ABSTRACT
Two cryoprotectant solutions, including trimethylamine oxide (TMAO) and dimethyl sulfoxide (DMSO), and several loading durations were used to evaluate the cryopreservation of the shoot tip of Eriobotrya plants. The best results for regrowth (59.91%) were obtained from 10% TMAO compared to 10% DMSO as cryoprotectant, although nonsignificant differences were found for survival between the two cryoprotectants. We detected pronounced effects of loading duration on survival and regrowth rates of shoot tips. The maximum regrowth (56.36%) was observed at 9 h of loading duration. The cryoprotectants and loading durations greatly affected the regrowth of Eriobotrya shoot tips, and TMAO could be introduced as a nontoxic and efficient cryoprotectant. These results could lay a foundation for the cryopreservation of Eriobotrya.
ABSTRACT
AIM: To transfer hepatitis E virus (HEV) ORF2 partial gene to tomato plants, to investigate its expression in transformants and the immunoactivity of expression products, and to explore the feasibility of developing a new type of plant-derived HEV oral vaccine. METHODS: Plant binary expression vector p1301E2, carrying a fragment of HEV open reading frame-2 (named HEV-E2), was constructed by linking the fragment to a constitutive CaMV35s promoter and nos terminator, then directly introduced into Agrobacterium tumefaciens EHA105. With leaf-disc method, tomato plants medicated by EHA105 were transformed and hygromycin-resistant plantlets were obtained in selective medium containing hygromycin. The presence and integration of foreign DNA in transgenic tomato genome were confirmed by Gus gene expression, PCR amplification and Southern dot blotting. The immunoactivity of recombinant protein extracted from transformed plants was examined by enzyme-linked immunosorbant assay (ELISA) using a monoclonal antibody specifically against HEV. ELISA was also used to estimate the recombinant protein content in leaves and fruits of the transformants. RESULTS: Seven positive lines of HEV-E2-transgenic tomato plants confirmed by PCR and Southern blotting were obtained and the immunoactivity of recombinant protein could be detected in extracts of transformants. The expression levels of recombinant protein were 61.22 ng/g fresh weight in fruits and 6.37-47.9 ng/g fresh weight in leaves of the transformants. CONCLUSION: HEV-E2 gene was correctly expressed in transgenic tomatoes and the recombinant antigen derived from them has normal immunoactivity. Transgenic tomatoes may hold a good promise for producing a new type of low-cost oral vaccine for hepatitis E virus.
