Cloning And Functional Analysis Of Promoters OsABAM16p And OsN1p Of Rice And Resistance Of Hrfl Transgenic Rice To Ustilaginoidea Virens

To isolate the plant's genes up-regulated after rice inoculated by Magnaporthe grisea, we used suppression subtractive library-based approach and got 15 differentially expressed genes. Semi-quantitative RT-PCR results showed that OsABAM16 and OsN1 were up-regulated markedly. It is suggested that OsABAM16 and OsN1 may play an important role during rice-M. grisea interactions. We analysed about 1000bp promoter region of OsABAM16 and OsN1 by promoter prediction software PLACE and PlantCARE and found that there were many conservative elements of high plant promoter and a variety of cis-elements, such as box-W1, EIRE, MBS and TCA.To make a cloning and functional analysis of these promoters, the 1003bp putative promoter region of OsABAM16 gene was amplified and substituted for the 35S promoter in vector pBI121 which had GUS or GFP to construct two new plant expression vectors pBIABAM16p and pBIMGp. At the same time, the 1089bp putative promoter region of OsN1 gene was isolated from the genomic DNA of Nipponbare.5 terminal deletions with a length of 881,489 and 245bp were fused with gus gene, and plant expression vectors pBIN0.9p,pBIN0.5p and pBIN0.2p were constructed respectively. All of vectors were transformed into Agribacterium tumefaciens EHA105 by the freeze-thaw method.The vectors were transformed into the rice calli through Agrobacterium-mediated transformation. Genomic integration and gene expression were determined by PCR, PCR-Southern blot and GUS activity staining. The results showed that the gene was integrated into the genome of transgenic rice.The promoter activity, tissus specificity and pathogen inductivity were analyzed as well. The promoter OsABAM16p drived GFP expressed in onion epidermis cells. Analysis of GUS activity showed that the gus expressed in root higher than in stem and blade. GUS activity was significantly increased in transgenic plants inoculated with M.grisea. After spraying with 5mM SA or 0.5mM MeJA, GUS activity in leaves of the transgenic plants was 3.2 and 2.8 times respectly higher than untreat control.Analysis of GUS activity showed that OsN1p (881bp) drived the gus expressed in transgenic callus. GUS activity analysis revealed that the callus which had pBIN0.2p unstained, indicating that the minimal promoter of OsNl is 489bp. GUS activity was significantly increased in transgenic plants (pBIN0.9p) when they inoculated with M.grisea. 6 hours after spraying with 5mM SA or 0.5mM MeJA, GUS activity in leaves of the transgenic plants (pBIN0.9p) was 5.9 and 2.4 times respectly higher than untreat control.It was confirmed that M.grisea, SA and MeJA were inductive factors for OsABAMl6 and OsNl promoter. The functional analysis of OsNlp and OsABAM16p were helpful to understand the mechanisms of gene expression and to utilize in generation of transgenic plants.Nine hrfl transgenic plants and untransgenic cultivar R109 were tested for resistance to Ustilaginoidea virens by inoculating with conidia. Injection inoculation in greenhouse showed that transgenic plants B12-2m, B12, HTRP2 and NJH12 were more resistant. Field tests in Nanjing of Jiangsu Province and Qianshan of Anhui Province confirmed that NJH12 was resistant to U.virens in the field with control effect 65%.