Transformation Of Tobacco (Nicotiana Tabacum) With Baculovirus Chitinase And Evaluation Of Its Expression And Effectiveness In Improving Fungal Resistance

A chitinase gene encoded within the genomic DNA of a nucleopolyhydrosis baculovirus from Autograph California was amplified by polymerase chain reaction (PCR), inserted into the plasmid vector pROK2, and transformed into Escherichia coli XL1 blue. The recombinant plasmid pROK2 DNA were mobilized into Agrobacteria tumefaciens LBA4404 using a freeze-thaw for transformation method. The chitinase gene was transformed into tobacco leaf disks by A. tumefaciens infection. Infected leaf discs were subsequently cultured for shoot induction. Rooting was induced on MS medium containing Kanamicin sulfate (50g/ml). The results of PCR and Western blot assays showed that the chitinase gene was expressed in flue-cured tobacco varieties CF80, K326 and Xanthi-nc. Enzyme activity was confirmed using a radiological assay before fluoromctric quantification.Confocal laser scanning microscopy of foliar sections from a chitinase-expressing N. tabacum Xanthi-nc transformant were embedded in (butyl methyl methacrylate) and stained with a polyclonal serum raised against the chitinase/fluor-conjugated secondary (IgG). A antibody detection system showed that chitinase was largely restricted to the vascular tissue with a few "accumulation hot spots " in chloroplasts.To, T1 , T2 , T3 generation plants inoculated with Colletotrichum nicotianae Averna several lines displayed resistance towards the fungus. Inoculating the detached leaves with Alternaria aUernala (Fries) Keissler also detected high resistance lines.Whether the exo-chitinase gene is stably inherited in the transgenic tobacco lines was studied by using MS medium containing Km 100mg/L, and Western blot. The results showed that the exo-chitinase gene is stable in most transgenic tobacco lines.A sensitive method for detecting chitinase gene expression in tobacco plants and cured tobacco leaves is described in this report. A medium containing Kanamycin is first used to screen the transgenic tobacco seeds and seedlings. Western blotting further verified the presence of the chitinase gene in the experimental samples. We studied the method of semi-nested PCR for testing transgenic tobacco expressing a chitinase gene. Tobacco leaf samples taken from transgenic tobacco plants were cured under certain high temperatures for about one week, and then tested for the presence of the chitinase transgene by semi-nested PCR also. PCR products were further analyzed by digestion with Hind[l\ restriction endonuclease to verify the presence of the chitinase transgene.The presence or absence of the baculovirus-derived chitinase in transformed leaf tissue was not observed visually to modify lesions in leaves that had been mechanically inoculated with a nepovirus (cherry leaf roll or arabis mosaic). Heliolhus virescens (second instar) larvae fed on leaf tissue expressing chitinase were not impaired cither in their development to pupation or in their feeding behavior in comparison with their counterparts which had consumed similar amounts of untransformed leaf tissue from tobacco seedlings.