【作者】 单立莉；

【导师】 张敏；

【作者基本信息】 延边大学 ， 动物营养与饲料科学， 2009， 硕士

【摘要】 本课题采用半定量RT-PCR法,检测了不同生长阶段金华猪和长白猪（35d、80d、125d）背最长肌中肌肉生长抑制素（Myostatin、MSTN）、生肌调节因子（MyoD）和肌肉成肌素（Myogenin、MyoG）的mRNA水平,探讨了他们的基因表达随动物体重增加的变化规律,以及在肌肉沉积过程中的调控作用。根据Genebank登陆的MSTN、MyoD、MyoG和内标β-actin基因序列设计引物,分别以背最长肌组织RNA为模板,经反转录和PCR,克隆得到的相应基因片段（登录号分别为NM 214435、SSU12574和NM₀01012406）同源性分别为97.0%、97.8%和93.6%。在此基础上,进一步探讨了PCR体系中适宜的退火温度、MgCl₂浓度和循环次数,构建了适合于相应基因序列的优化半定量RT-PCR法,用于不同生长阶段金华猪和长白猪背最长肌组织MSTN、MyoD和MyoG基因mRNA水平的测定。试验所用的金华猪和长白猪由浙江省东阳市金华猪保种场提供,按照品种分两个组,每组18头（28日龄断奶）预试期7天;正试期90天,金华与长白猪在相同的条件下饲养。分别于35,80和125日龄时采集背最长肌样品,测定瘦肉率,探讨MSTN、MyoD和MyoG基因表达与肌肉沉积的关系。试验结果表明,猪背最长肌组织中MSTN、MyoD和MyoG基因表达均呈现一定的生物学发育规律:（1）在两种猪的不同生长阶段（35日龄,80日龄,125日龄）MSTN mRNA都被检测到,其表达量在出生后35日龄时水平较高,而后,随着年龄体重的增加而显著减少（P＜0.05）。但是,在整个试验期间,品种间无显著差异（P＜0.05）。（2）在两种猪的不同生长阶段（35日龄,80日龄,125日龄）MyoD mRNA都被检测到,MyoD基因在金华猪背最长肌中的表达量随着年龄体重的增加而增加,80日龄到125日龄阶段增加平缓。而MyoD基因在长白猪背最长肌中的表达量则与金华猪的恰恰相反,长白猪在出生后35日龄时水平较高,而后,随着年龄体重的增加而减少（P＜0.05）。在整个试验期间,品种间差异显著（P＜0.05）。35日龄时品种间差异极显著（P＜0.01）。（3）在两种猪的不同生长阶段（35日龄,80日龄,125日龄）MyoG mRNA都被检测到,金华猪背最长肌中MyoG基因的表达35日龄时较低,而后随着年龄和体重的增加而增加,但不同年龄阶段MyoG基因表达差异不显著。长白猪背最长肌中基因的表达则与金华猪相反,35日龄时较高,而后随着年龄和体重的增加而减少;同样,不同年龄阶段MyoG基因表达差异不显著。但是,在整个试验期间,35日龄品种间差异显著（P＜0.05）。本试验探讨了MSTN、MyoD和MyoG基因与肌肉沉积的关系,结果表明,MSTN基因表达水平与胴体瘦肉率呈反比;MyoD基因表达水平在金华猪背最长肌中随着年龄和胴体瘦肉率的增加而增加呈正比变化,而长白猪背最长肌中的表达恰好相反;MyoG基因变化规律与MyoD基因变化规律相似。从而MSTN、MyoD和MyoG基因对猪肌肉沉积具有显著影响。MSTN、MyoD和MyoG基因mRNA水平随着猪日龄的变化和显著变化,其中80日龄到125日龄阶段表达量变化平缓,表明在猪的整个生长阶段中肌细胞在不断的分化,在生长后期分化程度减缓。更多还原

【Abstract】 The subject of this semi-quantitative RT-PCR method to detect the different growth stages of Jinhua and Landrace pigs （35d, 80d, 125d） the longest back muscle in Myostatin （Myostatin, MSTN）, myogenic regulatory factor （MyoD） And muscle-myoblasts （Myogenin, MyoG） of the mRNA level, to explore their gene expression with the animal’s weight changes, as well as muscle deposition in the process of regulation.According to the Genebank landing MSTN, MyoD, MyoG standard andβ-actin gene sequence design primer, respectively, to back up muscle tissue RNA as a template, and RT-PCR, cloned the corresponding gene fragment （registry, were NM₂14435, SSU12574 and NM₀01012406） homology were 97.0%, 97.8% and 93.6%. On this basis, to further explore the PCR system suitable annealing temperature, MgCl₂ concentration and the number of cycle, suitable for the construction of the corresponding gene sequence optimization of semi-quantitative RT-PCR method for the different stages of growth and Landrace pigs Jinhua back most Long muscles MSTN, MyoD and MyoG gene mRNA level of determination.Test used by the Jinhua and Landrace pigs from the city of Jinhua in Zhejiang Province Dongyang pig farms to provide security, according to the varieties in two groups of 18 （28-day-old weaned） pre-trial period of 7 days is the 90-day trial period, Jinhua Landrace and in keeping under the same conditions. At 35, 80 and 125 days old when collecting samples of muscle back up, lean determination to explore MSTN, MyoD and MyoG gene expression and muscle deposition.Test results show that the pig back up muscle tissue MSTN, MyoD and MyoG gene expression showed a certain degree of development of the law of biology:（1） two pigs in different stages of growth （35-day-old, 80-day-old, 125-day-old） MSTN mRNA were detected, and its expression in the 35-day-old after birth when a higher level, then, with age, body weight The increase in a significant reduction （P <0.05）. However, throughout the trial period, the species no significant difference （P <0.05）.（2） two pigs in different stages of growth （35-day-old, 80-day-old, 125-day-old） MyoD mRNA were detected, MyoD gene in Jinhua pig muscle back up in the expression with age, body weight increases 80-day-old to the 125-day-old increase in the flat stages. The MyoD gene in Landrace muscle back up in the expression with the Jinhua pig On the contrary, Landrace at the age of about 35 days when a high level, then, with age, weight gain and reduced （P <0.05）. Throughout the trial period, a significant difference between the varieties （P <0.05）. 35-day-old differences between varieties at a very significant （P <0.01）.（3） two pigs in different stages of growth （35-day-old, 80-day-old, 125-day-old） MyoG mRNA were detected, Jinhua pig muscle back up in MyoG gene expression at a lower 35-day-old, and then with the Age and body weight increases, but different age MyoG gene expression differences are not significant. Landrace longest back muscle in gene expression with the Jinhua pig On the other hand, when the 35-day-old high and then as age and weight gain reduced; Similarly, different age MyoG gene expression differences are not significant. However, throughout the trial period, the 35-day-old marked differences between varieties （P <0.05）.This test explored MSTN, MyoD and MyoG gene and the relationship between muscle deposition, the results show that, MSTN gene expression and lean carcass was inverse; MyoD gene expression in Jinhua pig muscle back up with the age of lean meat and carcass The rate of increase was proportional to the change, and Landrace muscle back up in the expression of the opposite; MyoG gene variation and gene variation is similar to MyoD. Thus MSTN, MyoD and MyoG gene on muscle swine have a significant impact on the deposition.MSTN, MyoD and MyoG gene mRNA level of day-old pigs with the changes and significant change in the 80-day-old stage to the 125-day-old moderate changes in theexpression of that pig in the whole growth stage of the muscle cell differentiation in the constant, the growth in The degree of differentiation in the late slow down.更多还原