Identification And Characterization Of OsWRKY7 And OmWRKY7

Rice is an important food crop worldwide.Bacterial blight(BB)is an vascular disease caused by Xanthomonas oryzae pv.Oryzae(Xoo).It seriously affects the rice yield and grain quality when disease outbreaks.Identification and characterization new genes of disease resistance and generate disease-resistant varieties is an effective way to control the disease.The wild rice is a natural gene pool for rice genetic improvement.It has been reported that Oryza meyeriana has a stronger resistance to various disease than cultivated rice.For rapid discovery of new disease resistant genes,we generated a full-length cDNA libraries with Xoo treated and mock inoculated O.meyeriana leaves in this study.Clones from the library were then sequenced and a homologous gene of OsWRKY7,OmWRKY7,was chosen for further study.The CDS of OmWRKY7 were cloned by RT-PCR according to the sequence results and the OsWRKY7 was also cloned with cDNA sample of cultivated rice(Oryza sativa L.ssp.Japonica cv.Nipponbare)for comparison.In plants,WRKY gene family members play a variety roles in disease resistance,the function characterization of two homologous genes,OsWRKY7 and OmWRKY7,should help us better understand the role of WRKY genes in BB resistance in rice.1.The expression pattern of OmWRKY7 and OsWRKY7Subcellular localization analysis confirmed that OsWRKY7 and OmWRKY7 were nuclear-localized genes.To investigate the tissue expression patterns of OsWRKY7,qRT-PCR was performed.The result show that OsWRKY7 was expressed in leaf,root,stem and flower,and have a high expression level in leaf.To investigate the expression pattern of WRKY7 under different plant growth regulators and environment stress,wide type plants(Nipponbare)were treated with seven different plant growth regulators(PGRs).For environmental stress experiments,rice plants were subjected to salt stress(200 mM NaCl),drought stress(100 g/1 PEG6000).The leaf and root sample were harvested 24 h after treatment,and the gene expression level were investgated by qRT-PCR.The result show that the gene expression level in leaf was higher than the control when treated with IAA.It also showed a strong induction in root when treated with KT.In addition,the gene expression levels in both leaf and root samples were found significantly higher under salt stress compared with control samples.All these findings suggest that WRKY7 gene is involved in IAA,KT signaling and the response to some abiotic stresses.2.The promoter analysis of OsWRKY7To determine the expression pattern of OsWRKY7,the putative promoter was isolated and fused with the GUS reporter gene and the japonicatype rice Nipponbare was used for the transformation.Histochemical detection of GUS activity showed that the WRKY7 gene express highly in root,leaf and flower.The analysis also showed that WRKY7 can be indunced by the treatment of KT,IAA and the Xoo inoculation.These results suugest that WRKY7 is involved in plant hormones signaling and the Xoo defence response signaling.3.Overexpression and bacterial blight resistance analysisOverexpression of target gene nowadays has become an useful method for gene function research.OsWRKY7 and OmWRKY7 were amplified and transformed into mature embryos developed from seeds of a japonica cultivar Nipponbare.Positive T0 and T1 transgenic plants were selected by PCR analysis,and positive T1 plants were used for bacterial inoculation.Lesion lengths in Om WRKY7 overexpression plants were shorter than that in the susceptible wild type,suggesting that the expression levels of this gene may correlate with resistance to BB.