EP1147199A1 - Nucleic acid sequences encoding isoflavone synthase - Google Patents

Nucleic acid sequences encoding isoflavone synthase

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Publication number
EP1147199A1
EP1147199A1 EP00907017A EP00907017A EP1147199A1 EP 1147199 A1 EP1147199 A1 EP 1147199A1 EP 00907017 A EP00907017 A EP 00907017A EP 00907017 A EP00907017 A EP 00907017A EP 1147199 A1 EP1147199 A1 EP 1147199A1
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European Patent Office
Prior art keywords
plant
xaa
nucleic acid
acid sequence
seq
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German (de)
English (en)
French (fr)
Inventor
Gary M. Fader
Woosuk Jung
Brian Mcgonigle
Joan T. Odell
Xiaodan Yu
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EIDP Inc
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EI Du Pont de Nemours and Co
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Publication of EP1147199A1 publication Critical patent/EP1147199A1/en
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

  • 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
  • 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 and to participate in the defense responses of legumes against phytopathogenic microorganisms (Dewick, P. M.
  • Isoflavonoids have also been reported to have physiological activity in animal and human studies. For example, it has been reported that the isoflavones found in soybean seeds possess antihemolytic (Nairn, M., et al. (1976) J. Agric. Food Chem. 24A 11A-X 11), antifungal (Nairn, M., et al. (1974) J. Agr. Food Chem. 22:806-810), estrogenic (Price, K. R. and Fenwick, G. R. (1985) FoodAddit. Contam.
  • Soybean seeds contain three types of isoflavones in four different forms: the aglycones, daidzein, genistein and glycitein; the glucosides, daidzin, 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.
  • isoflavonoids The content of isoflavonoids in soybean seeds 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 A 184-1192; Wang, H. and Murphy, P. A. (1994) J. Agric. Food Chem. ⁇ 2:1674-1677).
  • isoflavonoid 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) Plant Cell 7:1085-1097).
  • Proteins that are greater than 55% identical are designated as members of the same subfamily, while P450s that are 97% identical, or greater, are assumed to be allelic variants of the same gene (Chappie, C. (1998) Annu. Rev. Plant Physiol. Plant Mol. Bio. ⁇ 9:311-343).
  • isoflavonoids The physiological activities associated with isoflavonoids in both plants and humans makes the manipulation of their contents in crop plants highly desirable. For example, increasing levels of isoflavonoid in soybean seeds would increase the efficiency of extraction and lower the cost of isoflavone-related products sold today for use in either reduction of serum cholesterol or in estrogen replacement therapy. Decreasing levels of isoflavonoid in soybean seeds would be beneficial for production of soy-based infant formulas where the estrogenic effects of isoflavonoid are undesirable. Raising levels of isoflavonoid phytoalexins in vegetative plant tissue could increase plant defenses to pathogen attack, thereby improving plant disease resistance and lowering pesticide use rates.
  • isoflavone synthase is the central reaction in pathways producing isoflavonoids, identification of this functional gene is extremely important, and its manipulation via molecular techniques is expected to allow production of soybeans and other plants with high, stable levels of isoflavonoid.
  • Introduction of the isoflavone synthase gene in non- legume crop species including, but not limited to, corn, wheat, rice, sunflower, and canola could lead to synthesis of isoflavonoids.
  • the expression of isoflavonoids would confer to these species disease resistance and/or properties which produce human/livestock health benefits.
  • Substrates for isoflavone synthase may be limiting for synthesizing very high levels of isoflavonoids in soybean, or for synthesizing isoflavonoids in non-legumes. It is desirable to increase the flux of metabolites through the phenylpropanoid pathway to provide additional amounts of substrate to those occurring naturally. Different stress conditions such as UV irradiation, phosphate starvation, prolonged exposure to cold, and chemical (such as herbicide) treatment can cause activation of the phenylpropanoid pathway. While these treatments may produce the desired substrate availability, it is more desirable to have a genetic means of activating the phenylpropanoid pathway.
  • genes encoding certain transcription factors can regulate the expression of various genes that encode enzymes of the phenylpropanoid pathway. These include, but are not limited to, the CI myb-type transcription factor of maize and the AmMyb305 of Antirrhinum majus.
  • the CI myb-type transcription factor of maize in conjunction with the myc-type transcription factor R, activates chalcone synthase and chalcone isomerase genes (Grotewold, E., et al. (1998) Plant Cell 10:121-140).
  • the Antirrhinum majus AmMyb305 activates the phenylalanine ammonia lyase promoter (Sablowski, R. W., et al. (1994) EMBO J.
  • Transcription factors such as these may be expressed in host plant cells to activate expression of genes in the phenylpropanoid pathway thereby increasing the encoded enzyme activities and the flux of compounds through the pathway. Increases in the precursors to substrates of isoflavone synthase would enhance the production of isoflavonoids.
  • the instant invention relates to isolated nucleic acid sequences encoding isoflavone synthase.
