| Pig litter size is an important economic index in pig production, and ovaries of mammals are one of the important reproductive organs, the higher ovulation rate would increase high litter size. However, the heritability of pig litter size traits is relatively low (about 0.1), therefore, new ways to improve pig litter size have been exploring. Transcriptome (RNA-seq) and small RNA sequencing (microRNA-seq) identify all genes and miRNAs for specific cell or organization in some functional status. MiRNAs are kinds of important non-coding small RNA, which mainly results in target-specific post-transcriptional repression. Integrated analysis of mRNA and miRNA can accurate and rapid screen out key genes, miRNA and GO terms/KEGG pathways affecting litter size, promoting molecular regulation mechanism study on pig litter size.Yorkshire pigs with high (YH) and low (YL) litter sizes were used in this study, the following experiments were carried out:(1) we assessed gene expression differences between the ovaries of YH and YL pigs using Solexa sequencing technology, revealling ovarian gene expression, analyzing the transcriptome characteristics of YH and YL porcine ovaries, searching differentially expressed gene in transcriptome of YH and YL porcine ovaries, the GO enrichment and KEGG pathway analysis were performed on DEG, to explore the potential biological function of the target DEG;(2) We compared the ovarian miRNAs of YH and YL pigs using Solexa sequencing technology, analysing the miRNAome characteristics of YH and YL porcine ovaries, searching differentially expressed miRNAs in miRNAome of YH and YL porcine ovaries, the GO enrichment and KEGG pathway analysis were performed on DEM, to explore the potential biological function of the target DEM;(3)Integrating analyse of transcriptome and miRNAome to reveal DEG and DEM correlation expression in YH and YL porcine ovaries, up mRNA-down miRNA and down mRNA-up miRNA are correlation analysis, in order to find out the effects of litter size of the important nodes (gene and miRNA and go entries/KEGG pathway);(4) Q-PCR validation of the RNA-seq and miRNA-seq results, mRNA-miRNA correlation analysis, and the 4 miRNA (miR-126-3p, miR-126-5p, miR-130b and miR-145-5p) for tissue expression profile analysis.The main points are indicated as follows:(1) A total of 17,485 and 19,178 genes were obtained from the YH and YL libraries, respectively. Four hundred and two genes were expressed only in YH,2095 genes were expressed only in YL, and 17083 genes were co-expressed in both libraries. A total of 1243 genes were differentially expressed between the two groups, in which 897 genes were upregulated and 346 genes were downregulated in the YH group. After analyzing the function of these genes, we found 11 DEGs (CO1, GPX3, MSMB, COX3, TIMP1, CYTB, STAR, HSD3B, CYP11A1, SCARB1 and HSD17B7) that may be relevant to the prolificacy of pigs. The GO enrichment and KEGG pathway analysis were performed on 1,243 DEG, Steroid biosynthesis and Ovarian steroidogenesis may play important role in litter size traits in pigs;(2) A total of 327 and 320 miRNAs were obtained from the YH and YL libraries, respectively. Thirty miRNAs were expressed only in YH,23 miRNAs were expressed only in YL, and 297 miRNAs were co-expressed in both libraries. A total of 37 miRNAs were differentially expressed between the two groups, in which 21 miRNAs were upregulated and 16 miRNAs were downregulated in the YH group. After analyzing the function of these genes, we found 8 DEM (miR-26a, miR-129a-5p, miR-224, miR-99a, let-7c, miR-181c, miR-214 and miR-21) that may be relevant to the prolificacy of pigs. The GO enrichment and KEGG pathway analysis were performed on target genes of DEM, Steroid biosynthesis and Ovarian steroidogenesis may play important role in litter size traits in pigs. Peroxisome were involved in the significantly enriched pathway in the YH and YL libraries;(3)Integrating analyse of mRNA and miRNA, a total of 39 DEGs and 10DEMs were obtained from up mRNA-down miRNA by PPI analysis, and enrichment in 10 go term/KEGG pathway; a total of 30 DEGs and 15 DEMs were obtained from up mRNA-down miRNA by PPI analysis, and enrichment in 10 go term/KEGG pathway. To obtain an important node were analyzed. MiR-26a targeting DHCR24 gene enriched to Steroid biosynthesis pathway; miR-129a-5p targeting IGF1 gene enriched Ras signaling pathway pathway, these important nodes may play an important role in litter size traits;... |
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