金定鸭胚胎期骨骼肌发育模式及MRFs家族基因表达变化研究

MRFs (Myogenic regulatory factors,MRFs)家族包括肌分化因子MyoD、肌细胞生成素(myogenin)、生肌决定因子Myf5和Myf6,它们参与肌肉发生发育的各个环节.研究采用Real-time PCR方法研究了金定鸭胚胎期13、17、21、25和27日胚龄MRFs家族基因在骨骼肌中的表达变化,并分析其表达变化与胚重、骨骼肌发育的相关性.金定鸭胚重及骨骼肌重发育模式显示胚重在胚胎期呈持续增加趋势,但胸肌在胚胎中后期显示出较腿肌滞后的增重变化.MRFs家族基因在胚胎期骨骼肌中均能检测到表达,MyoD mRNA在21日胚龄前腿肌中的表达量高于胸肌,而在胚胎发育后期(25及27日胚龄)胸肌中表达量渐渐高于腿肌.胸、腿肌中myogenin mRNA的表达与胚重、胸、腿肌发育重量变化均呈强的负线性相关.上述研究结果首次揭示了鸭胚胎期骨骼肌中MRFs家族基因表达具有显著的时空性,推测MyoD mRNA在胸、腿肌中的表达变化可能与胸、腿肌增重表型差异有关,研究结果为进一步深入研究MRFs家族基因在胚胎期骨骼肌发生过程及其调控机理中的功能提供一定的理论依据.     

鸭胚骨骼肌生肌调节因子MyoD1和Myf5发育性变化研究

本研究采用Real-timePCR分析生肌调节因子MyoD1和Myf5在不同鸭品种(高邮鸭和金定鸭)胚胎期及初生早期(13、17、21、25、27日胚龄和出雏后7日龄)骨骼肌发育中的表达模式以及表达差异。结果表明,MyoD1mRNA和Myf5mRNA在两个品种鸭胸肌早期发育中表现出一致的表达规律,均呈"波浪"型;在腿肌早期发育中均呈近似"反√"型的表达趋势,在27日胚龄时表达量最低,出生后7日龄表达量又上升,但MyoD1mRNA的表达量仅有小幅增加,与27日胚龄的表达量差异不显著(P>0.05),而Myf5mRNA的表达量有大幅增加,与27日胚龄的表达量差异极显著(P<0.01),胸肌和腿肌中两基因的mRNA表达量在同一胚龄不同品种间差异均不显著。结果提示:骨骼肌中MyoD1和Myf5基因表达具有显著的时空性,但不存在品种差异,为进一步深入研究MyoD1和Myf5基因在胚胎期骨骼肌发生过程及其调控机理中的功能提供一定的理论依据。     

鸭发育早期骨骼肌NFATc3 mRNA差异表达及其与生长性状的相关性研究

为研究不同品种鸭胚胎期和出雏早期骨骼肌中NFATc3基因mRNA表达规律及其与生长发育的相关性,运用荧光绝对定量PCR技术检测了生长速度差异较大的高邮鸭和金定鸭13、17、21、25、27胚龄和7日龄胸肌和腿肌中NFATc3 mRNA的表达水平。结果表明胸肌和腿肌NFATc3 mRNA在两鸭品种中均具有相似的表达模式,不存在品种效应,胸肌和腿肌NFATc3 mRNA的表达均是13胚龄时表达量最高,显著高于其它各胚龄或日龄(P0.05);相关性分析结果表明两个品种鸭骨骼肌NFATc3 mRNA表达量与其组织重、体重均呈极显著负相关(P0.01),而与肌肉组织中IGF-Ⅰm RNA的表达量呈极显著正相关(P0.01)。鸭骨骼肌中NFATc3主要在早期的肌纤维生成中起作用,与IGF-Ⅰ可能共同参与了对骨骼肌生长发育的调节。     

