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大鼠睾丸特异表达基因{sl Ube1}的分离鉴定及生物学特征

杜颖, 刘美玲, 贾孟春

北京协和医学院研究生院.北京 100730；国家人口计生委科学技术研究所生殖内分泌室.北京 100081

摘要

该研究采用抑制性消减杂交(suppression subtractive hybridization, SSH)和cDNA快速扩增(rapid amplification of cDNA ends, RACE)技术从大鼠A型精原细胞和粗线期精母细胞中成功克隆出大鼠泛素激活酶(ubiquitin-activating enzyme)基因{sl Ube1}(GenBank登录号EF690356)。该基因序列全长3433 bp，其中开放阅读框有3171 bp，编码一个含1 057个氨基酸的蛋白质。Blast比对显示，{sl Ube1}与小鼠泛素激活酶基因{sl Ube1y1}的同源性为93%，与人泛素激活酶基因{sl UBE1}的同源性为82%。{sl Ube1}基因编码的蛋白质含泛素激活酶信号位点和泛素激活酶活化位点，这些位点也存在于人类和小鼠的泛素激活酶1中。RT--PCR分析显示，{sl Ube1}在睾丸中大量表达，而在心、肝、脾、肺、肾、肌肉、脑、卵巢中没有表达。荧光定量PCR分析不同生精细胞中{sl Ube1}的表达，显示{sl Ube1}在A型精原细胞中大量表达，在粗线期精母细胞、圆形精子细胞和支持细胞中微弱表达。以上结果提示，{sl Ube1}是大鼠睾丸特异表达基因，可能通过参与泛素/蛋白酶体途径来影响精子发生。

关键词： 泛素激活酶; 精子发生; 抑制性消减杂交; cDNA快速扩增; 逆转录-聚合酶链反应; 荧光定量PCR

Identification and characterization of a spermatogenesis--related gene {sl Ube1} in rat testis

Du Ying, Liu Meiling, Jia Mengchun

Peking Union Medical College Postgraduate School.Beijing 100730；China

Abstract

A gene that could potentially involved in spermatogenesis was identified and characterized by using suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE) with total RNA from type A spermatogonia and pachytene spermatocytes of rat. This gene consists of 3433 base pairs (bp) with a complete open reading frame (ORF) of 3171 bp and encodes a putative protein containing 1057 amino acids. The nucleotide sequence displays a 93% identity to mouse ubiquitin-activating enzyme E1, Chr Y 1 (Ube1y1) and an 82% identity to human ubiquitin-activating enzyme E1 (UBE1). The putative protein of this gene contains an ubiquitin-activating enzyme signature 1 and an ubiquitin-activating enzyme active site, which is also existed in mouse ubiquitin-activating enzyme E1, human ubiquitin-activating enzyme E1 et al. So we named this gene as Rattus norvegicus ubiquitin-activating enzyme E1 (Ube1). The sequence of Ube1 was submitted to GenBank and the accession number is EF690356. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that Ube1 was specifically expressed in testis, while its expression was not detected in heart, brain, spleen, lung, liver, muscle, kidney and ovary. Comparison of the expression of Ube1 in different developmental stages of testis and Sertoli cells indicated that Ube1 was expressed more highly in spermatogonia than in spermatocytes, spermatids and Sertoli cells. In conclusion, Ube1 is a gene encoding rat ubiquitin-activating enzyme E1 and specifically expressed in testis, which might play a key role in ubiquitin system and influence spermatogenesis.

Key words: ubiquitin-activating enzyme;Spermatogenesis;suppression subtractive hybridization;rapid amplification of cDNA ends;RT-PCR;real-time PCR

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引用本文：

杜颖, 刘美玲, 贾孟春. 大鼠睾丸特异表达基因{sl Ube1}的分离鉴定及生物学特征[J]. 生理学报 2008; 60 (3): 382-390.

Du Ying, Liu Meiling, Jia Mengchun. Identification and characterization of a spermatogenesis--related gene {sl Ube1} in rat testis. Acta Physiol Sin 2008; 60 (3): 382-390 (in Chinese with English abstract).