Cloning And Analysis Of Sod, Cat And Mpeg Gene From Small Abalone Haliotis Diversicolor Supertexta

Two genes belonging to the anti-oxidation defense system, superoxide dismutase (sod) and catalase (cat), one disease-resistance related gene, macrophage expressed protein (mpeg), and a house keeping gene, 28S rRNA have been cloned from small abalone Haliotis diversicolor supertexta by RT-PCR and RACE in this project. Real-time PCR was used to analyze the expression spectrums of sod, cat and mpeg gene under pathogen challenge and pollutant exposure.The results showed that:(1) Genomic sequence of the sod gene contains five extrons and four introns. The first and the fourth intron obey the GT-AG rule. The full length of sod cDNA is 984 bp. Its 5' UTR is 5 bp, ORF is 465 bp, and the 3'UTR is 514 bp. This cDNA encodes a 154 amino acids protein. Homology analysis indicated that this sequence is highly similar to other shellfish sod genes. Phylogenetic analysis demonstrated that the sod gene of H. diversicolor supertexta clusters with other mollusk sod genes, and occupies an independent lineage distinct from the sod genes in fish and mammals. Real-time PCR analysis reveals that after infection by Vibro parahaemolyticus for 8 hour, 24 hour, 48 hour and 96hour, the level of sod gene expression in the hepatopancreas of H. diversicolor supertexta have not changed compared with control group. In TBT exposure groups of 2 hour,12 hour,48 hour and 192hour, the sod gene expression level in hepatopancreas of H. diversicolor supertexta were same as control group. However, the sod gene expression level of 12 hour group was much lower than that of the control group.(2) A cat cDNA gene partial fragment of 2,312 bp has been obtained. the expression of cat gene in hepatopancreas of H. diversicolor supertexta injected with V. parahaemolyticus were significantly lower than that of the control group at 24 hour; the expression of cat in hepatopancreas of H. diversicolor supertexta exposed to TBT were significantly lower than that of the control group at 2 hour post-exposure.(3) The full length of immunity correlation gene mpeg is 2,781 bp. Its 5 ' UTR is 252 bp, ORF is 2,187 bp, and 3 ' UTR is 342 bp. This gene codes for a protein of 728 amino acids. The homology analysis indicated that it has very high similarity with the mpeg genes of H. corrugate, and H.rufescens. Phylogenetic analysis demonstrated that the mpeg genes of H. diversicolor supertexta, H. corrugate, H. rufescens were clustered in a branch, and the mpeg genes of M.musculus, R.norvegicus and B.taurus were clustered in another branch, which is consistent with the systematic biological relationship. Expression of mpeg in hepatopancreas of H. diversicolor supertexta injected with V. parahaemolyticus was most significantly higher than the control group at 8 hour and 96 hour There was not a significant difference in expression of mpeg from the control group under TBT exposure.(4) A 1,389 bp 28SrRNA gene fragment, which has laid the foundation for real- time PCR analysis has also been cloned in this project.