MYLPF基因在肉牛肌肉生长过程中的功能研究

试验旨在探究快肌肌浆球蛋白可调节磷酸化轻链（myosin light chain,phosphorylatable,fast skeletal muscle,HUMMLC2B或MYLPF）基因在布莱凯特黑牛各组织中的表达情况,以及布莱凯特黑牛与鲁西黄牛背最长肌中MYLPF基因表达量的变化规律,为探讨其与肌肉生长发育关系奠定基础。以布莱凯特黑牛和鲁西黄牛为研究对象,利用实时荧光定量PCR检测其背最长肌、心脏、肺脏、肝脏、脾脏等10个组织中MYLPF基因的表达情况,并对布莱凯特黑牛和鲁西黄牛2、6、10和12月龄4个不同时期背最长肌中MYLPF基因的表达量变化进行研究。结果表明,MYLPF基因在背最长肌中高表达,在心脏中少量表达,在其他组织中几乎不表达。MYLPF基因在2、6、10和12月龄牛背最长肌中均高表达,但随着年龄的增加MYLPF基因的表达量逐渐降低,2月龄的表达量最高,极显著高于其他3个时期（P<0.01）,12月龄表达量最低。4个时期布莱凯特黑牛背最长肌中MYLPF基因的表达量均高于鲁西黄牛,其中10月龄差异极显著（P<0.01）,2和12月龄差异显著（P<0.05）,6月龄差异不显著（P>0.05）。综上结果表明,背最长肌中MYLPF基因可能对骨骼肌生长发育起调节作用,可为研究肉牛骨骼肌生长发育机理和阐明肌肉生长的分子机制提供参考。     

microRNA-155在长白猪和通城猪骨骼肌发育过程中的表达分析

本研究旨在分析miR-155在长白猪(瘦肉型)和通城猪(脂肪型)骨骼肌发育过程中的表达变化,以了解其在猪肌肉发育中的重要作用。选用长白猪和通城猪不同发育时期(胚胎期12个时期,出生后10个时期)骨骼肌为材料,利用Stem-Loop RT-PCR方法检测miR-155的表达变化。此外,本研究还分析了miR-155在成年通城猪各组织的表达。结果,miR-155在长白和通城猪骨骼肌发育过程中差异表达呈现不同的表达模式。在通城猪中,miR-155在小肠和肺中表达量较高,在心脏和胎盘中度表达,而在其他组织中表达较低。结果提示,miR-155可能参与猪肌细胞增殖和分化,从而影响猪骨骼肌发育,与猪肌肉的表型有关。     

广西隆林黄牛HDACs家族基因调控肌肉发育的表达分析

牛的肌肉发育是涉及众多基因表达及网络式调控的复杂过程。目前,研究牛肌肉发育的分子调控机理并不完善,因此,挖掘与牛肌肉发育相关的基因可为进一步阐明隆林黄牛肌肉发育的分子机制奠定基础。本研究以隆林黄牛为研究对象,运用实时定量PCR(RT-qPCR)技术检测出生后6月龄和18月龄背最长肌中HDACs家族基因的表达情况,并分析HDACs家族基因在牛成肌细胞增殖、分化阶段中的表达情况。结果表明:在2个不同生长阶段的肌肉组织中,HDAC7、HDAC8、HDAC10基因表达无显著差异,其余HDACs家族基因的表达都显著上调,其中,HDAC11在18月龄肌肉组织的表达量最高(P0.000 1),且HDACs家族基因在牛成肌细胞增殖过程保持高表达水平。由此推测,HDACs基因在黄牛骨骼肌发育过程中可能具有重要的调控作用,可作为后续骨骼肌发育研究中的表观遗传修饰的候选基因进行深入研究。     

鲁西黄牛皮肤成纤维细胞分离与培养的研究

研究了鲁西黄牛耳皮肤成纤维细胞的分离与培养、纯化方法及生长特征,获得了传代培养的鲁西黄牛皮肤成纤维细胞和上皮细胞.鲁西黄牛耳皮肤细胞在体外培养时呈贴壁生长,其原代或早期传代培养物由上皮样细胞与成纤维细胞混合组成.传代培养混合生长的细胞用0.25%胰蛋白酶液37℃下消化处理3～4 min,脱壁悬浮的主要为成纤维细胞.采用反复消化法在传代过程中将二者逐步分离纯化,获得了可正常传代的鲁西黄牛耳上皮细胞系和成纤维细胞系.通过绘制生长曲线研究了鲁西黄牛耳皮肤成纤维细胞的增殖规律.传代培养至12代的鲁西黄牛皮肤成纤维细胞染色体倍性正常.利用第6代成纤维细胞作核供体克隆试验结果证明重构胚体外发育正常,囊胚发育率为27.3%.     

