Identification Of Non-coding RNAs And Functional Genes For Milk Production Traits In Liver Of Dairy Cattle Across Different Lactation Periods Using Transcriptomics Technologies

Milk performance traits have been considered as the most important economic traits.Liver and mammary gland are among the most prominent organs during lactation in dairy cows.However,the regulation mechanism between liver and mammary gland has not been stated very clearly.With the rapid advancement of omics technologies,it has been extensively used to study functional genomics in human,animals and plants.In this thesis,we performed transcriptomics and bioinformatics to identify the functional genes and non-coding RNAs related to milk production traits using the liver samples of three Chinese Hotstein cows in three periods?dry period,early lactation period,peak of lactation period?.Section 1:Identification of functional genes affecting milk production traits using RNA-seqCuffdiff software was used to identify differentially expressed genes?DEGs?among three periods.Totally,665 DEGs were found in three comparisons,including 270 altertive splicing DEGs,of which,160 genes have specific altertive splicing events in different periods.We predicted 15 DEGs as promising candidate genes affecting liver metabolism,ABCG5,ABCG8,ACADVL,APOM,CYP2C87,CYP2U1,CYP7A1,PC,HADHA,HAO2,PPP2R2B,RAB2A,KLF6,SLC22A7 and IRF6.Section 2:Screening of lncRNAs related to mik production traits by RNA-seqBased on the section 1 RNA-seq data,we totally identified 1,704 lncRNAs,including 33 differentially expressed lncRNAs in three comparisons.We set 100kb of lncRNAs as threshold and predicted 76 cis-target genes,and identified 852 trans-target genes which co-expression level were higher than 0.95.Then,we constructed differentially expressed lncRNA-DEGs networks and found two cis-target genes?AK4,MFSD2?and seven trans-target genes?IGF-1,IGFBP3,SCD,EGR1,LPIN1,KLF6 and GADD45B?could be promising candidate genes.Section 3:Identification of miRNAs influencing milk production traits using small RNA-seqTotally,we identified 1,046 miRNAs,including 446 known miRNAs and 600 novel miRNAs.Of these,57 miRNAs were found differentially expressed in three comparisons?early lactation vs.dry period,peak of lactation vs.dry period,peak of lactation vs.early lactation period?.All targets were predicted by miRanda and RNAhybrid software,and GO and KEGG functional enrichment analysis were performed using KOBAS software.It was found that these target genes involved in fatty acid metabolism,carbohydrate metabolism,protein metabolism and PPAR,AMPK signaling pathways.We predicted 21 functional genes were targeted by miRNAs:LIPH,FGFR4,ASCL1,IGF1R,EGR1,PPP1R3C,IRF6,PPP2R2B,GPX3,APOA4,DUSP1,PKLR,CYP7A1,INHBE,IRF9,GADD45B,CYP26A1,KLF6,PLIN3,LPIN1 and INPP1.Section 4:Intergrated ananlysis of transcriptome and construct ceRNA regulation networks related to milk production traitsA new regulatory mechanism builds a large-scale regulatory network among transcriptome,where lncRNAs act as competing endogenous RNAs?ceRNAs?to sponge miRNAs to regulate the expression of miRNA target genes post-transcriptionally.Based on the differentially expressed genes,IncRNAs and miRNAs,we constructed ceRNA networks.Subsequently,a total of 41 ceRNA pairs?lncRNA-mRNA?sharing 11 miRNAs were constructed including 30 differentially expressed genes and nine lncRNAs.Importantly,12 genes among them were presented in our large metabolic networks,and predicted to influence the lipid metabolism through insulin,PI3K-Akt,MAPK,AMPK,mTOR,and PPAR signaling pathways,thus,these genes were considered as the most promising candidates for milk fat formation:ARNTL,COL6A1,IGF1R,NGFR,INHBE,FGFR4,PKLR,FADS2,NROB2,S1PR1,SLC22A7 and PPP2R2B.Besides,non-conding RNAs that interacted with 12 functinal genes were thought to be important RNAs that could influence milk production traits,including seven IncRNAs?XYLOC-054981,XLOC₁242646,XLOC₁667881,XLOC₁359929,XLOC₈22557,XLOC₇92712,XLOC₁655255?and eight miRNAs?bta-miR-2887,bta-miR-1247-5p,novel_chrl1₄209,novel_chr24₂2303,novel_chrl3₆810,novel_chr5₃1133,novel_chr18₁3489,novel_chrX₃8392?.