The Mutant Construction And Physiological Function Analysis Of NdhK Subunit In Synechocystis Sp. PCC6803

Two decades ago, a novel cyanobacterial photosynthetic membrane proteincomplex, NADPH dehydrogenase (NDH-1), was discovered in the thylakoidmembrane, and is essential for CO2uptake, cyclic electron transport aroundphotosystem I and cellular respiration. So far, the NDH-1complex consists of at least18subunits, i.e., NdhA to NdhS.To screen the mutants related on NDH-1complex, we transformed WT cells with atransposon-bearing library, thus tagging and inactivating many genes, and thencultured the mutant cells under high light conditions. We isolated two mutants, whichgrew slowly on the plate under high light but similarly to WT under moderate growthlight. PCR results showed that both mutants were tagged in the same gene, slr1280,i.e., ndhK gene. To investigate the physiological roles of NdhK subunit, we firstconstructed an ndhK gene inactivation mutant by using the homologousrecombination strategy. Main points were listed as follows: the homologousrecombination vector, pUC-ndhK, was constructed, and then this vector wastransferred into wild-type of Synechocysitis sp. PCC6803by using the naturaltransformation method. Further, after several subcultures, the transformations wereexamined by using the PCR and Western immunoblotting. The experimental resultsindicated that the kanamycin coding sequence had successfully inserted into theconservative region of ndhK gene, and completely inhibited the expression of ndhKgene. Therefore, we successfully obtained an ndhK gene inactivaition mutant, ndhK.Next, we analyzed the physiological functions of NdhK subunit in cyanobacteria. The results showed that the inactivation of NdhK subunit remarkably reduced the rate ofcyclic electron transport around photosystem I which mediated by NDH-1complex asassessed by the chlorophyll fluoresece parameters, redox changes of P700+and there-reduction of P700+; further, such reduction was not the result of the changes inaccumulation and assembly of NDH-1complex. To prove the results above, wecultured the WT and ndhK under high light condition. The results showed that thendhK retarded growth rate compared to the wild type. Further, a series offluorescence parameters declined in ndhK.Taking all these results together, we conclude that the inactivation ofcyanobacterial NdhK subunit reduced the rate of cyclic electron transport aroundphotosystem I for the first time. Further, such reduction was not the result of thechanges in accumulation and assembly of NDH-1complex.