| Cyanobacteria are autotrophic prokaryotes performing oxygenic photosynthesis similar to that of higher plants. Synechocystis sp. strain PCC 6803 (Synechocystis 6803) is a unicellular cyanobacterium, and its complete genome is first characterized in phothosynthetic organisms. Moreover, this unicellular cyanobacterium possesses many characteristics, incuding 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. Also, the products that exogenous genes expressed in cyanobacteria are not easy to generate the inclusion bodies, and most of cyanobacteria and their extract are non-toxic to the humans and animals. On the basis of these charactericts, cyanobacteria are found to be the perfect model algae for studing the genetic engineering. Although 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.The aims of this study were to enhance the expression levels of exogenous gene, enhanced green fluorescent protein (eGFP) gene, in Synechcosystis 6803 by changing different regulatory elements. These intresting findings are listed as follows:First of all, two homologous recombination vectors, P-groESL and P-groESL-SD, were constructed by inserting the groESL promoter gene, indued by temperature, originated from Synechococcus 7942, and the groESL promoter gene and SD sequence (i.e., GGAGAG) in the upstream of egfp, respectively. Subsequently, these 2 vectors were transferred to Synchocystis 6803 to correspondingly generate 2 transgenic algae, Pg and Pg-s. Further, 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 egfg in Synechocystis 6803 were at 42℃for 30 min. Under the optimal conditions, comparisons of the expression levels of egfp in Pg, Pg-s strains indicated that in addition to SD sequence in Pg-s enhanced 1.6 times than that Pg and the application of groESL promoter gene and the SD sequence in Pg-s enhanced 2.0 times than P0 strain. Taken together, these results indicated that the groESL-SD tandem was more efficient for the expression of egfp in Synechocystis 6803.Secondly, a homologous recombination vector, P0-cpcβ, was constructed by inserting the cpcβpromoter gene in the upstream of eGFP gene. Subsequently, the vector was transferred to Synechcysits 6803, by using nature transformation system, to generate the transgenic alga, Pc. Further, the expression levels of exogenous gene, egfp, in Pc were investigated under different light intensities, time points and CO2 concentrations by using immuoblotting. The results indicated that the optimal conditions for expressing the exogenous gene, egfp, were on exposure of Pc cells to the light of 10μEm-2·s-1 for 4 h with low CO2 concentration.Thirdly, on the basis of the gene chip data and bioinformatics analyses, the promoter region (330-bp) in the ndhC-K-J operon was determined preliminarily. Next, a homologous recombination vector, P0-330, was constructed by inserting the 330-bp predicted promoter in the upstream of eGFP gene, and then this vector was transferred into Synchocystis 6803 cells to generate transgenic alga, P330. Further, the expression levels of exogenous gene, egfp, in P330 were investigated under different growth phases, time points under high light, and CO2 concentrations by using immuoblotting. The results indicated that the maximum levels for the egfp expression were obtained under the exposure of P330 cells to high light and low CO2 for 4 h. Comparison of the expression levels of egfp in P330 and Pg-s (control), it was found that the expression amounts in P330 was approximately 4.2 times than that in Pg-s, and 8.2 times than that in P0; and attained 1.8% in total protein, indicting that a novel potential promoter, P330, is more efficient for expressing exogenous genes in cyanobacteria than other, identified promoters such as groESL, cpcβ.In this study, on the basis of the construction of the homologous recombination platform, the effects of many regulatory elements, including groESL promoter originated from Synechococcus 7942, cpcβpromoter originated from Synechococcus 7002, SD sequence, and novel potential promoter, P330, contained in the ndhC-K-J operon, on the expression levels of exogenous gene, egfp, were analyzed. Based on the results of immunoblotting, we found that all these regulatory elements can enhance the expression levels of exogenous gene in Synechocystis 6803 with different degrees. Of these regulatory elements, the predicted novel promoter in the ndhC-K-J operon is the most efficient for expressing the exogenous gene in cyanobacteria. These results will futhrer help in solving the low expression levels of exogenous gene in cyanobacterial host, and establishing the exprimental basis for obtaining efficiently the drugs by applying cyanobacterial genetic engineering.
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