中国农业科学 ›› 2014, Vol. 47 ›› Issue (3): 574-585.doi: 10.3864/j.issn.0578-1752.2014.03.017

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

Cdc42蛋白在牛卵母细胞中的表达分布及对其成熟的影响

 刘扬1, 银凤霞银凤霞2, 白春玲白春玲2, 魏著英魏著英2, 昝林森昝林森1, 李光鹏李光鹏2   

  1. 1、西北农林科技大学动物科技学院/国家肉牛改良中心,陕西杨凌 712100;
    2、内蒙古大学生命科学院/国家转基因动物研究中心,呼和浩特 010070
  • 收稿日期:2013-05-17 出版日期:2014-02-01 发布日期:2013-09-24
  • 通讯作者: 李光鹏,E-mail:gpengli@imu.edu.cn
  • 作者简介:刘扬,E-mail:liuyang1738@163.com
  • 基金资助:

    国家“十二五”转基因生物新品种培育重大专项(2011ZX08007-002)、国家科技支撑计划项目(2011BAD28B04-03)、国家“863”计划项目(2011AA100307—02和2013AA102505)、国家“973”计划项目(2012CB722306)、国家自然科学基金项目(31272411)

Expression and Localization and Effect of Cdc42 Protein During the Maturation of Bovine Oocyte

 LIU  Yang-1, YIN  Feng-Xia-Yin-Feng-Xia-2, BAI  Chun-Ling-Bai-Chun-Ling-2, WEI  Zhu-Ying-Wei-Zhu-Ying-2, ZAN  Lin-Sen-Zan-Lin-Sen-1, LI  Guang-Peng-Li-Guang-Peng-2   

  1. 1、College of Animal Science and Technology/National Beef Cattle Improvement Center , Northwest A&F University, Yangling 712100, Shaanxi;
    2、College of Life Science/National Research Center for Animal Transgenic Biotechnology, Inner Mongolia University, Hohhot 010070
  • Received:2013-05-17 Online:2014-02-01 Published:2013-09-24

摘要: 【目的】探明Cdc42蛋白在牛卵母细胞成熟过程中的表达及分布规律,并且研究抑制Cdc42活性对于牛卵母细胞成熟的影响。【方法】首先通过收集体外成熟不同时间(0—24 h)的牛卵母细胞,用Western blotting的方法检测Cdc42蛋白在牛卵母细胞中的表达量变化情况,通过免疫细胞化学的方法检测Cdc42蛋白在牛卵母细胞中的分布及定位情况。根据GenBank公布的牛Cdc42基因(GenBank登录号:NM_001046332.1)的mRNA序列设计引物,并在正向和反向引物的末端分别加上KpnⅠ和EcoRⅠ酶切位点,用PCR的方法扩增牛Cdc42基因的CDS区,将PCR产物和pMD19T载体连接得到野生型的克隆载体pMD19T-Cdc42WT。用定点突变引物以pMD19T-Cdc42WT为模板进行PCR反应,得到Cdc42的显性负性突变体Cdc42T17N的克隆载体pMD19T-Cdc42T17N。将pMD19T-Cdc42WT和pMD19T-Cdc42T17N分别酶切后,将Cdc42WT(775 bp)和Cdc42T17N(775 bp)片段分别连接到pcDNA3.1(+)的多克隆位点区域,得到相应的原核表达载体pcDNA3.1(+)-Cdc42WT和pcDNA3.1(+)-Cdc42T17N。用体外转录的方法分别得到Cdc42WT和Cdc42T17N的cRNA,用显微注射的方法将相应的cRNA分别注入到牛GV期卵母细胞质内,检测其成熟率的变化。【结果】①Cdc42蛋白分别在牛卵母细胞中成熟培养0、4、8、12、16、20以及24 h时表达量没有明显差异,但是其分布规律随着减数分裂的进行发生着动态的变化:Cdc42蛋白在卵母细胞皮质区域集中分布的模式在生发泡(germinal vesicle,GV)期卵母细胞中很少出现,而在第一次减数分裂中期(metaphase Ⅰ,MⅠ)卵母细胞中出现得最多也最为明显;Cdc42蛋白在染色体附近的皮质富集的现象开始出现于前中期(pro-metaphase Ⅰ,pMⅠ),并且出现频率从pMⅠ期到第2次减数分裂中期(metaphase Ⅱ,MⅡ)逐渐增多;Cdc42蛋白和纺锤体重叠定位的现象在第1次减数分裂后期(anaphase Ⅰ,AⅠ)到末期(telophaseⅠ,TⅠ)的卵母细胞中占有有很高比例,而到MⅡ期时这种现象又很少出现。②从牛卵母细胞的总cDNA中扩增Cdc42基因得到785 bp的片段,构建的牛Cdc42的野生型及其显性负性突变体的克隆载体pMD19T-Cdc42WT和pMD19T-Cdc42T17N经酶切鉴定和以及测序比对后,符合预期结果,即成功克隆了牛野生型Cdc42基因并得到其显性负性突变体Cdc42T17N;③构建的相应的原核表达载体pcDNA3.1(+)-Cdc42WT和pcDNA3.1(+)-Cdc42T17N经酶切鉴定和测序比对,符合预期结果,经过体外转录均得到一个3079 bases和987 bases的RNA片段,符合预期结果,即成功构建了相应的原核表达载体并获得相应的cRNA;④与正常成熟培养组和注射Cdc42WTcRNA的牛卵母细胞相比,注射Cdc42T17NcRNA的成熟率显著降低(P<0.05)。【结论】卵母细胞在卵泡发育过程中就已经积累了足够的Cdc42蛋白,而在减数分裂过程中Cdc42可以在卵母细胞的皮质以及纺锤体的位置发挥作用,而且正常的Cdc42活性是牛卵母细胞完成成熟过程并排出第一极体所必需的。