高邮鸭和金定鸭胸肌早期发育及MyoD1基因表达分析

 
采用qPCR分析生肌调节因子MyoD1基因在高邮鸭和金定鸭胚胎期及初生早期(13、17、21、25、27胚龄和出雏后7日龄)胸肌发育中的表达模式以及与胚胎和胸肌发育的相关性。结果表明,MyoD1 mRNA在两个品种鸭胸肌早期发育中表现出一致的表达规律,均呈“波浪形”,在13胚龄时表达量相对较高,17胚龄时下降,21胚龄时上升到最高,随后下降,出雏后又维持较高水平;品种间比较结果显示除了在25胚龄时金定鸭胸肌中MyoD1 mRNA表达量稍低于高邮鸭,在其他所检测的胚龄/日龄中,金定鸭胸肌中MyoD1 mRNA表达量均高于高邮鸭胸肌中的表达量(P>0.05);高邮鸭胸肌MyoD1 mRNA表达与胚胎和胸肌发育无显著相关性,而金定鸭胸肌中MyoD1 mRNA的表达与其胚胎和胸肌发育呈强负相关(P胚重=0.048;P胸肌重=0.006)。MyoD1基因参与鸭胚胎期及出雏早期胸肌的发育,胸肌中MyoD1基因表达分析研究为进一步深入研究MyoD1基因在胚胎期胸肌发生过程及其调控机理中的功能提供一定的理论依据     

略阳乌鸡肌肉组织中肌苷酸合成酶系相关基因表达规律的研究

选取胚龄为8、11、15、18、21 d、出雏后3、6、120 d的略阳乌鸡为研究对象,探讨肌肉中肌苷酸合成酶系相关基因的表达规律。采用实时荧光定量PCR检测不同阶段的肌肉组织中PAICS、PPAT、GART和ADSL四种基因的表达水平。结果表明,PPAT、GART、PAICS、ADSL基因在胚胎期都大量表达,PAICS基因在胚胎早期(胚龄第8天)表达量最高,PPAT和GART在胚胎中期(胚龄第11天)表达量最高,ADSL基因的表达随着胚龄的增加呈上升趋势直到成年。另外,腿肌组织中PAICS、PPAT、GART、ADSL基因的表达水平在幼年期和成年期基本一致;但胸肌中四种基因表达量都明显高于腿肌,说明胸肌中肌苷酸的合成量可能会高于腿肌。     

肌纤维发育相关基因在不同地方鸡种早期生长发育中的表达分析

为研究肌纤维发育相关基因在不同地方鸡种的表达调控作用,选用1日龄体重相近的汶上芦花鸡、济宁百日鸡和鲁西斗鸡,分别在1日龄、1～5周龄采集同一部位的胸肌、腿肌,应用实时荧光定量PCR测定Myf5、Myf6、MyoD1和MSTN等基因的表达量。结果显示:汶上芦花鸡胸肌中MSTN基因1周龄表达量显著高于其他周龄(P0.05),鲁西斗鸡腿肌中MSTN基因4周龄表达量显著高于其他周龄(P0.05),济宁百日鸡胸肌中MSTN基因4周龄和5周龄表达量显著高于其他周龄(P0.05),腿肌中4周龄显著高于其他周龄(P0.05);汶上芦花鸡腿肌Myf6基因2周龄的表达水平明显高于其他周龄(P0.05),济宁百日鸡胸肌中Myf6基因5周龄时表达量显著低于鲁西斗鸡和汶上芦花鸡(P0.05);4～5周龄时,济宁百日鸡腿肌MyoD1基因的表达水平最低,在3周龄时,汶上芦花鸡胸肌MyoD1基因表达水平显著高于其他周龄(P0.05);在0和1周龄时,鲁西斗鸡中胸、腿肌Myf5基因表达水平均高于汶上芦花鸡和济宁百日鸡。研究表明Myf5、Myf6、MyoD1和MSTN基因在3个地方鸡种胸肌、腿肌中均呈现出不同的表达调控模式。     

“花山麻鸡”CPT1A基因时空表达规律研究

试验旨在阐明"花山麻鸡"CPT1A基因的组织表达和时序表达规律,以胚胎期到上市日龄的"花山麻鸡"为试验材料,在不同发育时间点采集胸肌、腿肌、心脏、肝脏组织样品,利用实时荧光定量方法分析CPT1A基因在不同组织不同发育阶段表达规律。在相同发育阶段不同组织差异表达结果显示:在胚胎期(9、12、16日胚龄)和上市日龄(9周龄),CPT1A基因在肝脏的表达量显著高于在心脏、胸肌和腿肌的表达量,在出生后1日龄,CPT1A在心脏表达量最高,1周龄时,CPTA在肝脏的表达量最低;5周龄时,CPT1A在腿肌的表达量最高;7周龄时,CPT1A在腿肌和肝脏的表达量显著高于胸肌和心脏。在同一组织不同发育阶段差异表达结果显示,CPT1A在胸肌、腿肌、肝脏和心脏不同发育阶段表达规律与表达量是不同的。研究表明CPT1A基因在胸肌、腿肌、肝脏和心脏相同发育阶段表达量不同,CPT1A基因在胸肌、腿肌、肝脏和心脏织不同发育阶段表达规律与表达量不同。     

