GENE AMPLIFICATION IN GLYPHOSATE TOLERANT TOBACCO CELLS
GENE AMPLIFICATION IN GLYPHOSATE TOLERANT TOBACCO CELLS
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DOI：
10.1016/0168-9452(90)90186-r
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发表时间：
1990-01-01
期刊：
影响因子：
5.2
通讯作者：
WELLER, SC
中科院分区：
文献类型：
--
作者：
GOLDSBROUGH, PB;HATCH, EM;WELLER, SC
以烟草（Nicotiana tabacum L.）已经选择了细胞系在通常致死浓度的草甘膦（高达20 mM）存在下生长。在耐受细胞中，5-烯醇式莽草酸3-磷酸合酶（EPSPS）的活性被草甘膦抑制，该酶的活性升高。然而，从耐受细胞分离的EPSPS仍然被除草剂抑制。EPSPS的活性增加是耐受细胞中酶水平增加的结果，如通过免疫印迹法测定的，而不是酶活性的变化。用矮牵牛EPSPS cDNA的RNA印迹证明了耐受性水平与EPSPS mRNA的稳态水平之间的相关性。EPSPS mRNA表达的增加是由于烟草基因组中编码该酶的至少两个基因的扩增。基因扩增的程度也随着草甘膦耐受性水平的增加而增加。在不存在草甘膦的情况下生长的选定细胞具有升高的EPSPS mRNA丰度并保持一些基因扩增，表明选择导致稳定的遗传修饰。
A series of tobacco (Nicotiana tabacum L.) cell lines have been selected for growth in the presence of normally lethal concentrations of glyphosate, up to 20 mM. Activity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), which is inhibited by glyphosate, is elevated in tolerant cells. However, EPSPS isolated from tolerant cells is still inhibited by the herbicide. Increased activity of EPSPS is the result of increased levels of the enzyme in tolerant cells, as determined by immunoblotting, rather than a change in enzyme activity. RNA blots with a petunia EPSPS cDNA demonstrate a correlation between levels of tolerance and steady state level of EPSPS mRNA. Increased expression of EPSPS mRNA results from amplifcation of at least two genes encoding this enzyme in the tobacco genome. The degree of gene amplification also increases with level of glyphosate tolerance. Selected cells grown in the absence of glyphosate have an elevated EPSPS mRNA abundance and maintain some gene amplification, indicating that selection has resulted in a stable genetic modification.