| Tibetan cashmere goat is a famous cashmere goat breed in China.The cashmere fibre diameter is finer among domestic cashmere goat breeds,which is a kind of natural animal fiber with higher economic value than other animal fibers.However,in recent years,with the transformation of domestic animal husbandry feeding methods and the impact of the mutton price have caused slack for the grassroots breeding work,resulting in the tendency of the cashmere fibre diameter to become coarser,which has seriously affected the cashmere quality and causes certain economic losses to farmers and herdsmen.At present,there are few studies on Tibetan cashmere goats cashmere fiber diameter trait,and its regulatory mechanism is still unclear.Therefore,it is particularly important to carry out the research on the fiber diameter trait of Tibetan cashmere goats.So from the perspective of genetics,which factors play an important regulatory role in the formation of the cashmere fiber diameter trait for Tibetan cashmere goats? In view of this,this study uses Tibetan cashmere goats with different cashmere fiber diameters as experimental materials,and usesRNA-seq,Label-free,molecular biology and bioinformatics methods to conduct study on cashmere fiber diameter trait,and selecting the key regulatory factors related to cashmere fiber diameter trait,which will provide data support for elucidating the molecular regulation mechanism of the cashmere fiber diameter traits and the cultivation of fine-type new strain of Tibetan cashmere goats.1.Selection of transcription factors related to cashmere fiber diameter trait based on DE lncRNA and DE mRNATranscriptome sequencing technology was used to analyze the skin tissues of the scapula of 4 fine cashmere groups(F)and 4 coarse cashmere groups(C).A total of 470 lncRNAs and 29119 mRNAs were detected.80 DE lncRNAs and 384 DE mRNAs were selected.The predicted target genes of DE lncRNAs were analyzed by GO,KEGG and lncRNA-target gene interaction network,and it was found that the 8 predicted target genes of lncRNA ENSCHIT00000009853 and MSTRG.16794.17 were related to the cashmere fiber diameter.GO and KEGG analysis of DEGs revealed that FOSB,CXCL10,COL4A3 BP,CXCL9,FOS,KRT85,KRT33 A,CXCL11,NOTCH2,NOTCH3 are involved in the differentiation and development of skin and hair follicles.Building an interactive network for DEGs found that PMSD3,PMSD11,PMSD14,and SRC are at the core nodes.Randomly select 6 DE lncRNAs and 6 DE mRNAs for q RT-PCR verification,and the results are consistent with the trend ofRNA-seq data,indicating that the lncRNA and mRNA sequencing results are credible.2.Selection of transcription factors related to cashmere fiber diameter traits based on DE miRNAUsing transcriptome technology,miRNA sequencing was performed on the skin tissues of 4 F-group and 4 C-group in Tibetan cashmere goats.A total of 545 miRNAs were detected,including 426 known miRNAs and 119 unknown miRNAs.GO analysis of the 12 DE miRNAs predicted target genes revealed that the target genes were significantly enriched in biological processes such as multicellular biological development,animal organ development,and axon development(p <0.05).KEGG analysis showed that target genes were significantly enriched in the B cell receptor signaling pathway,NOTCH signaling pathway,T cell receptor signaling pathway and other signal pathways related to hair follicle development(p <0.05).By constructing a DE miRNA prediction target gene-KEGG network,it was found that 11 prediction target genes of 8 DE miRNAs were annotated to 5 signal pathways related to skin formation and hair follicle development,including NOTCH,MAPK,PI3K-Akt,WNT,and TGF-?,and so on.Five miRNAs were randomly selected for q RT-PCR verification,and the results were consistent with the trend ofRNA-seq data,indicating that the miRNA sequencing results were credible.3.Selection of cashmere fiber diameter trait-related proteins based on Label-free proteomics technologyUsing label-free proteomics technology to analyze the skin tissues of 3 F-group and 3 C-group in Tibetan cashmere goats,34327 unique peptides and 7162 proteins were detected.Among the 29 DEPs identified,20 known proteins and 9 unknown proteins were identified.Compared with group C,5 proteins were up-regulated in Fgroup and 24 were down-regulated.GO analysis of DEPs found that DEPs were mainly enriched in GO items such as the regulation of biological processes,the organization of extracellular matrix,the organization of extracellular structure,the process of alcohol metabolism,and the process of cellular carbohydrate metabolism;KEGG analysis found that DEPs were mainly enriched in lysozyme Body,ECM-receptor interaction,PI3K-Akt signaling pathway,adhesion spot signaling pathway.Through protein interaction network analysis,it is found that 4 proteins are at the core nodes of the interaction network,including GC,VTN,APOH and CPB2.It is speculated that these4 proteins play a certain regulatory role on cashmere fiber diameter trait.Three DEPs were randomly selected for protein expression verification by Western blotting technology.The results showed that the expression levels of VTN,GLB1 and AEBP1 in the skin tissues of C-group were higher than those of F-group,which was consistent with the trend of Label-free data,indicating that the protein group sequencing data was reliable.4.Conjoint analysis of transcriptome and proteome data and functional verification of chi-mi R-105aBased on the analysis of the co-expression network of lncRNA-DEG,it is found that lncRNA MSTRG.17532.2 is highly correlated with the expression levels of NOTCH2 and NOTCH3 genes.Based on the LncRNA-DEG-miRNA interaction network analysis,it is found that 2 DE lncRNAs,4 DE miRNAs and 11 DEGs are constructed in the network;among them,chi-mi R-105 a has a negative regulatory relationship with the predicted target genes ETV6 and PIP5K1B;chi-mi R-767 and lncRNA ENSCHIT00000009853 have a common target gene SELE.Based on the DEG-DEP interaction network analysis,it is found that 13 DEPs and 49 DEGs are constructed in the network.15 DEGs and DEPs have a direct interaction relationship.Among them,CALD1,GLB1 and IMPA1 proteins have more direct interaction genes.Speculating these three proteins play an important regulatory role in the interaction network.Through the dual luciferase reporter system,overexpression and interference methods,it was found that chi-mi R-105 a can negatively regulate the expression of the target gene ETV6 in the Dermal papilla cells of cashmere goats.It indicates that chimi R-105 a may be a potential regulator of the transcription factor ETV6 gene,which play an important role in the formation of cashmere fiber diameter trait in Tibetan cashmere goats.In summary,Based on the combined analysis of transcriptome and proteomics,this study selected 14 candidate genes(FOSB,CXCL10,COL4A3 BP,CXCL9,FOS,KRT85,KRT33 A,CXCL11,NOTCH2,NOTCH3,PMSD3,PMSD11,PMSD14,SRC)and 4 important genes Of non-codingRNAs(lncRNA ENSCHIT00000009853,MSTRG.16794.17,MSTRG.17532.2,chi-mi R-105a)and 7 key proteins(GC,VTN,APOH,CPB2,CALD1,GLB1,IMPA1)and the biology process of cashmere fiber diameter trait is closely related.It was verified that chi-mi R-105 a can negatively regulate the ETV6 gene on the Dermal papilla cells of cashmere goat hair.It is speculated that chi-mi R-105 a can be used as a potential regulator of the transcription factor ETV6 gene.The above research results will lay the foundation for elucidating the regulation mechanism of Tibetan cashmere goat cashmere fiber diameter trait formation,and also provide important regulatory factors for the breeding of fine-type new strain of Tibetan cashmere goats and molecular marker-assisted selection. |
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