佛手瓜鲨烯合酶基因克隆及酶分子结构分析

发布时间：2018-01-29 23:51

本文关键词： 佛手瓜鲨烯合酶基因克隆结构分析　出处：《安徽农业科学》2016年32期 　论文类型：期刊论文

【摘要】：[目的]克隆佛手瓜鲨烯合酶(SQS)基因,并基于克隆的SQS序列分析该酶的分子结构,为佛手瓜生物活性成分的利用提供理论依据。[方法]根据SQS基因5'末端保守序列和3'race方法扩增的佛手瓜SQS基因3'末端序列设计佛手瓜SQS基因克隆引物。扩增的佛手瓜SQS基因片段插入p GEM-T Easy Vector后经DNA测序。应用生物信息学方法分析克隆序列的开放阅读框架(ORF)、酶蛋白的疏水性/亲水性和蛋白质磷酸化位点,预测酶蛋白的二级结构、结构域、三级结构和跨膜区。以布朗葡萄藻为outgroup,应用MEGA 5.0进行UPGMA算法构建系统树,并进行1 000次的Bootstrap测试。[结果]佛手瓜SQS由417氨基酸残基构成,等电点为6.983。构象预测提示该酶二级结构以α螺旋为主,是一种只有三级结构的单体酶,其活性中心是主要由几个α螺旋围绕形成的穴状结构,酶蛋白肽链中具有1个跨膜区。结构域分析表明SQS属于异戊二烯合酶家族。磷酸化位点分析显示该酶共有17个磷酸化位点,其中,S48位于与酶活性中心相关模体中,而S196为陆生植物SQS基因的正选择位点。以佛手瓜SQS基因ORF序列构建系统发生树得到的系统发生分类结果与形态学分类结果一致。[结论]佛手瓜SQS是由417氨基酸残基构成的单体酶,具有1个跨膜区和17个磷酸化位点,其中,S48和S196可能是佛手瓜SQS酶活性调节的关键性磷酸化位点。
[Abstract]:[Objective: to clone squalene synthase (squalene synthase) gene from papaya and analyze its molecular structure based on cloned SQS sequence. [Methods based on the conserved 5'terminal sequence of SQS gene and the 3'terminal sequence of SQS gene amplified by 3race method, cloned primers of SQS gene were designed. The amplified SQS gene of Fructus papaya was amplified. Fragment insertion p. GEM-T Easy Vector was sequenced by DNA. An open reading framework for the analysis of cloned sequences by bioinformatics (. ORF). The hydrophobic / hydrophilic and protein phosphorylation sites of the enzyme protein were predicted, and the secondary structure, domain, tertiary structure and transmembrane region of the enzyme protein were predicted. MEGA 5.0 was used to construct the system tree with UPGMA algorithm, and 1 000 Bootstrap tests were carried out. [Results SQS was composed of 417 amino acid residues and the isoelectric point was 6.983.Conformational prediction indicated that the secondary structure of the enzyme was 伪 helix, which was a monomeric enzyme with only tertiary structure. Its active center is a cavernous structure formed mainly by several 伪 -helix. SQS belongs to isoprene synthase family. Phosphorylation site analysis showed that there were 17 phosphorylation sites in the enzyme. S48 is located in motifs associated with enzyme active centers. S196 is the positive selection site of SQS gene in terrestrial plants. The phylogenetic classification results obtained from the phylogenetic tree constructed from the ORF sequence of SQS gene are consistent with the morphological classification results. [Conclusion SQS is a monomeric enzyme composed of 417 amino acid residues with one transmembrane region and 17 phosphorylation sites. S48 and S196 may be the key phosphorylation sites for the regulation of SQS activity.
【作者单位】：广西医科大学生物化学与分子生物学教研室;桂林医学院医药生物技术重点实验室;
【基金】：国家自然科学基金项目(31260069) 广西高等学校优秀中青年骨干教师培养工程资助项目
【分类号】：S642.9
【正文快照】： 佛手瓜(Sechium edule Swartz)为葫芦科多年生宿根性草质藤本植物,其果实肉质细嫩,富含蛋白质、碳水化合物、维生素、胡萝卜素及钙、磷、铁、硒等营养物质,是具有保健作用的珍稀蔬菜[1]。研究表明,佛手瓜根、茎、叶和果实含有叶酸、泛酸、烟酸、黄酮类和皂甙类等生物活性物质[

【相似文献】