Disease Resistance Genetics And Regulation Of Agronomic Traits Of Transgenic Rice With Enhanced Disease Resistance

The rice sheath blight (sb) caused by Rhizoctonia solani Kuhn, rice blast caused by Magnaporthe grisea and bacterial blight (bb) disease caused by Xanthomonas oryzaepv oryzae (Xoo) are the most destructive diseases in rice. In particular, there is no genetic resource resistant to sheath blight, and the complex genetic diversity of M. oryzae, genetic transformation with chitinase and glucanase genes to enhance resistance to those fungal provides an alternative approach. In the previous work of our laboratory, the rice chitinase gene (RCH10) and the alfalfaβ-1,3-glucanase gene (AGLU1) were tandem-inserted into the transformation vector pBI101 under the control of the 35S promoter with its enhancer sequence to generate double-defense gene expression cassette pZ100. The pZ100 cassette was transformed into rice (cv. Taipei 309) by Agrobacterium-mediated transformation, transgenic plants enhanced resistance to both sheath blight under greenhouse condition. Over-expression of OsNPR1 gene (OsNPR1-OX), a key regulator in plant systemic acquired resistance (SAR), transgenic plants increased resistance to bacterial blight. Both Z100 and OsNPR1-OX plants appeared stunt and seed setting reduction.In this study, we analysed genetic stability of Z100 transgene, and possible mechanism of development modification in Z100 and OsNPR1-OX plants.More than 160 Z100 independent transformants were obtained and confirmed by PCR and GUS analysis. The segregation ratio of some T₁ transgenic rice plants showed 3:1 and 15:1 with Mendelian inheritance pattern, but other lines didn't, indicating that the transgene inserted into rice chromosomes with a complex and casualness. Wefurther observed 100% the NPTII marker gene in the offspring (T₃), but RCH10 genewas lost at 15% cases and AGLU1 gene at 5% cases during progeny generating. Northern blotting analysis of inheritable progenies revealed similar levels of the RCH10 and AGLU1 transcripts in the same individuals. Disease resistance to both sheath blight and blast was enhanced in open field inoculation an evaluation valuable to rice breeding. OsPR1-b was stronger induced in the Z100 transgenic plants.Immunogold detection revealed that RCH10 and AGLU1 were located mainly in the chloroplasts in the transgenic plant, and they were delivered to the vacuole and cell wall after 24 h after infection by R. solani, suggesting that these antifungal proteins might gather and execute action in these subcellular compartments. The results probably suggest a novel mechanism for defense protein function during pathogens attack, we propose that following pathogen invasion, defense protein maybe trigged and moved to the attacked sites to play antifungal function.The expression and activity of chitinase and glucanase were investigated in transgenic plant resistant to sheath blight and the wild-type (WT) inoculated with R. solani, the results indicated that two enzymes in transgenic plant have samilar transcription levels among infection phase, and activities were significantly higher than that of WT. Membrane lipid peroxidation and antioxidant enzymes were measured in transgenic rice and the wild-type (WT) inoculated with R. solani. The results indicated that malondialdehyde (MDA) generation was induced by pathogen infection. Compared with the WT, the MDA content in the transgenic plants was significantly lower with or without pathogen inoculation. Superoxide dismutase (SOD) activity was significantly stimulated by pathogen in transgenic rice compared to the WT. The results suggest that MDA content and SOD activity might play roles in resistance enhanced by the transgene.We also observed that transgenic seeds displayed lower germination ratio and seedling vigor, and seedlings were more sensitive to exogenous GA application compared with the WT. Gene expression detected by RT-PCR showed that GID1, SLR1, D1 and GID2, the key GA signaling genes had the expression levels same to the WT, but the GA catabolism GA2oxl gene transcription level was significantly higher in the Z100 plants than that in WT plants. These results indicated that plant growth and development stunt might attributed to deactivation of bioactive GA by the transgenes, GA2oxl expression would decrease the level of endogenous bioactive GA.In order to get a fundamental sight into the OsNPR1-mediated development abnormality, a rice whole-genome chip representing 51 279 units was used to profile globle gene expression patterns in OsNPR1-OX transgenic plants, we obtained 968 differentially expressed in OsNPR1-OX genes versus WT, the statistical data indicated that 796 genes exhibited up-regulated and 172 genes were down-regulated, and some genes were involved in the plant development.These studies provide molecular clues of the defense transgene-mediated development modification, and target genes for the further studies.