斑节对虾Pellino基因的克隆及其在不同胁迫条件下的表达分析
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摘要
Pellino蛋白家族,是一类高度保守的E3泛素连接酶,在泛素化和先天性免疫中发挥重要作用。该研究通过RACE技术得到斑节对虾(Penaeus monodon)泛素连接酶Pellino基因的c DNA全长。该基因序列全长1 961 bp,编码区序列长1 299 bp,编码432个氨基酸,5'非编码区(UTR)为89 bp,3'UTR为573 bp。通过qRT-PCR技术,研究了PmPellino基因在斑节对虾不同组织中的表达水平,并研究了其在不同浓度氨氮胁迫下和不同微生物刺激下的表达情况。结果显示,PmPellino基因在各组织中均有表达,在鳃组织中表达量最高。急性氮氮胁迫后,PmPellino在肝胰腺中的表达量显著上调(P<0.01),但在鳃中的表达被抑制(P<0.01)。哈维氏弧菌(Vibrio harveyi)可显著激活PmPellino在鳃中的表达,抑制其在肝胰腺中的表达。鳗弧菌(V. anguillarum)可显著抑制PmPellino在肝胰腺中的表达,而对PmPellino在鳃中的表达无显著影响。金黄色葡萄球菌(Staphylococcus aureus)可以显著激活PmPellino在肝胰腺和鳃中的表达。结果表明PmPellino可以激活免疫应答通路,在免疫防御中发挥重要作用。
Pellino protein is a member of highly conserved E3 ubiquitin ligase which plays an important role in ubiquitination and innate immunity. In this study, we cloned and identi?ed a crustacean Pellino from Penaeus monodon(PmPellino). The full-length cDNA of PmPellino consisted of 1 961 bp with an 89 bp 5'UTR, a 573 bp 3'UTR and a 1 299 bp open reading frame encoding 432 amino acids. PmPellino mRNA was detected in all the tissues examined by real time PCR(highest in gill). The expression of PmPellino in hepatopancreas was significantly up-regulated after acute ammonia nitrogen stress(P<0.01), but was inhibited in gill(P<0.01). Vibrio harveyi could significantly activate the expression of PmPellino in gill and inhibit its expression in hepatopancreas.V. anguillarum could significantly inhibit the expression of PmPellino in hepatopancreas, but there was no sigficant difference in gill with the control group. Staphylococcus aureus could significantly activate the expression of PmPellino in both hepatopancreas and gill. The results indicate that PmPellino might play an important role in the immunity of P. monodon.
Pellino protein is a member of highly conserved E3 ubiquitin ligase which plays an important role in ubiquitination and innate immunity. In this study, we cloned and identi?ed a crustacean Pellino from Penaeus monodon(PmPellino). The full-length cDNA of PmPellino consisted of 1 961 bp with an 89 bp 5'UTR, a 573 bp 3'UTR and a 1 299 bp open reading frame encoding 432 amino acids. PmPellino mRNA was detected in all the tissues examined by real time PCR(highest in gill). The expression of PmPellino in hepatopancreas was significantly up-regulated after acute ammonia nitrogen stress(P<0.01), but was inhibited in gill(P<0.01). Vibrio harveyi could significantly activate the expression of PmPellino in gill and inhibit its expression in hepatopancreas.V. anguillarum could significantly inhibit the expression of PmPellino in hepatopancreas, but there was no sigficant difference in gill with the control group. Staphylococcus aureus could significantly activate the expression of PmPellino in both hepatopancreas and gill. The results indicate that PmPellino might play an important role in the immunity of P. monodon.
引文
[1]SCHAUVLIEGE R,JANSSENS S,BEYAERT R.Pellino proteins:novel players in TLR and IL-1R signalling[J].J Cell Mol Med,2007,11(3):453-461.
[2]GROSSHANS J,SCHNORRER F,NUSSLEIN-VOLHARD C.Oligomerisation of Tube and Pelle leads to nuclear localisation of Dorsal[J].Mech Dev,1999,81(1/2):127-138.
[3]陈方如,郝飞.泛素连接酶Pellino1蛋白研究进展[J].免疫学杂志,2015(7):633-636.
[4]MOYNAGH P N.The Pellino family:IRAK E3 ligases with emerging roles in innate immune signalling[J].Trends Immunol,2009,30(1):33-42.
[5]JIANG Z,JOHNSON H J,NIE H,et al.Pellino 1 is required for interleukin-1(IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4(IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6(TRAF6)complex[J].J Biol Chem,2003,278(13):10952-10956.
