【作者】 王彦芳；

【导师】 李奎；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2004， 博士

【摘要】 在过去很长一段时间里，猪育种的研究重点一直是提高瘦肉率，加快生长速度等，但是单纯追求高产使猪的抗病性降低，因为许多抗性性状和生产性状呈负相关。随着人民生活水平的提高，畜牧生产中已开始高度重视猪的健康、抗病力和猪肉品质，因为健康并充满活力的猪将大大减少用药量及猪肉的药物残留，增加市场竞争力。这就使得研究者们相继提出了从分子水平揭示疾病发生的原因，从而利用遗传学方法从遗传本质上提高动物对病原的抗性。 蛋白酶体激活因子PA28(Proteasome activator 28)和PA700(Proteasome activator 700)基因家族是一个研究热点，因为它们与MHC Ⅰ类分子介导的抗原提呈有密切关系，与机体的免疫能力直接相关。本研究利用生物信息学与分子生物学技术相结合的方法，克隆和定位了这两个家族的12个基因(PA28家族的2个基因和PA700家族的10个基因)，取得了如下结果： 1．结合猪EST数据库的信息和cDNA末端快速扩增(RACE)的方法，分离并鉴定了猪蛋白酶体激活因子PA28α和PA28β基因(PSME1和PSME2)的全长cDNA序列，GenBank数据库收录号分别为AF527990和AF527991。利用PCR的方法，克隆了PSME1和PSME2基因的基因组DNA全长序列并进行了序列分析，发现这两个基因有相似的基因结构，都由11个外显子和10个内含子组成。 2．用RT-PCR的方法，我们对PSME1和PSME2基因进行了组织表达谱分析。结果发现这两个基因在所检测的8个组织中均有表达，但在小肠和肾脏中的表达量相对低一些。 3．利用电脑克隆、RACE和RT-PCR的方法相结合，克隆了蛋白酶体激活因子PA700相关基因PSMC1、PSMC4和PSMC5共3个基因的全长cDNA序列，以及PSMC2和PSMD4基因的完整编码区(coding sequence，CDS)。进行了蛋白质序列的推导和功能域的预测。 4．根据猪EST数据库的已有数据，利用PCR的方法，分离了PSMC1-PSMC6和PSMD2-PSMD5共10个基因的基因组DNA片段并经测序确认。其中7个基因的序列已经提交给GenBank，获得了收录号。克隆了PSMC5基因的全长基因组DNA序列，发现该基因由12个外显子和11个内含子组成，所有的内含子和外显子的拼接位点都符合GT-AG规则。 5．用SCHP和IMpRH克隆板，对PSME1和PSME2基因进行了染色体区域定位和精细定位，结果表明这两个基因均被定位在猪7q12—q23，与T细胞受体因子α(T cell receptor alpha，TCRA)基因紧密连锁，LOD值分别为10.82和11.65。 6．用IMpRH克隆板，对PA700基因家族的10个基因进行了精细定位，结果表明，它们分布在8条不同的染色体上。PSMC1定位在7q；PSMC2定位于猪9号染华中农业大学博士学位论文色体;PSMC3基因定位于SsCZpl6;psMe4基因定位于sse6qlx一qxZ;PsMes和PsMD3基因都被定位于ssc12P;PsMc6和PsMDS基因均位于猪1号染色体的长臂;PsMDZ基因位于Ssc13q41;PsMD4荃因被定位在ssC4q21一q23。LOD值均大于8.仇 7.对琳28的2个基因和PA7OO的10个基因共12个基因进行了SNPs的检测，结果发现了10个可用PCR.R卫LP方法检测的SNPS，分别是PSMEI签因第8内含子的sphl·即LP(560C一560T);pSMCZ基因第5内含子的HPall一RFLp(279G一幻零);气PSMC3基因第3内含子的Mval·好LP(2 84G一18认);PSMCS基因第4内含子的Tail.RFLP(639G一639C)、第3内含子的D日el·RFLP(397A一397T)和第6外显子的Sael一RFLP(309C一309G)以及Munl.RFLP(120T一120C);PS刻吧6-基因第5内含子的M饰卜补LP(42A一42G);PSMD3基因第10外显子的Rsal一盯LP (286G一286C);PSMD4基因第9内含子的Hin61.R卫LP(703G一703A)。在PSMDZ基因中发现了用SSCP方法检测的多态性(10lC一10lT)。 8.对上述10个可用PCR·即LP方法检测的SNPs在二花脸、梅山猪、藏猪、清平猪、杜洛克和大白猪中进行群体遗传学分析，卡方检验结果表明，除了PSMC6和PSMD4基因，所有基因座的基因型频率在不同的品种中存在较大的差异。 9.在试验杂交群体(L X YT;YXLT)的上下代中检侧了这10个SNPs的遗传方式，均呈共显性遗传方式。 10.在荷兰学者赠送的资源家系中，对Ps坷FI基因第八内含子SPhl一孙LP的多态性与部分生产性状进行了关联分析，结果发现在不同基因型的个体中，猪断奶重存在显著差异(P<0.05)。 n.在我室与通城县畜牧局合作组建的试验群体中，采用方差分析的最小二乘分析法，对上述SNP，(除了PSM它1基因，PSMC弓基因介il-RFLP、Dde 1.R卫LP和P SMDZ基因)与部分生产性状(如:初生至上市平均日增重、达90kg或75kg的日龄、屠宰率、肌肉pH值等)、部分免疫性状(如:白细胞数、红细胞数、20周龄时血清中IgG的含量等)进行了初步的关联分析，结果发现，PSMCZ和PSMC6基因座不同基因型个体之间的性状没有显著的差异，PSMC3基因座不同基因型个体之间的三点平均背膘厚、红细胞数和红细胞压积存在极显著的差异(P<0.01)，PSMCS基因座对猪的6一7肋间的背膘厚、三点平均膘厚、板油率和平均血细胞容积有不同程度的影响，而PSMD3基因座不同基因型个体之间的平均血细胞容积存在显著差异(P<0.05)，PSMD4基因座不同基因型个体之间的肌肉pH值有显著的差异(P<0 .05)。更多还原

