家兔肌肉生长抑制素基因多态性及其遗传效应分析

以9个肉兔纯系及其37个杂交组合为实验材料,采用测序和单链构象多态(SSCP)的分析方法探讨了肌肉生长抑制素(Myostatin,MSTN)基因的全部3个外显子区及部分5’调控区的遗传多态性,结果发现该基因5’调控区476位点存在1处T→C的单碱基突变,外显子区域没有发现突变位点。关联分析表明,该突变有增加活体重,提高胴体重、前腿重、背腰重、后腿重和皮重,同时降低肌肉滴水损失和熟肉率等有利遗传效应(P<0.05),结果提示位于MSTN基因上游调控区的该处突变有利于家兔的体躯发育,可资开发为用于家兔肉用性能选育的分子标记。     

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。     

Molecular characterization of myostatin‐like genes expressed highly in the muscle tissue from Morotoge shrimp,Pandalopsis japonica

Myostatin is one of the transforming growth factor (TGF)‐β family members and plays inhibitory roles in the development and growth of muscle in mammals. Mammalian myostatins have been studied intensively, considering its medical and industrial potential use. Still, limited information is available about myostatin homologues in crustaceans. In the present study, we isolated for the first time cDNA that encodes for myostatin‐like protein (Pj‐MSTN) from Morotoge shrimp, Pandalopsis japonica. The putative mature peptide of Pj‐MSTN was composed of 109 amino acids, which contains an additional amino acid residue compared with mammalian myostatins. Pj‐MSTN exhibited 32% amino acid sequence identity and 52% similarity to human myostatin. Multiple sequence alignment analysis indicated that Pj‐MSTN shared the conserved proteolytic cleavage site (RXXR) for its maturation and nine cysteine residues for disulphide bridges. These results suggest that Pj‐MSTN has conserved the three‐dimensional structure of TGF‐β family members in vertebrates. Phylogenetic analysis suggests that Pj‐MSTN is a primitive form of vertebrate myostatin and GDF11. The expression of Pj‐MSTN was not just identified in muscular tissues, suggesting that Pj‐MSTN functions differently from mammalian myostatin. Ablation of the X‐organ/sinus gland complex significantly reduced the expression of Pj‐MSTN in most tissues, suggesting its potential association with moulting.     

Metabolic responses of juvenile GIFT strain of Nile tilapia (Oreochromis niloticus) to dietary L‐tryptophan supplementation

A 60‐day indoor feeding trial was conducted to evaluate the effects of dietary tryptophan supplementation on growth performances, whole‐body chemical composition, expression of muscle growth‐related genes (MyoD, myogenin and myostatin), and haematological and biochemical responses of juvenile genetically improved farmed tilapia (GIFT). Five corn–soy‐based isonitrogenous and isoenergetic diets were formulated to contain graded levels of dietary tryptophan (2.6, 3.2, 3.7, 4.2 and 4.8 g/kg of diet). Each diet was randomly assigned to triplicate groups of 30 fish (5.3 ± 0.1 g) per experimental unit, which were fed thrice a day (9:00, 13:00 and 17:00 hr). Maximum growth performances and feed utilization were observed in fish fed tryptophan at 3.7 g/kg of diet. There was no significant (p > .05) effect on whole‐body composition and amino acid profile by dietary tryptophan supplementation. However, significant (p < .05) differences were observed in plasma metabolites and the mRNA expression of MyoD, myogenin and myostatin. Serum cortisol level was found significantly lowest in fish fed tryptophan at 3.7 g/kg of diet. Second‐order polynomial regression analysis of weight gain and nitrogen gain against dietary tryptophan levels indicated that the optimum dietary tryptophan requirement for maximum growth and feed utilization of juvenile GIFT tilapia was 3.8 g/kg of diet.     

ENU诱变草鱼家系生长特性及肌肉生长抑制素基因SNP筛选的研究

经过175 d养殖,对4个N-乙基-N-亚硝基脲(ENU)诱变草鱼家系进行生长对比,采用显著性比较,偏相关分析和因子分析统计方法对草鱼体质量、全长、体长、头长、体高、尾柄长、尾柄高、体厚性状进行分析,进而运用双向测序法对MSTN1、MSTN2基因在具有明显差异家系中进行SNP位点筛选。试验结果显示,家系4的生长速度明显大于家系3,家系4的8个性状明显大于其他3个家系;家系1中体长、尾柄长、体厚与体质量密切相关,家系2中体长、头长、体厚与体质量密切相关,家系3中全长、体长、尾柄高与体质量密切相关,家系4中全长、体长、体厚与体质量密切相关。由此可知,家系4和家系3具有明显的生长差异。对于MSTN1基因,家系3在465位点C/G、467位点G/A,家系4在465位点C/G均发生错义突变;对于MSTN2基因,家系3和4在912位点C/T均发生同义突变,家系3在1027位点G/A、家系4在366位点A/G均产生错义突变,位于非编码区1390位点A/T和1401位点G/A在两个家系均发现SNP位点。试验结果表明,MSTN1、MSTN2基因的不同SNP位点与ENU诱变草鱼的生长性状存在紧密联系。     

