WDR45L大鼠同源基因编码区cDNA的克隆及生物信息学分析

Clone and bioinformatic analysis of rat WDR45L coding cDNA

目的:克隆肿瘤相关基因WDR45L的大鼠同源基因,并进行初步的生物信息学分析。方法:以人WDR45L同源氨基酸序列(CAJ57996.1)作为查询序列,利用TBLASTN和BLASTN软件搜索数据库中与大鼠WDR45L基因同源的cDNA和EST序列。把检出序列组装为连续体(Contig),在UCSC程序上进行EST连续体序列的拼接和基因组序列与EST序列的双重校对。克隆基因采用RT-PCR测序证实,并进行核酸与蛋白质特性的生物信息学分析。结果:综合利用基因组和EST序列信息成功克隆了大鼠WDR45L基因(GenBank登记号:AM182327和NM_001039587.1)。RT-PCR测序结果与计算机克隆结果完全一致。生物信息学分析显示:WDR45L基因定位于染色体10q32.3区,含11个外显子和10个内含子,ORF为1035bp,编码344个氨基酸;在种属中高度保守;WDR45L基因在胃癌、结肠癌、乳腺癌、卵巢癌、肾癌和胰腺癌等恶性肿瘤中表达上调。结论:综合利用基因组和EST序列信息是更为科学的计算机克隆方法,进一步深入研究WDR45L基因的功能具有重要生物学意义。

OBJECTIVE： To clone and identify the rat gene homologous to cancer-related gene WDR45L and to perform the perform primary bioinformatics analysis. METHODS： Rat cDNAs or expressed sequence tags （ESTs） derived from the putative rat ortholog were searched for with the Tblastn or Blastn programs with amino-acid sequence of human WDR45L protein used as a query sequence. Complete sequence of rat WDR45L eDNA was determined by EST assembling and revised by genomic and EST sequences. The cloned gene was verified by RT-PCR sequencing and the bioinformatics analysis on its DNA and protein characteristics were performed. RESULTS： By combining with genome and EST sequence information, rat WDR45L homolog gene was successfully cloned and verified by RT-PCR sequencing. WDR45L eDNA sequence has been deposited with the DDBJ/EMBL/ GenBank Data Libraries under accession number AM182327 and designated as rat WDR45L gene reference eDNA sequence under accession number NM_001039587. 1. Rioinformatic analysis showed that rat WDR45L gene was located at chromosome 10q32. 3 consisting of 11 exons and 10 introns. The predicted open reading frame （ORF） is 1 035 bp encoding a 344-amino-acid. The WDR45L proteins were extremely conserved. WDR45L gene up-regulated in several malignancies including stomach, large intestine, breast, ovary, kidney and pancreatic cancers. CONCLUSION： In silico cloning by combining with genome and EST sequence is a more robust gene cloning method, and to further study this gene＇s function is of biological significance.