pIRES2-EGFP-MyoG真核表达载体的构建和鉴定

为了构建能够表达日本和牛生肌素(myogenin,MyoG)的真核细胞表达载体pIRES2-EGFP-MyoG,试验从日本和牛成纤维细胞基因组中扩增出MyoG基因,插到带有EGFP和内部核糖体转入位点(IRES)的真核细胞表达载体pIRES2-EGFP中,经酶切鉴定正确后转染鲁西黄牛胎儿成纤维细胞,用RT-PCR和Western-blot检测MyoG基因的表达情况。结果表明:真核表达载体pIRES-EGFP-MyoG构建成功,且在鲁西黄牛胎儿成纤维细胞中得到了有效表达。说明真核表达载体中的MyoG能够在鲁西黄牛胎儿成纤维细胞中成功表达。     

表观遗传修饰在骨骼肌细胞增殖分化过程中的研究进展

骨骼肌是肌肉的主要构成部分，骨骼肌细胞发生增殖和分化的过程都是肌肉发育的基础，直接影响着家养动物的产肉性能。研究发现表观遗传修饰作用对骨骼肌细胞增殖分化具有重要的调控作用，表明该遗传修饰作用对家养动物肌肉发育具有重大的意义。作者从DNA甲基化对骨骼肌细胞增殖分化影响、组蛋白乙酰化所含因子调控基因选择表达作用、非编码RNA调控和染色体重塑作用所起的影响等方面分别介绍了表观遗传在骨骼肌细胞增殖分化过程中的研究进展，简述了不同修饰方式和不同作用因子对骨骼肌增殖和分化两个过程的影响。同时也回顾了前人在研究骨骼肌增殖分化过程所用到的方法和手段，进而分析了表观调控作用因子在骨骼肌生长过程中所起到的作用。旨在进一步阐述表观遗传修饰在骨骼肌增殖和分化过程中所起到的重要作用，增强对骨骼肌增殖分化调控过程的了解，为和动物生产实际相结合提供参考途径，同时也为骨骼肌生长发育等分子调控提供更多参考素材。     

CaN/NFAT信号通路相关基因在鸡骨骼肌中的表达规律及关联分析

旨在探讨CaN/NFAT信号通路相关基因在鸡生长发育早期不同表型骨骼肌中的表达规律以及各基因之间的调控联系。利用荧光定量PCR技术检测隐性白羽鸡2种表型骨骼肌(腓肠肌外侧头和趾长伸肌)中CaN/NFAT信号通路相关基因(CnAα、CnB1、NFATc3、MEF2C和PPARGC1A)在出生后不同生长阶段(0、1、3、5、7和9周龄)的相对表达,并对这些基因的表达进行相关性分析。结果表明,除CnB1基因外,其他4个基因mRNA在2种表型骨骼肌中的表达趋势均较相似;2种表型骨骼肌中5个基因的表达从7周龄到9周龄均呈上升的趋势;同周龄2种表型骨骼肌相比,除腓肠肌外侧头PPARGC1A基因各周龄表达均高于趾长伸肌外,其他4个基因表达均是在3周龄时腓肠肌外侧头高于趾长伸肌,而在7和9周龄时则低于趾长伸肌;相关分析结果表明,除CnAα与NFATc3、CnAα与PPARGC1A基因的表达在2种表型肌肉中呈负相关外,其余各基因表达之间均呈正相关。提示,鸡骨骼肌CnAα、CnB1、NFATc3、MEF2C和PPARGC1A基因表达发育模式具有年龄和表型特异性,可能参与调控鸡骨骼肌肌纤维的生长和类型转换。     

