黄颡鱼抗菌肽Hepcidin基因的克隆和表达分析  

Molecular Cloning of an Antimicrobial Peptide Hepcidin Gene from Yellow Catfish (Pelteobagrus fulvidraco) and Its Expression Analysis

作　　者：沈文英[1,2] 李卫芬[1] 雷凯[1] 吴兵兵[1] 祝尧荣[2] 

机构地区：[1]浙江大学饲料科学研究所,教育部动物分子营养学重点实验室,杭州310029 [2]绍兴文理学院生命科学学院,绍兴312000

出　　处：《农业生物技术学报》2009年第6期972-978,共7页Journal of Agricultural Biotechnology

基　　金：浙江省重点科技项目(No.:G20060837)资助

摘　　要：用RT-PCR方法,从黄颡鱼(Pelteobagrus fulvidraco)肝脏中克隆获得了Hepcidin(heppatic bactericidal protein)cDNA序列,并对其进行序列测定,分析了Hepcidin基因在不同组织中的表达差异和不同细菌感染后对其表达的影响。结果表明,黄颡鱼Hepcidin cDNA片段(GenBank登录号:EU257703)长为282bp,编码93个氨基酸,由信号肽(23个氨基酸)、前肽(45个氨基酸)和成熟肽(25个氨基酸)组成,成熟肽含有8个保守的半胱氨酸(Cys)残基,可形成4个链内二硫键。与已报道的其它鲶形目鱼类Hepcidin核苷酸序列的同源性为80%～82%,推导的氨基酸序列的同源性为69%～71%。RT-PCR半定量分析显示,黄颡鱼Hepcidin基因在不同组织中普遍表达,其中肝脏表达水平最高,头肾、肠道、肌肉、脑、胃、鳃、心脏、卵巢和肾脏表达次之,皮肤和脾脏表达较低。感染嗜水单胞菌(Aeromonas hydrophila)和腊样芽胞杆菌(Bacillus cereus)的黄颡鱼,肝脏Hepcidin mR-NA水平显著升高(P<0.05)。Antimicrobial peptides （AMPs） are regarded as important components of the host innate immune system and play crucial roles in host defense against microbial invasion. Hepcidin（heppatic bactericidal protein） is an antimicrobial peptide and iron regulatory molecule primarily in liver. One Hepcidin gene was amplified from the liver of yellow catfish （Pelteobagrus fulvidraco） by RT-PCR. The length ofHepcidin cDNA fragment （GenBank accession No. EU257703 ） was 282 bp with an ORF ranging from 1st to 279th bp, which encoded a peptide of 93 amino acids. According to the Blast result in GenBank database, the cDNA nucleotides and the deduced amino acid sequence of ORF share 80%-82% and 69%-71% identity with those of other species in Siluriformes, respectively. The prepropeptide contained a signal peptide （23 amino acids）, a prodomain （45 amino acids） and a mature peptide（25 amino acids）. The predicted 25-amino acid Hepcidin mature peptide included 8 conserved cysteine residues which were proposed to form four disulphide bonds. RT-PCR demonstrated that the Hepcidin gene was expressed in a wide range of tissues of yellow catfish. And the Hepcidin transcripts were highly abundant in liver, abundant in head kidney, intestine, muscle, brain, stomach, gill, heart, ovary and trunk kidney, less abundant in skin and spleen, respectively. Challenge of yellow catfish with Aeromonas hydrophila and Bacillus cereus significantly elevated Hepcidin mRNA levels in liver. The new member of Hepcidin discovered in this study can contribute to the research of immune function and mechanism in yellow catfish.

关 键 词：黄颡鱼 HEPCIDIN基因 CDNA克隆 序列分析 组织表达 细菌感染 

分 类 号：S188[农业科学—农业基础科学]