A DNA fragment encoding the hepatitis B virus surface antigen was amplified from a positive blood (hepatitis B) sample and introduced into the pET 32c prokaryotic expression vector. The gene encoding the HBV surface protein antigen was introduced into pCAMBIA 3300, and immobilized into Agrobacterium tumefaciens strain LBA4404. Cotyledonary leaf sections of Cucumis sativus (cucumber) cv 'Swarnamukhi' were cocultivated with Agrobacterium harboring the binary vector pCAMBIA 3300 carrying the HBV surface antigen gene driven by the CaMV35S promoter and the herbicide resistance gene phosphinothricin. Putative transformed shoots were induced on a Murashige and Skoog (MS) medium containing phosphinothricin, and these were then rooted on MS basal medium supplemented with 1 mg/L Indole 3-butyric acid (IBA). Integration of the T-DNA into in putative transgenic plants was confirmed by PCR and Southern blot analyses. RT-PCR and Northern blot analyses were conducted to determine RNA expression. Levels of expression in transgenic plants were confirmed by Western blot analysis, and quantification of the protein was determined by enzyme linked immuno assay (ELISA). Molecular mass of the recombinant protein was measured by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Mass Spectrometry.

The isolation and characterization of fruit specific promoters are critical for the manipulation of nutritional value and agronomic quality of fruits by genetic engineering and also opened a new era in edible vaccine technology. Expansins are proteins that induce loosening of individual plant cells by disrupting the non-covalent interactions between cellulose and hemicellulose microfibrils and hence have role in growth programs including fruit ripening. We report the identification of an expansin gene (CsExp) from Cucumis sativus that exhibits high levels of mRNA abundance and is specifically expressed in ripened fruit. The promoter region of CsExp also contains elements responsible for its fruit specific expression. Transient expression studies of the CsExp promoter were conducted with particle bombardment, followed by GUS histochemical assay and real time PCR. CaMV35S promoter was used as the positive control in all these experiments. Clear fruit specificity was observed for CsExp promoter in all the experiments. Thus CsExp promoter from Cucumber is a good candidate to target expression of the foreign genes to engineer fruit specific traits.