Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (5): 987-1001.doi: 10.3864/j.issn.0578-1752.2015.05.17

• ANIMAL SCIENCE·VETERINARY SCIENCERE • Previous Articles     Next Articles

Gene Cloning and Expression Analysis of 5-HT Receptors in Silkworm (Bombyx mori)

LI Hai-yin, LI Yan, CHEN Xi, CHEN Peng, CHEN Ping   

  1. College of Biotechnology, Southwest University, Chongqing 400716
  • Received:2014-09-11 Online:2015-03-01 Published:2015-03-01

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

[1]    Green A R. Neuropharmacology of 5-hydroxytryptamine. British Journal of Pharmacology, 2006, 147: S145-S152.
[2]    de Pontes A L B, Engelberth R C G J, da Silva Nascimento E, Cavalcante J J C, de Oliveira Costa M S M. Serotonin and circadian rhythms. Psychology & Neuroscience, 2010, 2: 217-228.
[3]    Rousselle J C, Plantefol M, Fillion M P, Massot O, Pourrias B, Fillion G. Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells. Naunyn- Schmiedeberg’s Archives of Pharmacology, 1998, 358: 279-286.
[4]    秦鹃鹃, 刘正华, 梁艳, 刘鹰, 张黎明, 李云峰. 5-羟色胺及其受体与抑郁症. 国际药学研究杂志, 2012, 39(5): 409-413.
Qin J J, Liu Z H, Liang Y, Liu Y, Zhang L M, Li Y F. Serotonin, its receptors and depression. Journal of International Pharmaceutical Research, 2012, 39(5): 409-413. (in Chinese)
[5]    Berger M, Gray J A, Roth B L. The expanded biology of serotonin. Annual Review of Medicine, 2009, 60: 355-366.
[6]    Johnson O, Becnel J, Nichols C D. Serotonin receptor activity is necessary for olfactory learning and memory in Drosophila melanogaster. Neuroscience, 2011, 192: 372-381.
[7]    Norville K, Sweeney S T, Elliott C J H. Postmating change in physiology of male Drosophila mediated by serotonin (5-HT). Journal of Neurogenetics, 2010, 24: 27-32.
[8]    Csaba G. Presence in and effects of pineal indoleamines at very low level of phylogeny. Experientia, 1993, 49: 627-634.
[9]    Rapport M M, Green A A, Page I H. Serum vasoconstrictor (serotonin): iv. isolation and characterization. Journal of Biological Chemistry, 1948, 176: 1243-1251.
[10]   Rapport M M. Serum vasoconstrictor (serotonin). The presence of creatinine in the complex: a proposed structure of the vasoconstrictor principle. Journal of Biological Chemistry, 1949, 180: 961-969.
[11]   Nichols D E, Nichols C D. Serotonin receptors. Chemical Reviews, 2008, 108: 1614-1641.
[12]   Erspamer V, Asero B. Identification of enteramine, the specific hormone of the enterochromaffin cell system, as 5-hydroxytryptamine. Nature, 1952, 169: 800-801.
[13]   宋清, 张晓文, 张绒. 5-羟色胺受体及亚型的研究现状. 甘肃医药, 2010, 29(1): 1-4.
Song Q, Zhang X W, Zhang R. Research of serotonin receptors and its subtypes. Gansu Medical Joumal, 2010, 29(1): 1-4. (in Chinese)
[14]   常菊花, 何月平. 昆虫5-羟色胺受体的研究进展. 安徽农业科学, 2014, 42(10): 2913-2914.
Chang J H, He Y P. Research progress of 5-serotonin receptor in insect. Journal of Anhui Agricultural Sciences,2014, 42(10): 2913-2914. (in Chinese)
[15]   Tierney A J. Structure and function of invertebrate 5-HT receptors: a review. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2001, 128: 791-804.
[16]   Witz P, Amlaiky N, Plassat L, Maroteaux L, Borrelli E, Hen R. Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase. Proceedings of the National Academy of Sciences of the United States of America, 1990, 87: 8940-8944.
