香鱼Apo-14基因的克隆、序列分析及其mRNA表达与盐度适应性的关系  

Gene cloning and sequencing of Apo-14 gene in ayu(Plecoglossus altivelis) and the effects of salinity change on its mRNA expression

作　　者：曹霄[1] 陈炯[1] 史雨红[1] 陆新江[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,浙江宁波315211

出　　处：《台湾海峡》2011年第2期203-209,共7页Journal of Oceanography In Taiwan Strait

基　　金：国家973计划前期专项资助项目(2008CB117015);宁波市科技局资助项目(2007A31004;2010A610001);宁波大学研究生科研创新基金资助项目(NG09JLA030)

摘　　要：14 kDa载脂蛋白(Apo-14)是鱼类一种特有的载脂蛋白,是鱼类高密度脂蛋白(HDLs)的重要成分,其生物学功能目前还不明确.本研究采用简并引物,从香鱼(Plecoglossus altivelis)肝脏cDNA文库中筛选香鱼Apo-14基因,该基因cDNA序列全长905个核苷酸,开放阅读框编码一个有144个氨基酸,分子量约为15.9 kDa的蛋白.香鱼Apo-14基因mRNA主要在香鱼肝脏中表达,在肠、肾和鳃中也有少量表达.同源性分析揭示,香鱼Apo-14与已知鱼类中虹鳟(Oncorhynchus mykiss)Apo-14最相似,氨基酸序列同源性为70%.qRT-PCR表明,咸淡水处理组(盐度为10)的肝、肠、肾、鳃中,该基因mRNA均比淡水处理组(盐度为0)明显下调(p<0.05),揭示载脂蛋白Apo-14可能与盐度适应相关.Apolipoproteins are important structural components of plasma lipoproteins and have been shown to participate in lipoprotein assembly,secretion,processing,and catabolism.14 kDa apolipoprotein（Apo-14）,a specific apolipoprotein in fish,is an important component of high-density lipoproteins,but its biological functions remain unclear.Apo-14 was amplified by a degenerate primer and then sequenced and analyzed using BLASTX,SignalP 3.0,NetNGlyc 1.0,YinOYang 1.2,NetPho 2.0,ClustalW,and MEGA 4.1 software.Sixty specimens of healthy ayu were acclimatized to laboratory conditions for two weeks before experiments.The fish were then subjected to a progressive salinity change to 10（BW） in four tanks or to zero（FW） in another four tanks over a period of 3 weeks.RT-PCR was employed to analyze the mRNA expressions in different tissues,and qRT-PCR was employed to analyze the effects of salinity change on the mRNA expression.In this study,a full-length cDNA clone of the Apo-14 gene,905 bp in size,encoding a protein of 144 amino acids with a molecular weight of 15.9 kDa,was isolated from the fish ayu cDNA library.The ayu Apo-14 amino acid sequence includes a signal peptide of 18 amino acids,a 33-codon block and 22 amino acid repeats,which is predicted to form amphipathic α-helical and be able to transport lipids and other hydrophobic molecules.RT-PCR analysis showed that Apo-14 was only transcribed in the liver,intestine,kidney and gill and not in the muscle,spleen,and brain.The mRNA level of ayu Apo-14 was highest in the liver.Sequence comparison and phylogenetic analysis showed that ayu Apo-14 was most similar to that of rainbow trout（Oncorhynchus mykiss）;about 70% amino acid sequence identity.qRT-PCR analysis demonstrated that the expressions of Apo-14 mRNA were significantly decreased in ayu liver,intestine,kidney and gill tissues when the fish were transferred from fresh water to brackish water.It is suggested that apo-14 transcription might be down-regulated when subjected to salinity change and related to osm

关 键 词：海洋生物学 Apo-14 克隆 序列分析 盐度适应 QRT-PCR 

分 类 号：P735[天文地球—海洋生物学]