MOLECULAR CLONING, ANTIBODY PREPARATION AND EXPRESSION ANALYSIS OF ASCORBATE PEROXIDASE IN HYDRA SINENSIS

In order to explore the origin and physiological function of ascorbate peroxidase (APX) gene of Hydra sinensis, a full-length cDNA of APX gene of H. sinensis was isolated by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of APX gene was 1357 bp, containing 107 bp 5′UTR (Untranslated region), 146 bp 3′UTR and a 1104 bp open reading frame (ORF) which encodes a polypeptide of 367 amino-acid residues with a molecular weight of 40.79 kD. BLAST (Basic Local Alignmen Search Tool) showed that most of homologous protein sequences of APX in H.sinensis belonged to the plant kingdom. Phylogenetic results by Maximum Likelihood (ML) method and Bayesian analyses revealed that homologous sequences of APX from plant and animal kingdom each formed a single cluster. The ORF of APX gene was subcloned into plasmid pET-GST and transferred into Escherichia coli BL21 (DE3). The recombinant GST-APX fusion protein mainly expressed in the form of soluble were immunized New Zealand rabbits get the polyclonal antibody against APX for Western blotting assay (WB). H. sinensis was cultured (0, 4, 8, 12, 16, 20 and 24 hours per day with illumination intensity 2000 lx) for 30 days, which up-regulated APX expression under longer illumination time (more than 16 hours per day). Due to continuous photosynthetic activity, a large number of reactive oxygen species (ROS) accumulating in symbiotic green algae could spread to the host cell (hydra cell), and the upregulation of APX expression in H. sinensis may mediate the removal of intracellular ROS.