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 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.

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 expression of swine myostatin gene and its application in animal immunization trial

We have amplified swine myostatin (MSTN) gene by reverse transcription poly-merase chain reaction (RT-PCR) and cloned it into pGEM-T Easy vector. The cloned swine MSTN gene consists of 1128 nucleotides, which has been submitted to GenBank (acquired reg-istered code- AY448008). The cloned swine MSTN gene was successfully expressed in E. coli without the first 25 amino acids. Crude extraction of the expressed recombinant MSTN protein was used to immunize mice to investigate the effects on their bodyweights. We show here that the body weights of the immunized mice were higher than that of the controls, even though the difference was not significant. Surprisingly, the progenies of the immunized mice also were heavier than the controls. Especially at day 3, the average body weight of the immunized mice was 10.5% higher than that of the controls , which is significant (p < 0.05).

The single nucleotide polymorphisms of the chicken myostatin gene are associated with skeletal muscle and adipose growth

Myostatin, a new member of the TGF-β superfamily, is predominantly expressed in skeletal muscle cells and functions as a negative regulator of skeletal muscle growth in animals. Recently, we have reported three single nucleotide polymorphisms (SNPs) in the chicken myostatin gene. Herein, we investigate the association of those SNPs with the production traits in a F2 chicken line derived from Broilers crossing to Silky with the least square analysis. The results show that the BB and AA genotypes are strongly associated with abdominal fat weight (AFW), abdominal fat percentage (AFP), and birth weight (BW) (P < 0.05). Breast muscle percentage (BMP) of the AA type is higher than that of the AB type. The breast muscle weight and breast muscle percentages of F2 individuals have significant difference between CC and DD genotypes (P < 0.05). Breast muscle weight (BMW) of EF birds is higher than that of EE birds (P < 0.05). In this report, we present the first genetic evidence to show that chicken myostatin not only plays an important role in controlling skeletal muscle growth and differentiation, but also may be involved in regulation of adipose growth in chicken.

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(488 bp,371 bp and 1779 bp,respectively) and two introns(390 bp 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 information will be useful for studying the transcriptional regulation of myostatin in fish.

鸡Myostatin基因单核苷酸多态性的群体遗传学分析

肌肉生长抑制素是控制骨骼肌生长发育的重要细胞因子 ,采用PCR SSCP和测序的方法发现了 5个位于Myostatin基因 5′-和 3′ -调控区的单核苷酸多态性位点 ,对北京油鸡、白耳鸡、石歧杂、矮小黄鸡、小型黄鸡、惠阳胡须鸡、隐性白羽鸡、海兰、AA鸡等不同鸡种的该单核苷酸多态性分析结果表明 :Myostatin基因的 5′调控区引物P6 0 P6 1扩增片段多态性是由 3个核苷酸的改变而产生的 [分别是G→A(30 4位 )、A→G(32 2位 )、C→T(334位 ) ],引物P93 P94扩增片段的多态性是由G→A(16 7位 )突变造成的 ,引物P117 P118PCR扩增片段多态性是由T→C(177位 )造成的。 3′调控区引物P80 P81扩增片段多态性是由第 72 6 3位A突变为T造成的 ,引物P76 P77扩增片段多态性是由A→G(6 935位 )造成的。不同鸡种群体遗传学分析表明 ,5′ -调控区引物P6 0 P6 1扩增片段多态性位点在北京油鸡的基因型频率分布与其他的品种有很大的差异 ,其BB基因型频率为 0 70 0 ,AA基因型频率仅为 0 0 33,而其他鸡种中以A基因占优势 ;对于引物P93 P94 ,品种间的基因型频率差异极显著 (P <0 0 1) ,北京油鸡和AA鸡的EE型频率低于其他品种 ,白耳鸡和海兰蛋鸡中以EE型为主 ,其频率高于其他品种 ;3′ -调控区引物P80 P81多态性位点在 9个鸡种中?

牛Myostatin基因单核苷酸多态性分析

Myostatin基因即肌肉生长抑制素,是一种肌肉生长的负调控因子。应运PCR-SSCP和测序的方法对中国秦川牛、南阳牛以及国外引入品种皮埃蒙特牛双肌基因的第三外显子进行了多态性分析。结果表明:第三外显子938处G→A的单核苷酸的突变造成了南阳牛、皮埃蒙特牛第三外显子扩增片段多态性,秦川牛则不然。

