SNP Genotyping of Myostatin (MSTN) Gene through Taq Man Probe Assay and Association Study between MSTN Genotypes and Growth Traits of Tan Sheep

To establish a rapid, accurate and economical real-time PCR assay system based on TaqMan probe technology for the detection of genetic variations of single nucleotide polymorphisms （SNPs） in myestatin （MSTN） gene, a pair of TaqMan probes were designed on the polymorphism loci of MSTN gene and used in PCR reaction system for SNP genotyping. Meanwhile, an association study was performed between MSTN genotypes and growth traits of Tan sheep, including birth weight, weaning weight, 3-month weight, and 6-month weight. The results showed that rs417816017 locus of MSTN gene in Tan sheep had two genotypes ： YY and XY. The individuals with genotype XY had a growth advantage over the ones with genotype YY. The results indicate that TaqMan probe-based real-time PCR assay can be used to detect the genotype of MSTN gene, which will provide candidate genes for breeding of Tan sheep.

Sequencing Analysis of Myostatin Gene （MSTN） for Meat Cattle in Mongolia

In developed countries, study on special or candidate genes, which are useful for identifying species, breed and productivity of livestock, was conducted at high level and the results have already been used in practice. Such advanced technology and innovation that we are facing is necessary to adopt in Mongolia. In this study, the myostatin gene （MSTN） was investigated as a candidate gene for meat animal in Mongolian breeds of cattle. The conventional phenol-chloroform method and FavorPrepTM tissue DNA extraction kit were used for DNA isolation, and the polymerase chain reaction （PCR） and sequencing analysis were used for further study. The nucleotide sequences of MSTN gene from Selenge, Kazakh white head breeds and Mongolian cattle were sequenced and reported on the DDBJ/EMBL/GenBank database （LC142726, LC146648, LC146649）, and Selenge breed showed the result of single nucleotide mutation in MSTN gene.

Quantitative Study on Expression of MSTN Gene in Different Tissues of Tibetan Sheep at Different Ages

[Objective] The aim was to investigate the difference in MSTN gene expression in different tissues of Tibetan sheep at different ages.[Method] According to the sequence（NM_001009428.1）published in GenBank,a pair of specific primers was designed to amplify part of cDNA sequence of MSTN by using QRT-PCR technique.The relative expression level of MSTN gene in rennet stomach,rumen,leg muscle and cardiac muscle of Tibetan sheep at different ages were analyzed.[Result] After normalization with β-actin gene,the relative expression level of MSTN gene in the 6-month-old Tibetan sheep was the highest and it was 2.52 times than that in 12-month-old Tibetan sheep（P0.05）,the relative expression level of MSTN gene in leg muscle was the highest among all tissues and it was 3 984.78 times than that in rumen（P0.01）.[Conclusion] The results established theoretical foundation for the correct use of MSTN antibody.

MSTN基因编辑湖羊胫腓肌的转录组分析

[目的]本文旨在探索肌肉抑制素基因(myostatin,MSTN)调控肌肉生长发育的分子机制。[方法]以3月龄MSTN基因编辑湖羊(试验组)和野生型湖羊(对照组)胫腓肌为对象,采用PCR和Western blot方法验证MSTN编辑湖羊编辑形式,在此基础上开展转录组测序和分析,并用real-time PCR方法对差异表达基因进行结果验证。[结果]对照组和试验组共有149个差异表达基因,其中65个基因上调,84个基因下调,GO注释和KEGG富集分析表明,差异表达基因显著富集在氧化磷酸化、果糖和甘露糖代谢、生热作用、FOXO和AMPK信号通路,表明MSTN基因可能通过以上信号通路参与湖羊生长发育调控。[结论]MSTN基因编辑湖羊可能通过FOXO和AMPK、氧化磷酸化、果糖和甘露糖代谢、生热作用等与动物代谢相关的信号通路调控其生长发育。

