Expression Analysis Of Promoters Of Resistance And Defense Related Genes And Functional Identification Of PIANK1 In Rice

Rice (Oryza sativa) is one of the most important crops worldwide. The yield of rice is often reduced because of bacterial blight, fungal blast and other diseases. It is an effective method to cultivate new resistant plants by combination with biotechnology and conventional breeding. The promoter plays a key role for RNA transcription, and regulates gene expression. Research on expression pattern of promoters of important genes in the signaling pathways can contribute to clarify the molecular mechanism of disease resistance in plants. Up to now, a number of R genes are separated and cloned. Evidence is accumulating that they play key roles in disease resistance in plants. But, there is less reported about the expression regulation of R genes. Moreover, there is a limited recognition about the expression regulation of disease resistance related genes. So, in the paper, the characteristic of the promoters of ABI2?Xal?Xa21 and DRERF1 were analyzed. On another hand, OsPIANK1, an ANK repeats containing protein gene, was induced by Xanthomonas oryzae pv.Oryzae and Magnaporthe oryzae. Its expression and function was deeply researched. The main results are as follows:1. The results of histochemical GUS staining showed the activity of GUS driven by OsABI2 promoter was highest in roots especially in meristem, and lowest in leafs.There was significant expression in germination buds as well. The GUS activity induced by promoter of OsABI2 in T1 generation was determined by quantitative assay. The results showed that GUS activity was increased significantly by stresses(wounding, M.oryzae and drought) and hormones treatments (ABA, MeJA and SA). It was concluded that OsABI2 played a role in regulation of development, biotic and abiotic stresses.2. GUS activity driven by Xa21 promoter in roots was higher than in stems and leaves, especially strong in filling post. GUS activity was controlled by developmental phase, which was weak in leaf 3 stage, later progressively increased to leaf 8-9 stage.GUS activity driven by Xa21 promoter could be increased by methyl jasmonate and wounding treatment, and not influenced by drought, salicylic acid and abscisic acid, it suggested that Xa21 promoter might function in organ-, tissue- and developmental specific fashions. X421-mediated disease resistance depended onjasmonate signal pathway.3. GUS activity driven by Xal promoter in roots was higher than in stems and leaves, especially strong in filling post of roots. GUS activity was increased significantly treated with MeJA, and increased slightly with SA and ABA. These results suggested that the resistance of Xal was related to its tissue-specific expression in filling post. MeJA played a critical role in activity regulation of Xa1 promoter.4. GUS activity driven by DRERF1 promoter was significantly.increasd treated with SA and MeJA, and increased with blast fungus infection. The results indicated that DRERF1 promoter could respond to blast fungus as a induced promoter, DRERF1 participate the SA- and MeJA- mediated disease defense signaling pathway.5. OsPIANK1 promoter could drive a gradual.increasd GUS activity treated with infection of the rice blast fungus and wounding. It meaned that OsPIANK1 promoter was an inducible promoter response to blast fungus. Several stress related elemens were found in 1985 bp of promoter region, such as TCA-element, JERE, GCC. GUS activity driven by serial deletions of OsPIANK1 promoter was analysed. The results showed that -1101 bp and -563 bp were sufficient for GUS gene activation by MeJA and SA respectively. TCA-element possibly was necessary element for OsPIANKl response to SA. JERE, GCC and CGTCA-box possibly were elements response to MeJA.The result of subcellular localization indicated the OsPIANK1-GFP fusion protein was localized in nucleus. Overexpression of OsPIANK1 cDNA in transgenic rice enhanced the resistance against Magnaporthe oryzae. OsPIANK1 could induce several disease defense related genes (NHlU PR1b, PR5, PAL, AOS2) in SA and JA signaling pathways. These results suggested that OsPIANK1, which standed upsteam of NH1, regulated rice resistance to M. oryzae and that its function was likely associated with the SA- and JA-pathways.