PGAM2基因启动子区变异鉴定及其与猪肉系水力的相关性研究

发布时间：2018-04-16 07:13

本文选题：系水力 + PGAM2　；参考：《南京农业大学》2016年硕士论文

【摘要】：系水力(water holding capacity,WHC)是指猪肉在施加外力的条件下保持水分的能力,是衡量猪肉质性状的重要标准之一。提高系水力可以避免猪肉中水分的大量流失,减少生产者在经济利益上的损失,同时可以改善猪肉的品质和风味。糖酵解途径是影响系水力高低的关键因素之一,磷酸甘油酸变位酶(phosphoglycerate mutase,PGAM2)、磷酸化酶激酶(phosphorylase kinase gamma 1,PHKG1)和乳酸脱氢酶(lactate dehydrogenase A,LDH4)基因编码糖酵解途径中的关键酶,并且研究表明这三个基因与多个肉质性状密切相关,它们可能通过糖酵解途径影响到系水力。因此,本实验以PGAM2、PHKG1和LDHA基因作为候选基因,筛选启动子区的单核苷酸多态性(Single nucleotide polymorphism,SNP)位点,研究基因启动子区SNP位点对基因表达水平的影响,及其与系水力的相关性,旨在寻找与系水力相关的分子遗传标记。本实验首先选取了系水力高组和低组各14个样品,通过对三个候选基因表达水平的分析,发现PGAM2和PHKG1基因的表达与系水力水平呈负相关(p0.05)。将高组和低组的DNA样品进行混池,通过比较测序,在PGAM2启动子区发现一个突变位点c.-360 TC,其等位基因在高组和低组之间的分布呈显著差异。通过等位基因特异性PCR技术(AS-PCR)对PGAM2基因c.-360TC位点在170头三元杂交猪中进行了基因分型,获得CC,TT和CT三种基因型。统计分析表明,CC型个体的系水力显著低于TT型个体(p0.05)。基因表达分析表明,CC型个体PGAM2基因表达水平显著高于TT型个体(p0.05)。通过双荧光素酶活性分析同样显示,CC型的启动子活性显著高于TT型(p0.05)。通过转录因子预测,发现SNP位点c.-360C可以结合CREB,TAXCREB和v-Maf三种转录因子,c.-360T可以结合LTSM一种转录因子。最后,通过对不同基因型样品中乳酸含量的测定和pH值的比较得出,与TT型比较,CC型样品中含有较高的乳酸含量和较低的pH值(p0.1)。结论:本研究在糖酵解关键基因PGAM2启动子区筛选到猪肉系水力相关的突变位点c.-360TC,不同基因型个体间PGAM2基因表达水平存在显著差异。研究结果提示,该突变位点可作为提高猪肉系水力的遗传标记。
[Abstract]:Water holding capacity (holding) is the ability of pork to maintain water under external force, which is one of the important criteria to measure the quality of pork.Improving the hydraulic capacity of pork can avoid the loss of water in pork, reduce the loss of producers' economic benefits, and improve the quality and flavor of pork at the same time.Glycolysis pathway is one of the key factors that affect the hydraulic capacity of the strain. Phosphoglycerate mutase PGAM2, phosphorylase kinase gamma 1PHKG1) and lactate dehydrogenase ALDH4 gene encode glycolysis pathway, and the key enzymes of glycolysis are phosphoglycerate mutase (PGAM2), phosphoglycerate mutase (PGAM2) and lactate dehydrogenase (lactate dehydrogenase ALDH4).The results showed that the three genes were closely related to many fleshy traits, and they might affect the hydraulic capacity of the lines through glycolysis.Therefore, PGAM2PHKG1 and LDHA genes were used as candidate genes to screen single nucleotide polymorphismSNPs of promoter region, and to study the effect of SNP locus in promoter region on gene expression level, and the correlation between PGAM2PHKG1 and LDHA gene expression.The aim of this study was to search for molecular genetic markers related to the water power of the line.In this experiment, 14 samples of high and low water power groups were selected. By analyzing the expression levels of three candidate genes, it was found that the expression of PGAM2 and PHKG1 genes was negatively correlated with the hydraulic level of the lines.A mutation locus c.-360 TC was found in the promoter region of PGAM2, and the distribution of alleles was significantly different between the high and low groups by comparing and sequencing the DNA samples of high and low groups.The c.-360TC loci of PGAM2 gene were genotyped in 170 ternary crossbred pigs by allele-specific PCR technique. Three genotypes of PGAM2 TT and CT were obtained.Statistical analysis showed that the hydraulic capacity of CC type individuals was significantly lower than that of TT type individuals.Gene expression analysis showed that the expression level of PGAM2 gene in CC type individuals was significantly higher than that in TT type individuals.Double luciferase activity analysis also showed that the promoter activity of CC type was significantly higher than that of TT type (P 0.05).Based on the prediction of transcription factors, it was found that SNP locus c.-360C could bind to three transcription factors, CREB-TAXCREB and v-Maf, which could bind to a transcription factor of LTSM.Finally, through the determination of lactic acid content and the comparison of pH value in different genotypes, it was concluded that compared with TT type, CC type samples contained higher lactic acid content and lower pH value (p 0.1).Conclusion: in this study, the hydraulic related mutation site c.-360TC was screened from the PGAM2 promoter of the key gene of glycolysis, and the expression level of PGAM2 gene was significantly different among the individuals with different genotypes.The results suggested that the mutation site could be used as a genetic marker to improve the hydraulic capacity of pork lines.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S828

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