俄罗斯鲟β-防御素基因的克隆及表达分析
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摘要
β-防御素是一类富含半胱氨酸的阳离子抗菌肽,在脊椎动物免疫系统中发挥重要作用。本研究基于转录组Solexa测序结果,利用PCR和荧光定量PCR技术对俄罗斯鲟(Acipenser gueldenstaedti)β-防御素基因进行了克隆和表达模式分析。结果显示,俄罗斯鲟β-防御素c DNA片段长度为333 bp,包含开放阅读框(Open reading frame,ORF)213 bp,推测编码71个氨基酸;SMART分析显示,该基因包含23个氨基酸的信号肽、1个防御素-β-2结构域和6个保守的半胱氨酸残基,此结构与其他物种极为相似,在进化中比较保守。同源比对发现,俄罗斯鲟β-防御素与鱼类的相似度最高,为54%~60%。β-防御素基因在健康俄罗斯鲟11种组织(肝、肠、脾、头肾、胃、鳃、血液、脑、皮肤、肌肉和性腺)中均有表达,其中,在性腺和皮肤中表达量最高;病原菌嗜水气单胞菌(Aeromonas hydrophila)感染后可强烈影响β-防御素基因在6种免疫组织(肝、肠、脾、头肾、血液和鳃)中的时空表达。其中,在头肾中的上调表达最明显,在感染后72 h为表达峰值,达到0 h表达量的700倍。在脾、血液和肠中均有几倍至十几倍的上调,而在肝中整体呈现下调表达趋势。研究表明,β-防御素基因参与了俄罗斯鲟的免疫应答过程,本结果为俄罗斯鲟的免疫调控和病害防治研究奠定了基础。
β-defensins are cysteine, cationic, antibacterial peptides that play very important roles in the vertebrate immune system. In this study, the c DNA fragment of β-defensin gene from Acipenser gueldenstaedti was cloned using a PCR method based on the Solexa sequencing results of gonad transcriptome, and gene expression was performed by q RT-PCR. The results showed that c DNA of β-defensin was 333 bp in length and contained an open reading frame(ORF) with 213 bp, which was deduced to encode a precursor peptide of 71 amino acids consisting of a signal peptide of 23 amino acid residues and a mature peptide of 48 amino acid residues. Russian sturgeon β-defensin possessed six conserved cysteine residues, forming three disulfide bridges at C38~C66, C44~C60 and C48~C67, and containing 1 defensin-β-2 domain. This structure is very similar to other species and conserved in evolution. Homologous alignment revealed that Russian sturgeon β-defensin had the highest sequence identity with fish species, about 54%~60%. q RT-PCR analysis showed the β-defensin transcript is constitutively expressed in 11 tissues(liver, intestine, spleen, head-kidney, gill, blood, brain, skin, muscle and gonad, stomach) in healthy fish, with highest expression in the gonads and skin. Artificial infection of A. gueldenstaedti with Aeromonas hydrophila resulted in a significantly upregulated expression in the intestine, spleen, head kidney, blood, and gill while downregulating expression in the liver. The most significant upregulated expression and the peak level at 72 h reached 700 times baseline in the head-kidney. The results indicate Acipenser gueldenstaedti β-defensin genes are involved in the immune response.
β-defensins are cysteine, cationic, antibacterial peptides that play very important roles in the vertebrate immune system. In this study, the c DNA fragment of β-defensin gene from Acipenser gueldenstaedti was cloned using a PCR method based on the Solexa sequencing results of gonad transcriptome, and gene expression was performed by q RT-PCR. The results showed that c DNA of β-defensin was 333 bp in length and contained an open reading frame(ORF) with 213 bp, which was deduced to encode a precursor peptide of 71 amino acids consisting of a signal peptide of 23 amino acid residues and a mature peptide of 48 amino acid residues. Russian sturgeon β-defensin possessed six conserved cysteine residues, forming three disulfide bridges at C38~C66, C44~C60 and C48~C67, and containing 1 defensin-β-2 domain. This structure is very similar to other species and conserved in evolution. Homologous alignment revealed that Russian sturgeon β-defensin had the highest sequence identity with fish species, about 54%~60%. q RT-PCR analysis showed the β-defensin transcript is constitutively expressed in 11 tissues(liver, intestine, spleen, head-kidney, gill, blood, brain, skin, muscle and gonad, stomach) in healthy fish, with highest expression in the gonads and skin. Artificial infection of A. gueldenstaedti with Aeromonas hydrophila resulted in a significantly upregulated expression in the intestine, spleen, head kidney, blood, and gill while downregulating expression in the liver. The most significant upregulated expression and the peak level at 72 h reached 700 times baseline in the head-kidney. The results indicate Acipenser gueldenstaedti β-defensin genes are involved in the immune response.
