Screening Of MiRNAs Related To Buffalo Fat Deposition And Their Effects On Fat Deposition

MicroRNA,approximately 18～24 nucleotides in length,is single-stranded non-coding small RNA which regulates the post transcriptional expression of genes in animal growth and development.The intramuscular fat content directly affects the tenderness and flavor of beef,while the intramuscular fat content of buffalo is lower than that of yellow beef,so its taste is far less than that of yellow cattle,which seriously affects the meat production of buffalo in China.In order to excavate the miRNAs which were related to buffalo fat deposition,potential miRNAs for the improvement of buffalo quality were obtained.In this experiment,Xinyang buffalo was used as material to construct a small RNA sequencing library of subcutaneous adipose tissue in different developmental stages of buffalo.Combined with high-throughput sequencing technology and bioinformatics analysis method,miRNAs in different developmental stages were screened.Target genes of Candidate miRNAs were predicted and miRNAs-target gene interaction pairs associated with fat deposition are screened.The sequencing results were verified by qRT-PCR technique and tissue expression profiling was performed.Overexpression of miRNAs that may regulate buffalo fat deposition by agomiR,and its effect on differentiation of bovine adipocytes;and the interaction of miRNA-target gene was verificated by dual luciferase reporter system.The aspect provides a new theoretical basis for molecular breeding and meat breeding in Xinyang buffalo.The main work and results of this study are as follows:1.High-throughput sequencing was carried out to construct a small RNA sequencing library of subcutaneous adipose tissue of Xinyang buffalo juvenile cattle(6months old)and adult cattle(30 months old),then 807 miRNAs were identified,in which355 were newly discovered miRNAs.Expression analysis indicated that there were 282 miRNAs differentially expressed in the two libraries.With age,148 miRNAs were up-regulated and 134 miRNAs were down-regulated.Target gene prediction of differentially expressed miRNAs and screening of target genes that may be associated with fat deposition,and finally screening for bta-miR-195-THRSP,bta-miR-195-EGR1,bta-miR-24-3p-DGAT2 andnovel＿miR-7＿28313-SREBF1 Interacting against miRNA-target genes that may be involved in adipose differentiation.2.The expression of candidate miRNAs were detected by qRT-PCRand the results are consistent with sequencing results.The tissue expression profiling showed that bta-miR-195 was highly expressed in heart,lung and adipose tissue;bta-miR-192 was highly expressed in liver tissue;bta-miR-24-3p was highly expressed in heart tissue,in muscle High expression in adipose tissue;novel＿miR-7＿28313 is highly expressed in the kidney and highly expressed in liver and adipose tissue.The expression levels of bta-miR-195,bta-miR-192 and novel＿miR-7＿28313 were significantly expressed in the process of differentiation and maturation of adipocytes in vitro culture,which indicating that they were invovled fat regulation.3.Overexpression of bta-miR-195 and bta-miR-192 in bovine primary adipocytes inhibited lipid accumulation in adipocytes,inhibited the expression of adipogenic marker genes PPARγ and CEBPα,and inhibited the expression of target genes THRSP and EGR1.The dual luciferase reporter assay showed that bta-miR-195 binds to the THRSP3’ UTR and down-regulates luciferase activity,which demonstrated that bta-miR-195 can targeting THRSP.