The Research On Resistance Of Chimeric Gene Composed Of Chitin Receptor And Xa21Against Rice Fungus Deseases

Fungal disease is the earliest and most types of research in plant diseas. Blast and sheath blight of rice, caused by fungus are the representative diseases resulted in the most severe reduction of rice (Antony et al.) out put. The cultivation of resistant varieties was considered as the most effective and economical option for management to control diseases. The resistant genes with great value for production apllication were scarce, so the modification of cloned gene will be an effective and convenient method to breed resistant cultivars. This will greatly promote the agricultural production and eliminate the pollution of the pesticide in disease control.In this study, we plan to use a promoter of rice, low expressional level in seed but high expressional level in leaf and stem at whole growth stage, to express a chimeric gene composed of extracellular LMYs and transmembrane portions (Antony et al.)of a receptor for the chitin elicitor OsCERK1、OsCEBIP and the intracellular domain of XA21(named respectively:OsCERK1-Xa21、OsCEBiP-Xa21), the chimeric gene is transferred into the rice by plant expression vector, then through the strategy of selection of hybrid and the resistant test in field to breed the safe transgenic rice with resistance to fungus at whole growth stage. Furthermore, though the molecular identification and resistance analysis of T1transgenic generations, confirmed that the resistance is indeed caused by genetically modified (gm), and the chimeric genes could stable genetic to future generations.The main results are as follows:1. The plant expression vectors pCEBiP-Xa21and pOsCERK1-Xa21were successfully constructed using for rice transformation.2. The two expression vectors were transferred into TP309rice callus, respectively, by using of agrobacterium transformation system, after selection, differentiation,33lines and41lines of transgenic plants were obtained, respectively. The transgenic plants were transplanted in panddy field, and after detection by PCR to To transgenic lines,23lines of transgenic OsCEBiP-Xa21rice were detected, and38lines of transgenic OsCERK1-Xa21rice were detected.3. The expression at transcription level of transformants was detected by RT-PCR,10lines of positive transformants from9lines were identified. Thus it suggests that the foreign gene is expressed at the level of mRNA.4. To transgenic lines were further verified by the identification of resistance to blast and sheath blight of rice,7lines of transformants from10lines were identified with only a few disease spot, thus, the transgenic plants have abilities to improve the resistance and tolerance to rice blast in some extent; The10lines identified with lesion length were lower than TP309conrtrol, thus, the transgenic plants have abilities to improve the resistance and tolerance to sheath blight of rice in some extent.5. T1transgenic lines of OsCEBiP-Xa21and OsCERK1-Xa21were growing15lines and17lines, respectively, a total of247plants and379plants, after detection by PCR, the positive transformants were185plants and278plants, respectively, in which13lines of them were accord with3:1ratio Mendel’s Law of segregation. This proved that OsCEBiP Xa21and OsCERK1-Xa21genes could stable genetic to future generations.6. Extraction protein of rice leaf for Western blot analysis, results showed that the relative molecular weight of65kD has a specificity of antibody belt, with the expected OsCERK1-Xa21protein size is the same, thus, the target protein was successful expression.7. T1transgenic lines were further verified by the identification of resistance to rice blast. Results showed that the T1transgenic plants have abilities to improve the resistance and tolerance to blast and sheath blight of rice in some extent.8. T1transgenic lines were further investigated by the three major agronomic characters, including plant height、tillers per plant、panicle length. Results showed that panicle length two transgenic lines was no significant differences compared with TP309controls; plant height and tillers per plant both compared with TP309controls were changed, but the result was not caused by genetically modified (gm), may be tissue culture, planting environment, etc.