鸡Myostatin基因单核苷酸多态性及其对屠体性状的遗传效应分析

以180只3个品系的温岭草鸡为材料,采用PCR-RFLP方法对鸡MSTN基因外显子1的2个多态位点进行研究,并分析对屠体性状的遗传效应。Bsh1236Ⅰ识别G(2100)A突变,产生MN和NN2种基因型,MspⅠ识别G(2109)A突变,产生AA、AB和BB3种基因型,联合2个位点分析出现了5种基因型。基因型频率在品系间的χ2检验表明差异均不显著(P>0.05)。方差分析显示不同基因型的屠宰率有显著或极显著的差异(P<0.01或P<0.05)。多重比较显示:杂合型MN的腹脂重和屠宰率显著(P<0.05)高于突变型NN;杂合型AB的胸肌重和胸肌率显著(P<0.01或P<0.05)高于基因型AA,基因型AA的腹脂重和腹脂率都极显著(P<0.01)高于突变型BB,在腿肌重性状上,BB型显著(P<0.05)低于AA型和AB型;2个位点联合分析时,NA/MA基因型的腹脂重、腹脂率和胸肌率均极显著(P<0.01)高于或低于其他基因型。

鹅Myostatin基因单核苷酸多态性检测及群体遗传分析

肌肉生长抑制素是TGF-β超家族的新成员,其主要在骨骼肌中特异性表达,且对骨骼肌的生长发育起负调控作用。本研究采用PCR-SSCP和DNA测序的方法检测了鹅Myostatin基因的单核苷酸多态性,并对浙东白鹅、五龙鹅、扬州鹅、朗德鹅、莱茵鹅、豁眼鹅、吉林农大白鹅、狮头鹅、皖西白鹅、籽鹅等不同品种(系)鹅单核苷酸多态性进行了群体遗传分析。结果表明:(1)在检验的12对Myostatin基因的引物中,发现3对引物的扩增片段具有多态性,分别为启动子区域T769G、C543T的突变,内含子1的A1 632G突变。(2)不同鹅品种(系)群体遗传学分析表明,启动子区域引物P3扩增片段多态性位点在各群体中等位基因A的频率均高于B,且等位基因频率均高于0.77,表现为优势等位基因;引物P4扩增片段多态性位点在扬州鹅、朗德鹅、狮头鹅群体中等位基因D的频率均高于C的频率,等位基因D的频率分别为0.625 0、0.548 8、0.647 4,而在其余群体中均表现为等位基因C的频率高于D的频率。

家鹅Myostatin基因3-′UTR序列分析

利用家鹅Myostatin基因第3外显子设计上游引物、家鸡Myostatin3-′UTR设计下游引物,PCR扩增并克隆出家鹅该基因3-′UTR长1 064bp DNA序列。该序列具有一个polyA加尾信号序列(TAATAAA),并富含连续的TTTT高保守区。分析家鹅与其他12个物种该序列的同源性发现,其与家鸡的同源性最高,达87.1%,与鱼类的最低(<11%)。以13个物种Myostatin3-′UTR构建的分子进化树表明,3′UTR能够为动物进化提供一定信息,但适用于较低分类阶元。

猪myostatin基因5’调控区的酶切多态性分析

用 PCR- RFLPs分析方法 ,对长白、大白、杜洛克、军牧 号、民猪 5个猪种的 myostatin基因 5’调控区进行了酶切多态性分析。结果表明 ,长白、大白、杜洛克以等位基因 T为主 ,检测的 4 0头军牧 号的等位基因全部是 T,民猪 3种基因型均有 ,且频率近乎相等。统计分析表明 ,3种基因型的分布在长白、大白、杜洛克、军牧 号间差异不显著 ,而民猪同这

鹅Myostatin基因编码区核苷酸多态性研究

利用鹅Myostatin基因3个外显子设计4对引物,分析扬州鹅、皖西白鹅和五龙鹅3个品种鹅的Myostatin基因编码区的单核苷酸多态性。结果表明:鹅该基因编码区高度保守,仅第3外显子的第263 bp处检测到由A→C的单碱基"沉默"突变,该突变位点仅检测到AA、AB 2种基因型,且杂合子频率极低(2.5%),皖西白鹅群体未能检测到AB型个体。对五龙鹅群体的初步分析发现,AB型个体具有较高的产肉性能,但因所检测到的杂合子个体数较少,故该位点各基因型与生产性能间的关系有待于进一步研究证实。

赤眼鳟myostatin基因的克隆与组织表达分析

以赤眼鳟(Squaliobarbus crriculus)肌肉总RNA为模板,采用RT-PCR结合RACE方法,获得其myostatin(肌肉生长抑制素)基因全长为2 214 bp的cDNA序列,其包含了1个含有1 128个核苷酸的开放性阅读框(ORF),共编码375个氨基酸。赤眼鳟与其他物种myostatin的特征性一致。其编码区序列与鲤鱼同源性最高,为96.99%,与其他鱼类的同源性为76%～81%,与哺乳类和鸟类的同源性较低,为63%左右。赤眼鳟Myostatin蛋白的氨基酸序列与其他物种同源性较高,尤其在C端生物活性区,高度的保守性反映了该基因受到了高度的进化限制以及功能的重要性。半定量RT-PCR分析表明,myostatin基因在除肝脏外的其他组织都有表达,在肌肉和脑中的表达量较高,此结果表明赤眼鳟myostatin基因除了对肌肉生长发育有调控作用以外,还可能在机体中发挥其他功能。

