【摘要】：豬是中國最重要的肉類食用動物,豬肉的品質(zhì)是重要經(jīng)濟性狀。影響豬肉品質(zhì)的因素有很多,例如嫩度、肌內(nèi)脂肪、肉色等。肌纖維類型與這些因素都有著直接的關(guān)系,是目前豬肉品質(zhì)研究的熱點,所以發(fā)現(xiàn)肌纖維類型轉(zhuǎn)化的調(diào)控機理,對于提高豬的肉質(zhì)性狀有重要的意義。過氧化物酶體增殖受體γ輔激活因子α(Peroxisome proliferator-activated receptor gamma coactivator 1 alpha,PGC1α)是一種轉(zhuǎn)錄因子共激活因子,能有效地誘導(dǎo)線粒體的生物合成和細胞呼吸功能,是調(diào)控肌纖維類型轉(zhuǎn)化的主要因子之一。本研究在轉(zhuǎn)基因豬模型中驗證了利用MCK啟動子超表達PGC1α基因后對骨骼肌肌纖維類型轉(zhuǎn)化的影響,并對轉(zhuǎn)PGC1α基因豬和野生型豬進行了轉(zhuǎn)錄組測序,進一步尋找PGC1α基因引起表型變化的調(diào)控機理。本研究的主要結(jié)果如下:1.在前期制備的原代轉(zhuǎn)PGC1α基因豬的基礎(chǔ)上,對轉(zhuǎn)基因豬群進行擴繁。分別對3月齡和6月齡的轉(zhuǎn)基因豬進行了屠宰實驗,通過PCR的方法進行陽性鑒定。觀察了兩個月齡豬的肌肉形態(tài),結(jié)果顯示腓腸肌和背最長肌的顏色都稍有變紅。對6月齡的轉(zhuǎn)基因豬和野生型豬進行了性狀的測定,發(fā)現(xiàn)轉(zhuǎn)基因豬肌肉滴水損失顯著降低,其他與肉質(zhì)相關(guān)的一些性狀指標也產(chǎn)生了差異,如肉色評分、大理石紋評分、肌內(nèi)脂肪和嫩度等升高了,但未達到顯著水平。2.開展了免疫組化和ATP酶染色的實驗,結(jié)果顯示氧化型肌纖維(Ⅰ型肌纖維)的數(shù)目顯著增多,酵解型肌纖維(ⅡB型肌纖維)的數(shù)目則顯著減少。HE染色結(jié)果表明肌纖維的密度和數(shù)目有所增加,而肌纖維的橫截面積卻減小了。這些都說明了PGC1α的超表達能夠使酵解型肌纖維向氧化型肌纖維轉(zhuǎn)化,同時引起肌纖維密度和數(shù)目的增多,橫截面積的減少。3.建立了3月齡和6月齡轉(zhuǎn)基因豬和野生型豬的PGC1α基因表達譜,發(fā)現(xiàn)PGC1α基因在較多組織中都有表達量的上調(diào),例如腎、肝、胃、腹脂、肌肉組織等,其中在腓腸肌和背最長肌達到了顯著水平,驗證了PGC1α超表達載體上的MCK啟動子的功能具有肌肉組織特異性。此外,在腎、肝中也顯著超表達。對3月齡和6月齡轉(zhuǎn)PGC1α基因豬的腓腸肌和背最長肌進行了轉(zhuǎn)錄水平和蛋白水平的檢測,發(fā)現(xiàn)氧化型肌纖維相關(guān)基因(MYHCⅠ、MYHCⅡA、Myog、Myoglobin、TNNI1和CS等)都上調(diào)表達,而酵解型肌纖維相關(guān)基因(MYHCⅡB、Myod、TNNI2等)則下調(diào)表達。一些PGC1α基因調(diào)控的下游基因也發(fā)生了變化,其中上調(diào)的有MEF2C、CPT-1B、NRF-1、PPARγ和FABP3,下調(diào)的有FOXO1和Myostatin。4.對3月齡轉(zhuǎn)基因豬和野生型豬的腓腸肌進行了轉(zhuǎn)錄組測序,共檢測到了371個差異表達基因(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 緯 coactivator 偽 (Peroxisome proliferator-activated receptor gamma coactivator 1 alpha,PGC1 偽) 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 偽 gene on skeletal muscle fiber type transformation were verified in transgenic pig model, and transcriptional sequencing of PGC1 偽 gene transgenic pig and wild type pig were carried out. To further explore the regulatory mechanism of phenotypic changes caused by PGC1 偽 gene. The main results of this study are as follows: 1. On the basis of the primary transgenic PGC1 偽 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 偽 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 偽 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 偽 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 偽 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 偽 gene were detected. It was found that oxidative muscle fiber related genes (MYHC 鈪，

本文編號：2285381