集胞藻PCC6803铜离子诱导表达平台的构建  

CONSTRUCTION OF COPPER-INDUCED GENE EXPRESSION PLATFORM IN SYNECHOCYSTIS SP. PCC6803

作　　者：高宏[1] 唐蜻[1] 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

出　　处：《水生生物学报》2007年第2期240-244,共5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(KSCX-2-SW-332)资助

摘　　要：在集胞藻PCC6803中,基因敲除是研究基因功能的最直接有效的方法,但是对于某些生存必需的基因则无法通过这种方法获得突变株。为研究集胞藻PCC6803中此类基因的功能,在其基因组中构建了一个petE基因启动子(PpetE)控制的铜离子诱导表达的平台。将集胞藻PpetE装配在lacZ报告基因的上游,通过同源双交换整合到这种蓝藻的基因组中。通过调节培养基中铜离子的浓度发现,lacZ的表达能够人为控制。特别是当铜离子浓度在6—400nmol/L范围时,LacZ活力随铜离子浓度增加呈S型增长关系。利用这个铜离子诱导表达平台,可以控制某些必需基因的表达:提供铜离子维持细胞生存;而撤去铜离子时则关闭基因的表达,可以观察其对生命活动的影响。In Synechocystis sp. PCC6803, gene knock-out is the most straightforward and effective method to reveal the physiological function of a gene. Nevertheless, insertion mutants could not be generated for those genes essential to survival. In order to elucidate the function of such genes in Synechocystis sp. PCC6803, a copper-induced gene expression platform was constructed using the promoter of petE （ PpetE）. PpetE from Synechocystis sp. PCC6803 and the beta-galactosidase gene （lacZ） from E. coli GM48 were cloned respectively by doing polymerase chain reaction （PCR）, and the PpetE was positioned upstream of lacZ. The PpetE-lacZ construct was integrated into the genome of Synechocystis sp. PCC6803 via homologous recombinations. The expression of beta-galactosidase gene was found to be controllable by adjusting the concentration of Cu^2＋ in medium. In a range from 6 to 400nmol/L, Cu2~ induced the expression of beta-galactosidase in an S-shaped curve, but when the concentration of Cu^2＋ in medium was below 6nmol/L or above 400nmol/L, the activity of beta-galactosidase was either too low to be detected or too high to be regulated. This copper-induced gene expression platform can be used in the control of some indispensable genes in Synechocystis sp. PCC6803 ： cells survived in the presence of Cu^2＋ , so that the physiological effect of a gene could be observed when Cu^2＋ is removed.

关 键 词：集胞藻 PpetE LACZ 铜离子 

分 类 号：Q344[生物学—遗传学]