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中国生物工程杂志

China Biotechnology
China Biotechnology  2010, Vol. 30 Issue (09): 31-35    DOI: Q784
    
Construction of the ndhO Gene Inactivation Mutant of the Cyanobacterium Synechocystis sp. Strain PCC 6803 by Using the Homologous Recombination Strategy and its Molecular Identification
LONG Zong-juan,ZHAO Jiao-hong,WEI Lan-zhen,WANG Quan-xi,MA Wei-min
College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China
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Abstract  

Cyanobacterial NADPH dehydrogenase (NDH-1) is an important photosynthetic membrane protein complex, and is essential to CO2 uptake, cyclic electrontransport around photosystem I and cellular respiration. The enzyme accepts electrons from NADPH and consists of at least 15 subunits, i.e., NdhA to NdhO.The NdhO is a newly identified subunit, and little is known regarding its roles in cyanobacteria. To obtain the ndhO gene inactivation mutant, the homologous recombination vector, pUC-ΔndhO, was constructed, and then this vector was transferred into wild type Synechocystis sp. strain PCC 6803 by using the natural transformation method. Further, after several subcultures, the transformations were examined by using the 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, an ndhO gene inactivation mutant, ΔndhO, has successfully been obtained, and it will further help us to reveal the roles of NdhO subunit in the NDH-1 complex and in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.



Key wordsNADPH dehydrogenase complex      ΔndhO      Synechocystis sp. strain PCC 6803     
Received: 12 April 2010      Published: 25 August 2010
Corresponding Authors: Weimin Ma     E-mail: wma@shnu.edu.cn
Cite this article:

LONG Zong-Juan, DIAO Jiao-Gong, WEI Lan-Zhen, WANG Quan-Chi, MA Wei-Min. Construction of the ndhO Gene Inactivation Mutant of the Cyanobacterium Synechocystis sp. Strain PCC 6803 by Using the Homologous Recombination Strategy and its Molecular Identification. China Biotechnology, 2010, 30(09): 31-35.

URL:

https://manu60.magtech.com.cn/biotech/Q784     OR     https://manu60.magtech.com.cn/biotech/Y2010/V30/I09/31

[1] Berger S, Ellersiek U, Steinmuller K. Cyanobacteria contain a mitochondrial complex Ihomologous NADHdehydrogenase. FEBS Lett, 1991, 286(12): 129 132. 
[2] Berger S, Ellersiek U, Kinzelt D, et al. Immunopurification of a subcomplex of the NAD(P)Hplastoquinoneoxidoreductase from the cyanobacterium Synechocystis sp. PCC6803. FEBS Lett, 1993, 326(13): 246250. 
[3] Ogawa T. A gene homologous to the subunit2 gene of NADH dehydrogenase is essential to inorganic carbon transport of Synechocystis PCC 6803. Proc Natl Acad Sci USA, 1991, 88(10): 42754279. 
[4] Mi H, Endo T, Schreiber U, et al. Electron donation from cyclic and respiratory flows to the photosynthetic intersystem chain is mediated by pyridine nucleotide dehydrogenase in the cyanobacterium Synechocystis PCC 6803. Plant Cell Physiol, 1992, 33(8): 12331237. 
[5] Mi H, Endo T, Ogawa T, et al. Thylakoid membranebound pyridine nucleotide dehydrogenase complex mediates cyclic electron transport in the cyanobacteria Synechocystis PCC 6803. Plant Cell Physiol, 1995, 36(4): 661668. 
[6] Pieulle L, Guedeney G, CassierChauvat C, et al. The gene encoding the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of Synechocystis PCC6803. FEBS Lett, 2000, 487(2):272276. 
[7] Ma W, Deng Y, Ogawa T, et al. Active NDH1 complexes from the cyanobacterium Synechocystis sp. strain PCC 6803. Plant Cell Physiol, 2006, 47(10): 1432 1436. 
[8] Ma W, Mi H. Effect of exogenous glucose on the expression and activity of NADPH dehydrogenase complexes in the cyanobacterium Synechocystis sp. strain PCC 6803. Plant Physiol Biochem, 2008, 46(89): 775779. 
[9] Ma W, Deng Y, Mi H. Redox of plastoquinone pool regulates the expression and activity of NADPH dehydrogenase supercomplex in Synechocystis sp. strain PCC 6803. Curr Microbiol, 2008, 56(2): 189193. 
[10] Steinmüller K, Ley A C, Steinmetz A A, et al. Characterization of the ndhCpsbGORF157/159 operon of maize plastid DNA and of the cyanobacterium Synechocystis sp. PCC6803. Mol Gen Genet, 1989, 216(1): 6069. 
[11] Steinmüller K. Nucleotide sequence and expression of the ndhH gene of the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol, 1992, 18(1): 135 137. 
[12] Ellersiek U, Steinmuller K. Cloning and transcription analysis of the ndh (AIGE) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803. Plant Mol Biol, 1992, 20(6): 10971110. 
[13] Schluchter W M, Zhao J, Bryant D A. Isolation and characterization of the ndhF gene of Synechococcus sp. strain PCC 7002 and initial characterization of an interposon mutant. J Bacteriol, 1993, 175(11): 33433352. 
[14] Prommeenate P, Lennon A M, Markert C, et al. Subunit composition of NDH1 complexes of Synechocystis sp. PCC 6803: identification of two new ndh gene products with nuclearencoded homologues in the chloroplast Ndh complex. J Biol Chem, 2004, 279(27): 2816528173. 
[15] Battchikova N, Zhang P, Rudd S, et al. Identification of NdhL and ssl1690 (NdhO) in NDH1L and NDH1M complexes of Synechocystis sp. PCC 6803. J Biol Chem, 2005, 280(4): 25872595. 
[16] Allen M M. Simple conditions for growth of unicellular bluegreen algae on plates. J Phycol, 1968,4(1): 14. 
[17] Cai Y P, Wolk C P. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entra Pinsertion sequences. J Bacteriol, 1990, 172(6): 31383145. 
[18] Sambrook J, Fitsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual. NY: Cold Spring Harbor Laboratory Press, 1989.1656. 
[19] Williams J G K. Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering method in Synechocystis 6803. Methods Enzymol, 1988, 167:766–778. 
[20] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5269): 680685.

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