绿尾虹雉(Lophophorus lhuysii)Toll样受体5基因的克隆和适应性进化分析
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摘要
【目的】获得绿尾虹雉(Lophophorus lhuysii)Toll样受体5基因(TLR5)编码区序列,并对其编码区蛋白的特征及适应性进化进行分析。【方法】以绿尾虹雉基因组DNA为模板,采用PCR扩增克隆获得TLR5基因编码区序列。使用MEGA、PAML等软件对其序列特征及选择压力进行分析。【结果】绿尾虹雉TLR5基因编码区序列全长2 583 bp,共编码860个氨基酸,以亮氨酸含量最高(133/860),色氨酸含量最低(11/860)。编码蛋白为典型的Ⅰ型跨膜结构,包括富含LRRs结构域的胞外区、跨膜区和胞内TIR结构域。绿尾虹雉TLR5基因极其保守,与雉鸡(P. colchicus)的同源性最高(97.75%),与小白鹭(E. garzetta)同源性最低(85.17%)。采用位点-特异模型在胞外区共检测到3个正选择氨基酸位点(263F, 280K和645I)。【结论】绿尾虹雉TLR5基因编码区受到较强的纯净化压力,胞外区LRRs结构域可能为识别病原微生物的区域。
【Objective】The objective of this study was to obtain the full coding DNA sequence(CDS) and to explore the mechanisms for the adaptive evolution of toll-like receptor 5(TLR5) gene in the Chinese monal(Lophophorus lhuysii).【Method】The Chinese monal genomic DNA was used as a template to amplify and clone the TLR5 gene by PCR. The sequences characterization and adaptive evolution of TLR5 gene encoded protein were analyzed by MEGA and PAML software.【Result】The results showed that the full coding DNA sequence(CDS) of Chinese monal TLR5 was 2 583 bp,encoding 860 amino acids, among which leucine(133/860) occupied the highest content and tryptophan(11/860) with the lowest content. The encoded protein of TLR5 was a typical type I transmembrane receptors, which were composed of a extracellular leucine-rich repeats(LRRs),a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor(TIR) signaling domain, and the entire coding area is subject to strong purification selection. Homology analysis showed that the TLR5 gene of Chinese monal had relatively conservative, and the closest relationship with Phasianus colchicus(97.75%),the lowest relationship with Egretta garzetta(85.17%). Additionally, three amino acid sites(263 F,280 K and 645 I) were found in the extracellular domain under positive selection by site-specific model of PAML software.【Conclusion】The coding regions of the Chinese monal TLR5 gene were subject to strong purification pressures. It is speculated that the extracellular LRRs domain of the Chinese monal TLR5 gene may play an important role for identifying pathogenic microorganisms.
【Objective】The objective of this study was to obtain the full coding DNA sequence(CDS) and to explore the mechanisms for the adaptive evolution of toll-like receptor 5(TLR5) gene in the Chinese monal(Lophophorus lhuysii).【Method】The Chinese monal genomic DNA was used as a template to amplify and clone the TLR5 gene by PCR. The sequences characterization and adaptive evolution of TLR5 gene encoded protein were analyzed by MEGA and PAML software.【Result】The results showed that the full coding DNA sequence(CDS) of Chinese monal TLR5 was 2 583 bp,encoding 860 amino acids, among which leucine(133/860) occupied the highest content and tryptophan(11/860) with the lowest content. The encoded protein of TLR5 was a typical type I transmembrane receptors, which were composed of a extracellular leucine-rich repeats(LRRs),a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor(TIR) signaling domain, and the entire coding area is subject to strong purification selection. Homology analysis showed that the TLR5 gene of Chinese monal had relatively conservative, and the closest relationship with Phasianus colchicus(97.75%),the lowest relationship with Egretta garzetta(85.17%). Additionally, three amino acid sites(263 F,280 K and 645 I) were found in the extracellular domain under positive selection by site-specific model of PAML software.【Conclusion】The coding regions of the Chinese monal TLR5 gene were subject to strong purification pressures. It is speculated that the extracellular LRRs domain of the Chinese monal TLR5 gene may play an important role for identifying pathogenic microorganisms.
引文
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[9]唐卓,杨建,刘雪华,等.利用红外相机研究卧龙国家级自然保护区绿尾虹雉的活动规律[J].四川动物,2017,36(5):582-587.
