Molecular Cloning And Functional Analysis Of Related Resistance Genes Induced By Sheath Blight In Rice

Rice is the most important grain crop in the world. Howerver, the disaster of sheath blight has caused rice production drop at some extent in part of China. In our previous work, 52 differential expressed cDNAs from rice induced by sheath blight infection were identified through suppression subtractive hybridization. Among them, 16 clones had homology with known annotated genes, including 10 defense response related genes. In this study the study functions of these genes were investigated.First, RT-PCR was used to analyze the expression pattern of these 10 clones in rice plants challenged by Rhizoctonia solani. The results showed that the expressions of clones 4D7, 5A5, 7G3 were reduced, and the expression of clones 4B11, 8A1 and 5J12 were induced, but no obvious in the clone 2B4, 4C7, 9B12, 1C5．One of the differential clone 4B11 contains an inserts of 305bp, by BLAST at GenBank, 4B11 shows 96% similarities to genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit in rice. Another clone 8A1with an inserts of 505bp has 98% similarities to genes encoding Ubiquitin-conjugating enzyme E2 in rice. Homologous cloning method was used to obtain the full length sequence of 4B11 and 8A1, they were designated as OsRBCS and OsUBC2, respectively.To elucidate the role of these two genes in rice disease resistance, the expression patterns of OsRBCS and OsUBC2 in rice after induced by JA, ABA PEG were first analyzed. RT-PCR results showed that there were basal expression in rice for these two genes. However, OsRBCS was quickly activated by JA, but with ABA and PEG treatments, the result is opposite. The expression of OsUBC2 kept the same after JA and ABA treatments, but little increased after PEG treatment. These results suggested that the inducible expression of OsRBCS is associated not only with sheath blight infection, but also with the stress response in rice, OsUBC2 is probably associated with drought stress response.To better understand the function of OsRBCS and OsUBC2 in disease resistance response, the OsRBCS and OsUBC2 coding region were cloned into a plant transgenic binary vector pCAMBIA1301-Ubiquitin. And OsRBCS was transferred to rice though Agrobacterium tumefaciens. 9 transgenic rice plants were obtained. The staining test showed that the GUS gene expressed in all parts of transgenic plants.In a summary, our research will facilitate the understanding of molecular interaction between rice and sheath blight fungus.