猪UBXNI基因启动子区突变鉴定及其与系水力相关性的研究
发布时间：2018-08-20 19:15
【摘要】：肌肉细胞的形态与结构主要是由细胞骨架维持,对系水力的维持有着相当重要的作用。RhoA(Ras同源蛋白家族成员A)基因可以通过RhoA-ROCK信号途径影响应力纤维的形成过程和维持其稳态,影响肌动蛋白细胞骨架重组,使得细胞形态发生变化,蛋白结合水平升高,促进骨架蛋白收缩,对系水力(water holding capacity,WHC)具有潜在调节作用。另外有研究表明,UBXN1(UBX结构域包含蛋白1)作为一种泛素修饰调控蛋白,可以通过促进肌纤维蛋白质的降解进而影响到系水力。UBXN1除了包含一个UBA结构域以外,其C端还包含一个UBX结构域,此结构域与泛素具有一定的同源相似性,它可以与泛素链结合,参与介导泛素蛋白酶体途径,从而可以调节肌纤维蛋白质的降解。肌纤维蛋白的降解一方面可以减少胴体屠宰后肌肉细胞的收缩,防止过多的水分流失,另一方面降解的蛋白提供了水分储存的空间,对于系水力水平都有提高。因此本实验将RhoA和UBXN1作为候选基因,研究这两个基因启动子区域的单核苷酸多态性(Single nucleotide polymorphism,SNP)对基因表达水平以及肌肉系水力水平的影响。本实验首先选取高低组WHC样品各16个,用荧光定量PCR检测两个候选基因在系水力高低组中的表达水平,发现两个基因在系水力高低组的表达水平均有差异。再将高低组样品分别进行DNA混池,将RhoA基因和UBXN1基因启动子区2000bp序列测序,进行高低组序列对比。结果在UBXN1基因启动子区得到了在WHC高组和低组中等位基因分布模式有差异的SNP位点c.-379TG。采用PCR-SSCP技术对170头杜长大三元杂交猪的c.-379TG位点进行大群基因分型,经过性状关联分析后发现TT型的系水力显著高于GG型(p0.01),随后对两组基因型各9个个体的UBXN1表达水平进行定量分析,结果发现TT基因型的UBXN1基因表达水平显著高于GG型(p0.05)。用双荧光素酶活性分析两种不同基因型的启动子活性差异,报告结果显示TT型启动子活性高于GG型,二者差异显著(p0.05)。最后我们对SNP位点c.-379TG进行了转录因子在线预测,发现该位点的两个等位基因的转录因子结合情况不同。通过对UBXN1基因启动子区SNP的研究为提高猪肉系水力水平提供了理论参考依据。
[Abstract]:The morphology and structure of muscle cells are mainly maintained by cytoskeleton. RhoA (member of Ras homologous protein family A) gene can influence the formation of stress fibers and maintain its homeostasis through RhoA-ROCK signaling pathway. It can affect the recombination of actin cytoskeleton, change the cell morphology, increase the level of protein binding, promote the contraction of skeleton protein, and play a potential role in regulating the hydrodynamic (water holding capacity (WHC). In addition, some studies have shown that UBXN1 (UBX domain containing protein 1), as a ubiquitin modified regulatory protein, can promote the degradation of myofibrillar proteins and thus affect the waterpower of the line. UBXN1 contains a UBA domain. The C-terminal also contains a UBX domain, which is homologous to ubiquitin. It can bind to the ubiquitin chain and mediate the ubiquitin proteasome pathway, thus regulating the degradation of myofibril proteins. On the one hand, the degradation of myofibrillar protein can reduce the contraction of muscle cells after carcass slaughter, and prevent excessive water loss, on the other hand, the degraded protein can provide water storage space, and improve the hydraulic level of the system. Therefore, RhoA and UBXN1 were used as candidate genes to study the effects of single nucleotide polymorphisms (SNP) in the promoter region of these two genes on gene expression and muscle hydraulic level. In this experiment, 16 samples of high and low WHC were selected, and the expression level of two candidate genes was detected by fluorescence quantitative PCR, and it was found that the expression levels of two candidate genes were different in the high and low hydraulic groups of the lines. The RhoA gene and UBXN1 gene promoter region 2000bp sequence were sequenced, and the high and low group sequences were compared. Results in the promoter region of UBXN1 gene, the SNP locus c.-379TG, which had different distribution patterns of alleles in high and low WHC groups, was obtained. Large group genotyping of c.-379TG loci in 170 Duroc crossbred pigs was carried out by using PCR-SSCP technique. It was found that the hydraulic capacity of TT genotype was significantly higher than that of GG type (p0.01), and the UBXN1 expression levels of 9 individuals with TT genotype were analyzed quantitatively. The results showed that the UBXN1 gene expression level of TT genotype was significantly higher than that of GG genotype (p0.05). Double luciferase activity was used to analyze the difference of promoter activity between two genotypes. The results showed that the promoter activity of TT type was higher than that of GG type, and the difference was significant (p0.05). Finally, we predicted the transcription factors of SNP locus c.-379TG on line, and found that the two alleles of SNP locus had different transcription factor binding. The study on SNP of UBXN1 gene promoter provides a theoretical reference for improving the hydraulic level of pork line.