Myf6基因在不同地方鸡种早期生长发育中的表达分析

为了探究Myf6基因在京海黄鸡和金茅黄鸡体内的差异表达规律，研究其在两个品种鸡中可能发挥的作用机制，试验采用实时荧光定量PCR技术对Myf6基因在不同品种鸡不同组织中的表达情况进行了检测。结果表明：京海黄鸡和金茅黄鸡公母鸡胸肌和腿肌中的Myf6基因相对表达量均比较高，且极显著高于脾脏(P<0.01);京海黄鸡心脏组织中的Myf6基因相对表达量显著高于脾脏(P<0.05),金茅黄鸡心脏组织中的相对表达量极显著高于脾脏(P<0.01);两个品种鸡的其余组织中Myf6基因相对表达量与脾脏相比差异不显著(P>0.05)。京海黄鸡公母鸡胸肌和腿肌组织中Myf6基因的表达规律基本一致，仅在4周龄胸肌组织中有稍微下降的趋势，但总体上呈现上升的趋势；腿肌组织中的相对表达量比较稳定，从0周龄到16周龄呈现上升趋势，16周龄时达到最高水平。金茅花鸡公母鸡胸肌和腿肌组织中Myf6基因的相对表达量从0周龄到4周龄呈上升趋势，8周龄有稍微下降的趋势，之后又呈现上升的趋势。在两个品种中，无论是公鸡还是母鸡，胸肌中Myf6基因的相对表达量始终显著高于腿肌(P<0.05)。说明Myf6基...     

白羽王鸽胚胎期胸肌肌纤维发育规律及相关基因表达分析

实验旨在研究白羽王鸽胚胎期胸肌肌纤维发育规律，并分析肌纤维发育相关基因在胸肌发育过程中的表达变化规律。采集鸽不同孵化时间胸肌组织，利用石蜡切片技术研究胸肌肌纤维发育规律，利用RT-qPCR技术检测肌纤维发育相关基因在胸肌发育过程中的表达变化规律。结果显示，鸽肌纤维在12胚龄时仍处于成肌细胞增殖和分化的混合期，14胚龄时肌细胞融合程度进一步上升，16胚龄时肌细胞增殖和分化基本完成，至出雏时肌纤维已基本发育成熟。在鸽胚胎发育过程中，胸肌肌纤维直径逐渐增加，肌纤维密度呈现先增加后降低的趋势。基因表达结果显示，Myf6表达量随着胚龄的增加不断上升，至出雏达到最大表达量；MyoD呈现先下降后上升的趋势；MyoG表达呈现持续下降的表达趋势，至出雏达到最低表达量；MSTN 12～14胚龄表达量下降，14胚龄后显著回升，至出雏达到最大表达量。综上所述，本研究结果有助于深入理解鸽骨骼肌发育规律，为进一步研究白羽王鸽骨骼肌生长发育调控机理提供理论参考。     

CPT1B基因在灵山香鸡上的时空表达规律研究

本实验旨在阐明CPT1B基因在灵山香鸡上的组织表达和时序表达谱,为研究鸡体内脂肪代谢提供一定的基础资料。选取胚胎期到性成熟时的灵山香鸡为实验材料,在不同发育时间点(9、12、16胚龄,1日龄,1、3、 5、7、10、13、16周龄)采集胸肌、腿肌、心脏、肝脏组织样品,利用实时荧光定量方法分析CPT1B基因在不同组织不同发育阶段的表达规律。结果显示:CPT1B在不同发育阶段4种组织中表达差异性不同,在胚胎期和1日龄,CPT1B基因在肝脏的表达量最高;5、7、10、13周龄时,CPT1B在胸肌的表达量最高;CPT1B基因在4种组织不同发育阶段变化趋势不一致,其在肝脏表达量呈现下降趋势,而在腿肌、胸肌和心脏表达量呈上升趋势,在胸肌的各个发育阶段上升趋势最显著;CPT1B虽然属于肌肉亚型CPT,但不是每个发育阶段在肌肉组织中的表达量最高,在胚胎期,鸡肝脏是CPT1B参与脂肪酸β-氧化的主要场所,而在出雏后到性成熟时期胸肌是CPT1B参与脂肪酸β-氧化的主要场所。     

