Identification Of The Curly Fleece Related Genes And Their Potential Function Analysis Base On Multi-Omics Data In Chinese Tan Sheep

Tan sheep(Ovis aries)is one of the Chinese indigenous breeds.It is renowned for its production of high quality pelts.The Tan lamb's(at one-month-old)pelt is characterized by natural white color and lustrous curly fleece.However,this unique phenotype disappears gradually with age and the mechanisms behind the phenomenon are still unclear.Using the high-throughput sequencing and integrating analysis of mult-omics data from Tan sheep,we hope to find the key candidate genes related to curly fleece formation.Then,we obtained some preliminary results showed below:1)In this study,we identified differentially expressed genes 67 between two growth stages of Tan sheep through suppressive subtractive hybridization(SSH).Analysis of GO and KEGG showed that 4.08%of DEGs were involved in hair growth in lamb subtractive cDNA library.Finally,we obtained 20 DEGs(such as KRT71 and KRT83)related to curly fleece formation in Tan sheep.2)We obtained the clean reads 371 million by high-throughput BS-seq.The clean reads obtained mapped uniquely to 92.6%of the cytosine residues in the reference genome.In total 1,367 significant DMRs represented 2.7%of annotated genes within the current genome assembly.There were 26 DMRs located within the promoter region of annotated genes,of which 5 genes(KRT71,CD44,TIFA,ITGBL1 and SOX6)related to the curly fleece formation directly and undirectly.3)To further explore the cruly fleece formation in Tan sheep,we identified 49 differentially expressed miRNAs in Tan sheep at both stages with miRNA-Seq(12 known miRNAs participated in the curly fleece growth),of which 28 are more abundant and 21 less abundant in lambs than in adults.And their target genes were predicted,most of them involved in the metabolic MAPK and AMPK signaling pathways.4)The promoter activity assay and EMSA were used to identify the core region of KRT71 promoter,and we found that upstream-439bp of KRT71 promoter was the core region to regulate the differential expression of KRT71 gene at two stages of Tan sheep.And we detected the expression of KRT71 protein was higher in one-month-old Tan sheep than that in 48-month-old.Then,the DNA methylation levels of this promoter region(from-475 to-435 bp)was detected,the results showed that DNA methylation level in 48-month-old Tan sheep was significantly higher than that in one-month-old.We speculated that differential modification of DNA methylation in KRT71 promoter region might be an important reason for the difference of fleece phenotypes at two stages of Tan sheep.5)KRT83 gene is another important gene that we obtained by integrating analysis to influence the curly fleece formation in Tan sheep.Using cell transfection experiment and EMSA,we found that transcription factor CAP1 was an important regulator for the different expression of KRT83 gene at two stages of Tan sheep.In addition,the result of miRNA interference showed that oar-miR-432 was another major regulator for KRT83 protein expression.Based on the above results,we concluded that the effect of the KRT83 gene on curly fleece formation is accomplished by pre-transcriptional and post-transcriptional regulation with CAP1 and oar-miR-432.In this study,we used multi-omics technology and analysis of candidate genes to elucidate the reasons related to curly fleece formation of Tan sheep.Our results provid new ideas and ways for further reveal the molecular mechanism of curly fleece formation in Tan sheep.