经白色念珠菌胁迫后家蝇新型抗菌肽AMP17表达模式的研究
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摘要
目的:探讨家蝇幼虫经白色念珠菌Candida albican胁迫后,AMP17基因时空表达谱的变化。方法:显微注射法将白色念珠菌导入家蝇幼虫体内,饲养3 h、6 h、12 h、24 h、36 h、48 h后,提取各组整虫RNA,采用实时荧光定量PCR分析AMP17的时间表达情况;选取经胁迫后3 h、12 h、48 h的幼虫,解剖获得体壁、脂肪体、马氏管、中肠、唾液腺、气管、血淋巴,同上分析AMP17的空间表达情况。以注射PBS为对照组,家蝇RPS18为内参基因,设3个生物重复。结果:注射白色念珠菌后,实验组幼虫的AMP17基因表达量较对照组明显上调,高表达一直持续到12 h,24 h明显下降,但48 h表达量又突然大幅度升高。不同组织中实验组与对照组比较,3 h时体壁表达量最高,上调6. 59倍(P<0. 01),其次是气管上调4. 74倍;感染12 h后,体壁表达量依然最高,上调了5. 54倍(P<0. 01),其次为血淋巴,上调了1. 87倍(P<0. 05);感染48 h后,体壁表达量上调了10. 98倍(P<0. 05);唾液腺上调了7. 28倍(P<0. 05)。结论:经白色念珠菌胁迫后,AMP17基因的表达量出现了明显的上调,提示该基因参与了家蝇应对真菌侵染的免疫反应。
Objective: To investigate the spatial and temporal expression profile of AMP17 gene in Musca domestica larvae after stress by Candida albican. Methods: Candida albicans infects Musca domestica larvae. After feeding for 3 h,6 h,12 h,24 h,36 h,and48 h,RNA of each group was extracted and real-time fluorescence quantitative PCR was used to analyze the temporal expression of AMP17. The larvae at 3 h,12 h and 48 h after stress were selected to obtain body wall,fat body,malpighian tubule,midgut,salivary gland,trachea and hemolymph. The spatial expression of AMP17 was analyzed as above. Injected PBS as control group,Musca domestica RPS18 as an internal reference gene with 3 biological replicates. Results: After injection of Candida albicans,the expression level of AMP17 gene in experimental group larvae was significantly higher than that in the control group. The high expression lasted until 12 h and decreased significantly at 24 h,but the expression level suddenly increased sharply at 48 h. Compared with the control group,the expression of body wall was the highest at the 3 h,which was 6. 59 times higher( P<0. 01),followed by 4. 74 times the trachea uptake;after 12 h,the body wall expression was still the highest,up 5. 54 times( P<0. 01),followed by hemolymph,increased 1. 87-fold( P<0. 05); after 48 h of infection,body wall expression was up-regulated by 10. 98-fold( P< 0. 05); salivary glands were up-regulated by7. 28-fold( P<0. 05). Conclusion: After being challenged by Candida albicans,the expression of AMP17 gene was significantly upregulated,suggesting that this gene is involved in the immune response of Musca domestica against fungal infection.
Objective: To investigate the spatial and temporal expression profile of AMP17 gene in Musca domestica larvae after stress by Candida albican. Methods: Candida albicans infects Musca domestica larvae. After feeding for 3 h,6 h,12 h,24 h,36 h,and48 h,RNA of each group was extracted and real-time fluorescence quantitative PCR was used to analyze the temporal expression of AMP17. The larvae at 3 h,12 h and 48 h after stress were selected to obtain body wall,fat body,malpighian tubule,midgut,salivary gland,trachea and hemolymph. The spatial expression of AMP17 was analyzed as above. Injected PBS as control group,Musca domestica RPS18 as an internal reference gene with 3 biological replicates. Results: After injection of Candida albicans,the expression level of AMP17 gene in experimental group larvae was significantly higher than that in the control group. The high expression lasted until 12 h and decreased significantly at 24 h,but the expression level suddenly increased sharply at 48 h. Compared with the control group,the expression of body wall was the highest at the 3 h,which was 6. 59 times higher( P<0. 01),followed by 4. 74 times the trachea uptake;after 12 h,the body wall expression was still the highest,up 5. 54 times( P<0. 01),followed by hemolymph,increased 1. 87-fold( P<0. 05); after 48 h of infection,body wall expression was up-regulated by 10. 98-fold( P< 0. 05); salivary glands were up-regulated by7. 28-fold( P<0. 05). Conclusion: After being challenged by Candida albicans,the expression of AMP17 gene was significantly upregulated,suggesting that this gene is involved in the immune response of Musca domestica against fungal infection.
