| Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases in the world. Development of resistance varieties is one of the most economical and effective way to control the disease. However, Xoo always have pathogenic differentiation, therefore, it is important to develop varieties with broad spectrum and durable resistance.Y73is a progeny of asymmetric somatic hybridization from an elite japonica rice cultivar (Dalixiang) and a wild rice (Oryza meyeriana) and shows high resistance to bacterial blight. By optiming AAM suspension culture medium and temperature, the transformation of Y73was raised to66.5%. Using Agrobacterium-mediated transformation, we explored the expression pattern of Agrobacterium infection-related gene preliminarily. Meanwhile, in microarry assay the expression of a NBS-LRR gene was found induced after inoculation with Xanthomonas oryzae pv.oryzae in Y73. Due to its similarity to Rpl in maize, the CDS of the gene was cloned and named OsRP1L1(Oryza sativa Rp1-like1). A series of experiments were carried out to investigate gene functions. The major points are as follow.1. Obtained three homozygotes transgenic rice lines (over-expression) by Agrobacterium-mediated transformation, which is stably expressed in progeny.2. In order to get the potential interactors of OsRP1L1, the BiFC (bimolecular fluorescence complementation) vectors were constructed. The results indicated that there didn’t have significant any self-interactions in tobacco transient expression system. Now, we are preparing to screen the interactors with Y2H (yeast two-hybrid) system.3. Our data showed that the expression of OsRP1L1was repressed by jasmonic acid and salicylic acid high salt and high temperature treatment, and the expression of OsRP1L1can be induced slightly by GA24h after treatment. All these findings suggest that OsRP1L1is involved in both growth-regulator-induced signaling and the response to some abiotic stresses. Microarray data showed that there are26genes which has2-fold difference compared with wild type, among them25genes were up-regulated, and only one gene were down-regulated. By searching against public databases, we noticed that there were seven genes co-regulated by OsRP1L1and OsbZIP46. In conclusion, OsRP1L1have a potential function in rice defence signal transduction.4. Different Xoo strains were used in the inoculation experiments, and the results revealed that the disease resistance of rice to particular races were influenced by over-expression of OsRP1L1.5. The OsRP1L1::sGFP fusion expression vector was constructed, and then used for plant transformation. With vibration slice technology and confocal microscopy, we found that green fluorescent mainly concentrated in the nucleus. So we speculated that OsRP1L1mainly localized in the nucleus.Furthermore, to accelerate the efficiency of identification in Agrobacterium-mediated transformation, the HPT-mCherry fusion marker was developed. The major results are as follow:1. The vector which contains HPT-mCherry fusion maker was constructed by using overlaping PCR.2. Using Agrobacterium-mediated transgenic experiments, we confirmed that HPT-mCherry fusion maker can be used to screen positive transgenic callus, and accelerate the efficiency of identification.3. The PCR identification results were consistent with the results observed in fluorescence microscope assay. Absolute quantification PCR also revealed that mCherry-HPT fusion tag can be efficiently-expressed in transgenic callus and plants. Thus, proves that the fusion maker can improve the efficiency of transgenic identification. |
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