Screening And Verifying The Differentially Expressed Genes And Mirna In Skeletal Muscle Of Hu And Dorper×hu Sheep F1

Hu sheep is an unique breed in our country,because of the directional selection,performance of hu sheep is low.Therefore,further study of molecular mechanism of sheep skeletal muscle growth and development,expounds the regulating mechanism of miRNA and target genes in muscle growth and the formation of quality traits,screening the differentially expressed genes and miRNA and regulating genes expression in skeletal muscle of Hu and DorperxHu sheep F1,will be able to provide a new idea for molecular improvement of performance of hu sheep,as well as lay a theoretical foundation for the breeding new strains of hu sheep.In this experiment,eight Hu Sheep and eight DorperxHu sheep F1 with 6 months were selected,gene chip and high-sequencing technologies were selected to screen the differentially expressed genes and miRNA in BFM,LDM and PMM of Hu and DorperxHu F1.The vascular smooth muscle contraction pathway was chosen as the study target for further analysis from the results of KEGG pathway of 2 times up or down differential expression genes.In addition,ACTG2 full length mRNA sequences of sheep was cloned and two Alternation splicings in 5,UTR were found.The following is the results of this research:(1)The result of the sequencing of miRNA and real-time fluorescent quantitative for miRNA:The result of the sequencing of miRNA show that the 2 times up and down differential expression miRNA in BFM of Hu Sheep and DorperxHu sheep F1 have 4(Novel-58,Novel-49b*,miR-628,miR-2285k*)and 3(miR-744,miR-708,Novel-13b)respectively,there are 26 and 44 respectively in PMM,3(oar-miR-299-5p,Novel-13b,Novel-25)and 2(Novel-11,oar-miR-409-5p)respectively in LDM;Two times up and down differential expression miRNA in BFM vs LDM of Hu sheep have 35 and 40 respectively,there are 25 and 31 respectively in BFM vs PMM,19 and 14 respectively in PMM vs LDM;Two times up and down differential expression miRNA in BFM vs LDM of DorperxHu sheep F1 have 55 and 58 respectively,there are 29 and 45 respectively in BFM vs PMM,15 and 12 respectively in PMM vs LDM.The result of real-time fluorescent quantitative for miRNA showed that the expressions of oar-miR-299-5p in male 6-month Hu Sheep was significantly higher than DorperxHu sheep F1(P<0.01).consistent with the result of miRNA sequencing;At the same time,a system of stem-loop RT-PCR method of quantitative detecting miRNAs:miR-1,miR-133 and oar-miR-299-5p found in Hu Sheep and DorperxHu sheep LDM were established.(2)The result of gene chip and real-time fluorescent quantitative for genes:The result of gene chip show that the 2 times up and down differential expression genes in BFM of Hu Sheep and DorperxHu sheep F1 have 11 and 40 respectively,there are 31 and 28 respectively in PMM,50 and 34 respectively in LDM;Two times up and down differential expression genes in BFM vs LDM of Hu sheep have 24 and 15 respectively,there are 13 and 12 respectively in BFM vs PMM,1 and 5 respectively in PMM vs LDM;Two times up and down differential expression genes in BFM vs LDM of DorperxHu sheep F1 have 16 and 15 respectively,there are 44 and 11 respectively in BFM vs PMM,7 and 32 respectively in PMM vs LDM.The result of clustering analysis show that the 2 times up and down differential expression genes participate in tissue development,regulation of cell proliferation and skeletal muscle thin filament assembly.The vascular smooth muscle contraction pathway was chosen as the study target for further analysis from the results of KEGG pathway of 2 times up or down differential expression genes.There are six 2 times up and down differential expression genes,involved myosin light chain kinase activity,calmodulin-dependent protein kinase activity,structural constituent of cytoskeleton and other molecular features;actin cytoskeleton,smooth muscle contractile fiber,striated muscle thin filament and other cellular component;participate in skeletal muscle thin filament assembly,vascular smooth muscle contraction,cell death and other biological processes.The full length mRNA sequences of ACTG2 in musule of Hu Sheep and DorperxHu sheep F1 was cloned.Two length(1317 bp and 1299 bp)sequences of ACTG2 was be found,and the distinction of two length sequences was that the 5' UTR with two alternation splicings(65 bp and 83 bp).Meanwhile,the alternation splicings in 5' UTR have been found are inconsistent with NCBI(XM 004006081).In this study,two alternation splicings in 5' UTR of ACTG2 was be found for the first time.The result of real-time fluorescent quantitative for MYL3^ ACTG2 and ACTG2-AS showed that the expressions of MYT3 in BFM of male and total 6-month Hu Sheep was higher than LDM(P<0.05),the expressions of MYL3 in BFM of total 6-month Dorper×Hu sheep F1 was significantly higher than LDM(P<0.01);The expressions of MYL3 in BFM of total 6-month Hu Sheep was higher than total 6-month Dorper×Hu sheep F1(P<0.05).The expressions of ACTG2 in the varieties or the organizations was non-significant.The expressions of ACTG2-AS in LDM of female 6-month DorperxHu sheep F1 was higher than BFM(P<0.05);The expressions of ACTG2-AS in BFM of male 6-month Hu sheep was higher than DorperxHu sheep F1(P<0.05),and the percentage of the expressions of ACTG2-AS for total expressions of ACTG2 was significantly higher than DorperxHu sheep F1(P<0.01).