The Genetic Engineering Disease Resistance Of Rice Blast Resistance Genes Conserved Structural

Rice is one of main food crops in China, the yield of rice of is very important to the nation's food security. But in the process of rice growth, the yield and quality of rice often be affected by the stress of rice disease. Rice-blast diseases, one of the major diseases in rice, can infection and disease on any time in the process of rice growth. The most economic and efficient way to control rice disease and insect pests, is using genetic engineering means to get broad spectrum, high efficiency, durable resistance, depend on rice blast resistance genes.Plants have evolved structurally conserved resistance proteins containing a N-terminal coiled-coil (CC) or Toll/interleukin-1 receptor (TIR) domain, a central nucleotide-binding (NB) domain and C-terminal leucine-rich repeats (LRRs). And LRR usually participates in pathogens'recognition and NB-LRR protein function switch. Then the NB-ARC structure domain usually appears the phenomenon of immunity, so that the R gene cause immune response. But research on flax rust resistant protein L6 and L7 structure function of TIR domain have shown that structure of TIR domain enough to trigger cell death and mediated immune responses. The barley powdery mildew resistance protein MLA10 its CC domain contain a highly conserved EDVID structure, and single CC domain or MLA10 fragments which contain of the CC domain can trigger cell death. The rice blast resistance genes also encode CC/TIR-NB-LRR protein. However, there are not any record that the CC domain alone of these proteins is sufficient to activate immunity.In our preliminary investigation, we found that the CC domain alone of Pi36 can trigger cell death in Nicotiana benthamiana and rice protoplasts, and its ectopic expression from pathogen-inducible promoter confers resistance against powdery mildew in Arabidopsis. In this work, based on CC domain alone of Pi36 can generate phenomenon of autoimmunity. we expressed the CC domain alone of Pi36 in rice, in order to explore the relationship between the activation and the immune response of rice, and reveal the mechanism of rice blast resistance genes mediated immune response, and find new ways to get resistance resource buy gene engineerings. Data from this project should provide insight into the molecular mechanism of Pi36-meidated resistance against rice blast disease and reveal the potential value of Pi36 CC domain in engineering broad-spectrum disease-resistant rice via biotechnology. Preliminary results obtained the following:1. According to the preliminary investigation, We successful amplified the divided resistance genes fragments, got Pi36CC (1-134aa) PCR. Then constructed these chimeric genes into the plant expression vector of pCAMBIA1300, and use rice blast induced promoter pPBZ1, and to join the GFP as marker gene. Final we got a contain pPBZ1::Pi36 CC (1-143) rice expression vector.2. We Choosing japonica rice varieties TP309 as receptors materials, conducted rice genetic transformation, have got transgenic To generation plants. Then transplanting rice to genetically modified (gm) special field. We got homozygous T2 generation plants use the methods of hygromycin resistanc selection and target gene PCR selection.3. Inoculating gene transgenic plants with rice blast fungals, the results show that the gene transgenic plants confer resistance to rice blast. The least lesion number and lesion size in gene transgenic plants showed significant reduce comparing with the WT. These results seemed to be due to the highest expression level of Pi36 CC in this line. Therefore, our data indicate that ectopic expression of Pi36 CC can indeed lead to disease resistance in rice.4. Resistant phenotypes are usually accompanied by the up-regulation of defense-related genes, thus, we examined the transcription of some defense-related genes in gene transgenic plants. As anticipated, the gene transgenic plants displayed significantly higher expression of OsPR1 and OsPR5 than WT. Meanwhile, PTI-related genes OsKS4 and OsNAC4 was significantly higher expressed in gene transgenic plants, suggesting that the rice blast defense responses mediated by Pi36 CC domain appear to be involved in PTI signaling. In summary, upon the control of pathogen inducible promoter pPBZ, the Pi36 CC transgenic lines showed rice blast induced defense responses and enhanced resistance rice blast.