鳡肌肉生长抑制素(MSTN)基因的克隆及其组织特异性表达分析

以鳡肌肉总RNA为模板,采用RTPCR和RACE的方法,获得了鳡肌肉生长抑制素(myostatin,MSTN)基因的3个重叠片段,测序后拼接得到2 177 bp全长cDNA序列,其包含了5′端非翻译区的89个核苷酸和3′端非翻译区1 052个核苷酸以及1 125个核苷酸的开放性阅读框,翻译编码375个氨基酸,其中前22个氨基酸为鳡MSTN的信号肽。鳡MSTN具有MSTN的共同特征,有蛋白酶水解位点RIRR和在C端生物活性区含9个保守的半胱氨酸残基。核苷酸和氨基酸同源性分析发现鳡MSTN与厚颌鲂、草鱼和鲤等鲤形目鱼类的同源性较高,与鲈形目的同源性较低,与哺乳动物和鸟类的同源性最低;系统发育分析表明鳡MSTN与厚颌鲂亲缘关系最近。 半定量RT-PCR分析表明,该基因在肌肉和脑中表达量最高,在心肌中也有较高表达,在肝脏、肠、鳃和肾等组织中未见明显表达,此结果表明鳡MSTN基因除对肌肉生长发育有调控作用以外,可能还有其他功能。     

鲁莱黑猪肌肉生长抑制素基因5′调控区单倍型多样性及育种应用

肌肉生长抑制素（myostatin,MSTN）是肌肉生长发育的负调控因子,该基因的缺失或突变会导致动物机体肌肉总量和瘦肉率的增加。本试验对142头鲁莱黑猪MSTN基因5′调控区3个单核苷酸多态性位点（SNPs）进行了分析,结果表明,在鲁莱黑猪MSTN基因5′调控区435、447和879位点存在多态性,共检测到AA、AB、BB、AC、BC、CC、CD、AD和BD 9种基因型及435A-447G-879T（A）,435G-447A-879T（B）、435A-447A-879A（C）和435A-447A-879T（D）4种单倍型,其中CC、BC和AC基因型及C单倍型在猪群中占绝对优势。哈代温伯格平衡检验结果显示,在这3个位点群体显著偏离平衡状态（P〈0.005）;3个单核苷酸多态性位点所产生的等位基因均表现出连锁不平衡。依据其遗传效应,在建系时,提高B和A单倍型的频率有望提高鲁莱黑猪的生长速度,而提高C单倍型的频率可保持鲁莱黑猪的优质和高繁特性。     

鸽MSTN基因mRNA表达的发育性变化及组织差异研究

通过研究公、母鸽肉质性状候选基因肌肉抑制素(Myostatin,MSTN)在不同组织、不同发育阶段的表达变化规律,为研究鸽肉质性状的遗传调控机理提供依据。采用荧光探针RT-PCR,定量分析MSTN mRNA在公、母鸽的多个组织、多个发育时间点的表达量。RT-PCR结果表明,MSTN mRNA表达量在3个生理时期表现出先降低后升高的变化趋势,母鸽峰值出现较早。公鸽MSTN mRNA表达量最高的组织为肾脏,睾丸中表达丰度最低,青年期肝脏、心肌MSTN mRNA表达量极显著高于其它时期的表达量,但在睾丸中没有检测到基因的表达。对不同生理时期母鸽MSTN mRNA的表达量比较后发现,青年期MSTN mRNA表达最高,显著高于其它时期。MSTN mRNA表达量受到组织、发育阶段和品种影响,该基因的表达在物种之间存在差异。     

牛肌肉抑制素前肽对牛肌卫星细胞增殖的影响

通过PCR扩增目的基因,构建真核表达载体,利用PEI转染牛肌卫星细胞,转染后48与72 h通过CCK-8与流式细胞术检测细胞增殖的变化,通过RT-PCR检测目的基因的表达,通过RT-PCR与Western blot检测下游基因P21,CDK2的表达变化.结果表明,在两个时间点,实验组细胞活力明显升高(P<0.01),G0/G1期细胞所占的比例明显下降(P<0.01),S期细胞所占比例明显上升(P<0.01),MSTN前肽基因有大量表达,P21表达下降,CDK2表达上升.结果揭示,在牛肌卫星细胞中,MSTN前肽通过下调P21,上调CDK2的表达量促进细胞增殖.旨在揭示牛MSTN前肽基因对牛肌卫星细胞(MDSC)增殖的影响,为后续的转基因牛试验提供理论依据.     

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. Thehomology 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.