【作者】 王林杰；

【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， 博士

【摘要】 在影响猪肌肉品质的诸多因素中,遗传因素是影响肉质的重要的方面。动物体内糖原的含量直接影响到肌肉的品质性状,比如屠宰后的肌肉pH值、肉色、系水力、肌肉嫩度等。由于骨骼肌的糖原代谢过程与肌肉的品质性状存在着密切的关系,所以对骨骼肌糖原合成和分解代谢过程的研究一直受到人们的关注,并且研究猪肌肉糖原代谢的过程有助于揭示猪肌肉品质性状形成的分子机理。因此本研究选取了一个重要的糖原代谢通路IGF1介导的P13K-AKT-GSK3β信号通路,对该通路中的重要基因进行了分离克隆,并且在体外分离培养的猪骨骼肌卫星细胞中研究insulin对这些基因的作用和调控机制。主要结果如下：1.分离克隆猪肌肉类型糖原合成酶(GYS1)和肝脏类型糖原合成酶(GYS1)基因,猪GYS1和GYS2在中间具有一个同源性较高的催化功能域，而其C-端和N-端则同源性较低,存在大量的可以调节其活性的磷酸化位点。2.猪GYS1在成年各个组织都表达,在肌肉和心脏组织表达量最高。GYS2则在肝脏高表达,在皮下脂肪组织微弱表达,其他组织不表达。免疫组化检测发现GYS1在骨骼肌的肌肉纤维膜中表达,而GYS2则主要在肝细胞的胞质中表达。3.分别选取了GYSl基因的启动子中的一个SNP位点1432(T/C)和第五外显子中的一个SNP位点1152(C/T),利用限制性片段长度多态性的方法进行了基因分型。两个位点分别与屠宰后45分钟背最长肌pH值,股二头肌pH值和半膜肌pH值三个性状存在显著或者极显著相关。4.分离克隆猪GSK3α和GSK3β基因,猪GSK3α和GSK3β蛋白都包含一个丝氨酸／苏氨酸蛋白激酶结构域,并且在猪GSK3α蛋白的N端有一个富含甘氨酸的序列。5.GSK3β基因的不同转录本的鉴定。我们分别在5’UTR和3’UTR各设计一条引物在猪不同组织的混合cDNA库中进行扩增,然后随机挑选32个阳性克隆子进行测序。发现猪GSK3β基因存在5个转录本,分别命名为GSK3β1、GSK3β2、GSK3β3、GSK3β4和GSK3β5。6.通过定量PCR,.对猪GSK3β的五个转录本在大白猪成年和胚胎期的不同组织中的表达进行了定量分析。7.将猪GSK3β基因不同转录本的编码区构建到绿色荧光蛋白融合表达载体pEGFP-N1中并转染入猪肾细胞系PK-15。猪GSK3β1-GFP、GSK3β2-GFP和GSK3β5-GFP融合蛋白主要分布于细胞质中,而GSK3β4-GFP融合蛋白主要分布于细胞核中。8.猪骨骼肌卫星细胞的分离培养及其鉴定。采用qRT-PCR方法检测肌卫星细胞特异表达的标记基因pax7及卫星细胞分化过程中表达的成肌调控因子myogenin。采用肌卫星细胞特异表达的标志蛋白desmin的抗体进行免疫细胞化学染色,结果表明,本研究分离得到的是猪骨骼肌卫星细胞。9.在分化的猪骨骼肌卫星细胞中添加外源胰岛素,观察猪GSK3β基因不同转录本的表达变化规律。10. GYS1基因在猪骨骼肌卫星细胞开始分化后,表达都呈现上调,并且在分化72 h时基因的表达量最高,添加外源胰岛素不能改变mRNA水平的表达量。11.在分化的猪骨骼肌卫星细胞中添加外源胰岛素,发现可以改变GSK3β和GS的磷酸化水平,并且具有时间依赖性。12.利用基因组步移的方法获得了猪GYSl和GYS2基因启动子区域,这两个基因的启动子区域发现多种潜在的转录因子结合位点,包括PPARs、HNF4α、NF-kappaB和CREB。13.利用insulin对转染pGYS1-175/+60,pGYS1-513/+60和pGYS1-1651/+60后的猪肾细胞(PK-15)进行处理,不同启动子的活性有显著下降。14.将GYS1启动子pGYS1-175/+60和不同GSK3β的蛋白表达形式的超表达载体pcDNA3.1+-GSK3β1,GSK3β2,GSK3p3和GSK3β5进行共转染。超表达3SK3P5可以使CYS1启动子的活性升高,但是没有达到显著水平。超表达GSK3β1,GSK3β2和3SK3β3可以使GYS1基因启动子的活性显著下降。15.采用定量PCR的方法对本实验室利用基因芯片在大白猪和梅山猪中筛选出的骨骼肌发育不同时期和品种间差异表达基因(AMPD1和ALP)进行验证。并分离克隆猪AMPD1和ALP基因,分析其在骨骼肌不同发育阶段和不同肌纤维类型的表达模式。更多还原