引文
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[2]张福梅,李明生.抗菌肽在免疫防御和抗肿瘤中的作用[J].国外医药:抗生素分册,2017,38(6):284-288.Zhang FM,Li MS.Antibacterial peptides'roles in immune defense and anti-tumorogenesis[J].World Notes Antibiotics,2017,38(6):284-288.
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[11]Matskevich AA,Quintin J,Ferrandon D.The Drosophila PRRGNBP3 assembles effector complexes involved in antifungal defenses independently of its Toll pathway activation function[J].Eur J Immunol,2010,40(5):1244-1254.
[12]Iijima R,Kurata S,Natori S.Purification,characterization,and c DNA cloning of an antifungal protein from the hemolymph of Sarcophaga peregrina(flesh fly)larvae[J].J Biol Chem,1993,268(16):12055-12061.
[13]吕丁丁,侯成香,耿涛,等.家蚕感染球孢白僵菌后4种抗菌肽基因的表达分析[J].南方农业学报,2016,47(11):1952-1957.Lv DD,Hou CX,Geng T,et al.Expression of four antimicrobial peptide genes in Bombyx mori infected with Beauveria bassiana[J].J Southern Agriculture,2016,47(11):1952-1957.
[14]黄玉霞,耿涛,侯成香,等.白僵菌诱导家蚕抗菌肽enbocin1基因的表达特征及产物的抗真菌活性[J].蚕业科学,2016(4):619-626.Huang YX,Geng T,Hou CX,et al.Expression patterns of antimicrobial peptide gene enbocin1 in bombyx mori induced by Beauveria bassiana and its antifungal activity[J].Sci Sericulture,2016(4):619-626.
[15]杨雪,唐婷,王一丽,等.家蝇新型抗菌肽Muscin的基因克隆、表达模式及抑菌活性[J].昆虫学报,2015,58(6):617-624.Yang X,Tang T,Wang YL,et al.Gene cloning,expression profiling and anti-microbial assay of Muscin,a novel antmicrobial peptide in the housefly(Musca domestica)[J].Acta Entomologica Sinica,2015,58(6):617-624.
[16]修江帆,魏川川,陈明明,等.不同诱导条件下家蝇三龄幼虫免疫相关基因的表达研究[J].生物技术通报,2014,(6):120-127.Xiu JF,Wei CC,Chen MM,et al.Expression Analysis of Immune genes at different induction conditions of third instar larvae of Musca domestica[J].Biotechnol Bulletin,2014,(6):120-127.
[17]Fu P,Wu J,Gao S,et al.The recombinant expression and activity detection of MAF-1 fusion protein[J].Scientific Rep,2015,5(5):14716.
[18]国果,吴建伟,付萍.家蝇幼虫分泌型抗菌肽对大肠埃希菌抑制作用[J].中国公共卫生,2012,28(10):1318-1340.Guo G,Wu JW,Fu P.Anti-Escherichia coli effect of antibacterial peptides from Musca domestica larvae secretion[J].Chin J Public Health,2012,28(10):1318-1340.
[19]Jiangfan X,Tao W,Yu W,et al.Histological observation and expression patterns of antimicrobial peptides during fungal infection in musca domestica(Diptera:Muscidae)larvae[J].Braz Arch Biol Technol,2016,59(6):1-13.
[20]陶如玉,李妍,马慧玲,等.家蝇AMP17基因的克隆及其分子特性和表达模式研究[J].中国病原生物学杂志,2017,11:1079-1083.Tao RY,Li Y,Ma HL,et al.Bioinformatic analysis,Cloning and expression patterns of Musca domestica AMP17 gene[J].J Pathogen Biol,2017,11:1079-1083.
[21]Guo G,Tao R,Li Y,et al.Identification and characterization of a novel antimicrobial protein from the housefly Musca domestica[J].Biochem Biophy Res Commun,2017,490(3):746-752.
[22]金小宝,王艳,朱家勇,等.家蝇生长发育各阶段抗菌肽基因表达情况的研究[J].中国寄生虫学与寄生虫病杂志,2008,26(3):187-190.Jin XB,Wang Y,Zhu JY,et al.Expression analysis of antibacterial peptide genes at different development stages of Musca domestica[J].Chin J Parasitol Parasitic Dis,2008,26(3):187-190.
