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.     

肌生成抑制素基因研究进展

肌生成抑制素是 1 997年发现的骨骼肌生长发育负调控因子 ,肌生成抑制素不仅在骨骼肌中表达 ,还可在心肌和浦肯野纤维等多种组织中表达。研究表明 ,大鼠骨骼肌在体内的再生 ,对 MSTN m RNA的表达表现出明显的时间依赖性 ,并且 m RNA的表达不必由功能性神经支配。通过对不同品种双肌表型牛 MSTN基因的研究 ,发现其骨胳肌增大的共同特点是肌生成抑制素基因发生了突变 ,如布鲁牛 MSTN基因在编码区外显子 3发生 1 1 bp的缺失 ,导致MSTN此位点后的阅读框架改变 ,并在此点后的 1 4个密码子处终止了阅读框架 ,从而使MSTN被截短 ,MSTN蛋白活性区域消失 ,活性丧失 ,结果肌肉大量增加。目前 ,已分别对人、牛和猪的 MSTN基因进行了定位研究 ,猪肌生成抑制素基因定位研究表明 ,猪 MSTN基因位于1 5号染色体上。     

Growth factors controlling muscle development.

The enlarged muscles of certain breeds of cattle, such as the Belgian Blue, have been shown to result from a marked increase in the number of normal sized muscle fibers. Originally insulin-like growth factors (IGFs) were implicated in this myofiber hyperplasia, as IGFs have been shown to stimulate myoblast proliferation as well as maintain fiber differentiation. Recently it has been reported that mice lacking a myostatin gene, a member of the TGFbeta superfamily, have enhanced skeletal mass resulting from increased muscle fiber number and size. Mutations in this gene have been found in double-muscled cattle, indicating that myostatin is an inhibitor of muscle growth. Myostatin is expressed early in gestation and then maintained to adulthood in certain muscles. Myostatin expression in bovine muscle is highest during gestation when muscle fibers are forming and some of the myogenic regulatory factors have elevated expression over the same period as myostatin. Molecular expression of the IGF axis does not differ between Belgian Blue and normal muscled cattle, and IGF-II mRNA is increased throughout formation of secondary fibers in both breeds. However, myostatin and MyoD expression in muscle differ between normal and hypertrophied muscle cattle breeds. This evidence strongly suggests that lack of myostatin is associated with an increase in fiber number which then results in a marked increase in potential muscle mass in double-muscled cattle.     

阳原驴MSTN基因生物信息学分析及组织表达研究

【目的】试验旨在分析阳原驴肌生长抑制素(myostatin,MSTN)基因CDS序列特征及其在不同生长发育时期和不同组织中的表达水平。【方法】采集6、12、18、24月龄阳原驴的血样及不同组织样,提取其基因组DNA及不同组织样总RNA。利用PCR技术扩增阳原驴MSTN基因CDS序列,并对其进行生物信息学分析。利用实时荧光定量PCR技术检测MSTN基因在阳原驴不同生长发育时期背最长肌、腿肌和18月龄不同组织(心脏、肝脏、脾脏、肺脏、肾脏)中的表达水平。【结果】阳原驴MSTN基因CDS序列长1 128 bp,编码375个氨基酸,与马的核苷酸序列相似性最高(99.9%),编码蛋白属于不稳定的亲水性蛋白,含有转化生长因子-β(TGF-β)超家族结构域,不含跨膜区,存在1个信号肽区域,包含6个O-糖基化位点、1个N-糖基化位点和34个磷酸化位点,主要分布于细胞核中(39.1%),二级结构中无规则卷曲占比最高(50.93%)。MSTN基因在18月龄阳原驴背最长肌和腿肌中的表达水平显著高于6、12、24月龄(P＜0.05);MSTN基因在18月龄阳原驴不同组织中均有表达,其中背最长肌中的表达水平显著高于其他组织(P＜0.05),其次是腿肌、肺脏、脾脏、肾脏和肝脏,心脏中的表达水平最低。【结论】试验成功扩增出阳原驴MSTN基因CDS序列,MSTN基因在阳原驴不同生长发育时期背最长肌和腿肌中的表达水平均以18月龄最高,且在18月龄不同组织中以背最长肌和腿肌中表达水平较高。研究结果为进一步探讨MSTN基因在阳原驴骨骼肌生长发育中的作用机制提供参考。     