关键词: Cdc42 , 免疫细胞化学 , 显性负性突变体 , 体外转录 , 显微注射

Abstract: 【Objective】 This study is designed to ascertain the expression and localization of Cdc42 protein during bovine oocyte maturation, and to clarify the effect of Cdc42 inhibition on maturation of bovine oocytes. 【Method】 Bovine oocytes in vitro matured for different times (0-24 h) were collected. Western blotting and immunocytochemical technique were adopted to detect the expression and distribution of Cdc42 protein from oocytes collected at different stages of in vitro maturation. A pair of primers with restriction enzyme sites of KpnⅠand EcoRⅠat the ends of forward primer and reverse primer respectively, was designed based on the mRNA sequence of bovine Cdc42 which was obtained from Genbank (accession No.: NM_001046332.1). The CDS of bovine Cdc42 gene was amplified by PCR, and then ligated with pMD19T vector to construct pMD19T-Cdc42WT, the cloning vector wild type of Cdc42. A pair of site-directed mutagenic primers was used in a PCR to obtain pMD19T-Cdc42T17N, the cloning vector of the dominant negative mutant of Cdc42. Both pMD19T-Cdc42WT and pMD19T-Cdc42T17N were digested by restriction endonuclease to obtain the fragment Cdc42WT (775 bp) or Cdc42T17N (775 bp). The obtained fragments were ligated to the multiple cloning site of pcDNA3.1(+) to construct the prokaryotic expression vectors. The cRNA of Cdc42WT and Cdc42T17Nwas synthesized by in vitro transcription, and then micro injected into the cytoplasm of bovine GV stage oocytes. The maturation rate of injected oocytes was detected. 【Result】 The expression level of Cdc42 protein remained constant over the whole bovine oocytes maturation process, but the distribution pattern varied as the meiosis proceeded. The distribution pattern that Cdc42 protein gathering at the cortex region was hardly found in oocytes of germinal vesicle (GV) stage, but occurred frequently and remarkably in metaphase Ⅰ (MⅠ) oocytes. The distribution pattern that Cdc42 protein gathering at the cortex region nearing meiosis apparatus rather than the other region of cortex was found in the oocytes at the stage of pro-metaphase Ⅰ (pMⅠ), and growingly frequent till to the stage of metaphase Ⅱ(MⅡ). The percentage of the pattern that Cdc42 overlaps to spindle was higher in the oocytes at the stage of anaphase Ⅰ (AⅠ) to telophaseⅠ (TⅠ), but went down when it comes to the MⅡ stage. Fragments of 785 bp were obtained via amplifying Cdc42 gene from the total cDNA of bovine oocytes. The cloning vector of wild type Cdc42 and the dominant negative mutant pMD19T-Cdc42WT and pMD19T-Cdc42T17N accorded with anticipation through the identification of restriction endonuclease digestion and sequence alignment. So bovine wild type Cdc42 gene and mutant Cdc42T17N were cloned successfully. The constructed prokaryotic expression vectors pcDNA3.1 (+) -Cdc42WT and pcDNA3.1 (+) -Cdc42T17N accorded with anticipation through the identification of restriction endonuclease digestion and sequence alignment, RNA fragments of 3079 bases and 987 bases were obtained by in vitro transcription in which the vector pcDNA3.1 (+) -Cdc42WT and pcDNA3.1 (+) -Cdc42T17N were used as the templates. The maturation rate of Cdc42T17N injected group was lower compared with Cdc42WT injected group and non-injected group (P < 0.05). 【Conclusion】 It was concluded that Cdc42 protein might be accumulated in the cytoplasm of oocytes during the follicular development, and was functional at the region of cortex and spindle during miosis of bovine oocyte. The normal activity of Cdc42 was necessary to bovine oocyte maturation and extrusion of first polar body.

Key words: Cdc42, immunocytochamical, dominant negative mutant, in vitro transcription, micro injection