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俄罗斯鲟β-防御素基因的克隆及表达分析
白 莉1,2, 陈亚东3,2, 夏永涛4, 许式见4, 胡 谋4, 沙珍霞3
1.大连海洋大学水产与生命学院 大连 116023;2.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 青岛 266071;3.青岛大学生命科学学院 青岛 266071;4.杭州千岛湖鲟龙科技股份有限公司 杭州 311701
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。
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
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基金项目:
Cloning and Expression Analysis of β-Defensin Gene from Russian Sturgeon (Acipenser gueldenstaedti)
BAI Li1,2, CHEN Yadong3,2, XIA Yongtao4, XU Shijian4, HU Mou4, SHA Zhenxia3
1.College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023;2.Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.College of Life Sciences,Qingdao University, Qingdao 266071;4.Hangzhou Qiandaohu Xunlong Sci-Tech Co. Ltd. Hangzhou 311701
Abstract:
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.
Key words:  Acipenser gueldenstaedti  β-Defensin  Gene cloning  Gene expression  Aeromonas hydrophila