/L的MeJA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MeJA对其调控的分析,为进一步研究GpSS1基因的功能和MeJA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。
[Key word]
[Abstract]
Objective To clone Gynostemma pentaphyllum squalene synthase gene (GpSS1) and analyze its sequence and expression pattern as well as its regulation in response to MeJA. Methods Primers were designed based on the sequence of GpSS (GenBank accession numbers: FJ906799) and GpSS1 was cloned by using RT-PCR method. Physicochemical properties and transmembrane regions of the deduced GpSS1 protein were predicted via Protparam and Tmpred programs, respectively. Conserved domains involved in catalytic activity of SS enzyme were identified using MotifScan and multiple sequence alignment was achieved using the BioEdit software. Expression pattern of GpSS1 and its regulation by MeJA were analyzed by quantitative real-time RT-PCR. Results GpSS1 contains an open reading frame of 1 254 bp encoding a putative protein of 417 amino acids. The protein has an aspartate-rich motif and an endoplasmic reticulum membrane anchoring region in addition to three conserved domains involved in catalytic activity of SS enzyme. The expression level of GpSS1 in young leaves was the highest, followed by that in old leaves, and the lowest was in rhizomes. The expression of GpSS1 was significantly upregulated in G. pentaphyllum leaves sprayed with different concentration of MeJA. The greatest upregulation of GpSS1 occurred in G. pentaphyllum leaves treated with 50 μmol/L MeJA. In both young and old leaves, the transcription of GpSS1 gradually increased and then decreased to varying degrees at 6-96 h after MeJA treatment. However, the expression level of GpSS1 was always higher in young leaves than that in old leaves. Conclusion The cloning of GpSS1 and analysis on the expression regulated by MeJA will be helpful for further elucidating the function of GpSS1 and its mechanism regulated by MeJA, as well as for improving the quality and content of gypenosides.
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[基金项目]
国家自然科学基金资助项目(31260039)