Complementary Transformation Of Rice Mutant With OsWRKY10, And Its Preliminary Function Study In Transgenic Tobacco

WRKY transcription factors, which appear to exist in plants exclusively, are involved in responses to pathogen, abiotic stresses, and in regulation of diverse developmental or physiological processes. They have become major areas of research focus in plant molecular biology. A novel gene encoding WRKY transcription factor, named OsWRKY10, was isolated from a rice cDNA library by PCR strategy with primers designed according to the flanking sequence of T456 cloned by a Tail-PCR. T456 is a delayed-in-development mutant screened from the T-DNA insertion mutant population (ZH11).In this study, G3 (full sequence of OsWRKY10 with 2kb-length of its promoter) was inserted into a pCambia1301 vector for constructing the plant expressing vector pCam-G3. Then, this vector together with another plant expressing vector pCHF3, carrying a selectable marker of NPT â…¡, were transformed into T456 by particle bombardment. Three independent transgenic rice plants were acquired, and confirmed by PCR. Two distinct phenotypes were showed in To transgenic rice plants. One line was similar to T456, while the other two lines were more similar to ZH11. It was presumed that the morphology of T456 was controlled by a pair of recessive mutant gene, according to the differentiation of T456 self-crossed progeny. This suggests that the phenotype of T456 was caused by the expression variation of OsWRKY10.In order to study the function of OsWRKY10 gene more clearly, we overexpressed this gene in tobacco. Two plant expressive vectors, pCamU-OsWRKY10-GFP (containing a fusion gene of OsWRKY10 and green fluorescent protein, GFP) and pCamU-OsWRKY10, were constructed and transformed into tobacco by A. tumefanciens-mediated method. 16 and 14 independent transgenic tobacco plants were obtained respectively, and detected by PCR method. The result showed that the genes were integrated into the genomes of tobacco plants. Green fluorescence was visualized under blue light excitation in cells of roots in transgenic tobacco plants by fluorescence microscopy. This study revealed that OsWRKY10 was exclusively localized in the nuclear, suggesting that it was a regulator within the nucleus.