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[11]曾思敏,周材权,杨本清,等.笼养状态下绿尾虹雉的鸣声行为特征[J].西华师范大学学报(自然科学版),2010,31(1):8-15.
[12]余翔,陈俊橙,王彬,等.四川小寨子沟国家级自然保护区绿尾虹雉种群密度调查与栖息地评价[J].四川动物,2017,36(4):361-367.
[13]陈冬梅,何松,张敬.笼养绿尾虹雉血液生理生化指标分析[J].动物学杂志,2018,53(3):354-359.
[14]陈冬梅,何松,韦毅,等.不同营养水平对绿尾虹雉血液生理生化指标的影响[J].饲料研究,2017(6):11-16.
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[18]BROWNLIE R,ALLAN B.Avian toll-like receptors[J].Cell Tissue Res,2011,343(1):121-130.
[19]WARREN W C,CLAYTON D F,ELLEGREN H,et al.The genome of a songbird[J].Nature,2015,464(7289):757-762.
[20]MATSUSHIMA N,TANAKA T,ENKHBAYAR P,et al.Comparative sequence analysis of leucine-rich repeats(LRRs)within vertebrate toll-like receptors[J].BMC Genomics,2007,8(1):124-144.
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[22]WLASIUK G,NACHMAN M W.Adaptation and constraint at toll-like receptors in primates[J].Mol Biol Evol,2010,27(9):2172-2186.
[23]MIZEL S B,SNIPES J A.Gram-negative flagellin-induced selftolerance is associated with a block in interleukin-1 receptor-associated kinase release from toll-like receptor 5[J].J Biol Chem,2002,277(25):22414-22420.
[24]HAYASHI F,SMITH K D,OZINSKY A,et al.The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5[J].Nature,2001,410(6832):1099-1103.
[25]FEUILLET V,MEDJANE S,MONDOR I,et al.Involvement of Toll-like receptor 5 in the recognition of flagellated bacteria[J].P Natl Acad Sci USA,2006,103(33):12487-12492.
[26]KAWAI T,AKIRA S.TLR signaling[J].Cell death and differentiation,2006,13(5):816.
[27]QIANG F,PAN Z,GENG S,et al.Molecular cloning,characterization and expression of goose Toll-like receptor 5[J].Mol Immunol,2012,52(3/4):117-124.
[28]HAWN T R,VERBON A,LETTINGA K D,et al.A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to Legionnaires′disease[J].J Exp Med,2003,198(10):1563-1572.
[29]KEESTRA A M,DE ZOETE M R,VANAUBEL R A,et al.Functional characterization of chicken TLR5 reveals species-specific recognition of flagellin[J].Mol Immunol,2008,45(5):1298-1307.
[30]IQBAL M,PHILBIN V J,WITHANAGE G S,et al.Identification and functional characterization of chicken toll-like receptor 5reveals a fundamental role in the biology of infection with Salmonella enterica serovar typhimurium[J].Infect Immun,2005,73(73):2344-2350.
[31]詹祥江,张正旺,吴爱平,等.基于线粒体细胞色素b基因序列的虹雉属鸟类的系统发育关系[J].动物学研究,2003,24(5):337-342.
[32]MA L L,ZHANG X Y,YUE B S,et al.Complete mitochondrial genome of the Chinese Monal pheasant Lophophorus lhuysii,with phylogenetic implication in Phasiani dae[J].Mitochondr DNA,2010,21(1):5-7.
[33]SAMBROOK J,RUSSELL D W.Molecular cloning:a laboratory manual,3rd edn[M].New York:Cold Spring Harbor Laboratory Press,2001.
[34]ALCAIDE M,EDWARDS S V.Molecular evolution of the tolllike receptor multigene family in birds[J].Mol Biol Evol,2011,28(5):1703-1715.
[35]YANG Z,WONG W S,NIELSEN R.Bayes empirical bayes inference of amino acid sites under positive selection[J].Mol Biol Evol,2005,22(4):1107-1118.
[36]ZHANG J,NIELSEN R,YANG Z.Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular leve[lJ].Mol Biol Evol,2005,22(12):2472-2479.
[37]SCHNARE M,BARTON G M,HOLT A C,et al.Toll-like receptors control activation of adaptive immune responses[J].Nature Immunology,2001,2(10):947-950.
