【作者】 赵永贞；

【导师】 曹斌云； 李奎；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 随着人类基因组计划的完成以及功能基因组学研究的不断深入,与人类生理和病理相关的大量的新基因被证实。然而,对于相对滞后的猪基因组研究而言,与之相应的基因并没有被发现。因此,本研究根据比较基因组学的原理,利用生物信息数据库资源,分离定位了猪MMP-19和MMP-23基因,并对其基因结构和功能进行了初步研究,取得了如下结果:1.通过电子克隆、普通RT-PCR和RACE相结合的技术获得了猪MMP-19的全长cDNA序列和MMP-23基因的全长CDS,通过同源基因序列比对对分离的基因进行了鉴定,推导出了相应基因编码的氨基酸序列并对其基本生化性质和功能域进行了预测。2.获得了猪MMP-19和MMP-23基因的DNA全长序列,并进行了基因结构分析。结果显示,MMP-19基因总长度约6340bp,由9个外显子和8个内含子组成;MMP-23基因总长度约3726 bp,由8个外显子和7个内含子组成。两个基因所有内含子的剪切都符合GT/AG规则。3.通过Real-time PCR技术对MMP-19和MMP-23基因在不同品种猪的不同组织中的表达进行了分析。结果表明,MMP-19基因在不同品种猪的肝脏中的相对表达量较高,在脾和卵巢中适度表达,在其他组织中的表达量较低;比较MMP-19基因在两个品种不同组织的表达发现,该基因在肝、脾和卵巢中的表达量在品种间存在差异,在通城猪的表达量明显高于长白猪;MMP-23基因在两个品种的心和卵巢中都有较高的表达,在脾、腹脂、输卵管、大肠、小肠、肺、舌和胃中适量表达,在其他组织中的表达量较低,但该基因在心和卵巢中的表达模式在不同品种间存在较大差异,具体表现为该基因在长白猪心脏中的表达量高于卵巢,在通城猪则相反。4.通过Real-time PCR技术对MMP-19和MMP-23基因在不同品种猪不同发育阶段背最长肌中的表达进行了分析。结果表明,两个基因在两个品种的表达模式完全一致,基因的表达量随发育胚胎发育呈上调。5.获得了猪MMP-23基因的约1400bp的5’端上游序列,经过多种软件的综合分析发现,该区段内存在典型的CpG岛型启动子,没有TATA框和CAAT框。CpG岛序列长506bp,GC含量达70.75,在该区域内富含转录因子结合位点,包括Sp1、Myod、Snail、MZF1、c-Ets、c-FOS等因子的结合位点。6.用猪×仓鼠辐射杂种板对猪MMP-19和MMP-23基因进行了染色体精细定位,结果显示,猪MMP-19基因位于5号染色体,与微卫星标记DK紧密连锁,LOD值为18.02;猪MMP-23基因位于8号染色体,与微卫星标记Sw2521紧密连锁,LOD值为11.69.7.通过测序在猪MMP-19基因发现了15个SNP位点。在3个品种的猪群(通城、大白和长白)中,利用Mse? -RFLP对位于第5外显子203bp处的多态进行了检测,并分析了不同品种中的基因频率和基因型频率。8.在7个品种的猪群(通城、五指山、贵州、巴马、莱芜、大白和长白)中,利用PCR-RFLP的方法分别对MMP-23基因第3外显子第131bp处的Mbo?-RFLP多态和第4外显子第150bp处的Bsh1236I-RFLP多态进行了检测,并分析了不同品种中的基因频率和基因型频率。9.以本试验室与通城县畜牧局种畜场合作建立的实验群体(3个纯种群,大白、长白和通城以及2个3元杂种群,大长通和长大通)为材料,分析猪MMP-19和MMP-23基因的多态位点与部分生产性状和部分免疫性状的关联。结果显示:MMP-19基因第5外显子第203bp处的多态位点不同基因型个体在白细胞数和血清中免疫球蛋白含量以及眼肌高度方面差异显著(P<0.05);MMP-23基因第3外显子第131bp处的多态位点不同基因型个体在血红蛋白浓度和血细胞平均血红蛋白含量方面差异显著(P<0.05);MMP-23基因第4外显子第150bp处的多态位点不同基因型个体在胸背结合处背膘厚度差异显著(P<0.05)。更多还原

【Abstract】 With achievement of human genome project and increasing deepen of gene function research, a lot of new genes were identified and characterized. However, the pig genome research is hysteretic, and the genes homologous of pig and human were not identified. Pig MMP-19 gene and MMP-23 gene were isolated and mapped by combining with bioinformatics and biotechnology in this research. Furthermore, the gene structure and function of these two genes were analyzed. These main results are as follows:1. Full length cDNA of pig MMP-19 gene and entire coding region of pig MMP-23 gene were isolated by combination with e-PCR, RT-PCR, and RACE. These two genes were identified by blasting with the homologous genes of human. The amino acid sequences of MMP-19 and MMP-23 were deduced and the domain of corresponding protein was predicted.2. The full-length DNA sequences of MMP-19 gene and MMP-23 gene were obtained and the genomic structures are further analyzed. The results showed MMP-19 gene spanned about 6340bp which composed of 9 exons and 8 introns. Pig MMP-23 gene spanned about 3726 bp which comprised of 8 exons and 7 introns. All the introns of the two genes were spliced by the GT/AG rule.3. The expression profiles of MMP-19 gene and MMP-23 gene in different tissues of Tongcheng and Landrace pig were analyzed using real-time PCR. The results showed MMP-19 were most highly expressed in the different pig breeds liver. Moderate expression was observed in spleen and ovary. There was a