The genetic diversity analysis in the donkey myostatin gene

Myostatin（MSTN）gene negatively controls skeletal muscle development and growth,variations of which play an important role in the regulation of skeletal muscle growth in mammals.However,study on genetic polymorphism of MSTN gene in donkey is limited.In this study,we screened the single nucleotide polymorphsims（SNPs）of MSTN gene in 13 Chinese donkey breeds.Four novel SNPs（g.229T〉C,g.872A〉G,g.2014G〉A,and g.2395C〉G）were detected and genotyped by sequencing and polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP）methods.Six haplotypes（H1–H6）were analyzed,which indicated abundant haplotype diversities in Chinese donkeys.The haplotype H1 was the most dominant and ancient in all breeds.Xinjiang donkey displayed the highest haplotype diversity.The Neighbour-Joining（NJ）tree of MSTN gene among different species was constructed.The clustering result of nine species was consistent with the fact of species differentiation.Our results will provide a reliable theoretical basis for the preservation,exploration and utilization of Chinese donkey genetic resources.

Dietary Methionine Affect Meat Qulity and Myostatin Gene Exon 1 Region Methylation in Skeletal Muscle Tissues of Broilers

Dietary amino acids imbalance will result in stunted broiler performance and deteriorated meat quality, which are involved in various biochemical cycles in vivo. In this study, the effects of dietary methionine on meat quality and methylation of myostatin exon 1 were investigated. Drip loss of the broilers fed with diet of high methionine levels （0.2%） increased from （6.3 ± 0.1）% （control group） to （10.1 ± 1.0）%, and the muscle shearing force increased from （22.8 ± 1.9） N （control group） to （26.3 ±2.3） N. Moreover, many CpG sites were found at the myostatin exon 1 region （nucleotides 2 360-2 540 bp）. To further understand the regulation of broiler myostatin expression, the methylation status of broiler myostatin exon 1 and its mRNA expression were analyzed. At the myostatin exon 1 region where CG enriches （nucleotides 2 360-2 540 bp）, the percentages of methylation were 46 and 84% in low Met and high Met content groups after 55-d feeding, respectively. In skeletal muscle tissues, the exon 1 hypermethylation status of myostatin gene was found to be negatively correlated with the gene expression. These results suggested that methylation of this gene is a dynamic process, which plays a dominant role in regulating gene expression for development of individuals.

Cloning and Characterization of Largemouth Bass(Micropterus salmoides) Myostatin Encoding Gene and Its Promoter

Myostatin or GDF-8, a member of the transforming growth factor-β （TGF-β） superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass （Micropterus salmoides）. The myostatin encoding gene consisted of three exons （488bp, 371 bp and 1779bp, respectively） and two introns （390bp and 855 bp, respectively）. The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 （MEF2）, serum response factor （SRF）, activator protein 1 （AP1）, etc., and muscle-specific Mt binding site （MTBF） were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This infunnation will be useful for studying the tran- scriptional regulation of myostatin in fish.

Myostatin gene promoter:structure,conservation and importance as a target for muscle modulation

Myostatin(MSTN) is one of the key factors regulating myogenesis. Because of its role as a negative regulator of muscle mass deposition, much interest has been given to its protein and, in recent years, several studies have analysed MSTN gene regulation. This review discusses the MSTN gene promoter, focusing on its structure in several animal species, both vertebrate and invertebrate. We report the important binding sites considering their degree of phylogenetic conservation and roles they play in the promoter activity. Finally, we discuss recent studies focusing on MSTN gene regulation via promoter manipulation and the potential applications they have both in medicine and agriculture.

MSTN^(-/-)牛肌肉miRNA测序及生物信息学分析

为了探寻参与调控动物骨骼肌生长发育的肌肉生长抑制素(myostatin,MSTN)基因的相关miRNA,试验活体采集了5头MSTN基因敲除(MSTN^(-/-))鲁西黄牛(试验组)和5头野生型鲁西黄牛(对照组)的腿臀肌肉,提取肌肉总RNA进行miRNA测序,筛选两组鲁西黄牛肌肉样品间差异表达的miRNA,分析miRNA序列的碱基偏好性,并预测显著差异表达miRNA的靶基因,然后对靶基因进行GO功能和KEGG信号通路富集分析,最后利用实时荧光定量PCR在MSTN基因干扰牛骨骼肌卫星细胞模型上验证miRNA测序数据的准确性。结果表明:miRNA序列第一位碱基的偏好性因长度不同而不同,且不同位置碱基具有偏好性;试验组和对照组样品共鉴定到761个差异表达miRNA,其中有12个显著差异表达miRNA(miR-2285l、miR-496、miR-12006、miR-6533、miR-450b、miR-6524、miR-410、miR-335、miR-2447、miR-147、miR-33a、miR-654),其中试验组有9个较对照组高表达的miRNA(miR-2285l、miR-12006、miR-6533、miR-450b、miR-410、miR-335、miR-147、miR-33a、miR-654)和3个低表达miRNA(miR-496、miR-6524、miR-2447);预测到5969个显著差异表达miRNA的靶基因和9280个靶位点;靶基因主要富集在细胞内部、膜结合细胞器、蛋白结合、生物过程的正向调节等相关功能和卡波氏肉瘤病毒感染、胞吞作用、MAPK信号通路等代谢通路;验证试验中的6个miRNA(miR-6533、miR-450b、miR-2285l、miR-654、miR-12006、miR-496)的表达量变化均与测序结果的变化趋势一致。说明12个显著差异表达miRNA可能参与了MSTN基因调控牛骨骼肌生长发育过程。

