Expression Analysis Of LncRNA In Sheep Skeletal Muscle And Protein Encoding Ability Analysis Of LncRNA-MRLN Gene

Objective:Skeletal muscle is an important part of muscle and has important economic value.The growth and development of skeletal muscle is regulated by a variety of genes.By exploring key factors and important components in skeletal muscle development,studys can help researchers better understand the regulation mechanism of sheep skeletal muscle development.Long non-coding RNAs,as a new RNA in non-coding RNAs,regulateed skeletal muscle development at epigenetic,transcriptional,and post-transcriptional levels,but few reports have been reported.Therefore,this study analyzed the expression of long non-coding RNAs in sheep embryos and adult stages,which will further enrich the database of sheep non-coding RNA and lay the foundation for the subsequent breeding of meat sheep.Methods:Kazakh skeletal muscles in embryonic and adult stages were harvested and total RNA was extracted.Illumina HiSeq 2500 high-throughput sequencing technology was used to obtain new and differential expression long non-coding RNAs in skeletal muscle at two developmental stages.The accuracy of high-throughput sequencing was verified by qRT-PCR and DNA sequencing.The targeted gene of long non-coding RNA was predicted by co-localization with mRNA,and the functionally enriched analysis of differentially expressed long non-coding RNA was performed.The long non-coding RNA fusion expression vector was constructed by molecular biology techniques;the protein coding ability of long non-coding RNA was verified by cell biology.Results:1.A total 199,001,830 of clean reads was obtained by high-throughput sequencing,and the quality of the data obtained by filtration was reliable.Compared with the sheep genome,the overall alignment rate was over 80%.2.A total of 511 annotated lncRNA and 1055 new lncRNA were obtained after splicing assembly and a series of screenings.3.Compared with the sheep reference genome,lncRNA was evenly distributed on the positive and negative strands of the chromosome,with no obvious preference.92.5% of lncRNA was lincRNA.The average length of lncRNA was 1788 nt.The average number of exons was 2.3,and lncRNA with 2 exons accounted for 78%.The average length of the ORF of lncRNA was 126 nt,and the ORF length of 57% of lncRNA was concentrated at 90-150 nt.4.404 differentially expressed lncRNA were obtained in embryonic and adult Kazakh sheep.Among them,359 lncRNA were up-regulated in embryonic Kazakh skeletal muscle,while 45 lncRNA were up-regulated in skeletal muscle of adult Kazakh sheep.Ten of differentially expressed lncRNA were selected for RT-PCR and DNA sequencing,and the alignment was identical.The ten of lncRNA were quantified by qPCR and the results were consistent with the RNA-seq data.The authenticity of the RNA-seq data is further demonstrated.5.Bioinformatics analysis found that 511 lncRNA annotated targeted 2587 genes,and 1055 new lncRNA targeted 2795 genes.One of the lncRNA targeted multiple mRNAs,or multiple lncRNA targeted the same mRNA.6.GO enrichment analysis revealed that the lncRNA obtained by screening was associated with protein kinase activity,muscle structural components,tubulin binding,and myofibrils.The KEGG pathway enrichment analysis found that the candidate lncRNA obtained by screening may be involved in the regulation of metabolism,disease,cell communication and endocrine regulation.However,multiple genes were enriched into signaling pathways involved in muscle development,such as the Wnt signaling pathway and the calcium signal transduction pathway.7.A total of 61 up-regulated expression levels and 6 down-regulated lncRNA were screened for muscle development by constructing lncRNA-miRNA-mRNA interaction network map,and these lncRNA have one or more miRNA binding sites.And some lncRNA can adsorbed at least three miRNA.8.The lncRNA-MRLN fusion expression vector was constructed and was verified on mouse myoblasts,the result shown that lncRNA-MRLN can encode protein and was conserved in different species.Conclusion:1.Using high-throughput sequencing,a total of 1055 candidate lncRNAs were screened and distinguished from mRNA.2.Thousands of lncRNA were found in the skeletal muscle of Kazakh sheep using second-generation high-throughput sequencing,and their expression levels were different at two stages.Through target gene prediction and functional enrichment analysis,it was found that some lncRNA may be involved in the growth and development of skeletal muscle,which provided the valuable data for further understanding the molecular regulation mechanism of muscle growth and development from the perspective of lncRNA.3.The study was verified that lncRNA-MRLN gene can express proteins by molecular cloning and cell biology,which reshapes the understanding that lncRNA can not encode proteins.