MiRNAs And Their Target Genes For Regulating Milk Quality In Dairy Cow Mammary Gland

Milk quality of dairy cow are regulated by many factors, in recent years, it was found that non-coding RNA may be involved in the regulation of lactating in dairy cow.In this study, small RNA-seq technology combined with RNA crosslinking immunoprecipitation sequencing(CLIP-Seq) technology are used to research and identificate miRNAs and their key target genes that associated to the milk quality from Chinese Holstein cows mammary gland.(1) Identification and separation the candidate mi RNA related to milk quality using small RNA-seq technologyThe study was based on different experimental groups of cow mammary gland which have different milk quality, one was milk of high quality lactation cows group(H) and other was lactating cows of low milk quality group(L), first, using small RNA high-throughput sequencing technology screening of different expression of mi RNAs in each experimental groups of mammary gland. Analysed new sequencing had obtained with two mi RNA predictive software mi REvo and mir Deep2. The results showed 56 miRNA differentially expressed in different tissues of cow milk quality(P <0.05, FDRq <0.05); Then bioinformatics methods were used to predict target gene of differentially expressed miRNAs, and using Gene Ontology(GO) and signaling pathway enrichment analyse the fouction of these target genes. Bioinformatics target genes screening res ult, we found that the target genes such as CSN3, SCD, LALBA and DGAT2 had been reported to interrelate with milk protein, milk fat synthesis. Most predicted target genes of mi RNA in biological function involved in protein aggregation and fat metabolism, m ammogenesis and differentiation and immune function, these target genes were mainly concentrated in the signaling pathway MAPK, signaling pathway glycerol phosphate lipid metabolism, signaling pathway hypoxia-inducible factor-1 and phospholipids inositol 3-kinase-protein kinase B. The results suggest that mi RNAs play a role in milk milk quality regulation by targeting mainly glucose metabolism, fat metabolism, protein metabolism, apoptosis and immune-related gene pathway.(2) Application of RNA binding protein immunoprecipitation and CLIP-Seq technology validation target genes of mi RNA regulation related tomilk qualityTo validate the result of mi RNA target gene that related to milk quality predictied by bioinformatics, we continued to use high-quality dairy cow(H) and low milk quality lactation cow(L) of the mammary gland as subjects. The experimental samples was acquired by application of RNA-binding protein immunoprecipitation technique, 81 differentially expressed target genes of miRNA regulation(P<0.05, FDRq <0.05) were obtained through CLIP-Seq analysis. It showed enrichment in protein metabolism, fat metabolism, mammogenesis and other biological processes in these 81 differentially expressed target genes of miRNA by GO analysis and pathway analysis.(3) Bioinformatics analysis of target genes and integrated analysis of the experimental results verifyBy intergrated analysis of target genes bioinformatics analysis and experimental verify, 26 intersection of target genes were selected, then these 6 gene construct miRNA-Gene-pathway and miRNA-Gene-GO interaction network diagram, found 6 target genes( FABP3,TGFβ3,CACNA1 G, ACSM1,GSS and CHAD) are the important candidate gene in milk protein and milk fat synthesis; bta-mi R-423-3p, bta-miR-2449, bta-miR-2378, bta- mi R-2382*, bta-mi R-3604 and bta-miR-324 regulate over 6 intersection of target genes, suggesting that these miRNA are the key mi RNA affect on milk protein and milk fat synthesis.