3品种鹅Myostatin基因3′-调控区SNPs群体遗传学分析

利用已测得的鹅Myostatin基因3′-调控区核苷酸序列设计5对PCR-SSCP引物,对扬州鹅、皖西白鹅以及五龙鹅3品种家鹅该区域内单核苷酸多态性进行了分析,并探索了单核苷酸多态性与生产性能之间的关系。结果表明,检测到A→G(88位)和G→T(353位)2个单碱基突变位点。88位处,E等位基因为优势等位基因,具有EE、EF两种基因型,以扬州鹅杂合子频率最高(0.550),皖西白鹅30个个体仅有一例EF型;353位处,G为优势等位基因,具有GG、GH和HH 3种基因型,3品种均具有较高的GG基因型频率、较低的HH基因型频率。屠体性能分析发现,EF基因型家鹅具有更高的屠体性能;扬州鹅、皖西白鹅GG型个体的腿肌重和腿肌率显著高于HH型个体(P<0.05),五龙鹅腿肌重、腿肌率具有GG型(GH型(HH型的趋势,但差异不显著(P>0.05)。

Myostatin基因打靶载体的构建及猪胎儿成纤维细胞Myostatin基因的敲除

目的:用缺口修复等技术构建Myostatin(肌肉生长抑制素,MSTN)基因打靶载体,并对大白猪胎儿成纤维体细胞进行转染,获得基因敲除细胞。方法与结果:首先构建用于MSTN基因同源长臂(LA)的抓捕载体,然后在大肠杆菌内利用Red同源重组系统介导的缺口修复,从含大白猪MSTN基因座的细菌人工染色体上亚克隆9.9 kb的LA到抓捕载体上,经过部分序列测定,同源性为100%;通过PCR获得1.4 kb的同源短臂(SA);将LA和SA连入载体pLOXP,构建含有neo和tk正负筛选标记基因的MSTN基因打靶载体pLOXP-MSTN-KO;将线性化的pLOXP-MSTN-KO通过电转染整合到大白猪胎儿成纤维细胞基因组中,利用G418和丙氧鸟苷进行药物筛选,获得抗性细胞克隆890个,通过PCR和DNA测序鉴定获得基因敲除的细胞克隆4个。结论:构建了有效的MSTN基因打靶载体,通过转染获得基因敲除细胞,为利用体细胞核移植制备MSTN基因敲除猪奠定了基础。

东北马鹿Myostatin基因编码序列的克隆及序列分析

根据Myostatin基因的保守性,选择牛（Bos taurus cattle登录号：AB076403）的Myostatin序列为模板进行引物设计,首次从东北马鹿基因组中扩增出Myostatin序列,扩增产物连接pMD18-T载体,克隆出3个外显子片断。根据5-′GU-AG-3′规则和同源基因比对拼接出完整的编码序列,全长为1 128 bp（发表到GenBank登录号：EF629535）,Myostatin蛋白前体由375个氨基酸组成。同源比较结果表明：Myostatin在进化过程中具有高度保守性,东北马鹿与猪Myostatin编码序列同源性最高达到98%,鸟类与哺乳动物间Myostatin编码序列同源性为85%~89%,鱼类与其他动物间编码序列同源性为60%-67%。Myostatin编码序列能够真实反映远缘物种的进化关系,可以作为远缘物种进化分析较理想的标记。

绵羊Myostatin基因敲除载体的构建

根据Myostatin基因突变可导致肌肉量激增而产生"双肌"表型的特点,构建绵羊Myostatin基因置换型敲除载体。利用LA-PCR技术成功地扩增得到绵羊Myostatin基因同源臂序列,其中同源长臂4.9 kb,包括全部的exon1,intron1,exon2及部分启动子和大部分intron2;同源短臂1.1 kb,包括部分exon3和3′非翻译区序列,将二者连入PloxpII正负筛选敲除骨架载体,利用骨架载体上Neo基因替代Myostatin基因的exon3,从而成功构建专门针对Myostatin第3外显子区域缺失的置换型敲除载体PloxpⅡ-OVIS-MSTN。酶切和测序鉴定证明载体构建正确,为后续获得绵羊Myostatin基因缺失型体细胞株奠定试验基础。