肌肉抑制素与“双肌”表型关系的研究进展

肌肉抑制素是特异性表达于骨骼肌的一种细胞因子,属于TGF-β超家族成员,可以抑制骨骼肌的发育,并同多种疾病有关。肌肉抑制素在不同物种间高度保守,其抑制功能的丧失可以导致骨骼肌显著增生,即动物产生"双肌"表型。具有"双肌"表型的动物则表现出肌肉产量显著增加、饲料转化率高、肌肉嫩度增加、脂肪含量少、而脂肪酸组成中不饱和脂肪酸比例显著增加等优秀性状,这些性状是动物育种计划中的理想育种目标。本文从"双肌"表型的起源及特点、肌肉抑制素的发现及功能等方面对肌肉抑制素与"双肌"表型关系的研究进展进行了综述。     

Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro

Myostatin is an important negative regulator of muscle growth and development. Natural mutations of the myostatin gene cause a double muscling phenotype in beef cattle, pigs and sheep. Therefore, it is feasible to produce a high growth domestic breed by generating a transgenic animal with a mutation, deletion or knockout of the myostatin gene. Our objective was to introduce a subtle mutation of G to A 281-bp upstream of the 3' untranslated region (3'UTR) end of the myostatin gene in Poll Dorset fetal myoblast cells in vitro. Fetal myoblast cells were isolated from fetuses at day 50 of gestation from Poll Dorset sheep and transfected with linear gene-targeting vector pMSTN-A using electroporation. We obtained seven gene-targeted cell colonies with homologous recombination, which were positive as confirmed by PCR, Southern blot. The Western blot analysis result demonstrated that the myostatin protein expression in positive colonies is lower than that of negative ones. These results strongly suggest that we successfully mutated the myostatin gene of Poll Dorset ovine fetal myoblast cells and the mutation can effectively downregulate the myostatin protein expression.     

Role of serum myostatin during the lactation period

Myostatin, also known as GDF-8 (Growth/Differentiation Factor-8), is a member of the TGF-beta superfamily that negatively regulates skeletal muscle mass in mammals. Mutation of the myostatin gene in mice, cattle, and humans causes a massively developed skeletal muscle, characterized by muscle hypertrophy and hyperplasia. Although myostatin is predominantly expressed in skeletal muscle tissue, several recent studies have shown the presence of myostatin protein in blood and suggested a possible role for circulating myostatin in the regulation of skeletal muscle mass. In the present study, we examined changes in the levels of active form myostatin (13 kDa) in serum after birth by Western blot analysis to predict the role of serum myostatin in early postnatal muscle growth in the rat. Interestingly, the amount of active form myostatin in serum increased after birth and then decreased along with ageing after weaning. To clarify the role of increased serum myostatin during the postnatal period, we administrated follistatin, an inhibitor of myostatin activity, into postnatal rats intraperitoneally just after birth. Follistatin-administration during the postnatal period caused selective hypertrophy of type II muscle fibers in the soleus muscle. These results demonstrate that myostatin in serum acts on skeletal muscle and negatively regulates early postnatal muscle growth.     

三黄鸡ST3Gal6基因的半定量RT-PCR及生物信息学分析

本研究旨在对三黄鸡ST3Gal6基因进行组织表达谱和生物信息学分析。参考三黄鸡ST3Gal6基因序列设计引物,采用PCR技术克隆三黄鸡ST3Gal6基因序列,并利用半定量RT-PCR进行组织表达谱分析;同时对其进行生物信息学分析。结果表明,克隆的三黄鸡ST3Gal6基因全长1169 bp,含有1059 bp的完整CDS编码区,编码352个氨基酸。其CDS编码区的核苷酸序列与人、黑猩猩、牛、大鼠、蟾ST3Gal6基因对应序列的同源性分别为62%、62%、61.9%、59%、54.4%。组织表达谱分析表明,ST3Gal6基因在各组织均不同程度地表达,其中在大脑表达量很高,肺脏中最低。生物信息学预测ST3Gal6蛋白结构发现,三黄鸡的ST3Gal6蛋白存在2个跨膜螺旋结构域,同时预测ST3Gal6存在22个磷酸化位点和1个特异性蛋白激酶磷酸化位点。     

cDNA encoding sequences for myostatin and FGF6 in sea bass (Dicentrarchus labrax, L.) and the effect of fasting and refeeding on their abundance levels