猪CFL2基因启动子的克隆与序列分析

CFL是真核生物肌动蛋白结合蛋白家族的成员。CFL2主要在哺乳动物的骨骼肌和心肌表达,在肌肉纤维的形成过程及肌肉的再生起重要作用。为了深入研究猪CFL2基因转录的调控机制,本研究利用染色体步移法首次获得了CFL2基因起始密码子上游约2.5 kb区域的序列。应用相关生物信息学软件对该序列进行分析,预测了其启动子核心区域及转录起始点,发现在起始密码子上游-508 bp和-453 bp两处存在TATA-box,另外还发现了GC-box、CAAT-box;该序列还包含SP1、AP1、AP2、GATA-1等转录因子结合位点。本试验结果为进一步研究该基因表达调控与肌肉发育的关系奠定了基础。     

不同营养水平小尾寒羊体内AMPK表达调控的研究

本研究旨在通过免疫组织化学及Realtime PCR来研究不同营养水平条件下磷酸蛋白激酶AMPK和P-AMPK在小尾寒羊体内的分布情况及AMPKa亚基表达量的变化。研究结果表明,AMPKα亚基在下丘脑、肝、心、肾、脾、骨骼肌、十二指肠中分布,而P-AMPK在下丘脑、肝、心、肾、骨骼肌、十二指肠等组织中分布;低营养组AMPKαlmRNA相对表达量上调的组织有下丘脑、肝、心、骨骼肌、脂肪,禁食组AMPKαltaRNA相对表达量上调的组织有骨骼肌、脂肪。低营养组AMPKa2mRNA相对表达量上调的组织有下丘脑、心、骨骼肌、脂肪,禁食组AMPKa2mRNA相对表达量上调的组织有骨骼肌、脂肪、心、下丘脑。说明能量摄入水平对小尾寒羊体内AMPKαmRNA表达丰度有影响,并且AMPK对能量摄入水平的调节具有组织特畀性。     

牛骨骼肌特异性启动子的筛选

分别构建含有牛骨骼肌α-肌动蛋白（α-actin）启动子、牛骨骼肌肌球蛋白轻链2(mylpf)启动子及牛肌酸激酶(ckm)启动子的荧光素酶报告基因表达质粒。将3种荧光素酶表达质粒分别与含有海肾荧光素酶的质粒共转染牛成肌细胞和牛成纤维细胞。经双荧光素酶检测得到转染成肌细胞,72 h后,α-actin启动子的表达量显著高于mylpf启动子和ckm启动子,转染成纤维细胞72 h后,这3种启动子的表达量和空载体相近。结果表明,α-actin启动子在成肌细胞中的表达效率高于mylpf启动子和ckm启动子;3种启动子在成纤维细胞中的表达量与空载体相近,证明了这3种启动子的骨骼肌特异性。通过对骨骼肌特异性启动子的研究,为外源基因在骨骼肌组织的特异性表达提供了试验依据,为进一步研究转基因肉牛奠定了基础。     

猪各组织器官内肌生成抑制素基因mRNA表达谱的研究

用Trizol试剂法提取军牧一号猪的骨骼肌、心肌、肝、肾、肺、脾、胰、脂肪的总RNA做RT-PCR和嵌套PCR,结果在军牧一号猪骨骼肌、心肌和脂肪组织内检测到了肌生成抑制素mRNA的表达,而在其他组织中未检测到.     

TIMP3基因在长白猪和通城猪不同组织及不同时期骨骼肌中的表达分析

采用定量PCR(Quantification PCR, QPCR)方法,比较分析了基质金属蛋白酶3(TIMP3)基因在长白猪和通城猪间的组织表达谱,同时比较分析了其在这2个猪种胚胎期(33, 45, 55, 65, 70和90 d)和出生后(0, 9, 30, 60, 120和160 d)骨骼肌生长发育过程中的差异性表达。组织表达谱结果表明,TIMP3基因在不同猪种的各组织器官中广泛表达,除在腿肌和胰腺中有所差异外,TIMP3基因在两个猪种间的组织表达谱基本是一致的,在肺脏和子宫中表达最高,在背肌、脾脏和胃中表达较高,在肠、心脏、肾脏和肝脏中表达最低。与出生后阶段相比,TIMP3基因在2个猪种胚胎期均显著高表达(P＜0.05),但其表达变化趋势在2个猪种间存在差异;与在长白猪胚胎期相比,该基因在通城猪胚胎期中的高表达水平维持在一个更大的范围内。本试验结果提示,TIMP3基因可能参与不同类型猪种骨骼肌生长发育异步性的调控,影响到不同类型猪种的产肉性状。     