[6]BUTLER M P,HANLY J A,MOYNAGH P N.Pellino3 is a novel upstream regulator of p38 MAPK and activates CREB in a p38-dependent manner[J].J Biol Chem,2005,280(30):27759-27768.
[7]XIAO H,QIAN W,STASCHKE K,et al.Pellino 3b negatively regulates interleukin-1-induced TAK1-dependent NFκB activation[J].J Biol Chem,2008,283(21):14654-14664.
[8]LI C Z,CHAI J T,LI H Y,et al.Pellino protein from Pacific white shrimp Litopenaeus vannamei positively regulates NF-kappa B activation[J].Dev Comp Immunol,2014,44(2):341-350.
[9]RAVICHANDRAN G,KUMARESAN V,ARASU M V,et al.Pellino-1 derived cationic antimicrobial prawn peptide:bactericidal activity,toxicity and mode of action[J].Mol Immunol,2016,78:171-182.
[10]韩坤煌,张子平,王艺磊,等.Cyclin-CDK-CKI及UPP参与生殖调控及在甲壳动物性腺发育中的研究进展[J].生物技术通报,2010(7):48-54.
[11]STRICKLAND E,HAKALA K,THOMAS P J,et al.Recognition of misfolding proteins by PA700,the regulatory subcomplex of the 26 S proteasome[J].J Biol Chem,2000,275(8):5565-5572.
[12]VIDYA R,GIREESH-BABU P,PANI PRASAD K.White spot syndrome virus manipulates ubiquitin gene expression in Penaeus monodon[J].Indian J Virol,2013,24(1):82-84.
[13]SUDHAKARAN R,OKUGAWA S,MEKATA T,et al.Deciphering the DNA repair protein,Rad23 from kuruma shrimp Marsupenaeus japonicus:full-length cDNA cloning and characterization[J].Lett Appl Microbiol,2011,53(1):63-72.
[14]CESAR J R,YANG J.Expression patterns of ubiquitin,heat shock protein 70,α-actin andβ-actin over the molt cycle in the abdominal muscle of marine shrimp Litopenaeus vannamei[J].Mol Reprod Dev,2007,74(5):554-559.
[15]PREECHAPHOL R,LEELATANAWIT R,SITTIKANKEAWK,et al.Expressed sequence tag analysis for identification and characterization of sex-related genes in the giant tiger shrimp Penaeus monodon[J].J Biochem Mol Biol,2007,40(4):501-510.
[16]周发林,陈劲松,黄建华,等.斑节对虾谷氨酸脱氢酶基因克隆及氨氮胁迫对其时空表达的影响[J].中国水产科学,2016,23(6):1236-1246.
[17]LI F,XIANG J.Recent advances in researches on the innate immunity of shrimp in China[J].Dev Comp Immunol,2012,39(1/2):11-26.
[18]黄建华,李永,杨其彬,等.斑节对虾家系氨氮耐受性的比较[J].南方水产科学,2012,8(6):37-43.
[19]KOO J G,KIM S G,JEE J H,et al.Effects of ammonia and nitrite on survival,growth and moulting in juvenile tiger crab,Orithyia sinica(Linnaeus)[J].Aquacult Res,2005,36:79-85.
[20]郑微云,翁恩琪.环境毒理学概论[M].厦门:厦门大学出版社,1993:57-60.
[21]SCHAUVLIEGE R,JANSSENS S,BEYAERT R.Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling:a role as novel RING E3-ubiquitin-ligases[J].FEBS Lett,2006,580(19):4697-4702.
[22]CIECHANOVER A,BEN-SAADON R.N-terminal ubiquitination:more protein substrates join in[J].Trends Cell Biol,2004,14(3):103-106.
[23]张译尹,李培志,廖锐,等.过表达pellino-1在Kupffer细胞内毒素耐受时对TRAF3泛素化及MAPK信号通路的调控[J].中山大学学报(医学科学版),2017,38(5):658-664.
[24]李永.斑节对虾人工选育及氨氮对其免疫解毒代谢影响[D].上海:上海海洋大学,2012:9.
[25]HONG M L,CHEN L Q,SUN X J,et al.Metabolic and immune responses in Chinese mitten-handed crab(Eriocheir sinensis)juveniles exposed to elevated ambient ammonia[J].Comp Biochem Physiol C,2007,145(3):363-369.
[26]DEEPIKA A,SREEDHARAN K,PARIA A,et al.Toll-pathway in tiger shrimp(Penaeus monodon)responds to white spot syndrome virus infection:evidence through molecular characterisation and expression profiles of MyD88,TRAF6 and TLRgenes[J].Fish Shellfish Immunol,2014,41(2):441-454.
