【作者】 张荣萍；

【导师】 王继文； 陈洁；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2016， 博士

【摘要】 鸟类骨骼肌发育与其它哺乳动物基本一致,其成肌细胞数量在胚胎期就基本固定下来,出壳后肌肉质量的获得主要依赖于蛋白质沉积,体现在肌纤维的增粗和增长上。水禽骨骼肌发育在不同品种、组织间均表现出明显差异,目前关于这些差异的研究主要集中在表型特征测定或者单个基因检测等方面,而未对引起这些差异的分子机制进行系统性研究。本研究以两个表型差异明显黑鹜鸭、北京鸭胸肌和腿肌为研究材料,通过比较胚胎期和出壳后不同生长时期鸭骨骼肌发育特点,并检测影响肌肉生长的部分基因表达情况,明确黑鹜鸭和北京鸭骨骼肌发育表现出品种、组织差异,利用RNA-seq高通量测序技术,筛选出鸭骨骼肌发育品种、组织特异性的关键基因和信号通路,并在细胞水平上探讨差异表达基因—Pαx3调节鸭成肌细胞增殖和分化的分子机制。本研究主要结果如下：(1)北京鸭胚胎期胸肌和腿肌均重于黑鹜鸭,且腿肌重表现出显著的品种差异：在同一鸭品种间腿肌发育均显著快于胸肌(P<0.05)。(2)参与调控肌肉发育的基因(IGF-1、PI3K、AKT、Pax3和Pax7)在品种间和组织间均表现出不同的发育表达模式,从分子水平证实鸭胚胎期骨骼肌发育表现出品种、组织差异。(3)北京鸭出壳后胸肌比腿肌有更高的肌纤维密度和细胞核密度(P<0.05)；反之,腿肌较胸肌有更高的肌纤维直径和横切面积(P<0.05)。(4)调控蛋白质合成(mTOR和S6K)和蛋白质降解(FoxO1、MuRF1和MAFbx)的相关基因在北京鸭胸肌和腿肌之间表达水平显著不同,且在胸肌中表达高于腿肌(P<0.05),暗示胸肌较腿肌有更高的蛋白质代谢能力。(5)卫星细胞频率(Pax3和Pax7)在胸肌和腿肌上均随着周龄的增加而降低,但在胸肌和腿肌间差异不显著(P>0.05)。通过检测caspase-3活性和观察细胞核分裂情况发现,鸭胸腿肌组织之间细胞凋亡情况差异也不显著(P>0.05。(6)利用RNA-Seq技术在黑鹜鸭和北京鸭腿肌和胸肌组织中均获得超过1.2G的干净序列读数。通过分析发现,鸭胸肌和腿肌组织差异表达基因338个,远高于品种间差异表达基因88个,在黑鹜鸭和北京鸭上的组织特异性差异表达基因数量分别为246和258个,而在胸肌和腿肌上的品种特异性差异表达基因数量分别是54和20个。(7) KEGG通路分析发现focal adhesion、PI3K-AKT和ECM-receptor interaction相关通路是两个品种的骨骼肌发育表现出组织差异的主要分子原因,而ribosomes可能是腿肌和胸肌发育表现出品种差异涉及到的一个主要信号通路。(8)构建了一条能够高效地(>70%)沉默鸭Pax3基因表达水平的shRNA干扰载体。(9) shRNA沉默Pax3后鸭成肌细胞的增殖和分化能力显著地降低,同时,肌源性因子MyoD、Myf5和MyoG的表达也显著下调(P<0.05),暗示Pax3对鸭成肌细胞生长有着正向调控功能。(10)在沉默Pax3后的培养基中添加叶酸,发现叶酸能显著增加肌源性因子(MyoD和Myf5)和Pax7的表达(P<0.05),从而缓解鸭成肌细胞被抑制的增殖和分化能力。综上,本研究从分子水平证实鸭胚胎期骨骼肌发育表现出品种和组织差异,发现蛋白质代谢能力可能是造成胸肌和腿肌发育差异的重要原因之一。鸭骨骼肌发育组织差异表达基因数量(338个)远多于品种差异表达基因数量(88个),focal adhesion、 PI3K-AKT和ECM-receptor interaction相关通路是黑鹜鸭和北京鸭骨骼肌发育表现出组织差异的主要分子原因,而ribosomes可能是影响腿肌和胸肌发育表现出品种差异的主要信号通路,证明Pax3能正向调控鸭成肌细胞增殖和分化。以上研究为骨骼肌发育品种、组织差异调控机制解析奠定了基础。更多还原

