Isolation And Functional Characterization Of Systemic Acquired Resistance Related Gene OsMOS3in Rice

Rice is one of the main grain crops in our country. Due to blast, sheath blight and bacterial blight, the yield and quality of rice have been seriously affected. In this research, using rice genome information and bioinformatics, one broad-spectrum disease resistance gene OsMOS3was cloned in rice. Second, we used transgenic technology to realize over-expression, RNAi and fusion expression of OsMOS3. Through transgenic rice to bacterial blight pathogen resistance study and salicylic acid content analysis showed that the OsMOS3took part in broad-spectrum disease resistance function. Detailed results are as follows:1. OsMOS3and mir interference fragment cloningFirst, using NCBI blast, we got a homologous sequence of Arabidopsis AtMOS3gene in rice genome, named OsMOS3. And using rice cDNA as template and specific designed primers, we amplified OsMOS3A segment,1475bp, OsMOS3B segment,1543bp, and finally through the overlapping PCR (overlap) we developed OsMOS3full-length cDNA (A+B),3018bp. According to the features of domain analysis in OsMOS3, we selected a conservative and contained a functional domain sequence as an interference fragment mir,250bp. Then we constructed recombinant plasmids: CPB-OsMOS3, CPB-2F and P1302-OsMOS3-GFP.2.35S-OsMOS3-GFP subcellular localizationVia gene gun,35S-OsMOS3-GFP fusion expression vector was transformed into onion epidermal cells, and then used fluorescence microscopy to detect subcellular localization of35S-OsMOS3-GFP. The result showed that OsMOS3instantaneous located in the nucleus.3. T0generation of transgenic plant positive testTransformed by Agrobacterium EHA105, we got3strains of OsMOS3over-expression transgenic plants,1strain of OsMOS3-RNAi transgenic plant. The PCR on amplification of selected markers HPT gene showed that the4strains of transgenic plants were positive plants. Then we analyzed semi quantitative expression of OsMOS3. Gel results showed that OsMOS3expression level in OsMOS3over-expression plant was significantly higher than that in the original control plants, however, OsMOS3-RANi transgenic plants displaied that OsMOS3gene expression was silencing. The results indicated that the OsMOS3and interference fragment mir had been transform into the plant and regular expression. 4. T0generation of transgenic plant resistance detectionBy detection of vivo free salicylic acid (free SA) contents in OsMOS3over-expression transgenic plants, OsMOS3-RNAi transgenic plants and the original control plants, the results showed that free SA in OsMOS3over-expression transgenic plants were significantly higher than those in control plants and RNAi plants By Xoo pathogen incubation of OsMOS3over-expression transgenic plants, OsMOS3-RNAi transgenic plants and the original control Nipponbare in seeding stage and booting stage. The results showed that the disease resistance of OsMOS3over-expression transgenic plant was obviously improved however, OsMOS3-RNAi was disease sensitive.To sum up, OsMOS3took part in SAR signal pathway by SA accumulation in plants, and improving the disease resistance of whole plants.