| Fibroblast Growth Factor 8(FGF8) is a member of FGFs family, which was purified from a conditioned medium of SC-3 cells stimulated with testosterone. Sequence comparison indicates that FGF8 is highly conserved between humans and mice and is most homologous to FGF18 and FGF17 among the FGF family members. It is a unique secreted signaling molecule in several adult and developing tissues and plays a crucial role in development of some ectodermal organs like midbrain. Furthermore, the FGF8 is also efficient in healing organic wound, culturing the neural stem cells and induction of dopaminergic neurons.In this study, a full length cDNA of grass carp FGF8 was cloned and characterized from total RNA of grass carp brain by RT-PCR and RACE methods, and analyzed by bioinformatics. The finding may be helpful to reveal the pathology mechanism of serious disease in fish and understand physiological mechanism of cells growth and tissue development during embryonic development of Ctenopharyngodon idellus.1. Total RNA was isolated from grass carp brain. The first strand cDNA was synthesized in the catalysis of AMV reverse transcriptase using oligo dT-3 sites adaptor primer as a primer and the total RNA as a template. Then the polymerase chain reaction (PCR) was performed in the catalysis of Taq DNA polymerase using the first strand cDNA as a template and the synthetic oligonucleotides primers as primer (94 degrees C, 60 s; 48 degrees C, 60 s; 72 degrees C, 90 s; 40 cycles). After PCR amplification, the products were cloned into an E. coli expression vector (DH5a). The result showed that the cDNA of grass carp FGF8 is 887 bp, which includes a 5'-untranslated region (5'UTR,100bp), the start codon (ATG), the stop codon (TGA), and 3'-untranslated region (3'UTR,97 bp). The cDNA sequence analysis revealed an open reading frame of 687 bp coding for a protein of 210 amino acids including 22 amino acids of the signal peptide and 188 amino acids of the nature peptide. A polyadenylation signal, AATAAA, was also present 29 base upstream from the poly A tail. Compared with zebrafish and Mexican tetra, the homology of nucleotide sequence for grass carp FGF8 cDNA is 96.7% and 90.0%, respectively, in the coded region, indicating that the experimental result is sure.2. The full-length grass carp FGF8 pro-polypeptide of 210 amino acids including 40 basic amino acids and 16 acidic amino acids. The signal peptide sequence is MRLIPSRLSYLFLHLFAFCYYA, and its cleavage site is located between Ala22 and Gln23. Grass carp FGF8 protein is a secreted protein since the typical character of secreted protein is that its N-domain contains a signal peptide. There are two N- glycosylation in the sites of Asn37 and Asnl36, respectively, which were Asn-Phe-Thr and Asn-Tyr-Thr. The molecular weight (Mw) of grass carp FGF8 protein is 24.68 kDa. Its isoelectnic point (pI) is 10.93. The protein contains four main secondary structures: a-helix,β-sheet,β-turn and random coil.3. Analysis of phylogenetic tree showed that Cyprinidae fishes FGF8 proteins are highly similar. These FGF8 proteins have 210 amino acids and exhibit high homology more than 97.1%. Similarly, the grass carp FGF8 protein also has 210 amino acids and shares 97% homology with zebrafish and Mexican tetra. Furthermore, amino acid sequence of grass carp FGF8 protein also has higher homology with other organism, such as chicken, spotted salamander, silurana tropicalisand axolotl (82.4% 86.1% 82.9% 81.0% amino acid homology, respectively), but there is lowest homology with ciona intestinalis (only 28.2%). All of results suggested that Cyprinidae fishes FGF8 is highly conservated in the evolutional process.
|
PDF Full Text Request