| 摘要: |
| 【目的】研究泛素（Ub）在牛孤雌胚胎发育过程中的表达定位及与荧光标记线粒体分布的关联性。【方法】将人工合成Ub基因插入真核表达载体pVenus的BamHⅠ和Hind Ⅲ酶切位点之间，构建重组质粒pVenus-Ub，采用脂质体2000介导重组质粒pVenus-Ub转染Hela细胞，经荧光显微镜观察和PCR检测，探讨重组质粒pVenus-Ub在Hela细胞中的表达情况；用体外转录试剂盒将pVenus-Ub转录为cRNA并注射牛卵母细胞，选择成熟卵母细胞并将其经离子霉素孤雌激活后，在荧光显微镜下观察Ub的表达和定位情况；同时，用JC-1荧光染料对牛卵母细胞线粒体进行染色，观察线粒体的分布，探讨Ub定位与线粒体分布的相关性。【结果】重组载体pVenus-Ub能在Hela细胞中表达；Ub蛋白在牛体外成熟卵母细胞及孤雌发育过程中均有表达，分布于整个胞质中，并在皮质及核区分布较集中；成熟卵母细胞及早期孤雌胚胎中Ub蛋白与线粒体共定位。【结论】成功建立了Ub基因的真核表达体系及孤雌胚胎表达体系，且在牛孤雌胚胎发育过程中Ub的定位及与线粒体的分布相互关联。 |
| 关键词: 泛素 体外转录 显微注射 牛卵母细胞 孤雌激活 线粒体JC-1染色 |
| DOI： |
| 分类号: |
| 基金项目:陕西省自然科学基金项目(2009JM3008) |
|
| Correlation between ubiquitin gene localization and mitochondrial distribution in bovine parthenogenetic embryo |
|
MA Miaomiao, LI Wei, JING Yong, et al
|
|
Northwest A&F University
|
| Abstract: |
| 【Objective】This study aimed to locate ubiquitin gene during development of bovine parthenogenetic embryonic and its correlation with mitochondrial distribution.【Method】Plasmids pVenus-Ub were recombined by inserting artificial synthesis ubiquitin gene into pVenus between BamHⅠ and Hind Ⅲ before being transfected into Hela cells by Lipofectamine 2000 Reagent.Then,fluorescence microscope and PCR were used to observe the expression of pVenus-Ub in Hela cells.In addition,pVenus-Ub was transcribed to cRNA using in vitro transcription kit to cRNA,and injected into bovine oocytes.The expression and localization of oocyte were observed with fluorescence microscope after mature oocytes were activated by ionomycin.Meanwhile,the relationship between ubiquitin and mitochondrial distribution of stained oocyte mitochondria with JC-1 fluorescence dye were observed.【Result】The recombinant vector can be expressed in Hela cells.Ubiquitin was found in bovine oocytes in vitro mature and parthenogenetic process throughout the cytoplasm and the most concentrated areas were cortex and nucleus,where ubiquitin and mitochondria were co-located.【Conclusion】Ubiquitin eukaryotic and parthenogenetic embryos expression system was successfully established.And it was confirmed that distribution of ubiquitin genes and mitochondria were connected during oocyte maturation and early embryonic development. |
| Key words: Ubiquitin in vitro transcription microinjection bovine oocytes parthenogenetic activation mitochondria JC-1 staining |
