脂多糖对家蚕幼虫Toll样受体基因BmToll9-2表达的影响
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摘要
【目的】Toll样受体是昆虫Toll信号传导途径的一个重要组成成员,对激活昆虫机体对入侵病原微生物的先天免疫应答起着至关重要的作用。本研究旨在探究脂多糖对家蚕Bombyx mori幼虫中Toll样受体基因BmToll9-2基因表达的影响。【方法】利用RT-PCR分析BmToll9-2基因在家蚕大造品系4龄和5龄幼虫表皮、脂肪体、马氏管、中肠、神经、丝腺、胸部肌肉共7种组织中的表达特征;将脂多糖和大肠杆菌Escherichia coli注射到5龄幼虫体腔内,诱导其免疫系统响应,采用实时荧光定量PCR分析注射后不同时间BmToll9-2基因在中肠中的表达水平。【结果】RT-PCR结果表明,BmToll9-2基因在家蚕4龄幼虫的脂肪体、马氏管、神经和丝腺中高度表达,在5龄幼虫脂肪体、中肠、马氏管和神经中均有较高表达。实时荧光定量PCR结果显示,注射脂多糖能诱导5龄幼虫中肠中BmToll9-2基因转录,在注射后6 h时的诱导效果最好;注射大肠杆菌也能诱导BmToll9-2基因转录,在注射后3和6 h时的诱导效果最好。【结论】家蚕幼虫BmToll9-2基因在脂多糖和大肠杆菌处理后上调表达,提示BmToll9-2受体可能参与昆虫Toll样受体对脂多糖和细菌的识别过程。
【Aim】Toll-like receptors are important members in Toll signaling pathway in insects and play an important role in activating the innate immune response to invading pathogenic microorganisms.This study aims to explore the influence of lipopolysaccharide( LPS) on the expression of the Toll-like receptor gene BmToll9-2 in larvae of the silkworm,Bombyx mori.【Methods】The expression profiles of BmToll9-2 in epidermis,fat body,Malpighian tubules,midgut,brain,silk glands and thoracic muscles of the 4th and 5th instar larvae of B.mori strain Dazao were detected by RT-PCR.LPS and Escherichia coli were respectively injected into the body cavity of the 5th instar larvae to induce their immune response,and the expression levels of BmToll9-2 in the midgut at different time after injection were detected by real-time quantitative PCR.【Results】RT-PCR results revealed that BmToll9-2 was highly expressed not only in the fat body,Malpighian tubules,brain and silk glands of the 4th instar larvae of B.mori,but also in the fat body,midgut,Malpighian tubules and brain of the 5th instar larvae.The real-time quantitative PCR results showed that injection of LPS could induce the transcription of BmToll9-2 in the midgut of the5 th instar larvae,and the best induction effect was at 6 h after injection.Injection of E.coli could also induce the transcription of BmToll9-2 in the midgut of the 5th instar larvae,and the best induction effect was at 3 and 6 h after injection.【Conclusion】Expression of BmToll9-2 in B.mori larvae is up-regulatedafter injection of LPS and E.coli,suggesting that BmToll9-2 receptor may be involved in the recognition process of Toll-like receptors to LPS and E.coli in insects.
【Aim】Toll-like receptors are important members in Toll signaling pathway in insects and play an important role in activating the innate immune response to invading pathogenic microorganisms.This study aims to explore the influence of lipopolysaccharide( LPS) on the expression of the Toll-like receptor gene BmToll9-2 in larvae of the silkworm,Bombyx mori.【Methods】The expression profiles of BmToll9-2 in epidermis,fat body,Malpighian tubules,midgut,brain,silk glands and thoracic muscles of the 4th and 5th instar larvae of B.mori strain Dazao were detected by RT-PCR.LPS and Escherichia coli were respectively injected into the body cavity of the 5th instar larvae to induce their immune response,and the expression levels of BmToll9-2 in the midgut at different time after injection were detected by real-time quantitative PCR.【Results】RT-PCR results revealed that BmToll9-2 was highly expressed not only in the fat body,Malpighian tubules,brain and silk glands of the 4th instar larvae of B.mori,but also in the fat body,midgut,Malpighian tubules and brain of the 5th instar larvae.The real-time quantitative PCR results showed that injection of LPS could induce the transcription of BmToll9-2 in the midgut of the5 th instar larvae,and the best induction effect was at 6 h after injection.Injection of E.coli could also induce the transcription of BmToll9-2 in the midgut of the 5th instar larvae,and the best induction effect was at 3 and 6 h after injection.【Conclusion】Expression of BmToll9-2 in B.mori larvae is up-regulatedafter injection of LPS and E.coli,suggesting that BmToll9-2 receptor may be involved in the recognition process of Toll-like receptors to LPS and E.coli in insects.
