公开(公告)号：US06486382B1

公开(公告)日：2002-11-26

申请号：US09214909

申请日：1999-12-20

申请人： William Gordan-Kamm ,  Dorothy A. Pierce ,  Benjamin Bowen ,  Dennis Bidney ,  Margit Ross ,  Christopher Scelonge ,  Michael D. Miller ,  Gary Sandahl ,  Lijuan Wang

发明人： William Gordan-Kamm ,  Dorothy A. Pierce ,  Benjamin Bowen ,  Dennis Bidney ,  Margit Ross ,  Christopher Scelonge ,  Michael D. Miller ,  Gary Sandahl ,  Lijuan Wang

IPC分类号： C12N1582

CPC分类号： C12N15/8216 ,  C07K14/43595 ,  C12N15/8209 ,  C12N15/8212 ,  C12N15/8213

摘要： A method for the production of transgenic plants is provided in which a vector carrying a gene encoding the green fluorescent protein is introduced into cells, the cells are screened for the protein and transformed cells are selected and regenerated. The cellular toxicity of the green fluorescent protein is circumvented by regulating expression of the gene encoding the protein or directing the protein to a subcellular compartment where it is not toxic to the cell. DNA constructs are provided for cell transformation in which the expression of a gene encoding the green fluorescent protein is placed under the control of an inducible promoter. In addition, DNA constructs are provided in which a nucleotide sequence encoding the green fluorescent protein is operably linked to a signal sequence which directs the expressed protein to a subcellular compartment where the protein is not toxic to the cell. Oxidative stress to plant cells transformed with GFP also can be ameliorated by transforming cells with an expression vector comprising genes encoding GFP and an oxygen scavenger enzyme such as superoxide dismutase. The toxicity of GFP in transformed plants can be eliminated by excising the screenable marker gene following detection of transformed cells or sectors. The FLP/FRT system is used in conjunction with GFP as a visible marker for transformation and FRT excision. A nucleotide sequence optimized for expression of the green fluorecent protein in plants is also provided.

摘要翻译： 提供了一种用于生产转基因植物的方法，其中将携带编码绿色荧光蛋白的基因的载体导入细胞，筛选细胞以进行蛋白质，并选择和再生转化的细胞。 绿色荧光蛋白的细胞毒性通过调节编码蛋白质的基因的表达或引导蛋白质转移到对细胞无毒性的亚细胞区域来避免。 提供DNA构建体用于细胞转化，其中将编码绿色荧光蛋白的基因的表达置于诱导型启动子的控制之下。 此外，提供了DNA构建体，其中编码绿色荧光蛋白的核苷酸序列可操作地连接到将表达的蛋白质导向亚细胞区域的信号序列，其中蛋白质对细胞无毒性。 用GFP转化的植物细胞的氧化应激也可以通过用包含编码GFP和氧清除酶如超氧化物歧化酶的基因的表达载体转化细胞来改善。 通过在检测到转化的细胞或部分后切除可筛选标记基因，可以消除GFP在转化植物中的毒性。 FLP / FRT系统与GFP结合使用，作为转化和FRT切除的可见标记。 还提供了优化用于植物中绿色荧光蛋白表达的核苷酸序列。