| Hepatitis B virus (HBV) infection is one of the most widespread viral infections of humans and causes acute and chronic hepatitis and hepatocellular carcinoma. Vaccination is the best method to prevent HBV infection. At present, HBV genetic engineering vaccine mainly chooses yeast as basic carrier. With the development of "edible" vaccines based on transgenic plants, HBsAg gene has been also expressed in many plants. Cherry tomato {Lycopersicon.Esculentum var. cerasiforme), which is a subspecies of common tomato varieties, is an annual or perennial herb of the Nightshade (Solanaceae), tomato Genus (Lycopersicon) and tomato species. It has delicious taste and rich nutrition, and especially can be eaten raw as a fruit. Therefore it is an ideal candidate crop for the production and delivery of oral vaccines. In this work, we constructed a plant expression vector carrying hepatitis B surface antigen gene (HBsAg/adr), transformed cherry tomato mediated by Agrobacterium tumefaciens, and obtained transgenic plants with an inserted HBsAg gene encoded the small protein. During the transformation, we accidentally obtained a triploid mutant and analyzed its growth characteristics (including physio-biochemical characteristics, resistance and morphogenesis changes).1 Construct of Agrobacterium transformation with pCAMBIA1301/HB and expression of HBsAg in cherry tomatoPlant expression vector pCAMBIA1301/HB, in which HBsAg S gene was driven by the CaMV 35S promoter and flanked downstream by the NOS-terminator, was constructed. PCR identification and Southern hybridization confirmed the stable integration of the HBsAg S fragment into the cherry tomato genome. ELISA assay showed that HBsAg in transgenic cherry tomato was 100.36ng/g FW in leaves and 127.54ng/g FW in fruits, implying that recombinant HBsAg had natural epitope. The part was published in vaccine (2010, vol.28 No.46).2 The physiological and biochemical characteristics of HBsAg-transgenic cherry tomato mutant in the greenhouseBy Hgy screening, PCR identification, Southern blotting detection and flow cytometry, a transgenic cherry tomato mutant was obtained. The mutant was triploid, grew well, and its seeds were sterile. In our work, we detailedly analyzed morphological and physiological characteristics of the transgenic mutant in the greenhouse. The results suggested that the mutant exhibited morphological, cytological and physiological variation. By PCR identification, HBsAg gene was stably expressed in the transgenic mutant grown in the greenhouse. Moreover, the chromosome of the mutant were found to be triploid (n=36) by flow cytometric analysis. Furthermore, the mutant has obvious physiological alterations (such as higher chlorophyll and protein content, better water retention abilities and membrane permeability, stronger antioxidative enzyme activities), as compared to the control. The result was accepted for publication in Russian journal of genetics and would be published in 2011 vol.47 No.8.3 The physiological and biochemical responses of HBsAg-transgenic cherry tomato mutant callus under salt stressSalinity is an important factor in affecting plant growth. Studies on physiological and biochemical responses of transgenic plants under salt stress can understand the ability of salt or adversity resistance of plant during the growth and development. In our work, the effects of NaCl stress of diferent gradients on physiological indexes of transgenic cherry tomato callus were studied. The results showed that:1) When transgenic cherry tomato mutant callus was processed by NaCl, its semi-lethal concentration was 80mmol/L. The threshold value of salt tolerance in transgenic cherry tomato mutant callus was 80mmol/L-100mmol/L.2) Under salt stress, with the increase of NaCl concentration gradient, the activity of SOD, POD, CAT and APX in mutant callus first increased and then decreased. When NaCl concentration was above 40mmol/L, the activities of antioxidant enzymes in mutant callus began to be affected by salt stress, but CAT activity changed little. Under the same NaCl concentration, the activities of antioxidant enzymes in the mutant callus were all higher than those of the non-transgenic cherry tomato callus, which indicated that the mutant had better salt tolerance.3) With the gradual increase of NaCl concentration, H2O2 content, MDA content, soluble sugar content, proline content, and relative conductivity of the mutant callus presented a persistent elevation. But under the same NaCl concentration, compared with the non-transgenic cherry tomato callus, the mutant callus had lower H2O2 content, MDA content and relative conductivity, higher soluble sugar and proline content, which indicated that the mutant had stronger adversity resistance.4) With the increase of NaCl concentration, chlorophyll content of the mutant callus showed a downward trend. And under the same treatment, the mutant callus had higher chlorophyll a, chlorophyll b and total chlorophyll content than the non-transgenic cherry tomato callus, which indicated that the mutant callus had higher photosynthesis. These results showed that the salt resistant ability of the mutant callus is improved clearly, which was closely related to the changes of SOD, POD and APX activities and the accumulation of soluble sugar.4 The physiological and biochemical responses of HBsAg-transgenic cherry tomato mutant seedling in vitro under salt stressIn our work, the effect of NaCl stress of diferent gradients on physiological indexes of transgenic cherry tomato callus mutant seedling in vitro was analyzed, which was expected to provide the resistance research of the mutant in the greenhouse with earlier foundation. The results showed that:1) When transgenic cherry tomato mutant seedling in vitro was processed by NaCl, its