引文
Beutler B,2004.Inferences,questions and possibilities in Toll-like receptor signaling.Nature,430(6996):257-263.
Cheng TC,Zhang YL,Liu C,Xu PZ,Gao ZH,Xia QY,Xiang ZH,2008.Identification and analysis of Toll-related genes in the domesticated silkworm,Bombyx mori.Dev.Comp.Immunol.,32(5):464-475.
Chintapalli RT,Hillyer JF,2016.Hemolymph circulation in insect flight appendages:physiology of the wing heart and circulatory flow in the wings of the mosquito Anopheles gambiae.J.Exp.Biol.,219:3945-3951.
Evans JD,Aronstein K,Chen YP,Hetru C,Imler JL,Jiang H,Kanost M,Thompson GJ,Zou Z,Hultmark D,2006.Immune pathways and defence mechanisms in honey bees Apis mellifera.Insect Mol.Biol.,15(5):645-656.
Garbutt JS,Reynolds SE,2012.Induction of RNA interference genes by double-stranded RNA;implications for susceptibility to RNAinterference.Insect Biochem.Mol.Biol.,42(9):621-628.
Guo YM,Dai ZY,1996.The progress of insects antibacterial peptides.Progr.Biotechnol.,16(3):24-27.[郭玉梅,戴祝英,1996.昆虫抗菌肽的研究进展.生物工程进展,16(3):24-27]
Hetru C,Hoffmann JA,2009.NF-kappa B in the immune response of Drosophila.Cold Spring Harb.Perspect.Biol.,1(6):a000232.
Hou Y,Zhao P,Liu HL,Zou Y,Guan J,Xia QY,2007.Proteomics analysis of fat body from silkworm(Bombyx mori).Chin.J.Biotechnol.,23(5):867-872.[侯勇,赵萍,刘鸿丽,邹勇,官建,夏庆友,2007.家蚕脂肪体蛋白质组学研究.生物工程学报,23(5):867-872]
Huang ZJ,Deng XJ,Chen FY,Mu PQ,Yang WY,Zhong YJ,Xia QY,Cao Y,2007.Analysis on the protein expression profile in midgut from silkworm,Bombyx mori,during larva pupae metamorphosis.Sci.Seric.,33(2):207-215.[黄志君,邓小娟,陈芳艳,母培强,杨婉莹,钟仰进,夏庆友,曹阳,2007.家蚕中肠组织在变态发育不同时期的蛋白表达差异分析.蚕业科学,33(2):207-215]
Huang ZR,Chen JW,1990.Some advances in the research of insect defense mechanism.Bull.Biol.,(10):9-10.[黄自然,陈劲伟,1990.昆虫防御机制研究的一些进展.生物学通报,(10):9-10]
Imamura M,Yamakawa M,2002.Molecular cloning and expression of a Toll receptor gene homologue from the silkworm,Bombyx mori.Biochim.Biophys.Acta,1576(3):246-254.
Klein DC,Skjesol A,Kers-Rebel ED,Sherstova T,Sporsheim B,Egeberg KW,Stokke BT,Espevik T,Husebye H,2015.CD14,TLR4 and TRAM show different trafficking dynamics during LPSstimulation.Traffic,16(7):677-690.
Lavine MD,Chen G,Strand MR,2005.Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includes.Insect Biochem.Mol.Biol.,35(12):1335-1346.
Lavine MD,Strand MR,2002.Insect hemocytes and their role in immunity.Insect Biochem.Mol.Biol.,32(10):1295-1309.
Li YS,2013.The molecular Mechanism Underlying Resistance against Entomopathogenic Fungi by Protease Inhibitors in Bombyx mori.Ph D Dissertation,Southwest University,Chongqing.[李游山,2013.家蚕蛋白酶抑制剂抵御昆虫致病性真菌入侵的分子机理研究.重庆:西南大学博士学位论文]
Liu J,Kolliopoulou A,Smagghe G,Swevers L,2014.Modulation of the transcriptional response of innate immune and RNAi genes upon exposure to ds RNA and LPS in silkmoth-derived Bm5 cells overexpressing Bm Toll9-1 receptor.J.Insect Physiol.,66:10-19.
Liu J,Smagghe G,Swevers L,2013.Transcriptional response of Bm Toll9-1 and RNAi machinery genes to exogenous ds RNA in the midgut of Bombyx mori.J.Insect Physiol.,59(6):646-654.
