摘要
【目的】克隆获得中华蜜蜂(Apis cerana cerana)Acer OBP14基因序列,并对其蛋白结构进行预测,分析其在不同组织和发育阶段的时空表达差异,为该基因的功能研究提供参考。【方法】以中华蜜蜂全组织c DNA为模板利用RT-PCR技术扩增和克隆获得Acer OBP14 c DNA全长序列,并提交至Gen Bank数据库;利用多种生物信息学软件预测分析其编码蛋白的理化特性和结构特征;采用MEGA5.2软件中的邻位相连法(neighbor-joining,NJ)构建Acer OBP14及其同源膜翅目昆虫OBPs的系统发育树;通过实时荧光定量PCR技术分析Acer OBP14在1、5、10、15、20、25和30日龄中华蜜蜂不同组织(触角、头、胸、腹、足和翅)中m RNA的表达情况。【结果】克隆获得了中华蜜蜂气味结合蛋白基因Acer OBP14的c DNA序列(Gen Bank登录号为KT24648)。Acer OBP14的开放阅读框(ORF)长度为408 bp,编码135个氨基酸,预测其蛋白分子量为15.08 k D,理论等电点为5.44,有信号肽,无跨膜结构,且第18—127位氨基酸之间存在一个昆虫气味结合蛋白家族PBP-GOBP superfamily保守结构域;存在多个比较明显的疏水区域,可能是脂溶性气味分子的结合位点;含有4个保守的半胱氨酸位点,属于Minus-C OBP亚家族。亚细胞定位分析表明,Acer OBP14属于分泌型蛋白,主要集中在内质网-高尔基体-质膜分泌途径上。Acer OBP14蛋白具有7个α螺旋,α1—α6属于典型OBPs核心螺旋,α7位于C端且暴露在蛋白表面。氨基酸同源序列比对结果显示,Acer OBP14与西方蜜蜂Amel OBP14的氨基酸序列一致性最高,达到86%;其次是大蜜蜂Ador GOBP56a-like,一致性为55%。系统进化树分析表明,同为蜜蜂属的中华蜜蜂Acer OBP14、西方蜜蜂Amel OBP14、大蜜蜂Ador GOBP56a-like、中华蜜蜂Acer OBP21和小蜜蜂Aflo GOBP56d-like聚为一个分支,切叶蜂属的苜蓿切叶蜂Mrot GOBP56a-like、侧沟茧蜂属的毁侧沟茧蜂Mdem GOBP83a-like、壁蜂属的角额壁蜂Ocor OBP3和熊蜂
【Objective】The objective of this study is to clone the c DNA sequence of Acer OBP14 from Apis cerana cerana, predict the protein structure, explore its expression profiles in different tissues and at different developmental stages of A. c. cerana, and to provide a fundamental evidence for the future study of the physiological function of this gene.【Method】 The full-length c DNA sequence of Acer OBP14 was cloned from the entire tissues of A. c. cerana by RT-PCR and submitted to the Gen Bank database. Physiochemical properties and structure characteristics of the deduced amino acid sequence were analyzed by multiple bioinformatics methods. Phylogenetic tree between Acer OBP14 and its homologous OBPs from Hymenoptera insects was constructed using neighbor-joining of MEGA5.2. The expression levels of Acer OBP14 m RNA in different tissues（antenna, head, thorax, abdomen, legs and wings） at 1, 5, 10, 15, 20, 25 and 30 days were detected by real-time PCR.【Result】The full-length c DNA sequence of Acer OBP14 was obtained（Gen Bank accession number is KT24648）. The open reading frame（ORF） of Acer OBP14 is 408 bp, encoding a protein of 135 aa with an estimated molecular weight of 15.08 k D and p I of 5.44. The encoding protein has a signal peptide and no transmembrance structure, possesses a PBP-GOBP superfamily domain between 18-127 residues, exists multiple obviously hydrophobic regions binding liposoluble odorant and contains four conserved cysteins, suggesting that it belongs to the Minus-C OBP subfamily. Subcellular localization results showed that Acer OBP14 was located on the secretory pathway about endoplasmic reticulum, golgi and plasmalemma which belongs to the secretory protein. Acer OBP14 possesses 7 α-helixs, among which the α1-α6 belong to the core helixs of classical OBPs and α7 is exposed to the surface of protein at the C-terminus. The results of amino acid sequence alignment showed that Acer OBP14 was homologous to Amel OBP14 with 86% amino acid sequence identity, and then had 55%
出处
《中国农业科学》
CAS
CSCD
北大核心
2016年第19期3852-3862,共11页
Scientia Agricultura Sinica
基金
国家自然科学基金(31272513
31502021)
国家现代农业蜂产业技术体系建设专项资金(cars-45-syz10)