金茅黑鸡miR-206表达及靶基因预测分析

为了研究miR-206的组织表达分布及其在鸡骨骼肌生长发育中的表达规律，预测其可能的作用机制，本研究采集4周龄金茅黑鸡的心脏、肝脏、脾脏、肺脏、肾脏、胸肌、腿肌和腹脂8种组织及2、6、10、14、16周龄胸肌和腿肌组织，利用实时荧光定量PCR技术检测miR-206在8种组织及不同周龄胸肌和腿肌中的表达，利用生物信息学方法预测其靶基因并进行功能注释。结果显示，miR-206在公、母鸡胸肌和腿肌中表达量均极显著高于其他组织（P < 0.01），在腹脂、心脏、肝脏、脾脏、肺脏和肾脏中低表达，为鸡骨骼肌特异性表达miRNA；miR-206均在公、母鸡腿肌和胸肌组织生长早期（2周龄）表达最高，之后表达量逐渐下降。靶基因预测和功能分析发现，miR-206共有356个靶基因，其中21个与肌肉生成相关，多个靶基因已知参与调控肌肉生成，包括配对框7（paired box 7，Pax7）、胰岛素样生长因子1（insulin-like growth factor 1，IGF1）、脑衍生神经营养因子1（brain-derived neurotrophic factor 1，BDNF1）、视黄酸受体（retinoic acid receptor beta，RARB）和卷曲类受体7（frizzled class receptor 7，FZD7）基因。此外，发现miR-206靶基因富集到多条已知参与调控骨骼肌生成的信号通路，包括MAPK、Wnt、肌动蛋白骨架调控和黏着斑激酶信号通路。结合表达数据推测，miR-206可能通过靶向调控Pax7、IGF1、BDNF1、RARB、FZD7基因和MAPK、Wnt等信号通路参与调控鸡肌肉生成。本研究揭示了金茅黑鸡miR-206的组织表达分布及其在鸡骨骼肌生长过程中的表达规律，并预测了其调控骨骼肌生成的可能作用机理，为进一步研究miR-206在鸡肌肉生长发育中的作用机理和表达调控奠定了基础。     

Study on the Function of MYLPF Gene of Muscle Growth in Cattle

This study was aimed to investigate the expression level of myosin light chain,phosphorylatable,fast skeletal muscle (MYLPF) gene in the tissues of Black cattle, and its expression level in the longissimus dorsi muscle of Black cattle and Luxi cattle, and to lay the foundation for the study of its relationship with muscle growth and development. The MYLPF gene expression level in 10 tissues of longissimus dorsi, heart, lung, liver and spleen and so on were detected by Real-time quantitative PCR, and then the changes of MYLPF gene expression level in longissimus dorsi of Black cattle and Luxi cattle at 2, 6, 10 and 12 months of age were studied. The results showed that MYLPF gene was highly expressed in the longissimus dorsi, lowly expressed in the heart, and the other tissues were almost not expressed. The MYLPF gene expression level in the longissimus dorsi was high in 2, 6, 10 and 12 months, with the increase of age,the MYLPF gene expression level decreased gradually, the expression level of 2 months of age was the highest, which was extremely significantly higher than that of the other three periods (P<0.01). The MYLPF gene expression level in the longissimus dorsi of Black cattle was higher than that of Luxi cattle in four period, which in 10 months of age were extremely significantly different (P<0.01), 2 and 12 months of age had significant difference (P<0.05), the difference in 6 months of age was not significant (P>0.05). The results suggested that MYLPF gene in the longissimus dorsi might play a regulatory role in the growth and development of skeletal muscle, which might provide a reference for studying the mechanism of growth and development of skeletal muscle and the molecular mechanism of muscle growth.     

