【作者】 梁磊；

【导师】 娜仁花；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖学， 2010， 硕士

【摘要】 哺乳动物的受精过程,是一个精密而有序的细胞之间的相互作用的过程,其最终的结果就是精卵间通过质膜融合作用,成为受精卵。目前,科学家们虽然通过基因组学理论和蛋白质组学技术在精子表面上发现了数量可观的与受精相关的分子,但是精卵融合过程是在顶体反应发生之后才进行的,其中精子和卵细胞的质膜必须通过相匹配的受体结合,而这个引发精卵融合的分子机制究竟是怎样运作目前还不清楚。在这些已经获得的与精卵融合过程相关的分子中,只有近期在精子上发现的IGSF(免疫球蛋白超家族)新成员Izumo基因,经过基因敲除的小鼠实验,证明它是精卵融合所必需的。内蒙古白绒山羊作为一种内蒙古地区培育多年的家畜,是羊毛和羊绒的主要来源之一。由于Izumo基因的发现对于了解绒山羊的受精机制有重要的指导意义,为了研究其在内蒙古白绒山羊精卵融合中的分子机制,本实验根据GenBank中已发表的牛的Izumo核苷酸序列,设计并合成了扩增绒山羊Izumo基因的1对引物,实验采用RT-PCR(逆转录聚合酶链式反应)扩增获得了Izumo基因部分序列,并对Izumo基因进行了克隆和序列分析。在此基础之上,以得到的绒山羊Izumo部分序列为模板,采用RACE(快速扩增cDNA末端)技术,扩增了雄性成年内蒙古白绒山羊睾丸组织中Izumo基因的全长序列,并钓取了该基因的CDS(编码区)。另外本实验还制备了地高辛标记的绒山羊Izumo cDNA探针,用利用RNA Northern印记技术,对内蒙古白绒山羊Izumo RNA进行分析,从而测定总RNA中编码Izumo基因的mRNA分子的大小和表达丰度。1、通过RT-PCR技术,获得484 bp的Izumo基因cDNA序列,与牛的Izumo基因同源性高达98%。2、通过RACE技术,获得1536 bp的全长序列,钓取到1035 bp的Izumo基因编码区序列,该编码区编码344个氨基酸。3、通过对绒山羊睾丸和附睾的头、体、尾部组织Izumo基因mRNA的RT-PCR组织表达鉴定,以及对该基因mRNA的Northern印迹鉴定,结果睾丸和附睾的头部、体部、尾部组织均显示阳性条带。4、本研究为进一步进行绒山羊Izumo基因在精卵融合过程中的功能研究奠定了基础。更多还原

【Abstract】 Mammalian fertilization process is a precise and orderly process of interaction between cells, the end result is that sperm-egg membrane fusion between the adoption of the role of a fertilized egg. At present, the scientists although the theory and through genomics proteomics technology found in the sperm surface fertilization associated with a significant number of molecules, but the sperm-egg fusion process is after the acrosome reaction carried out, in which sperm and egg plasma membrane must match the receptor binding, and this lead to what the molecular mechanism of sperm-egg fusion is unclear how it will operate. Has been obtained in these processes associated with sperm-egg fusion molecules, only recently discovered in the sperm of IGSF (immunoglobulin superfamily) new members of Izumo gene knockout mice after experiments prove that it is sperm-egg fusion necessary.Inner Mongolia White Cashmere goats for many years as a foster livestock in Inner Mongolia, is the main source of wool and cashmere, one of the research on the Izumo gene reported in this paper are first. Since the discovery of Izumo gene for understanding the mechanism of fertilization and goats have important guiding significance, in order to study in Inner Mongolia White Cashmere goats in the molecular mechanism of sperm-egg fusion, this study has been published under the GenBank, the nucleotide sequence of bovine Izumo, designed and synthesized goats Izumo gene amplification primers 1, experiment using RT-PCR (reverse transcription polymerase chain reaction) amplification of the Izumo gene sequences, and Izumo gene was cloned and sequenced. In this basis, to get some goats Izumo sequence as a template, using RACE (rapid amplification of cDNA ends) technology, the first expansion of the adult male testis Inner Mongolia Cashmere goats in Izumo gene sequences, and fishing took the gene CDS (coding region). Also this experiment was prepared digoxigenin labeled goat Izumo cDNA probe technology with the use of RNA Northern imprint on the Inner Mongolia White Cashmere goats Izumo RNA analysis, and thus the determination of total RNA, mRNA encoding Izumo gene expression of molecular size and abundance.1. By RT-PCR technique, the Izumo gene was 484 bp cDNA sequence, and the cattle Izumo gene homology as high as 98%.2. By RACE technology to obtain 1536 bp full-length sequence, fishing access to the 1035 bp of the Izumo gene coding sequence, the coding region encoding 344 amino acids.3. Through the goat testis and epididymis head, body, tail tissue Izumo gene mRNA in the RT-PCR identification of tissue expression, and mRNA of the gene in the Northern blotting results of testis and epididymis of the head, body, tail tissue show positive bands.4. This study further goats Izumo sperm-egg fusion in the process of gene function in the foundation.更多还原