权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

开发水稻靶向系统以研究表观遗传基因表达。

基本信息

批准号：
11660011
负责人：
TERADA Rie
金额：
$ 2.3万
依托单位：
National Institute for Basic Biology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660011/
关键词：
targeting rice Agrobacterium transformation diphtheria toxin protein negative selection marker DT-A chain gene Waxy DFR ターゲッティング

项目摘要

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

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一个水稻靶向系统，以研究水稻WESTERN和DFR（二氢黄酮醇-4-还原酶）基因的表观遗传表达。由于基因靶向是通过同源重组诱导的，因此靶向载体除了阳性（潮霉素磷酸转移酶（hph））和阴性选择标记物之外还包括WFR或DFR基因的同源序列及其侧翼序列。从粳稻中克隆了WAF 1和DFR基因及其侧翼序列。Shimokita和ver. Murasakiine分别。通过使用这些克隆，构建了针对WFR和DFR基因座的靶向载体。小鼠基因打靶中通常使用正选择和负选择来选择出罕见引起的同源重组。 ...更多信息大量的非法重组我们选择白喉毒素（DT）A链基因作为阴性选择标记。为了处理大量的转化事件，我们已经建立了一个高效的农杆菌转化系统。通过优化农杆菌接种参数，改进后的方法转化率比常规方法提高了10倍左右。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。观察到DT基因转化水稻愈伤组织的死亡率超过99%。在此基础上，利用农杆菌介导的高效转化体系，通过正反向筛选，将WESTERN打靶载体应用于基因打靶。一次诱导500 ~ 2000个转化事件，平均存活50 ~ 60个愈伤组织。为了发现基因打靶事件，这些存活的愈伤组织的PCR分析正在进行中。这些结果有力地表明，所开发的转化方法和上述正-负选择的组合很有可能成为水稻植物基因打靶的强大系统。少