Cloning And Expression Of (6-4) Photolyase Gene From D.salina

UV radiation induced two major classes of pyrimidine dimmers:the pyrimidine (6-4) pyrimidone photoproduct (6-4 product) and the cyclobutane pyrimidine dimmer(CPD). Many organisms produce enzymes, termed photolyases, that specifically bind to these damage products and split the via a UV-A/blue light-dependent mechanism(photoreactivation), thereby reversing the damage. These two photolyase are specific for either CPDs(CPD photolyase) or 6-4 products (6-4 photolyase). A gene that expresses a protein with 6-4 photolyase activity in vitro, was recently cloned from high organisms(Arabidopsis thaliam , Drosophila melanogaster , Danio rerio , Xenopus laevis and Homo sapiens).We report here the cloning of a homolog of this gene from Dunaliella salina . This cloned gene produces as protein with photolyase activity when expressd in Escherichia coli. It' s the first cDNA code (6-4)photolyase found in low orgnism(alga), and this study is important for the reseach of 6-4 photolyase. The methods and analysis as bellows:1. This EST was analyzed by method of BLAST, and the conclusion suggests that the sequence probably be a partial cDNA that can code one of protein family of photolyase/ blue light photoreceptor.2. Amplify the 3' sequence of EST by 3' -RACE, the result is 2575bp , and the open reading frame (ORF) is 18OObp, coding 600 AA , thenanalyze this 3' UTR by bioinfomatice methods, including BLAST, ORF analysis, conservative analysis and phylogenetic analysis. The sequence probably code a (6-4) phtolyase.3. Vector pGEX-4T-l/6-4 was constructed by inserting the BamHI/XhoI ORF sequence of (6-4) phtolyase into the vector pGEX-4T-l. The E. coli strain JM109 was transformed with resultant plasmid pGEX-4T-1/6-4.4. The transformation was induced with IPTG, then the total protein from cell extract was analyzed by electrophoresis on a 8% SDS-PAGE in order to validate the GST fusion protein, and the fusion protein is about 90kD.5. After induced with IPTG, JM109 with pGEX-4T-l/6-4 was radiated by UV for 30 seconds, cultivate these E. coli with light or without light. The survival rate proved the gene of D. salina (6-4) photolyase has photoreactivation.