[38]WERLING D,JANN O C,OFFORD V,et al.Variation matters:TLR structure and species-specific pathogen recognition[J].Trends Immunol,2009,30(3):124-130.
[39]LIU Y P,WU G S,YAO Y G,et al.Multiple maternal origins of chickens:out of the Asian jungles[J].Mol Phylogenet Evol,2006,38(1):12-19.
[40]RUAN W,WU Y,ZHENG S J.Different genetic patterns in avian Toll-like receptor(TLR)5 genes[J].Mol Biol Rep,2012,39(4):3419-3426.
[41]XIONG D,SONG L,PAN Z,et al.Molecular cloning,characterization,and functional analysis of pigeon(Columba livia)Tolllike receptor 5[J].Poultry Sci,2018,97(11):4031-4039.
[42]LIU L,BOTOS I,WANG Y,et al.Structural basis of toll-like receptor 3 signaling with double-stranded RNA[J].Science,2008,320(5874):379-381.
[2]约翰·马敬能,卡伦·菲利普斯,何芬奇.中国鸟类野外手册[M].长沙:湖南教育出版社,2000:31-32.
[3]CHEN Y H.Conservation priority for endemic birds of mainland China based on a phylogenetic framework[J].Chinese Birds,2013,4(3):248-253.
[4]何芬奇,卢汰春,芦春雷,等.绿尾虹雉的繁殖生态研究[J].生态学报,1986,6(2):100-106.
[5]张涛.甘肃白水江自然保护区绿尾虹雉分布与生态的初步观察[J].动物学杂志,1995(4):25-28.
[6]WANG B,XU Y,RAN J H.Predicting suitable habitat of the Chinese monal(Lophophorus lhuysii)using ecological niche modeling in the Qionglai Mountains,China[J].Peerj,2017,5(7):3477-3497.
[7]钟雪,窦亮,杨本清,等.笼养绿尾虹雉繁殖季节的日行为节律和时间分配[J].四川动物,2013,32(4):521-525.
[8]窦亮,徐雨,耿秋扎西,等.笼养绿尾虹雉炫耀行为节律及天气因素对该行为的影响研究[J].四川动物,2015,34(3):399-402.
[9]唐卓,杨建,刘雪华,等.利用红外相机研究卧龙国家级自然保护区绿尾虹雉的活动规律[J].四川动物,2017,36(5):582-587.
[10]卢汰春,何芬奇,卢春雷.绿尾虹雉叫声的声谱分析[J].生态学报,1986,6(1):89-90.
[11]曾思敏,周材权,杨本清,等.笼养状态下绿尾虹雉的鸣声行为特征[J].西华师范大学学报(自然科学版),2010,31(1):8-15.
[12]余翔,陈俊橙,王彬,等.四川小寨子沟国家级自然保护区绿尾虹雉种群密度调查与栖息地评价[J].四川动物,2017,36(4):361-367.
[13]陈冬梅,何松,张敬.笼养绿尾虹雉血液生理生化指标分析[J].动物学杂志,2018,53(3):354-359.
[14]陈冬梅,何松,韦毅,等.不同营养水平对绿尾虹雉血液生理生化指标的影响[J].饲料研究,2017(6):11-16.
[15]MORISALO D,ANDERSON K V.Signaling pathways that establish the dorsal-ventral pattern of the drosophila embryo[J].Annu Rev Genet,2003,29(29):371-399.
[16]ADEREM A,ULEVITCH R J.Toll-like receptors in the induction of the innate immune response[J].Nature,2000,406(6797):782-787.
[17]BELL J K,MULLEN G E,LEIFER C A,et al.Leucine-rich repeats and pathogen recognition in Toll-like receptors[J].Trends Immunol,2003,24(10):528-533.
[18]BROWNLIE R,ALLAN B.Avian toll-like receptors[J].Cell Tissue Res,2011,343(1):121-130.
[19]WARREN W C,CLAYTON D F,ELLEGREN H,et al.The genome of a songbird[J].Nature,2015,464(7289):757-762.
[20]MATSUSHIMA N,TANAKA T,ENKHBAYAR P,et al.Comparative sequence analysis of leucine-rich repeats(LRRs)within vertebrate toll-like receptors[J].BMC Genomics,2007,8(1):124-144.
