Transformation Of Ds/Ac Transposon In Rice And Analysis Of Nulear Localization Of WRKY Gene Of Rice

With the completion of the sequence of the genome of rice, the most important task of rice Functional Genomics is the construction of rice mutant library. Transponson tagging system is the primary method for rice mutant library. A number of genes that do not express or express weakly under normal condition will be activated driven by Ds transponson containing 4 35 promoter. Obviously, utilization of Ds transponson containing 4x35 promoter will promote the research of rice functional genomics.We developed a rapid and high-efficient rice transformation technique mediated by Agrobacterium tumefaciens and used it to construct mutant population. Mature embryos of rice (Oryza saliva L. subsp. Japonica cv. Xiushui 11) were used to initiate embryogenic calli. Calli were co-cultivated with A. tumefaciens strain EHA105, which harbored Ti plasmid pDsBar!300H or plasmid NeaAc. pDsBar!300H carried maize Ds (Dissociation), Bar gene, Hpt gene and four 35S enchancers. NeaAc carried maize Ac transposase. We combined T-DNA tagging with transposon tagging and constructed transposon activativation tagging. Thirty-four independent transforming Ds lines and twenty-eight independent transforming Ac lines were obtained, among which 85 percent independent lines were identified as transgenic plants by molecular technological analysis. Most transforming Ds lines were one or two site insertion. In some TI generations, some plants were morphological mutants, including etiolation, high stalk and forked leaf.WRKY genes are a large type of transcription factors, which have been founded only in plant. This gene family may be very important in the regulation of disease-resistance and developement of plant. OsWrky gene has been cloned througth an elicitor induced rice (IR-72) cDNA library in our laboratory. Green fluorescent protein (GFP) is a new reporter gene and is widely used in plant research. OsWrky gene was fused with GFP gene, and constructed into pCambia1301 under the control of an ubiquitin promoter. The plasmid (pCU-OsWrky-GFP) was transformed into onion epidermal cells by particle bombardment. Bright green fluorescence was visualized only in the nuclear with bombardment of pCU-OsWrkyGFP by fluorescence microscopy, whereas the ubiquitin controlled GFP plasmid showed the whole cell distribution of GFP. The result suggested that OsWrky gene might function as atranscription factor in the nuclear.