Cloning, Prokaryotic Expression And Expression In Skin Analysis Of The KAP Gene Family In Tianzhu White Yak

Yak is famous for the body wool and cashmere. Yaks are the only kind of cattle producing wool and cashmere. Tianzhu yak is special breed which has white cashmere, this cashmere is easy dyeing. As the cashmere’s major protein, in the growth process of the cashmere the KAP is very important. This experiment aims to study KAP proteins family for screening major gene controlling wool and cashmere growth to do some groundwork. This study selected four genes KAP1.1, KAP3.1, KAP 11.1 and KAP 13.1 and researchs them. According to the sequence of the cattle four genes reported in Genbank design corresponding primers, PCR method was used the to amplify four genes in Tianzhu white yak, to obtain the complete sequence of CDS region in these genes using TA cloning technology. Bioinformatics was used to analyse them. The genes were cloned to obtain objective fragment into the expression vector and expressed the proteins in E.coli. The Real-time PCR technology was used to detect Tianzhu white yak’s four genes of cashmere growth differential expression between telogen and anagen in skin tissues.These are results:(1) TA clone method were used to to get the KAP1.1, KAP3.1, KAP11.1 and KAP 13.1 genes complete coding sequence (CDS). The length of the coding region of KAP1.1 gene complete 501bp, the encoded a protein of 166 amino acids; it belongs to the hydrophilic protein, and it has High sulphur keratin-associated protein complete family domain, it contains six phosphorylation sites, has the very important biological fuction in mitochondria, it has the four secondary structures of alpha helix, extended strand, Beta turn and random coil. The KAP3.1 gene CDS region is 297bp, it encode 98 amino acids; it belongs to the hydrophilic protein, and it has Keratin, high sulphur matrix protein complete family domain, it contains 5 phosphorylation sites, has the very important biological fuction in nuclear and cytoplasmic, it has the three secondary structures of extended strand, Beta turn and random coil. The length of the coding region of KAP 11.1 gene complete 480bp, the encoded a protein of 159 amino acids; it belongs to the hydrophilic protein, it has Keratin-associated complete family domain, it contains 18 phosphorylation sites, has the very important biological fuction in mitochondria, it has the two secondary structures of extended strand and random coil. The length of the coding region of KAP13.1 gene complete 495bp, the encoded a protein of 164 amino acids; it belongs to the hydrophilic protein, it has Keratin-associated complete family domain, it contains 25 phosphorylation sites, has the very important biological fuction in nuclear, it has the three secondary structures of extended strand, Beta turn and random coil.(2) The formation of prokaryotic expression vector with pET28a plasmid vector and KAP genes family, the genes were succeeded to express in E.coli of prokaryotes. Through the SDS-PAGE analysis results showed that the KAP 1.1, KAP3.1, KAP 11.1 and KAP 13.1 results same as the expected. The Western Blot method was used to validate the results of SDS-PAGE analysis. Through these experiments showed to obtain the KAP protein family, and it was proved that the cloning genes were the target genes, it provided support for the following test sample.(3) Real-time quantitative PCR method was used to analyze the expression level in KAP genes family of Tianzhu White Yak’s skin in anagen and telogen. In the anagen of skin, KAP 1.1 and KAP 13.1 had no significant difference (P>0.05), but the other genes had extremely significant difference (P<0.01). And KAP1.1 gene had the highest expression quantity in anagen of skin, KAP11.1 had the lowest expression quantity. In the telogen of skin, all of the KAP genes family had extremely significant differences (P<0.01). The KAP 13.1 gene had the highest expression level in telogen of skin, but the KAP3.1 gene had the lowest in telogen of skin. By comparing the KAP family between in anagen and telogen, expression quantity result was that KAP3.1 gene in anagen was significantly higher than it in telogen, the other three genes are in anagen than them in telogen. It showed that the KAP3.1 gene played a promoting role in cashmere growth. The KAP 13.1 gene expression had more difference than the other two genes between telogen and anagen, so the KAP 13.1 gene played a more important role in inhibiting growth in Tianzhu white yak’s skin.