【Abstract】 Meat quality is controlled by many factors and genetic is the mainly factor influencing on pork quality. The glycogen content in muscle affects the meat quality traits of pigs that include ultimate pH、color measures、tenderness and cooking loss.The glycogen metablism has a relationship with meat quality, So many researchers focus on the glycogen metabolism. It will be useful for further investigation on the molecule mechanism of the pork quality. In this study, we focus on the important glycogen metabolism signalling transduction pathway (IGF1-PI3K-AKT-GSK3β).The important genes in this pathway were obtained. Moreover, their potential involvement in the mechanism of action of insulin is discussed in porcine satellite cells.The main results are as follows:1.The full-length of coding sequence of porcine GYS1 and GYS2 genes were cloned. Porcine GYS1 and GYS2 showed the highest homology toward their catalytic domains in the central region. The main structural differences between GYS1 and GYS2 isoforms lie in the N and C-terminal regions, which contain the known phosphorylation sites.2.GYS1 was detected in all eleven tissues, and GYS2 was only detectable by RT-PCR in liver and fat. Fibres showing granular cytoplasmic accumulations of GYS1 are seen in porcine muscle, and porcine liver sections showed strong positive staining within the hepatocyte cytoplasm.3.Association analyses revealed that both the GYS1 Hin6I and MvaI polymorphisms had significant associations (p<0.05) with pH of M. longissimus dorsi (pHLD), pH of M.biceps femoris (pHBF) and pH of M. semipinalis capitis (pHSC) at post slaughter 45 minutes.4. The full-length of coding sequence of porcine GSK3αand GSK3βgenes were cloned. The difference in size is due to a glycine-rich extension at the N-terminus of GSK-3α. Although highly homologous within their kinase domains (98% identity), the two gene products share only 36% identity in the C-terminal residues.5.During the cloning of porcine GSK3β, sequencing of 32 clones from different tissues cDNA pool revealed five forms of GSK3βmRNA. We named GSK3β1,GSK3β2, GSK3β3,GSK3β4,GSK3β5 respectively.6.The expression level of different GSK3βisoforms in different embryo and adult tissues were detected by qRT-PCR.7.The cellular locations of each GSK3βisoform were determined by fluorescence and confocal analysis of PK15 cells transiently transfected with different GSK3β-GFP vectors respectively. GSK3β1-GFP、GSK3β2-GFP and GSK3β5-GFP were all found to localize in cytoplasm, however, GSK3β4-GFP displayed a nuclei distribution.8.Porcine myogenic satellite cell culture and identification. The pax7 and myogenin expression profile was used to for monitoring the capacity of satellite cell progeny to proliferate and differentiate.9.The effect of insulin on the expression of different porcine GSK3βtranstripts.10. The expression level of porcine GYS1 is up-regulated in the porcine satellite cells from proliferation to differentiation, but can not be induced significantly during differentiation by insulin.11.Insulin promotes phosphorylation of GSK3βand dephosphorylation of GS in differentiated porcine satellite cells.12.The promoter regions of porcine GYS1 and GYS2 genes were obtained by genomics walking. The promoter of porcine GYS1 contained several putative transcription factor binding sites (PPARs, HNF4a, NF-kappaB and CREB).13.Treatment with insulin resulted in a dramatic reduction in reporter gene activity of the deletion fragment of GYS1 promoter.14. The overexpression of GSK3P5 isoforms increases the activity of pGYS1-175/+60, whereas GSK3β1,GSK3β2 and GSK3β4 induce the activity of pGYS1-175/+60.15.To assess the validity of the microarray approach to identify differentially expressed genes (AMPD1 and ALP),qRT-PCR was performed. We also cloned and characterized (AMPD1 and ALP) from porcine muscle. Moreover, we analyze the expression patterns of them during skeletal muscle development and in four different muscles contained different muscle fibre types.更多还原