Liu JB,2014.Immune Response of Different Pathogenic Microorganisms to the Main Immune Signal Pathway in Bombyx mori.MSc Thesis,Soochow University,Suzhou,Jiangsu.[刘佳宾,2014.家蚕主要免疫信号通路对不同病原微生物的感染应答.江苏苏州:苏州大学硕士学位论文]
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method.Methods,25(4):402-408.
Luna C,Wang X,Huang Y,Zhang J,Zheng L,2002.Characterization of four Toll related genes during development and immune responses in Anopheles gambiae.Insect Biochem.Mol.Biol.,32(9):1171-1179.
Medzhitov R,Janeway CAJr,1997.Innate immunity:the virtues of a nonclonal system of recognition.Cell,91(3):295-298.
Merkling SH,van Rij RP,2013.Beyond RNAi:antiviral defense strategies in Drosophila and mosquito.J.Insect Physiol.,59(2):159-170.
Nüsslein-Volhard C,Lohs-Schardin M,Sander K,Cremer C,1980.Adorso-ventral shift of embryonic primordia in a new maternal-effect mutant of Drosophila.Nature,283(5746):474-476.
Takeda K,Akira S,2004.TLR signaling pathways.Semin.Immunol.,16(1):3-9.
Tanaka H,Ishibashi J,Fujita K,Nakajima Y,Sagisaka A,Tomimoto K,Suzuki N,Yoshiyama M,Kaneko Y,Iwasaki T,Sunagawa T,Yamaji K,Asaoka A,Mita K,Yamakawa M,2008.A genomewide analysis of genes and gene families involved in innate immunity of Bombyx mori.Insect Biochem.Mol.Biol.,38(12):1087-1110.
Wang CX,2014.Immune pattern of PGRP and Cecropin genes in Bombyx mori midgut and fatbody.J.Shanxi Agric.Univ.(Nat.Sci.Ed.),34(2):109-113.[王传旭,2014.PGRP和Cecropin基因在家蚕中肠和脂肪体中的免疫模式研究.山西农业大学学报(自然科学版),34(2):109-113]
Wang GH,Xia QY,Cheng DJ,Duan J,Zhao P,Chen J,Li Z,2008.Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by q RT-PCR.Insect Sci.,15(5):405-413.
Wang NP,Liu FR,Li SY,1993.Advances in epidermal immunity.Chin.J.Dermatoven.,(2):116-118.[汪南平,刘辅仁,李世荫,1993.表皮免疫研究进展.中国皮肤性病学杂志,(2):116-118]
Wang X,Quinn PJ,2010.Lipopolysaccharide:biosynthetic pathway and structure modification.Prog.Lipid Res.,49(2):97-107.
Wu S,Zhang X,Chen X,Cao P,Beerntsen BT,Ling E,2010.Bm Toll9,an arthropod conservative Toll,is likely involved in the local gut immune response in the silkworm,Bombyx mori.Dev.Comp.Immunol.,34(2):93-96.
Xue MQ,2014.The Response of Immune System to Bacterial Infection of Diamondback Moth(Plutella xylostella)in the Midgut.MSc Thesis,Fujian Agriculture and Forestry University,Fuzhou.[薛敏倩,2014.小菜蛾中肠免疫系统对细菌侵染的响应.福州:福建农林大学硕士学位论文]
Yamamoto-Hino M,Goto S,2016.Sptzle-processing enzymeindependent activation of the Toll pathway in Drosophila innate immunity.Cell Struct.Funct.,41(1):55-60.
Zhang ZY,2011.Gene Expression Analysis of Central Nervous System and Immune System of Locust migratory.MSc Thesis,Chongqing University,Chongqing.[张正一,2011.东亚飞蝗中枢神经系统和免疫系统基因表达分析.重庆:重庆大学硕士学位论文]
Zhao XH,2013.Primary Research on mi RNAs in Malpighian Tubules from the Silkworm Larvae(Bombyx mori)Response to Bm NPVInfection.MSc Thesis,Jiangsu University of Science and Technology,Zhenjiang,Jiangsu.[赵新慧,2013.家蚕马氏管响应Bm NPV感染相关mi RNA的初步研究.江苏镇江:江苏科技大学硕士学位论文]
Zhong X,Xu XX,Yi HY,Lin C,Yu XQ,2012.A Toll-Sptzle pathway in the tobacco hornworm,Manduca sexta.Insect Biochem.Molec.Biol.,42(7):514-524.
