Molecular Cloning And Functional Analysis Of EREBP-type Transcription Factor Gene OsBIERFs And An F-box Gene OsHIHN-n11

In the previous studies, we demonstrated that benzothiadiazole (BTH) could induce rice resistance against blast, bacterial leaf blight and sheath blight, based on which over 200 differential expressed cDNA were cloned and identified through suppression subtractive hybridization. Among them, BIHI-n2 with inserts of 567bp showed significant similarities to plant genes encoding known EREBP transcription factors. In order to elucidate the biological function of the putative ERF gene and the role in rice disease resistance, this study cloned and identified full-length cDNA of the gene and designated as OsBIERFl. In addition, A bioinformatics-based approach was used for cloning of OsBIERF2~4.To obtain the full-length cDNA of OsBIERFl gene, the 5'-ends and 3'-endsflanking the sequence in the clone BIHI-n2 was amplified by PCR using phage DNAprepared from a rice cDNA library as template. The 1613bp long sequence contained anopen eading frame (ORF) of 1200bp was retrieved through sequencing and assembling,which predicts to encode a protein of 399 amino acids contained a conserved ERFdomain. OsBIERF2~4 genes were cloned using bioinformatics-based approach. TheOsBIERFl cDNA sequence was used to search the rice genomic sequence database forputative ERF protein-encoding genes and cDNA sequences of the putative ERF geneswere predicted. The predicted ERF gene cDNA sequences were then used to search riceESTs (expressed sequence tag) and putative cDNA sequences of the ERF genes wereassembled. The specific primers were designed According to the assembled cDNAsequences of the ERF genes and used to amplified the full-length cDNA of OsBIERF2~4by PCR. The full-length cDNA sequences of OsBIERF2, OsBIERFS and OsBIERF4 were1116bp, 1199bp and 1214bp, respectively, with predicted ORF of 708bp, 912bp and987bp, respectively. The genes predict to encode proteins of 235, 303 and 328aarespectively and all of them also contain the conserved ERFdomain. Alignment and phylogenetic tree analysis revealed that OsBIERFs belong to different classes of plant ERF proteins. OsBIERF3 and OsBIERF2 are grouped into class I and class II respectively, OsBIERFl and OsBIERF4 are members of the Class IV. Recombinant OsBIERF3 protein expressed in and purified from E. coli was bound to the GCC box cis-element sequence for the EREBP transcriptional factors. Subcellular localization analysis revealed that the OsBIERF3 protein was localized in the nucleus in the plant cells.In the analysis of the expression patterns of OsBIERFs in BTH-induced disease resistance response and in the compatible/incompatible interactions between rice and the blast fungus Magnaporthe grisea, we observed Expression of OsBIERFl, 0sBIERF3 and 0sBIERF4. They were induced by treatments with benzothiadiazole and salicylic acid, chemical inducers capable of inducing disease resistance response in rice. In the benzothiadiazole-treated rice seedlings, expression of OsBIERFl, OsBIERF3 and 0sBIERF4 was further induced by infection with Magnaporthe grisea, the rice blast fungus, as compared with those in water-treated seedlings. OsBIERFl and OsBIERF3 were activated in an incompatible interaction but not in compatible interaction between rice and M. grisea. Moreover, OsBIERF1, OsBIERF3 and OsBIERF4 were also up-regulated by various abiotic stress including salt, clod, drought and wounding. These results suggest that OsBIERF proteins may participate in different signaling pathways that mediate disease resistance response and stress responses to abiotic factors in rice.To better understand function of OsBIERF3 in disease resistance response, we performed a functional analysis of OsBIERF3 in transgenic tobacco plants. The OsBIERF3 coding region was cloned into a plant transformation binary vector CHF3pp2p212 under control of the CaMV 35S promoter. A total of 11 independent transgenic tobacco lines were obtained through the Agrobacterium-mediated leaf disc transformation and confirmed by PCR. Southern analysis of the transgenic lines showed that the OsBIERF3 gene was integrated into the tobacco genome. Northern...