Establishment Of A Copper-regulated Gene Expression Platform And A Novel Insertional Mutagenesis Method In Synechocystis Sp. PCC6803

Synechocystis sp. PCC6803 is a unicellular cyanobacterium that is able to grow autotrophically using light energy or heterotrophically on glucose, can be naturally transformed by exogenous DNA. It is one of the research model species for genetic and molecular studies of cyanobacteria. In this study, a copper-induced gene expression platform was constructed in Synechocystis sp. PCC6803 and used to investigate a thylakoid membrane biogenesis gene vipp1, and a novel insertional mutagenesis method was developed and used to inactivate 93 genes of unknown functions but highly conserved among cyanobacteria and a higher plant.The main researches and results are in three parts:1) Construction of copper-induced gene expression platform in Synechocystis sp. PCC6803. The promoter of petE gene was cloned from Synechocystis sp. PCC6803 and a copper-induced gene expression platform was constructed using lacZ as the reporter gene. As seen with the result, the PpetE-driven expression of lacZ was regulated by the concentration of copper in the medium.2) Functional studies of vipp1 gene in Synechocystis sp. PCC6803. A vipp1 mutant (PpetE-vipp1) was constructed on the basis of copper-induced expression platform, in which the expression of vipp1 was controlled by the copper. The results indicated that vipp1 was essential for the viability of Synechocystis PCC6803. It was probably involved in thylakoid membrane biogenesis or maintenance, and indirectly affected the oxygen evolution, photosynthetic pigments synthesis and photosynthetic electron transport in the cyanobacterium.3) Insertion of genes of unknown functions and highly conserved. By similarity search, 93 genes of unknown functions were identified to be conserved among cyanobacteria and Arabidopsis. Insertional mutants of each of these genes were constructed. Of these mutants, 56% could not be completely segregated, suggesting that these genes might be essential for the survival of Synechocystis sp. PCC6803.The main conclusions of this study are:1) The copper induced gene expression platform is a useful tool for investigating the gene function in Synechocystis sp. PCC6803.2) The gene vipp1 is essential for the viability of Synechocystis sp. PCC6803.3) About half of the genes of unknown functions and highly conserved among cyanobacteria and plants are probably essential for the viability of Synechocystis sp. PCC6803.