越南油茶环阿屯醇合酶基因的克隆及表达分析

Cloning and Expression Analysis of Cycloartenol Synthase Gene in Camellia vietnamensis

为研究越南油茶(Camellia vietnamensis T.C.Huang ex Hu)中环阿屯醇合酶(cycloartenol synthase,CAS)基因的分子作用机制,本研究采用同源克隆的方法克隆得到环阿屯醇合酶基因全长并命名为CvCAS,对其进行生物信息学分析及表达模式研究。结果表明,CvCAS全长2411 bp,包括一个2277 bp完整的开放阅读框,编码758个氨基酸,与茶(Camellia sinensis)、中华猕猴桃(Actinidia chinensis var.chinensis),刺楸(Kalopanax septemlobus)和人参(Panax ginseng)的CAS蛋白序列相似度均在85%以上,系统发育树分析显示CvCAS与茶(Camellia sinensis)和向日葵(Helianthus annuus)的CAS蛋白序列聚为一支。预测其蛋白分子质量为86.6 kD,理论等电点为6.20,无跨膜螺旋区,属于不稳定亲水性蛋白。序列比对分析表明,CvCAS具有氧化鲨烯环化酶(OSCs)超家族典型结构位点(DCTAE),三萜合成酶高度保守结构位点(DGSWYGSWGVCFTYG)和OSCs稳定结构保守位点(QW)。CvCAS基因在种子不同成熟期及植株不同组织部位均有表达,其中种子的表达量最高,且在九月与十月的表达量显著高于其他月份。本研究结果为越南油茶三萜皂苷等活性成分合成研究提供理论依据。

In order to study the molecular mechanism of the cycloartenol synthase(CAS)gene in Camellia vietnamensis T.C.Huang ex Hu,this study used homologous cloning technology to clone the full-length of cycloartenol synthase gene,named CvCAS,and conduct bioinformatics analysis and expression pattern research on it.The results showed that Cv CAS had a total length of 2411 bp,including a complete open reading frame of 2277 bp,encoding 758 amino acids,which had above 85%similarity with the CAS protein sequence of C.sinensis,Actinidia chinensis var.chinensis,Kalopanax septemlobus and Panax ginseng.Phylogenetic tree analysis showed that CvCAS clustered with the CAS protein sequences of tea(C.sinensis)and sunflower(Helianthus annuus).It was predicted that the molecular weight of the CvCAS protein was 86.6 kD,the psI was 6.20.The enzyme was an unstable hydrophilic protein,which had no transmembrane helix region.Sequence alignment analysis showed that Cv CAS had oxidosaualene cyclase(OSCs)superfamily typical motif(DCTAE),triterpene synthase highly conserved motif(DGSWYGSWGVCFTYG)and OSCs stable structural motif(QW).CvCAS gene was expressed in different maturity stages of seeds and different tissues of plants.The expression level of seeds was the highest,which the expression level in September and October was significantly higher than that in other months.The results of this study provide theoretical basis for the synthesis of active ingredients such as triterpene saponins from C.vietnamensis.