【摘要】 为进一步研究SQS基因在鹿角灵芝三萜合成途径中的作用机制及茉莉酸甲酯(MeJA)对GaSQS基因的表达调控,采用RT-PCR结合RACE技术,克隆鹿角灵芝鲨烯合酶基因GaSQS,采用生物信息学方法对该基因及其编码蛋白进行数据分析,采用MeJA对GaSQS基因诱导表达。结果显示,该基因全长1 969 bp,开放阅读框(ORF)长度为1 515 bp,编码504个氨基酸,分子量为58.2 ku,具有1个跨膜结构域和3个保守区,为无信号肽的亲水性蛋白;GaSQS蛋白二级结构包括α-螺旋(59.52%)、无规则卷曲(28.37%)、延伸链(8.53%)和β-转角(3.57%);系统进化分析显示,鹿角灵芝GaSQS蛋白与灵芝SQS蛋白(ABF57213.1)具较高的同源性(99%);String蛋白互作网络分析显示,GaSQS蛋白互作的蛋白质共有10个,均为三萜、甾醇等萜烯类生物合成过程的重要物质;qRT-PCR分析表明,鲨烯合酶(SQS)的表达量与产量在原基和子实体初期高于其他发育阶段;在不同浓度MeJA诱导下,SQS蛋白均能高效表达,在200μmol/L时达到峰值效果最佳。更多还原

【Abstract】 For further study on the mechanism of GaSQS gene in triterpene synthesis pathway of Ganoderma lucidum and the regulation of methyl jasmonate(MeJA) on GaSQS gene expression, the paper cloned the GaSQS gene from Ganoderma amboinense with RT-PCR combined RACE technologies, then using bioinformatics to analyze the GaSQS gene and its coding protein, and used MeJA to induce the expression of GaSQS gene and so on. The results showed that the full-length of SQS gene was 1 969 bp, its ORF was 1 515 bp in length, which encoded 504 amino acids, and its molecular weight was 58.2 ku. GaSQS protein had a transmembrane domain and three conserved regions, which was a hydrophilic protein with no signal peptide; its secondary structure of GaSQS protein includes the four structures, namely α-helix(59.52%),random coil(28.37%),extended strand(8.53%) and β-turn(3.57%);The phylogenetic relationship analysis showed that SQS protein from Ganoderma amboinense was closely related to Ganoderma lucidum(ABF57213.1),its similarity coefficient was 99%. Analysis of protein interaction network by String software showed that, there were ten proteins interacting with SQS protein, all of which were important substances in terpene biosynthesis process, such as the biosynthesis of triterpenoids and sterols; the analysis of qRT-PCR showed that the expression level and yield of squalene synthase(GaSQS) were all higher in primordium and early stage of fruiting body than other stages; GaSQS protein could be induced efficiently under the different concentrations of MeJA,and the peak effect was at 200 μmol/L.更多还原