集胞藻PCC6803中ORF sll0260对于维持基本生命活动的必要作用  

THE ESSENTIAL ROLE OF ORF SLL0260 IN MAINTENANCE OF BASIC LIFE ACTIVITIES OF SYNECHOCYSTIS SP. PCC6803

作　　者：罗泉[1,2] 徐旭东[1] 

机构地区：[1]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072 [2]中国科学院研究生院,北京100049

出　　处：《水生生物学报》2009年第5期789-794,共6页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金重点项目(30330030);中国科学院知识创新工程方向性项目(KSCX2-SW-332)资助

摘　　要：集胞藻(Synechocystis sp.)6803的未知功能基因中有很多是细胞的基本生命活动所需要的,这些基因插入失活往往会导致细胞死亡,因而得不到分离完全的突变株,难以进行遗传学研究。构建突变株以铜离子调控的启动子PpetE来控制此类未知功能基因的表达则可能获得完全分离。构建PpetE-sll0260突变株并对sll0260必要作用进行研究。在完全分离的突变株中,去除铜离子可关闭sll0260的表达。此时,突变株生长受到严重抑制,色素含量大为降低,类囊体膜结构破坏,光合作用消失,呼吸能力下降。这些结果表明该基因对于维持集胞藻6803的基本生命活动来说是必需的。亚细胞定位研究显示sll0260编码一个膜蛋白,位于质膜和外膜混合物中。Sll0260可能作为某些离子的转运蛋白起作用,或者直接与类囊体膜的发生过程相关。In the cyanobacterium Synechocystis 6803, many genes of unknown function are essential for basic fife activities. Insertion inactivation of these genes could lead to loss of viability, and the mutants could not be completely segregated, consequently, genetic studies are hard to perform with these genes. However, to construct mutants expressing these genes from a copper-regulated promoter PpetE may result in complete segregation. To find out the physiological effects of the investigated gene, copper may be removed from the medium to turn off the gene expression or adjusted to certain low concentrations to lower the gene expression level. This report presents the construction of the PpetE-sll0260 mutant and the study on the essential role of sll0260 in Synechocystis 6803. Upstream of the PpetE promoter was an omega cassette with stem-loop structures at both ends to terminate background transcriptions. The mutant was completely segregated in the presence of cupric ion as shown with PCR detection. In the mutant, the expression of sll0260 was fully turned on in the presence of 320nmol/L cupric ion, but turned off after removal of cupric ion from the medium. The copper regulation of sll0260 by PpetE was confirmed by Western blot analysis. Upon copper deprivation, the growth of the PpetE-sll0260 strain was seriously inhibited, pigments were greatly reduced, thylakoid membranes were destructed, oxygenic photosynthesis ceased and respiration was remarkably lowered. Under the same conditions, the wild type strain showed almost no change in growth and physiological characters. All these results suggested that sll0260 should be essential for the maintenance of basic life activities of Synechocystis 6803. Based on Western blot analysis, sll0260-encoded protein was localized to the total membrane fraction. Further analysis with purified thylakoid membranes and a mixture of cytoplasmic and outer membranes showed that the sll0260-encoded membrane protein （Sll0260） was located in either cytoplasmic or outer membrane or both,

关 键 词：集胞藻6803 sll0260 PpetE 膜蛋白 

分 类 号：Q344.1[生物学—遗传学]