| Object:Sheep raising is an important part of China's agriculture,Sheep can not only provide meat products,but also provide high-quality fine wool raw materials for wool textile industry to solve the shortage of high-quality wool supply.Chinese merino is fine wool varieties with the best quality in China.Wool develop from hair follicles,and the development of secondary hair follicles determined the economic value of wool.So it's very important to illuminate the mechanism of wool follicles.With the appearance and development of new generation sequencing technology,people have done many research on fine wool sheep and cashmere goats.Many reports have studied the involvement of these microRNAs in the regulation of hair follicle development,In particular,the related reports on microRNAs at different stages of hair follicle development in sheep provide a good reference for this study.The development of follicles begin from fetal period,In this study,the key time nodes of hair follicle development were obtained from the histomorphological observation of skin follicles in different branching stages of fetal period.The differences of microRNAs expression in different stages of hair follicle development were further analyzed,and the microRNAs that can actually regulate the major target genes were excavated.The results were deeply and carefully studied through molecular biology experiments.It laid a theoretical foundation for revealing the growth and development of secondary hair follicles and wool,and provided a reference for molecular breeding of fine wool sheep.At the same time,it provides ideas for controlling the periodic growth of hair follicles and improving wool yield and quality.Methods:(1)Choosing thirty-three Chinese merino sheep(Xinjiang military reclamation type)with gestational age of 45 days,55 days,65 days,75 days,85 days,95 days,105 days,115 days,125 days,135 days and 145 days,and picking three from each developmental stage to collect lateral skin tissue,one piece for microRNAomic analysis,and the other one was preserved in 4% polyformaldehyde to make paraffin sections of fetal skin hair follicle for histomorphological observation.(2)The Illumina HiSeq2500 System sequencing platform was used to sequence microRNAs,and analyze differentially expressed microRNAs at different developmental stages of skin hair follicle,predict known microRNAs,conserved microRNAs and new microRNAs;bioinformatics was used to predict target genes,GO enrichment,KEGG pathway analysis;and screening key microRNAs at important stages of hair follicle development.According to the results of microRNAomics analysis and the morphological characteristics of hair follicle development.(3)18 differentially expressed microRNAs were validated by qPCR.(4)Constructed recombinant plasmids containing microRNA and target gene Psicheck-2 Luciferase double luciferase reporter gene,and transfected into 293 T cells respectively.The target gene of microRNA was confirmed by luciferase activity.(5)Culture primary sheep fibroblasts,and transfect miRNA mimics with NC,the effect of protein and mRNA expression level of target gene of microRNA was identified by Western blot and QPCR.(6)PCR and DNA sequencing techniques were used to detect the genetic polymorphisms of 260 individuals in the Chinese merino sheep and multiple suffolk at the miRNA-377 locus.Results:(1)By observing the follicular tissue sections of 11 developing fetuses,it was found that the epidermis,dermis and subcutaneous connective tissue gradually formed from 45 days of gestational age.By 55 days of gestational age,the skin structure had developed completely;at the 65 d,the primary hair follicles began to form vesicles and gradually increased.By 75 days the primary hair follicles had formed;and the secondary hair follicles began on 85 days of gestational age.The number of primary hair follicles increased rapidly.At 105 days of gestational age,the primary secondary follicles began to differentiate into secondary follicles.(2)The densities of primary and secondary hair follicles of 85-145 d were calculated.The results showed that the density of secondary hair follicles increased with gestational age as the gestational age increased.Calculating the 85-145 d S/P value in the fetal period gradually increases with the increase of gestational age.(3)By Illumina high-throughput sequencing,179 new microRNAs and 152 known microRNAs were detected in 11 stages of fetal development in sheep,of which 37 differentially expressed microRNAs were associated with secondary follicle morphogenesis.(4)Hierarchical cluster analysis was carried out by using MEV software v4.6.The differentially expressed microRNAs in 11 hair follicle development stages were clustered into 2 categories and 4 subcategories.The two categories included primary and secondary hair follicle development critical stages of 45-95 days in fetal period,and hair follicle growth acceleration stages of 105-145 days.The four subgroups included hair follicle developmental stages of 45,55 and 65 days,75,85 and 95 days,105,115 and 125 days,135 and 145 days.Cluster analysis results were consistent with the key period of hair follicle(HF)growth and development.(5)There were 37 differentially expressed microRNAs,including 19 up-regulated microRNAs and 18 down-regulated microRNAs,during secondary follicular development from 75 to 85 days.Novel-126 was significantly down-regulated and microRNA-196 b was significantly up-regulated at the critical stage of secondary follicular development.(6)GO and Pathway analysis showed that PRL signaling pathway and platelet activation pathway played an important role in the formation of secondary hair follicles.Oar-mir-133,oar-mir-376c-3p,mir-196b*,mir-2285 l,mir-504,mir-873,Novel-8*,Novel-17,Novel-20 and Novel-64 genes are candidate genes for secondary hair follicle development.(7)Double luciferase reporting system was used to detect the regulatory effect of microRNA767 on target genes.The results showed that co-transfection of mimic microRNA767 and wild-type plasmid DDX5 significantly inhibited luciferase activity in 293 T cells(P < 0.05).mir-767 could regulate the expression of DDX5 gene.The co-transfection of oar-mir-377 mimic and wild-type plasmid SLC224A2 significantly inhibited the activity of luciferase in 293 T cells(P < 0.05),suggesting that oar-mir-377 could regulate SLC24A2.Gene expression.(8)Blank control(NC)and microRNA mimics were transfected into sheep skin fibroblasts,respectively.Western Blot was used to detect the regulation of microRNA-767 and oar-microRNA-377 on the protein levels of their target genes DDX5 and SLC24A2;QPCR was used to detect the regulation of microRNA-767 and oarmicroRNA-377 on the mRNA levels of their target genes DDX5 and SLC24A2.The results showed that microRNA-767 significantly reduced the protein and mRNA expression of DDX5 gene;oarmicroRNA-377 significantly decreased the protein and mRNA expression of SLC24A2 gene.(9)It was confirmed by PCR sequencing that the T?C base mutation was detected at 276 bp upstream of the oar-miR-377 core sequence,and distributed in Chinese Merino sheep(Xinjiang Junken type),and multiple-child Suffolk varieties.The results of linear model analysis showed that there was a significant correlation between the genotype effect and the fineness dispersion coefficient(P<0.05),and the variety effect was significantly correlated with the fiber diameter,fineness standard deviation and fineness dispersion coefficient(P<0.01).Correlation analysis showed that the wool fiber diameter of CC genotype individuals was large,and the fineness dispersion coefficient was significantly larger than that of TT genotype individuals(P<0.01).In conclusion,we get these results in this study: 85 days is the key period of secondary hair follicle development,PRL signaling pathway and platelet activation pathway play important roles in secondary hair follicle formation.The genes :oar-miR-133?oar-miR-376c-3p?miR-196b*? miR-2285 l?miR-504?miR-873?Novel-8*?Novel-17?Novel-20?Novel-6 are key genes in the secondary hair follicle development,among them,microRNA-767 promotes secondary hair follicle development by down-regulating DDX5 gene expression.Oar-mir-377 promotes secondary hair follicle development by down-regulating the expression of SLC24A2 gene.The CC genotype of oar-miR-377 has a large wool fiber diameter,therefore,TT and TC genotype individuals are selected in fine wool sheep breeding. |
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