Detection And Analysis Of Polymorphism At The Sheep Skeletal Muscle-specific MiRNA Among Different Flocks

Sheep husbandry plays an important role in breeding industry. Sheep farming can get a variety of economic benefits,in which meat performance is the most important economic traits.Different sheep species have different meat production performance. Molecular mechanism of muscle development have carried out a lot of research. Some of the genes involved in muscle development and their mechanisms have been found, while the mechanisms controlling sheep muscle differentiation and proliferation are still unclear.Micro RNA is one kind of small molecule that is about 18~26nt of endogenous non coding RNA. Mi RNA can regulate m RNA’s expression through being combined with 3’untranslated region of the m RNA, which regulate the function of animals and plants developed. According to the research, Mi R-1, mi R-133 and mi R-206 were expressed specifically in skeletal muscle and myocardium, and were closely related to muscle development.Therefore, this thesis takes mi R-1, mi R-133 and mi R-206 and their precursor gene as the object of study to explore their expression and biological function in skeletal muscle development of sheep.1. Collecting sheep embryos which have respectively developed 45 days, 65 days, 85 days, 105 days and 125 days, and collect their hind legs skeletal muscle samples to extract RNA, to explore the expression of mi R during the different stages of the embryonic stages of the sheep with fluorescence quantitative PCR technology.2. Using PCR-SSCP method to detect the polymorphisms at pre-mi R-133, pre-mi R-1 and pre-mi R-206 among four breeds with differences in meat production performance(Texel Sheep, Chinese Merino Fine Wool Sheep, Altai Sheep and Hu Sheep),the result shows that there’s a A/G transversion at position 194 bp of mi R-133 precursor downstream sequence.what’s more, the SNP is significantly associated with meat production performance. The GG genotype is preponderant genotype in Texel Sheep, GG genotype and G allele frequencies in Texel were reached 66.74% and 0.86 in Texel, respectively, 15 times and 4 times in Chinese Merino. Prediction of secondary structure showed that, The SNP loci significantly changed the secondary structure of pre-mi R-133, and the free energy value reduced by 3.2k J/mol.3. Building the expression vector containing mi R-133 precursor gene mutation sequences and import the vector into Hela celsl, detecting the expression of mi R-133 in cells after 48 hours. The results showed that GG genotype could significantly improve the expression of mi R-133 in the cells.The experimental results may help to cultivate excellent meat sheep breeds, and thus promoting the meat performance of sheep and sheep breeding improvement.