山鸡椒LcGPPS表达模式及其与LcGGPPS蛋白互作分析  

Gene Expression Pattern of LcGPPS and Its Interaction with LcGGPPSs in Litsea cubeba

作　　者：曹佩 陈益存[1] 高暝[1] 郭浩波 汪阳东[1] 

机构地区：[1]中国林业科学研究院亚热带林业研究所,浙江杭州311400 [2]美国田纳西大学生物化学系,田纳西州诺克斯维尔tn37996

出　　处：《林业科学研究》2017年第6期1050-1058,共9页Forest Research

基　　金：国家自然科学基金项目(31370576)

摘　　要：[目的]鉴定与山鸡椒精油合成关键酶香叶基二磷酸合酶(LcGPPS)的互作蛋白,为揭示山鸡椒精油主要成分单萜物质的合成机理奠定基础。[方法]通过本地Blast搜索山鸡椒果实转录组数据库,采用RT-PCR克隆山鸡椒GPPS基因(编码异质GPPS小亚基的LcGPPS.SSU1)和山鸡椒GGPPS基因(LcGGPPS1和LcGGPPS3);利用qRTPCR分析其组织特异性表达模式,通过蛋白互作预测和酵母双杂实验验证LcGPPS.SSU1分别与LcGGPPS1和LcGGPPS3的互作关系。[结果]克隆得到LcGPPS.SSU1、LcGGPPS1和LcGGPPS3 3条基因序列,基因表达模式分析表明,LcGPPS.SSU1特异性地在花和果实中高水平表达;蛋白结构互作预测结果显示,LcGPPS.SSU1可以分别与LcGGPPS1和LcGGPPS3互作形成异质二聚体;经过酵母双杂交实验验证发现,仅LcGGPPS3能与LcGPPS.SSU1发生互作。[结论]LcGPPS.SSU1通过与LcGGPPS3蛋白发生互作从而在山鸡椒萜类合成途径中发挥作用,为深入研究山鸡椒萜类合成机制提供参考。[Objective] To identify the interacted proteins of geranyl diphosphate synthase （GPPS） and provide theoretical bases for illuminating the mechanism of monoterpene biosynthesis in Litsea cubeba. [Method] The transcriptome database of fruits of L. cubeba was searched by basic blast tool. RT-PCR was used to clone GPPS genes （LcGPPS.SSU1 encoding the small subunit of heteromeric GPPS） and GGPPS genes （LcGGPPS1 and LcGGPPS3）. qRT-PCR was conducted to analyze the expression patterns. In addition, prediction of three-dimensional structure models and yeast two-hybrid system were used to verify the protein interaction. [Result] LcGPPS.SSU1, LcGGPPS1 and LcGGPPS3 were cloned. The expression pattern analysis showed LcGPPS. SSU1 specifically expressed in flower, flower bud and fruit in a significant high level. The prediction of protein interaction showed that both LcGGPPS1 and LcGGPPS3 could interact with LcGPPS.SSU1, while the result of yeast two-hybrid system showed that only LcGGPPS3 can interact with LcGPPS.SSU1. [Conclusion] LcGPPS.SSU1 can interact with LcGGPPS3 to form heterodimer to function in the terpenoids biosynthesis in L. cubeba, which provides knowledge for addressing the mechanism of terpenoids biosynthesis pathway in L. cubeba.

关 键 词：山鸡椒 萜类合成 GPPS GGPPS 异质二聚体 蛋白互作 表达模式 

分 类 号：S718.46[农业科学—林学]