To enhance the antifungal response of litchi, transferring rice chitinase gene under a maize-ubiquitin promoter along with its first intron into the zygotic embryos via Agrobacterium tumefaciens-mediated transformatio...To enhance the antifungal response of litchi, transferring rice chitinase gene under a maize-ubiquitin promoter along with its first intron into the zygotic embryos via Agrobacterium tumefaciens-mediated transformation generated transgenic plants. After co-cultivation for 2 days with recombinant Agrobacterium, zygotic embryos were transferred onto Murashige and Skoog (MS) medium consisted of MS salts and Gamborg (B5) vitamins with 2 mgl-1 2, 4-dichlorophenoxyacetic acid (2, 4-D), 50 gl-1 sucrose and 8 gl-1 agar supplemented with 25 mgl-1 hygromycin and 400 mgl-1 cefotaxime. Embryos were selected passing through a series of MS modified media and the antibiotic resistant transgenic plantlets were analyzed. The integration and stability of the transgene was confirmed by PCR, RT-PCR, Southern blotting and by Western blot analyses. The transgenic plants exhibited higher chitinase activity than the non-transformed plants. The chitinase activity was also examined using the native polyacrylamide in-gel assay. These analyses indicated that the foreign gene was translated into the protein of expected molecular weight that showed chitinase activity. Following in-vitro inoculation of die-back, leaf spots and blight pathogen (Phomopsis sp.), the transgenic plants showed delayed onset of the disease and smaller lesions. The transgenic plants were adapted to the greenhouse and did not show any phenotypic alterations.展开更多
文摘To enhance the antifungal response of litchi, transferring rice chitinase gene under a maize-ubiquitin promoter along with its first intron into the zygotic embryos via Agrobacterium tumefaciens-mediated transformation generated transgenic plants. After co-cultivation for 2 days with recombinant Agrobacterium, zygotic embryos were transferred onto Murashige and Skoog (MS) medium consisted of MS salts and Gamborg (B5) vitamins with 2 mgl-1 2, 4-dichlorophenoxyacetic acid (2, 4-D), 50 gl-1 sucrose and 8 gl-1 agar supplemented with 25 mgl-1 hygromycin and 400 mgl-1 cefotaxime. Embryos were selected passing through a series of MS modified media and the antibiotic resistant transgenic plantlets were analyzed. The integration and stability of the transgene was confirmed by PCR, RT-PCR, Southern blotting and by Western blot analyses. The transgenic plants exhibited higher chitinase activity than the non-transformed plants. The chitinase activity was also examined using the native polyacrylamide in-gel assay. These analyses indicated that the foreign gene was translated into the protein of expected molecular weight that showed chitinase activity. Following in-vitro inoculation of die-back, leaf spots and blight pathogen (Phomopsis sp.), the transgenic plants showed delayed onset of the disease and smaller lesions. The transgenic plants were adapted to the greenhouse and did not show any phenotypic alterations.
