Study Of The Transgenic Cyanophyta Without Antibiotic Resistance Markers

Blue green algae are photoautotrophic organisms with many special properties, it has abundant nutrition; Meanwhile, blue green algae have unique nature on cell's organization,metabolizability,heredity, etc., and it has a lot of advantages as genetic engineering receptor. However the existence of the marker genes in the transgenic production are surplus, even harmful, Enhanced Green Fluorescent Protein (EGFP) was confirmed to be safe. In this study we research transgenic blue green algae without antibiotic resistance markers by using EGFP gene and FACS Vantage SE.First part of this study, the upstream sequence P_cpcB of the cpcB gene from Spirulina platensis was obtained by PCR and subcloned into the5' MCS of pEGFP, thus the plasmid pCEGFP was constructed, the 1.2kb fragment containing P_cpcB and EGFP gene were subcloned into plasmid pPKE2 which contained small plasmid pPbS of Plectonema boryanum and the shutter plasmid pCGPKE2 with EGFP marker was construced, digest pCGPKE2 with Pst I to excise kanamycin resistance gene(Km), finally we got the strain including pCGP which didn't contain antibiotic resistance marker by FACS Vantange SE. The recombinant plasmids were directly transformed into Synechocystis sp.PCC6803 respectively, then using FACS Vantage SE to pick out the transgenic Synechocystis sp.PCC6803 with EGFP marker, then observe the transformant under the fluorescent microscope, the result indicated that we successfully obtained the transgenic Synechocystis sp.PCC6803.The second part of this study, the 1.2kb fragments from pCEGFP were cloned into the homologous recombinant plasmid pKRET4, thus the plasmid pCGKT with EGFP marker was constructed, and then got 4.2kb fragments which contained homologous recombinant gene R(recA gene),P_cpcB and EGFP marker gene, these fragments were transformed into Spirulina plantensis through ultrasonic treatment. because of the influence of various kinds of factors, we didn't obtain the transformant.In this study we combined EGFP marker gene and FACS technology for the first time, we constructed the shutter plasmid and homologous recombinant plasmid without antibiotic resistance marker gene, these two plasmids were transformed into Synechocystis sp.PCC6803 and Spirulina platensis separately, then by FACS Vantage SE and the fluorescent micro to obtain the transformant with EGFP marker . It's the first time to get the transgenic Synechocystis sp.PCC6803 without antibiotic resistance marker. This research could provide experience to other researcheres to develop transgenic blue green algae without antibiotic resistance marker and lay a certain foundation further.