Effects Of Androgen On KAP11.1 And KAP24.1 Gene Expression In Hetian Sheep And Karakul Sheep

【Objective】The purpose of this study was to clone and express KAP11.1 and KAP24.1 gene of Hetian sheep and Karakul sheep,to explore the effect of different androgen concentrations on protein expression,and to compare the expression of the two genes in sheep skin hair follicles,so as to provide theoretical basis for further study of KAP gene function and its effect on wool quality.【Method】Three kinds of sheep lateral skin follicles were used as research materials,and the primers were designed according to the sheep KAP11.1 gene and KAP24.1 gene in Gen Bank.（1）The expression of KAP11.1 and KAP24.1 genes in different sheep skin follicles was detected by q PCR using 18S r RNA as housekeeping gene;（2）The KAP11.1 gene and KAP24.1 gene were amplified by PCR.The cloning plasmids p MD19-T-KAP11.1 and p MD19-T-KAP24.1 were constructed and identified.The digested gene fragment was combined with p ET-28a vector to form prokaryotic expression vector and identified again.The vector was imported into BL21 E.coli to obtain the target fragment by SDS-PAGE.The protein was verified by Western Blot.The KAP11.1 gene and KAP24.1 gene sequences were analyzed;（3）The KAP11.1 and KAP24.1 genes were amplified by PCR with the primers designed by removing the termination codon.The PCR products were linked to p MD19-T vector and identified.The target fragments were recovered and ligated with p EGFP-N1 vector again.The eukaryotic expression vectors of KAP11.1 and KAP24.1 genes were constructed and transfected into Hela cells.SDS-PAGE and Western Blot were used to detect the expression of KAP11.1 and KAP24.1 genes.The transfected cells were treated with different concentrations of androgen to explore the changes of protein expression.【Results】（1）The expression of KAP11.1 gene in skin follicles of mountain-type Hetian sheep（HS）and Karakul sheep（KL）was significantly higher than that of plain-type Hetian sheep（HP）（P<0.01）.There was no significant difference between mountain-type Hetian sheep（HS）and Karakul Sheep（KL）（P>0.05）.KAP24.1 gene expression in mountain-type Hetian sheep（HS）was significantly different from that in plain-type Hetian sheep（HP）（P<0.01）,and was significantly different from that in Karakul Sheep（KL）（P<0.05）.The expression of Karakul Sheep（KL）was significantly higher than that of plain-type Hetian sheep（HP）（P<0.05）;（2）The CDS sequence of KAP11.1 gene of three sheep was 480 bp,encoding 159 amino acids.The CDS sequence of KAP24.1 gene was 759 bp,encoding 252 amino acids.Both KAP11.1 and KAP24.1 were unstable proteins,and the secondary structure was mainly composed of random coils.The KAP11.1 and KAP24.1 prokaryotic expression proteins with molecular weight of 19 KDa and 30 KDa were obtained;（3）Eukaryotic expression protein KAP11.1 with molecular weight of 47 KDa and KAP24.1 with molecular weight of 58 KDa were obtained.When androgen concentration was 10^-8mol/L,the expression levels of KAP11.1 in three sheep were significantly different from those in control（P<0.01）,KAP24.1 there was significant difference between Hetian sheep and the control（P<0.01）,and Karakul Sheep and the control（P<0.001）.【Conclusion】（1）The expression levels of KAP11.1 and KAP24.1 genes were the highest in mountain-type Hetian sheep,followed by Karakul sheep,and the lowest in plain-type Hetian sheep.（2）Prokaryotic expression proteins KAP11.1 and KAP24.1 were successfully obtained,which were unstable proteins.（3）The eukaryotic expression proteins KAP11.1 and KAP24.1 were successfully obtained.When the androgen concentration was 10^-8mol/L,the protein expression reached the highest level.When the concentration was too high or too low,the protein expression decreased.