利用同源重组构建蓝藻集胞藻6803ndhO基因突变株及其分子鉴定

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体，参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸。迄今为止，人们在蓝藻细胞中已鉴定出15种NDH-1复合体亚基(NdhA-NdhO)。然而，人们对NdhO亚基的研究尚不够，至今未见有反向遗传学等方面的研究。在通过构建同源重组载体、自然转化和多次继代筛选后，对转化子进行了PCR和蛋白免疫印迹鉴定。结果表明，卡那霉素基因已成功地插入到ndhO基因的保守区域，并完全破坏了ndhO基因的蛋白表达，从而获得了ndhO基因缺失的突变株，为进一步研究NdhO亚基对NDH-1复合体的稳定性和生理功能等奠定了实验基础。

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

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.