猪myostatin基因不同突变体的功能研究

发布时间：2019-03-28 06:10

【摘要】：肌肉生长抑制素(myostatin)是转化生长家族-β(TGF-β)中的一员,对于肌肉的生长和发育起负调控的作用。在本研究中,我们制备了四种转基因小鼠,即分别表达猪myostatin错义突变体(pm MS),C313Y,猪myostatin切割位点的突变体(RSRR),猪myostatin的野生型前肽(pp MS)以及它的突变体形式(D75A)来探究它们对肌肉生长的影响。而且我们还通过在小鼠体内注射猪myostatin的前肽和它的突变体来抑制小鼠内源的myostatin,从而探究它们对于肌肉生长的影响。此外,我们还发现在pm MS的转基因公鼠中肌肉没有出现增长,但是在pm MS和pp MS的转基因母鼠以及pp MS的转基因公鼠中肌肉却出现了增长。为了探索为何在pm MS和pp MS的转基因公鼠中出现肌肉生长的差异,我们对肌肉中雄激素受体(AR)和它的突变体AR45通过Western blot进行了检测。所有的研究结果如下:(1)四种转基因小鼠通过PCR,RT-PCR,q RT-PCR和Southern Blot等方法进行了鉴定,结果显示四种转基因小鼠均制备成功。(2)除了pm MS转基因雄鼠没有肌肉增多表型,其余的转基因小鼠肌肉均出现了增多表型。注射猪myostatin的前肽和其突变体的小鼠相比于注射PBS的小鼠肌肉也出现了明显增多。(3)通过AR和AR45的蛋白表达量计算两者灰度值的比例(AR45/AR)得出在pm MS转基因雌鼠和野生型雌鼠之间AR45/AR没有差异,但是在pm MS雄鼠和野生型雄鼠之间则具有较大的差异。这些数据解释了为何pm MS的转基因雄鼠和雌鼠肌肉生长存在差异。与此同时,研究结果显示AR和AR45的蛋白表达在pp MS的雄鼠中与pm MS的雄鼠正好相反,这对于我们的推测起到支持作用,即在pm MS的雄鼠中高水平的myostatin成熟肽导致AR45蛋白的高表达、AR的低表达进而补偿了由于外源突变myostatin所引起的肌肉增长。总之,这些研究结果为日后通过人工操控myostatin的表达或改变其活性来制备高瘦肉率的育种猪和治疗肌肉疾病的转基因猪生物医学模型提供了科学依据和理论基础。
[Abstract]:Myostatin (myostatin) is a member of transforming growth family-尾 (TGF- 尾), which plays a negative role in muscle growth and development. In this study, we prepared four kinds of transgenic mice, namely, expressing porcine myostatin missense mutant (pm MS), C313Y and pig myostatin cleavage site mutant (RSRR), respectively. Porcine myostatin's wild-type propeptide (pp MS) and its mutant form (D75A) were used to explore their effects on muscle growth. We also investigated their effects on muscle growth by injecting porcine myostatin propeptide and its mutants into mice to inhibit endogenous myostatin, in mice. In addition, we also found that there was no muscle growth in pm MS transgenic male mice, but in pm MS and pp MS transgenic female mice and pp MS transgenic male mice muscle growth appeared. In order to explore the differences in muscle growth between pm MS and pp MS transgenic male mice, we detected androgen receptor (AR) and its mutant AR45 in muscle by Western blot. All the results are as follows: (1) four transgenic mice were identified by PCR,RT-PCR,q RT-PCR and Southern Blot. The results showed that all four transgenic mice were successfully prepared. (2) except for pm MS transgenic male mice, no muscle enhancement phenotype was found in the muscle of the other transgenic mice. Compared with the mice injected with PBS, the prepeptide of porcine myostatin also increased significantly. (3) the ratio of gray value (AR45/AR) between AR and AR45 was calculated by calculating the ratio of gray value (AR45/AR) between the two mutants. (3) the transformation in pm MS was obtained by calculating the ratio of gray value between the two groups (AR45/AR). There was no difference in AR45/AR between the genetic female and the wild-type female. However, there was significant difference between pm MS male and wild type male. These data explain why pm MS transgenic male and female mice differ in muscle growth. At the same time, the results suggest that the expression of AR and AR45 proteins in pp MS males is the opposite of that in pm MS males, which supports our conjecture. That is, the high level of mature peptide of myostatin in male mice of pm MS leads to the high expression of AR45 protein, and the low expression of AR in turn compensates the muscle growth caused by exogenous mutation myostatin. In conclusion, these results provide a scientific basis and theoretical basis for the preparation of high lean-meat breeding pigs and transgenic pig biomedical models for treating muscle diseases by artificially manipulating the expression of myostatin or changing its activity in the future.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S828

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