[21]HASHIMOTO C,HUDSON K L,ANDERSON K V.The Toll gene of Drosophila,required for dorsal-ventral embryonic polarity,appears to encode a transmembrane protein[J].Cell,1988,52:269-279.
[22]WLASIUK G,NACHMAN M W.Adaptation and constraint at toll-like receptors in primates[J].Mol Biol Evol,2010,27(9):2172-2186.
[23]MIZEL S B,SNIPES J A.Gram-negative flagellin-induced selftolerance is associated with a block in interleukin-1 receptor-associated kinase release from toll-like receptor 5[J].J Biol Chem,2002,277(25):22414-22420.
[24]HAYASHI F,SMITH K D,OZINSKY A,et al.The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5[J].Nature,2001,410(6832):1099-1103.
[25]FEUILLET V,MEDJANE S,MONDOR I,et al.Involvement of Toll-like receptor 5 in the recognition of flagellated bacteria[J].P Natl Acad Sci USA,2006,103(33):12487-12492.
[26]KAWAI T,AKIRA S.TLR signaling[J].Cell death and differentiation,2006,13(5):816.
[27]QIANG F,PAN Z,GENG S,et al.Molecular cloning,characterization and expression of goose Toll-like receptor 5[J].Mol Immunol,2012,52(3/4):117-124.
[28]HAWN T R,VERBON A,LETTINGA K D,et al.A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to Legionnaires′disease[J].J Exp Med,2003,198(10):1563-1572.
[29]KEESTRA A M,DE ZOETE M R,VANAUBEL R A,et al.Functional characterization of chicken TLR5 reveals species-specific recognition of flagellin[J].Mol Immunol,2008,45(5):1298-1307.
[30]IQBAL M,PHILBIN V J,WITHANAGE G S,et al.Identification and functional characterization of chicken toll-like receptor 5reveals a fundamental role in the biology of infection with Salmonella enterica serovar typhimurium[J].Infect Immun,2005,73(73):2344-2350.
[31]詹祥江,张正旺,吴爱平,等.基于线粒体细胞色素b基因序列的虹雉属鸟类的系统发育关系[J].动物学研究,2003,24(5):337-342.
[32]MA L L,ZHANG X Y,YUE B S,et al.Complete mitochondrial genome of the Chinese Monal pheasant Lophophorus lhuysii,with phylogenetic implication in Phasiani dae[J].Mitochondr DNA,2010,21(1):5-7.
[33]SAMBROOK J,RUSSELL D W.Molecular cloning:a laboratory manual,3rd edn[M].New York:Cold Spring Harbor Laboratory Press,2001.
[34]ALCAIDE M,EDWARDS S V.Molecular evolution of the tolllike receptor multigene family in birds[J].Mol Biol Evol,2011,28(5):1703-1715.
[35]YANG Z,WONG W S,NIELSEN R.Bayes empirical bayes inference of amino acid sites under positive selection[J].Mol Biol Evol,2005,22(4):1107-1118.
[36]ZHANG J,NIELSEN R,YANG Z.Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular leve[lJ].Mol Biol Evol,2005,22(12):2472-2479.
[37]SCHNARE M,BARTON G M,HOLT A C,et al.Toll-like receptors control activation of adaptive immune responses[J].Nature Immunology,2001,2(10):947-950.
[38]WERLING D,JANN O C,OFFORD V,et al.Variation matters:TLR structure and species-specific pathogen recognition[J].Trends Immunol,2009,30(3):124-130.
[39]LIU Y P,WU G S,YAO Y G,et al.Multiple maternal origins of chickens:out of the Asian jungles[J].Mol Phylogenet Evol,2006,38(1):12-19.
[40]RUAN W,WU Y,ZHENG S J.Different genetic patterns in avian Toll-like receptor(TLR)5 genes[J].Mol Biol Rep,2012,39(4):3419-3426.
[41]XIONG D,SONG L,PAN Z,et al.Molecular cloning,characterization,and functional analysis of pigeon(Columba livia)Tolllike receptor 5[J].Poultry Sci,2018,97(11):4031-4039.
[42]LIU L,BOTOS I,WANG Y,et al.Structural basis of toll-like receptor 3 signaling with double-stranded RNA[J].Science,2008,320(5874):379-381.