Nucleic acid fragments encoding isoflavone synthase

a technology of isoflavone synthase and nucleic acid sequence, which is applied in the field of plant molecular biology, can solve the problems of unstable 2-hydroxyisoflavanone intermediate, difficult cloning of plant p450s by traditional protein purification strategies, and difficult to develop soybean or other plant lines with consistently high levels of isoflavon

Inactive Publication Date: 2006-10-26
FADER GARY +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the 2-hydroxyisoflavanone intermediate was described as unstable and could convert directly to genistein.
Cloning of plant P450s by traditional protein purification strategies has been difficult, as these membrane-bound proteins are often very unstable and are typically present in low abundance.
To date, however, it has proven difficult to develop soybean or other plant lines with consistently high levels of isoflavonoid.
Substrates for isoflavone synthase may be limiting for synthesizing very high levels of isoflavonoids in soybean, or for synthesizing isoflavonoids in non-legumes.

Method used

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example 1

Microsome Preparation from Elicitor-Treated Soybean Hypocotyls and Elicitor-Treated Cell Suspension Culture

Elicitor Treatment of Soybean Seeds

[0165] Soybean seeds were placed on a bed of vermiculite (5 to 6 cm thick) and covered with a layer of vermiculite about 2 cm thick. Seeds were germinated for five days in a growth chamber until the average length of hypocotyls reached to about 3 to 4 cm. The growth chamber was kept at a cycle that consisted of a 14 h light period at 25° C. and a 10 h dark period at 21° C. Illumination was supplied from cool white fluorescent and incandescent lamps that provide a photon flux density of 450 μEm−2s−1. Soybean hypocotyls were pulled out from the vermiculite bed and were placed on wet paper towels. The soybean hypocotyls were divided into two groups: one of the groups was treated with elicitor and the other was not treated.

[0166] Elicitor treatment was conducted as follows. The epidermal surfaces of the hypocotyls were opened using a razor bla...

example 2

Development of Isoflavone Synthase Assay

[0169] An assay to measure isoflavone synthase activity was developed using either of the two substrates of isoflavone synthase, (±) naringenin (4′,5,7-trihydroxyflavanone; Sigma, N-5893) or liquiritigenin monohydrate (4′,7-dihydroxyflavanone; Indofine, 02-1150S), dissolved in 80% ethanol. The reaction mixture was prepared at room temperature and consisted of 100 μM naringenin or liquiritigenin, 80 mM K2HPO4, 0.5 mM glutathione (Sigma, G-4251), 20% w / v sucrose, and 30 to 150 μg of microsome preparation. The reaction mixtures were preincubated for 5 minutes without NADPH (synthesis of genistein and daidzein requires NADPH as a co-factor). The volume of microsomes and substrate added to any one reaction did not exceed 5% and 1%, respectively, of the total reaction volume. A typical reaction volume was 250 μL. The reaction was started by the addition of 40 nmol of NADPH per each 100 μL of final reaction volume. The pH of the reaction mixture was...

example 3

Composition of Soybean cDNA Library, Isolation and Sequencing of cDNA Clone

[0176] A cDNA library was prepared using mRNAs from soybean seeds that had been allowed to germinate for 4 hours. The library was prepared in Uni-ZAP™ XR vector according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). Conversion of the Uni-ZAP™ XR library into a plasmid library was accomplished according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript. cDNA inserts from randomly picked bacterial colonies containing recombinant pBluescript plasmids were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences or plasmid DNA was prepared from cultured bacterial cells. Amplified insert DNAs or plasmid DNAs were sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams, M. D. et al...

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Abstract

This invention relates to an isolated nucleic acid sequence encoding isoflavone synthase. The invention also relates to the construction of chimeric sequences encoding all or a substantial portion of the enzymes, in sense or antisense orientation, wherein expression of the chimeric sequence results in production of altered levels of the enzyme in a transformed host cell.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60 / 117,769, filed Jan. 27, 1999, U.S. Provisional Application No. 60 / 144,783, filed Jul. 20, 1999, and U.S. Provisional Application No. 60 / 156,094, filed Sep. 24, 1999.FIELD OF THE INVENTION [0002] This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid sequences encoding isoflavone synthase and their use in producing isoflavones. BACKGROUND OF THE INVENTION [0003] Isoflavonoids represent a class of secondary metabolites produced in legumes by a branch of the phenylpropanoid pathway and include such compounds as isoflavones, isoflavanones, rotenoids, pterocarpans, isoflavans, quinone derivatives, 3-aryl-4-hydroxy-coumarins, 3-arylcoumarins, isoflav-3-enes, coumestans, alpha-methyldeoxybenzoins, 2-arylbenzofurans, isoflavanol, coumaronochromone and the like. In plants, these compounds are known to be involved in interactions with other organisms...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01H5/00C12N9/10C12N1/00C12N5/04A01H1/00C12N15/82
CPCC12N9/0077C12P17/06C12N15/825C12N15/8243
Inventor FADER, GARYJUNG, WOOSUKMCGONIGLE, BRIANODELL, JOANYU, XIAODAN
Owner FADER GARY
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