【作者】 李家连；

【导师】 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2001， 硕士

【摘要】 抑制消减杂交技术(Suppression Subtractive Hybridizition，SSH)是以抑制PCR为基础的一种高效寻找差异表达基因的新方法，而通城猪和大白猪是各具优点，具有代表性的中外猪种，本文以SSH筛选两品种猪基因组差异，旨在寻找导致这两个品种猪性状表现差异不同的分子遗传基础，从而为猪的育种提供一条新的途径和方法。 本实验共得到如下结果： 1．建立了在猪中运用SSH进行分子生物学研究的方法。本课题率先在国内将SSH运用于猪的分子生物学研究，并得到了经初步鉴定的差异片段。 2．扩增得到基因组差异条带。通过对基因组酶切产物加接头，两次杂交后，进行两次扩增，得到两品种猪基因组差异条带。 3．克隆扩增产物建立了消减文库。将第二次PCR产物插入pGE-T Easy Vector得到消减文库。 4．初步鉴定了一些插入片段：通过EcoR Ⅰ酶切，得到400~600bp的插入片段，对酶切产物进行PCR扩增。得到与酶切的插入片段大小相近的扩增片段。 5．点杂交鉴定了一些差异片段。 6．本研究认为SSH可以在猪的分子生物学研究中得到很好应用。本课题所得到的结果，还有待进一步鉴定。得到差异片段后寻找差异基因、差异基因的功能及其在生产中的应用还有待进一步研究。更多还原

【Abstract】 Suppression Subtractive Hybridization (SSH) Based on Suppression PCR is a highly effective method for finding differently expressed genes in the genome of animals. Tongcheng and Laige White pigs are famous indigenous and foreign pig breeds with different excellent traits, so were used as experimental materials. In these study SSH was used to investigate the genome differcce, i.e molecular basis for the different traits between the two breeds, thereby to provide a new method for swine breeding.The results are as follow:1. A method for using SSH to study swine molecular biology was established. Using this method establish differently expressed fragments were obtained.2. Genome different bands were gained by PCR amplification. After adding adaptor and two times of hybridization, genome different bands between the two breeds were obtained by two times of PCR amplification.3. A subtractive library was built by cloning the amplified products. The library was built by inserting the second PCR products into pGEM-T Easy Vector.4. Some inserted fragments were initially identified After Eco R I digestion some fragments between 400 to 600bp were obtained, some fragments similar with those of the inserted fragments were gained by PCR amplification using the digested products as template.5. Some different fragments was identified by dot blot6. We came to a conclusion that SSH could be effectively used in the studies of swine molecular biology. The fragments obtained in this studies awaited further identification, meanwhile the jobs of finding new genes through differently expressed fragment, the function of the differently expressed genes and their application in animal production also awaited deepening and broadening.更多还原