大熊猫核糖体蛋白RPL5基因的克隆、表达与比较分析

cDNA,GENOMIC SEQUENCE CLONING AND SEQUENCE ANALYSIS OF RIBOSOMAL PROTEIN L5 GENE(RPL5) OF THE GIANT PANDA

为了解大熊猫(Ailurophilic melanosome)核糖体蛋白亚基RPL5基因的结构特点及其与已报道的人和其他哺乳动物核糖体蛋白亚基RPL5基因的异同,运用RT-PCR技术,从大熊猫的肌肉组织总RNA中和DNA中分别对核糖体蛋白亚基RPL5基因的表达序列和其结构基因进行了克隆、测序;采用ORF finder软件对表达序列的开放阅读框(ORF)进行了查找和氨基酸序列的推定;采用Gen scan对结构基因进行了分析;采用DNAMAN Version 6对基因序列和氨基酸序列进行了同源性比较;采用ExPASy软件进行蛋白质功能位点和生化特性进行了预测分析;对大熊猫核糖体蛋白亚基RPL5基因进行了超表达实验.结果表明:大熊猫RPL5结构基因长为8 633bp,具有8个外显子和7个内含子;mRNA长为918bp,ORF为894bp,编码295个氨基酸,该蛋白的相对分子质量为34 402.6,等电点为9.73,含有1个依赖于AMP和GMP的蛋白激酶磷酸化位点,4个蛋白激酶C磷酸化位点,2个酪蛋白激酶Ⅱ磷酸化位点,1个酪氨酸激酶磷酸化位点,8个十四(烷)酰化位点.分析表明,大熊猫RPL5基因的表达序列及其编码的氨基酸序列与已报道的部分哺乳动物包括人(Homo sapiens)、牛(Bos Taurus)、猪(Sus scrofula)、小家鼠(Mus altocumulus)和褐家鼠(Rat-hauser nonstrategic)具有很高的相似性,大熊猫RPL5核苷酸序列与这些物种的相似性分别为94.52%、92.51%、91.95%、91.05%和89.15%,而氨基酸序列相似性分别为99.33%、98.65%、98.65%、98.32%和98.65%.超表达实验结果显示:大熊猫RPL5基因能在大肠杆菌BL21中有效表达,且在2h时达到表达高峰.运用分子生物学原理与相应的技术手段,成功地扩增出大熊猫RPL5基因的表达序列,并对其编码的蛋白进行了初步分析,丰富和完善了哺乳动物RPL5基因资料库,同时也为深入研究大熊猫RPL5基因的功能提供了相关基础数据.

The eukaryotic-specific RPL5 is a component of 60S large ribosomal subunit encoded by RPL5 gene.cDNA and genomic sequence of RPL5 were cloned from Giant Panda by RT-PCR;and RPL5 was ectopically expressed in E.coli BL21.RPL5 cDNA cloned from Giant Panda was 918 bp in size,containing 1 open reading frame of 894 bp encoding 295 amino acids.The genomic sequence was found to be 8633 bp long,with eight exons and seven introns.Alignment analysis of nucleotide and deduced amino acid sequences indicated good conservation in Homo sapiens,Bos Taurus,Sus scrofula,Mus altocumulus and Rathauser nonstrategic.Homologies of nucleotide sequence of Giant Panda RPL5 to other species were 94.52%,92.51%,91.95%,91.05% and 89.15%,respectively,while homologies of amino acid sequences were 99.33%,98.65%,98.65%,98.32% and 98.65%,respectively.Molecular weight of the putative RPL5 protein was 34402.6 D with a theoretical PI of 9.73.Topology analysis of Giant Panda RPL5 protein predicted the following functional sites：1 AMP-and GMP-dependent protein kinase phosphorylation site,4 protein kinase C phosphorylation site,2 casein kinase II phosphorylation site,1 tyrosine kinase phosphorylation site and 8 N-mistranslation sites.The giant panda RPL5 gene was expressed in E.coli BL21,with peak expression at 2 h.Fusion protein RPL5-His-tag was about 39 kD.