The Study Of Sheep Wool Follicle Cortex Gene KAP13-1 Regulating Hair Shaft Assembly

Sheep wool shaft mainly consisiting of Keratins and Keratin associated proteins can be divided into cuticle,cortex and medulla.The cortex,as the main structure of the wool shaft,plays an important role in physical features of wool shaft,where most of the kertains and kertain associated proteins are expressed.Thus,the proteins in the cortex control the assembly and traits of wool shaft.However,the functions large amount of sheep cortex proteins exert are still mysterious.Our previous study found that sheep KAP13-1(.sKAP13-1)was highly expressed in the wool follicle,suggesting the potentially essential role it played.As a result,transgenic mouse overexpressing sKAP13-1 in cortex was constructed and protein interaction was carried out to further investigate the functions sKAP13-1 had in the wool shaft formation and determination of the physical features.Here are the results:1.Generation and identification of transgenic mosue overexpressing sKAP13-1 specifically in hair cortex(1)The activity of sKAP13-1 was detected in wool shaft cortex with the sheep KAP13-1 antibody synthesized by ourselves.Previously sKAP11-1 promoter sequence has been demonstrated to be able to drive exogenous gene to specifically express in hair shaft cortex.In our study,sheep KAP11-1 promoter sequence was utilized to gerenate transgenic mouse overexpressing sheep KAP13-1 and GFP specifically in hair cortex,which was named as pKAPll-1-sKAP13-1-2A-ZSG-Plus,(Plus mouse).It was showed that the activity of sKAP13-1 and GFP was strictly confined to the hair cortex of Plus mouse.The expression of sKAP13-1 and GFP driven by sKAP11-1 promoter sequence depended on the progress of hair follicle growth cycle,with strong singal in anagen and no activity in both catagen and telogen.The results indicate that we have successfully constructed transgenic mouse with the overexpression of sKAP13-1 in hair shaft cortex,which can be applied to the further research.(2)It was revealed that Plus mouse entered the regular hair follicle cycle and also normally owned four types of pelage hair shaft,including Guard,Awl,Zigzag and Auchene.Therefore,sKAP13-1 has no influence in regulating hair follicle growth cycle and is unable to alter the types of hair shaft.Subsequently,the pelage hair shaft was collected from Plus mouse,pKAPll-1-ZSG mouse overexpressing GFP in hair cortex and wild type mouse respectively,among which the diameter of Guard hair shaft was measured and statistically analyzed.It revealed that great difference was found between male and female mouse in same type of mouse.The diameter of Guard hair shaft from male mouse is bigger than that from female mouse.Furthermore,Guard hair shafts from male and female Plus mouse are thinner than counterpart of pKAP11-1-ZSG and wild type mouse,respectively.Therefore,the data indicate that sKAP13-1 can improve the finess of the hair shaft.2.Identifiying Keratins interacting with sKAP13-1(1)The sequence analysis of sKAP 13-1 revealed two fundamental pentapeptide quasi-repeats of the form A,C-C-X-P-X and B,C-C-X-S/T-S/T,which showed high homology among different speicies.Large amount of random coil stuructures were predicated in the 3-Dimension structure of KAP13-1.As a result,KAP13-1 was likely to interact with keratins through disulphide bonds or hydrogen bonding.(2)GST-Pull down technique was established by using positive interacting proteins,human KAP2-1 and human K86.Subsequently,sheep K33,K34,K39 and K86 were selected as candidates interacting with sKAP13-1 according to the expression pattern in sheep wool follicle.The results of GST-Pull down showed that there were no interactions between sKAP13-1 and candidate proteins,sheep K33,K34,K39 and K86.Thus,sKAP13-1 might form complexes with other sheep keratins to fullfil its function.Consequently,our study elucidated that sKAP13-1 was expressed in cortex of sheep wool follicle.The transgenic mouse specifically overexpressing sheep KAP13-1 in hair follicle cortex was generated successfully.Although no influence of sKAP13-1 was found in the types of hair shaft and hair follicle cycle,it could improve the fineness of the wool shaft significantly,which might rely on the interaction between sKAP13-1 and other sheep keratins,apart from sK33,sK34,sK39 and sK86 through forming disulfide bond or hydrogen bond.