牙鲆mstn基因SNPs位点多态性与生长性状的关联分析

为研究牙鲆(Paralichthys olivaceus)肌肉生长抑制基因(mstn)多态性,开发其分子辅助育种新标记,采用重测序方法对120尾牙鲆(3个双克隆杂交家系:60尾;3个雌核发育家系:60尾)的mstn基因进行单核苷酸多态性(Single nucleotide polymorphism,SNP)位点筛选,并对不同基因型个体的生长性状进行多重比较及SNP标记验证。结果显示,共检测到7个SNPs位点,均为颠换型突变;外显子区3个(Exonl 1:G2063A;Exonl 3:C3883T,C4009A),为同义突变;内含子区3个(Intron 1:A2444T;Intron 2:T3816C,A3832C);3'端非编码区1个(3'UTR:C4564A)。SNPs与生长性状关联分析结果表明,A3832C和C4564A标记位点与牙鲆体质量、体长、体高等生长性状显著相关(P<0.05),其中A3832C位点AA基因型和C4564A位点AC基因型在生长上表现出优势,A3832C位点CC和C4564A位点AA、CC基因型在生长上表现出劣势,其他5个SNPs位点对牙鲆生长性状影响不显著(P>0.05)。将A3832C和C4564A标记位点在牙鲆生长快速群体(F:60尾)和生长缓慢群体(S:60尾)中验证,验证结果与多重比对结果一致,表明2个SNPs位点具有较高的稳定性。综上所述,研究筛选出了牙鲆mstn基因中与生长性状显著相关的A3832C和C4564A两个标记位点,为牙鲆分子辅助育种提供了理论参考。

Study on the Polymorphisms of Porcine Myostatin Gene in Promoter Region by PCR-RFLPS

In order to further study functions of the porcine myostatin gene, we analyzed the polymorphisms of porcine myostatin gene in promoter region among different breeds including Yorkshire, Landrace, Duroc, Junmu, Min pig and Sanjiang white pig by PCR-RFLPs. The allele T dominated in the imported lean-type pig breeds such as Yorkshire, Landrace and Duroc. No allele A was detected in Junmu and Sanjiang white pig, and the frequencies of three genotypes were about equal in Min pig. The result using X2 analysis showed that the distribution of three genotypes was related to pig breeds.

Cloning and Sequence Analysis on 3′ Coding Region of Wild Boar and Cross Bred Pig Myostatin Gene

Myostatin, with a highly conservative gene among breeds is a negative regulator of muscle. The 3′ coding regions of wild boar and crossbred pig myostatin were cloned by RT-PCR and sequenced respectively. The homology of the nucleotide sequence between wild boar and crossbred pig was 100% and there was no difference in this region compared with pig myostatin gene of Genbank. This indicated that there was not change of gene sequence in this region during the evolution processes.

