Construction of externally controllable gene promoters in plants

植物外部可控基因启动子的构建

基本信息

批准号：
07554044
负责人：
WATANABE Akira
金额：
$ 12.8万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

This research has been conducted to obtain a good externally controllable promoter to be used in the analysis of gene functions in plants. To do this we first isolated cDNA clones for genes that are expressed in plants under very specific conditions. These conditions included those deprivation of thiamine from culture media for tobacco BY-2 cells, dark treatment of Arabidopsis leaves and application of salicylic acid to tobacco plants. We could isolate several clones by a direct-display PCR procedure, but the expression of the genes complimentary to those cDNA sequences were not very specific, ie.the transcripts were found even in the presence of the vitamin. We were successful to find several genes that are expressed by a manner very specific to the second conditions. Three out of more than a dozen genes found to be dark-inducible were selected for rapid and specific expression after careful northern hybridization analysis. Their promoter regions were isolated and fused with a reporter gene (firefly luciferase). The fused genes were introduced into cultured BY-2 cells and looked at the expression after deprivation of sugar from the culture media, because we noticed in the northern analysis that the dark-induced expression was abolished if the leaves were fed with sucrose at 10mM.The transformed cells were very sensitive to sugar starvation in respect to the expression of the reporter gene. As the expression is very rapid, strong and reversible, and the control the promoter can be done by a very mild and naturally occurring stimuli, it appears promising as a tool for the analysis of functions of genes not favorable for normal growth, and for agricultural use for crop improvement as well.

已经进行了这项研究，以获得良好的外部控制启动子，用于分析植物的基因功能。为此，我们首先分离出在非常具体条件下在植物中表达的基因的cDNA克隆。这些条件包括从培养基中剥夺毒含量的烟草By-2细胞，对拟南芥叶片的深色处理以及将水杨酸应用于烟草植物。我们可以通过直接显示PCR程序分离几个克隆，但是与这些cDNA序列相称的基因的表达不是很具体，即，即使在维生素存在下，也发现了转录本。我们成功地找到了几个基因，这些基因表达了一种非常针对第二条件的方式。在仔细的北部杂交分析后，选择了十多个基因中的三个，以快速而特异性的表达选择。他们的启动子区域被隔离并与记者基因（萤火虫荧光素酶）融合。将融合的基因引入了培养的BY-2细胞中，并在培养培养基中剥夺糖后的表达，因为我们在北部分析中注意到，如果在10mm处用蔗糖喂食叶子，则废除了黑暗诱导的表达。转化的细胞对糖颗粒非常敏感，对糖颗粒对记者基因的表达非常敏感。由于表达非常快速，很强和可逆，并且可以通过非常温和且天然发生的刺激来完成启动子，因此作为分析基因功能不利于正常生长的基因功能，以及用于农作物改善的农业用途的工具，它似乎很有希望。