小鼠睾丸生精细胞凋亡相关基因mTSARG3的克隆  

Molecular Cloning of mTSARG3 Gene Related to Apoptosis in Mouse Spermatogenic Cells

作　　者：刘刚[1] 卢光琇[1] 傅俊江[1] 刘上峰[1] 邢晓为[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,长沙410078

出　　处：《生物化学与生物物理学报》2003年第12期1133-1139,共7页

基　　金：国家重点基础研究发展规划项目 ( 973计划 ) (No .G19990 5 5 90 1)资助~~

摘　　要：从在小鼠隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 4 4 5 37)出发 ,利用GeneScan软件分析该片段所在染色体基因组序列 ,获得一个包含该EST的新基因序列。设计该基因特异性引物从小鼠睾丸cDNA文库中进行PCR扩增 ,分离出小鼠睾丸生精细胞凋亡相关基因mTSARG3(GenBank登录号为AF4 192 92 )。该基因定位于小鼠 7号染色体 7E1 E2区带 ,全长为 11kb ,cDNA全长为 132 8bp ,包含 8个外显子 ,编码由 316个氨基酸组成的、分子量为 36kD的蛋白质。该蛋白质含有DnaJ区和DnaJ- c区 ,与热激蛋白 4 0家族多种蛋白质有较高相似性 ,其中与小鼠DJB4 - MOUSE在 336aa的范围内有 4 6 %的相似性 ,属热激蛋白 4 0家族新成员。多组织RT PCR和Northern印迹结果显示 ,该基因在小鼠睾丸组织高表达 ,转录本大小约为 1.35kb ;Southern杂交结果显示 ,该基因在小鼠正常睾丸和隐睾组织无缺失和重排。实验结果证明成功克隆到了一个小鼠睾丸生精细胞凋亡相关基因mT SARG3。Beginning from a mouse EST (GenBank Accession No: BE644537) which was significantly changed in cryptorchidism and represented a novel gene, GeneScan program was performed and a predicted mouse novel gene full-length cDNA sequence containing the BE644537 sequence was attained. Gene-specific primers were designed for PCR in mouse testis cDNA library. The sequencing result of the PCR product showed that we obtain a new gene mTSARG3(GenBank Accession No: AF419292) whose full cDNA length is 1328 bp containing 8 exons and 7 introns. The predicted open reading frame encodes a protein of 316 amino acid residues. The protein encoded by the new gene is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. mTSARG3 protein displays a 46% identity in a 336-amino-acid overlap with DJB4 —MOUSE protein. The result of RT-PCR analysis and Northern blot showed that mTSARG3 is specifically expressed in mouse testis. Southern blot showed that there were no loss and rearrangement in cryptorchid. Based upon all these observations, it is considered that the function of the new gene is related to inhibit mouse testis spermatogenesis apoptosis.

关 键 词：小鼠 睾丸 生精细胞 凋亡 基因 mTSARG3 克隆 PCR 热激蛋白 隐睾 

分 类 号：Q78[生物学—分子生物学]