Development of a rice targeting system towards a study of epigenetic gene expression.

为了研究基因转化引起的表观遗传现象的分子机制，基因打靶系统将成为在不改变基因位点的情况下操纵基因的有力工具。本研究的目的是建立水稻靶向系统，以研究水稻糯性基因和二氢黄酮醇-4-还原酶基因的表观遗传表达。由于基因打靶是通过同源重组诱导的，因此打靶载体除了包括阳性(潮霉素磷酸转移酶，HPH)和阴性选择标记外，还包括蜡质或DFR基因及其侧翼序列的同源序列。克隆了水稻蜡质和DFR基因及其侧翼序列。Shimokita和Ver.Murasakiine分别为。利用这些克隆构建了针对WAXY和DFR基因座的靶向载体。正选择和负选择通常用于小鼠基因打靶，以选择出…之间罕见的同源重组更多数量的非法重组。我们选择白喉毒素(DT)A链基因作为负选择标记。为了处理大量的转化事件，我们建立了一个高效的农杆菌转化系统。通过对农杆菌接种条件的优化，获得了比普通方法高10倍左右的转化效率。利用水稻高频转化方法，分析了DT基因负选择标记对水稻愈伤组织的致死效应。转DT基因水稻愈伤组织的死亡率可达99%以上。基于这些高效的农杆菌转化系统和正反向选择，我们将蜡质打靶载体应用于基因打靶。在一次实验中诱导的500~2000个转化事件中，平均有50~60个愈伤组织存活。为了找出基因打靶事件，对这些存活的愈伤组织进行了PCR分析。这些结果有力地表明，上述转化方法和正负选择方法的结合，很有可能成为水稻基因打靶的有力系统。较少

In order to study molecular mechanism of epigenetic phenomena caused by gene transformation, gene targeting system will serve as a powerful instrument for manipulation of genes without changing the loci of the genes in question. The objective of research is to develop a rice targeting system towards a study of epigenetic gene expression of rice Waxy and DFR (dihydroflavonol-4-reductase) genes. Since gene targeting is induced through homologous recombination, targeting vector includes homologous sequences of Waxy or DFR genes with its flanking sequences in addition to a positive (hygromycin phosphotransferase (hph)) and negative selection markers. The Waxy and DFR genes and its flanking sequences were cloned from Japonica rice ver. Shimokita and ver. Murasakiine respectively. By using these clones targeting vectors for Waxy and DFR loci were constructed. Positive and negative selection is generally used in mouse gene targeting to choose out rarely caused homologous recombination among a … More huge number of illegitimate recombinations. We have chosen the diphtheria toxin (DT) A chain gene as a negative selection marker. In order to treat a huge number of transformation events we have established a highly effective transformation system via Agrobacterium. Compared to general method about 10 times higher frequency of trans formation was obtained by our improved method, in which parameters about Agrobacterium inoculation were optimized. By using the high frequency rice transformation method the lethal effect for rice calli of the negative selection marker of DT gene was analyzed. More than 99% of mortality of rice calli transformed with the DT gene was observed. Based on these highly effective transformation system via Agrobacterium and the positive and negative selection, the Waxy targeting vector was applied for gene targeting. Average 50〜60 calli were survived among 500〜2000 transformation events induced in one time of experiment. In order to find out gene targeting event PCR analysis of these survived calli is on going. These results strongly suggest that the combination of the developed methods of transformation and of positive negative selection described above, have much possibility to be a powerful system for gene targeting of rice plant. Less