  • this invention relates to nucleic acid sequences that are complementary to nucleic acid sequences encoding isoflavone synthase.
  • the nucleic acid sequences may be of genomic or cDNA origin and may contain introns.
  • the instant invention relates to chimeric genes encoding isoflavone synthase or to chimeric genes that comprise nucleic acid sequences that are complementary to the nucleic acid sequences encoding the enzyme, operably linked to suitable regulatory sequences, wherein expression of the chimeric genes results in production of levels of isoflavone synthase in transformed host cells that are altered (i.e., increased or decreased) from the levels produced in untransformed host cells.
  • the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an isoflavone synthase that is operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the enzyme in the transformed host cell.
  • the transformed host cell can be of eukaryotic or prokaryotic origin, and includes 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 synthase in a transformed host cell comprising transforming a host cell with a chimeric gene comprising a nucleic acid sequence (cDNA or genomic DNA) encoding an isoflavone synthase operably linked to suitable regulatory sequences and 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 isoflavone synthase in the transformed host cell.
  • the altered levels of isoflavone synthase may be higher due to overexpression, or may be lower due to cosuppression or anti sense suppression.
  • a further embodiment of the instant invention is a method for increasing the amount of one or more isoflavonoids in a host cell.
  • the method comprising the steps of transforming a host cell with a chimeric gene comprising a nucleic acid sequence encoding an isoflavone synthase operably linked to suitable regulatory sequences and 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 an amount of isoflavonoids in the transformed host cell that is greater than the amount of isoflavonoids that are produced in a cell that is not transformed with the chimeric gene.
  • a further embodiment of the instant invention is a method for decreasing the amount of one or more isoflavonoids in a host cell.
  • the method comprising the steps of transforming a host cell with a chimeric gene comprising a nucleic acid sequence encoding all or a substantial portion of an isoflavone synthase operably linked to suitable regulatory sequences and 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 an amount of isoflavonoids in the transformed host cell that is less than the amount of isoflavonoids that are produced in a cell that is not transformed with the chimeric gene.
  • 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 for obtaining a nucleic acid sequence encoding all or substantially all of an amino acid sequence encoding isoflavone synthase.
  • a still further embodiment of the instant invention concerns a transformed host cell comprising a chimeric gene encoding isoflavone synthase and at least one chimeric gene encoding a transcription factor that can regulate expression of one or more genes in the phenylpropanoid pathway.
  • the invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
  • a further embodiment is a method of increasing the amount of one or more isoflavonoids in a host cell comprising transforming a host cell with a chimeric gene having a nucleic acid sequence encoding an isoflavone synthase operably linked to suitable regulatory sequences and with at least one chimeric gene having a nucleic acid sequence encoding a transcription factor that regulates expression of genes in the phenylpropanoid pathway, and growing the transformed host cell under conditions that are suitable for expression of the chimeric genes wherein expression of the chimeric genes result in production of an amount of one or more isoflavonoids in the transformed host cell that is greater than the amount of the isoflavonoids that are produced in a cell that is not transformed with the chimeric genes.
  • the invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
  • a further embodiment of the present invention is a method of altering the level of isoflavonoids in a plant cell that is transformed with a chimeric isoflavone synthase gene comprising exposing said cell to a phenylpropanoid pathway-altering agent.
  • the phenylpropanoid pathway-altering agent may be a transcription factor or stress, for example. Stress includes and is not limited to ultraviolet light, temperature, pressure, phosphate level, and herbicide treatment.
  • the transcription factors may be a CI myb-type transcription factor of maize and a myc-type transcription factor R, or a chimera containing the maize R region between the CI DNA binding domain and the CI activation domain. BIOLOGICAL DEPOSIT
  • the following transformed yeast strain and vector plasmid have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, and bears the following designation, accession number and date of deposit.
  • ATCC American Type Culture Collection
  • Figure 1 depicts the phenylpropanoid metabolic pathway, and illustrates particularly the biosynthesis of isoflavonoids.
  • Figure 2 A and B presents the results of HPLC analyses of naringenin standards.
  • Figure 2A presents the absorption spectra recorded at 260 nm and
  • Figure2B presents the absorption spectra recorded at 280 nm.
  • Figure 3 A and B presents the results of HPLC analyses of genistein standards.
  • FIG. 3 A presents the abso ⁇ tion spectra recorded at 260 nm and Figure 3B presents the absorption spectra recorded at 280 nm.
  • Figure 4 A and B presents the results of HPLC analyses of genistein and naringenin from microsomes derived from elicitor-treated soybean hypocotyls. Absorption spectra was recorded at 260 nm ( Figure 4A) and 280 nm ( Figure 4B). Naringenin and genistein peaks are indicated.
  • Figure 5 A and B presents the results of HPLC analyses of genistein and naringenin from microsomes derived from non-treated soybean hypocotyls. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 5A) and 280 nm ( Figure 5B). Naringenin and genistein peaks are indicated.
