OVERPRODUCTION BY GENE AMPLIFICATION OF THE MULTIFUNCTIONAL AROM PROTEIN CONFERS GLYPHOSATE TOLERANCE TO A PLASTID-FREE MUTANT OF EUGLENA-GRACILIS

被引:13
作者
REINBOTHE, S [1 ]
ORTEL, B [1 ]
PARTHIER, B [1 ]
机构
[1] INST PLANT BIOCHEM,O-4050 HALLE,GERMANY
来源
MOLECULAR & GENERAL GENETICS | 1993年 / 239卷 / 03期
关键词
AROM PROTEIN; EUGLENA-GRACILIS (PLASTID-FREE MUTANT); GENE AMPLIFICATION; GLYPHOSATE TOLERANCE; SHIKIMATE PATHWAY;
D O I
10.1007/BF00276940
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cells of the plastid-free mutant line of Euglena gracilis var. bacillaris, W10BSmL, can be adapted to glyphosate [N-(phosphonomethyl)glycine] by gradually increasing the concentration of the herbicide in the culture medium. The molecular basis of glyphosate tolerance is the selective ca. ten-fold overproduction of the multifunctional arom protein catalyzing steps 2-6 in the pre-chorismate pathway. Determination of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (E.C.2.5.1.19), shikimate:NADP+ oxidoreductase (E.C.1.1.1.25) and shikimate kinase (E.C.2.7.1.71) activities after non-denaturing gel electrophoresis, in combination with two-dimensional separations, revealed an increase in all three enzyme activities associated with overproduction of a 165 kDa protein in cells adapted to 6 mM glyphosate. Further evidence for an involvement of the multifunctional arom protein in aromatic amino acid synthesis in the plastid-free W10BSmL cells was obtained by Northern hybridization with ARO1-, aroA-, aroL- and aroE- specific Saccharomyces cerevisiae gene probes encoding the entire arom protein or parts of the EPSP synthase, shikimate: NADP+ oxidoreductase and shikimate kinase domains, respectively. Overproduction in adapted relative to control cells of a 5.3 kb transcript that cross-hybridized with all of the different probes could be demonstrated. The elevated content of the arom transcript correlated with a selective amplification of two out of five genomic sequences that hybridized with the S. cerevisiae ARO1 gene probe in Southern blots. One of the amplified genomic fragments is assumed to encode the previously identified monofunctional 59 kDa EPSP synthase, which is thought to be an organellar protein, that accumulates to a certain extent in its enzymatically active precursor form of 64.5 kDa in the plastid-free W10BSmL cells.
引用
收藏
页码:416 / 424
页数:9
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