Cloning And Preliminarily Functional Analysis Of OsOGCP, OsDXR And OsRPA3a Genes Related To Rice Blast Resistance

Rice is one of the most important food crops in the world. Excavating disease resistance genes and developing durable disease resistance rice varieties have been the hotspot for breeding till now. Based on the analysis of transcriptomics data of japonica variety Yunyin induced by Magnaporthe oryzae, we screened three genes associated possibly with rice blast resistance, (1) the first gene was AK068090, temporarily named OsOGCP encoding mitochondrial carrier protein; (2) the second gene was AF367205, temporarily named OsDXR encoding 1-deoxy-D-x-ylulose 5-phosphate reductoisomerase; (3) the third gene was AK070400, temporarily named OsRPA3a encoding nucleic acib binding protein with OB-fold domain. In order to determine the function of these three genes, we cloned and carried out their expression analysis from blast resistance variety Yunyin and blast susceptible variety LTH. The main results were as follows:1. The sequences including coding region and the promoter region for those three genes were cloned, with cDNA as template from Yunyin and LTH. The results indicated that the coding region of OsOGCP, OsDXR, OsRPA3a and the promoter region of OsRPA3a had no difference between Yunyin and LTH, and they were all consistent with the login sequence at NCBI. The homology of sequence for promoter region of OsOGCP and OsDXR between Yunyin and LTH was 95.98% and 98.78% respectively.2. The full-length cDNA sequences of these three genes were cloned by RACE technology, respectively. Then the corresponding phylogenetic analysis was carried out using MEGA5.1, in order to initially analyze the function of these three genes respectively. The results indicated that the relationship between the gene OsOGCP and the gene encoding 2-oxoglutarate carrier protein in Panicum miliaceum was near with each other, the relationship between the gene OsDXR and the gene encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase in Alpinia officinarum was near with each other, the relationship between the gene OsRPA3a and the gene encoding retrotransposon protein in Zea mays was near with each other.3. The expression level was analyzed on these three genes infected by Magnaporthe oryzae from Yunyin by real-time PCR at different time point. As time went on, the expression of these three genes had some changes. So we believed that these three genes might be associated with rice blast resistance.4. The vectors of these three genes including plant expression vectors, RNAi interference expression vectors and plant subcellular localization vectors were constructed and transformed into blast resistance variety Yunyin. Then obtained the positive plants so as to carry out the research on their functional analysis and resistant mechanisms.