利用同源重组构建蓝藻集胞藻6803ndhM基因突变株及其分子鉴定  

Construction of the ndhM gene inactivation mutant of the cyanobacterium synechocystis sp. strain PCC 6803 by using the homologous recombination strategy and its molecular identification

作　　者：龙宗娟[1] 赵娇红[1] 魏兰珍[1] 王全喜[1] 马为民[1] 

机构地区：[1]上海师范大学生命与环境科学学院,上海200234

出　　处：《广东农业科学》2010年第12期14-16,23,共4页Guangdong Agricultural Sciences

基　　金：国家自然科学基金(30770175);国家"973"计划项目(2009CB118500);教育部重点项目(209045);上海市教委科研创新重点项目(08ZZ67)

摘　　要：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,人们已在蓝藻细胞中鉴定出15种NDH-1复合体亚基(NdhA-NdhO)。然而,人们对NdhM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。研究结果表明,壮观霉素基因已成功地插入到ndhM基因的保守区域,并完全破坏了ndhM基因的蛋白表达,从而获得了ndhM基因缺失的突变株,为进一步研究NdhM亚基对NDH-1复合体的稳定性和生理功能等奠定了试验基础。Cyanobacterial NADPH dehydrogenase（NDH-1） is an important photosynthetic membrane protein complex,and is essential to CO2 uptake,cyclic electron transport around photosystem I and cellular respiration.This enzyme accepts electrons from NADPH and consists of at least 15 subunits,i.e.,NdhA to NdhO.The NdhM is a newly identified subunit,and little is known regarding its roles in cyanobacteria.To obtain the ndhM gene inactivation mutant,the homologous recombination vector,pUC-ΔndhM,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 ndhM gene,and completely inhibited the expression of ndhM encoding gene.Therefore,an ndhM gene inactivation mutant,ΔndhM,had successfully been obtained in this study,and it would further help us to reveal the roles of NdhM subunit in the NDH-1 complex and in the unicellular cyanobacterium Synechocystis sp.strain PCC 6803.

关 键 词：NDH-1复合体 ndhM基因缺失突变株 集胞藻6803 

分 类 号：Q949.2[生物学—植物学]