甜瓜鲨烯合酶基因克隆及酶分子结构分析

Gene cloning and molecular characteristics of squalene synthase from Cucumis melo

关键词:  甜瓜, 鲨烯合酶, 基因克隆, 序列分析

Key words:  Cucumis melo, squalene synthase, gene cloning, sequence analysis

摘要:

葫芦素类是主要分布于葫芦科植物中具有多种医药活性的四环三萜类化合物,目前药用葫芦素原料主要从甜瓜蒂中提取。该研究从甜瓜中克隆葫芦素类合成关键酶——鲨烯合酶(SQS)的基因,并对其序列进行了生物信息学分析。结果表明:DNA测序和BLAST RefSeqGene分析表明,克隆的甜瓜SQS基因片段具有完整的该酶基因开放阅读框架(ORF)序列。ORF分析显示,甜瓜SQS由417氨基酸残基构成,等电点为7.56。对推衍的甜瓜SQS氨基酸序列分析结果提示,该酶二级结构以α螺旋为主。结构域预测结果表明,SQS属于异戊二烯合酶家族,具有法呢酰基二磷酸及镁离子的结合位点。三级结构预测提示,甜瓜SQS为单体酶,其活性中心主要由几个α螺旋围绕形成的穴状结构。磷酸化位点分析显示,S⁴⁸处于酶活性中心相关⁴⁷VSRSF⁵²的模体中,而S¹⁹⁶是正选择位点,提示这两处磷酸化位点可能是甜瓜SQS酶活性调节的关键部位。以甜瓜SQS基因ORF序列构建系统发生树的系统发生分类结果与形态学分类结果一致。该研究结果为葫芦素类的生物合成调控研究提供了新的线索和实验依据。

Abstract:

Cucurbitacins(Cus)are tetracyclic triterpenoid compounds which exist mainly in cucurbitaceous plants and have various pharmaceuticals-like actions. The medicinal raw materials of Cus are currently extracted mainly from the pedicellus melo. In this study, the gene of squalene synthase(SQS), the key enzyme for Cus biosynthesis, from Cucumis melo was cloned, and the amino acid sequence of the enzyme derived from its nucleotide sequence was analyzed with bioinformatics methods. DNA sequencing and BLAST RefSeqGene analysis indicated that the cloned fragment of the C. melo SQS gene contained a complete open reading frame(ORF)of the gene. ORF Finder analysis showed that the SQS of C. melo was constituted of 417 amino acid residues with an isoelectric point of 7.56. Analysis of the deduced amino acid sequence suggested that the main type of secondary structure of the enzyme was α helix. Domain prediction study indicated that the SQS belonged to the isoprenoid biosynthase superfamily possessing the banding sites of farnesyl diphosphate and magnesian ion. Prediction of the tertiary structure suggested that the SQS was a monomeric enzyme with a cave-like activity center formed by α helixes. Protein phosphorylation analysis indicated that the phosphorylation site S⁴⁸ located in the activity center-related motif ⁴⁷ VSRSF⁵² and S¹⁹⁶ was a positive selection site, suggesting that both S⁴⁸ and S¹⁹⁶ were critical sites for the regulation of SQS activity. The phylogenetic classification based on the phylogenetic tress constructed with the ORF sequence of the SQS gene showed that the result was in accordance with that of morphologic classification. Therefore, this study provides new clues and reference for research of the regulation of Cus biosynthesis.