[17]   Saudou E, Boschert U, Amlaiky N, Plassat J L, Hen R. A family of Drosophila serotonin receptors with distinct intracellular signaling properties and expression patterns. The Embo Journal, 1992, 11(1): 7-17.
[18]   Dacks A M, Reale V, Pi Y L, Zhang W J, Dacks J B, Nighorn A J, Evans P D. A characterization of the Manduca sexta serotonin receptors in the context of olfactory neuromodulation. PLoS One, 2013, 8(7): e69422.
[19]   Vleugels R, Lenaerts C, Baumann A, Broeck J V, Verlinden H. Pharmacological characterization of a 5-HT1-type serotonin receptor in the red flour beetle, Tribolium castaneum. PLoS One, 2013, 8(5): e65052.
[20]   Schlenstedt J, Balfanz S, Baumann A, Blenau W. Am5-HT7: molecular and pharmacological characterization of the first serotonin receptor of the honeybee (Apis mellifera). Journal of Neurochemistry, 2006, 98: 1985-1998.
[21]   Watanabe T, Sadamoto H, Aonuma H. Identification and expression analysis of the genes involved in serotonin biosynthesis and transduction in the field cricket Gryllus bimaculatus. Insect Molecular Biology, 2011, 20(5): 619-635.
[22] Pietrantonio P V, Jagge C, McDowell C. Cloning and expression analysis of a 5HT7-like serotonin receptor cDNA from mosquito Aedes aegypti female excretory and respiratory systems. Insect Molecular Biology, 2001, 10(4): 357-369.
[23]   Zhan S, Merlin C, Boore J L, Reppert S M. The monarch butterfly genome yields insights into long-distance migration. Cell,2011,147: 1171-1185.
[24]   Gatellier L, Nagao T, Kanzaki R. Serotonin modifies the sensitivity of the male silkmoth to pheromone. The Journal of Experimental Biology, 2004, 207: 2487-2496.
[25]   Hill E S, Okada K, Kanzaki R. Visualization of modulatory effects of serotonin in the silkmoth antennal lobe. The Journal of Experimental Biology, 2003, 206: 345-352.
[26]   French A S, Simcock K L, Rolke D, Gartside S E, Blenau W, Wright G A. The role of semtonin in feeding and gut contractions in the honeybee. Joumal of Insect Physiology, 2014, 61: 8-15.
[27]   Orchard I. Semtonin: a coordinator of feeding-related physiological events in the blood-goring bug, Rhednius prolixus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology,, 2006, 144: 316-324.
[28]   Blenau W, Thamm M. Distribution of serotonin (5-HT) and its receptors in the insect brain with focus on the mushroom bodies. Lessons from Drosophila melanogaster and Apis mellifera. Arthropod Structure & Development, 2001, 40: 381-394.
[29]   吴顺凡, 郭建洋, 黄佳, 叶恭银. 昆虫体内章鱼胺和酪胺的研究进展. 昆虫学报, 2010, 53(10): 1157-1166.
Wu S F, Guo J Y, Huang J, Ye G Y. Advances in insect octopamine and tyramine. Acta Entomologica Sinica, 2010, 53(10): 1157-1166. (in Chinese)
[30]   Ayala F J, Rzhetsky A, Ayala F J. Origin of the metazoan phyla: Molecularclocks confirm paleontological estimates. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95: 606-611.
[31]   Filip M, Bader M. Overview on 5-HT receptors and their role in physiology and pathology of the central nervous system. Pharmacological Reports, 2009, 61: 761-777.
[32]   Theodore W H, Wiggs E A, Martinez A R, Dustin I H, Khan O I, Appel S, Reeves-Tyer P, Sato S. Serotonin 1A receptors, depression, and memory in temporal lobe epilepsy. Epilepsia, 2012, 53(1): 129-133.
[33] Hedlund P B, Sutcliffe J G. Functional, molecular and pharmacological advances in 5-HT7 receptor research. Trends in Pharmacological Sciences, 2004, 25(9): 481-486.
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