發(fā)布時間：2018-04-16 07:13

  本文選題：系水力 + PGAM2��； 參考：《南京農業(yè)大學》2016年碩士論文

【摘要】：系水力(water holding capacity,WHC)是指豬肉在施加外力的條件下保持水分的能力,是衡量豬肉質性狀的重要標準之一。提高系水力可以避免豬肉中水分的大量流失,減少生產者在經濟利益上的損失,同時可以改善豬肉的品質和風味。糖酵解途徑是影響系水力高低的關鍵因素之一,磷酸甘油酸變位酶(phosphoglycerate mutase,PGAM2)、磷酸化酶激酶(phosphorylase kinase gamma 1,PHKG1)和乳酸脫氫酶(lactate dehydrogenase A,LDH4)基因編碼糖酵解途徑中的關鍵酶,并且研究表明這三個基因與多個肉質性狀密切相關,它們可能通過糖酵解途徑影響到系水力。因此,本實驗以PGAM2、PHKG1和LDHA基因作為候選基因,篩選啟動子區(qū)的單核苷酸多態(tài)性(Single nucleotide polymorphism,SNP)位點,研究基因啟動子區(qū)SNP位點對基因表達水平的影響,及其與系水力的相關性,旨在尋找與系水力相關的分子遺傳標記。本實驗首先選取了系水力高組和低組各14個樣品,通過對三個候選基因表達水平的分析,發(fā)現PGAM2和PHKG1基因的表達與系水力水平呈負相關(p0.05)。將高組和低組的DNA樣品進行混池,通過比較測序,在PGAM2啟動子區(qū)發(fā)現一個突變位點c.-360 TC,其等位基因在高組和低組之間的分布呈顯著差異。通過等位基因特異性PCR技術(AS-PCR)對PGAM2基因c.-360TC位點在170頭三元雜交豬中進行了基因分型,獲得CC,TT和CT三種基因型。統(tǒng)計分析表明,CC型個體的系水力顯著低于TT型個體(p0.05)。基因表達分析表明,CC型個體PGAM2基因表達水平顯著高于TT型個體(p0.05)。通過雙熒光素酶活性分析同樣顯示,CC型的啟動子活性顯著高于TT型(p0.05)。通過轉錄因子預測,發(fā)現SNP位點c.-360C可以結合CREB,TAXCREB和v-Maf三種轉錄因子,c.-360T可以結合LTSM一種轉錄因子。最后,通過對不同基因型樣品中乳酸含量的測定和pH值的比較得出,與TT型比較,CC型樣品中含有較高的乳酸含量和較低的pH值(p0.1)。結論:本研究在糖酵解關鍵基因PGAM2啟動子區(qū)篩選到豬肉系水力相關的突變位點c.-360TC,不同基因型個體間PGAM2基因表達水平存在顯著差異。研究結果提示,該突變位點可作為提高豬肉系水力的遺傳標記。
[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.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S828

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本文編號：1757808