MyoD and Myf6 gene expression patterns in skeletal muscle during embryonic and posthatch development in the domestic duck (Anas platyrhynchos domestica)

The MyoD and Myf6 genes, which are muscle regulatory factors (MRFs), play major roles in muscle growth and development and initiate muscle fibre formation via the regulation of muscle‐specific gene translation. Therefore, MyoD and Myf6 are potential candidate genes for meat production traits in animals and poultry. The objective of this study was to evaluate MyoD and Myf6 gene expression patterns in the skeletal muscle during early developmental stage of ducks. Gene expression levels were detected using the quantitative RT‐PCR method in the breast muscle (BM) and leg muscle (LM) at embryonic days 13, 17, 21, 25, 27, as well as at 1 week posthatching in Gaoyou and Jinding ducks (Anas platyrhynchos domestica). The MyoD and Myf6 gene profiles in the two duck breeds were consistent during early development, and MyoD gene expression showed a ‘wave’ trend in BM and an approximate ‘anti‐√’ trend in LM. Myf6 gene expression in BM showed the highest level at embryonic day 21, which subsequently decreased, although remained relatively high, while levels at embryonic days 13, 17 and 21 were higher in LM. The results of correlation analysis showed that MyoD and Myf6 gene expression levels were more strongly correlated in LM than in BM in both duck breeds. These results indicated that different expression patterns of the MyoD and Myf6 genes in BM and LM may be related to muscle development and differentiation, suggesting that MyoD and Myf6 are integral to skeletal muscle development.     

Association Analysis between Differences of GnRHR-Ⅱ Gene Expression with Reproduction Traits Heterosis in Duck Populations

The objective of this study was to clone the sequence of duck type Ⅱ gonadotropin releasing hormone receptor (GnRHR-Ⅱ) gene, and to analyze the association of its expression levels with reproduction trait heterosis. RT-PCR method was used to clone GnRHR-Ⅱ gene fragments, and the expression levels during early laying days and laying fastigium period in Gaoyou duck, Jinding duck and crossed populations were tested by Real-time PCR method. Sequencing and homology analysis showed that the cloned duck GnRHR cDNA was about 500 bp in length, and it belonged to the avian type Ⅱ GnRHR isoforms. It was 96% identical to the cGnRHR-Ⅱ sequence reported in domestic chicken. From the early laying days to laying fastigium period, expression levels of GnRHR-Ⅱ gene in pituitary increased for Jinding duck, Gaoyou duck and crossed populations, with the highest expression levels in Jinding×Gaoyou hybrid group (P<0.01) . The expression levels of GnRHR-Ⅱ gene in hypothalamus also increased for Jinding duck, Gaoyou duck, however, it decreased in two crossed populations. The expression levels of GnRHR-Ⅱ gene in ovary were significantly higher for Jinding duck and two crossed populations than Gaoyou duck (P<0.01), but it decreased during laying fastigium period. Compared to Jinding duck and Gaoyou duck breeds, the two crossed populations had positive heterosis in 42-week production and 42-week egg fertilization percent, and negative heterosis in the first laying age and 42-week fertilized egg hatching rate traits. It suggested that the short-lived higher expression of ovary GnRHR-Ⅱ gene before early laying days be correlated with heterosis of the first laying age and 42-week production traits, and its higher expression levels in pituitary could aid to keep laying fastigium.     

鸭卵巢组织基因差异表达与繁殖性状杂种优势率相关性分析

采用mRNA差异显示技术检测了产蛋高峰期高邮鸭、金定鸭及正反交组合群体卵巢组织中的基因差异表达,并分析了差异表达模式与繁殖性状杂种优势率的相关性.研究结果表明:杂交组合在开产日龄、42周龄产蛋数性状上表现出明显的杂种优势,杂种优势率>±10%;16对引物组合共扩增出326条cDNA片段,能够重现的有248条,重现率为76.03%,发现7种差异表达模式;相关性分析显示开产日龄、42周龄产蛋数杂种优势率与杂种超强表达模式呈极显著正相关(P<0.01),与杂种特异表达呈极显著负相关(P<0.01);共显性表达模式与开产日龄杂种优势率呈显著正相关(P<0.05);7种差异表达模式与42周龄种蛋受精率和受精蛋孵化率杂种优势率2个指标之间并不存在显著相关性(P>0.05).     