little expression in other tissues analyzed. Expression profiles of Tongcheng MMP-19 in different tissues was compared with those of Landrace pig’s, the results revealed the expression level of Tongcheng MMP-19 gene in liver, spleen and ovary is higher than those of Landrace. MMP-23 is highly expressed in the different pig breeds heart and ovary. Moderate expression is observed in spleen, oviduct, large intestine, small intestine, lung, tongue and stomach. There is a little expression in other tissues analyzed. However, the expression of this gene in heart and ovary between Tongcheng pig and Landrace pig was different. The expression level in heart is higher than that in ovary of Landrace pig, and the reverse results were found in Tongcheng pig.4. Temporal expression profiles of porcine MMP-19 and MMP-23 gene for development of skeletal muscle in Tongcheng and Landrace pigs were detected by real-time PCR, the results showed the expression mode of two genes in different pig breeds were completely same. 5. The 5’upstream sequence about 1400bp of pig MMP-23 was obtained. There was a classical CpG island type promoter, but without TATA box and CAAT box in this region. The length of CpG was 506bp, and the GC content reached 70.75%. This region contained many transcription factor binding sites, including Sp1+, Myod+, Snail+, MZF1-, c-Ets+, c-FOS, GC box+.6. Porcine MMP-19 and MMP-23 genes were physical mapped by the RH panel. MMP-19 gene was located on SSC5, which is closely linked to marker DK with LOD score threshold 18.02. MMP-23 gene was located on SSC8, which is closely linked to marker Sw2521 with LOD score threshold 11.69.7. The total of 15 SNPs were found in MMP-19 gene by sequencing. The polymorphisms at 203th bp of exon 5 of porcine MMP-19 gene was detected in Tongcheng pigs, Landrace, and Large Yorkshire, respectively using Mse?–RFLP, and genotypic and allelic frequencies in different breeds were determined.8. The Mbo?-RFLP polymorphisms at 131th bp of exon 3 and Bsh1236I-RFLP polymorphisms at 150th bp of the exon 4 in MMP-23 gene were detected in Tongcheng pigs, Wuzhishan mini-pigs, Guizhou mini-pigs, Bama mini-pigs, Laiwu pigs, Landrace, and Large Yorkshire using PCR–RFLP, and genotypic and allelic frequency were analyzed among different breeds.9. The partial polymorphic sites of MMP-19 and MMP-23 gene were genotyped in the experimental pig population constructed under the cooperation of our lab and animal husbandry bureau of Tongcheng county, which consisted of Tongcheng, Large White, Landrace and three-way crossbreds, Landrace×(Large White×Tongcheng) and Large White×(Landrace×Tongcheng). Association between genotypes, phenotypes of economic traits and immunological traits were analyzed, the results as followed: there were significant associations between genotype of polymorphisms at 203th bp of exon 5 of MMP-19 gene with content, leukocyte counts and loin-muscle height of serum immunoglobulin G; there were significant associations between genotype of polymorphisms at 131th bp of exon 3 of MMP-23 gene with concentration and content of hemoglobin; there were significant associations between genotype of polymorphisms at 150th bp of the exon 4 of MMP-23 gene with backfat thickness.更多还原