【作者】 赵振华；

【导师】 王杏龙；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 胚胎发育一直是生命科学研究的主题之一。从一个单细胞发育成一个多细胞的个体,是许多基因和蛋白质在时间和空间上顺序表达的结果,核内基因的遗传信息控制着细胞质内蛋白质的合成,细胞质中的调控因子又反过来控制以后的发育时期中核内遗传信息的表达,胚胎内这种核与质之间的矛盾,相互作用和相互制约,推动着发育过程的进行。通过卵母细胞的体外成熟、体外受精、体外培养的方法和常规同期发情、超数排卵处理,获取山羊体外培养的成熟卵母细胞、2细胞、4细胞和8～16细胞胚胎、桑椹胚和囊胚及体内发育的2细胞、4细胞和8～16细胞胚胎、桑椹胚和囊胚。运用mRNA差异显示技术和蛋白质相关技术,分析胚胎发育不同阶段基因表达和蛋白质组的变化,从分子水平了解山羊胚胎发育过程中基因和蛋白质整体变化规律,探讨不同发育阶段基因表达的模式和胚胎发育机理,以期为哺乳动物胚胎早期发育基因表达的分子调控机制研究提供参考。mRNA差异显示共筛选到了44条分别在不同时期胚胎特异表达的片段,其中成熟卵母细胞4条,2细胞期7条(体内5条,体外2条),4细胞期8条带(体内5条,体外3条),8～16细胞期14条带(体内11条,体外3条),桑椹胚和囊胚9条带(体内8条,体外1条),全程表达2条(体内1条,体外1条),经过测序及比对分析,表明有10条带与已知的功能基因或调控基因相似,其余条带为未知功能基因或无相似基因。其中:S12在卵母细胞中特异性表达与蛋白激酶基因具有较高的同源性(98%),蛋白激酶对卵母细胞成熟过程中基因转录、调控和表达调控起到重要作用;Y62序列在2细胞胚胎中特异性表达与磷酸酶基因具有较高的同源性(98%),磷酸酶通过信号传递,对细胞的分裂活动具有重要的调控作用;E66序列在2细胞胚胎中特异性表达与动力球蛋白基因具有较高的同源性(95%),动力球蛋白为胚胎发育提供能量,从而对早期胚胎发育过程中的基因转录调控和表达调控起到重要作用;Y72序列4细胞胚胎中特异性表达与表皮生长因子受体基因具有较高的同源性(89%),EGF通过刺激胚胎细胞分裂增殖,而影响早期胚胎的生长发育;Sg78序列在4细胞和8-16细胞胚胎中特异性表达与牛醛氧化酶(脱氢酶)基因具有较高的同源性97%,E8序列在8-16细胞胚胎中特异性表达与氨基乙酸脱氢酶基因具有较高的同源性(97%),氧化酶(脱氢酶)通过调控ATP的合成,提供细胞活跃代谢以及DNA去甲基化和核内基因组再程序化时的能量需要;S81序列在8-16细胞胚胎中特异性表达与连接蛋白基因具有较高的同源性(97%),连接蛋白促进卵裂球间缝隙连接,促进胚胎的致密化和囊胚的形成;S97序列在桑椹胚和囊胚中特异性表达与老鼠白血病抑制因子(LIF)受体基因具有较高的同源性(95%),LIF是调节胚胎发育、启动胚胎着床过程的重要细胞因子,在妊娠识别过程中起到了重要作用;Y97序列在桑椹胚和囊胚中特异性表达与生长激素(GH)基因具有较高的同源性(91%),GH是调节调节母体的妊娠生理,影响生殖激素的分泌,从而推动胚胎生长、分化;Sg6789为早期胚胎发育各时期均表达的基因,其序列与肥胖基因(ob)转录翻译的Leptind蛋白具有较高的同源性(96%),Leptin作用于卵母细胞的成熟以及早期胚胎的分裂,参与早期胚胎的生长发育过程。体内外胚胎蛋白经蛋白质电泳和染色,能够检测到的蛋白斑点数为195个,其中卵母细胞14个蛋白质斑点;2细胞胚胎检测到32个蛋白质斑点,其中体内16个,体外16个,匹配的蛋白斑点为15个,匹配率为93.8%;4细胞胚胎检测到39个蛋白质斑点,其中体内21个,体外18个,能匹配的蛋白斑点为16个,匹配率为76.9%;8~16细胞期检测到53个蛋白质斑点,其中体内29个,体外24个,能匹配的蛋白斑点为19个,匹配率为71. 7%;桑椹胚和囊胚时期57个蛋白质斑点,其中体内30个,体外27个,能匹配的蛋白斑点为20个,匹配率为70. 2%。在体内发育的胚胎蛋白质电泳图谱中有11个蛋白斑点在体外培养的胚胎蛋白质电泳图谱中不存在,推测这些蛋白与胚胎的发育阻滞有关。在山羊胚胎早期发育过程中始终能够检测到的位点有4个,卵母细胞的蛋白斑点在2细胞胚胎中全部能够找到,这些蛋白质在胚胎发育进程中起重要作用。体内外不同时期差异蛋白和差异基因表达显示:山羊受精卵在卵裂后就开始合成胚胎自身的mRNA和蛋白质;不同阶段基因表达和胚胎蛋白质的组成存在着差异,说明胚胎基因的表达是依照一定的时空性的,前一阶段基因的不表达,影响下一阶段基因的表达,从而影响胚胎蛋白的合成,影响胚胎解除阻滞进入生长发育阶段;呈阶段性表达的基因及其翻译的蛋白,在随后邻近的胚胎发育阶段中消失,显示这些基因和蛋白可能与相应胚胎的形体特征发育有关,或进一步影响邻近胚胎的形体特征发育;全程表达的基因和蛋白,参与胚胎植入前发育的全过程,说明这些基因可能是生命活动所必需的。本实验在分子水平上对特异性表达的基因和蛋白进行分析,从蛋白质的变化初步了解了山羊胚胎结构基因激活表达的实施,但对于各特异性表达的基因在染色体上的精确定位、结构以及特异蛋白斑点的功能等方面,尚需通过大量分析和实验进行进一步研究。更多还原

【Abstract】 Embryo development is one of the most important subjects in life science research. Embryogenesis represents the critical transition from the single-celled fertilized embryo to the mature and multicellular embryo by undergoing a series of controlled cell divisions and cell differentiation events. It is a process of gene expression and protein regulation with time-space alternation. Nucleus gene determined translation progress of cytoplasm protein, on the contrary gene expression during embryogenesis and development stages was controlled by protein regulation factors. The impact between nucleolus and cytoplasm was promoted the development of embryo. The oocytes, 2-cell, 4-cell, 8~16-cell, morulae and blastocyst embryos of in vitro of goat were obtained through the process of oocytes matured in vitro (IVM), fertilized in vitro (IVF) and cultured in vitro (IVC). The 2-cell, 4-cell, 8~16-cell, morulae and blastocyst embryos of in vivo in goat were obtained using the way of estrus synchronization and superovulation. Silver staining mRNA differential display and SDS-PAGE were used in this study to analysis the variety of gene expression and proteome at developmental embryo. In order to establish the pattern of developmental gene expression and to understand the molecular mechanism of developmental goat preimplantation embryos, provide the academic theory of gene expression and control mechanism about mammal. Results as follow:44 different stage specific bands in total were screened. 5, 5, 11, 8 specific bands which only displayed in in vivo 2-cell, 4-cell, 8~16-cell, morulae and blastocyst stage were screened and 4, 2, 3, 3, 1 specific bands were displayed in in vitro oocytes, 2-cell, 4-cell, 8~16-cell, morulae and blastocyst. 2 specific bands were found all stage of embryo development. The sequencing and alignment results The sequencing and alignment results suggested that 10 differential expression bands were homologous with function genes or regulatory genes which already known from the GenBank. Other 34 differential expression bands were unknown genes.The specific band S12 which was expressed in oocytes was similar to protein kinase gene (98%). Protein kinase played an important role in regulation of gene transcription and expression in the progress of oocytes mature. The specific band Y62 which was expressed in 2-cell stage, was similar to phosphatas gene (98%). Cell divisions and cell differentiation were controlled by phosphates through signal transfer. E66 was 2-cell stage specific band and it was homologous with sapiens dynein gene (95%). Dynein regulated gene transcription and expression of preimplantation stage embryo in virtue of provided energy. Y72 was 4-cell stage specific band and it was homologous with epidermal growth factor receptor gene (89%). Effect of EGF on early embryos development in goat was stimulated cell divisions and differentiation. Sg78 was 4-cell and 8~16- cell stage specific band and it was homologous with aldehyde dehydrogenase gene (97%). E8 was 8~16-cell stage specific band and it was homologous with Glycine dehydrogenase gene (97%). Glycine dehydrogenase and aldehyde dehydrogenase were essential for ATP synthesis to provide energy for cell metabolism, DNA demethylation and genomic reprogramming. S81 was 8~16- cell stage specific band and it was similar to fibronection gene (97%). Fibronection was the regulator of connection between cleavage cells, it also can promoted come into being blastula. S97 was morulae and blastocyst specific band and it was homologous with leukemia inhibitory factor gene (95%). LIF is an important cytokine in early embryo development, which regulated fetation and pregnancy. The specific band Y97 which expressed in morulae and blastocyst was homologous with growth hormone gene (91%). It was promoted embryonic cell division and differentiation by the way of influence pregnancy physiology and reproductive hormones secretion. Sg6789 