Epigenetics Preliminary Study Of Cashmere Goat Hair Follicle Development Based On Methylation And LncRNA

Cashmere is the productof secondary hair follicle growing in the cortex appearance of goat.Cashmere has important economic value in livestock production and has the reputation of "diamond fiber" and "soft gold" due to the light, soft, warm characteristics of its fiber. The development of skin hair follicle directly affects the yield and quality of cashmere, so it will be of the great significance to improve the yield and quality of cashmere(fineness and length)and it is also the very important content in current cashmere goat breeding to study the regulatory mechanism of hair follicle development.The growth of cashmere has obvious cyclical changes and a variety of regulatory factors involved in this process including coding RNA and non-coding RNA. Long non-coding RNAs(lncRNAs) are a series of RNA molecules that do not encode proteins and have transcripts longer than 200 nt in length. LncRNAs play important roles in many biological processes. Previous study on skin tissue of cashmere goat via RNA-seq at anagen and telogen stages showed that lnc15479 differentially expressed between anagen and telogen and it was associated with hair follicle development genes, such as Hox, KAPs, Wnt2, FGF5 and BMPs and other related genes. In order to elucidate the mechanism of cashmere growth, the methylation level of Hoxc13 and other related genes Msx-2 and FGF5 was detected, then the relative expression of Hoxc13 and DNMTs(DNMT1, DNMT3 a, DNMT3b) genes was performed by quantitative real-time PCR(qRT-PCR) at anagen and telogen. In order to study the function of lnc15479 during hair follicle development, the targeted knockout system of lnc15479 on cashmere goat was constructed by CRISPR/Cas9 technology, which would provide technical support for the further study on lnc15479.The following results were obtained:(1) The methylation level of Hoxc13 gene showed that a CpG site in its promoter region was significantly higher in telogen than that in anagen(P<0.05), and the methylation level in promoter region of Msx-2 gene and exon 1 of FGF5 gene had no significant difference in different cashmere growth stages.(2) The qRT-PCR result showed that the relative expression of Hoxc13 was significantly higher in anagen compared with that in telogen(P<0.01). And the relative expression ofDNMTs in telogen was higher compared with that in anagen(P<0.01). But at the same stage,their expression had no difference. The results showed that the expression of DNMTs and the methylation level of Hoxc13 gene had similar trend at different stages. Therefore, the study would provide the evidence that DNMTs involve in methylation statement of Hoxc13 gene during hair periodic growth.(3)Two sgRNA target sites in the upstream and downstream of lnc15479 were designed and the CRISPR/Cas9 expression vector was constructed. The double fluorescent reporter vectors based on SSA repair mechanisms was then constructed to detect the cleavage efficiency of CRISPR/Cas9. The expression and report vectors were then co-transfected HEK293 T cells to detect the work efficiency of CRISPR/Cas9. The results showed that CRISPR/Cas9 vector was successfully constructed and its cleavage efficiency was about20%~30% from the red and green fluorescence by counting cells. The results would provide technical support for the further study on lnc15479.