Research Of Lnc-SEMT Function In The Process Of Enhancing Sheep Muscle Differentiation And Generation

LncRNA is a kind of length which is more than 200 nt and does not show the potential of protein coding RNAs. LncRNA participated in the X chromosome silence, genomic imprinting and chromatin modification and transcription activation, transcription interference, intranuclear transport, and other important regulatory process, involved epigenetic regulation, transcriptional regulation and transcription regulation in multiple levels and so on. Latest reports showed that lncRNA molecules can regulate the muscle function of mice and humans, but there is no report about the lncRNA on animal muscle differentiation regulation of development studies. This study filtrated a lncRNA molecular established (named Lnc-SEMT) which found by high-throughput sequencing screening. Lnc-SEMT can promote the sheep muscle cell differentiation, promote the formation into muscle, test analysis and it play an important regulatory role in skeletal muscle development. The study of this molecule is to clarify the molecular mechanism of animal muscle cell proliferation and differentiation, which is the great significance, and can provide for molecular assisted breeding the growth and development of new agents to provide scientific basis and technical support. The main results were as follows:1.The embryonic period of 90 days and 120 days of age sheep muscle samples for high-throughput sequencing a total of 1114 IncRNAs,466 IncRNAs differentially expressed, and among the 466 differentially expressed lncRNA, of which 157 IncRNAs rised,309 IncRNAs downregulated.2. Through the sequencing of 1114 IncRNAs chip verification, we found that there are 867 prediction of lncRNA exist, there are 119 IncRNAs expression rised, we got a lncRNA (named Lnc-SEMT) which rised in highly specific expression in skeletal muscle tissue by quantitativing screening of these 119 IncRNAs.3. The Inc-SEMT expression quantity was the highest and reduced after the birth of gradually, adult expression levels didn't change evidently by measuring different growth periods of sheep Inc-SEMT expression levels found in embryonic period of 120 days of age.4. By detecting the sheep myoblast lncRNA expression in the process of the growth of the quantity change,0 to 5 days in myoblast differentiation, with the growth of myoblast differentiation, MyoG and MyoD gene expression level increased as well.5. By building the Inc-SEMT high expression and interference vector, transfecting into sheep muscle cells, respectively,and by taking DAPI and MyHC test analysis, the results show that the established process of myoblast differentiation., The MyoG and MyoD gene expression changed with the Inc-SEMT expression and interference.6. By microinjection, five mutant Inc-SEMT sheep were got. Genetically modified (gm) established mutation of sheep Inc-SEMT expression level is 3.3 times that of the wild type sheep.7. Through the established counter rotating Inc-SEMT sheep after birth weight measurement, sheep found mutations in 3 months,6 months and 9 months weight was significantly higher than control group in the wild. Sheep slaughtering experiment mutations of carcass weight also is significantly higher than comparison group in the wild.8. Established by the counter rotating lnc-SEMT sheep for HE staining and immunofluorescence staining, and found mutations sheep muscle fiber cross-sectional area is significantly higher than the control group, in addition, the mutation of sheep 30 to 45 microns in diameter of fibers was significantly higher than that of control group, no obvious difference of the diameter of the fibers.9. Through application software, we established the predicted lnc-SEMT and potential binding sites of miR-125b.10. Established on lnc-SEMT in miR-125b binding sites of mutation, and transfection in vitro experiment was carried out, and we found that miR-125b is established with lnc-SEMT potential binding sites, established the combination of lnc-SEMT and miR-125b competitive IGF-2 at the same time, in order to realize the promoting the formation and into muscle myoblast differentiation.11. Established by the counter rotating lnc-SEMT sheep serum testing, established found lnc-SEMT and IGF-2 expression in sheep blood volume is significantly higher than comparison group in the wild.