myoD表达时间对肌纤维数量的影响及其在猪品种间表达时间差异的分子机制

MyoD is a crucial switch in regulating muscle-specific gene transcription, which has been proven to induce multiple cell types into myoblast and facilitate myoblast fusing into myotube. However, previous studies focused mainly on the expression (expression level) of myoD in regulating myoblast differentiation and myotube formation. In our latest study concerning three pig breeds differing in meat production, Wuzhishan pig with the least meat production showed myoD mRNA expression at 21 days post coitus (dpc), Lantang pig with moderate meat production showed modicum myoD mRNA expression while Landrace with the highest meat production showed almost no expression of myoD mRNA. Thus, we reasonably deduce that distinct expression time of myoD in different pig breed embryos probably determine the differentiation time of myoblast and the numbers of myofibers, which in turn, leading to the distinction in meat production after birth. Moreover, in line with our expectations, at 21 dpc, Wuzhishan pig showed higher level of transcription promoting histone modification H3K4me3 than transcription repressing histone modification H3K27me3, which could facilitate myoblast differentiation status. Meanwhile, Landrace showed balanced modification of H3K4me3 and H3K27me3, which could promote myoblast proliferation status. This research aim to control myoD expression time in vitro and myoD inducible mice model with or without myf5 expression in accompany with Chip and other validating methods to illuminate the mechanism of distinct myoD expression time in pig breeds differing in meat production. This study will not only help better understand the mechanism involved in pig breeds differing in meat production, but also provide scientific evidence to increase meat production using genetic mechanism of distinct myoD expression time in the future.

在肌肉特异基因转录调控中，MyoD起着总开关的作用。但以往研究主要集中于它的表达（量）对成肌细胞分化的调控，我们新近观察到受精后21天时，五指山小型猪已有myoD的表达，蓝塘猪仅有少量表达，而瘦肉型长白猪几乎无表达，提示myoD的起始表达时间可能决定了成肌细胞增殖与分化时间，进而决定肌纤维数量并最终影响产肉量。不仅如此，受精21天后，促进转录的H3K4me3在五指山猪的表达量比抑制转录的H3K27me3高，有助于促进分化；而在长白猪中两者平衡表达有助于成肌细胞增殖，这与预期完全相符，为此，本课题拟通过构建myoD可诱导表达并同时敲除myf5的小鼠模型证实是否可通过控制myoD的表达时间来影响肌纤维数量，并通过Chip等实验阐明myoD在产肉量不同猪种中起始表达时间差异的分子机制，为确立将myoD起始表达时间差异的遗传机制作为提高生猪产肉量新靶标提供科学依据。

中国地方猪虽然肉质好，但产肉量低，生产效益低下。解析产肉量的遗传调控机制被认为是遗传育种专业最为困难和最具挑战的任务之一，也是当今畜牧领域公认的重大科学问题。在肌肉特异基因转录调控中，MyoD起着总开关的作用。通过本项目的实施，我们在体内外证实了：地方猪由于MyoD起始表达早，使部分成肌细胞率先退出细胞周期而分化，由于没有更多的成肌细胞增殖，致使用于融合形成肌纤维的成肌细胞数量少，并最终导致产肉量减少。关于myoD早表达的调控机制，表观修饰在其中发挥着至关重要的作用，主要有以下几点：.1、在胚胎18天-28天，五指山猪成肌基因会更早去甲基化，有利于成肌基因早表达，导致成肌细胞较长白猪更早进入分化过程，其中去甲基化酶Tet1可使myogenin去甲基化。.2、胚胎28天-35天，组蛋白H3K4me3在五指山猪中富集程度高，而H3K27me3在长白猪中高度富集，使得五指山猪种成肌基因包括MyoD较早激活，开启成肌分化，而长白猪成肌调控基因的表达受到抑制。利用敲除鼠模型进一步证实了在不同猪种胚胎期差异表达组蛋白修饰基因EZH2、UTX、Mll1、KDM4A均可对成肌基因MyoD或myf5进行调控。.3、与长白猪相比，地方猪肌前体细胞中Notch信号的活性较低也是其表达MyoD较早的一个重要原因。当Notch信号活性较高时（如在长白猪中），可以抑制myoD的表达，从而阻止成肌细胞退出细胞周期，维持细胞的增殖。.4、首次提出并证实了猪的成肌细胞分化发生在细胞接触之时，并且需要发生部分细胞的凋亡，从而介导成肌细胞融合形成肌管乃至肌纤维，在这其中，ZFP422- EphA7调控轴发挥着重要作用。.综上，本项目证实了MyoD表达时间对肌纤维数量的影响，并阐明了MyoD在产肉量不同猪种中起始表达时间差异的分子机制，为确立将MyoD表达时间作为提高生猪产肉量新靶标提供了坚实的科学依据。