The Investigation On Cloning And Expression Regulation Of Egfp Gene In Synechocystis Sp. Strain PCC 6803

Since 1968 genetic recombinant phenomenon was found in cyanobacteria, more and more people committed to the cyanobacteria genetic engineering. With the development of cyanobacteria genetic engineering, many exogenous genes were introduced to cyanobacteria. It mainly involved environmental governance, metabolic, nutrition and health products, genetic engineering drugs,and so on. However, the inefficient expression for exogenous gene restricted the development and application of cyanobacteria genetic engineering.There are many factors affect gene expression in cyanobacteria. Such as, gene copy number, genetic code preference, promoter, terminator, antisense RNA technology and protein expression strategies. In this study, we choose different promoter and modify SD sequence, in order to improve the exogenous gene expression system of cyanobacteria and increase it's expression efficiency.Synechocystis sp. strain PCC6803 is an unicellular cyanobacteria. It has faster growth rate and need simple culture condition. It's cell structure is simple and genetic background is clear. It is facilitate operation and able to grow by either autotrophically or heterotrophically in special culture condition. It is an ideal biology to produce exogenous protein in large-scale bioreactor photosynthetic. Therefore, Synechocystis sp. strain PCC6803 is a very good receptor for cyanobacteria gene engineering.We choosed a fragment close with 3'terminal from cupA gene of Synechocystis sp. strain PCC6803 as the upstream integration platform(up) and another fragment linked to cupA gene as the downstream integration platform(down), and inserted efficient inducible promoter, egfp(enhanced green fluorescent protein gene), kanamycin resistance marker, constructed a homologous recombination double exchange integration platform.On the basis of the integration platform, we replaced the promoter and modified SD sequence, and gained six transformation vectors with promoter and one transformation vector with no promoter. The transformation vectors were transformed into the wild-type Synechocystis sp. strain PCC6803 cell. We gained seven transgenic cyanobacteria after the kanamycin screening. They contain the red-induced Pcpcβ(Transgenic pk-PcpcβSynechocystis PCC6803 and Transgenic pk-Pcpcβ2 Synechocystis PCC6803), the temperature-induced PgroESL(Transgenic pk-PgroESL Synechocystis PCC6803 and Transgenic pk-PgroESL2 Synechocystis PCC6803), the light-induced PpsbA(Transgenic pk-PpsbA Synechocystis PCC6803 and Transgenic pk-PpsbA2 Synechocystis PCC6803) and a non-promoter (Transgenic pk-P0 Synechocystis PCC6803).According to the promoter's features, we designed gradient induced conditions to induce EGFP(enhanced green fluorescent protein) expression, and analysised expression efficiency by detecting EGFP fluorescence activity. Under the 488 nm excitation light, EGFP will eradiate green fluorescent. And the fluorescent from the active algal cells can be detected by using laser scanning confocal microscope directly. By comparing the test results of EGFP, we can judge the exogenous gene expression efficiency of the transgenic cyanobacteria.The main experimental results or conclusions including, 1)constructed six transformation vectors with promoter; 2)constructed one non-promoter transformation vector; 3)obtained seven transgenic cyanobacterium through natural transformation; 4)the PgroESL can increased the expression of EGFP after temperature-induced; 5)SD sequence modified can also up-regulated the expression of EGFP.