The Functional Study Of Rice WRKY68 In Xa21-mediated Resistance Response

Background: Now nearly half of the world population depends on rice for food. Bacterial leaf blight is one of the most serious bacterial disease infection rice(Oryza sativa L.). The earliest cloned gene Xa21 confers a broad spectrum resistance against the bacterial blight. WRKY transcription factors as one of the largest family in plants play an important role in plant growth, metabolism, dealing with biotic and abiotic stress, oxidative aging regulation and so on. So it has a great significance on studying the function of WRKY transcription factor in the pathway of Xa21 mediated resistance to bacterial leaf blight.Research purposes: To detect the expression pattern of WRKY68 in the rice inoculated with Xoo and different growth stages with WRKY68 specific antibodies. The WRKY68 protein was expressed in vitro for analysis its binding properties with W-boxes in pathogenesis-related genes involved in Xa21-mediated resistance response. Constructing the RNAi vector of WRKY68 gene for getting transgenic plants which with the silenc of WRKY68 gene. From the genetics angle analysis the function of WRKY68 in the pathway of Xa21 mediated resistance to bacterial leaf blight.Materials and methods: Through forecasting the Os WRKY68 epitopes to synthetic peptide immune New Zealand white rabbits for preparation of polyclonal antibody of anti-WRKY68. Western blotting(WB) was used to detect the expression of WRKY68 protein in rice leaves after inoculated with Xoo and normal growth and development. The Microscale thermophoresis(MST) was used to test the affinity of WRKY68 protein and W-box which in the pathogenesis related gene. The raw data of Os WRKY68 transcriptional information was downloaded from Rice genome annotation project, Massively parallel signature sequencing(MPSS) and Rice gene coexpression database(Rice XPro) respectively. Domain prediction was carried out using the SMART software. The RNAi vector of WRKY68 gene was constructed and 4021 as transformation receptor in transformation.Results: WB was performed to examine the Os WRKY68 protein expression in the leaves at different time points after inoculation with Xoo to 4021. The results showed two clear bands, one was closed to the predicted molecular weight(32 k Da) of the target protein, which showed constant intensity, another one, designated at Os WRKY68+, was bigger than the prediction MW, which appeared at 3 days post inoculation, and its intensity increased with the inoculation time prolonged. To further understand Os WRKY68 protein expression patterns in the interactions between rice and Xoo, samples were collected at 0h, 3d and 5d after inoculation among resistance responses, susceptible and mock responses. Compared with the mock control, Os WRKY68 expression showed a similar pattern in resistance and susceptible reaction, the induced band appeared in the late stage, but not detectable in mock. MST showed the dissociation constant(Kd) was 25 n M for Os WRKY68 with W-1b, when the core sequence of W-box was mutated, the Kd of Os WRKY68 with m W-1b was 353 n M. Os WRKY68 binds to both W-10 a and m W-10 a with similar Kd(33 n M for W-10 a and 39 n M for m W-10 a respectively). Additionally Os WRKY68 does not bind with W-1a and BS65, whether the W-box core sequences mutated or not. The leaves in different growth stage of rice was detected showed that a single band consistent with predicted molecular weight of Os WRKY68 with constant intensity. Transcription data showed that Os WRKY68 gene was transcripted in rice leaves, inflorescence, anther, pistil, seeds, endosperm and embryo, the high transcriptional signal was detected in pistil. Os WRKY68 transcriptional levels were slightly down-regulated after inoculation with Xoo, but transcriptional levels no obvious changed when challenged with M. grisea. Os WRKY68 transcriptional levels were induced at 14 days seedling leaves by cold, drought and Na Cl treatments and enhanced by abscisic acid(ABA) treatment in root. The successful of construction RNAi vector of WRKY68 gene and the transgenic plants were obtained.Conclusion: The induced band of rice after inoculation and the combination of WRKY68 with W-box probe, illustrated that WRKY68 plays an imporent role in the Xa21-mediated bacterial blight resistance response via regulation the downstream PR1 b and PR10 a. WRKY68 proteins were constitutively expressed in different growth period reveal that it also has a function in normal growth and development. The obtaining of transgenic plants was prepared for analysis the function of WRKY68 from genetics.