Research On Acquiring Target Genes Of MiRNA Expressed At A High Level In Sheep Skeletal Muscle By A High Throughput Way And Function Of Partial Target

To investigate mi RNAs and their profiling in sheep skeletal muscle, further understand their expression patterns in sheep different skeletal muscle development stages and detect their targets, then finally help to breed sheep with great skeletal muscle production ability. Here we first applyed microarray to detect the miRNA in skeletal muscle of Suffolk sheep, then further investigated mi RNAs which expressed specifically or at a high level from 4 aspects:(1) Using PCP-SSCP to detect genetic variations of miRNA genes which expressed at a high level in sheep skeletal muscle in four sheep breeds with different muscle production performance, and analyse the relationship of genetic variations and production performance, then chosen 7 genetic variations that greatly affected the stability of stem-loop structure of primary miRNA to research their fuctions in miRNA biogenesis applying cell assay and qRT-PCR.(2) Detect the expression patterns of miRNAs, which expressed specifically or at a high level in sheep skeletal muscle during fetal period, including 40 d(E40), 60 d(E60), 80 d(E80), 100 d(E100), 120 d(E120) and new born, 6 months old and 1 year old, then analyse their functions in sheep skeletal muscle development.(3) Build target cDNA library of miRNA that expressed higly in sheep skeletal, and obtain their targets in skeletal muscle of fetal(E60) and adult period of sheep.(4) Investigate effect of miR-133 on proliferation of sheep skeletal muscle satellite cells and expression of 4 candidate target genes when miR-133 is overexpressed or inhibitored, and analyse its functions in proliferation of sheep skeletal muscle satellite cells.Based on above research, we got some important results below:(1) Using microarray, 261 miRNAs were detected in sheep skeletal muscle, 58 of them were already discovered before in sheep, and the rest 203 were novle. The expression levels of these 261 miRNA were greatly different in sheep skeletal muscle, and 22 of them were expressed at a very high level, and equated to 89% of the total miRNA.(2) A total of 49 genetic variations, which included 41 single nucleotide polymorphisms(SNPs) and 8 deletions/insertions, were identified in four sheep breeds. Three variations were further researched in a larger sample set, including five sheep breeds with different meat production performances. We found that the genotype and allele frequencies of the CCC deletion/insertion in pri-miR-133 a were significantly related to the sheep meat production trait.89.8% genetic variations can influence miRNA biogenesis and increase or decrease the levels of mature miRNAs, in accordance with the energy and stability change of hair-pin secondary structures.(3) The expression of these 22 mi RNAs varied during sheep skeletal muscle development and growth, but their expression patterns were greatly different. If the expression of miRNAs in 1 year old sheep were chosen as control, these 22 mi RNAs could be sorted into 4 categories according to their expression pattern. During fetal period, most miRNA showed two lowly expressed period, that were the fortieth day to sixtieth day and one hundred and twentieth day of fetal stage.(4) Build two target cDNA libraries of miRNA with high expression levels in sheep skeletal muscle. Total 500 clones of target cDNA library were sequenced, and got 246 and 239 valid sequences, the positive clone rate were 85% and 79% respectively. There were 132 and 124 ESTs could match with unigene of NCBI, and they composed 53.66% and 51.88% of target gene cDNA library.(5) If miR-133 was overexpressed in sheep skeletal muscle satellite cells, the proliferation of cells were inhibitored significantly, but the mRNA levels of miR-133 target genes did not changed at early stage of this period. The result of Western Blot showed that, in sheep skeletal muscle satellite cells, miR-133 can target Rhoa,CDC42,TAGLN2 genes and knockdown their protein levels, but miR-133 did not regulate MSN gene in sheep skeletal muscle satellite cells. Two target sites were located in CDS sequence of Rhoa and TAGLN2 gene respectively, and the rest were located in 3’UTR of genes.Depending on above research, the miRNA profiles and their target genes in sheep skeletal muscle were known more comprehensively, and their functions were further understood. Our work would help to further the understanding the molecular network in skeletal muscle growth and development, and help to breed sheep with perfect meat production performance, finally promote the sheep breeding in the future.