端粒酶催化亚基基因在小鼠胚胎发育早期的转录活性  

Gene Transcription of Mouse Telomerase Catalytic Subunit in Preimplantation Embryos

作　　者：王宇[1] 姚慧[1] 安晓荣[1] 陈永福[1] 苟克勉[1] 

机构地区：[1]中国农业大学生物学院农业生物技术国家重点实验室,北京100094

出　　处：《生物化学与生物物理进展》2005年第3期282-287,共6页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30270647); 北京市自然科学基金项目(5012007); 农业生物技术国家重点实验室开放课题;国家高技术"863"计划项目(2002AA206311) 资助~~

摘　　要：用RT-PCR引物分别扩增成年昆明(KM) 小鼠睾丸、脾脏、肾脏、肝脏和胸腺组织的总RNA发现,端粒酶催化亚基基因tert在这些组织中都有转录,目标产物正确组装到PMD 18-T载体后测序,结果与已知cDNA序列一致. PMSG/hCG超数排卵方法获得KM小鼠成熟卵母细胞和CZB溶液体外培养的胚胎(KM ♀×KM♂),用酸性Tyrode蒺s溶液消化透明带后,采用巢式RT-PCR,同时分析tert基因和持家基因hprt的转录发现,对于单个样品来说, 全部卵母细胞(15 h-post hCG,10/10) 都存在hprt转录本,其中,只有40%(4/10) 还同时存在tert转录本. 原核形成初期(20 h-post hCG,6/6) 和原核晚期(30 h post-hCG,8/8) 的受精卵,以及发育至2-C早期的胚胎(35 h-post hCG,7/7) 都不转录tert基因,只有hprtmRNA存在;2-C晚期(50 h-post hCG)时,两个基因同时转录(4/8)和一个基因单独转录(4/8)的胚胎各占50%;从4-C阶段(65 h-post hCG,4/4)开始,包括8-C阶段(75 h-post hCG,4/4),桑椹胚阶段(93 h-post hCG,4/4),直至囊胚阶段(118 h-post hCG,4/4),所有的胚胎都同时转录tert和hprt基因,而且转录水平明显升高. 以20枚胚胎量为模板进行RT-PCR发现,原核早期,原核晚期的胚胎中仍然没有tert基因转录,只有hprtmRNA,但是,在2-C早期胚胎中同时检测到了hprt和tert两种mRNA. ?Telomerase is a ribonucleoprotein complex that elongates telomeres, allowing to stabilize the ends of chromosomes during cell proliferation. Here nested PCR method was used to describe the transcriptional patterns of mTERT (mouse telomerase reverse transcriptase) gene, encoding the catalytic subunit of mouse telomerase expressed both in murine adult tissues and preimplantation embryos cultured in CZB medium from zygote to blastocyst. The analysis revealed that the mTERT mRNA expression in adult tissues, such as testis, spleen, kidney, liver, and thymus. For single-copy zona-free oocyte or embryo samples, the tert transcript in 60% (6/10) of matured oocytes (15 h-post hCG) had been not found, while the transcripts of housekeeping gene hprt located in all matured oocytes (10/10). No tert mRNA was present in single-copy embryo samples at early (20 h-post hCG, 6/6), late (30 h post-hCG, 8/8) pronuclear stage, or early 2-C stages (35 h-post hCG, 7/7), although the hprt mRNA transcriptions were found during this period. Interestingly, the embryos at early 2-C stage expressed of tert and hprt gene together in multi-copy (20 embryos/sample) embryo samples. Half of single-copy embryos (4/8) shown expression of two genes simultaneous at late 2-C stage (50 h-post hCG), while the other half of them (4/8) only transcript either tert (2/4) or hprt gene (2/4). Expression of tert and hprt genes were detected simultaneous in all single-copy embryos developed at 4-C (65 h-post hCG, 4/4), 8-C (75 h-post hCG, 4/4), morulae (93 h-post hCG, 4/4) and blastocyst (118 h-post hCG, 4/4) stages and the level of transcription increased. These results suggested that tert gene was began to express in embryos at early 2-C stage during the process of embryonic genome activation and the level of transcription was increased during embryogenesis.

关 键 词：小鼠 卵母细胞 胚胎 tert基因 HPRT基因 端粒酶 RT-PCR 

分 类 号：Q27[生物学—细胞生物学]