Reverse genetics for rice in the Post-Genome

在水稻全基因组序列完成后，基因定位和基因标记是研究基因功能的有力工具。在这个项目中，我们已经有效地推进了这两个程序。(1)Gene_targeting：为了更广泛地应用我们最初开发的用于内源基因修饰的水稻GT程序，我们分别对乙醇脱氢酶基因c1和Adh2进行了新的GT实验。我们已经成功地进行了两个GT实验，并正在获得精确重组的详细分子证据。这一结果保证了我们的GT方法对水稻新基因的适用性。通过GT实验，我们改进了GT过程的每一步，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正负选择。此外，我们还研究了T-DNA转化后的同源重组（HR）过程。对11号染色体上Adh1和Adh2之间30kb片段的靶向切除将是本项目的另一个挑战。我们获得了假定的重组植株系。为了利用Cre/loxP位点特异性重组系统建立条件GT，我们试图通过引入Cre基因来去除目标蜡质位点内两个loxP序列两侧的阳性标记。我们初步的PCR分析表明，阳性标记可以从蜡质位点中去除，进一步的分析正在进行中。(2)基因标记：我们已经鉴定出了hAT超家族的非自主dna活性水稻转座子1 (nDart1)，它的转座子可以通过与含有主动自主元件aDart的系杂交诱导，并通过从aDart分离得到稳定。

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.