引文
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Qi Z,Xu W,Meng F,et al.Cloning and expression ofβ-defensin from soiny mullet(Liza haematocheila),with insights of its antibacterial mechanism.PLoS One,2016,11(6):e0157544
Ruangsri J,Kitani Y,Kiron V,et al.A novel beta-defensin antimicrobial peptide in Atlantic cod with stimulatory effect on phagocytic activity.PLoS One,2013,8(4):e62302
Yang ZG.Isolation and identification of Aeromonas hydrophila from sturgeons.Freshwater Fisheries,2001,31(5):43-44[杨治国.鲟鱼嗜水气单胞菌的分离鉴定.淡水渔业,2001,31(5):40-41]
Yin HB,Sun ZW,Sun DJ et al.Cytogenetic analysis of Acipenser gueldenstaedti Brandt.Journal of Fisheries of China,2006,30(2):181-184[尹洪滨,孙中武,孙大江,等.俄罗斯鲟的细胞遗传学分析.水产学报,2006,30(2):181-184]
Yu MJ,Sun LM,Chen YD et al.Cloning and expression of the c-Jun gene in immune response to pathogen challenge in half-smooth tongue sole(Cynoglossus semilaevis).Progress in Fisheries Science,2017,38(2):31-39[于孟君,孙璐明,陈亚东,等.半滑舌鳎(Cynoglossus semilaevis)c-Jun基因的克隆及免疫应答分析.渔业科学进展,2017,38(2):31-39]
Zhu S,Gao B.Evolutionary origin of beta-defensins.Developmental and Comparative Immunology,2013,39:79-84
Zou J,Mercier C,Koussounadis A,et al.Discovery of multiple beta-defensin like homologues in teleost fish.Molecular Immunology,2007,44(4):638-647
Zhu J,Wang H,Wang J,et al.Identification and characterization of aβ-defensin gene involved in the immune defense response of channel catfish,Ictalurus punctatus.Molecular Immunology,2017,85:256
Zhang W,Zhu LQ,Zhao FL,et al.Prongress onβ-defensin.Progress in Veterinary Medicine,2006,27(5):47-50[张伟,朱连勤,赵风立,等.β-防御素研究进展.动物医学进展,2006,27(5):47-50]
Casadei E,Wang T,Zou J,et al.Characterization of three novelβ-defensin antimicrobial peptides in rainbow trout(Oncorhynchus mykiss).Molecular Immunology,2009,46(16):3358-3366
Chen Y,Xia Y,Shao C,et al.Discovery and identification of candidate sex-related genes based on transcriptome sequencing of Russian sturgeon(Acipenser gueldenstaedtii)gonads.Physiological Genomics,2016,48(7):464
Dong JJ,Wu F,Ye X,et al.Β-defensin in Nile tilapia(Oreochromis niloticus):Sequence,tissue expression,and anti-bacterial activity of synthetic peptides.Gene,2015,566(1):23-31
Ellis AE.Innate host defense mechanisms of fish against viruses and bacteria.Developmental&Comparative immunology,2001,25(8-9):827-839
Guo M,Wei J,Huang X,et al.Antiviral effects ofβ-defensin derived from orange-spotted grouper(Epinephelus coioides).Fish&Shellfish Immunology,2012,32(5):828-838
Jin JY,Zhou L,Wang Y,et al.Antibacterial and antiviral roles of a fishβ-defensin expressed both in pituitary and testis.PLoSOne,2010,5(12):e12883
Liang T,Wang DD,Zhang GR,et al.Molecular cloning and expression analysis of two beta-defensin genes in the blunt snout bream(Megalobrama amblycephala).Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology,2013,166(1):91-98
Lu Y,Wang QL,Li M,et al.Expression analysis of TLR20 and TLR21 genes in channel catfish Ictalurus punctatus challenged by different bacteria and virus.Progress in Fisheries Science,2013,34(6):44-51[路飏,王启龙,李敏,等.斑点叉尾TLR20和TLR21基因在不同细菌和病毒感染后的表达特征.渔业科学进展,2013,34(6):44-51]
Qi Z,Xu W,Meng F,et al.Cloning and expression ofβ-defensin from soiny mullet(Liza haematocheila),with insights of its antibacterial mechanism.PLoS One,2016,11(6):e0157544
Ruangsri J,Kitani Y,Kiron V,et al.A novel beta-defensin antimicrobial peptide in Atlantic cod with stimulatory effect on phagocytic activity.PLoS One,2013,8(4):e62302
Yang ZG.Isolation and identification of Aeromonas hydrophila from sturgeons.Freshwater Fisheries,2001,31(5):43-44[杨治国.鲟鱼嗜水气单胞菌的分离鉴定.淡水渔业,2001,31(5):40-41]
Yin HB,Sun ZW,Sun DJ et al.Cytogenetic analysis of Acipenser gueldenstaedti Brandt.Journal of Fisheries of China,2006,30(2):181-184[尹洪滨,孙中武,孙大江,等.俄罗斯鲟的细胞遗传学分析.水产学报,2006,30(2):181-184]
Yu MJ,Sun LM,Chen YD et al.Cloning and expression of the c-Jun gene in immune response to pathogen challenge in half-smooth tongue sole(Cynoglossus semilaevis).Progress in Fisheries Science,2017,38(2):31-39[于孟君,孙璐明,陈亚东,等.半滑舌鳎(Cynoglossus semilaevis)c-Jun基因的克隆及免疫应答分析.渔业科学进展,2017,38(2):31-39]
Zhu S,Gao B.Evolutionary origin of beta-defensins.Developmental and Comparative Immunology,2013,39:79-84
Zou J,Mercier C,Koussounadis A,et al.Discovery of multiple beta-defensin like homologues in teleost fish.Molecular Immunology,2007,44(4):638-647
Zhu J,Wang H,Wang J,et al.Identification and characterization of aβ-defensin gene involved in the immune defense response of channel catfish,Ictalurus punctatus.Molecular Immunology,2017,85:256
Zhang W,Zhu LQ,Zhao FL,et al.Prongress onβ-defensin.Progress in Veterinary Medicine,2006,27(5):47-50[张伟,朱连勤,赵风立,等.β-防御素研究进展.动物医学进展,2006,27(5):47-50]