猪骨骼肌发育过程中免疫相关基因的表达分析

为探究猪骨骼肌发育过程中免疫相关基因的表达模式以及相关生物学功能,本研究根据蓝塘猪和长白猪骨骼肌10个发育时期的转录组数据,对各时期免疫相关基因的表达数目和表达量进行分析,鉴定了相邻发育时期之间以及同一时期品种间差异表达的免疫相关基因,并对后者进行功能注释分析。结果表明:在蓝塘猪与长白猪骨骼肌发育过程中存在1 129个表达的免疫相关基因;胚胎期35 d和49 d、胚胎期91 d和生后期2d这2组相邻时期之间的差异表达免疫相关基因数目最多;品种间差异表达免疫相关基因数目则在胚胎期49d以及生后期180d最多,且趋向于在蓝塘猪上调表达,并富集在NIK/NF-κB信号传导、细胞凋亡的负调控等生物学过程中。本研究表明免疫相关基因在猪骨骼肌发育过程中存在表达,长白猪与蓝塘猪骨骼肌生长发育差异可能与NF-κB介导的细胞凋亡有关。     

Differential expression of peroxisome proliferator-activated receptors alpha and gamma gene in various chicken tissues

The peroxisome proliferator-activated receptors (PPARs) are the members of superfamily of nuclear hormone receptors. A great number of studies in rodent and human have shown that PPARs were involved in the lipids metabolism. The goal of the current study was to investigate the expression pattern of PPAR genes in various tissues of chicken. The tissue samples (heart, liver, spleen, lung, kidney, stomach, intestine, brain, breast muscle and adipose) were collected from six Arber Acres broilers (8 weeks old, male and female birds are half and half). Semi-quantitative RT-PCR and Northern blot were used to characterize the expression of PPAR-alpha and PPAR-gamma genes in the above tissues. By semi-quantitative RT-PCR, the results showed the expression level of PPAR-alpha gene was higher in brain, lung, kidney, heart and intestine, medium in stomach, liver and adipose than in spleen, and it did not express in breast muscle. The expression level of PPAR-gamma gene was higher in adipose, medium in brain and kidney than in spleen, heart, lung, stomach and intestine, but it did not express in liver and breast muscle. Northern blot results showed that PPAR-alpha gene expressed in heart, liver, kidney and stomach, and the intensity of hybridization signal was the stronger in liver and kidney than in other tissues, however, PPAR-gamma gene only expressed in adipose and kidney tissues. The results of this study showed the profile of PPAR gene expression in the chicken was similar to that in rodent, human and pig. However the expression profile of chicken also have its own specific trait, i.e. compared with mammals, PPAR-alpha gene can not be detected in skeletal muscle and PPAR-gamma gene can be stronger expressed in kidney tissues. This work will provide some basic data for the PPAR genes expression and lipids metabolism of birds.     

Expression of Myogenic Regulatory Factors in Myocardium and Skeletal Muscle at Different Fetal Stages

The topic of this research was to investigate the expression pattern of myogenic regulatory factors (MRFs) in the myocardium and skeletal muscle of sheep at different fetal stages,the fetuses of Tan sheep at 60 (E60),70 (E70),80 (E80) and 90 days (E90) of gestation were selected as the research objects,and the myocardium and skeletal muscle were collected.The histogenesis of myocardium and skeletal muscle during fetal development were observed by HE and oil red staining.The expression patterns of MRFs in myocardium and skeletal muscle during fetal development and the expression differences of MRFs between myocardium and skeletal muscle at the same stage were tested by Real-time quantitative PCR.The results of HE staining showed that the microstructure of myocardium and skeletal muscle was very different,myocardial fibers crisscross into a network with high density,which were more mature;the density of skeletal muscle fibers were low with large gaps,the number of fibers from E60 to E90 increased gradually,and the structure fiber bundles was very clear.The results of oil red staining showed that there were no fat droplets stained red in myocardium and skeletal muscle tissues,which means that these two tissues form E60 to E90 did not have fat differentiation.The results of Real-time quantitative PCR showed that the expression of MYOG,Myf5 and Myf6 genes in myocardium decreased continuously from E60 to E90 day,but there was no MYOD gene expression.The expression of MYOD,MYOG and Myf6 genes in skeletal muscle from E60 to E90 increased gradually,among which the level of MYOG gene was the highest,and the expression of Myf5 gene fluctuated periodically.The above results indicated that the skeletal muscle was at the stage of rapid development from E60 to E90,however,the myocardial tissue had been roughly formed,which led to the expression of MRFs in skeletal muscle tissue was much higher than that in myocardial tissue,and MYOG gene played an important role in maintaining the rapid development of skeletal muscle.