Genetic Transformation Of Tomato(Lycopersicon Esculentum) With The Glucose Oxidase Gene From Aspergillus Niger And Its Resistance Mechanism To Fulvia Fulva

Plant develop a variety of defence mechanisms against pathogen attack. The formation of necrotic lesions in the infection site is associated with the co-ordinated induction of diverse defense responses such as cell wall reinforcement, synthesis of phytoalexins and accumulation of pathogen-related(PR) proteins. All these responses are related to the rapid production of active oxygen species(AOS) such as (O₂^-),·OH,OH^-and H₂O₂ during the early stage of plant-pathogen interaction. The oxidative burst is postulated to mediate by hypersensitive response. The AOS can act as microbicial reagents to inhibit pathogen growth and as signal molecules to trigger the expression of plant defense system, finally leading to the induction of systematic acquired resistance(SAR). Glucose oxidase(GO) catalyses the oxidation of β-D-glucose into gluconic acid ang hydrogen peroxide. A number of fungi produce GO as an antimicrobial metabolite for the biocontrol of antagonistic microorganisms because H₂O₂ effectively inhibits bacterial and fungal growth, triggering peroxidation of membrane and cytoplasm. H₂O₂ also contributes to plant cell wall strengthening and acts as a local signal for hypersensitive cell death and as diffusible signal for the induction of defensive genes in adjacent cells during plant-pathogen interactions. H₂O₂ has been implicated as a second messenger in SAR, and activates expression of plant defense genes, and finally lead to resistance to pathogen. This property of GO provides a new strategy for the screening of disease-resistant plants. A lot of tests showed that disease resistance to a wide range of pathogens were enhanced in transgenic plants. 1 Regeneration and optimization of transformation system in tomato Ten tomato lines were used to study effects of different hormones combination on callus and shoot regeneration. The optimistic combination selected is MS+ZT 2.0mg/L+IAA 0.5mg/L.The results indicated the formation rate of callus and shoot regeneration of explants in 10 tomato lines is different. It demonstrates the density of endogenous hormones in different genotype lines are different. The optimistic culture medium of rooting is MS plus 0.5mg/L IAA. Using regeneration system above, Agrobacterium transformation factors were tested. The best transformation condition is pre-cultivation for 2-3 days, Agrobacterium cells diluted to 30 times, infection for 20 minutes and co-cultivation for 2 days. 2 Vector construction of tomato transformation The plant expression vector pCPGO , comprising of a GO gene from Aspergillus niger driven by prp-1 promoter and containing a selectable marker bar gene for resistance to the herbicide phosphinothricin, was constructed. Five tomato inbred lines were transformed via Agrobacterium tumefaciens and 58 transgenic tomato plants were obtained. PPT resistance, PCR and Southern blot analysis showed that the GO gene was integrated into tomato genome. Northern analysis displayed expression of Go gene at transcriptional level. And the expression was verified by KI-Starch staining and measurement of H2O2 content. The transgenic tomato flowered normally. Whereas compared with wild-type plants, the seed set in transgenic tomato were fewer and the germination rate of seeds was less. T1 tomato plants infected with Fulvia fulva and Phytophthora infestans showed enhanced disease resistance, delayed disease symptoms and reduced leaf lesions to different extent. Resistance to Fulvia fulva has correlation with H2O2 content. 3 Resistance Mechanism to Fulvia fulva of Foreign Go in transgenic Tomato Superoxidase(SOD), peroxidase (POD), catalase (CAT), phenylalanine ammonialyase (PAL) and polyphenoloxidase(PPO) activities of three transgenic tomato lines and non-transgenic line (Ck) were studied after inoculation to determine resistance mechanism to Fulvia fulva in transgenic tomato. The results showed that SOD, POD, CAT, APX activities of transgenic tomato lines were much higher than that of non-transgenic tomato to some extent, and the peak of transgenic tomato lines was earlier than that of non-transgenic tomato line. Moreover, activities of PAL and PPO were increased and two peaks appeared in three transgenic tomato lines and only one peak in non-transgenic tomato line. Tomato plants infected with Fulvia fulva showed enhanced disease resistance, delayed disease symptoms and reduced leaf lesions to different extent. The isoenzyme of POD, SOD and PPO were analysed. The results showed that in T1 transgenic tomato lines a new POD isoenzyme band (Rf=0.930) and two new PPO isoenzymebands(Rf=0.338,0.831)emerged. Also two new POD isoenzyme bands(Rf=0.930,0.953) appeared in F1 transgenic tomato plants. However, the number of SOD isoenzyme bands hasn't changed. T2 plants of different genotype displayed significant difference in resistance to Fulvia fulva and Phytophthora infestans. So did Different plants in the same line. It suggested the highly segregation in T2 plants. In order to use resistance transgenic plants in future breeding, transgenic plants must be homologous through self-pollinatation. 4 The inheritance of transgenic tomato progenies The results showed the main agronomic characteristics have some difference in T1 plants. Compared with non-transgenic plants, average thousand grains weight, weight per fruit increased, whereas yield per plant, flowers per panicle, fruits per panicle, germination rate and seed setting decreased in T1 progeny. Agronomic characteristic difference of generations in a transgenic line is different, which indicated the foreign genes integrated had different effects on its expression. T3 progeny didn't show significant difference in agronomic characteristics. It indicates its difference in agronomic characteristics can be recovered through self-pollination. Resistance to PPT was evaluated in transgenic plants. The segregation ratios in T1 plants were 1:1, 3:1,1:0 and 0:1. These data may indicate the foreign Go genes in some transgenic plants is not stable or some transgenic plants are chimeric in To. The segregation ratio in T3 progeny showed 3:1 in most transgenic lines and homozygous transgenic plants were obtained. It implies that heredity of GO gene is single dominant gene. F1 plants by crossing GO transgenic lines with non-transgenic lines displayed resistance to PPT. Most F2 progeny showed a 3:1 segregation pattern, few showed a 1:1 segregation ratio. F3 progeny also gave the same result. This is due to the fact that number of F1 plants is not large enough or some seeds of F2 plants were not harvested, leading to lack of transgene. All BC1 plants gave a 1:1 segregation ratio. These data demonstrate inheritance of GO gene accords with a Mendelian manner. Northern blotting analysis of F1 generations indicated signals could be detected, suggesting GO genes have been expressed in generations. Resistance to Fulvia fulva and Phytophthora infestans of T2,T3 and F2 progeny was evaluated. The results showed all the progenies displayed different response to pathogen.