【作者】 张进隆；

【导师】 赵兴绪；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2008， 博士

【摘要】 正常附植前胚胎的发育需要经过受精卵的多次分裂、胚胎基因组的活化等重要阶段。附植前胚胎是胚胎发育分化的关键,研究胚胎在不同发育阶段mRNA表达模式,便于了解胚胎正常发育的重要信息。在相关研究报道的基础上,本研究通过建立单个胚胎mRNA差异显示技术,并利用该技术对体外培养的奶牛早期胚胎发育基因的表达以及卵巢在有无黄体情况下卵母细胞差异表达基因进行研究,了解奶牛体外培养的早期胚胎在特定发育阶段及卵母细胞基因特异表达的模式,对于阐明胚胎发育的内在机制,分析影响卵母细胞发育潜能的因素具有重要意义。1.单胚DDRT-PCR方法的建立选取体外培养的4细胞期胚胎,用酸性台氏液除去透明带,胚胎裂解液裂解胚胎,以单胚和多胚mRNA作模板,用锚定引物和随机引物进行RT-PCR扩增,用持家基因GAPDH特异引物扩增作对照,分别用琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳比较单胚与多胚扩增结果,结果表明,单胚能够取得与多胚同样的效果,从单胚扩增的GAPDH与预期的一致,因此建立了的单胚DDRT-PCR方法是有效的。2.黄体对卵母细胞差异表达基因的影响为了研究卵巢在有无黄体状态下卵母细胞基因表达的情况,从兰州屠宰场采集奶牛的卵巢,分离卵母细胞,以单个卵母细胞的mRNA作为模板,用随机引物和锚定引物、一步法RT-PCR扩增卵母细胞基因,聚丙烯酰胺凝胶电泳检测有黄体和无黄体卵巢卵母细胞表达基因,发现电泳条带之间无明显差异,选择唯一条差异条带回收、纯化PCR产物连接到T载体,阳性克隆鉴定后,进行测序,同源性比较分析,与体外培养的牛早期胚胎的一条序列具有很高的同源性。3.奶牛早期胚胎基因表达的研究选取体外培养的奶牛2～16细胞期胚胎,以单胚mRNA作模板,进行RT-PCR扩增,扩增产物凝胶电泳检测,在胚胎基因组激活期间有8条选择差异条带,回收、转化、克隆,对阳性克隆进行测序,用Blast软件进行同源性检索,E1与Oct-4mRNA同源,E2与类似于牛细胞周期蛋白激酶调节亚基1同源,E3与牛THAP结构域9同源,E4与鼠胚胎NbME13.5、14.5同源,E5与牛高迁移率族蛋白同源,E6与鼠10日龄胚胎cDNA同源、E7与类似于牛11染色体开放阅读框同源,E8与牛Sec61γ亚单位同源。这些基因与细胞增殖、分化,细胞周期调节有关,在早期胚胎发育中发挥重要作用。更多还原

【Abstract】 Summary:Normal preimpletation development is characterized several cleavage division of the fertilized egg, activation of zygotic transcription, compaction and blastocoel formation, knowledge of genes and characterization of their expression patterns at different embryonic stages will provide critical information on genes which are important for normal preimplantation development, on the basis of the related research, by means of establishing bovine single embryo DDRT-PCR, dentials studies on gene expression in bovine embryo in vitro cultured had been carried out , bovine oocyte genes expression from ovaries with or without corpus luteum was studied at the same time. This is very important to study the elucidate embryo intrinsic development mechanism and anylize oocyte development competency.1. Establishment single bovine embryo DDRT-PCRThe method of single embryo RT-PCR plays an important role on investigating preimplantation development-related genes. selecting 4 cell embryo in vitro cultured, zone pellucida was lysised with acidic Tyrodes solution and embryos were lysised with lysis buffer, using single embryo and multiple embryos mRNA as templet, PCR amplification with radom primer and oligo-(dT) , the house keeping gene GAPDH PCR amplification with specific primer as control, amplification products was seprated on agrose gel electrophoresis(AGE) and polyacrylamide gel electrophoresis(PAGE). Multiple embryo result shows that ,the GAPDH was detected successfully in single embryo, Thus, method of single embryo RT-PCR is effective.That is multiple embryo and single ones has the same effects.2. Effect of corpus luteum on bovine oocyte genes expressionThe purpose of this study is to detect bovine oocyte genes from ovaries with or without corpus luteum Ovries had been collected from Lanzhou slaughterhouse which were divided into functional corpus luteum and no corpus luteum ,separating oocyte from ovries, mRNA of single oocyte lysates as templet with oligod(T) and radom primer,the oocyte genes had been amplified successfully by one-step reverse transcription polymerase chain reaction (RT-PCR), amplified products were detected with polyacrylamide gel electrophoresis(PAGE), there was no obvious difference in gel electrophoresis result, selecting the only one different gel electrophoresis belt to recover and purify ,PCR products was cloned into T-vector through sequencing and homologies analyzing ,it’s Homologous with Early Embryo In Vitro Produced bemiv Bos taurus cDNA 3-mRNA sequence.3.Research on bovine preimpletation embryonic genesSelected 2-16 cell embryos in vitro cultured, using single embryo mRNA as templet, amplified products were detected with PAGE, there are 8 different belt in EGA stage ,different gel electrophoresis belt to recover and purify ,PCR products was cloned into T-vector through sequencing and homologies analyzing ,with Blast software 8 differential expression fragments were isolated from early bovine embryo, Homologous analysis indicated that,E1 sequence is Homologous with M.musculus mRNA for Oct-4 protein ;E2 sequence is Homologous with Bos taurus similar to Cyclin-dependent kinases regulatory subunit 1;E3 sequence is Homologous with Bos taurus THAP domain containing 9; E4 sequence is Homologous with mg98c01.r1 Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA ;E5 sequence is Homologous with Bos taurus high-mobility group box 2; E6 sequence is Homologous with Mus musculus 10 days embryo whole body cDNA ; E7 sequence is Homologous with Bos taurus similar to Chromosome 11 open reading frame; E8 sequence is Homologous with Bos taurus Sec61 gamma subunit (SEC61G) these genes related to cell split ,cleavage, cell recycle regulation, then these genes play important role in early embryo development.更多还原