Sequence and Bioinformatics Analysis on MSTN Gene of the Hybrid Grouper Derived from(Epinephelus fuscoguttatus×Epinephelus polyphekadion)

[Objectives]This study aimed to investigate the sequence structure and function of Myostatin(MSTN)gene in the hybrid grouper(Epinephelus fuscoguttatus,♀×Epinephelus polyphekadion,♂).[Methods]Genetic DNA samples were extracted from the caudal fins of the hybrid grouper and its parents to amplify their MSTN genes.Then,MSTN gene sequences were analyzed using bioinformatics tools to predict their protein structures and functions.[Results]The hybrid grouper and its parents shared the same MSTN gene structure,consisting of three exons and two introns.Nucleotide sequence of the gene could be translated into 376 amino acids,including an N-terminal signal peptide,a proteolytic processing site(RXXR motif),and nine conserved cysteine residues at C-terminal,which were the typical features of transforming growth factor beta(TGF-β)superfamily proteins.Alignment of protein sequence showed that MSTN was highly conserved between the hybrid grouper and its parents.Especially,exon 3,an important functional domain,exhibited a sequence similarity of 100%among them.In addition,four variable amino acid residues were detected in exon 2 at positions 141,153,185 and 186 in the hybrid grouper,but they did not affect the secondary structure of the protein.[Conclusion]These results will provide molecular information for future investigation on the growth and heterosis of hybrid grouper species,and on the roles of MSTN gene in regulating the growth traits of the hybrid grouper.

花斑裸鲤MSTN-1基因克隆及表达特性分析

花斑裸鲤是黄河上游重要的冷水性土著鱼类,其生长、发育缓慢,性成熟又较晚,故多年来其种群一直呈下降趋势。MSTN(肌肉生长抑制素)是一种对肌肉生长具有负调控作用的因子,通过克隆花斑裸鲤MSTN-1基因并检测其表达特性,为花斑裸鲤生长缓慢的分子调控机理提供基础资料。采用PCR、5′-RACE和3′-RACE法获得花斑裸鲤MSTN-1基因全长cDNA序列,采用qPCR检测MSTN-1基因在花斑裸鲤不同组织的表达特征和在不同鲤科鱼类肌肉组织中的差异性表达。花斑裸鲤MSTN-1基因的cDNA序列全长为2190 bp,其中ORF长1128 bp,5′UTR长96 bp,3′UTR长966 bp,共编码375个氨基酸。该蛋白是带有一个信号肽、无跨膜结构的不稳定亲水性分泌蛋白,主要分布在细胞核、线粒体和细胞质中。MSTN-1蛋白质二级结构以无规卷曲为主,存在2个TGF-β结构域:即TGF-β前肽区域(37—268 aa)和TGF-β功能区域(281—375 aa),有1个蛋白酶水解位点RIRR和9个位于TGF-β功能区域保守的半胱氨酸残基。氨基酸序列同源性分析表明,花斑裸鲤MSTN-1氨基酸序列与其他鲤科鱼类MSTN-1具有较高的相似性,而与禽类和哺乳动物的相似性较低。系统发育分析显示,花斑裸鲤MSTN-1与其他鲤科鱼类聚于同一进化支。qPCR检测结果表明,MSTN-1在花斑裸鲤的脑、肌肉、鳃、心、肝脏和肾脏组织中均有表达,但在脑和肌肉组织中表达量较高。该基因在不同鲤科鱼类肌肉组织中均有表达,在青海湖裸鲤肌肉组织中的表达量最高,其次在花斑裸鲤和黄河鲤鱼肌肉组织中。通过克隆获得花斑裸鲤MSTN-1基因cDNA序列,进行生物信息学分析和qPCR检测,MSTN-1在这几种土著鱼类肌肉组织中的特征性表达差异有助于进一步了解高原鱼类生长缓慢的分子机理。

MSTN基因多态性及其与兰州大尾羊生长性状关联分析

肌肉生长抑制素(Myostatin,MSTN)是肌肉生长的负调节因子,抑制了成肌细胞的增殖和分化。本文研究MSTN可选作兰州大尾羊生长性状候选基因的可能性,PCR扩增及PCR-SSCP技术检测536只兰州大尾羊MSTN基因多态性,并分析其群体分布规律。结果:(1)兰州大尾羊MSTN基因中检测到2个SNP位点,其中g.119286026C>A的等位基因型为CC和CA,突变位点CC基因型胸宽和胸围显著高于CA基因型(P<0.05);g.119286166A>G的等位基因型为AA和AG,突变位点AG基因型体斜长和体重显著高于AA基因型(P<0.05),两位点均呈现低多态性。(2)其中2个基因型组合(A1和C1)对兰州大尾羊生长性状具有显著影响,C1组基因型组合体斜长、胸围、胸宽和胸深均显著高于B1组基因型组合(P<0.05),在各基因型之间其他生长性状差异均不显著(P>0.05)。结果表明,MSTN基因多态性与兰州大尾羊的体斜长、体重、胸围和胸宽显著相关,可作为其生长性状的有效遗传标记位点。

