Improvement Of The Activation-tagging Vector For Rice Transformation And Construction Of Rice Activation-tagging Mutant Pool

Rice is the staple food of Asia where approximately half of the world's total population resides. Rice genomic sequencing project will be finished at the end of this year and functional genomics focusing on elucidating the biological function of genes. Rice mutant pool is the ideal material for the research of rice functional genomics, and activation-tagging is a powerful tool for the study of qualitative loci, hi this paper, we unproved the activation-tagging vector and transfer it to rice mediated by Agrobacterium- tumfacience to construct rice activation-tagging mutant pool. The main achievement in this paper are:1. The original activation-tagging vector, pSKI015, confers resistence to PPT. Because we found PPT selection of rice ^fficult, we used hygromycin for selection of rice and developed the activation-tagging vector pEnl305 which containing 4 X 35S enhancer for rice transformation.2. We used BamHI and SphI to digest the plasmid pEn!305 in the E.coli and Agrobacterium EHA105 which stored at 4C and -70 C, and find the CaMV35S enhancer is stable in E. cojli and Agrobacterium EHA105 at least for 3 months.3. We study the growing of callus affected by cefotaxime produced in China use this cefotaxime, an efficient Agrobacterium-medialed transformation protocol for rice was established and two transgenic rice pool was obtained.4. 4 × 35S enhancer was transferred into two japonica rice Ishikari-Shing and Nippon bare, respectively use this protocol. So far, more than 200 transgenic plants of Shishoubaimao and more than 100 transgenic plants of Nippon bare were regenerated. The exist and expression of the exogenous HPT gene in some transgenic plants was confirmed by PCR analysis and anti-hygromycin test. The exist of the 4 X 35S enhancer hi some transgenic plants was confirmed by PCR analysis.