Wrky Family Of Cloning Osddwrky Rice Genome Analysis

Two rice mutants (named Osddl and Osdd2), delayed in development for about one month were isolated by screening a rice insertional pool generated with Ac/Ds transposone system derived from maize. Flanking sequences of the insertion sites were isolated by inverse-PCR and Tail-PCR methods. Based on the Blast analysis and other studies, Osddl mutant was a multi-copy-insertion mutant, and one of the insertion sites was in an NPTII like transposase gene, whereas Osdd2, a single-copy-insertion mutant was disrupted at a gene encoded WRKY transcript factor. Because the phenotype of Osdd2 mutant was segregated with the Basta resistance, it suggested that the mutation was arose by disruption of the WRKY gene.Osdd2 gene existed as a single copy gene in the rice genome based on the result of Southern blot analysis. We took a PCR method to clone the cDNA of Osdd2 gene. The possible profile of transcription of the gene was predicted based on the PACs of RGP, and the primers were designed accordingly. PCR products amplified from an elicitor induced cDNA library were sequenced and analyzed. To our surprise, one of the products contained the WRKY domain, and the other not. But both had NLSs and many regulation sites such as for phosphorylation and glycosylation. The results suggested there were two possible splicing processes. On the other hand, the expression ofOsdd2 gene was not determined yet by Northern blot analysis of mRNA extracted from rice (cv. Zhonghua 11) at different growth phases. So, the promoter region ofOsdd2 gene was cloned and fused with a Gus gene, in order to speculate the Osdd2 expressing patterns. The chimeric gene was transformed into rice calli by particle bombardment. Many resistance calli were obtained, and Gus activity was detected.In order to confirm the causality of the mutant phenotype and Osdd2 gene, we took an approach of functional knockout ofOsdd2 gene, with the method RNAi (RNA interference). Two RNAi constructs were made to validate the function of Osdd2 gene. One contained the conserved WRKY domain and the other did not. At present, we obtained many transgenic rice lines with one of the plasmids.Meanwhile, we annotated the whole family of WRKY genes in rice genome. There were more than one hundred members in the family. WRKY genes of rice were found more complex and diverse than those in Arabidopsis. The WRKY genes of rice and Arabidopsis were not parallel at gene level but at subgroup level. This indicated the evolutionary variety of WRKY genes.