Functional Analysis For The Rice Transcription Factors BTB/TAZ In Disease Resistance By RNA Interference Technology

Transcriptional regulation is the main control form of a series of gene expression in stress response, signal transduction and disease resistance. Transcription factors are key components and play important roles in the processes of transcription regulation. BTB/TAZ transcription factor (BTB/TAZ TF) is a specific plant transcription factor containing BTB and TAZ domains. It can specifically interact with TAZ cis-acting elements and regulate gene expression.In order to reveal the function of Ostaz gene in disease resistance or susceptibility, a pair of primers was designed and was used to clone the frangment covering the TAZ-type domain of Ostaz gene(LOC_Os01g66890.1)by PCR reaction, then the cloned fragment was used to construction the RNAi vector which was transformated into rice(Oryzae sativa L.cv.Nipponbare)by Agrobacterium-mediated transformation method. Transgenic positive rice plants were detected by PCR and histochemical method GUS. Expression change of Ostaz gene in transgenic positive plants were analyzed by FQ-PCR. Finally, inoculation resistance appraisal of transgenic rice were detected by Xanthomonas oryzae pv. oryzae Jxo1 and Xanthomonas campestris pv.vesicatoria Xv5. The main results were summarized as below:1. Cloning of Ostaz gene frangment. A pair of specific primers was designed according to the Ostaz gene sequence, PCR amplification was performed on rice Nipponbare DNA template. Ostaz gene frangment was successfully cloned by PCR. After by agrose gel electrophoresis, the target fragments were obtained and were cloned into pMD18-T vector, the successful construction of recombinant positive plasmids were identified by PCR, enzymes digestion and sequeneing.2. Construction of plant RNA interference vectors. pMD18-T-Ostaz was digested by Sacâ… and Speâ… ,and then ligated into the same double-digested pTCK303 interference vector and reverse recombinant vector pTCK303-Ostaz- was successfully constructed. pMD18-T-Ostaz was digested by Kpnâ… and BamHâ… , and then ligated into the same double-digestedc pTCK303-Ostaz- vector and RNA interference vector pTCK303-Ostaz was successfully constructed. At last, the pTCK303-Ostaz interference vector were identified by PCR reaction, enzymes digestion and sequeneing, And then were transformated into rice by Agrobacterium by using Oryza sativa L.cv. Nipponbare. Ostaz silent resistant calli and plants were obtained.3. PCR detection of Ostaz silent resistant calli and plants. Hyg resistant calli were detected in the process of Agrobacterium transformation by GUS,PCR and HR phenotype.8 RNAi transgenic indepent plants were obtained and were detected by GUS and PCR analysis. The result showed that 8 transgenic Ostaz indepent plants were positive plants.4. FQ-PCR analysis of transgenic Ostaz gene plants. RNA was extracted from leaves of transgenic plants and reverse transcripted into cDNA. FQ-PCR analysis showed that the expression levels of Ostaz gene in RNAi transgenic plants were lower than these non-transgenic plants. It indicated that Ostaz gene transcription is supressed and silent in transgenic plants.5. Resistance or suscept identification of transgenic plants.Inoculated with Xanthomonas oryae pv.oryae Jxo1 and non-host Xv5 on transgenic and non-transgenic rice plants. The result showed that WT,vector and transgenic rices were susceptible to Jxo1. After inoculating with Xv5, Compared with WT and vector rice plants, the transgenic rice plants were susceptible. It showed that BTB/TAZ transcription factor may be one of the important components in non-host resistance of rice.