Abstract:

The construction of a site-specific integration and expressionvectorand its potential utility was reported in the spinach(Spinacia oleracea L.)chloroplast genetic transformation. According to the published chloroplast DNA sequence of spinach, the full length of rbcL gene and the 5′end part of accD gene,　1956bp and 1320bp respectively, were cloned through PCR technique and used as the homologous recombinant fragments in 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 what was desired. After E. coli was transformed by the vector pRAGA and excited by 488nm blue light, it was found that  E. coli emitted bright green fluorescence under a confocal laser scanning microscope, while the controlE.coli showed no fluorescence. This result indicated that the chloroplast sitespecific integration and expression vector pRAGA had been successfully constructed and could be used in prokaryotic chloroplast transformation of spinach.

Key words: spinach(Spinacia oleracea L.); chloroplast transformation; vector construction; site-specific integration; GFP gene