| The quality of wool is mainly determined by the characteristics of wool fibers.Wool fibers are composed of cuticle,cortex and medulla.The gene expressed in the cortex has an important influence on the properties of wool fiber.KAP13-1 was expressed in the wool follicle,and may determine the fineness of wool fiber.In the previous study of our lab,KAP11-1 promoter who specific expressed in the cortex of the wool cortex were constructed,and sheep KAP11-1 promoter sequence was utilized to gerenate transgenic mouse overexpressing sheep KAP13-1 and ZSG specifically in hair cortex,which was named as pKAP11-1-sKAP13-1-2A-ZSG-Plus,(Plus mouse).Hair fiber fineness in Plus mouse is thinner than that in wild type mouse.On the basis of this,we bred Plus mouse and generated three generations(G2-G4 generation).First,the stability of the genetic and expression of exogenous genes in Plus mouse was analyzed,and the hair follicle phenotype of Plus mouse was detected.Finally,bioinformatics prediction,EMSA,immunofluorescence and other techniques were used to identify transcription factors that regulate the KAP11-1 promoter,and SNPs were screened for the identified transcription factor NFAT5 in different sheep populations.The main results as follows: 1.The stability of exogenous genes expression in Plus mouse(1)Exogenous gene inserted in the intergenic region between the mouse Ermard gene and the delta-like protein 1 precursor gene on Chr17 and the intergenic region between Mbnl3 gene and Hs6st2 on ChrX.The insertion site was examined in 9 individuals of G2,G3,G4 Plus mouse and it was found that the insertion site of Plus mouse did not change when breeding.(2)The copy number of the exogenous gene in the genome were examined in these G2,G3 and G4 Plus mouse.There were about 6 copys in G2 generation,about 4 copys in G3 generation,about 2 copys in G4 generation.The copy number of the exogenous gene was reduced when breeding.(3)The expression levels of exogenous genes in these G2,G3 and G4 Plus mouse indicated that the expression level of the exogenous genes is positively correlated with copy number.And the green fluorescence in the hair of Plus mouse is visible.2.Measurement and analysis of Plus mouse hair follicle phenotypeThe following hair follicle phenotypes were determined:(1)primary hair follicle diameter,primary hair follicle DP area and primary hair follicle hair shaft diameter;(2)secondary hair follicle diameter and secondary hair follicle hair shaft diameter;(3)hair follicle density;(4)hair follicle medulla air space density.The primary hair follicle dermal papilla volume and hair follicle medulla air space density were not significantly different between Plus mouse and wild type mouse,but(1)the primary hair follicle diameter of wild type mouse was significantly larger than that of female Plus mouse(P=0.0377);(2)the secondary hair follicle diameter of wild-type mouse was significantly larger than that of Plus mouse(male: P=0.0139,female: P=0.0109);(3)the hair follicles density of female wild-type mouse was significantly lower than that of Plus mouse(P = 0.0078).Statistical analysis of the hair follicle phenotype demonstrate that KAP13-1 can change the hair follicle phenotype,indicating that KAP13-1 may influence the development of hair follicles.3.Research on transcription factors for KAP11-1 promoter regulationIt is predicted that the transcription factors NFAT5 anf KLF4 may bind to the-251 ~-148 bp region of KAP11-1 upstream,EMSA results indicated that NFAT5 bind on the-251 ~-148 bp region of KAP11-1 upstream,but KLF4 not bind to the region.The immunofluorescence results of NFAT5 indicated that NFAT5 was expressed in the cortex of Plus mouse.It was strongly expressed in hair follicles during the growth phase(P9 days,P15 days),but almost not expressed during rest period(P18)and regression(P21),which was consistent with the expression pattern of sheep KAP11.1 in hair follicles and the expression pattern of sKAP13-1 in Plus mouse.The results indicate that NFAT5 may play a role in the regulation of KAP11-1 expression.4.SNP polymorphism detection of transcription factor NFAT5 in different sheep populationsThrough the database,we predicted 30 SNPs in the sheep NFAT5 gene that can change amino acids,we determined 24 non-synonymous mutant SNPs on the NFAT5 gene in Chinese Merino(Xinjiang),German Merino and Tibetan Sheep population.Only three SNPs were detected variation in this three populations.The chi-square independence test found that the allele frequencies of the three SNP loci were not significantly in this three populations. |
