Genetic Improvement Of Rice For Disease Resistance And Salt Tolerance By Overexpression Of OsiWRKY And OPBP1, Two Different Transcription Factors

Plants are constantly subjected to a variety of biotic and abiotic stresses. Through evolution plant has developed a delicate mechanism to perceive external signals and to manifest adaptive response with proper physiological and morphological changes. Stress gene induction occurs primarily at the level of transcription, which is mediated by transcription factors. Through binding to conrresponding cis-acting element, a single transcription factor can influence the transcription of different target genes that allow plants to response optimally to their changing environment. ERF and WRKY are two transcription factors that unique to plants, and play an important role in regulating plant responses to stresses. We isolated a rice WRKY gene, OsiWRKY that may function in response of rice plants to pathogen attack. Using the yeast one-hybrid screening system, OPBP1 was cloned that specifically interact with the GCC-box in the osmotin promoter. Overexpression of the OPBP1 in transgenic tobacco enhanced salt tolerance in rice. In this study, OsiWRKY gene was constructed under the control of ubiquitin promoter into a binary vector for plant transformation, and introduced into rice by Agrobacterium-mediated method. Some of the transgenic plants showed a broad spectrum disease resistance. Meanwhile, we co-transformed the two different transcription factors, OPBP1 and OsiWRKY into rice by micropartical bombardment. Overexpression of the two genes in transgenic rice not only exhibited high resistance to pathogen but also showed improved tolerance to salt stress. Results were summarized as follows:1. We constructed the plant binary vector, in which the isolated OsiWRKY was under the control of maize ubiquitin promoter, and introduced into rice embyrogenic calli via Agrobactrium. 31 Hygromycin B-resistant plants were obtained. There are 90% of transgenic lines were PCR positive. DNA gel blots showed that the integration of the transfer gene into rice genome with low copy number(1~2). Further, the resultsof RNA gel bolts indicated the OsiWRKY gene overexpressed in transgenic plants. Genetic analyses showed that the foreign gene were transmitted to the progeny in Mendelian fashion. The transgenic rice lines overexpressed OsiWRKY were challenged by Magnaporthe. grisea and Xanthomonas. oryzae pv. oryzae, respectively. Compared with non-transgenic control, the transgenic plants showed an enhanced disease resistance against both the two pathogens.2. To determine OsiWRKY genes biological function, using double-stranded RNA interferance (dsRNAi) of OsiWRKY, our lab obtained a dsRNAi transgenic plants. In this study, we evaluated the disease resistance to Magnaporthe. grisea in the third generation (T2) of overexpression transgenic lines, the second generation (Tl) of dsRNAi transgenic lines and the control line. Both control and dsRNAi transgenic lines exhibited the same level of disease susceptibility to blast infection, respectively. But all overexpressed transgenic T2 lines showed enhanced resistance to blast, as indicated by significantly lesion numbers. These results demonstrated that the overexpression of OsiWRKY gene in rice may result in improved resistance to pathogen, and the enhanced disease resistance was consistent in Tl and T2 generation of overexpression transgenic plants. RNA gel blots showed that the OsiWRKY gene was expressed constitutively in T2 overexpression transgenic lines but not in control and Tl dsRNAi transgenic plants under normal growth conditions. Further, the endogenous level of OsiWRKY in dsRNAi lines was rather low than control and overexpression lines by inoculation of with Magnaporthe. grisea. These results indicate that the expression of endogenous OsiWRKY was blocked almost completely in dsRNAi transgenic lines under the induction condition. OsiWRKY gene might be as a positive factors involved in regulating gene expression for broad disease resistance of rice.3. Because OsiWRKY overexpression lines exhibited increased resistance to pathogens, we analyzed the expression of some pathogenesis-related (P...