Influence of recombinant duck follistatin protein on embryonic muscle development and gene expressions

Follistatin (FST) acts as a positive regulator of muscle development by inhibiting the activities and expression of myostatin. The recombinant duck FST protein was injected into hatching eggs and was also added to the medium of duck myoblast to study its role on duck embryonic muscle development and gene expressions. Duck embryo weight increased 3.49% (p > 0.05) in FST treatment group as compared with control group, but minor effects were found on leg or breast muscle weights of ducklings at 2 days post‐hatching (p > 0.05). Relative expression of Pax7 was upregulated in both leg and breast muscle tissues (p < 0.05), while MyoD was only upregulated in leg muscle (p < 0.05), and Myf5 was only upregulated in breast muscle (p < 0.05). Relative expression of myostatin was downregulated in both muscle tissues researched (p < 0.05). In vitro studies also showed some maker genes relevant to protein synthesis and degradation, cells’ proliferation and differentiation had significant changes in myoblasts after treated with FST. These results suggested that in ovo feeding of recombinant FST protein to duck hatching eggs had an effect on duck embryo development but have less roles on the duck embryonic muscle development.     

Molecular evolutionary analysis of the duck MYOD gene family and its differential expression pattern in breast muscle development

1. The objective of the research was to investigate the molecular evolutionary relationships between the duck myogenic determination factors (MYOD) gene family members and their roles in muscle development. 2. The four members of the duck MYOD gene family were cloned using RT-PCR, and their relative mRNA expression during duck muscle development was measured using qRT-PCR. 3. The results showed that MyoD and Myf5 clustered together, as did MyoG and MRF4 based on their complete amino acid sequence and the basic helix-loop-helix domain. Results of the evolutionary level analysis were consistent with that of the differential expression patterns during duck breast muscle development. As determined by qRT-PCR, MyoD and Myf5 were highly expressed in 22-day embryos, while MyoG and MRF4 expression was high in 14-day embryos. 4. We conclude that the entire MYOD gene family in the duck originated from a common ancestral gene and evolved after two duplication events. The roles of the MYOD gene family members in duck muscle development are similar to those in mammals.     

中国6个地方鸭品种蛋品质比较

本研究测定了处于产蛋高峰6个地方鸭品种同一天各30枚种蛋的蛋重、壳重、蛋黄重、蛋白重、膜重、蛋黄蛋白比、哈氏单位、蛋壳厚度、蛋黄色泽、蛋形指数、蛋壳强度等11项蛋品质的指标。结果表明:高邮鸭除哈氏单位稍低外,其他10项指标均比其他5个鸭品种高;11项指标主成分分析说明了蛋重是蛋品质的一个重要指标,6个鸭品种分别依特征值排序的结果中,由前到后顺序为:高邮鸭、金定鸭、莆田黑鸭、山麻鸭、攸县麻鸭、连城白鸭。研究结果为进一步开发利用品种资源提供了参考依据。     

猪骨骼肌卫星细胞中miR-24功能的初步研究及靶基因分析

为了探讨miR-24对猪骨骼肌发育的影响,对体外培养1日龄纯种长白公猪骨骼肌卫星细胞分别转染miR-24过表达载体和干扰片段,发现过表达miR-24后,成肌分化基因MyoD、Myogenin和Myf5表达量显著升高,骨骼肌卫星细胞分化趋势增强;干扰miR-24后,Myogenin的表达量显著降低;同时,靶基因预测结果显示Myogenin可能是miR-24的靶基因。     

北京鸭Myogenin基因部分序列的克隆及表达时间分析

利用4对引物分别对42、14日龄北京鸭胸肌组织RNA及出生后0日龄北京鸭腿肌组织和孵化22、15日龄胚胎腿肌RNA进行Myogenin基因的PCR反转录扩增，均没扩增出目的基因。资料分析表明在胚胎发育期内Myogenin基因可能只在成肌细胞分化的特定时间内表达，Myogenin基因也可在动物失去神经后或在体外培养的肌卫星细胞内表达。利用DNA扩增出了北京鸭Myogenin基因外显子1260bp部分序列，共编码86个氨基酸，编码氨基酸序列含有bHLH结构域，该结构与鸡、火鸡的同源性非常高。研究结果将为北京鸭Myogenin的表达、全序列的克隆及其分子标记的研究提供理论基础。