【作者】 朱银城；

【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， 2010， 硕士

【摘要】 根据梅山猪(Meishan)与大白猪(Large White)背最长肌组织比较蛋白质组学分析发现磷酸葡萄糖变位酶1(PGM1)在大白猪中高表达,因此PGM1可以作为影响猪肉品质的一个较好的候选基因。PGM同工酶是在糖类代谢过程中起着关键作用的一类酶。鉴于PGM1丰富的多态性使得它在遗传学和法医学上得到了广泛的应用。本研究的主要目的是为分子进化研究奠定基础,为实现分子育种寻找较好的分子标记并初步探索猪PGM1基因3’-UTR在转录后调控中的潜在作用。本研究利用分子生物学技术及生物信息学知识主要对猪PGM1基因进行了研究,研究结果如下：(1)在我们的研究中,首先从猪骨骼肌组织分离了一个普遍存在于各组织的PGM1亚型的cDNA,CDS为1,689bp,编码562个氨基酸,与人相应转录本的氨基酸序列相似性达99%。利用推断的猪PGM1氨基酸序列和另外选择的13个物种的氨基酸序列进行了系统树分析,这些物种包括了动物界、植物界和原核生物界,分析结果表明PGM1能较好地将各界生物分开,猪与牛的PGM1比与人的PGM1有着更高的相似性,猪与牛被很好的归为独立的树枝系,且与人在同一个大树枝系里。(2)先前比较蛋白质组学研究暗示PGM1在猪上可能存在翻译后修饰的差异,因为经二维凝胶电泳(2D-E)分离及质谱鉴定发现猪PGM1在蛋白胶上存在两个独立的点。然而,在我们的研究中未发现PGM1存在任何错义突变的现象更加支持这个推断。(3)位于猪PGM1基因编码区(CDS)的突变位点A420G、C462T和3’-UTR的突变位点G1693A分别由PCR-RFLP和PCR-SSCP鉴定。对CDS内突变位点分析表明A和C等位基因频率在中国地方猪种中显著高于进口的猪种；G和T等位基因频率在国外猪种中显著高于中国地方猪种。(4)在284头“大白猪×梅山猪”的F2代群体中对前两个位点与猪重要经济性状进行GLM过程,相关性分析结果表明C462T与猪瘦肉率(LMP)显著相关(P<0.05),经卡方(χ2)适合性检验表明该等位基因座在大白猪(χc2<χ20.05(df))与长白猪(χc2<χ20.05(df))群体中基因型分布没有偏离哈代温伯格平衡(HWE)。(5)我们也利用半定量PCR的方法研究了在不同组织中猪PGM家族包括PGM1、PGM2、PGM3和PGM5基因mRNA的表达模式,以及利用实时荧光定量PCR的方法研究了在不同肌肉组织中和不同时期背最长肌组织中猪PGM1基因mRNA的表达模式。在所有分析的组织中,结果表明PGM1在半腱肌中表达最低,在背最长肌中较其它组织表达高,我们推测在背最长肌中PGM总酶活力主要来源于PGM1。此外,据文献报道3’-UTR和miRNA结合会抑制蛋白质的翻译过程,于是我们成功构建了将猪PGM1基因完整3’-UTR插入到pEGFP-C1载体多克隆位点(MCS)的真核表达载体,这为下一步进行转录后调控研究打下了基础。更多还原

【Abstract】 Phosphoglucomutasel(PGM1) is up-regulated in Yorkshire muscles according to proteome analysis of porcine Longissimus Doris between Meishan and Yorkshire, Thus it is a good candidate gene affecting the pork quality. The isozymes of PGM are central to carbohydrate metabolism. PGM1 has also achieved prominence as a key marker in genetic linkage mapping and in forensic science. The aim of this work was to establish foundation for evolution research, to seek better SNPs for MAS and to investigate the potential function of 3’UTR of PGM1 on post-transcriptional level. In our research, methods of molecular biology and bioinformatics were used to study the PGM1 gene and the results are as follows:(1) Here we have isolated only one cDNA clone type corresponded to the ubiquitous transcript of PGM1 in porcine skeletal muscles, CDS is 1,689bp and encodes 562 amino acids, which is 99% similarity with the ubiquitous transcript of human PGM1. Putative amino acid sequence of Sus scrofa was closer to B.taurus PGM1 than H.sapiens on the basis of phylogenic tree among 14 selected species. These sequences spanned animal kingdom, plant and prokaryote.(2) It is implied that pig PGM1 has at least two different products since PGM1 was separated into two points in albumin gel by 2D-E during the previous studies. However, no missense mutation was detected for the diversity of swine PGM1. We suggested that the PGM1 have disparity in post-translational modification.(3) The samesense mutations A420G and C462T of which genotypes and allelic frequencies were analyzed in CDS and mutational site G1693A in 3’-UTR of PGM1 are confirmed by using PCR-RFLP and PCR-SSCP respectively. For the former two SNPs, the frequencies of the’A’ and’C allele were dominant in Chinese indigenous breeds.(4) The results of correlation analyses between genotype and traits, which were performed by GLM in 284 pigs of the’Yorkshire×Meishan’F2 resoure population, showed that C462T has significant correlation with lean meat percentage(P<0.05). Chi-square test indicated that no deviation from HWE was observed in Landrace(Xc2<χ20.05(df))and Yorkshire(χc2<χ20.05(df)) population.(5) We have also studied the temporal and spatial expression profile of the PGM superfamily except PGM4 by semiquantitative RT-PCR and quantitative real-time PCR(qRT-PCR). PGM1 has higher expression in longissimus Dorsi, the lowest in Semitendinosus. We speculate that the total enzymatic activity of PGM in longissimus Dor si come from PGM1.In addition, the complete 3’-UTR, which is possibly combined with miRNAs inhibiting the translation of swine PGM1 gene, was successfully inserted into the multiple cloning site(MCS) of pEGFP-C1 vector for the further study on post-transcriptional regulation.更多还原