Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Utility Patent
1999-02-10
2001-01-02
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S471000, C435S410000, C435S252300, C435S006120, C435S015000, C536S023200
Utility Patent
active
06168954
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding soybean glutathione-S-transferase (GST) enzymes involved in the detoxification of xenobiotic compounds in plants and seeds.
BACKGROUND OF THE INVENTION
Glutathione-S-transferases (GST) are a family of enzymes which catalyze the conjugation of glutathione, homoglutathione (hGSH) and other glutathione-like analogs via a sulfhydryl group, to a large range of hydrophobic, electrophilic compounds. The conjugation can result in detoxification of these compounds. GST enzymes have been identified in a range of plants including maize (Wosnick et al.,
Gene
(Amst) 76 (1) (1989) 153-160; Rossini et al.,
Plant Physiology
(Rockville) 112 (4) (1996) 1595-1600; Holt et al.,
Planta
(Heidelberg) 196 (2) (1995) 295-302), wheat (Edwards et al.,
Pestic. Biochem. Physiol.
(1996) 54(2), 96-104), sorghum (Hatzios et al.,
J. Environ. Sci. Health,
Part B (1996), B31(3), 545-553), arabidopsis (Van Der Kop et al.,
Plant Molecular Biology
30 (4) (1996), sugarcane (Singhal et al.,
Phytochemistry
(OXF) 30 (5) (1991) 1409-1414), soybean (Flury et al.,
Physiologia Plantarum
94 (1995) 594-604) and peas (Edwards R.,
Physiologia Plantarum
98 (3) (1996) 594-604). GST's can comprise a significant portion of total plant protein, for example attaining from 1 to 2% of the total soluble protein in etiolated maize seedlings (Timmermann,
Physiol. Plant
. (1989) 77(3), 465-71).
Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the nucleophilic attack of the thiol group of GSH to various electrophilic substrates. Their functions and regulation in plants has been recently reviewed (Marrs et al.,
Annu Rev Plant Physiol Plant Mol Biol
47:127-58 (1996); Droog, F.
J Plant Growth Regul
16:95-107, (1997)). They are present at every stage of plant development from early embryogenesis to senescence and in every tissue type examined. The agents that have been shown to cause an increase in GST levels have the potential to cause oxidative destruction in plants, suggesting a role for GSTs in the protection from oxidative damage. In addition to their role in the protection from oxidative damage, GSTs have the ability to nonenzymatically bind certain small molecules, such as auxin (Zettl, et al.,
PNAS
91: 689-693, (1994)) and perhaps regulate their bioavailability. Furthermore the addition of GSH to a molecule serves as an “address” to send that molecule to the plant vacuole (Marrs, et al.,
Nature
375: 397-400, (1995)).
GSTs have also been implicated in the detoxification of certain herbicides. Maize GSTs have been well characterized in relation to herbicide metabolism. Three genes from maize have been cloned: GST 29 (Shah et al.,
Plant Mol Biol
6, 203-211(1986)), GST 27 (Jepson et al.,
Plant Mol Biol
26:1855-1866, (1994)), GST 26 (Moore et al.,
Nucleic Acids Res
14:7227-7235 (1986)). These gene products form four GST isoforms: GST I (a homodimer of GST 29), GST IV (a heterodimer of GST 29 and GST 27), GST III (a homodimer of GST 26), and GST IV (a homodimer of GST 27). GST 27 is highly inducible by safener compounds (Jepson (1994) supra; Holt et al.,
Planta
196:295-302, (1995)) and overexpression of GST 27 in tobacco confers alachlor resistance to transgenic tobacco (Jepson, personal communication). Additionally Bridges et al. (U.S. Pat. No. 5,589,614) disclose the sequence of a maize derived GST isoform II promoter useful for the expression of foreign genes in maize and wheat. In soybean, herbicide compounds conjugated to hGSH have been detected and correlated with herbicide selectivity (Frear et al.,
Physiol
20: 299-310 (1983); Brown et al.,
Pest Biochem Physiol
29:112-120, (1987)). This implies that hGSH conjugation is an important determinant in soybean herbicide selectivity although this hypothesis has not been characterized on a molecular level.
Glutathione (the tripeptide &ggr;-glu-cys-gly, or GSH) is present in most plants and animals. However, in some plants from the family Leguminaceae the major free thiol is homoglutathione. For example, soybeans (
Glycine max
) have nearly undetectable levels of glutathione with the tripeptide homoglutathione (&ggr;-glu-cys-&bgr;-ala) apparently substituting for the same functions. Some herbicides are detoxified in soybeans by homoglutathione conjugation catalyzed by glutathione S-transferase (GST) enzyme(s).
Homoglutathione (hGSH) was originally detected in Phaseolus vulgaris and several other leguminous species (Price, C. A.,
Nature
180: 148-149, (1957)). The structure of hGSH (Carnegie, P. R.,
Biochemical Journal
89:471-478 (1963)) was determined to be the tripeptide &ggr;-glu-cys-&bgr;-ala. Homoglutathione has not been found in non-leguminous species. In plants from the family Legumaceae, the ratio of hGSH to GSH varies according to both species and tissue examined. In seeds and leaves of the tribe Vicieae, only traces of hGSH were found in addition to the main thiol GSH, whereas in roots the hGSH content exceeded the GSH content. The tribe Trifolieae contained both tripeptides and in the tribe Phaseoleae, hGSH predominated. In soybean (
Glycine max
), a member of the Phaseoleae, hGSH constitutes 99% of the free thiol in leaves and seeds and greater than 95% of the free thiol in soybean roots (Klapheck, S.,
Physiolgia Plantarum
74: 727-732 (1988)). As such, it is essential that soybean glutathione S-transferases be able to efficiently utilize hGSH.
Some efforts have been made to alter plant phenotypes by the expression of either plant or mammalian foreign GST genes or their promoters in mature plant tissue. For example, Helmer et al. (U.S. Pat. No. 5,073,677) teach the expression of a rat GST gene in tobacco under the control of a strong plant promoter. Similarly, Jepson et al. (WO 97/11189) disclose a chemically inducible maize GST promoter useful for the expression of foreign proteins in plants; Chilton et al. (EP 256223) discuss the construction of herbicide tolerant plants expressing a foreign plant GST gene; and Bieseler et al. (WO 96/23072) teach DNA encoding GSTIIIc, its recombinant production and transgenic plants containing the DNA having a herbicide-tolerant phenotype.
Manipulation of nucleic acid fragments encoding soybean GST to use in screening in assays, the creation of herbicide-tolerant transgenic plants, and altered production of GST enzymes depend on the heretofore unrealized isolation of nucleic acid fragments that encode all or a substantial portion of a soybean GST enzyme.
SUMMARY OF THE INVENTION
The present invention provides nucleic acid fragments isolated from soybean encoding all or a substantial portion of a GST enzyme. The isolated nucleic acid fragment is selected from the group consisting of (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:56; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:56; and (c) an isolated nucleic acid fragment that is complementary to (a) or (b). The nucleic acid fragments and corresponding polypeptides are contained in the accompanying Sequence Listing a
McGonigle Brian
O'Keefe Daniel P.
Achutamurthy Ponnathapu
E. I. du Pont de Nemours & Company
Kerr Kathleen
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