The Mutant Construction, Antibody Preparation And Physiological Function Analysis Of NdhO Subunit In Synecystic Sp. PCC 6803

Two decades ago, the fifth cyanobacterial photosynthetic membrane protein complex, NADPH dehydrogenase (NDH-1), was discovered in the thylakoid membrane, and is essential for CO2 uptake, cyclic electron transport around photosystem I and cellular respiration.So for, the NDH-1 complex consists of at least 17 subunits, i.e., NdhA to NdhP. Although the NdhO of these 17 Ndh subunits has been identified in 2005, its physiological function remains unknown.To investigate the physiological roles of NdhO subunit, we first constructed an ndhO gene inactivation mutant, i.e., AndhO by using the homologous recombination strategy. Main points were listed as follows:the homologous recombination vector, pUC-AndhO, was constructed, and then this vector was transferred into wild-type of Synechocysitis sp. PCC 6803 by using the natural transformation method. Further, after several subcultures, the transformations were examined by using the PCR, RT-PCR, and immunoblotting. The experimental results indicated that the kanamycin coding sequence had successfully inserted into the conservative region of ndhO gene, and completely inhibited the expression of ndhO gene. Therefore, we successfully obtained an ndhO gene inactivaition mutant, AndhO. Next, we prepared the polyclonal antibody of NdhO subunit. Namely, the encoding gene, ndhO, was PCR amplified from Synechocystis sp. strain PCC 6803, the expression plasmid pET32a(+)-ndhO was constructed and transformed into BL21(DE3)pLysS, and the expression of NdhO protein was induced by IPTG. After purification, the fusion protein pET-NdhO was used to immunize Japanese white rabbit to obtain the polyclonal antibody. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by immunoblotting. The results indicated that the NdhO polyclonal antibody possessed the high efficiency and specificity. On the basis of the above mentioned findings, we finally analyzed the different physiological functions of NdhO subunit in cyanobacteria. The results showed the inactivation of NdhO subunit remarkably increased the rate of cyclic electron transport around photosystem I as assessed by the chlorophyll fluoresece parameters; further, such incrase was not the result of the accumulation and assembly of NDH-1 complex.Taking together all these results, we conclude that the inactivation of cyanobacterial NdhO enhanced the rate of cyclic electron transport around photosystem I, which was distinctly different from that NdhO subunit in higher plants. To clarify the difference, the releated researches are under investigation.