Expression Profiles Of MRNA And MiRNA In The Muscle Of Tibetan Chickens And Preliminary Function Identification Of MiR-499-5p

Tibetan chickens are unique and precious in China,and their muscle development law is particular.Previous studies on Tibetan chickens mostly focused on development rules and correlation analysis between genetic polymorphisms and growth traits.There is no systematic research on the molecular mechanism of muscle development on Tibetan chickens.Therefore,in this study,leg muscle tissues of 120-day-old and 150-day-old Tibetan chickens were selected as the research materials.First,differentially expressed genes and miRNAs of leg muscle tissue at different developmental stages on Tibetan chickens were screened using high-throughput sequencing technology,and the sequencing results were verified by qRT-PCR.Then,among the differentially expressed genes and miRNAs,enrichment analysis was used to obtain significantly enriched signal pathways.The two were jointly analyzed to obtain an mRNA-miRNA regulatory network.Finally,the function of differentially expressed miR-499-5p was preliminarily identified using injection in vitro and high-throughput sequencing.The main results are as follows:1.Transcriptome sequencing of leg muscle tissue at different developmental stages on Tibetan chickenAfter filtering the original sequencing data,a total of 185 million clean reads were obtained from the two samples,and 78.08% and 76.66% of the clean reads were mapped to the chicken reference genome,respectively.Differential expression analysis was performed on the two samples.Compared with 120-day-old Tibetan chickens,there were 1 691 differentially expressed genes in leg muscle tissue on 150-day-old Tibetan chickens,of which 330 genes were up-regulated and 1361 genes were down-regulated.Among the differentially expressed genes,genes related to muscle development such as APOBEC2,MUSTN1,GDF-8,and SMYD1 were detected to be differentially expressed.5 differentially expressed genes were randomly selected and verified by qRT-PCR,and the results showed that their expression trends were consistent with the sequencing results.GO and KEGG analysis was performed on differentially expressed genes.GO enrichment analysis showed that among the top 10 entries for enrichment,immune-related items such as positive regulation of immune system processes and immune system processes were significantly enriched.KEGG analysis showed that PI3K-Akt and other signaling pathways were significantly enriched.The results indicate that differentially expressed genes may be involved in the regulation of muscle development through these pathways.2.Small RNA Sequencing of leg muscle tissue at different developmental stages on Tibetan chickenAfter filtering the original sequencing data,a total of 24 million clean reads were obtained from the two samples.The length of miRNA was analyzed on two samples,and it was found that miRNAs were mainly concentrated in 21~24 nt,and miRNAs of 22 nt were the most in two samples.Differential expression analysis revealed that compared with 120-day-old Tibetan chickens,there were 22 differentially expressed miRNAs in leg muscle tissue in 150-day-old Tibetan chickens,of which 9 miRNAs were up-regulated and 13 miRNAs were down-regulated.Among them,miR-499-5p,which was specifically expressed in muscle tissue,was significantly down-regulated.5 differentially expressed miRNAs were randomly selected and verified by qRT-PCR,and the results showed that their expression trends were consistent with the sequencing results.Target genes were predicted for differentially expressed miRNAs and KEGG analysis was performed on the target genes.It was found that 83 predicted target genes appeared on the signal pathways that the differentially expressed genes were significantly enriched.The differentially expressed genes and miRNAs were jointly analyzed to construct an mRNA-miRNA regulatory network.3.Preliminary identification of miR-499-5p functionAfter exogenous injection of miR-499-5p,gastrocnemius muscle tissue was collected for measurement of muscle fiber diameter and transcriptome analysis.The results showed that injection of miR-499-5p could significantly reduce the diameter of leg muscle fiber(P < 0.05).Sequencing results showed that 310 million and 320 million clean reads were obtained in the injection group and the control group,respectively.And the mapped rates on the chicken reference genome were 90.29% and 89.57%,respectively.Compared with the control group,the injection group had a total of 18 differentially expressed genes,of which 12 genes were up-regulated and 6 genes were down-regulated(P < 0.05).The two genes with the highest differential expression were FOS and EGR1.4 differentially expressed genes were randomly selected and verified by qRT-PCR,and the results showed that their expression trends were consistent with the sequencing results.GO enrichment analysis found that differentially expressed genes were significantly enriched in the transmembrane receptor protein serine/threonine kinase signaling pathway,lipid synthesis,and skeletal muscle regeneration.KEGG enrichment analysis results showed that differentially expressed genes were significantly enriched in the MAPK signaling pathway and Wnt signaling pathway.Studies have shown that injection of miR-499-5p can change muscle fiber diameter and affect muscle fiber characteristics.FOS,EGR1,CYR61 and WNT7 A may be involved in fat deposition and muscle development regulation through the above pathways under the action of miR-499-5p.In summary,the differences on genes and miRNAs expression in Tibetan chicken leg muscles at different developmental stages were explored and the function of key miRNA that affects leg muscle development of Tibetan chickens was preliminarily identified in this study.This study provided a theoretical basis for further understanding the particularity of muscle development in Tibetan chickens.