小鼠睾丸新基因TSEG-2的克隆及表达分析

Cloning and Expression of a Novel Mouse Testis Gene TSEG-2

目的:利用生物信息学手段克隆小鼠睾丸特异性基因TSEG-2。方法:从二级表达序列标签(EST)数据库ZooDDD中获得小鼠正常睾丸表达的EST,通过dbEST数据库检索出与其高度同源的EST序列,构建EST叠加群,Biolign软件拼接,GeneScan软件预测contigs对应的基因组序列中的外显子、内含子;针对开放阅读框设计引物序列,采用RT-PCR从小鼠睾丸组织中克隆新基因的cDNA,分析该基因在小鼠睾丸不同发育阶段、隐睾中及各脏器中的表达,并对测序结果进行生物信息学分析。结果:成功克隆了小鼠睾丸新基因TSEG-2,全长451bp,开放阅读框为267bp,编码88个氨基酸。RT-PCR证实该基因开放阅读框正确,TSEG-2在小鼠睾丸组织中高表达,在4、9、14、18、21、38日龄和2、6月龄小鼠睾丸组织中呈现规律性表达,在隐睾组织中表达减弱,且与小鼠其他cDNA无同源性,获得GenBank登录号EU079025。功能区分析发现,小鼠TSEG-2cDNA序列定位于染色体15qE3,为可溶性非分泌型蛋白,有2个可能的蛋白激酶C(PKC)磷酸化位点,1个酪蛋白激酶Ⅱ(CK2)磷酸化位点,并可能是一种核蛋白。结论:获得小鼠睾丸特异性表达基因TSEG-2,诱导小鼠隐睾发生的17-β雌二醇可引发基因TSEG-2的下调,为进一步研究该基因的生物学功能和表达调控奠定了基础。

Objective： To clone the mouse testis specific gene TSEG-2 via a bioinformatic approach. Methods： The expressed sequence tags （EST） in the normal mouse testis were obtained from the online EST database ZooDDD. Their highly homologous EST sequences were retrieved through the dbEST database to construct contigs and spliced with the biomedical software Biolign. The corresponding exons and introns within the genome sequences were predicted with the software GeneScan. Primers were designed according to the open reading frame. RT-PCR was applied in cloning the cDNA of the novel gene from the mouse testis tissue and analyzing its expression patterns in the undescended testis and various organ tissues as well as in different developmental stages of the mouse testis. The sequencing results of TSEG-2 underwent bioinformatic analyses. Results ： The novel mouse testis gene TSEG-2 was successfully cloned, with full-length sequence of 451 bp. The open reading frame was 267 bp, coding a protein of 88 amino acid residues, and demonstrated to be correct by RT-PCR. The expression of TSEG-2 was high in the mouse testis, regular in the testis cDNA samples of different postnatal days, and down-regulated in the cryptorchidism model. No obvious homology with other mouse cDNA was found for TSEG-2. The GenBank accession number EU079025 was achieved. Function prediction showed that mouse TSEG-2 was probably a soluble non-secretary protein located at chromosome 15qE3, or a nucleoprotein with 2 phosphorylation sites of protein kinase C （PKC） and 1 of casein kinase Ⅱ （CK2）. Conclusion ： A novel mouse testis specific gene TSEG-2 was successfully cloned, which could be down-regulated by cryptorchidism-inducible 17-6 estradiol. This has prepared the ground for further researches on the biological function and expression regulation of TSEG-2.