集胞藻PCC6803铜离子诱导表达平台的构建

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。

Construction of copper-induced gene expression platform in Synechocystis sp. PCC6803

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 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-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 Cu2+,so that the physiological effect of a gene could be observed when Cu2+ is removed.