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，基因靶向系统将成为一种不改变基因位点而操纵基因的有力工具。本研究的目的是建立水稻Waxy和DFR（二氢黄酮醇-4还原酶）基因表观遗传基因表达研究的靶向系统。由于基因靶向是通过同源重组诱导的，因此靶向载体包括Waxy或DFR基因的同源序列及其侧翼序列，以及阳性（潮霉素磷酸转移酶（hph））和阴性选择标记。从粳稻中克隆了Waxy和DFR基因及其两翼序列。Shimokita和ver。Murasakiine分别。利用这些克隆构建了Waxy和DFR基因座的靶向载体。小鼠基因靶向一般采用正选择和负选择，在大量的非正当重组中选择出很少引起同源重组的基因。我们选择白喉毒素（DT） A链基因作为阴性选择标记。为了处理大量的转化事件，我们通过农杆菌建立了一个高效的转化系统。改良方法对农杆菌接种参数进行了优化，其转化频率比常规方法提高了约10倍。采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。经DT基因转化的水稻愈伤组织死亡率达99%以上。基于这些高效的农杆菌转化体系和正、负选择，应用Waxy靶向载体进行基因靶向。试验一次诱导500 ~ 2000个愈伤组织，平均成活50 ~ 60个。为了找出基因靶向事件，对这些愈伤组织进行了PCR分析。这些结果有力地表明，将上述已开发的转化方法与正负选择方法相结合，很有可能成为一个强大的水稻基因靶向系统。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