俄罗斯鲟β-防御素基因的克隆及表达分析

Cloning and Expression Analysis of β-Defensin Gene from Russian Sturgeon (Acipenser gueldenstaedti)

中文关键词: 俄罗斯鲟  β-防御素  基因克隆  基因表达  嗜水气单胞菌

英文关键词: Acipenser gueldenstaedti  β-Defensin  Gene cloning  Gene expression  Aeromonas hydrophila

中文摘要:

      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cDNA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。

英文摘要:

      β-defensins are cysteine, cationic, antibacterial peptides that play very important roles in the vertebrate immune system. In this study, the cDNA 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 qRT-PCR. The results showed that cDNA 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%. qRT-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.