Function Analysis Of The Promoter Region And Coding Region Of Pib Gene By Transgenic Rice

Rice blast, caused by the fungus Magnaporthe grisea (Hebeert) Barr (anamorph Pyricularia grisea Saccardo), is the most devastating plant disease of cultivated rice (Oryza sativa L.) all over the world. Breeding for durable resistance to Magnaporthe grisea seems to be difficult since examples of long-lasting resistance are very rare. Plant genetic engineering is thus regarded as an approach for providing resistance to rice blast in rice varieties.The Pib gene is the first R gene conferring resistance to rice blast cloned via a map-based cloning strategy. The deduced amino acid sequence of Pib gene product contains a NBS-LRR motif. Interestingly, RNA gel blot analysis of the Pib gene revealed that their expression is regulated by environmental signals such as temperature, light, water availability and chemical treatments such as jasmonic acid, salicylic acid, ethylene and probenazole. The Pib gene was obtained from rice genome library and its DNA sequences kept the original state, which providing a good material for studying the function of resistance gene in molecular level.Four binary vectors, pNAR701, pNAR702, pNAR703 and pNAR704, which carrying the promoter region and coding region of Pib gene, sense and antisense DNA fragment of the third exon and the ORF of Pib gene respectively, were transferred into japomca rice cv. R109 and Kittake by an Agrobacterium-mediated method. And total 27 independent transgenic rice plants were obtained.All transgenic rice plants were confirmed by PCR, Southern blot and segregation of hygromycin resistance in T₀ seeds. Northern blotting analysis also confirmed the transcript of Pib gene in T₁ transgenic rice plants. Challenge inoculation of T₁ transgenic plants with Magnaporthe grisea race ZD₁ and ZG₁ at seedling stage and in intro leaves at heading stage, revealed that T₁ transgenic plants carrying Pib gene were more resistant than the untransformed wild type, while transgenic plants carrying the antisense fragment were more susceptible. The GUS fluorometric measurement was performed in pNAR604 T₂ transgenic rice plants.The results of GUS fluorometric measurement showed that after 24h dark treatment, the GUS activity increased in roots and leaves. What's more the GUS activity in roots was 7 times higher than that in leaves, which indicated the GUS expression was organic characteristic. Interestingly, there was an evident diurnal rhythm in roots and leaves with the lowest level of GUS activity at 15:00 and the highest at 03:00. Treated with differential temperatures through one day or in dark, the average GUS level changed followed the temperatures. Our findings suggested that the promoter of pib gene was regulated by different environmental factors.