甜瓜鲨烯合酶基因克隆及酶分子结构分析

发布时间：2019-06-15 17:13

【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BLASTRefSeqGene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,S~(48)处于酶活性中心相关~(47)VSRSF~(52)的模体中,而S~(196)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]:Cucurbitins are tetracyclic triterpenes mainly distributed in cucurbitaceae with many kinds of medical activity. at present, medicinal cucurbitins are mainly extracted from melon butts. In this study, the gene of squalene polymerase (SQS), a key enzyme of cucurbitine synthesis, was cloned from muskmelon and its sequence was analyzed by bioinformatics. The results showed that DNA sequencing and BLASTRefSeqGene analysis showed that the cloned muskmelon SQS gene fragment had a complete open reading frame (ORF) sequence of the enzyme gene. ORF analysis showed that muskmelon SQS was composed of 417amino acid residues, and the isoelectric point was 7.56. The results of SQS amino acid sequence analysis showed that the secondary structure of the enzyme was 伪 helix. The results of domain prediction show that SQS belongs to the isoprene enzyme family and has the binding sites of farinyl diphosphate and magnesium ions. The prediction of tertiary structure suggests that muskmelon SQS is a monomer enzyme, and its active center is mainly formed by several 伪 helix around the hole structure. The analysis of phosphorylation sites showed that S48 was in the motif of 47 VSRSF~ (52) associated with enzyme activity, and S196 was a positive selective site, suggesting that these two phosphorylation sites might be the key sites for the regulation of SQS enzyme activity in muskmelon. The results of phylogenetic classification based on the ORF sequence of muskmelon SQS gene were consistent with those of morphological classification. The results provide a new clue and experimental basis for the regulation of cucurbitins biosynthesis.
【作者单位】：桂林医学院生物化学与分子生物学教研室;广西医科大学生物化学与分子生物学教研室;
【基金】：国家自然科学基金(31260069) 广西高等学校优秀中青年骨干教师培养工程项目~~
【分类号】：S652;Q946

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