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

  • 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。

     

    Abstract: In Synechocystis sp. PCC6803, gene knock-out is the most straightforward and effective method to reveal the physio-logical 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 platformwas constructed using the promoter of petE ( ). 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 genomeof Synechocystis sp. PCC6803 via homologous recombinations. The expression of beta-galactosidase gene was found to be controllable by adjusting the concentration of Cu2+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 Cu2+in medium was below 6nmol/L or above 400nmol/L, the activity of beta-galactosidasewas 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 Cu2+, so that the physiological effect of a gene could be observed when Cu2+is removed.

     

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