【Abstract】 Pig breeding is always focus on improving the lean percentage, increasing the growth rate et al over the past long periods. However, seeking the high performance traits merely decreased the disease resistance traits because the many disease resistance and performance traits present the negative correlation. The heath ware, disease resistance and the quality have been now paid more attention on the pig breeding and production because the healthy and vigor pigs would decrease the drug use and drug retention, increasing the market competition. So, the researchers want to explore the molecular mechanism of diseases, therefore to improve the disease resistance using the genetic methods.Proteasomal activator PA28 and PA700 gene family is the important research hot point, not only because these genes are play the key role in the antigen presentation mediated by the MHC class I, but also they are related to the immune capacity, oncogenesis and the disease resistance. Combining of the in silico cloning and the biotechnology, the 12 genes, which include 2 genes of the PA28 activator and 10 genes of the PA700 activator, were cloned and physical mapped. The main results are as follows:1. Combining the EST data and the Rapid Amplification of cDNA ends (RACE) method, full-length cDNA of the pig PSME1 and PSME2 gene were isolated and identified. Both sequences has been submitted the GenBank database, the accession number are AF527990 and AF527991. Using the PCR, the full-length genomic DNA sequences of the PSME1 and PSME2 genes were cloned and the sequences were analyzed. These two genes have the similar gene structure, both with the 11 exons and 10 introns.2. The reverse transcriptase - polymerase chain reaction (RT-PCR) revealed that porcine PSME1 and PSME2 genes expressed in all 8 tissues studied. The bands obtained in small intestine and kidneys were weak in both cases, relatively.3. Combining the in silico cloning, RACE and RT-PCR method, the full-length cDNA sequence of PSMC1, PSMC4 and PSMC5 gene and the entire coding region of the PSMC2 and PSMD4 genes were cloned. The amino acids were also been deduced and the motifs were been predicted.4. The genomic DNA fragments of PSMC1, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6 PSMD2, PSMD3, PSMD4 and PSMD5 genes were cloned using the method of PCR. The sequences of these 10 genes were used in the BLAST searches against the ’nr’ database and the results showed that all the amplified sequences from pig DNAcorresponded to the gene expected. Of which, the sequences of 7 gene has been deposited to GenBank and got the accession numbers. Full-length genomic DNA sequence of pig PSMC5 gene has been isolated and this gene comprised by 12 exons and 11 introns. All the splice sites of the exon/intron conformed to the GT/AG rule.5.The pig X rodent somatic cell hybrid panel (SCHP) was used for regional assignment of both genes and the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of both genes. Statistical analysis revealed that both genes are located to the pig chromosome 7q 12-23 and closed to T cell receptor factor a (TCRA) with LOD scores of 10.82 and 11.65 respectively.6. 10 genes of PA700 gene family described above were physical mapped by the RH panel. Results showed that these 10 gene distribution on the 8 different pig chromosome. PSMC1 gene was located on SSC7, PSMC2 gene was mapped to SSC9, PSMC3 and PSMC4 genes were mapped to the SSC2pl6 and SSC6qll-ql2, respectively; Both PSMC5 and PSMD3 gene were located to the SSC12p; PSMC6 and PSMD5 gene were located to the SSClq; PSMD2 gene and the PSMD4 gene are assigned to the SSC13q41 and SSC4q21-23 respectively. All the results were creditable due to the LOD scores.7. Identification for mutations in the amplified fragments of these 12 genes (including 2 genes of PA28 and 10 genes of PA700 genes) were performed and the 10 SNPs were detected by PCR-RFLP, these loci are as follows: PSME1 gene Sphl-RFLP (560C-560T, intron8), PSMC2 HpaII-RFLP (279G-279C, intron更多还原