鸭胚骨骼肌不同组织Myf6基因表达的发育性变化研究

为研究Myf6基因对鸭肌肉发育的影响,本试验利用实时荧光定量PCR绝对定量技术检测Myf6基因在高邮鸭和金定鸭胚胎期第13、17、21、25、27天及出生后7日龄胸肌、腿肌发育过程中的表达量。结果显示,Myf6基因在2个品种的胸肌和腿肌中均有表达,在不同品种同一肌肉组织中的表达变化规律一致,而在不同肌肉组织中的表达模式不同,其中在胸肌组织中21胚龄时表达量最高,随后表达量下降,维持相对较高表达水平;在腿肌组织中13胚龄时表达量较高,17胚龄时达到最高,随后表达量有所降低,并在21胚龄后急速下降至极低水平,但其表达在胚胎期后期(27胚龄)及初生期(7日龄)又有缓慢上升趋势。上述结果表明,Myf6基因参与了胸、腿肌组织的发育,在胸、腿肌中表达模式不同,推测Myf6基因在胸、腿肌中的调控存在差异,可能与其胸、腿肌不同肌纤维的发育与分化有关。     

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.     

生肌调节因子及肌生成调控因素研究进展

生肌调节因子(myogenic regulatory factors,MRFs)是调控肌肉生成的重要基因,家族中包括MyoD、MyoG、Myf5和Myf6。MyoD能把多种类型细胞转化为成肌细胞,在肌肉特异基因转录调控中起着总开关作用;MyoG的表达能够控制成肌细胞融合的起始,促使成肌细胞增殖;Myf5协同MyoD在肌肉形成过程中发挥作用,而Myf6与MyoG共同控制肌肉分化。研究表明,MRFs家族基因的表达与肉用动物的产肉性能和肉质性状密切相关,遗传、营养和环境等因素对该家族基因的表达水平有显著影响。作者综述了MRFs家族的结构与功能、基因表达与活性调节以及营养、光照、温度、运动和活性物质等因素对该家族成员基因活性和肌生成的调控作用,以期为建立动物肌肉发育调控技术奠定理论基础。     

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     

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.     

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type‐specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin‐deficient double‐muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse‐transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC‐2x and ‐2a and less ‐slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster‐type muscles and on MyoD expression in slower‐type muscles, suggesting a possible muscle type‐specific effect of myostatin in skeletal muscle growth and maintenance.     

海南五指山猪Myf5与MyoD基因多态性分析

为研究海南五指山猪Myf5和MyoD基因的多态性分布情况,本试验通过PCR-RFLP方法对80头海南五指山猪进行了Myf5和MyoD基因型检测,并运用生物信息学方法对这两个基因的序列进行分析.结果发现,在Myf5基因第1外显子的HhaⅠ-RFLP位点上,五指山猪以CC和CG基因型为主;在Myf5基因第1外显子的Hsp92Ⅱ-RFLP位点上,五指山猪以AA基因型为主;在MyoD基因第1内含子的DdeⅠ-RFLP位点上,等位基因A在五指山猪中是优势等位基因.此外,Myf5和MyoD基因启动子区域和第1外显子处存在CpG岛.本研究得到海南五指山猪Myf5和MyoD基因的序列信息,为下一步五指山猪Myf5和MyoD基因的功能研究奠定基础.     

绵羊骨骼肌生长发育调控基因研究进展

骨骼肌发育是一个复杂的过程,其中包括肌细胞的形成与增殖、肌管和肌纤维的形成及最后的成熟过程。骨骼肌生长发育直接影响到绵羊的生产性能。随着测序技术的发展,更多人尝试从基因层面来阐述骨骼肌发育过程中系统的调控网络,挖掘与肉品质、产肉性能等重要功能性状相关的分子标记。肌细胞增强因子2(MEF2)是控制肌肉基因表达的保守且功能显著的转录因子,在调节肌肉生成、神经发育与分化等方面起作用。成肌细胞决定因子(myogenic differentiation,MyoD)基因家族调控肌细胞分化和骨骼肌系统的发育成熟。为了更深入地认识与绵羊骨骼肌生长发育相关的调控基因的功能,作者从骨骼肌的结构特点入手,重点介绍MEF2与MyoD基因家族的结构与功能,以及非编码RNA对骨骼肌中基因表达的调控作用。     

Polymorphism Analysis of Myf5 and MyoD Genes in Hainan Wuzhishan Pig

This study was aimed to investigate the distribution of genetic polymorphisms of Myf5 and MyoD genes in Hainan Wuzhishan pig.PCR-RFLP method was used to detect the Myf5 and MyoD of 80 Wushishan pigs.Some characters of Myf5 and MyoD genes were analyzed by bioinformatics tools.The main results were as follows at the HhaⅠ-RFLP locus of exon 1 in Myf5 gene,CC and CG genetypes were major in Wuzhishan pig.At the Hsp92Ⅱ-RFLP locus of exon 1 in Myf5 gene,AA genetype was major in Wuzhishan pig.At the DdeⅠ-RFLP locus of intron 1 in MyoD gene,allele A was predominant in Wuzhishan pig.In addition,bioinformatics analysis revealed that Myf5 and MyoD genes promoter and exon 1 region contained CpG islands.In this study,we obtained the sequences of Myf5 and MyoD genes which laid a foundation for further studying their functions in Hainan Wuzhishan pig.     

