利用转基因猪模型研究PGC1α对肌纤维类型转变的影响及其分子机制
发布时间：2018-10-21 14:29
【摘要】：猪是中国最重要的肉类食用动物,猪肉的品质是重要经济性状。影响猪肉品质的因素有很多,例如嫩度、肌内脂肪、肉色等。肌纤维类型与这些因素都有着直接的关系,是目前猪肉品质研究的热点,所以发现肌纤维类型转化的调控机理,对于提高猪的肉质性状有重要的意义。过氧化物酶体增殖受体γ辅激活因子α(Peroxisome proliferator-activated receptor gamma coactivator 1 alpha,PGC1α)是一种转录因子共激活因子,能有效地诱导线粒体的生物合成和细胞呼吸功能,是调控肌纤维类型转化的主要因子之一。本研究在转基因猪模型中验证了利用MCK启动子超表达PGC1α基因后对骨骼肌肌纤维类型转化的影响,并对转PGC1α基因猪和野生型猪进行了转录组测序,进一步寻找PGC1α基因引起表型变化的调控机理。本研究的主要结果如下:1.在前期制备的原代转PGC1α基因猪的基础上,对转基因猪群进行扩繁。分别对3月龄和6月龄的转基因猪进行了屠宰实验,通过PCR的方法进行阳性鉴定。观察了两个月龄猪的肌肉形态,结果显示腓肠肌和背最长肌的颜色都稍有变红。对6月龄的转基因猪和野生型猪进行了性状的测定,发现转基因猪肌肉滴水损失显著降低,其他与肉质相关的一些性状指标也产生了差异,如肉色评分、大理石纹评分、肌内脂肪和嫩度等升高了,但未达到显著水平。2.开展了免疫组化和ATP酶染色的实验,结果显示氧化型肌纤维(Ⅰ型肌纤维)的数目显著增多,酵解型肌纤维(ⅡB型肌纤维)的数目则显著减少。HE染色结果表明肌纤维的密度和数目有所增加,而肌纤维的横截面积却减小了。这些都说明了PGC1α的超表达能够使酵解型肌纤维向氧化型肌纤维转化,同时引起肌纤维密度和数目的增多,横截面积的减少。3.建立了3月龄和6月龄转基因猪和野生型猪的PGC1α基因表达谱,发现PGC1α基因在较多组织中都有表达量的上调,例如肾、肝、胃、腹脂、肌肉组织等,其中在腓肠肌和背最长肌达到了显著水平,验证了PGC1α超表达载体上的MCK启动子的功能具有肌肉组织特异性。此外,在肾、肝中也显著超表达。对3月龄和6月龄转PGC1α基因猪的腓肠肌和背最长肌进行了转录水平和蛋白水平的检测,发现氧化型肌纤维相关基因(MYHCⅠ、MYHCⅡA、Myog、Myoglobin、TNNI1和CS等)都上调表达,而酵解型肌纤维相关基因(MYHCⅡB、Myod、TNNI2等)则下调表达。一些PGC1α基因调控的下游基因也发生了变化,其中上调的有MEF2C、CPT-1B、NRF-1、PPARγ和FABP3,下调的有FOXO1和Myostatin。4.对3月龄转基因猪和野生型猪的腓肠肌进行了转录组测序,共检测到了371个差异表达基因(P-value
[Abstract]:Pig is the most important meat animal in China, and the quality of pork is an important economic trait. There are many factors affecting pork quality, such as tenderness, intramuscular fat, meat color and so on. The type of muscle fiber has a direct relationship with these factors and is the hot spot of pork quality research at present, so it is important to find out the regulation mechanism of muscle fiber type transformation for improving the meat quality of pigs. Peroxisome proliferative receptor 纬 coactivator 伪 (Peroxisome proliferator-activated receptor gamma coactivator 1 alpha,PGC1 伪) is a transcription factor coactivator, which can effectively induce mitochondrial biosynthesis and cell respiratory function. In this study, the effects of MCK promoter supergene expression of PGC1 伪 gene on skeletal muscle fiber type transformation were verified in transgenic pig model, and transcriptional sequencing of PGC1 伪 gene transgenic pig and wild type pig were carried out. To further explore the regulatory mechanism of phenotypic changes caused by PGC1 伪 gene. The main results of this study are as follows: 1. On the basis of the primary transgenic PGC1 伪 gene pigs prepared in the previous period, the transgenic pigs were expanded and propagated. Three and six month old transgenic pigs were slaughtered and identified by PCR. Muscle morphology of two month old pigs was observed. The results showed that both gastrocnemius and longissimus dorsi were slightly reddish. The traits of transgenic pigs and wild type pigs at 6 months of age were measured. The results showed that the muscle drip loss of transgenic pigs was significantly reduced, and some other traits related to meat quality, such as meat color score, marbling score, were also different. Intramuscular fat and tenderness increased, but did not reach significant levels. Immunohistochemistry and ATP enzyme staining were carried out. The results showed that the number of oxidized muscle fibers (type I muscle fibers) increased significantly. The results of HE staining showed that the density and number of muscle fibers increased, while the cross sectional area of muscle fibers decreased. These results suggest that the overexpression of PGC1 伪 can transform the fermentable muscle fibers into oxidized ones, increase the density and number of muscle fibers, and decrease the cross sectional area. The PGC1 伪 gene expression profiles of 3 and 6 month old transgenic pigs and wild type pigs were established. It was found that the expression