Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1997-09-17
2003-02-18
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S252500, C435S410000, C435S254110, C435S254200, C435S320100, C536S023200
Reexamination Certificate
active
06521433
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 enzymes involved in isoflavone biosynthesis in plants and seeds.
BACKGROUND OF THE INVENTION
Isoflavones represent a class of secondary metabolites produced in legumes by the phenylpropanoid metabolic pathway. The biosynthetic pathway for free isoflavones and their relationship with several other classes of phenylpropanoids is presented in FIG.
1
. Many of the enzymes involved in the synthesis of isoflavones in soybean have been identified and the genes in the pathway from phenylalanine ammonia lyase to chalcone synthase and chalcone reductase have been cloned. However, remaining soybean genes involved in synthesis (chalcone isomerase and isoflavone synthase), further metabolism (isoflavone reductase and vestitone reductase), and branch points of the isoflavone pathway that could compete for substrates (flavanone hydroxylase and flavonol synthase) heretofore have not been available.
Free isoflavones rarely accumulate to high levels in soybeans. Instead they are usually conjugated to carbohydrates or organic acids. Soybean seeds contain three types of isoflavones in four different forms: the aglycones daidzein, genistein, and glycitein; the glucosides diadzin, genistin, and glycitin; the acetylgucosides 6″-O-acetyldaidzin, 6″-O-acetylgenistin, and 6″-O-acetylglycitin; and the malonylglucosides 6″-O-malonyldaidzin, 6″-O-malonylgenistin, and 6″-O-malonylglycitin. It has been reported that the isoflavones found in soybean seeds possess antihemolytic (Naim, M. et al. (1976)
J. Agric. Food Chem.
24:1174-1177), antifungal (Naim, M. et al. (1974)
J Agr. Food Chem.
22:806-810), oestrogenic (Price, K. R. and Fenwick, G. R. (1985)
Food Addit. Contam.
2:73-106), tumor suppressing (Messina, M. and Barnes, S. (1991)
J. Natl. Cancer Inst.
83:541-546; Peterson, G. et al. (1991)
Biochem. Biophys. Res. Commun.
179:661-667), hypolipidemic (Mathur, K. et al. (1964)
J. Nutr.
84:201-204), and serum cholesterol lowering (Sharma, R. D. (1979)
Lipids
14:535-540) effects. These epidemiological studies indicate that when isoflavone levels are high in soybean seeds and in the subsequent commercial protein products made from the seeds, the dietary intake of these products provide many health benefits.
The content of isoflavones in soybean seeds, however, is quite variable and is affected by both genetics and environmental conditions such as growing location and temperature during seed fill (Tsukamoto, C. et al. (1995)
J. Agric. Food Chem.
43:1184-1192; Wang, H. and Murphy, P. A. (1994)
J. Agric. Food Chem.
42:1674-1677). In addition, isoflavone content in legumes can be stress-induced by pathogenic attack, wounding, high UV light exposure, and pollution (Dixon, R. A. and Paiva, N. L. (1995)
The Plant Cell
7:1085-1097). To date, it has proven difficult to develop soybean lines with consistantly high levels of isoflavones; moreover, lines reported to be low in isoflavone content produced normal levels of isoflavones when grown under standard cultural conditions (Kitamura, K. et al. (1991)
Jap. J. Breed.
41:651-654). The isolation and cloning of genes associated with synthesis and metabolism of isoflavones in soybean will afford the application of molecular techniques to achieve stable, high level accumulation of isoflavones.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding plant enzymes involved in isoflavone biosynthesis. Specifically, this invention concerns isolated nucleic acid fragments encoding the following soybean enzymes that catalyze steps in the biosynthesis of isoflavones from phenylalanine: chalcone isomerase, isoflavone reductase and vestitone reductase. In addition, this invention relates to nucleic acid fragments that are complementary to nucleic acid fragments encoding the listed soybean biosynthetic enzymes.
In another embodiment, the instant invention relates to chimeric genes encoding the isoflavone biosynthetic acid enzymes listed above or to chimeric genes that comprise nucleic acid fragments that are complementary to the nucleic acid fragments encoding the enzymes, operably linked to suitable regulatory sequences, wherein expression of the chimeric genes results in production of levels of isoflavone biosynthetic enzymes in transformed host cells that are altered (i.e., increased or decreased) from the levels produced in untransformed host cells.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an isoflavone biosynthetic enzyme operably linked to suitable regulatory sequences, the enzyme selected from the group consisting of chalcone isomerase, isoflavone reductase and vestitone reductase. Expression of the chimeric gene results in production of altered levels of the biosynthetic enzyme in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a plant isoflavone biosynthetic enzyme in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a soybean isoflavone biosynthetic enzyme selected from the group consisting of chalcone isomerase, isoflavone reductase and vestitone reductase, operably linked to suitable regulatory sequences; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of an isoflavone biosynthetic enzyme in the transformed host cell.
An additional embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or substantially all of an amino acid sequence encoding a plant chalcone isomerase, isoflavone reductase and vestitone reductase.
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E. I. du Pont de Nemours and Company
Slobodyansky Elizabeth
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