[23]王艳,金小宝,朱家勇,等.家蝇抗菌肽基因的表达模式研究[J].中国科学,2009,7:685-691.Wang Y,Jin XB,Zhu JY,et al.Study on the expression pattern of antibacterial peptide gene in Musca domestica[J].Sci China Press,2009,7:685-691.
[24]马晓娟,李亚明,胡翠美,等.家蚕免疫负调控分子BmFAF的功能[J].中国农业科学,2014,47(15):3085-3093.Ma XJ,Li YM,Hu CM,et al.Functional charaterization of BmFAFas an immune negative regulatory molecule in Silkworm(Bombyx mori)[J].Scientia Agricultura Sinica,2014,47(15):3085-3093.
[25]路永鹏,顾冀海,高一夫,等.肽聚糖识别蛋白PGRP-SC在家蝇肠道免疫中的功能分析[J].昆虫学报,2016,59(3):269-277.Lu YP,Gu JH,Gao YF,et al.Functional analysis of peptidoglycan recognition protein PGRP-SC in intestinal immunity of Musca domestica[J].Acta Entomologica Sinica,2016,59(3):269-277.
[26]王延稳,吕志强.家蚕肠道感染细菌后6种抗菌肽基因表达的变化[J].西北农林科技大学学报(自然科学版),2016,44(1):185-191.Wang YW,Lv ZQ.Expression of 6 antimicrobial peptide genes in silkworm,Bombyx mori after oral bacterial infection[J].J Northwest A&F Univ(Natural Science Edition),2016,44(1):185-191.
[27]Brennan CA,Anderson KV.Drosophila:the genetics of innate immune recognition and response[J].Ann Rev Immunol,2004,22(1):45.
[28]张杨,刘霁,杨杰,等.家蚕肽聚糖识别蛋白L1参与Imd信号通路[J].昆虫学报,2016,59(2):164-171.Zhang Y,Liu J,Yang J,et al.Peptidoglycan recognition protein L1 is Involved in the Imd pathway in the silkworm,Bombyx mori[J].Acta Entomologica Sinica,2016,59(2):164-171.
[29]魏川川,修江帆,胡亚,等.家蝇3种丝氨酸蛋白酶抑制剂(Serpin)基因的克隆、序列分析及表达模式[J].环境昆虫学报,2017,39(6):1334-1341.Wei CC,Xiu JF,Hu Y,et al.Cloning,sequence analysis and expression pattern of three serine protease inhibitors(Serpin)in Musca domestica[J].J Environmental Entomol,2017,39(6):1334-1341.
[30]肖海军,魏兆军,薛芳森.昆虫滞育关联热休克蛋白的研究进展[J].昆虫学报,2011,54(9):1068-1075.Xiao HJ,Wei ZJ,Xue FS.Progress in heat shock proteins(Hsps)related to insect diapause[J].Actas Entomologica Sinica,2011,54(9):1068-1075.
[31]Wu S,Zhang XF,He YQ,et al.Expression of antimicrobial peptide genes in Bombyx mori gut modulated by oral bacterial infection and development[J].Dev Comparative Immunol,2010,34(11):1191-1198.
[32]Boulanger N,Munks RJ,Hamilton JV,et al.Epithelial innate immunity.A novel antimicrobial peptide with antiparasitic activity in the blood-sucking insect Stomoxys calcitrans[J].J Biol Chem,2002,277(51):49921-49926.
[2]张福梅,李明生.抗菌肽在免疫防御和抗肿瘤中的作用[J].国外医药:抗生素分册,2017,38(6):284-288.Zhang FM,Li MS.Antibacterial peptides'roles in immune defense and anti-tumorogenesis[J].World Notes Antibiotics,2017,38(6):284-288.
[3]Cruciani RA,Barker JL,Zasloff M,et al.Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation[J].Proc Natl Acad Sci U S A,1991,88(9):3792.
[4]朱健,刘超群,刘瑞奇,等.抗菌肽免疫调节功能的研究进展[J].黑龙江畜牧兽医,2018,(1):77-79.Zhu J,Liu CQ,Liu RQ,et al.Research progress on immunomodulatory function of antimicrobial peptides[J].Heilongjiang Animal Sci Veterinary Med,2018,(1):77-79.
