| Narcissus tazetta L. var. chinensis Roem is one of the ten Chinese traditional flowers is a kind of perennial plant with beautiful color and sweet fragrance. And it is a favorite flower of common Chinese and a famed traditional export flower for a long time. Chinese narcissus has widely spread in China, including Zhangzhou, Putuo and Chongming, especially the Zhangzhou narcissus is the most fomous. But for the slowly multiplication severe degeneration of varieties and simplex color, the production of Chinese narcissus can not satisfy the demand of global market and its development has been seriously restricted. So it's imperative to develop new varieties with modern breeding methods for the narcissus industry.In order to modify the Narcissus flower color by genetic engineering, the expressinal plasmid of objective genes and regenerate system from callus after gene transformation are two key steps. In present study, we cloned flower color genes from the petal of Chinese narcissus, and constructed plant expressing plasmid vector(pCAM35S-PSY), after the establishment of an efficient gene transformation system, The anti-PSY(phytoene synthase) gene was introduced into Agrobacterium tumefaciens strain LBA4404 and then transferred to the callus tissue of Chinese narcissus.Some hygromycin-resistant shoots were identified with molecular analysis. The main results are as follow:(1) The RNA extracted from Narcissus flower petal was used for the isolation of the Psy, LycB and Pds genes by RT-PCR. The results of analysis of the coding sequences of the genes demonstrated that the gene of Psy, 1272 bp, encoded a protein of 423 amino acid;ZycB(Lycopene (3-cyclase), 1515bp, encoded a protein of 504 amino acid, and Pds(Phytoene desaturase), 1713 bp, encodes a protein of 570 amino acid(2) In order to expression the Psy in plant, A plant expression vecter was constructed with a binary vector pCAMBIA1301. The antisense gene fragment was amplified from the coding region of Psy gene having the sequence of 554 bp, degisted by BamH I and Xba I, then introduced to binary expressional vecter pCAMBIA1301 under the constitutive CaMV 35S promotor, and the resulting construction with antisense Psy was name d pCAM35S-PSY. Results from PCR amplification and restriction digestion showed that the fragment of antisense gene Psy was correctly introduced into pCAMBIA1301 plasmid and then the expression vector pCAM35S-PSY was successfully transferred into Agrobacterium.(3) Effect of hygromycin was studied on the regeneration of Narcissus Tazetta bulb-scales in vitro culture. An efficient gene transformation system is established by co-culturing the bulds pretreated for 7 days with Agrobacterium tumefaciens introducing the plasmid vector pCAMBIA1301. Explants from plantlets are the most sensitive to hygromucin, and 30 mg·L-1 hygromycin would inhibit the regeneration. The effect of acetosyringone(AS) on transformation of Chinese narcissus by Agrobacterium tumefaciens was tested in this papers. The results showed that added 100 mg·L-1 AS, then co-cultured 3days, the instant expression activity of gus gene reached 92.9%.(4) An efficient gene transformation system was established, and the optimum conditions for the transformation of antisense gene Psy fragment were as follow: After preculture of 7 days with the bulb-scales as the primary explants, the explants was infected with Agrobacterium tumefaciens strain LBA4404 contained with pCAM35S-PSY lasting 15-20 min. The coculture time was 3 days. Then the cocultured explants were inoculated into the regeneration MS medium containing 5 mg-L"1 BAP, 0.1 mg-L'1 NAA, 200 mg-L"1 Cef, 30 mg-L'1 Hyg, 3% sucrose and 0.7% agar to obtain the HygR shoot, and the shoots were transfer to fresh medium for growth and exchanged per 3 weeks. The results with PCR analysis showed that some tested lines carried the transgenes. the antisense gene fragment was susucesfully transferred into Narcissus tazetta L. var. chinensis Roem. The in-depth biochemical and molecular biological analysis was undertaken. The study on flower color and stability of this traits will be carried out in the field test in future.
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