【Abstract】 Myogenesis in avian has a similar development character with other vertebrates, the myoblast number is almostly fixed during the embryonic stage. After post-hatching, protein deposition is main contributor for muscle mass gain, a process displayed as the lengthen and thicken of myofibers. The priviously studies have domenstranted that muscle deposition capability exites differences among breeds and locations. To date, the resarch about the difference of muscle development between species or locations is mainly focus on phenotypic index or some single genes, but the detail molecular mechanism related to these differences is still unknown. Herein, this study employed the leg muslce and pectoral muslce of two phenotypic difference duck species as research materials, acrooding to compare the character of duck skeletal muscle development at different time point from embryo stage and post-hatch stage, to clear Heiwu and Peking duck skeletal development displayed species-specific and locations-specific. The RNA-Seq was used to identify differentially expressed genes related to leg muscle and pectoral muscle myogenesis in Heiwu and Peking ducks. In vitro, the function of Pax3 in on duck myoblast roliferation and differentiation was identified.The main results as following:(1) During duck embryo stage, compared to Heiwu duck. Peking duck has a quickly muscle growth ability, and leg muscle weight is significantly different between two duck species. Meanwhile, leg muscle has a higher development ability than pectoral muscle at the same duck species. (P<0.05).(2) Some genes(IGF-1,PI3K,AKT,Pax3 and Pax7) involved in muscle development have a different expression pattern among four skeletal muscles, suggested the difference of muscle development between locations and species is already exist at embryo stage.(3) During Peking duck post-hatching, compared to leg muscle fibers, pectoral muscle fibers have a higher density of myofiber and myonuclei. Verse vice, the diameter and cross-section area of myofiber have a higher number in leg muscle fiber than pectoral muscle fiber (P<0.05).(4) Both protein synthesis genes (mTOR and S6K) and protein degradation genes (FoxO1, MuRF1 and MAFbx) have a significantly different expression pattern between leg muscle and pectoral muscle, and pectoral muscle has a higher expression level than leg muscle (P<0.05), indicated pectoral muscle has a higher capability in protein metabolism than leg muscle.(5) With the week increase, satellite cell frequency (Pax3 and Pax7) decreased at both pectoral muscle and leg muscle, but it has no difference between two muscles (P>0.05). Based on the Hoechst-stained and the detection of caspase-3 activity, no obvious apoptosis is observed in both muscles (P>0.05).(6) In this study, all four skeletal muscle libraries obtain over 1.2G clean reads by using RNA-Seq. All data from this study demonstrated that obvious differences in gene expression between leg muscle and pectoral muscle (338 genes) and between Heiwu duck and Peking duck (88 genes). Intriguingly, more location-specific genes (246 for Heiwu duck and 258 for Peking duck) were identified compared with breed-specific gene (20 for leg muscle and 54 for pectoral muscle).(7) The KEGG results revealed that focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction are the three main pathways responsible for muscle development differences between leg and pectoral muscles, and only ribosomes is mainly involved in muscle development differences between Heiwu and Peking duck.(8) One shRNA is successfully constructed to target and silence the expression of duck Pax3(>70%).(9) After silencing Pax3, both the proliferation and differentiation of duck myoblast are significantly inhibited, by reducing the expression of myogenic genes (MyoD, Myf5 and MyoG) (P<0.05).(10) Adding folic acid to the medium of duck myoblast find that folic acid can significantly increase the expression of myogenic genes (MyoD and MyoG) and Pax7 (P<0.05), to rescue the duck myoblast proliferation and differentiation decreased by shRNA silencing Pax3.In all, this study proved that skeletal muscle development is different between species and locations during duck embryo, and protein metabolism signaling maybe is the main reason for muscle deposition difference between leg muscle and pectoral muscle, focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction are the three main pathways responsible for muscle development differences between leg and pectoral muscles, and ribosomes is thereason for muscle development differences between Heiwu and Peking duck. Pax3 has a positive effect on duck myoblast proliferation and differentiation. All these results are helpful for understand the molecular basis of skeletal muscle development difference between species and locations.更多还原