【摘要】：肌肉細(xì)胞的形態(tài)與結(jié)構(gòu)主要是由細(xì)胞骨架維持,對(duì)系水力的維持有著相當(dāng)重要的作用。RhoA(Ras同源蛋白家族成員A)基因可以通過RhoA-ROCK信號(hào)途徑影響應(yīng)力纖維的形成過程和維持其穩(wěn)態(tài),影響肌動(dòng)蛋白細(xì)胞骨架重組,使得細(xì)胞形態(tài)發(fā)生變化,蛋白結(jié)合水平升高,促進(jìn)骨架蛋白收縮,對(duì)系水力(water holding capacity,WHC)具有潛在調(diào)節(jié)作用。另外有研究表明,UBXN1(UBX結(jié)構(gòu)域包含蛋白1)作為一種泛素修飾調(diào)控蛋白,可以通過促進(jìn)肌纖維蛋白質(zhì)的降解進(jìn)而影響到系水力。UBXN1除了包含一個(gè)UBA結(jié)構(gòu)域以外,其C端還包含一個(gè)UBX結(jié)構(gòu)域,此結(jié)構(gòu)域與泛素具有一定的同源相似性,它可以與泛素鏈結(jié)合,參與介導(dǎo)泛素蛋白酶體途徑,從而可以調(diào)節(jié)肌纖維蛋白質(zhì)的降解。肌纖維蛋白的降解一方面可以減少胴體屠宰后肌肉細(xì)胞的收縮,防止過多的水分流失,另一方面降解的蛋白提供了水分儲(chǔ)存的空間,對(duì)于系水力水平都有提高。因此本實(shí)驗(yàn)將RhoA和UBXN1作為候選基因,研究這兩個(gè)基因啟動(dòng)子區(qū)域的單核苷酸多態(tài)性(Single nucleotide polymorphism,SNP)對(duì)基因表達(dá)水平以及肌肉系水力水平的影響。本實(shí)驗(yàn)首先選取高低組WHC樣品各16個(gè),用熒光定量PCR檢測兩個(gè)候選基因在系水力高低組中的表達(dá)水平,發(fā)現(xiàn)兩個(gè)基因在系水力高低組的表達(dá)水平均有差異。再將高低組樣品分別進(jìn)行DNA混池,將RhoA基因和UBXN1基因啟動(dòng)子區(qū)2000bp序列測序,進(jìn)行高低組序列對(duì)比。結(jié)果在UBXN1基因啟動(dòng)子區(qū)得到了在WHC高組和低組中等位基因分布模式有差異的SNP位點(diǎn)c.-379TG。采用PCR-SSCP技術(shù)對(duì)170頭杜長大三元雜交豬的c.-379TG位點(diǎn)進(jìn)行大群基因分型,經(jīng)過性狀關(guān)聯(lián)分析后發(fā)現(xiàn)TT型的系水力顯著高于GG型(p0.01),隨后對(duì)兩組基因型各9個(gè)個(gè)體的UBXN1表達(dá)水平進(jìn)行定量分析,結(jié)果發(fā)現(xiàn)TT基因型的UBXN1基因表達(dá)水平顯著高于GG型(p0.05)。用雙熒光素酶活性分析兩種不同基因型的啟動(dòng)子活性差異,報(bào)告結(jié)果顯示TT型啟動(dòng)子活性高于GG型,二者差異顯著(p0.05)。最后我們對(duì)SNP位點(diǎn)c.-379TG進(jìn)行了轉(zhuǎn)錄因子在線預(yù)測,發(fā)現(xiàn)該位點(diǎn)的兩個(gè)等位基因的轉(zhuǎn)錄因子結(jié)合情況不同。通過對(duì)UBXN1基因啟動(dòng)子區(qū)SNP的研究為提高豬肉系水力水平提供了理論參考依據(jù)。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S828

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