家蚕5-羟色胺受体基因的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2015.05.17

摘要/Abstract

摘要： 【目的】5-羟色胺（5-HT）在许多生理过程中扮演重要角色，克隆介导5-HT生理效应的家蚕5-HT受体及组织表达分析，为研究家蚕5-TH受体基因功能奠定基础。【方法】利用基因组数据及半定量real-time PCR技术，鉴定克隆4种家蚕5-HT受体基因; 应用生物信息学方法分析5-HT受体在物种间同源性并构建系统进化树; 利用半定量real-time PCR方法分析它们在幼虫和成虫不同组织的表达情况。【结果】根据预测基因序列，设计特异引物，克隆得到4种5-HT受体基因序列，分别命名为5-HT1ABm、5-HT1BBm、5-HT2Bm、5-HT7Bm（GenBank登录号KM236100—KM236103），其开放阅读框分别为1 395、1 341、1 881和1 497 bp，可编码464、446、626和498氨基酸。4种5-HT受体属于典型的G蛋白偶联受体。选择已报道的昆虫及脊椎动物的5-HT受体氨基酸序列进行比对并系统发生树构建，结果显示家蚕4种5-HT受体间的氨基酸序列相似度仅为30.4%。5-HT1A、5-HT1B、5-HT2、5-HT7 4种受体在昆虫中的相似性分别为45.4%、61.4%、48.4%、54.1%。另外，家蚕5-HT受体与其他昆虫甚至脊椎动物有较高的同源性，跨膜区的保守性比非跨膜区高。不同物种间的同一家族5-HT受体的相似性高于同一物种内不同家族5-HT受体的相似性，家蚕5-HT受体的进化关系与烟草天蛾最近。半定量real-time PCR结果显示，5-HT1ABm、5-HT1BBm在幼虫所有组织中均有表达。5-HT2Bm仅在幼虫的头、腹部神经索、精巢表达。5-HT7Bm在幼虫头部、腹部神经索、中肠、脂肪体、精巢、卵巢表达。5-HT1ABm、5-HT1BBm、5-HT7Bm在成虫中除雌性头部以外的组织中均有表达，而5-HT7Bm在雄蛾的生殖系统中表达量明显高于其他组织。5-HT2Bm在成虫中不表达。【结论】鉴定和克隆的4种家蚕5-HT受体基因，在昆虫和脊椎动物中具有较高的保守性，与烟草天蛾同源关系最近，它们在幼虫和成虫的组织表达模式具有多样性。

关键词: 家蚕, 5-HT受体, 克隆, 基因表达

Abstract: 【Objective】The objective of this study is to clone four kinds of 5-HT receptor genes and investigate their expression in different tissues of Bombyx mori, and to provide basic knowledge for further functional studies of these 5-HT receptor genes.【Method】Four 5-HT receptors were cloned based on genome database and using RT-PCR techniques. The bioinformatic method was used to analyze 5-HT receptor genes of B. mori and homology between species. The expression profiles of these genes in different tissues of larvae and adults were investigated by using semi-quantitative real-time PCR.【Result】Based on the predicted gene sequences, special primers were designed to clone the 5-HT receptor genes. Four 5-HT receptor genes 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm (GenBank accession number: KM236100-KM236103) were cloned. The open reading frame (ORF) of 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm was 1 395, 1 341, 1 881 and 1 497 bp, which encoded a polypeptide of 464, 446, 626 and 498 amino acids, respectively. They were typical of G protein-coupled receptors with seven transmembrane protein domains. The sequences were aligned and the phylogenetic tree of four 5-HT receptors was analyzed with other insects and vertebrates. Similarity of the amino acid sequence of 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm was only 30.4%. While, the similarity of 5-HT1A, 5-HT1B, 5-HT2 and 5-HT7 were 45.4%, 61.4%, 48.4%, and 54.1%, respectively in insects. In addition, 5-HT receptors had high homology and more conservative transmembrane region than non-transmembrane region in insects and vertebrates. The phylogenetic tree indicated that the same type receptors from different species got together, then the same receptor formed branch as species genetic relationships. The evolutionary relationships of 5-HT receptors in silkworm were close to Manduca sexta. The results of semi-quantitative real-time PCR showed that 5-HT1Abm, 5-HT1BBm expressed in all tissues in larvae, 5-HT2Bm only had expression in head, ventral chain and testis in larvae, 5-HT7Bm had expression in head, ventral chain, midgut, fat body, testis and ovary in larvae. 5-HT1ABm, 5-HT1BBm,and 5-HT7Bm expressedin other tissues except head in male of adults, whereas 5-HT7Bm in the reproductive system expression of male adults was significantly higher than other tissues. 5-HT2Bm did not express in adult.【Conclusion】Four 5-HT receptor genes were cloned in silkworm. They have more conservative in insects and vertebrates, and homologous relationships are close to M. sexta. The results of semi-quantitative real-time PCR showed that the tissue expression patterns were diverse.

Key words: Bombyx mori, 5-HT receptors, cloning, gene expression

李海银，李艳，陈曦，陈鹏，陈萍. 家蚕5-羟色胺受体基因的克隆及表达分析[J]. 中国农业科学, 2015, 48(5): 987-1001.

LI Hai-yin, LI Yan, CHEN Xi, CHEN Peng, CHEN Ping. Gene Cloning and Expression Analysis of 5-HT Receptors in Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2015, 48(5): 987-1001.

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