Studies On RNA Interference And Promoter Of Porcine MYOSTATIN Gene

Myostatin (myostatin, MSTN), also known as GDF-8 (growth differentiation factor 8), is a member of TGF-β(transforming growing factor-β) superfamily. It is a autocrine secretion factor of the muscle cells, which mainly specificitly expressed in skeletal muscle and negatively regulated growth and development of muscle. The knockout mice, muscle weight had increased about twice to the normal mice. RNA interference (RNAi) is a gene expression regulatory mechanism that specificitly degradation mRNA sequence of target gene, highly efficient, specific and rapid, etc., have become a effective method to investe gene function and expression regulation. Therefore, this study used RNA interference to reduce expression of myostatin in the pig fetal fibroblast cell line of overexpression, screen the best interference sequence. In this study, three pigs were selected myostatin gene mRNA interference sites were designed and synthesized three pairs of specific interfering shRNA sequence and a pair of interfering non-specific shRNA sequence then them were annealed to double-strand, finally cloned them into the vectors containing U6 promoter of interference vector pGenesil-1, the obtained DNA fragment containing four objectives recombinant plasmids were transiently transfected Myostatin overexpression cells with lipofectamine-mediated transfection. real time PCR was used to detect the expression levels of MSTN gene in the detection interference effect. The results showed that three pairs of specific sequences significantly decreased mRNA expression levels of myostatin gene. shRNA1, shRNA 2 and shRNA 3 inhibition rates were 69.82%, 80.97% and 87.52%, while shRNA0 also inhibited MSTN gene expression but the difference was not significant, three pairs of specific sequences interference levels were not significantly different .At the same time, Myostatin gene promoter was studied. Myostatin gene promoter was obtained from porcine genomic DNA by PCR method.. The promoter sequence with the reported GenBank sequence: AJ133580 and AY527153, respectively, the corresponding sequence similarity of 100% and 99.5%. And then eukaryotic expression vector MSTNPro-EGFP was constructed ; by transfection of C2C12 and porcine fibroblasts, myostatin gene promoter transcription activity were identified. The results show that the experiment successfully cloned porcine myostatin gene promoter and constructed eukaryotic expression vector, the promoter can start GFP expression in C2C12 cells, while not observed in fetus fibroblasts. Indicating that myostatin gene expression of muscle-specific transcription from the promoter specificity.