Sequential expression of myogenic regulatory factors in bovine skeletal muscle and the satellite cell culture

Myogenic regulatory factors (MRFs) are important in the control of skeletal muscle development. To understand myogenic regulation by MRFs in bovine adult muscle cells, their expressions, namely that of Myf5, MyoD, myogenin, and MRF4 in the biceps femoris muscle (BF) and in the satellite cell culture, were analyzed by RT-PCR. In the BF, all four MRFs were expressed and in particular, myogenin and MRF4 were strongly expressed, whereas Myf5 was faintly expressed. The satellite cells prepared from the BF expressed Myf5, but only a trace of MyoD, at day 9 of culture. During the growth of the cells to day 14, the MyoD and myogenin expressions gradually increased, and that of MyoD expression reached its maximum at the confluence of the culture. After induction of myogenic differentiation by a serum-free medium at day 14, Myf5 expression gradually decreased, and the up-regulated expression of MyoD was suppressed, whereas myogenin expression continued to increase sharply. Following the myogenin expression, MRF4 also drastically increased toward the myotube formation of the cells. When huge myotubes were formed at day 18, Myf5 was expressed at a low level, whereas the MyoD expression remained at a moderate level.     

Caprine sex affects skeletal muscle profile and MRFs expression during postnatal development

The important roles of myogenic regulatory factors (MRFs) have been well addressed in the process of mammalian skeletal myogenesis, while limited research has been performed in small ruminants. Furthermore, the effects of gender on the development of skeletal muscle and MRFs expression remain unknown. In this study, we identified the caprine Myf5, Myf6, MyoD and myogenin genes and quantified their expressions at six different postnatal time points by real‐time RT‐PCR. The sex has a marked effect on the expression differences of Myf5, MyoD and myogenin in the five investigated skeletal muscles, while minor influence on the expression difference of Myf6 except for Semitendinosus and Quadriceps femoris tissues (P < 0.001). The histological sections of muscles revealed a constant increase of muscle fiber diameter with aging but non‐significant differences between genders. We provide novel evidence for MRFs expression in age‐ and gender‐dependent manners, which will contribute to prioritizing these genes as potential candidate genes for trait‐associated study and provide a foundation for understanding the molecular control of skeletal muscle growth in goat species.     

五指山猪不同组织中Myf5与MyoD1基因的表达研究

 MRFs家族成员包括Myf5、MyoD1、Myf4和Mfy6,利用Real time PCR技术,检测Myf5、MyoD1基因在从出生到体成熟（30 d、210 d、360 d）五指山猪背部肌肉组织中的表达变化趋势,以及Myf5、MyoD1基因在体成熟五指山猪心脏、肝脏、肺脏、脾脏、肾脏、肌、胃和小肠以上组织中的mRNA表达水平。结果表明：Myf5和MyoD1基因在出生后五指山猪背部肌肉组织中的mRNA表达水平与五指山猪的生长年龄成正比（P<0.05）;Myf5及MyoD1基因在成年五指山猪以上8种组织中均有表达,其中在肌肉组织中相对表达量最高。     

Expression profiles and associations of muscle regulatory factor (MRF) genes with growth traits in Tibetan chickens

1. Muscle regulatory factors (MRFs), including Myf5, Myf6 (MRF4/herculin), MyoD and MyoG (myogenin), play pivotal roles in muscle growth and development. Therefore, they are considered as candidate genes for meat production traits in livestock and poultry.

2. The objective of this study was to investigate the expression profiles of these genes in skeletal muscles (breast muscle and thigh muscle) at 5 developmental stages (0, 81, 119, 154 and 210 d old) of Tibetan chickens. Relationships between expressions of these genes and growth and carcass traits in these chickens were also estimated.

3. The expression profiles showed that in the breast muscle of both genders the mRNA levels of MRF genes were highest on the day of hatching, then declined significantly from d 0 to d 81, and fluctuated in a certain range from d 81 to d 210. However, the expression of Myf5, Myf6 and MyoG reached peaks in the thigh muscle in 118-d-old females and for MyoD in 154-d-old females, whereas the mRNA amounts of MRF genes in the male thigh muscle were in a narrow range from d 0 to d 210.

