RNA-seq And Small RNA-seq Reveal Novel Functional Genes For Milk Production Traits And Bioinformatic Prediction Of Novel MicroRNAs In Bovine

By intergrated analysis of RNA-seq and Small RNA-seq from the mammary glands of four lactating Holstein cows with extremely high and low phenotypic values of milk protein and fat percentage, we identified critical genes and miRNAs related milk components and predicted novel miRNAs of bovine with comparative genomics and bioinformatic analysis. To determine the interaction between the target genes and miRNAs, the dual luciferase reporter system was launched.Section1:Transcriptional profiling of mammary gland using RNAsequencing.We used massive, parallel, high-throughput, RNA-seq to generate the bovine transcriptome from the mammary glands of four lactating Holstein cows with extremely high and low phenotypic values of milk protein and fat percentage. In total,31differentially expressed genes (P<0.05, false discovery rate q<0.05) between the high and low groups of cows were revealed. Gene ontology and pathway analysis demonstrated that the31differently expressed genes were enriched in specific biological processes with regard to protein metabolism, fat metabolism, cell viability/death/differentiation,activation of immune-related cells and mammary gland development (P<0.05). Integrated analysis of differential gene expression, previously reported quantitative trait loci, and genome-wide association studies indicated that TRIB3, SAA (SAA1, SAA3, and M-SAA3.2), VEGFA, PTHLH, and RPL23A were the most promising candidate genes affecting milk protein and fat percentage.Section2:Screening of the microRNA related to milk composition by small RNA-seq.To determine the regulation of milk composition traits, we investigated the microRNA (miRNA) expression profiles using the same samples as our previous RNA-Seq experiment.We identified a total of497known miRNAs (miRBase21.0) and49novel miRNAs among the reads. Of these,71miRNAs were found differentially expressed between the high and low groups (P<0.05, false discovery rate q<0.05);36were down-regulated and35were up-regulated.We predicted a total of12202target genes for the known and novel miRNAs. All targets were then processed by GO annotation analysis. It was found that these targetsinvolved in glucose metabolism, protein and Iipid metabolism, cell development and differentiation, and immune functions (P<0.01, false discovery rate (FDR) q<0.01).Section3:Intergrated analysis of small RNA-seq and RNA-seq.Integrated analysis of differentially expressed miRNAs and differentially expressed genes showed that22intersection genes which were both differentially expressed between the two groups and were targets of differentially expressed miRNAs were identified. Based on the results of Gene ontology and pathway analysis, TRIB3、SAA1、SAA3、M-SAA3.2、PTHLH、VEGFA and CDKN1A were the most promising candidate genes. Corresponding miRNAs for7intersection genes might be critical miRNAs related to milk components,including miR-1388-5p、miR-294、miR-106b、miR-29c、miR-190a and miR-125a(b) et al. To further verify the mechanism of miRNA and determine the relationship between target gene and miRNA, we carried out the dual-luciferase reporter gene assay on the candidate genes (TRIB3-. PTHLH and VEGFA) and the corresponding miRNAs.The results suggested significant interaction of miR-1388-5ps miR-2904with TRIB3, and of miR-29、miR-106b、miR-190a with PTHLH.Section4:Computational identification of novel microRNAs in bovine.Identification and discovery of miRNAs is essential to reveal the regulation network of miRNAs in the expression of candidate genes for important traits in domestic animals and their genetic mechanisms. According to the conservation of miRNAs sequence, we compared the known miRNAs in human, mouse, sheep, pig and dog with the bovine genome sequence (UMD3.1) in NCBI and a total of44novel miRNAs of bovine were obtained. Compared with the novel miRNAs which were found by small RNA-seq, we did not get the same miRNA.Gene ontology and KEGG pathway analyses showed that some target genes were enriched for functions such as protein metabolism and fat metabolism, e.g. TRIB3,CDKN1A et al.In this paper, we found7key genes about milk composition by RNA-seq, small RNA-seq and intergrated analysis. The functional verification test showed the regulation between miRNAs and target genes (TRIB3and PTHLH).