【作者】 吴潇；

【导师】 李奎； 赵书红；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2006， 博士

【摘要】 现代育种技术使猪的生产性能得到了显著的改良，然而随着人民生活水平的提高，对猪肉品质的要求也随之提高，在改进猪肉口味的同时要求减少药物残留。畜牧生产的重点因此包括两个方面：一方面提高瘦肉的生长速度、改良肉质；另一方面提高猪的抗病性，从而降低生产成本，提高畜产品的质量，与国际接轨。本研究利用生物信息学与分子生物学技术相结合的方法，克隆和定位了泛素—蛋白酶体途径的相关新基因，并对这些基因与部分生长性状和免疫性状进行了初步关联分析，取得了如下结果：1．以人的同源基因的cDNA序列为探针，从GeneBank中搜索猪的同源EST，由EST构成的连接群设计引物，从猪的基因组中分离克隆了34个基因片段。这34个基因是(1)20S蛋白酶体基因alpha亚基基因PSMA1，PSMA2，PSMA3，PSMA5，PSMA6，PSMA7；20S蛋白酶体基因beta亚基基因PSMB1，PSMB2，PSMB3，PSMB4，PSMB5，PSMB6，PSMB7，PSMB8，PSMB9，PSMB10；(2)19S蛋白酶体基因PSMD7，PSMD8，PSMD9，PSMD10，PSMD11，PSMD12，PSMD13，PSMD14；(3)信号酶体基因SGN1，SGN2，SGN3，SGN4，SGN5，SGN6，SGN7，SGN8；(4)泛素特异活化酶基因USP6，USP10。2．采用电脑克隆策略结合RT-PCR技术，获得了PSMB4，PSMB6，PSMB8，PSMB9，PSMB10基因的完整编码区序列(coding sequence，CDS)和PSMB4，PSMB6，PSMB8，PSMB10基因的基因组全长，进行了物种同源基因序列比对分析和蛋白质序列的推导及功能域的预测。3．采用RT—PCR和Q-PCR方法检测了PSMB4，PSMB6，PSMBS，PSMB10基因在通城猪的心、肝、脾、肺、肾、肌肉、脂肪7个组织中的表达差异，除了PSMB8在心脏和肌肉中不表达外，其余基因在所检测的组织中都有表达。4．用猪×仓鼠辐射杂种板将新分离的25个基因进行了染色体精细定位，结果如下：PSMA1定位在SSC2，PSMA2定位在SSC18，PSMA3定位在SSC14，PSMA5定位在SSC4，PSMA6定位在SSC17，PSMB2定位在SSC6，PSMB3定位在SSC12，PSMB4定位在SSC4，PSMB6定位在SSC12，PSMB7定位在SSC1，PSMB8定位在SSC7，PSMB9定位在SSC7，PSMB10定位在SSC6，PSMD8定位在SSC6，PSMD9定位在SSC14，PSMD10定位在SSCX，PSMD12定位在SSC12，PSMD13定位在SSC2，PSMD14定位在SSC15，SGN2定位在SSC1，SGN4定位在SSC8，SGN5定位在SSC4，SGN6定位在SSC3，USP6定位在SSC12，USP10定位在SSC6。5．运用PCR-RFLP结合DHLPC，PCR-SSCP方法，检测了PSMA1，PSMA6，PSMB4，PSMB6，PSMB8，PSMB10等6个基因10个位点的多态性，分析了不同猪种中的基因频率和基因型频率，以及各等位基因在不同猪品种中的分布差异。6．以本室与通城县畜牧局种畜场合作所建立的实验群体中的长白猪(22头)、大白猪(23头)、通城猪(58头)、长大通(27头)、大长通(26头)为材料，分析了PSMA1，PSMA6，PSMB4，PSMB6，PSMB8，PSMB10基因的DNA多态位点与部分生产性状和免疫性状的关联，结果发现：除PSMB4基因外，其余基因和部分生产性状、免疫性状均有一定的关联。更多还原