[5]王龙,冯群,高嘉敏,等.昆虫抗菌肽分类及在医学中应用[J].环境昆虫学报,2017,39(6):1387-1396.Wang L,Feng Q,Gao JM,et al.Classification of insect antibacterial peptides and its application in medicine[J].J Environmental Entomol,2017,39(6):1387-1396.
[6]Tonk M,Vilcinskas A.Themedical potential of antimicrobial peptides from insects[J].Curr Top Med Chem,2017,17(5):554-575.
[7]Boman HG,Nilsson-Faye I,PauI K.Insect immunity I.Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cynthia pupae[J].Infect Immun,1974,10(1):136-145.
[8]韩宝瑜.浅谈虫生真菌致病机制及昆虫活体防卫功能[J].生物安全学报,1997,3(1):110-111.Han BY.On the pathogenic mechanism of entomogenous fungi and the defense function of insects in vivo[J].Biosafety Newspaper,1997,3(1):110-111.
[9]Fullaondo A,Garcíasánchez S,Sanzparra A,et al.Spn1 regulates the GNBP3-dependent Toll signaling pathway in Drosophila melanogaster[J].Mol Cell Biol,2011,31(14):2960-2972.
[10]Lee H,Kwon HM,Park JW,et al.N-terminal GNBP homology domain of Gram-negative binding protein 3 functions as a beta-1,3-glucan binding motif in Tenebrio molitor[J].Bmb Reports,2009,42(8):506-510.
[11]Matskevich AA,Quintin J,Ferrandon D.The Drosophila PRRGNBP3 assembles effector complexes involved in antifungal defenses independently of its Toll pathway activation function[J].Eur J Immunol,2010,40(5):1244-1254.
[12]Iijima R,Kurata S,Natori S.Purification,characterization,and c DNA cloning of an antifungal protein from the hemolymph of Sarcophaga peregrina(flesh fly)larvae[J].J Biol Chem,1993,268(16):12055-12061.
[13]吕丁丁,侯成香,耿涛,等.家蚕感染球孢白僵菌后4种抗菌肽基因的表达分析[J].南方农业学报,2016,47(11):1952-1957.Lv DD,Hou CX,Geng T,et al.Expression of four antimicrobial peptide genes in Bombyx mori infected with Beauveria bassiana[J].J Southern Agriculture,2016,47(11):1952-1957.
[14]黄玉霞,耿涛,侯成香,等.白僵菌诱导家蚕抗菌肽enbocin1基因的表达特征及产物的抗真菌活性[J].蚕业科学,2016(4):619-626.Huang YX,Geng T,Hou CX,et al.Expression patterns of antimicrobial peptide gene enbocin1 in bombyx mori induced by Beauveria bassiana and its antifungal activity[J].Sci Sericulture,2016(4):619-626.
[15]杨雪,唐婷,王一丽,等.家蝇新型抗菌肽Muscin的基因克隆、表达模式及抑菌活性[J].昆虫学报,2015,58(6):617-624.Yang X,Tang T,Wang YL,et al.Gene cloning,expression profiling and anti-microbial assay of Muscin,a novel antmicrobial peptide in the housefly(Musca domestica)[J].Acta Entomologica Sinica,2015,58(6):617-624.
[16]修江帆,魏川川,陈明明,等.不同诱导条件下家蝇三龄幼虫免疫相关基因的表达研究[J].生物技术通报,2014,(6):120-127.Xiu JF,Wei CC,Chen MM,et al.Expression Analysis of Immune genes at different induction conditions of third instar larvae of Musca domestica[J].Biotechnol Bulletin,2014,(6):120-127.
[17]Fu P,Wu J,Gao S,et al.The recombinant expression and activity detection of MAF-1 fusion protein[J].Scientific Rep,2015,5(5):14716.
[18]国果,吴建伟,付萍.家蝇幼虫分泌型抗菌肽对大肠埃希菌抑制作用[J].中国公共卫生,2012,28(10):1318-1340.Guo G,Wu JW,Fu P.Anti-Escherichia coli effect of antibacterial peptides from Musca domestica larvae secretion[J].Chin J Public Health,2012,28(10):1318-1340.
[19]Jiangfan X,Tao W,Yu W,et al.Histological observation and expression patterns of antimicrobial peptides during fungal infection in musca domestica(Diptera:Muscidae)larvae[J].Braz Arch Biol Technol,2016,59(6):1-13.
