Study On Resistance To Sheath Blight For Transgenic GAFP Rice Lines

Rice is native to subtropical zone, which is one of the most important food crops in the world. It accounts for about half percent of cereal consumption in China. Sheath blight is one of the three most serious rice diseases worldwide. As the plant dwarfing, increasing plant density and fertilizer level, the disease aggravated progressively.The research on resistance to sheath blight disease develops rapidly and has become the most severe disease in part of southern rice-growing region of our country. An antifungal protein of gastrodia elata (GAFP) gene and the3homologous sequences have been cloned and added a guide peptide, constructed8plant expression vectors.They were transferred into Xudao3and R6547by Agrobacterium-mediated genetic transformation. Through the tissue culture get into seedlings, some transformants were obtained to determine if they improved on resistance to rice sheath blight disease by molecular detection for real transformant of single cope and field fungus inoculation for resistance identification. The major findings of this research are broadly the following aspects:1. Optimizing tissue culture system:(1) The choice of culture media for Xudao3. The calli of Xudao3were induced on N6culture medium, and they kept the growth strong with bright yellow and smooth surface. Its structure was dense. But the calli of Xudao3were induced on MS culture medium which color was dark yellow. Its surface was rough and watery. This condition goes against the subculture. So we choose N6culture medium.(2) The effect of adding ABA on rice callus induction. In comparison, adding certain concentration of ABA solution can help to inhibit excessive growth of the germ, makes callus more compact and hard, enhances the induced rate of callus.(3) The suitable concentration of G418for screening resistant calli. The concentration of selective agent for R6547callus was lower than that for Xudao3callus. G418concentration of the first screening medium on Xudao3is150mg/L, and the concentration of the second screening medium is250mg/L. G418concentration of the first screening medium on R6547is100mg/L, and the concentration of the second screening medium is150mg/L.2. Molecular detection of genetically modified rice plants. The GAFP’gene integration was tested by Southern Blot analysis. The transgenic transcription was verified by RT-PCR. Gene relative expression was detected by Real-time RT-PCR.3. Identification of resistance of genetically modified rice plants in fieldThe resistance of progenies of R6547with7expression vectors was tested, respectively.7vectors are G1、G2、SG2、G3、SG3、G4、SG4. The result showed that the resistance to rice sheath blight among all the T2generations with GAFP gene increased significantly compared with the control. The highest resistant transgenic line was transferred by SG2vector and alleviated1.15disease scale in2012.Because of obtained plants lately, T1populations with single copy of the transgene through SG1vector were screened to identify field resistance to sheath blight. The result showed that the resistance to rice sheath blight of positive lines increased significantly compared with the negative lines.