利用电子差异展示方法克隆人类睾丸特异性新基因  

Molecular cloning of a novel human testis-specfic gene TDRG1

作　　者：尹光明[1] 阳建福[1] 蒋先镇[1] 汤育新[1] 何乐业[1] 蒋志强[1] 钟狂飙[1] 曾青[1] 

机构地区：[1]中南大学湘雅第三医院泌尿外科,湖南长沙410013

出　　处：《南方医科大学学报》2009年第4期631-634,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30672090)

摘　　要：目的克隆一个新的人睾丸特异性基因。方法运用电子差异展示方法筛选人类睾丸特异表达新基因,获得有差异显示的代表新基因的克隆重叠群,挑选其中一个克隆重叠群Hs.180197进行多组织RT-PCR验证该重叠群在人睾丸中的表达。然后从包含该重叠群的IMAGE克隆出发,采用生物信息学方法克隆一个人类新基因的全长cDNA序列。结果新基因全长1197bp,开放阅读框为504～806bp,定位于6p21.1-p21.2,编码由100个氨基酸组成,相对分子质量为10000,等电点为6.81的一个蛋白,该蛋白与已知蛋白无同源性。克隆实验验证该基因阅读框完全正确,推测其可能与精子生成相关,暂命名为TDRG1(testis development related gene1),GenBank登录号为DQ168992。结论电子差异展示方法与实验验证相结合用于发现人类功能新基因是行之有效的。Objective To clone a novel human testis-specific gene TDRG1. Methods A new contig of expression sequence tags （ESTs） Hs. 180197 was identified from the testis libraries using digital differential display （DDD） to screen the novel human testis-specific gene. To validate the use of bioinformatics approaches in gene discovery, the ESTs Hs.180197, which was predicted to be testis specific, was chosen for further study. Reverse transcriptase-polymerase chain reaction （RT-PCR） was performed on different normal tissues to identify the expression of Hs. 180197 in human testis. Using bioinformatics methods and IMAGE cloning of this contig, the full-length cDNA sequence of the noval human gene was cloned. Results This novel gene was 1197 bp in length, located in chromosome 6p21.1-p21.2. The sequence of the open reading flame was 504-806 bp, as confirmed by RT-PCR and sequencing in human testis. The eDNA encodes a novel protein of 100 amino acids with a theoretical molecular weight of 10 000 and isoelectric point of 6.81. The sequence shares no significant homology with any known protein in the databases. Semi-quantitative RT-PCR analysis of multiple tissues further showed that the novel gene was expressed specifically in adult human testis. Considering a possible relation of this novel gene with the function of human testis, we named this new gene TDRG1 （testis development related gene 1, GenBank accession number： DQ168992）. Conclusion DDD combined with laboratory validation is an efficient method for identifying new human functional genes.

关 键 词：基因克隆 电子差异展示 逆转录聚合酶反应 睾丸 组织特异表达 

分 类 号：Q78[生物学—分子生物学]