KGF Gene In Antler Of Red Deer: Cloning, Sequence Analysis And Transcriptional Patterns

As a specific mitogen for epithelial cells, Keratinocyte Growth Factor (KGF or FGF-7), the seventh member of Fibroblast Growth Factors (FGFs) family, could induce proliferation, differentiation and motility, and enhance epithelial wound repair. To clarify the possible roles KGF may play in deer antler development and regeneration, for the first time in this thesis, KGF gene (Accession No.AY923858) was cloned from 90-day deer antler of red deer (Cervus elaphus) by two step RT-PCR method. Both KGF DNA and amino acids sequences of human, red deer, mouse, sheep, dog and pig, were used for following bioinformatic analysis and semi-quantitative RT-PCR method was further used to explore spacial transcriptional dynamics of such growth factors as KGF, IGF-I, IGF-2, BMP-2, BMP-4. Results showed:(1) KGF gene of red deer contains 585bp CDS region in length, which codes for a putative 194Aa polypeptide.(2)Bioinformatics analysis showed: the linear fit curve of KGF gene transition and transversion among the six species: y = — 0.44925+0.37278x (R~2 =0.86115);the ME trees that were reconstructed based on KGF gene and polypeptide sequence, induced that compared with other three species, the hereditary distance of red deer, sheep and pig should be close (bootstrap>86%), this result was consistent with the functional domains forecast result of KGF gene secondary structure;For any two species, neither isolectric point or charge at PH 7.0 of KGF was equal, thereinto, isolectric point(9.173) and charge at PH 7.0(10.706) of human was lowest, and the pig was highest, 9.436 and 12.733 separately;gene searching results: based on 2696 motifs in the Prosite data bank, KGF motifs were analysed. KGF included 46 motifs, thereinto, FGF family signatures, signal peptide related signatures, and metabolism related signatures were very important. In addition, transcript regulation related signatures were also involved, such as Leucine zipper pattern, Zinc finger RING-type signature, Zinc finger C2H2 type domain signature, Zinc finger PHD-type signature. It induced that KGF may participate in gene regulation process, however, to date there have not been any evidences to support this viewpoint;the types of KGF gene motifs varied in different species. Comparation of hKGF and dKGF showed that there was a 5'-nucleotidase signature 1 at 105-107aa site of hKGF, not a Casein kinase II phosphorylation site at 159-162aa site of hKGF, however dKGF was controversary, moreover, for both of them tyrosine kinase phosphorylation site had been varied. (3) The semi-quantity RT-PCR was used to study mRNA transcriptional model of KGF, IGF-1, IGF-2, BMP-2, BMP-4 on pestile (A), proximal growth tip (B), intermediant growth tip (C), distal growth tip (D), and middle regions (E-I) of the same antler tissue. The results showed: BMPs had no transcript on each site. The variation of IGF-1 and IGF-2 transcripts level could tend to be consistent on site A-I, however, KGF at site D and site G was different from IGF-1 and IGF-2. There was no IGF-1 transcript at proximal growth site (B). The total transcripts level of IGF-2 was higher than IGF-1.All results and data above could further provide important basement for studying the role of KGF in antler regeneration.