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Reverse genetics for rice in the Post-Genome

后基因组中水稻的反向遗传学

基本信息

批准号：
15380008
负责人：
TERADA Rie
金额：
$ 10.11万
依托单位：
National Institute for Basic Biology (2004-2005)Okazaki National Research Institutes (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Gene targeting (GT) and gene tagging are quite powerful tools for studying gene function, after the completion of the entire rice genomic sequence. We have effectively progressed both procedures in this project. (1)Gene_targeting : Towards the wider application of our rice GT procedure, which we firstly developed for endogenous gene modification, new GT experiments have been performed for the Alcohol dehydrogenase genes, c1 and Adh2, independently. We have succeeded both GT experiments and are obtaining detailed molecular evidence for the precise recombination. The results ensure the applicability of our GT procedure to a new rice gene. Through GT experiments, we have improved each step of our GT procedure including the vector construction and the targeting transformation consisting of a strong positive-negative selection depending on a large-scale Agrobacterium-mediated transformation. In addition, we have examined the processes of homologous recombination (HR) after the T-DNA transformation. It would be another challenge in this project that the targeted excision of a 30-kb segment between the Adh1 and Adh2 on chromosome 11. We have obtained the putative recombination plant lines. To establish a conditional GT using the Cre/loxP site-specific recombination system, we attempted to remove a positive marker flanked by two loxP sequences within the targeted waxy locus by introducing the Cre gene. Our preliminary PCR analysis indicated that the positive marker can be removed from the waxy locus, and further analysis is in progress. (2)Gene tagging : We have identified non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily, and its transposition can be induced by crossing with a line containing an active autonomous element, aDart, and be stabilized by segregating out of aDart.

基因打靶和基因标签技术是水稻全基因组测序完成后研究基因功能的有力工具。我们在这个项目中有效地推进了这两个程序。（1）基因打靶：为了更广泛地应用我们的水稻GT程序，我们首先开发了内源基因修饰，新的GT实验已经进行了乙醇脱氢酶基因，c1和Adh 2，独立。我们已经成功的GT实验，并获得详细的分子证据的精确重组。这些结果保证了我们的GT程序对一个新的水稻基因的适用性。通过GT实验，我们改进了GT程序的每一步，包括载体构建和靶向转化，包括依赖于大规模农杆菌介导的转化的强阳性-阴性选择。此外，我们还研究了T-DNA转化后的同源重组（HR）过程。该项目的另一个挑战是靶向切除11号染色体上Adh 1和Adh 2之间的30 kb片段。我们已经获得了推定的重组植物系。为了使用Cre/loxP位点特异性重组系统建立条件性GT，我们试图通过引入Cre基因来去除靶向蜡质基因座内的两个loxP序列侧翼的阳性标记。初步的PCR分析表明，阳性标记可以从蜡质基因座上去除，进一步的分析正在进行中。（2）基因标签：我们已经确定了非自主的基于DNA的活性水稻转座子1（nDart 1），属于hAT超家族，其转座可以通过与含有活性自主元件aDart的品系杂交来诱导，并通过分离出aDart来稳定。