band was found at all stage of embryo development and it homologous with leptin receptor-like protein (obese) gene (96%). Leptin had the effects on oocytes maturation, embryonic cell division and differentiation.The changes of embryo protein in different development stags from in vivo and in vitro were discussed in the present research. Through electrophoresis and staining about 195 protein spots were detected in the gel. 14 protein spots were detected in MII. 32 protein spots were detected in 2-cell stage, 16 in in vivo and 16 in in vitro, respectively. Match protein spots were 15, the matching rate of these two patterns reached 93.7%. 39 protein spots were detected in 4-cell stage, 21 in in vivo and 18 in in vitro, respectively. Match protein spots were 16, the matching rate of these two patterns reached 76.9%. 53 protein spots were detected in 8~16-cell stage, 29 in in vivo and 24 in in vitro, respectively. Match protein spots were 19, the matching rate of these two patterns reached 71.7%. 57 protein spots were detected in morulae and blastocyst stage, 30 in in vivo and 27 in in vitro, respectively. The matching rate of these two patterns reached 70.2%. 11 protein spots were not detected from embryo in in vitro culture, which suggested those protein spots connected with block of development. 4 protein spots were detected from 2-cell stage to blastocyst stage and 2 protein spots were detected from MII stage to blastocyst stage. No change of proteins during embryo development showed that it played an important role in cell divisions and differentiation.Through the comparative analysis of the patterns of protein and mRNA of goat embryo in different stages, the results showed as follow:The zygote genome of goat synthesized mRNA and protein at the stage when embryo was start cleavage. The gene expression and protein composing were difference in different developmental stage of goat embryo, it indicated that process of gene expression was regulation with time-space alternation. The potential ability of gene expression was determined by previous correlation gene expression. Cell divisions and differentiation was controlled by gene expression, and it also related with block of development. Research on the changes of special protein spots and mRNA appeared in each stage showed that the majority of these spots disappeared in the following contiguous stage of embryo development. The results suggested that these proteins and genes existed in the corresponding stages might be related with the development of morphological character of corresponding embryo, even with contiguous embryo. The special proteins and genes which were expressed at all stage were concerned with preimplantation embryo development. Those proteins and genes were necessary for embryo activity.This study on the molecule level of the hereditary basis provided important information for researching the mechanism of developmental embryo. However, further research on the function, structure and orientation of special expressed gene in the gel from developmental embryo is required.更多还原