【作者】 陈勇；

【导师】 苏永全；

【作者基本信息】 厦门大学 ， 海洋生物学， 2008， 硕士

【摘要】 抗菌肽(antimicrobial peptides,AMPs)是一类具有广谱抗微生物活性的天然小肽类物质,是先天免疫系统中重要的非特异性免疫因子。Piscidin一词最初来源于Silphaduang等从杂交斑纹鲈(hybrid stripedbass,Morone.chrysops×M.saxatils)肥大细胞分离出的一种肽类抗菌素,随后的研究发现,从鲆鲽类获得的抗菌肽pleurocidin,杂交斑纹鲈的moronecidin,欧洲狼鲈(Dicentrarchus labrax)的dicentracin,斜带石斑鱼(Epinepheluscoioides)的epinecidin-1等,都具有和piscidin十分相似的氨基酸序列及其多肽结构和功能,为α-螺旋、带正电荷、两性亲和多肽,可以统称为piscidin。本论文以我国重要的海水经济养殖鱼类——赤点石斑鱼(Epinephelus akaara)和大黄鱼(Pseudosciaena crocea)为研究对象,克隆得到两种鱼类的piscidin类抗菌肽基因,并分析其氨基酸序列组成和次级结构特点,与piscidin家族其他成员进行多序列比对,并建立系统进化树,研究结果如下:1、通过RT-PCR、3’RACE、5’RACE和测序等分子生物学技术,从赤点石斑鱼的头肾和脾脏扩增出piscidin-like抗菌肽序列,并进行序列分析和蛋白特性预测,结果显示赤点石斑鱼piscidin为带正电荷的两亲性α-螺旋结构,成熟肽碳末端具有强阳离子四肽的RRRH结构,与斜带石斑鱼成熟肽碳末端一致,这种结构有利于提高与细菌胞膜的结合能力,与piscidins家族其他抗菌肽序列相似性为59%-79%,一致性为45%-75%,其氨基酸序列和次级结构符合piscidin抗菌肽家族的特征,表明从赤点石斑鱼头肾和脾组织克隆得到的piscidin-like抗菌肽基因是piscidin基因变体,属于piscidins家族的新成员。2、通过RT-PCR、3’RACE、5’RACE和测序等分子生物学技术,从大黄鱼的头肾和脾脏扩增出piscidin-like抗菌肽序列,并进行序列分析和蛋白特性预测,结果显示大黄鱼piscidin成熟肽为带正电荷的两亲性α-螺旋结构,成熟肽碳末端没有石斑鱼类的RRRH结构,正电荷值比赤点石斑鱼piscidin低,优势域碳末端也没有鲈鱼类的XQQ模块,与piscidins家族其他抗菌肽序列相似性50-77%,一致性41-70%,其氨基酸序列和次级结构符合piscidin抗菌肽家族的特征,表明从大黄鱼克隆得到的piscidin-like抗菌肽基因是piscidin基因变体,属于piscidins家族的新成员。3、利用CLUSTALW软件对piscidin家族抗菌肽进行多序列比对,验证了其具有高度序列同源性,说明生物体结构与功能的高度统一。利用MEGA 4.0软件绘制了Piscidin抗菌肽家族与其它鱼类抗菌肽家族的系统进化树,验证了从赤点石斑鱼和大黄鱼克隆得到的基因属于鱼类piscidin抗菌肽基因,且piscidin家族抗菌肽基因自成一簇,与鱼类hepcidin抗菌肽家族相互区分。更多还原

【Abstract】 As possessing broad-spectrum bactericidal activity,antimicrobial peptides(AMPs) play the crucial roles in innate immune system in tested animals.Piscidin is initially described as a peptide antibiotic derived from mast cell of hybrid-striped bass,and the later researches revealed that pleurocidins from flat fishes,moronecidin from hybrid-striped bass,dicentracin from sea bass,epinecidin-1 from orange-spotted grouper,shared great similarity and identity in the properties of amino acid sequence and peptide secondary structure,representing asα-helix cationic amphipathic AMPs,which could properly be classified as piscidin family.In this study,two species of commercial maricultured fish in China,red-spotted grouper(Epinephelus akaara) and large yellow croaker (Pseudosciaena crocea),were taken as samples to be used to study on piscidin cDNA cloning,deduced amino acid sequence alignment,secondary structure analysis,and phylogenetic relationship.The results are mainly described as follows:1.By using RT-PCR,3’RACE,5’ RACE and sequencing,we amplified piscidin-like AMP cDNA from the kidney and spleen of red-spotted grouper.After the analysis on amino acid sequence and protein secondary structure,the peptide was identified as aα-helix cationic amphipathic propeptide,which strongly matched the main character of piscidin family.The RRRH motif in the C-terminal of mature peptide was found as that of epinecidin-1 and predicted to develop the electrostatic binding activity to negatively charged bacterial phospholipids.The similarity and identity between this peptide and other piscidin family members ranged respectively from 59%-79%and 45%-75%.Experimental evidence above demonstrated that the cloned cDNA is probably a new member in piscidin family. 2.Piscidin-like AMP cDNA from large yellow croaker was amplified by RT-PCR,3’RACE,5’RACE and sequencing.After the analysis on amino acid sequence and protein secondary structure,the peptide was identified as aα-helix cationic amphipathic propeptide,which strongly matched the main character of piscidin family.The shortages of RRRH motif which presented in the mature peptide C-terminal of epinecidin-1 as well as the XQQ motif in the prodomain C-terminal of moronecidin maked the piscidin of large yellow croaker related more closely to pleurocidins rather than other two above.The similarity and identity between this peptide and other piscidin family members ranged respectively from 50%-77%and 41%-70%.Experimental evidence above demonstrats that the cloned cDNA is probably a new member in piscidin family.3.Sequence alignment via the biosoft CLUSTALW confirmed the remarkable homology among piscidin family,the conserved domain extended from signal peptide through the mature peptide,which also indicated the harmony between structure and function in organisms.Phylogenetics relationship analysis via MEGA 4.0 supported the wide evolutionary conservation of piscidins in fish species,including both red-spotted grouper and large yellow croaker.The cluster of piscidin family clearly separated from hepcidin family in the agreement with the fact that of piscidins that are linear peptide whereas hepcidins are characterized by 4 intra chain bonds giving totally different 3D structures.更多还原