4. Correlation analysis suggested that gender had an influence on the relationships of MRF gene expression with growth traits. The RNA levels of MyoD, Myf5 genes in male breast muscle were positively related with several growth traits of Tibetan chickens (P < 0.05). No correlation was found between expressions of MRF genes and carcass traits of the chickens.

5. These results will provide a base for functional studies of MRF genes on growth and development of Tibetan chickens, as well as selective breeding and resource exploration.     

牛MRFs基因家族研究进展

肌肉调节因子（MRFs）基因家族又叫生肌决定因子（MyoD）基因家族,该家族编码4种肌肉特异性转录因子,分别是MyoD、MyoG、Myf5和Myf6,它们是控制骨骼肌生成的关键调节因子,共同控制肌肉的生成。研究表明,这些肌肉特异性转录因子与肌肉生长和肉质有着密切的关系。近年来,该家族的基因已成为肉用动物研究的热点,在牛上已有一些研究,其中包括遗传变异及功能研究等。在此,本文对牛MRFs家族基因结构与基因定位,MRFs家族的功能,MRFs家族基因遗传多态性研究的现状及其在肉牛育种中的应用进行了综述。     

水禽StAR基因克隆、表达及其对睾丸发育的影响

旨在获取水禽StAR基因的结构和组织表达规律，初步了解其对水禽睾丸发育的影响。本研究选取山麻鸭和狮头鹅的下丘脑和睾丸组织为材料，克隆StAR基因，并采用实时荧光定量PCR方法检测该基因在山麻鸭和狮头鹅不同组织中的表达规律，并进一步比较该基因在不同时期鹅睾丸组织中的表达差异。本试验克隆获得鸭StAR基因3个转录本（dSTAR-A、dSTAR-B和dSTAR-C），dSTAR-A和dSTAR-B编码序列相同，编码283个氨基酸，而dSTAR-C编码238个氨基酸；获得鹅STAR基因两个转录本（gSTAR-A和gSTAR-B），均编码283个氨基酸。比对分析发现，鸭和鹅StAR氨基酸序列与其他禽类的同源性较高，在物种间的保守性高。StAR基因在鸭和鹅的各个组织中均有表达，其中在睾丸中表达量最高，在腹脂、胸肌和腿肌也有较高的表达水平。比较不同时期公鹅的睾丸，发现该基因在3岁龄时表达水平极显著高于1和52日龄（P<0.01），且在成年公鹅繁殖期的表达水平极显著高于休产期（P<0.01）。结果表明，StAR基因可能是鹅睾丸发育所必需的关键基因，并参与成年公鹅繁殖性能的调控。     

7个猪种MyoD基因家族中3个基因外显子的SNPs检测分析

本研究利用PCR SSCP技术,以7个猪种为研究对象,根据GenBank上猪的MyoG、MyoD和Myf5基因序列设计了17对引物,对此三基因的外显子进行了SNPs检测。检测结果:MyoG和MyoD两个基因均未检测到多态;在Myf5基因的3个外显子上都检测到了多态:第1外显子上有1个点突变G→C;第2外显子上有3个点突变C→A、A→G、G→A;第3外显子上,3387bp处有1个碱基A缺失,3417bp处有3个碱基T缺失,3443bp处有1个点突变T→C。Myf5基因的高度多态性,说明7个猪种含有丰富的遗传变异信息,具有很高的选择潜力。     

Lentivirus-mediated RNA interference of myostatin gene affects MyoD and Myf5 gene expression in duck embryonic myoblasts

1. The aim of this study was to construct lentivirus-mediated short hairpin RNA (shRNA) vectors targeting the duck MSTN gene and investigate whether these vectors can affect the development of duck primary cultured embryonic myoblasts.

2. MSTN mRNA levels in the myoblasts were detected using quantitative real-time polymerase chain reaction (PCR), cell proliferation was assessed by MTT assays and cell differentiation was assayed by photography.

3. MSTN mRNA levels in PLL3.7-MSTN-shRNA1, PLL3.7-MSTN-shRNA2 and PLL3.7-MSTN-shRNA3 lentivirus-mediated shRNA groups were reduced by 61.6%, 76.9% and 79.1%, respectively, compared to control cells.

4. Down-regulation of MSTN in duck embryonic myoblasts stimulated cell proliferation and inhibited differentiation, accompanied by a greater than twofold down-regulation of MyoD expression and up-regulation of Myf5 expression.

5. These results revealed that silencing of MSTN changes the development of duck embryonic myoblasts by regulating the expression level of MyoD and Myf5 genes.