FGF10 And Spry4 Gene Cloning And Expression In Skin Of Sheep

In this study, the lateral skin of fetal chainese merino sheep,which on 55、75、85、105 and 135 days of gestation,were used in the research object. Firstly,cloning and eukaryotic expression the CDS of FGF10 and Spry4 gene. And then,use Quantitative Real-time PCR to explore regulation of transcription for the gene of Spry4 and FGF10 above four periods. Then use HE staining method to observe development of the situation in different stages at fetal sheep skin. Finally using immunohistochemical to locate FGF10 and Spry4 protein expression in the process of hair follicle development, thus to explore the role of FGF10 and Spry4 in hair follicle development regulation. Through the above three kinds of research methods, the conclusion is as follows:1.Total RNA of skin from Merino sheep was applied for transcriptional cDNA, it was subsequently used as a template for amplification of Spry4 and FGF10 gene covering its complete coding region. PCR products were cloned into zero PCR@ TM-Blunt vector and sequenced.Then Spry4 gene was subcloned to pcDNA3.0 eukaryotic expression vector, the construct was transfected into 293 T cells and the recombinant Spry4 and FGF10 was identified by western blot. The sequence of the FGF10 and Spry4 nucleic acid and amino acid were analyzed using different software. The results revealed that obtained Spry4 gene of Merino sheep(GenBank Accession number:KP280032). Western Blot showed that the size of the expressed of Spry4 and FGF10 protein is about 36 kDa and 24 kDa, which is consistent with the expected. It was consistent with the expected size of protein.2.In the development of Chinese merino wool bursa FGF10 and Spry4 expression law of five different developmental periods. FGF10 genes from Shen 55 days to 75 days, when they arrive in 85 days express a higher amount of which reached the peak, from 85 days to 85 days to express quantity drops rapidly to express quantity quite with 55 days. Express content increases slightly to 135 days. Spry4 gene expression quantity in 55 days pregnancy table top, then keep falling, until the 85- day dropped to a lower level, rapid rise to 105 days, rose to almost and the expression of 55 days equal quantity. Amount to 135 days of pregnancy expression to a minimum.3.The primary hair follicle primordia was formed at the embryonic age of 75 days. FGF10 and Spry4 Expression in epidermis and the primary hair follicle primordia. 85 days into primary follicles continue to leather direction, surrounded by the secondary follicle form. Where FGF10 expression mainly in epidermis and secondary follicles. And Spry4 in skin layer, primary follicles and secondary follicles are to express. When embryonic development to 105 days of pregnancy, FGF10 staining obviously becomes shallow, and quantitative presented the same trend. Its main expressed in cuticular layer. At the same time, Spry4 has a broader range of expression. Within the epidermis layer, primary and secondary follicles were expressed. Embryonic development to 135 days, the primary follicles and secondary follicles basic development completely, the expression of FGF10 and Spry4 area, within the epidermis and hair follicles had express inner root sheath and outer root sheath. FGF10 and Spry4 express parts are almost the same as the gestation day 135 when at 8 weeks after birth.