【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
[Abstract]:The morphology and structure of muscle cells are mainly maintained by cytoskeleton. RhoA (member of Ras homologous protein family A) gene can influence the formation of stress fibers and maintain its homeostasis through RhoA-ROCK signaling pathway. It can affect the recombination of actin cytoskeleton, change the cell morphology, increase the level of protein binding, promote the contraction of skeleton protein, and play a potential role in regulating the hydrodynamic (water holding capacity (WHC). In addition, some studies have shown that UBXN1 (UBX domain containing protein 1), as a ubiquitin modified regulatory protein, can promote the degradation of myofibrillar proteins and thus affect the waterpower of the line. UBXN1 contains a UBA domain. The C-terminal also contains a UBX domain, which is homologous to ubiquitin. It can bind to the ubiquitin chain and mediate the ubiquitin proteasome pathway, thus regulating the degradation of myofibril proteins. On the one hand, the degradation of myofibrillar protein can reduce the contraction of muscle cells after carcass slaughter, and prevent excessive water loss, on the other hand, the degraded protein can provide water storage space, and improve the hydraulic level of the system. Therefore, RhoA and UBXN1 were used as candidate genes to study the effects of single nucleotide polymorphisms (SNP) in the promoter region of these two genes on gene expression and muscle hydraulic level. In this experiment, 16 samples of high and low WHC were selected, and the expression level of two candidate genes was detected by fluorescence quantitative PCR, and it was found that the expression levels of two candidate genes were different in the high and low hydraulic groups of the lines. The RhoA gene and UBXN1 gene promoter region 2000bp sequence were sequenced, and the high and low group sequences were compared. Results in the promoter region of UBXN1 gene, the SNP locus c.-379TG, which had different distribution patterns of alleles in high and low WHC groups, was obtained. Large group genotyping of c.-379TG loci in 170 Duroc crossbred pigs was carried out by using PCR-SSCP technique. It was found that the hydraulic capacity of TT genotype was significantly higher than that of GG type (p0.01), and the UBXN1 expression levels of 9 individuals with TT genotype were analyzed quantitatively. The results showed that the UBXN1 gene expression level of TT genotype was significantly higher than that of GG genotype (p0.05). Double luciferase activity was used to analyze the difference of promoter activity between two genotypes. The results showed that the promoter activity of TT type was higher than that of GG type, and the difference was significant (p0.05). Finally, we predicted the transcription factors of SNP locus c.-379TG on line, and found that the two alleles of SNP locus had different transcription factor binding. The study on SNP of UBXN1 gene promoter provides a theoretical reference for improving the hydraulic level of pork line.
【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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