Optimization Of Culture Conditions For Transgenic Synechocystis And The Removal Of Selective Marker Gene

This research was carried on the transgenic Synechocystis with paralichthys olivaceus growth hormone gene which was constructed by Dr. Zang. The transgenic Synechocystis with the growth hormone, can obviously speed up the fish growth, and may use for the development of the promising fish food chemical additive. After the successful constructing of transgenic algae, high density culture is the key factor for practical application of the transgenic Synechocystis.Marker genes are effectively used for selection for transgenic plants. The existence of kanamycin resistant gene in transgenic Synechocystis is mainly to choose the transgenic Synechocystis as a selective marker. The growth hormone of Paralichthys olivaceus is cloned and expressed stably in Synechocystis, therefore the kanamycin resistant gene is not needed to act as the selective markers anymore in the production. Therefore, by rejecting the kanamycin resistant gene from Synechocystis through the gene removal method we can eliminate the influence of kanamycin resistant gene.In this thesis, we have examined the composition of medium, culture temperature, light intensity on the growth of transgenic Synechocystis with paralichthys olivaceus growth hormone gene. By analyzing the effect of nitrate, phosphate and carbonate content in the medium , we conducted single factor experiments and got the applicable range of concentration of NaNO3,K2HPO4 ,Na2CO3 was 1.0～1.5 g/L, 0.04～0.06g/L, 0.02～0.04g/L respectively. The orthogonal test based on the three nutrient factors was conducted in order to obtain the optimal combination of NaNO3, K2HPO4, and Na2CO3. The NaNO3, K2HPO4 and Na2CO3 concentrations were 1.25g/L, 0.04 g/L and 0.04 g/L respectively. (The rest of the components were the same as BG-11 medium).On the basis of optimal combination of nutrients, the orthogonal test was designed to search for culture conditions of temperature, light intensity, pH. The results of our experiment demonstrated that the optimal medium conditions for transgenic algae were determined as follow: temperature 30℃, light intensity 60μmol·m-2·s-1, pH 8.0. Under these optimal culture conditions, the biomass of the transgenic alga increased effectively and the specific growth rate of the transgenic Synechocystis reached to 20.64. This paper provides the basis to achieve high density and amplificatory culturing of transgenic Synechocystis.Based on the plasmid pZGH constructed by the laboratory which including Synechocystis sp.pcc6803 homologous recombination fragment groESL, isiAB promoter, paralichthys olivaceus growth hormone gene and kanamycin resistant gene. According to the 5'and 3'sequence of kanamycin resistant gene, the plasmid which includes homologous recombination fragment of groSEL but without kanamycin resistant gene was obtained by using the restriction enzymes NotΙand XhoΙ. By means of nature transformation, we can delete the kanamycin resistant gene from the transgenic Synechocystis through homologous recombination.