| Chloroplast contains the entire machinery for the process of photosynthesis and their genetic system. Compared with nuclear transformation, chloroplast genetic engineering has shown considerable advantages, such as lower environmental risks, the ability to accumulate large amounts of foreign protein, simultaneous expression of multiple transgenes, and so on. Now there have been several crops whose chloroplast genomes have been engineered, such as tobacco, tomato, potato, cotton, carrot, oilseed rape, soybean and so on. However, the chloroplast transformation of spinach (Spinacia oleracea L.), a very important leafy vegetable, has not been developed. Because of a large number of chloroplasts in the large leaves of spinach, the transplastomic spinach would be used as a promising green bioreactor in the production of vaccines, therapeutic proteins and other important materials. Here we study chloroplast transformation of spinach for the first time in the world, which will lay the foundation for the improvement of spinach and development of bioreactor of spinach. The paper has got the results below:1. We constructed a site-specific integration and expression vector for spinach choloroplast transformation, and confirmed the potential utility of the vector in spinach chloroplast by introducing the vector into E. coli. According to the published chloroplast DNA sequence of spinach, rbcL gene and the 5'end part of accD gene were cloned through PCR technique and used as the homologous recombinant fragments in the vector construction. The selectable marker aadA gene (encoding aminoglycoside 3'-adenylytransferase and conferring resistance to spectinomycin and streptomycin) and the reporter gene GFP (encoding green fluorescent protein) were controlled by the promoter Prrn and the terminator psbA3' from tobacco. aadA expression cassette and gfp expression cassette were cloned and placed between two homologous recombination fragments to obtain the site-specific integration and expression vector pRAGA for spinach chloroplast transformation. The results of restriction enzyme analysis of obtained vector were in accordance with the desired. After E. coli was transformed by the vector pRAGA and excited by 488nm blue light, it was found that the E. coli emitted bright green fluorescence under confocal laser scanning microscope. This result indicates that the chloroplast site-specific integration and expression vector pRAGA has been constructed successfully and can be used in prokaryotic chloroplast transformation of spinach.2. We studied the tissue cultures of spinach by using six genotypes of spinach including JDY, QN, CQ, FT, DY and HB-1. The callus induction and differentiation of leaves, hypocotyls and cotyledons of spinach JDY and the cotyledons of spinach QN, CQ, FT, DY and HB-1 on several media supplemented with different hormones were investigated. The research indicated that all explants of spinach JDY can produce a great deal of calli, but only explants of cotyledons cultured on medium supplemented with 1mg/L 6-BA and 0.4mg/L NAA could form shoots. Cotyledons of other spinach genotypes can also produce calli, but the percentage of callus induction is lower than that of spinach JDY. Shoots were obtained from cotyledons of QN cultured on medium containing 1mg/L 6-BA and 0.4mg/L NAA. However, the explants of cotyledons of other four kinds of spinaches did not produce shoots.3. The site-specific integration and expression vector pRAGA were introduced into calli of spinach through particle bombardment. The calli bombarded by pRAGA were cultured on medium without antibiotic about one week after the bombardment. Then the bombarded calli were transferred onto the medium supplemented with 80 mg/L of streptomycin for the first round of selection for one month and 100mg/L of streptomycin for the second round of selection for another month. The selection experiments are now ongoing.
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