青钱柳鲨烯合成酶(CpSS)全长基因的克隆、分析与表达

Cloning, Analysis and Expression of the Squalene Synthase in Cyclocarya paliurus

为研究和调控青钱柳三萜代谢,克隆了青钱柳鲨烯合成酶(CpSS)全长基因,研究该基因的特征和蛋白结构,以及青钱柳悬浮培养细胞在黑曲霉诱导子作用下该基因的表达变化情况。本文以青钱柳悬浮细胞RNA为模板,采用RT-PCR和RACE技术克隆出全长为1 667 bp的CpSS基因cDNA序列,该序列包含一个1 245 bp的完整ORF,编码414个氨基酸;CpSS序列与胡桃鲨烯合成酶基因的相似度最高,相似度达到97%,与白桦、葡萄、大豆、甘草、可可5个物种的相似度都在85%以上。序列分析表明,CpSS蛋白属于不稳定亲水蛋白,具有典型的鲨烯合成酶保守区和结构域,存在于内质网膜中,由多个α螺旋组成空穴状结构,该结构与大多数植物鲨烯合成酶蛋白的空间结构相似。实时荧光半定量RT-PCR检测结果表明,在黑曲霉诱导子作用下,悬浮培养细胞CpSS基因表达量显著增加,在诱导后60 h时的表达量最高。本文可为深入研究青钱柳三萜代谢途径及真菌诱导子的诱导作用机制提供参考。

In order to understand and regulate the triterpinoid metabolism, the full length sequence of Cyclocarya paliurus squalene synthetase(CpSS) gene was cloned, and the characters and protein structure of this gene were further studied, as well as its’ expression level in the suspended cultured Cyclocarya paliurus cells induced by Aspergillus niger elicitor. Taking Cyclocarya paliurus cell RNA as template, the full length CpSS sequence of 1 667 bp was cloned using RT-PCR and RACE technologies, which contained a complete ORF with 1 245 bp encoding 414 amino acids. The sequence similarities between Cyclocarya paliurus and walnut, grapes, soybeans, licorice, cocoa were more than 85%, especially walnut with the highest similarity up to 97%. Sequence analysis showed that the protein encoded by CpSS was an unstable hydrophilic protein, which had a typical conserved region and domain of squalene synthase and located in the endoplasmic reticulum. There is a cavity in CpSS protein molecular formed by several α-helixes, which was similar to the majority of plant squalene synthase protein. The determination results of real-time fluorescence semi-quantitative RT-PCR indicated that the expression of CpSS gene increased significantly in suspended cultured Cyclocarya paliurus cells induced by Aspergillus niger elicitor, and the expression peaked at the 60 h after inducement. Our study provides a reference for further investigations on the triterpenoid metabolism of Cyclocarya paliurus and the inducement mechanism of fungal elicitor.