[20]陶如玉,李妍,马慧玲,等.家蝇AMP17基因的克隆及其分子特性和表达模式研究[J].中国病原生物学杂志,2017,11:1079-1083.Tao RY,Li Y,Ma HL,et al.Bioinformatic analysis,Cloning and expression patterns of Musca domestica AMP17 gene[J].J Pathogen Biol,2017,11:1079-1083.
[21]Guo G,Tao R,Li Y,et al.Identification and characterization of a novel antimicrobial protein from the housefly Musca domestica[J].Biochem Biophy Res Commun,2017,490(3):746-752.
[22]金小宝,王艳,朱家勇,等.家蝇生长发育各阶段抗菌肽基因表达情况的研究[J].中国寄生虫学与寄生虫病杂志,2008,26(3):187-190.Jin XB,Wang Y,Zhu JY,et al.Expression analysis of antibacterial peptide genes at different development stages of Musca domestica[J].Chin J Parasitol Parasitic Dis,2008,26(3):187-190.
[23]王艳,金小宝,朱家勇,等.家蝇抗菌肽基因的表达模式研究[J].中国科学,2009,7:685-691.Wang Y,Jin XB,Zhu JY,et al.Study on the expression pattern of antibacterial peptide gene in Musca domestica[J].Sci China Press,2009,7:685-691.
[24]马晓娟,李亚明,胡翠美,等.家蚕免疫负调控分子BmFAF的功能[J].中国农业科学,2014,47(15):3085-3093.Ma XJ,Li YM,Hu CM,et al.Functional charaterization of BmFAFas an immune negative regulatory molecule in Silkworm(Bombyx mori)[J].Scientia Agricultura Sinica,2014,47(15):3085-3093.
[25]路永鹏,顾冀海,高一夫,等.肽聚糖识别蛋白PGRP-SC在家蝇肠道免疫中的功能分析[J].昆虫学报,2016,59(3):269-277.Lu YP,Gu JH,Gao YF,et al.Functional analysis of peptidoglycan recognition protein PGRP-SC in intestinal immunity of Musca domestica[J].Acta Entomologica Sinica,2016,59(3):269-277.
[26]王延稳,吕志强.家蚕肠道感染细菌后6种抗菌肽基因表达的变化[J].西北农林科技大学学报(自然科学版),2016,44(1):185-191.Wang YW,Lv ZQ.Expression of 6 antimicrobial peptide genes in silkworm,Bombyx mori after oral bacterial infection[J].J Northwest A&F Univ(Natural Science Edition),2016,44(1):185-191.
[27]Brennan CA,Anderson KV.Drosophila:the genetics of innate immune recognition and response[J].Ann Rev Immunol,2004,22(1):45.
[28]张杨,刘霁,杨杰,等.家蚕肽聚糖识别蛋白L1参与Imd信号通路[J].昆虫学报,2016,59(2):164-171.Zhang Y,Liu J,Yang J,et al.Peptidoglycan recognition protein L1 is Involved in the Imd pathway in the silkworm,Bombyx mori[J].Acta Entomologica Sinica,2016,59(2):164-171.
[29]魏川川,修江帆,胡亚,等.家蝇3种丝氨酸蛋白酶抑制剂(Serpin)基因的克隆、序列分析及表达模式[J].环境昆虫学报,2017,39(6):1334-1341.Wei CC,Xiu JF,Hu Y,et al.Cloning,sequence analysis and expression pattern of three serine protease inhibitors(Serpin)in Musca domestica[J].J Environmental Entomol,2017,39(6):1334-1341.
[30]肖海军,魏兆军,薛芳森.昆虫滞育关联热休克蛋白的研究进展[J].昆虫学报,2011,54(9):1068-1075.Xiao HJ,Wei ZJ,Xue FS.Progress in heat shock proteins(Hsps)related to insect diapause[J].Actas Entomologica Sinica,2011,54(9):1068-1075.
[31]Wu S,Zhang XF,He YQ,et al.Expression of antimicrobial peptide genes in Bombyx mori gut modulated by oral bacterial infection and development[J].Dev Comparative Immunol,2010,34(11):1191-1198.
[32]Boulanger N,Munks RJ,Hamilton JV,et al.Epithelial innate immunity.A novel antimicrobial peptide with antiparasitic activity in the blood-sucking insect Stomoxys calcitrans[J].J Biol Chem,2002,277(51):49921-49926.