Zou Y,2012.Proteomics of Malpighian Tubules and the Functional Analysis of Important Enzymes,Bomyx mori.Ph D Dissertation,Southwest University,Chongqing.[邹勇,2012.家蚕马氏管蛋白质组学及重要酶类的功能分析.重庆:西南大学博士学位论文]
Cheng TC,Zhang YL,Liu C,Xu PZ,Gao ZH,Xia QY,Xiang ZH,2008.Identification and analysis of Toll-related genes in the domesticated silkworm,Bombyx mori.Dev.Comp.Immunol.,32(5):464-475.
Chintapalli RT,Hillyer JF,2016.Hemolymph circulation in insect flight appendages:physiology of the wing heart and circulatory flow in the wings of the mosquito Anopheles gambiae.J.Exp.Biol.,219:3945-3951.
Evans JD,Aronstein K,Chen YP,Hetru C,Imler JL,Jiang H,Kanost M,Thompson GJ,Zou Z,Hultmark D,2006.Immune pathways and defence mechanisms in honey bees Apis mellifera.Insect Mol.Biol.,15(5):645-656.
Garbutt JS,Reynolds SE,2012.Induction of RNA interference genes by double-stranded RNA;implications for susceptibility to RNAinterference.Insect Biochem.Mol.Biol.,42(9):621-628.
Guo YM,Dai ZY,1996.The progress of insects antibacterial peptides.Progr.Biotechnol.,16(3):24-27.[郭玉梅,戴祝英,1996.昆虫抗菌肽的研究进展.生物工程进展,16(3):24-27]
Hetru C,Hoffmann JA,2009.NF-kappa B in the immune response of Drosophila.Cold Spring Harb.Perspect.Biol.,1(6):a000232.
Hou Y,Zhao P,Liu HL,Zou Y,Guan J,Xia QY,2007.Proteomics analysis of fat body from silkworm(Bombyx mori).Chin.J.Biotechnol.,23(5):867-872.[侯勇,赵萍,刘鸿丽,邹勇,官建,夏庆友,2007.家蚕脂肪体蛋白质组学研究.生物工程学报,23(5):867-872]
Huang ZJ,Deng XJ,Chen FY,Mu PQ,Yang WY,Zhong YJ,Xia QY,Cao Y,2007.Analysis on the protein expression profile in midgut from silkworm,Bombyx mori,during larva pupae metamorphosis.Sci.Seric.,33(2):207-215.[黄志君,邓小娟,陈芳艳,母培强,杨婉莹,钟仰进,夏庆友,曹阳,2007.家蚕中肠组织在变态发育不同时期的蛋白表达差异分析.蚕业科学,33(2):207-215]
Huang ZR,Chen JW,1990.Some advances in the research of insect defense mechanism.Bull.Biol.,(10):9-10.[黄自然,陈劲伟,1990.昆虫防御机制研究的一些进展.生物学通报,(10):9-10]
Imamura M,Yamakawa M,2002.Molecular cloning and expression of a Toll receptor gene homologue from the silkworm,Bombyx mori.Biochim.Biophys.Acta,1576(3):246-254.
Klein DC,Skjesol A,Kers-Rebel ED,Sherstova T,Sporsheim B,Egeberg KW,Stokke BT,Espevik T,Husebye H,2015.CD14,TLR4 and TRAM show different trafficking dynamics during LPSstimulation.Traffic,16(7):677-690.
Lavine MD,Chen G,Strand MR,2005.Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includes.Insect Biochem.Mol.Biol.,35(12):1335-1346.
Lavine MD,Strand MR,2002.Insect hemocytes and their role in immunity.Insect Biochem.Mol.Biol.,32(10):1295-1309.
Li YS,2013.The molecular Mechanism Underlying Resistance against Entomopathogenic Fungi by Protease Inhibitors in Bombyx mori.Ph D Dissertation,Southwest University,Chongqing.[李游山,2013.家蚕蛋白酶抑制剂抵御昆虫致病性真菌入侵的分子机理研究.重庆:西南大学博士学位论文]
Liu J,Kolliopoulou A,Smagghe G,Swevers L,2014.Modulation of the transcriptional response of innate immune and RNAi genes upon exposure to ds RNA and LPS in silkmoth-derived Bm5 cells overexpressing Bm Toll9-1 receptor.J.Insect Physiol.,66:10-19.
Liu J,Smagghe G,Swevers L,2013.Transcriptional response of Bm Toll9-1 and RNAi machinery genes to exogenous ds RNA in the midgut of Bombyx mori.J.Insect Physiol.,59(6):646-654.
