The Study On Screening Of Donkey Muscle Development-related MRNA,miRNA And LncRNA Based On RNA-seq

The livestock and poultry meat is mainly composed of skeletal muscle,which has multiple types of muscle fiber that affects meat quality,especially the increase in the number and volume of muscle fibers.Therefore,exploring the molecular mechanism of development of skeletal muscle has remarkable guiding significance for increasing the yield of meat and improving the quality of meat.A mushrooming number of studies have shown that non-codingRNA(ncRNA)play an important role in regulating the life activities of organisms such as embryonic development,tissue differentiation,and organ formation.Also,it plays an indispensable role in the regulation of muscle development.Although the non-codingRNA expression profiles pattern of muscle have been reported in some animals,they have not been reported and studied less in equine muscle tissue.Donkey meat has high nutritional value and is very popular in the Chinese market.The Dezhou donkey is a well-known breed in China.It is characterized by tall body,well structure,nice appearance,resistance of crude feed,good disease resistance,and high meat yield.Therefore,in this study Dezhou donkey was chosen as research object,to reveal the effect of non-codingRNA on the muscle development by analysing skeletal muscle transcriptomes of Dezhou donkeys at different developmental stages(2 months and 24 months of age)using Illumina technology.The non-codingRNA(lncRNA,miRNA)and mRNA of differential expression in skeletal muscle were verified by RT-PCR and the target genes of differently expressed miRNA(miR-1)were screened by dual-luciferase reporter system,which can be used for the breeding of meat producing donkey.The main results of this study are shown as follows:1.Identification and characterization of lncRNAs and mRNAs in Dezhou donkey muscle tissue at different development stagesIn this section,a total of 37,004 lncRNAs were identified on the longest dorsal muscle of the Dezhou donkey in two periods byRNA sequencing.After differential expression analysis,a total of 183 lncRNAs and 628 mRNAs with differential expression were screened.Functional enrichment analysis revealed that differentially expressed lncRNAs and mRNAs were participated in polysaccharide binding,receptor binding,and AMPK signaling pathways for energy metabolism.Target genes analysis of lncRNA showed that lnc-008278,lnc-017241,lnc-030153,and lnc-033914 may regulate the expression of DCN,ITM2 A,MUSTN1,and ARRDC2 and then affect muscle development.These results were confirmed by RT-PCR,which were consisted with sequencing results.2.Identification and characterization of miRNAs in Dezhou donkey muscle tissue at different development stagesIn this section,a total of 536 miRNAs(422 known miRNAs and 112 novel miRNAs)were identified on the longest dorsal muscle of the Dezhou donkey in two periods by smallRNA sequencing.This length of most miRNAs were concentrated in 21-23 nt.After differential expression analysis,we found 45 differential miRNAs.Functional enrichment analysis of differential miRNA target genes revealed that the MAPK signal pathway and Ras signal pathway may be related to muscle development.Through target gene analysis of miRNA,it was found that novel-204-COL6A2,ADSSL1-miR-370-ARRDC2,miR-125a-3p-MUSTN1,and miR-328-PIK3R1 may be related to muscle development.The results were also confirmed by RT-PCR.3.Verification of miR-1 and their target genesIn this section,IGF-1,TMSB4 X,EFS1,TPM3,PLXDC2 and ZZZ3 were predicted as the target gene of miR-1 by the software of miRanda.the wild-type and mutant-type plasmid of target genes were successfully constructed for dual-luciferase reporter system,and the dual-luciferase reporter experiments showed that the target genes of miR-1 were TPM3 and ZZZ3.In summary,a large number of miRNA,lncRNA and mRNA have been identified in the muscle tissue of Dezhou donkeys,which will provide the theoretical base for the molecular breeding of meat trait in Dezhou donkey.