Study On Antarctic Algae Chlamydomonas Sp. ICE-L Photolyase Gene And Its Moleculer Mechanism Of DNA UV-damage Repair

Photoreactivation repair(PRR) is an important DNA damage repair mechanism for organisms. It is necessary for keeping hereditary continuity and integrated genetical structure. Photoreactivation repair means DNA photolyase repair the ultraviolet radiation damaged DNA using light as driving force. Photolyase plays an extremely important role in DNA photoreactivation repair.Antarctic ice algae are lots categories of microalgae living in Antarctic sea ice, sea ice edge and sea water. They are significant primary producers of Antarctic region. Antarctic ice algae long time survival from Antarctic high ultraviolet radiation environment, photolyase undertakes the role of repairing DNA damages. Photolyase photoreactivation is the key mechanism for Antarctic ice algae resisting ultraviolet enhancement and adapting to Antarctic high ultraviolet radiation conditions. It is necessary to study Antarctic ice algae photoreactivation repair on molecular level. Antarctic ice algae Chlamydomonas sp. ICE-L is a eukaryotic and single cell algae. Chlamydomonas sp.ICE-L is also the most common and representative algae among Antarctic area. Study on Chlamydomonas sp. ICE-L photolyase can lay foundations for Antarctic living circumstances and other species research.This paper was carried out by following steps. Antarctic algae Chlamydomonas sp.ICE-L photolyase gene PHR2 was cloned by PCR and RACE methods, and then bioinformatics analysis were performed on PHR2. The expression regulating mechanisms of Chlamydomonas sp.ICE-L photolyase gene PHR2 were intensively studied by quantitative real-time PCR(q RT-PCR). Recombinant of prokaryotic expression vector p ET-28a-PHR2 was constructed and transformed to E.coli BL21(DE3) to express PHR2 protein and verify the PHR2 photolyase function. Optimization of the PHR2 protein expressing conditions was conducted by single factor experiment, Box-Behnken design and response surface methodology(RSM). Quadratic regression formula of impact factors was established. Ni-NTA affinity chromatography was utilized to purify the proteins. Edman degradation was used in analyzing PHR2 protein N-terminal sequence. Chlamydomonas sp. ICE-L PHR2 photolyase in vivo and in vitro repair activities were assayed.The full-length of Antarctic algae Chlamydomonas sp.ICE-L photolyase PHR2 c DNA were 2 651 bp, encoding 579 amino acids. The molecular weight(MW) was predicted to be 64.3 KDa. The amino acid sequence of PHR2 was 68% homologous with PHR2 of C. reinhardtii. q RT-PCR results showed that the expression of PHR2 gene increased under high light and ultraviolet stress conditions. This indicated that PHR2 was sensitive to high light and ultraviolet stress. SDS-PAGE revealed that the molecular weight of the target protein was similar to theoretic molecular weight. The target protein was expressed as soluble protein mainly existed in supernate. Response surface methodology confirmed the optimal expressing conditions were p H 6.98, salinity 1.4%, IPTG concentration 0.53 mmol/L and inducing temperature 22 °C. Expression of the protein under optimal conditions was paralleled with calculated quantity. Target protein content increased after purification and high purity target protein was acquired. N-terminal sequence analysis indicated that the amino acids from N-terminal to C-terminal were D/Q/T/A-P-K-R-T, basically conformed to theoretic sequence. The target protein was confirmed to be PHR2 photolyase. In vivo and in vitro activity assays proved that, expressed PHR2 photolyase can effectively repair the DNA ultraviolet radiation damage.