【Abstract】 Nowadays, the production performance of pig has been improved by modern breeding technie. However, the more standard of living enhance, the more demand for quality of pork: good taste and few drug retention. So, pig breeding has two foci: one is improving lean growth rate and meat quality, the other is increasing the disease resistance to reducing growth cost. Only by excellent quality, the pig breeding industry of China can compete with other countries for world market.In this research, the porcine 20S proteasome related new genes were isolated and mapped by combining bioinformatics and biotechnology. Furthermore, association between genes and traits was analyzed. The main results are as follows:1. Pig ESTs were identified through standard BLAST analysis by using cDNA sequences of human homologous genes. Highly conserved pig ESTs were assembled to EST contigs. And primers selected from the contigs were used to isolate 34 novel porcine gene fragments from pig genomic DNA. The 34 novel porcine genes were as follows:(1) 20S proteasome alpha subunit genes PSMA1, PSMA2, PSMA3, PSMA5, PSMA6, PSMA7; beta subunit genes PSMB1, PSMB2, PSMB3, PSMB4, PSMB5, PSMB6, PSMB7, PSMB8, PSMB9, PSMB10; (2) 19S proteasome subunit genes PSMD7, PSMD8, PSMD9, PSMD10, PSMD11, PSMD12, PSMD13, PSMD14; (3) signalsome genes SGN1, SGN2, SGN3, SGN4, SGN5, SGN6, SGN7, SGN8; (4) ubiquitin specific protease genes USP6, USP10.2. Combining the in silico cloning and RT-PCR method, the entire coding region of the PSMB4, PSMB6, PSMB8, PSMB9, PSMB10 genes were obtained as well as the full length genomic DNA of PSMB4, PSMB6, PSMB8, PSMB9, PSMB10. The sequences were analyzed with species homologous genes and amino acid sequences were deduced to forecast functional domains.3. Expression profiles of PSMB4, PSMB6, PSMB8, PSMB10 genes of Tongcheng pigs were analysis by semi-quantitative RT-PCR assay and realtime-PCR. Except for PSMB8 gene unexpressing in heart and muscle, other genes are widely expressed in tissues of heart, liver, spleen, lung, kidney, muscle and fat. 4. 25 novel porcine genes were physical mapped by the RH panel. Results were showed as below: PSMA1 gene was located on SSC2, PSMA2 gene was located on SSC18, PSMA3 gene was located on SSC14, PSMA5 gene was located on SSC4, PSMA6 gene was located on SSC17, PSMB2 gene was located on SSC6, PSMB3 gene was located on SSC 12, PSMB4 gene was located on SSC4, PSMB6 gene was located on SSC12, PSMB7 gene was located on SSC1, PSMB8 gene was located on SSC7, PSMB9 gene was located on SSC7, PSMB10 gene was located on SSC6, PSMD8 gene was located on SSC6, PSMD9 gene was located on SSC14, PSMD10 gene was located on SSCX, PSMD12 gene was located on SSC12, PSMD13 gene was located on SSC2, PSMD14 gene was located on SSC15, SGN2 gene was located on SSC1, SGN4 gene was located on SSC8, SUN5 gene was located on SSC4, SGN6 gene was located on SSC3, USP6 gene was located on SSC12, USP10 gene was located on SSC6.5. Studies on DNA polymorphisms of PSMA1, PSMA6, PSMB4, PSMB6, PSMB8 and PSMB10 genes by PCR-RFLP combining DHLPC revealed ten polymorphic sites. Allele frequencies among different pig populations were determined.6. The all polymorphic sites were genotyped in the experimental pig population constructed under the cooperation of our lab and animal husbandry bureau of Tongcheng county (include 22 Landrace pigs, 23 Large White pigs, 58 Tongcheng pigs, 27 Landrace♂×(Large White×Tongcheng)♀pigs and 26 Large White♂×(Landrace×Tongcheng)♀pigs). Association of genotypes with phenotypes of production traits and immune traits were analyzed. The results showed that there are associations between the genotype of PSMA1, PSMA6, PSMB6, PSMB8, PSMB10 genes with some production traits and some immuno traits except PSMB4 gene.更多还原