Screening Of Cashmere Trait Related Genes And Functional Verification Of ELOVL3 And FA2H Genes In Jiangnan Cashmere Goats

The economic value of cashmere is mainly reflected in two aspects: cashmere production and cashmere fineness.It is important to explore the internal molecular process of hair follicle development in cashmere goats to improve hair yield and quality.Understanding the mechanism of interaction and induction of communication between epithelial cells and dermal cells is a key scientific problem to analyze the formation and regeneration of cashmere fiber diameter.The specific functions of m RNA and lnc RNA in the development and circulation of secondary hair follicle(SHF)have not been extensively analyzed,and transcriptome sequencing is considered to be an ideal method to explore gene expression variation.Therefore,high-throughput sequencing was used to study the regulatory roles of key m RNA and lnc RNA in hair follicle development and regeneration.To provide reference data for solving the above scientific problems.The main research results are as follows:(1)Transcriptome sequencing was performed on skin tissues of eight Jiangnan cashmere goats based on cashmere finness.A total of 479 differentially expressed m RNAs(DE m RNAs)and 565 differentially expressed lnc RNAs(DE lnc RNAs)were detected in skin tissues of Jiangnan cashmere goats in coarse and fine groups.DE lnc RNA predicted 12,554 target genes.Five protein interaction networks were constructed for DE m RNA encoding proteins,among which WNT5 A and IGFBP4 were constructed as key genes in the network.Five DE m RNAs and three DE lnc RNAs were selected for q RTPCR analysis,and the results were consistent with RNA-seq data.(2)Skin tissues of the SHF of Jiangnan cashmere goats were stained by H.E staining.Transcriptome sequencing was performed on the skin tissues of SHF during anagen,catagen and telogen stages of Jiangnan cashmere goats.There were 199 DE m RNAs and 256 DE lnc RNAs at different stages of SHF.DE lnc RNAs predicted 947 target genes.MMP9,AWAT1 and ACSM3 are the core nodes of the protein interaction network.Five DE m RNAs and three DE lnc RNAs were selected for q RT-PCR analysis,and the RNA-seq data showed basically the same trend.(3)In the fineness grouping,combined with the predicted results of DE m RNA and DE lnc RNA target genes,we found that 467 differentially expressed target genes were predicted by 565 DE lnc RNAs.Compared with the results of this study,18 common DE m RNA were found in the two breeds.Based on the above analysis,we believe that FBLN5,ELN,SOX18,SOX4,PTCH2,COL5A1 and FA2 H are considered to be related to the development of skin hair follicles.In periodic grouping,combined analysis of DE m RNA and DE lnc RNA target genes showed that 36 DE lnc RNAs predicted 28 differentially expressed target genes;Compared with this study,26 common DE m RNAs were found in shaanxi White cashmere goats.Based on the above analysis,we believe that VASN,LGR6,FOS,ELOVL3,MMP9,KRT2 and KRT4 genes are related to the hair follicle cycle.(4)In this study,dermal papilla cell(DPC)was isolated from the SHF of Jiangnan cashmere goats.CD133,Versican and α-SMA were positive in cell immunofluorescence identification,indicating that relatively pure DPC was obtained.The expression level of ELOVL3 and FA2 H gene were found to be consistent with transcriptome sequencing results by q RT-PCR,and ELOVL3 and FA2 H protein could be expressed on DPC by immunofluorescence.In the ELOVL3 gene study,CCK-8 showed that interference of ELOVL3 expression could significantly inhibit DPC activity(P<0.01),while overexpression of ELOVL3 could significantly increase DPC activity(P<0.01).Using EDU technique to detect overexpressed ELOVL3 can promote the proliferation of DPC.Flow cytometry was used to detect that interfering ELOVL3 expression inhibited DPC from G1 phase to S phase,and overexpression of ELOVL3 gene promoted DPC from G1 phase to S phase.In the FA2 H gene study,CCK-8 showed that FA2 H gene interference significantly decreased cell viability ratio(P<0.05).Overexpressed FA2 H gene was detected by EDU technique to promote DPC proliferation.Flow cytometry was used to detect overexpressed FA2 H gene to promote DPC transition from G1 phase to S phase.(5)QRT-PCR showed that compared with the control group,the expression levels of ELOVL3,CIDEA and ELOVL1 genes in DPC were significantly down-regulated(P<0.05),and the expression levels of NCMAP,ELOVL4 and FGF5 genes were extremely significantly down-regulated(P<0.01).The expression levels of ACER3 and FGF7 were significantly up-regulated(P<0.05),and the expression levels of SHH,FGF10 and CTNNB1 were extremely significantly up-regulated(P<0.01).Overexpression of FA2 H gene resulted in extremely significant down-regulation of FGF5 gene expression in DPC(P<0.01),and extremely significant up-regulation of ELOVL3,ASAH2,ACER3,DIO2,BSCL2 and BMP2 expression(P<0.01).Based on the above studies,some key genes are associated with cashmere traits,hair follicle development and SHF cycle,including FBLN5,ELN,SOX18,PTCH2,COL5A1,FA2 H,VASN,LGR6,FOS,ELOVL3,MMP9,KRT2 and KRT4 genes.Function verification of candidate genes showed that FA2 H gene could regulate the expression of ELOVL3 gene,and overexpression of FA2 H and ELOVL3 genes could promote the proliferation of DPC in cashmere goats’ secondary hair follicles.In conclusion,the results of this study provide an experimental basis for clarifying the regulation mechanism of cashmere fineness and secondary hair follicle circulation in cashmere goats,and also provide a theoretical basis for the breeding of new breeds/strains of high quality and high yield cashmere goats in southern Xinjiang.