MRNA And LncRNA Induced By Exogenous Melatonin That Regulate Cashmere Growth In Cashmere Goat Skin

Fully understanding the mechanism of melatonin promoting goat cashmere growth remains a challenge.In order to study which mRNA and Long non-coding RNA(lncRNA)in cashmere goat skin were recruited by exogenous melatonin at transcriptional level to promote cashmere growth during non-producing period,which mRNA and lncRNA undergo alternative splicing(AS)at post transcriptional level were involved in the regulation of cashmere growth.Six Hanshan white cashmere goats were selected and randomly assigned to control group and melatonin treatment group with three replicates in each group.Take the natural year as the experiment period,skin samples were collected monthly for RNA sequencing(RNA-Seq)analysis.1.RNA-Seq data was analyzed using Bowtie,Top Hat,Cufflinks,Cuffmerge,Cuffdiff,Cuffcompare,CPAT and CPC software,2024 differentially expressed mRNAs(DE-mRNA)were identified,329 differentially expressed lncRNAs(DE-lncRNA)were newly identified,it was found that the number of potential subtypes of known transcripts lncRNAs and intergenic lncRNAs was higher,and other types was fewer.2.Principal component analysis(PCA)showed that 2024 DE-mRNAs and 329 DElncRNAs responded to the growth cycle of the secondary hair follicles in cashmere goat skin.3.Weighted gene co-expression network analysis(WGCNA)method was used to screen the gene modules regulating goat cashmere growth.Multiple analysis methods of bioinformatics were used to further determine the candidate gene set regulating cashmere growth.(1)The weighted gene co-expression networks of 2024 DE-mRNAs and 329 DElncRNAs in cashmere goat skin under normal physiological conditions and exogenous melatonin induction conditions were constructed respectively,which reduced their dimensions into 8 and 10 gene modules in the control and treatment group,and Hippo target gene module was found.(2)The interaction between modules and the correlation between modules and samples were quantified,it was found that the Hippo gene module in the control group was positively correlated with the samples in the growth period of secondary hair follicles(August to December),the synergistic interaction between the gene of Hippo module,oxidative phosphorylation module and cell cycle module in the treatment group was positively correlated with the samples of two cashmere growing periods(April to July and August to December).The internal relationship of 45 GO terms or KEGG pathways by time course in the control gene module regulating goat cashmere growth was revealed,and 44 GO terms or KEGG pathways in the treatment group was revealed.(3)The co-expression network of 159 mRNA and 24 lncRNA in the candidate gene set regulating goat cashmere growth in the control group was revealed,191 mRNA and 49 lncRNA in the treatment group was revealed.The candidate gene set of the control group was highly expressed during the normal cashmere growing period(August to December),the treatment group was highly expressed during two cashmere growing periods(April to July and August to December).The 11 genes annotated to Hippo signal pathway,8 genes annotated to skin development,9 genes annotated to cell cycle,11 genes annotated to intermediate filament,4 genes annotated to oxidative phosphorylation and 14 lncRNAs strongly related to the above 43 genes in the candidate gene set of control and treatment group were highly expressed during cashmere growing period.4.The biological function of candidate gene set regulating cashmere growth was scanned from three different angles: Gene set enrichment analysis(GSEA),Gene Set Variation Analysis(GSVA),quantitative correlations between candidate gene set and cashmere growth cycle,64 hub genes,22 transcription factors and 11 transcription cofactors of the candidate gene set were identified.5.A total of 715 differential alternative splicing events(DASE)of cashmere goat skin transcriptome mRNA were identified by r MATS software,SE and MXE were the main AS way,569 DASE of lncRNA were identified,SE and RI were the main AS way.(1)There was a significant positive correlation of PSI(Percent spliced-in)levels between different development stages of cashmere goat hair follicle in the control group,and between the two cashmere growing stages in the treatment group,exogenous melatonin increased the probability of AS of DE-mRNA.(2)DASE occurred in 67 of 2024 DE-mRNAs and 7 of 329 DE-lncRNAs,the 74 DAStranscripts were most distributed in Hippo target gene modules in the control group and more in cell cycle target gene modules in the treatment group.(3)The AS regulatory network composed of 220 mRNAs and 63 lncRNAs in cashmere goat skin in the control group was constructed,DAS-transcripts among them interacted with35 mRNAs and 18 lncRNAs of candidate gene set regulating cashmere growth.The AS regulatory network composed of 207 mRNAs and 41 lncRNAs in cashmere goat skin in the treatment group was constructed,DAS-transcripts among them interacted with 24 mRNAs and 5 lncRNAs of candidate gene set.(4)The As regulatory network was mainly involved in cellular response to interleukin-4,positive regulation of cell differentiation,ECM-receptor interaction,PI3K-Akt signaling pathway,c GMP-PKG signaling pathway,cortisol synthesis and secretion,melanogenesis.To summarize our studies explained the co-expression regulator relationship of mRNA,lncRNA and TF from the perspective of gene set at the omics level,the biological functions of the 59 lncRNAs were annotated in cashmere goat skin,enriched our knowledge about mRNA,lncRNA and TF of promoting cashmere growth,which provided a new thinking to annotate lncRNA in Livestock.Determined that Hippo signal pathway was the key pathway to regulate cashmere growth,provided reference for further investigations on the mechanism of melatonin promoting cashmere growth in goat.The AS of mRNA and lncRNA during the development of skin follicle in cashmere goat was recognized,the potential interaction network of AS occurred in mRNA and lncRNA of cashmere goat skin transcriptome was constructed,it provided a new potential insight into the effect of AS regulation on melatonin promoting cashmere growth.