Fish have the ability to compensate for set-backs in growth as a result of fasting. When food levels are restored, growth in these fish can increase over and above normal rates. This phenomenon, known as “compensatory growth”, has been studied with respect to enhancing food conversion efficiency. However, the mechanisms by which food intake activates an increase in somatic growth, and especially in muscle growth, are not well understood. In this study, we report first on the isolation of two complete cDNAs sequences encoding sea bass (Dicentrarchus labrax) myostatin and fibroblast growth factor 6 (FGF6), which have been shown to be major genetic determinants of skeletal muscle growth. The open reading frames of myostatin (376 amino acids) and FGF6 (209 amino acids) showed 97–63% and 87–62% sequence identity with other vertebrate myostatins and FGF6s, respectively. We also report on the expression profile of myostatin and FGF6 in sea bass skeletal muscle in response to different feeding regimens, as quantified by real-time RT-PCR. Nutritional status significantly influenced the myostatin expression levels in muscle, inducing an up-regulation during fasting and a down-regulation during the recovery from fasting, whereas the muscular FGF6 mRNA levels were not significantly affected by the feeding status of the animals. These findings suggest that myostatin has an inhibitory role in muscle growth in response to different feeding regimens, whereas FGF6 is not involved in the muscle compensatory growth induced by refeeding.     

西门塔尔牛肌肉生成抑制素基因编码序列的克隆与序列分析

本研究根据Genbank中牛肌肉生成抑制素基因序列设计引物，在设计的引物两端分别加上限制性内切酶BarnHI和EcoRI的识别位点序列。利用RT—PCR技术从西门塔尔牛肌肉组织的总RNA中扩增出MSTN基因的CDNA序列，扩增出1125bp片段，该片段与pMD18-T载体连接，转化JM109感受态细胞，所得阳性克隆进行酶切和PCR鉴定，并进行了测序分析，得到的克隆序列与设计的序列基本一致，表明成功地克隆了西门塔尔牛的肌肉生成抑制素基因的蛋白编码序列。     

鲁西黄牛MSTN基因上游序列多态性对转录表达的影响

肌肉生长抑制素（myostatin,MSTN）基因是转化生长因子-β（transforming growth factor-β,TGF-β）超家族的新成员。为了研究鲁西黄牛MSTN基因上游序列多态性与转录表达的关系,本试验提取鲁西黄牛腿肌组织细胞的基因组,扩增出MSTN基因上游序列,构建进化树并通过基因测序确定其上游序列中存在单核苷酸多态位点,多态位点位于起始密码子上游805 bp处。构建表达载体,在体外对C2C12细胞进行转染,从而验证MSTN基因上游序列多态位点对转录的影响。结果显示,MSTN基因上游序列中单个核苷酸的改变会影响其下游基因的表达水平。推测在体内MSTN基因上游序列中单核苷酸多态性能够影响基因的转录活性,在调节基因转录的过程中起着重要作用。     

会理黑山羊肌肉生长抑制素基因编码序列的克隆与多样性分析

为探究会理黑山羊遗传分化状况,试验参考普通山羊肌肉生长抑制素（MSTN）基因设计引物,采用PCR方法扩增、克隆会理黑山羊MSTN基因编码区,并与其他物种相应基因编码区核苷酸序列进行比对分析。结果表明,会理黑山羊MSTN基因完整编码序列长度为1128 bp,由外显子1（372 bp）、外显子2（375 bp）和外显子3（381 bp）组成;会理黑山羊与建昌黑山羊、黔北麻羊同源性最高,均达97.4%,聚为一簇,亲缘关系最近,与牛、恒河猴、狗等物种的同源性最低,亲缘关系远;会理黑山羊MSTN基因编码区共编码375个氨基酸,会理黑山羊MSTN基因编码的各种氨基酸含量相差较大,其中以亮氨酸含量最丰富,达8.80%。本研究为会理黑山羊的遗传资源保护、开发与利用提供了分子遗传学研究基础。     

秦川牛生长抑制素基因克隆及原核表达研究

 试验旨在通过基因工程方法获得重组肌肉生长抑制素蛋白。提取秦川牛的骨骼肌总RNA,根据基因库中海福特牛肌肉生长抑制素基因序列设计合成特异性引物,引物两端分别加上Bam HⅠ和Xho Ⅰ酶切位点及保护碱基,用RT-PCR方法扩增肌肉生长抑制素全长基因,并将其克隆到pMD18-T载体上,测序后经Bam HⅠ和Xho Ⅰ双酶切,将目的片段插入到PGEX-4T-1中,构建原核表达载体PGEX-4T-1-M,将重组表达质粒转化至Rosetta(DE3)中诱导表达。结果表明,扩增的秦川牛肌肉生长抑制素全长基因1 128 bp,克隆载体经过DNA序列测定,所得基因与GenBank上发表的一致;成功构建原核表达载体PGEX-4T-1-M,经IPTG诱导,表达出了GST-M融合蛋白,用GST标签抗体做Western blotting印记证明产物大约69 ku,与预期大小相符,并在Rosetta（DE3）菌中成功的进行融合蛋白表达。     