of PGC1 伪 gene was up-regulated in many tissues, such as kidney, liver, stomach, abdominal fat, muscle tissue, etc. In the gastrocnemius muscle and the longissimus dorsi muscle, the function of the MCK promoter on the PGC1 伪 overexpression vector was proved to be muscle-specific. In addition, in the kidney, liver is also significantly overexpression. The transcriptional and protein levels of gastrocnemius and longissimus dorsi in 3-month-old and 6-month-old pigs with PGC1 伪 gene were detected. It was found that oxidative muscle fiber related genes (MYHC 鈪，
本文编号：2285381
[Abstract]:Pig is the most important meat animal in China, and the quality of pork is an important economic trait. There are many factors affecting pork quality, such as tenderness, intramuscular fat, meat color and so on. The type of muscle fiber has a direct relationship with these factors and is the hot spot of pork quality research at present, so it is important to find out the regulation mechanism of muscle fiber type transformation for improving the meat quality of pigs. Peroxisome proliferative receptor 纬 coactivator 伪 (Peroxisome proliferator-activated receptor gamma coactivator 1 alpha,PGC1 伪) is a transcription factor coactivator, which can effectively induce mitochondrial biosynthesis and cell respiratory function. In this study, the effects of MCK promoter supergene expression of PGC1 伪 gene on skeletal muscle fiber type transformation were verified in transgenic pig model, and transcriptional sequencing of PGC1 伪 gene transgenic pig and wild type pig were carried out. To further explore the regulatory mechanism of phenotypic changes caused by PGC1 伪 gene. The main results of this study are as follows: 1. On the basis of the primary transgenic PGC1 伪 gene pigs prepared in the previous period, the transgenic pigs were expanded and propagated. Three and six month old transgenic pigs were slaughtered and identified by PCR. Muscle morphology of two month old pigs was observed. The results showed that both gastrocnemius and longissimus dorsi were slightly reddish. The traits of transgenic pigs and wild type pigs at 6 months of age were measured. The results showed that the muscle drip loss of transgenic pigs was significantly reduced, and some other traits related to meat quality, such as meat color score, marbling score, were also different. Intramuscular fat and tenderness increased, but did not reach significant levels. Immunohistochemistry and ATP enzyme staining were carried out. The results showed that the number of oxidized muscle fibers (type I muscle fibers) increased significantly. The results of HE staining showed that the density and number of muscle fibers increased, while the cross sectional area of muscle fibers decreased. These results suggest that the overexpression of PGC1 伪 can transform the fermentable muscle fibers into oxidized ones, increase the density and number of muscle fibers, and decrease the cross sectional area. The PGC1 伪 gene expression profiles of 3 and 6 month old transgenic pigs and wild type pigs were established. It was found that the expression of PGC1 伪 gene was up-regulated in many tissues, such as kidney, liver, stomach, abdominal fat, muscle tissue, etc. In the gastrocnemius muscle and the longissimus dorsi muscle, the function of the MCK promoter on the PGC1 伪 overexpression vector was proved to be muscle-specific. In addition, in the kidney, liver is also significantly overexpression. The transcriptional and protein levels of gastrocnemius and longissimus dorsi in 3-month-old and 6-month-old pigs with PGC1 伪 gene were detected. It was found that oxidative muscle fiber related genes (MYHC 鈪，
本文编号：2285381
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