Cloning Of Tumor Suppressor Gene P53 And Study Of The Relation Between The P53 Gene And Lymphocystis Disease In Cultured Japanese Flounder (Paralichthys Olivaceus)

p53 gene is an important kind of tumor suppressor genes, which is lost or mutated in about 50% of human tumours. So far, p53 gene has been cloned in a lot of kinds of fishes but no report of Japane flounder. cDNA full-length of p53 in Japanese flounder was cloned using RACE in this study. The length of p53 gene in Japanese flounder is 1798bp, which is consisting of 238bp-5'UTR, 1095bp Open Readin Frame, TGA terminator and 432bp-3'UTR. The fragment contains a single open reading frame coding for a 365-amino acid protein. Results of sequences comparison demonstrated that the p53 protein of Japanese flounder is highly homologous to that of Japanese medaka and European flounder but low to that of human, zebrafish, chicken, Xenopus and rainbow trout. Moreover, a highly divergence is observed between fish p53 proteins. Although the predicted flounder amino acid sequence is rather divergent from other species, it nevertheless displays the main features found in all p53 proteins, i.e. (i) five highly conserved amino acid clusters; (ii) an acidic and hydrophobic N terminus; (iii) a hydrophilic and charged C terminus; and (iv) a penultimate serine residue. In addition, RT-PCR analyses revealed a similar pattern of expression for Japanese flounder in lymphocystis divease virus-infected and healthy flounder.The p53 gene was also cloned from Japanese flounder using PCR techniques. Sequence analysis revealed that: the flounder p53 gene is 5208 bp in length and consists of 11exonsand 10 introns; the first exon is non-encoding region; the five conserved domains are located in the 2, 4, 5, 7and 8 exons. The structure of the flounder p53 gene is similar to that of human, mouse, zebrafish and Japanese medaka, which demonstrates that the structure of p53 protein is conserved in evolution.The polymorphism of the 5-8 exons in flounder p53 gene was also detected using PCR-RFLP, based on the sequences of cloned p53 DNA sequence. Results showed that a polymorphism exists in the Bglâ…¡restriction site of exon 6 of the flounder p53 gene at codon 187. The allele that can be cut by Bglâ…¡is defined as A and that can not be cut is defined as a. It was found that the genotype of this site is AA in all detected health flounder while is Aa in all infected flounder. Therefore, it is highly possible that the polymorphism in the Bglâ…¡restriction site of exon 6 of the flounder p53 gene at codon 187 is related to the susceptibility of flounder to Lymphocystis Disease. Meanwhile, there are a large mount of polymorphisms in the 5-b exons of flounder p53 gene. Polymorphisms caused by the insertion and deletion of sequences were also found in introns 5 and 6. Furthermore, no gene mutation was found in the p53 cDNA in tumour.