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。     

猪肌肉生长抑制素基因启动子的克隆与鉴定

利用PCR方法从猪基因组DNA中扩增了1.2 kb的肌肉生长抑制素(myostatin)基因启动子序列。并进一步以绿色荧光蛋白(GFP)为报告基因,构建了真核表达载体MSTNPro-EGFP;通过转染C2C12小鼠骨骼肌成肌细胞和猪成纤维细胞,对猪myostatin基因启动子的转录调控活性进行鉴定。结果表明:猪myostatin基因启动子可以启动GFP在C2C12细胞中的转录和表达,而将猪MSTNPro-EGFP载体转染猪胎儿成纤维细胞后并未观察到GFP的表达,说明myostatin基因表达的肌肉特异性源于启动子的转录特异性。     

牛RPLP1基因的克隆和生物信息学分析

采用RT-PCR法克隆牛RPLP1基因,利用生物信息学分析软件对该基因进行生物信息学分析,应用半定量RT-PCR方法对该基因的组织表达谱进行分析。结果表明,牛RPLP1基因全长为502 bp,包含345 bp的编码区序列,编码114个氨基酸;拓扑预测表明,RPLP1蛋白可能存在3个酪蛋白激酶Ⅱ磷酸化位点,2个豆蔻酰化位点,含有一个Ribosomal-P1保守结构域;牛RPLP1基因在多种组织中均表达,在骨骼肌、肝脏和心肌中表达量较高。     

猪TIMP-2基因克隆、序列特征和表达分析

本研究分析了五指山试验用小型猪TIMP-2基因的分子特性和可能的生物学功能.将人TIMP-2 mR-NA全长序列在猪ESTs数据库中检索获得同源性较高的ESTs,用电子克隆结合RT-PCR方法克隆获得包含完整CDS区的猪TIMP-2基因cDNA序列,应用生物信息学方法分析了其核苷酸序列及其编码蛋白质分子结构特性,应用RT-PCR研究该基因在猪不同组织和发育时期的特异表达情况.结果,猪TIMP-2 cDNA序列全长790 bp,包括663 bp的完整开放阅读框,编码221个氨基酸.多序列比对和系统进化分析表明,该基因编码蛋白质与人(97%)、大鼠(97%)、小鼠(97%)等物种TIMP-2蛋白质具有较高的同源性.蛋白质序列预测分析发现,猪TIMP-2氨基酸序列理论等电点(pI)为7.65,相对分子质量(MW)为24.5 ku,它包括27AA的前导序列和194AA的成熟肽段,其前导序列比其它物种多1个亮氨酸(Leu).结构功能预测发现其具有1个在种间高度保守的NTR_TIMP结构域和N端VIRAK保守序列,序列中存在的12个半胱氨酸(Cys)可以形成6对二硫键结构.表达谱分析表明TIMP-2基因在猪的多个组织和不同发育时期的表达量存在较大差异,在睾丸、垂体、胃、大肠、卵巢、子宫和90 d胚胎骨骼肌中高表达;而在心脏、小肠、脑、肝脏、成年猪骨骼肌中表达量极低;在肾脏、胸腺、脾、甲状腺、33 d胚胎中中度表达.结果表明该基因在发生组织发育和重构活动相对活跃的组织器官和时期表达水平较高,亦符合其固有生物学功能.     

牛肌肉生成抑制素基因真核表达载体的构建及其在COS-7细胞中的表达

将构建的牛pMD 18-T-MSTN克隆载体与真核表达载体pef-dhfr1a酶切,回收牛MSTN目的片段及pef-dhfr1a载体,构建了牛MSTN基因的真核表达质粒pef-dhfr1a-MSTN,然后转染COS-7细胞,将牛MSTN成熟蛋白编码序列在COS-7细胞中进行了表达.提取转染细胞的总RNA,采用RT-PCR和Western-blotting方法,分别从mRNA水平和蛋白质水平上检测到了牛MSTN基因在COS-7细胞中的表达产物,证明已经成功构建出该基因的真核表达载体.