Inactivation And Functional Analysis Of Genes Encoding Superoxide Dismutases In Synechocystis Sp. PCC6803and Synechococcus Sp. CC9311

Reactive oxygen species (ROS) are produced inevitably in photosynthetic organisms through photosynthesis. Accumulation of ROS in cells will make damage to the cellular components, but superoxide dismutases exist in plants and cyanobacteria as the first defence to scavenge ROS to protect the cells. In this paper, two cyanobacterial strains (Synechocystis sp. PCC6803and Synechococcus sp. CC9311) was introduced, and the research progress of superoxide dismutases was summarized. Inactivation and fuctional analysis of gene encoding FeSOD in Synechocystis sp. PCC6803was studied, and the methods of knocking out gene encoding NiSOD in Synechococcus sp. CC9311were tried.In this work, two ways were tried to knock out the gene encoding FeSOD (slr1516). First, the gene was inactived by insertion of kanamycin resistance cassette through homologous recombination. Under low light, the mutant slr1516::CK2was cultivated with BG11added with NaHC03, BSA and Leu. After transferring for several generations maintained with kanamycin, the PCR detection showed that there were kanamycin insertions in the genome, but wide type copies still existed. This result suggested sir1516could not be knocked out directly by insertion inactivation. So we tried another way using the promoter PpetE which is regulated by Cu2+to control the expression of the downstream gene. Under30℃,45μmol photons m"2s"1condition, the growth of PpetE-slr1516strain was significantly supressed cultured in the medium without Cu2+addition, and the colour of the strain turned yellow, which suggested that slr1516was essential to the growth of Synechocystis sp. PCC6803and inactivation of slr1516was lethal. When the concentration of Cu2+was reduced to1/32(10nM) of control (BG11medium), the electron transport rate (rETR) and maximal PSII photochemistry efficiency (Fv/Fm) of PpetE-slr1516strain were decreased significantly, indicating that the activity of PSII was influenced when the expression of FeSOD was decreased. In addition, under Cu2+-absent condition,PpetE-slr1516strain was more sensitve to light intensity. The growth of PpetE-slr1516under high light (45μmol photons m-2s-1) was depressed significantly compared to low light (30μmol photons m-2s-1), suggesting FeSOD plays an important role in relieving oxidative damage.As to inactivation of gene encoding NiSOD (sync0755), several ways were tried to transform the plasmid vector to Synechococcus sp. CC9311. Finally, suicide vector pHSZ9was transferred to Synechococcus sp. CC9311through conjugation and exconjugants were attained. Exconjugants could grow stably under antibiotic resistance. PCR detection showed that the vector had integrated into the genome, but the wide type copies still existed, and the protein it encoded was reduced, suggesting that sync0755might be essential to the growth of Synechococcus sp. CC9311and could not be knocked out directly.