Effects On The Foreign Egfp Gene Regulated By Promoter In Cyanobacteria

Cyanobacteria are autotrophic prokaryotes performing oxygenic photosynthesis, which possesses many characteristics, the natural DNA transformation system, simple cell structure, fast growth, strong adaptability, and obtaining the useful organic products with little expenditure of energy and resources. Though the development of gene project and the deeply research of cyanobacteria molecular biology, the expressed vector were constructed in cyanobacteria by recombinant DNA molecular technique. Cyanobacteria are not virulent and have high nutrition value compared with others receptor system. Therefore, cyanobacteria are found to be the perfect model algae for studing the genetic engineering. Since 1984, the pRL shuttle vector was constructed and transfered from E.coli to nitrogen-fixing filamentous cyanobacteria. Many expression plasmids of cyanobacterial cells have been extensively reported, the expression levels of foreign genes have not been optimized. Therefore, how to enhance exogenous gene expression levels in cyanobacterial cells remains unclear.Synechocystis sp. strain PCC6803 (hereafter Synechocystis 6803) is a unicellular autotrophic photosynthesis cyanobacterium and its complete genomic sequences have been obtained. It is a useful tool to study the genetics engineering of cyanobacterium.Firstly, two homologous recombination vectors, Pg and Pg-s were constructed by inserting the groESL promoter gene, and SD sequence in the upstream of egfp, respectively. Subsequently, the expression levels of exogenous gene, egfp, in Pg and Pg-s were investigated under different growth phases, temperatures, and time points by using immuoblotting. The results indicated that the optimal conditions for the expression of egfp in Synechocystis 6803 were at 42°C for 30 min. These results indicated that the groESL-SD tandem was more efficient for the expression of egfp in Synechocystis 6803.Secondly, the promoter region (330-bp) in the ndhC-K-J operon was determined preliminarily and inserted in the upstream of egfp gene, and then this vector was transferred into Synchocystis 6803 cells to generate transgenic alga, P330. The level of foreign gene in P330 was analyzed in different growth stages, different high-light induced time and carbor dioxide concentration. The results indicate that eGFP has the highest amounts in high-light with 4h at low CO2 condition by this unknown promoter. And was associated with increased expression level in P330 as compared to Pg-s.In this study, the effects of many regulatory elements, including groESL promoter originated from Synechococcus 7942, SD sequence, and novel potential promoter, P330, contained in the ndhC-K-J operon, on the expression levels of exogenous gene, egfp, were analyzed. We found that all these regulatory elements can enhance the expression levels of exogenous gene in Synechocystis 6803 with different degrees. These results will futhrer help in solving the low expression levels of exogenous gene in cyanobacterial host.