一个新的人类睾丸特异性基因-TDRG1的cDNA克隆及表达分析

发布时间：2018-10-29 08:34

【摘要】：越来越多的研究表明，，生精障碍是导致男性不育的重要因素之一。精子生成是一个复杂的细胞发育与分化的过程，受一个由多个基因参与的基因网络的高度调控，这些基因可能既存在性染色体上，也存在常染色体上。分离生精相关基因是男性不育研究中的一个重要课题，研究和发现这些基因，对于阐明精子发生的机制具有重要的生理和病理学意义。 本课题运用了一种发现人类新基因的新策略“数据库消减杂交”(Digital Differential Display，DDD)——即通过计算机对不同文库进行比较来发现在统计学意义上存在明显转录差异基因的定量分析方法，结合RT-PCR、Northern杂交等实验验证成功克隆了一个人类睾丸组织特异性表达的新基因，并探讨了其与精子发生的关系。本研究分为2个部分，其主要实验方法及实验结果如下： 第一部分：数据库消减杂交方法克隆人类睾丸组织特异性表达新基因TDRG1 1 新基因的电子克隆 美国NCBI的Unigene数据库中拥有大量的表达序列标签(EST)，它们来源于不同组织、不同细胞、不同病理状态下所构建的cDNA文库，已成为人们寻找新基因的重要标志物。Digital DifferentialDisplay(DDD)是一种利用Unigene数据库中的ESTs进行数种平行材料之间的比较的方法。运用该方法，我们进行了两种平行材料之间的比较：挑选9个人类睾丸组织来源的cDNA序列文库作为检测子，并将之归为pool A；将76个人类其他组织或细胞株来源的cDNA文库序列作为驱赶子，并将之归为pool B；通过pool A：pool B之间的比值来计算倍数差异，并以≥10倍的差异为筛选标准，经数据库杂交筛选获得在统计学意义上存在差异显示的克隆重叠群。 在获得的29个全新克隆重叠群中，Hs.180197是其中一个其差异表达最显著的克隆重叠群，生物信息学分析显示其代表一个新基因，我们将之作为本课题的研究重点。Hs．180197包括3个EST，运用EST拼接工具进行序列匹配、整合，获得了一个新的未知序列contig1。将contig1与EST Human数据库比较，发现该序列包含另
[Abstract]:More and more studies show that spermatogenic disorder is one of the important factors leading to male infertility. Spermatogenesis is a complex process of cell development and differentiation, which is highly regulated by a network of genes involved in multiple genes, which may exist on both sex and autosomal chromosomes. The isolation of spermatogenic genes is an important subject in the study of male infertility. The study and discovery of these genes have important physiological and pathological significance in elucidating the mechanism of spermatogenesis. In this study, a new strategy for the discovery of new human genes, "Database subtractive Hybridization" (Digital Differential Display,), was used. DDD)-A quantitative analysis of genes with significant transcriptional differences in statistical significance by comparing different libraries with a computer, combined with RT-PCR, A novel gene specifically expressed in human testis was successfully cloned by Northern hybridization and its relationship with spermatogenesis was discussed. This study is divided into two parts. The main experimental methods and results are as follows: part 1: cloning of human testis specific expression gene TDRG1 by database subtractive hybridization Electronic cloning of a new gene Unigene database of NCBI in the United States has a large number of expressed sequence tags (EST), They come from cDNA libraries constructed in different tissues, different cells, different pathological states, . Digital DifferentialDisplay (DDD), an important marker for searching for new genes, is a method to compare several parallel materials using ESTs in Unigene database. Using this method, we compared two kinds of parallel materials: 9 cDNA sequences from human testis were selected as detectors and classified as pool A; The 76 cDNA library sequences from other human tissues or cell lines were used as the banding vectors and classified as pool B; The multiple difference was calculated by the ratio of pool A:pool B, and the screening criterion was the difference of 鈮

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