| The growth and development and periodic changes of hair follicles were complex molecular regulation processes that were regulated by a variety of factors.Lnc RNA and mi RNA are newly discovered non-coding RNA.They are not translated into proteins but regulate various cellular and biological processes.The interaction between mi RNA,lnc RNA and m RNA constitutes a complex regulatory relationship.More and more studies had reported the role of mi RNA and lnc RNA in the development and periodic growth of hair follicles,but there were few studies on the regulatory network of hair follicle growth and development.Therefore,this experiment used bioinformatics methods to analyze the regulatory relationship between lnc RNA,mi RNA,and m RNA in the development of Subo merino wool sac by mining the transcriptome data of Subo merino sheep skin tissue samples.Construct the lnc RNA-mi RNA-m RNA molecular regulatory network,further analyzed the function of the target genes of the regulatory network,screened and find the key genes in the nc RNA regulatory network related to hair follicle development and verify it.(1)Construct the lnc RNA-mi RNA-m RNA regulatory network related to the development of Submerino wool follicles at different stages,and conduct functional analysis of the target genes in the lnc RNA-mi RNA-m RNA regulatory network,screen the key genes in the regulatory network,found that the target genes TGFB2 and NOTCH1 are involved in the biological process of hair follicle morphogenesis(GO:0031069).(2)Fluorescence quantitative results showed that the expression trends of mi R-133,mi R-433-3p,and NOTCH1 were opposite.The expression trend of TCONS＿00008348 is opposite to that of mi R-154b-3p,mi R-485-3p,and mi R-539-3p.The expression trends of mi R-154b-3p,mi R-485-3p,and mi R-539-3p were opposite to the target genes MAPKAPK3,DUSP1,and PTPRR.TCONS＿00008348was consistent with the expression trend of MAPKAPK3,DUSP1,and PTPRR.Therefore,it could be concluded that TCONS＿00008348 had a targeting relationship with mi R-154b-3p,mi R-485-3p,mi R-539-3p,and the target genes MAPKAPK3,DUSP1,PTPRR.(3)The dual luciferase reporter system showed that the firefly luciferase activity value of NOTCH1-WT+mi R-133 mimics was significantly lower than that of NOTCH1-WT+NC mimics group.Compared with NOTCH1-WT+mi R-433-3p mimics and NOTCH1-WT+NC mimics group,the value of firefly luciferase activity also decreased significantly.This indicates that oar-mi R-133 and oar-mi R-433-3p can target the 3’UTR of the NOTCH1.(4)Bioinformatics analysis of the nucleotide and amino acid sequences of the NOTCH1 gene revealed that the sheep NOTCH1 gene is located on chromosome 3,and its CDS region is 9487 bp,which encodes 2541 amino acids.p I=4.95,which is a neutral protein.The instability coefficient is 52.17,and the protein is unstable.The sheep NOTCH1 protein contains a signal peptide,and its cleavage site probability between 21 and 22 is 0.5269.The number of transmembrane helix is 1,which is a transmembrane protein.It contains 8 O-glycosylation sites,9 N-glycosylation sites and 37 phosphorylation sites.Its secondary structure is composed of 70.64% random coils,12.71% alpha helices,and 16.65% extended chains.In summary,this study established a non-coding RNA molecular regulatory network related to the development of Subo merino wool follicles,closely linked mi RNA and lnc RNA with the growth and development of hair follicles,and discussed the growth and development of hair follicles from the transcriptomics level.By searching for genes related to the regulation of wool growth and development,the molecular mechanism of subo merino wool sac development was systematically explored. |