[27]WALTON D J.Stereochemistry of reduction of D-glyceraldehyde catalyzed by a nicotinamide adenine dinucleotide phosphate dependent dehydrogenase from skeletal muscle[J].Biochemistry,1973,12(18):3472-3478.
[28]陈劲松,周发林,江世贵,等.斑节对虾谷氨酰胺合成酶基因的克隆及氨氮胁迫对其时空表达的影响[J].上海海洋大学学报,2016,25(4):497-507.
[29]曾媛媛,陈曦飞,艾春香,等.氨氮胁迫对拟穴青蟹血淋巴生理生化指标及鳃和肝胰腺显微结构的影响[C]//中国甲壳动物学会第十一届年会暨学术研讨会论文摘要集.青岛:中国海洋湖沼学会,2011:107.
[30]LI C Z,CHEN Y X,WENG S P,et al.Presence of Tube isoforms in Litopenaeus vannamei suggests various regulatory patterns of signal transduction in invertebrate NF-κB pathway[J].Dev Comp Immunol,2014,42(2):174-185.
[31]JANG I H,CHOSA N,KIM S H,et al.A sp?tzle-processing enzyme required for toll signaling activation in Drosophila innate immunity[J].Dev Cell,2006,10(1):45-55.
[32]ZHANG S,LI C Z,YAN H,et al.Identification and function of myeloid differentiation factor 88(MyD88)in Litopenaeus vannamei[J].PLoS One,2012,7(10):e47038.
[33]HO S H,SONG Y L.Cloning of penaeidin gene promoter in tiger shrimp(Penaeus monodon)[J].Fish Shellfish Immunol,2009,27(1):73-77.
[34]韩明.果蝇先天性免疫Toll与Imd信号通路相关基因进化机制研究[D].南京:南京师范大学,2014:4-6.
[2]GROSSHANS J,SCHNORRER F,NUSSLEIN-VOLHARD C.Oligomerisation of Tube and Pelle leads to nuclear localisation of Dorsal[J].Mech Dev,1999,81(1/2):127-138.
[3]陈方如,郝飞.泛素连接酶Pellino1蛋白研究进展[J].免疫学杂志,2015(7):633-636.
[4]MOYNAGH P N.The Pellino family:IRAK E3 ligases with emerging roles in innate immune signalling[J].Trends Immunol,2009,30(1):33-42.
[5]JIANG Z,JOHNSON H J,NIE H,et al.Pellino 1 is required for interleukin-1(IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4(IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6(TRAF6)complex[J].J Biol Chem,2003,278(13):10952-10956.
[6]BUTLER M P,HANLY J A,MOYNAGH P N.Pellino3 is a novel upstream regulator of p38 MAPK and activates CREB in a p38-dependent manner[J].J Biol Chem,2005,280(30):27759-27768.
[7]XIAO H,QIAN W,STASCHKE K,et al.Pellino 3b negatively regulates interleukin-1-induced TAK1-dependent NFκB activation[J].J Biol Chem,2008,283(21):14654-14664.
[8]LI C Z,CHAI J T,LI H Y,et al.Pellino protein from Pacific white shrimp Litopenaeus vannamei positively regulates NF-kappa B activation[J].Dev Comp Immunol,2014,44(2):341-350.
[9]RAVICHANDRAN G,KUMARESAN V,ARASU M V,et al.Pellino-1 derived cationic antimicrobial prawn peptide:bactericidal activity,toxicity and mode of action[J].Mol Immunol,2016,78:171-182.
[10]韩坤煌,张子平,王艺磊,等.Cyclin-CDK-CKI及UPP参与生殖调控及在甲壳动物性腺发育中的研究进展[J].生物技术通报,2010(7):48-54.
[11]STRICKLAND E,HAKALA K,THOMAS P J,et al.Recognition of misfolding proteins by PA700,the regulatory subcomplex of the 26 S proteasome[J].J Biol Chem,2000,275(8):5565-5572.
[12]VIDYA R,GIREESH-BABU P,PANI PRASAD K.White spot syndrome virus manipulates ubiquitin gene expression in Penaeus monodon[J].Indian J Virol,2013,24(1):82-84.
[13]SUDHAKARAN R,OKUGAWA S,MEKATA T,et al.Deciphering the DNA repair protein,Rad23 from kuruma shrimp Marsupenaeus japonicus:full-length cDNA cloning and characterization[J].Lett Appl Microbiol,2011,53(1):63-72.
