| Liaoning Cashmere Goat is a breed with stable heritability and famous for cashmere.With the popularity of cashmere,the demand for cashmere is higher and higher.Although the yield of cashmere is very high,the overall trend is too coarse.Cashmere fineness plays a decisive role and is one of the important indexes to evaluate cashmere quality.In recent years,new progress has been made in the regulation of cashmere fineness by highthroughput sequencing and genome-wide association analysis.Key genes and pathways regulating cashmere fineness have been found,but the problem of cashmere fineness regulation has not been solved fundamentally.From the perspective of molecular biology,this study intends to screen out the key genes and related mechanisms that may regulate cashmere fineness,construct the ce RNA regulatory network and expression verification of the key gene KRT26,and analyze the correlation between cashmere fineness functional genes and cashmere production performance.The results are as follows:1.Isolation and expression of miRNA from skin tissue of Liaoning cashmere goatIn this study,the skin of Liaoning cashmere goat(LCG)was sequenced with highthroughput,and a clean reading of 14801303 was obtained,including 464 known miRNAs and 45 new miRNAs.Target gene enrichment analysis showed that miRNA target genes were enriched in KEGG pathway,which included PI3 K Akt signal transduction pathway and Wnt signal transduction pathway through progesterone oocyte maturation and endocytosis.The protein interaction analysis of target genes showed that PCNA,RPL11 and RPS13 were highly enriched,which may have an effect on the regulation of cashmere fineness.Twenty-eight miRNAs were randomly selected from three genotypes for q PCR verification.The results showed that mir-30a-5p,mir-30e-5p,mir-105 a and miR-101 may regulate cashmere fineness.Finally,we predicted the target genes of these 28 miRNAs,among which CHRM2,FGL2,KRT26 and GPR6 may play an important role in the regulation of cashmere fineness.2.Regulation network and expression verification of the key cashmere fineness gene KRT26In this study,miRNAs in LCG skin were firstly identified by RNA SEQ technique,and then the m RNAs,lncrnas and circrnas expressed in LCG and Inner Mongolia cashmere goat(MCG)skin were analyzed.Results: a total of 1222 differentially expressed m RNAs were identified in LCG and MCG skin,including 187 up-regulated and 1035 down regulated m RNAs;170 differentially expressed lncrnas and 32 differentially expressed circrnas were obtained.There were 67 up-regulated and 103 down regulated lncrnas,17 up-regulated and 15 down regulated circrnas.QPCR was used to further verify the key lncrnas,m RNAs,circrnas and miRNAs.In addition,miranda predicted the relationship between the regulatory networks of ce RNA among lncRNAs,circRNAs,miRNAs and m RNAs,and analyzed their potential regulatory roles by go and KEGG.Through the screening and analysis of the results,the ce RNA network regulating cashmere fineness was obtained.lncRNA-MSTRG.706.1,lncRNA-MSTRG.9947.1,lncRNA-MSTRG.5882.1,lncRNA-MSTRG.13983.1,lncRNAMSTRG.10487.1,lncRNA-MSTRG.11754.1,lncRNA-MSTRG.10615.1 and circ6691 and miR-105 a targets KRT26.Lnc RNA MSTRG14109.1 and circRNA452 compete with miRNA-2330 to regulate the expression of TCHH,KRT35 and JUNB.3.Correlation analysis of cashmere fineness function gene KRT26 and cashmere production performanceIn this study,the single nucleotide polymorphism(SNP)analysis of cashmere fineness candidate gene KRT26 was carried out.Seven polymorphic sites were detected in KRT26 gene: A310 T,G317A,G326 A,G410A,G502 A,T672C and C704 G.Six of them were effective loci: A310 T,G317A,G326 A,G410A,G502 A and T672 C.According to the relationship between gene substitution effect and cashmere yield,the correlation between KRT26 gene and cashmere fineness was further analyzed. |
