Construction Of Skin Cell-specific Expression Vector And Function Analysis Of KROX20 Gene In Tibetan Sheep

This study analyzed the coding region sequence of KROX20,a specific expression gene of Tibetan sheep skin,and its effect on the expression of intracellular signaling molecules,which laid the foundation forfurther studying the effect of KROX20 gene on the development of hair follicle development and hair growth in Tibetan sheep.Firstly,the experiment collected Tibetan sheep skin tissue,cloned the coding region sequence of KROX20 gene from the skin tissue,and analyzed the nucleotide and amino acid sequence of KROX20 gene using bioinformatics.Then,the recombinant skin cell-specific expression vector of KROX20 gene was constructed by Transfer-PCR?TPCR?method,and the constructed vector was used to transfect Dermal Papilla Cell?DPC?and the cell transfection conditions were explored and optimized.Finally,real-time quantitative PCR and Western blot technology were used to detect the expression of KROX20 gene and its downstream target gene IGFBP5 at the m RNA and protein levels.The main results obtained in this study are as follows:1.The KROX20 gene coding region sequence of Tibetan sheep was cloned,with a length of 1458 bp.Compared with the predicted sequence of the sheep KROX20gene?XM?02796224.1?,the sequence of this gene is 39 bp long in the CDS region?long at the beginning of the CDS?.The similarity of the matched sequence is 99.51%,and there are 7 base differences?excluding 39 bases?.The base,A,C,G and T of Tibetan sheep KROX20 gene sequence accounts for 18.90%,37.70%,26.30%and17.10%,respectively.The amino acid sequence deduced from the sequencing sequence of the Tibetan sheep KROX20 gene encodes 486 amino acids with a theoretical isoelectric point of 9.13 and a relative molecular weight of 51.37 k D.The sequence similarity to the sheep amino acid sequence?XP?027818049.1?is 99.85%,and there are 2 amino acid differences?excluding the 13-amino acid multi-code?.There are three C₂H₂zinc finger domains in the KROX20 amino acid sequence of Tibetan sheep,and their positions are at 350-374,380-401,and 408-429 of the protein,respectively.Similar zinc fingers exist in other animals,but the sequence homology is not high.2.Transfer-PCR?TPCR?method successfully constructed the recombinant skin cell-specific expression vector p CDs RED-KROX20 of KROX20 gene.The recombinant eukaryotic expression vector was transiently transfected into sheep dermal papilla cells?DPC?cultured in vitro,the transfection efficiency was about30.5%,the optimal transfection time was 48 h,and the optimal ratio of recombinant eukaryotic expression vector to liposome was listed as 1:3.3.Real-time quantitative PCR detection showed that indermal papilla cells?DPC?transfected with recombinant eukaryotic p CDs RED-KROX20,the KROX20gene was overexpressed by about 45 times,while the m RNA level of the target gene IGFBP5 downstream of the KROX20 gene was increased by about 7.5 times.Furthermore,Western blot was used to detect the expression of KROX20 and IGFBP5proteins.The expression levels of the two proteins were significantly increased.These results indicate that KROX20 gene may affect skin hair follicle development and hair growth through the IGF signaling pathway.