水稻基因组测序和注释分析.doc

水稻基因组测序及注释分析 Han Bing National Center for Gene Research, Chinese Academy of Sciences Establishment of a Rice Mutant Library by T-DNA Insertion Changyin Wu1, Wenya Yuan1, Guoxing Chen1, Xiangjun Li1, Dong Guo1, Jian Zhang1, Zhihui Chen1, Caishun Li1, Andrzej Kilian2, Juan Li1, Caiguo Xu1, Shiping Wang1 and Qifa Zhang1 1National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, China；2Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra, ACT 2601 Australia Rice (Oryza sativa L.) is an important crop worldwide and, with the availability of the draft sequence, a useful model for analyzing the genome structure of grasses. T-DNA tagging is one of the widely-used methods for generation of insertion mutants for gene functional analysis in plants. As a part of our rice functional genomics project, a powerful enhancer trap system carrying a GAL4/VP16 transactivator and a UAS-GUSPlus reporter cassette was employed in a high efficiency Agrobacterium-mediated rice transformation system to generate a library. We have currently obtained more than 35,000 independent transgenic plants. The transformants carried on average 2.0 copies of the T-DNA and 42% of the transformants had single copy insertion in this mutant library. GUS assay of different organs revealed various patterns of the reporter gene expression. Continued screening for GUS activity in leaves, roots and flowers of T1 families confirmed the stable transmission of expression patterns from the T0 to T1 generation, indicating that UAS in rice was not as sensitive to methylation as in tobacco and Arabidopsis. The system GAL4/VP16-UAS-GUSPlus will provide for the first time a powerful tool for the targeted expression of transgenes in an important monocotyledonous crop. The plasmid rescue and Tail-PCR strategies were used to isolate the T-DNA flanking sequence. Analysis of those sequences from