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Position: Home > Articles > Cloning and Expression Analysis of β-Defensin Gene from Russian Sturgeon (Acipenser gueldenstaedti) Progress in Fishery Sciences 2018 (5) 20-26

俄罗斯鲟β-防御素基因的克隆及表达分析

作  者：
白莉;陈亚东;夏永涛;许式见;胡谋;沙珍霞
单  位：
青岛大学生命科学学院;农业农村部海洋渔业可持续发展重点实验室中国水产科学研究院黄海水产研究所;杭州千岛湖鲟龙科技股份有限公司;大连海洋大学水产与生命学院
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
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。
译  名：
Cloning and Expression Analysis of β-Defensin Gene from Russian Sturgeon (Acipenser gueldenstaedti)
作  者：
BAI Li;CHEN Yadong;XIA Yongtao;XU Shijian;HU Mou;SHA Zhenxia;College of Fisheries and Life Science, Dalian Ocean University;Key Laboratory for Sustainable Development of Marine Fisheries,Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;College of Life Sciences,Qingdao University;Hangzhou Qiandaohu Xunlong Sci-Tech Co.Ltd.;
关键词：
Acipenser gueldenstaedti;;β-Defensin;;Gene cloning;;Gene expression;;Aeromonas hydrophila
摘  要：
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.

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