刺参核糖体蛋白L17基因(RPL17)全长cDNA克隆及组织表达  

Cloning and Expression Analysis of the Ribosomal Protein L17 Gene(RPL17 ) in Sea Cucumber(Apostichopus japonicus)

作　　者：王艳枫[1] 李霞[1] 秦艳杰[1] 

机构地区：[1]大连海洋大学辽宁省海洋生物资源与生境修复重点实验室/农业部北方海水增养殖重点实验室,大连116023

出　　处：《农业生物技术学报》2013年第5期562-569,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30371099);辽宁省教育厅创新团队(No.2007T015);辽宁省教育厅实验室专项(No.2008S064)

摘　　要：核糖体蛋白除组成核糖体、参与蛋白质合成之外,还作用于细胞的分裂、增殖、分化、凋亡、发育调控等过程。为了研究核糖体蛋白基因在刺参中的生理功能,本研究采用全长cDNA克隆技术首次从刺参(Apostichopus japonicus)体壁中克隆出核糖体蛋白L17基因(ribosomal protein L17,RPL17)的全长cDNA序列(GenBank登录号:JQ922561)。该基因cDNA全长643bp,包括33bp的5′-UTR,58bp的3′-UTR,开放阅读框552bp,编码183个氨基酸,编码的蛋白质相对分子量为21.5010kD,理论等电点(pI)为10.29,总平均亲水性(GRAVY)为-0.964,为不稳定的亲水蛋白。在NCBI网站上经Blastn和Blastx比对分析,发现该序列的核苷酸序列以及推导的氨基酸序列与其他物种的同源性均在70%以上,其中与紫海胆(Stron gylocentrotus purpuratus)的同源性分别为75%和81%。二级结构预测结果:α螺旋占36.61%;β折叠占7.65%;无规卷曲占42.08%;其余13.66%为延伸链。蛋白功能位点分析显示其含有1个核糖体蛋白L22信号位点和9个磷酸化位点,其中cAMP和cGMP依赖性蛋白激酶磷酸化位点3个,蛋白激酶C磷酸化位点4个,酪氨酸激酶Ⅱ磷酸化位点2个。构建的系统进化树显示,刺参与同为棘皮动物的紫海胆聚为一支,分子进化地位上关系最近,其次聚类顺序较近的为玻璃海鞘、非洲爪蟾,脊椎动物聚为一支,该结果与这些物种在生物学上的分类是较一致的。利用荧光定量PCR(qRT-PCR)技术检测RPL17基因在刺参各组织中的表达,结果表明,RPL17基因在体腔细胞中表达量最高,肠中次之,呼吸树中最低。体腔细胞中的表达量分别为肠、体壁、纵肌、呼吸树的5.67、21.63、43.25和346倍,与4种组织的差异均极显著(P<0.01)。另外,肠与呼吸树中的表达量差异显著(P<0.05),其余组织差异不显著(P>0.05)。本研究结果将为进一步研究刺参蛋白质合成、再生及多种生理活动的调控机制提供基础数据。Ribosomal protein has been proved to play an important role in protein synthesis, cell proliferation, apoptosis, development and some kinds of regulation. In order to investigate the physiological role of ribosomal protein gene in sea cucumber, full-length cDNA sequence of ribosomal protein L 17（RPL17）gene was cloned from the body wall of sea cucumber （Apostichopus japonicus） using molecular cloning method in this study. The results showed that the cDNA （GenBank accession No. JQ922561） length was 643 bp, including the 33 bp of the 5＇-UTR and 58 bp of the 3＇-UTR. The open reading frame was 522 bp, which encoding polypeptides of 183 amino acids, with a calculated relative molecular mass of 21.5010 kD and pI of 10.29. The RPL17 was a kind of hydrophilic and unstable protein, with grand average of hydropathicity （GRAVY） of - 0.964. By Blastn and Blastx searching in NCBI, the sea cucumber homologies of gene sequence and deduced amino acid sequence of RPL17 with other kinds of animals were over 70%, and the homologies between the sea cucumber and sea urchin （Strongylocentrotus purpuratus） were 75% and 81%, respectively. The predicted secondary structure of RPL 17 was consisted of four recondary structure elements, including 36.61% of alpha helix, 7.65% of beta turn, 42.08% of random coil and 13.66% of extended strand. The RPL17 contained 1 functional site （RPL22 signature site）and 9 phosphorylated sites （3 cAMP- and cGMP-dependent protein kinase phosphorylation sites, 4 Protein kinase C phosphorylation sites and 2 casein kinase II phosphorylation sites）. Phylogenetic relationship analysis, which was similar to the results of the biological classification, indicated that A. japonicus was firstly clustered with S. purpuratus, and also closely related with Ciona intestinalis and Xenopus laevis. And all vertebrates were clustered together. Real-time PCR was performed to analyze the expression profiling of RPL17, results showed that mRNA of RPL17 could be expressed in 5 different tissues in s

关 键 词：刺参 核糖体蛋白L17 基因克隆 表达 

分 类 号：S436.8[农业科学—农业昆虫与害虫防治]