[14]CESAR J R,YANG J.Expression patterns of ubiquitin,heat shock protein 70,α-actin andβ-actin over the molt cycle in the abdominal muscle of marine shrimp Litopenaeus vannamei[J].Mol Reprod Dev,2007,74(5):554-559.
[15]PREECHAPHOL R,LEELATANAWIT R,SITTIKANKEAWK,et al.Expressed sequence tag analysis for identification and characterization of sex-related genes in the giant tiger shrimp Penaeus monodon[J].J Biochem Mol Biol,2007,40(4):501-510.
[16]周发林,陈劲松,黄建华,等.斑节对虾谷氨酸脱氢酶基因克隆及氨氮胁迫对其时空表达的影响[J].中国水产科学,2016,23(6):1236-1246.
[17]LI F,XIANG J.Recent advances in researches on the innate immunity of shrimp in China[J].Dev Comp Immunol,2012,39(1/2):11-26.
[18]黄建华,李永,杨其彬,等.斑节对虾家系氨氮耐受性的比较[J].南方水产科学,2012,8(6):37-43.
[19]KOO J G,KIM S G,JEE J H,et al.Effects of ammonia and nitrite on survival,growth and moulting in juvenile tiger crab,Orithyia sinica(Linnaeus)[J].Aquacult Res,2005,36:79-85.
[20]郑微云,翁恩琪.环境毒理学概论[M].厦门:厦门大学出版社,1993:57-60.
[21]SCHAUVLIEGE R,JANSSENS S,BEYAERT R.Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling:a role as novel RING E3-ubiquitin-ligases[J].FEBS Lett,2006,580(19):4697-4702.
[22]CIECHANOVER A,BEN-SAADON R.N-terminal ubiquitination:more protein substrates join in[J].Trends Cell Biol,2004,14(3):103-106.
[23]张译尹,李培志,廖锐,等.过表达pellino-1在Kupffer细胞内毒素耐受时对TRAF3泛素化及MAPK信号通路的调控[J].中山大学学报(医学科学版),2017,38(5):658-664.
[24]李永.斑节对虾人工选育及氨氮对其免疫解毒代谢影响[D].上海:上海海洋大学,2012:9.
[25]HONG M L,CHEN L Q,SUN X J,et al.Metabolic and immune responses in Chinese mitten-handed crab(Eriocheir sinensis)juveniles exposed to elevated ambient ammonia[J].Comp Biochem Physiol C,2007,145(3):363-369.
[26]DEEPIKA A,SREEDHARAN K,PARIA A,et al.Toll-pathway in tiger shrimp(Penaeus monodon)responds to white spot syndrome virus infection:evidence through molecular characterisation and expression profiles of MyD88,TRAF6 and TLRgenes[J].Fish Shellfish Immunol,2014,41(2):441-454.
[27]WALTON D J.Stereochemistry of reduction of D-glyceraldehyde catalyzed by a nicotinamide adenine dinucleotide phosphate dependent dehydrogenase from skeletal muscle[J].Biochemistry,1973,12(18):3472-3478.
[28]陈劲松,周发林,江世贵,等.斑节对虾谷氨酰胺合成酶基因的克隆及氨氮胁迫对其时空表达的影响[J].上海海洋大学学报,2016,25(4):497-507.
[29]曾媛媛,陈曦飞,艾春香,等.氨氮胁迫对拟穴青蟹血淋巴生理生化指标及鳃和肝胰腺显微结构的影响[C]//中国甲壳动物学会第十一届年会暨学术研讨会论文摘要集.青岛:中国海洋湖沼学会,2011:107.
[30]LI C Z,CHEN Y X,WENG S P,et al.Presence of Tube isoforms in Litopenaeus vannamei suggests various regulatory patterns of signal transduction in invertebrate NF-κB pathway[J].Dev Comp Immunol,2014,42(2):174-185.
[31]JANG I H,CHOSA N,KIM S H,et al.A sp?tzle-processing enzyme required for toll signaling activation in Drosophila innate immunity[J].Dev Cell,2006,10(1):45-55.
[32]ZHANG S,LI C Z,YAN H,et al.Identification and function of myeloid differentiation factor 88(MyD88)in Litopenaeus vannamei[J].PLoS One,2012,7(10):e47038.
[33]HO S H,SONG Y L.Cloning of penaeidin gene promoter in tiger shrimp(Penaeus monodon)[J].Fish Shellfish Immunol,2009,27(1):73-77.
[34]韩明.果蝇先天性免疫Toll与Imd信号通路相关基因进化机制研究[D].南京:南京师范大学,2014:4-6.