semi-lethal concentration was 150mmol/L. The threshold value of salt tolerance in transgenic cherry tomato mutant seedling in vitro was 100mmol/L-200mmol/L.2) Under salt stress, the activity of SOD, POD, CAT and APX and proline content increased first but decreased then with the increase of NaCl concentration gradient. When NaCl concentration was above 60mmol/L, the activities of antioxidant enzymes began to be affected by salt stress. The content of proline reached the top under salt stress of 100mmol/LNaCl. Under the same NaCl concentration, the activities of antioxidant enzymes and proline content in the mutant seedling in vitro were all higher than those of the non-transgenic cherry tomato, which indicated that the mutant seedling in vitro had higher salt tolerance.3) With the gradual increase of NaCl concentration, H2O2 content, MDA content, soluble sugar content and relative conductivity of the mutant seedling in vitro increase enormously. But under the same NaCl concentration, compared with the non-transgenic cherry tomato seedling in vitro, the mutant seedling in vitro had lower H2O2 content, MDA content and relative conductivity.4) Salt stress treatment caused the remarkable decrease of chlorophyll content. Overall, with the increase of NaCl concentration, chlorophyll content of the mutant seedling in vitro showed a downward trend. And under the same treatment of NaCl concentration, the mutant seedling in vitro had higher chlorophyll a, chlorophyll b and total chlorophyll content than the non-transgenic cherry tomato seedling in vitro, which indicated that the mutant seedling in vitro had stronger photosynthesis. These results showed that the stronger salt resistant ability of the mutant seedling in vitro was closely related to the changes of SOD and APX activities, the accumulation of soluble sugar and praline, and the chlorophyll content variation.5 Morphology variation of HBsAg-transgenic cherry tomato mutant during the developmentBased on obvious variation of ploidy, physio-biochemical characteristics and resistance in HBsAg-transgenic cherry tomato mutant, the difference in morphogenesis both the mutant and non-transgenic cherry tomato was studied. The leaf explants of both the mutant and the control were cultured on Murashige and Skoog (MS) basal medium supplemented with 6-BA 1.Omg/L and IAA 0.05mg/L for callus induction. Morphology changes were observed in the process of explant differentiation and callus development by the technology of paraffin wax slice and electron microscope.Histological studies of the mutant explants at various developmental stages revealed that organogenic buds first appeared in the axillary position of mutant explants on the 21th cultured day and then somatic embryos formed in the same mutant explants after 35 days of culture. The result indicated that shoot organogenesis and somatic embryogenesis occurred together during the in vitro regeneration of transgenic cherry tomato mutant leaf explants treated by 6-BA combined with IAA. However, only somatic embryogenesis pathway was observed during the regneration of non-transgenic cherry tomato explant on the same culture condition.Further transmission electron microscopy and scanning electron microscopy indicated that there were significant changes in morphology and quantity of some organelles in the mutant callus cells. On the 7th day of culture, there was little cytoplasm or less organelles, except a great number of dense lipid bodies in the mutant callus cells. In the later stage, the changes of chloroplasts, Golgi bodies and mitochondria in the mutant cells had obvious difference compared with the control. The result was accepted for publication in Journal of Chinese Electron Microscopy Society (bimonthly) and and will be published in No.2 (2011).6 Physiological and biochemical changes during the development of HBsAg-transgenic cherry tomato mutantThe physio-biochemical changes are the basis of morphogenesis. Based on the development of organogenic buds and somatic embryos in the mutant, the changes of antioxidant enzyme activities and some physicochemical indexes (growth rate, soluble protein content, chlorophyll content and so on) in the mutant explant during the regeneration were studied. The result showed that SOD activities of the mutant had only one peak value on the 21st day. POD activities of the mutant had declined less sharply since the explants were cultured. MDA (Malonaldehyde) of the mutant showed a significant decreasing trend during the cultured 0-20 days. IAAO (IAA oxidase) activity of the mutant increased not sharply before cultured 35 days and subsequently fell steeply. It indicated that SOD and POD activities were closely related to early event of organogenesis; IAAO activity involved the formation process of adventitious buds.Except for a rapid decrease from the cultured 21st to 28th day, the overall growth rate of the mutant trend had been rising. This phenomenon was caused by the first appearance of organogenesis during its callus differentiation. And the change trend of soluble protein content in the mutant callus development was similar to that of the non-transgenic cherry tomato, and but their change stages were different. It indicated that soluble protein content was closely related to organogenesis. The result was accepted for publication in Acta Agriculturae Nucleatae Sinica (bimonthly) and and will be published in No.3 (2011).In conlusion, HBsAg/adr gene was successfully expressed in cherry tomato in our study. At the same time, a transgenic cherry tomato mutant was obtained by chance. And studies showed that the mutant had obvious variations in in many respects such as ploidy, physio-biochemical characteristics, resistance and morphogenesis pathway.
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