Polymorphisms of the CAPN1, CAST and MSTN Genes,and Their Association with Histological and TexturalParameters of Beef from Polish Holstein-Friesian Cattle

The purpose of this study was to analyse polymorphisms of the CAPN1, CAST and MSTN genes and their association with the microstructure of the Musculus longissimus thoracis (MLT) and textural parameters in bulls of the Holstein-Friesian breeds, black-and-white variety. The polymorphisms at the three loci: in position 6536 of the 3’UTR region of the CAPN1 gene, in position 230 of intron 5 in CAST gene, and in position 371 of the promoter region of the MSTN gene were analysed. Given the inconsequential genetic diversity at the analysed CAPN1 and MSTN loci in the animal sample, it was considered unreasonable to perform further statistical analyses aimed at determining associations between polymorphisms in these positions and meat characteristics. Based on an analysis of the CAST gene polymorphism, a significant association with certain histological and textural parameters was identified.

海子水牛MSTN基因多样性与系统分化研究

针对牛肌肉生长抑制素基因(MSTN)编码区设计引物,以PCR扩增及测序的方法,检测了19头海子水牛样本的MSTN基因多样性,并对其系统分化进行研究。结果显示:海子水牛群体内MSTN基因编码区含1 128个碱基对,所检样本中存在3个核苷酸多态位点,但仅含有2种基因单型。说明海子水牛MSTN基因分化水平较低。

猪肌生成抑制素(MSTN)基因cDNA克隆及序列分析

利用GenBank中猪肌生成抑制素(MSTN)编码序列设计引物,以湖北白猪背最长肌肌细胞总RNA为模板,利用RT-PCR技术,扩增出MSTN基因cDNA片段。该片段全长1277bp,包含猪MSTN基因的全部编码序列。将所得片段与pUCm-T载体连接,转化到DH5α大肠杆菌中,成功地筛选到阳性克隆,其质粒测序结果与文献报道的一致。

真鲷肌肉生长抑素（MSTN）基因的克隆及表达分析

采用同源克隆的策略,设计了3对引物,首次分离、克隆了2820bp的真鲷肌肉生长抑素（MSTN）基因组序列,该序列在GenBank上注册（注册登录号为AY965686）.经过剪接、比较,分析了真鲷MSTN基因的序列和结构特征.真鲷的MSTN基因具有3个外显子、2个内含子,整个开放阅读框编码着384个氨基酸,第1个外显子上有一个连续11个Q氨基酸的残基,C-末端具有9个保守的半胱氨基酸及一个RVRR的蛋白酶解加工位点.在第1、第2内含子和3＇非翻译区上分别具有一个微卫星重复序列.RT-PCR分析表明,MSTN基因在真鲷不同组织中都有表达,但表达量具有明显的差异;MSTN在肌肉中表达量最高,其次是脑、小肠、头肾、鳃和性腺,心脏、眼睛、肝脏中只有极少量表达,脾脏中没有检测到MSTN的表达.

藏系绵羊MSTN基因在不同年龄不同组织的表达定量研究

[目的]研究MSTN基因在藏系绵羊不同年龄、不同组织的表达差异。[方法]根据GenBank已发表序列(NM_001009428.1)设计1对引物,以藏系绵羊组织总RNA为模板,采用实时荧光定量PCR技术(QRT-PCR)分析了MSTN基因在不同年龄阶段的真胃、瘤胃、腿肌和心肌中的基因表达量。[结果]经过内参基因校正后,综合4个组织分析,MSTN基因在6月龄藏系绵羊的相对表达量最高,是12月龄藏系绵羊的2.52倍,是羔羊的2.24倍,是9月龄的1.30倍(P<0.05)。且MSTN基因在腿肌中的表达量最高,在其他组织几乎未表达,在腿肌中的表达量是其瘤胃的3984.78倍,是心肌的602.69倍(P<0.01)。[结论]为MSTN蛋白抗体的正确利用奠定了基础。