Liu JB,2014.Immune Response of Different Pathogenic Microorganisms to the Main Immune Signal Pathway in Bombyx mori.MSc Thesis,Soochow University,Suzhou,Jiangsu.[刘佳宾,2014.家蚕主要免疫信号通路对不同病原微生物的感染应答.江苏苏州:苏州大学硕士学位论文]
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method.Methods,25(4):402-408.
Luna C,Wang X,Huang Y,Zhang J,Zheng L,2002.Characterization of four Toll related genes during development and immune responses in Anopheles gambiae.Insect Biochem.Mol.Biol.,32(9):1171-1179.
Medzhitov R,Janeway CAJr,1997.Innate immunity:the virtues of a nonclonal system of recognition.Cell,91(3):295-298.
Merkling SH,van Rij RP,2013.Beyond RNAi:antiviral defense strategies in Drosophila and mosquito.J.Insect Physiol.,59(2):159-170.
Nüsslein-Volhard C,Lohs-Schardin M,Sander K,Cremer C,1980.Adorso-ventral shift of embryonic primordia in a new maternal-effect mutant of Drosophila.Nature,283(5746):474-476.
Takeda K,Akira S,2004.TLR signaling pathways.Semin.Immunol.,16(1):3-9.
Tanaka H,Ishibashi J,Fujita K,Nakajima Y,Sagisaka A,Tomimoto K,Suzuki N,Yoshiyama M,Kaneko Y,Iwasaki T,Sunagawa T,Yamaji K,Asaoka A,Mita K,Yamakawa M,2008.A genomewide analysis of genes and gene families involved in innate immunity of Bombyx mori.Insect Biochem.Mol.Biol.,38(12):1087-1110.
Wang CX,2014.Immune pattern of PGRP and Cecropin genes in Bombyx mori midgut and fatbody.J.Shanxi Agric.Univ.(Nat.Sci.Ed.),34(2):109-113.[王传旭,2014.PGRP和Cecropin基因在家蚕中肠和脂肪体中的免疫模式研究.山西农业大学学报(自然科学版),34(2):109-113]
Wang GH,Xia QY,Cheng DJ,Duan J,Zhao P,Chen J,Li Z,2008.Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by q RT-PCR.Insect Sci.,15(5):405-413.
Wang NP,Liu FR,Li SY,1993.Advances in epidermal immunity.Chin.J.Dermatoven.,(2):116-118.[汪南平,刘辅仁,李世荫,1993.表皮免疫研究进展.中国皮肤性病学杂志,(2):116-118]
Wang X,Quinn PJ,2010.Lipopolysaccharide:biosynthetic pathway and structure modification.Prog.Lipid Res.,49(2):97-107.
Wu S,Zhang X,Chen X,Cao P,Beerntsen BT,Ling E,2010.Bm Toll9,an arthropod conservative Toll,is likely involved in the local gut immune response in the silkworm,Bombyx mori.Dev.Comp.Immunol.,34(2):93-96.
Xue MQ,2014.The Response of Immune System to Bacterial Infection of Diamondback Moth(Plutella xylostella)in the Midgut.MSc Thesis,Fujian Agriculture and Forestry University,Fuzhou.[薛敏倩,2014.小菜蛾中肠免疫系统对细菌侵染的响应.福州:福建农林大学硕士学位论文]
Yamamoto-Hino M,Goto S,2016.Sptzle-processing enzymeindependent activation of the Toll pathway in Drosophila innate immunity.Cell Struct.Funct.,41(1):55-60.
Zhang ZY,2011.Gene Expression Analysis of Central Nervous System and Immune System of Locust migratory.MSc Thesis,Chongqing University,Chongqing.[张正一,2011.东亚飞蝗中枢神经系统和免疫系统基因表达分析.重庆:重庆大学硕士学位论文]
Zhao XH,2013.Primary Research on mi RNAs in Malpighian Tubules from the Silkworm Larvae(Bombyx mori)Response to Bm NPVInfection.MSc Thesis,Jiangsu University of Science and Technology,Zhenjiang,Jiangsu.[赵新慧,2013.家蚕马氏管响应Bm NPV感染相关mi RNA的初步研究.江苏镇江:江苏科技大学硕士学位论文]
Zhong X,Xu XX,Yi HY,Lin C,Yu XQ,2012.A Toll-Sptzle pathway in the tobacco hornworm,Manduca sexta.Insect Biochem.Molec.Biol.,42(7):514-524.
Zou Y,2012.Proteomics of Malpighian Tubules and the Functional Analysis of Important Enzymes,Bomyx mori.Ph D Dissertation,Southwest University,Chongqing.[邹勇,2012.家蚕马氏管蛋白质组学及重要酶类的功能分析.重庆:西南大学博士学位论文]