大鲵皮肤cDNA文库构建及Arpc5l基因cDNA序列和组织表达分析  

Construction of cDNA Library of Andrias davidianus Skin Tissue with Analyzing the cDNA Sequence and Expression of Arpc5l Gene

作　　者：王立新[1] 郑尧[1] 艾闽[1] 杨辉[1] 杨朝霞[1] 

机构地区：[1]西北农林科技大学动物科技学院水产科学系,陕西省农业分子生物学重点实验室,陕西杨凌712100

出　　处：《中国生物化学与分子生物学报》2011年第3期273-281,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：陕西省自然科学基金(No.2003C124);西北农林科技大学重点项目(No.01140513)~~

摘　　要：以健康大鲵背部皮肤为材料,采用Smart技术构建大鲵皮肤cDNA文库,经检测文库容量为1.50×106cfu,文库重组率为98%,插入片段平均长度约为1 kb.通过对大鲵皮肤cDNA文库的随机筛选,分离获得了大鲵肌动蛋白相关2/3复合物亚基-5样蛋白(Arp2/3 complex subunit 5-like,Arpc5l)基因cDNA序列.生物信息学分析表明,大鲵Arpc5l基因全长cDNA为699 bp,包含459 bp的开放阅读框,分子量为17 154.36,等电点(pI)为5.43;该基因编码的Arpc5l蛋白分别在7-29 AA处和44-66 AA处具有跨膜结构域及多种二级结构,且三级结构与Arp2/3 complex家族成员相似.实时定量PCR结果表明,该基因在大鲵皮肤、肌肉、肠、肝、脾、肺、胃以及肾等组织中广泛表达,且在肾、肠、肌肉中的表达量极显著高于其它组织(P<0.01).进化分析结果表明,大鲵Arpc5l氨基酸序列和小鼠(77%)等陆生动物同源性最高,和爪蟾同源性为73%,大鲵处于水生到陆生之间的过渡类型,且与陆生动物的亲缘关系更近.To clone Arp2 /3 complex subunit 5-like（Arpc5l） gene from Chinese giant salamander,an animal suitable to study genetic evolutionary relationship,and to analyze the characteristics of the functional gene by bioinformatics analysis,Arpc5l gene was isolated from skin cDNA library.It has been revealed that Arpc5l gene was related with cancer cell movement.A cDNA library of the skin tissue was constructed by using the isolated mRNA as the template during reverse transcription.The skin cDNA library of Chinese giant salamander was detected and sequenced by picking clones randomly.It is confirmed that the titer of the cDNA library was 1.50 × 106 cfu,the recombination rate was 98%,and the PCR results showed that the average size of insert segments was about 1 000 bp.Meantime,we cloned the Arpc5l cDNA from skin cDNA libraries using our own sequence as primers.However,the cDNA from skin cDNA library was 699 bp,containing an open reading frame（ORF） region,with 459bp.By bioinformatics software,we found that the molecular weight of Arpc5l was 17.15 kD and its pI was 5.43.The analysis of amino acids（AA） structure showed that the Arpc5l had two transmembrane domains,located at 7-29 AA and 44-66AA,respectively.The tertiary structure of the Arpc5l was highly homologous to Arp2 /3 complex family.Moreover,we were able to detect Arpc5l mRNA in 8 tissues of Andrias davidianus,including kindey,bowel,muscle,liver,lung,skin,spleen and stomach.The expression of Arpc5l mRNA was found to be significantly higher in kindey,bowel and muscle than other tissues by qRT-PCR（P 0.01）.Through searching in public database and sequence comparisons and assemblying 10 related sequences of Arpc5l and cytochrome c oxidase subunit,respectively,phylogenesis analysis of their genomic sequences was obtained.It revealed that functional gene Arpc5l of A.davidianus was the most closely related to terrestrial animals,just like Bos taurus,Homo sapiens,Mus musculus.However,the structural gene showed that COXⅡof A.davidianus was highly homologous t

关 键 词：大鲵 皮肤cDNA文库 大鲵肌动蛋白相关2/3复合物亚基-5样蛋白(Arpc5l)基因 组织表达 进化关系 

分 类 号：Q17[生物学—水生生物学]