  • Figure 6 A and B presents the results of HPLC analyses of genistein and naringenin from microsomes derived from elicitor-treated soybean cell suspension cultures. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 6A) and 280 nm ( Figure 6B). Naringenin and genistein peaks are indicated.
  • Figure 7 A and B presents the results of HPLC analyses of genistein and naringenin from microsomes derived from non-treated soybean cell suspension cultures. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 7A) and 280 nm ( Figure 7B). Naringenin peak is indicated.
  • Figure 8 A and B presents the results of HPLC analyses of genistein and naringenin in
  • Figure 9 A and B presents the results of HPLC analyses of genistein and naringenin in 75 ⁇ g of yeast microsomal proteins after 1 h incubation in the presence of NADPH cofactor. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 9A) and 280 nm ( Figure 9B).
  • Figure 10A and B presents the results of HPLC analyses of genistein and naringenin in 75 ⁇ g of yeast microsomal proteins after 2 h incubation in the presence of NADPH cofactor. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 10A) and 280 nm ( Figure 10B).
  • Figure 11A and B presents the results of HPLC analyses of genistein and naringenin in
  • Figure 12 A and B presents the results of HPLC analyses of genistein and naringenin in 75 ⁇ g of yeast microsomal proteins after 4 h incubation in the presence of NADPH cofactor. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 12A) and 280 nm ( Figure 12B).
  • Figure 13 A and B presents the results of HPLC analyses of genistein and naringenin in 75 ⁇ g of yeast microsomal proteins after 14 h incubation in the presence of NADPH cofactor. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 13 A) and 280 nm ( Figure 13B).
  • Figure 14A and B presents the results of HPLC analyses of genistein and naringenin in cofactor. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 14 A) and 280 nm ( Figure 14B).
  • Figure 15A and B presents the results of HPLC analyses of genistein and naringenin in 150 ⁇ g of yeast microsomal proteins after 40 minutes incubation in the presence of NADPH cofactor. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 15A) and 280 nm ( Figure 15B).
  • Figure 16A and B presents the results of HPLC analyses of genistein and naringenin in 75 ⁇ g of yeast microsomal proteins after 4 h incubation in the absence of NADPH cofactor. Abso ⁇ tion spectra was recorded at 260 nm ( Figure 16A) and 280 nm ( Figure 16B).
  • Figure 17 A and B presents a comparison of the abso ⁇ tion spectra recorded by a diode array detector of a genistein standard ( Figure 17 A; with an HPLC retention time of 3.128), and a reference spectrum (Figure 17B).
  • Figure 18A and B presents a comparison of the abso ⁇ tion spectra recorded by a diode array detector of the newly synthesized peak located at the retention time of 3J31 in the HPLC analysis of yeast microsomes incubated for 14 h in the presence of NADPH on Figure 18A and the reference spectrum on Figure 18B.
  • Figure 19A, B, C, D and E presents the electropositive mass spectrum obtained for the peaks observed by HPLC analysis of yeast microsome samples incubated with liquiritigenin.
  • Figure 19A corresponds to the peak at 273.2 m/z
  • Figure 19B corresponds to the peak at 271 m z
  • Figure 19C corresponds to "peak 2”
  • Figure 19D corresponds to liquiritigenin standard (the substrate)
  • Figure 19E corresponds to daidzein standard (the product).
  • Figure 20 depicts the plasmid map of pOY160.
  • Figure 21 depicts the plasmid map of pOY206.
  • Figure 22 depicts the plasmid map of pDP7951, having an ATCC accession No. PTA-371.
  • Figure 23 depicts the plasmid map of pOY162.
  • Figure 24 depicts the plasmid map of pKS93s.
  • Figure 25 depicts the distribution of the isoflavonoid content of 25 transgenic lines transformed with the isoflavone synthase sequence from clone sgslc.pk006.o20 and a control line. Bars represent the mean of three analyses for each line. The result of single factor ANOVA is presented along with the least significant difference (LSD) at P ⁇ 0.01. The asterisk above the bars represents those lines with mean isoflavonoid concentrations significantly lower than control (bars 1 through 6), or those lines with mean isoflavonoid concentrations significantly greater than control (bars 15 through 25) based on the LSD test at P ⁇ 0.01.
  • Figure 26 depicts the comparison of the rates of genistein and daidzein synthesis by microsomes of the yeast transformant GM1.
  • Samples representing incubation periods of 2, 4, 6, 8 and 10 h were analyzed by HPLC and the peak areas for genistein and daidzein were quantitated by calibration with authentic genistein and daidzein standards. Assays were repeated three times and the average amount of isoflavonoid synthesized at each time point was plotted, with vertical lines representing error bars.
  • Figure 27 presents the results of HPLC analyses of daidzein and liquiritigenin in extracts from BMS cells before incubation in the presence of NADPH cofactor (Panels A and B) and after 10 h incubation in the presence of NADPH cofactor (Panels C and D). Abso ⁇ tion spectra was recorded at 260 nm (Panels A and C) and 280 nm (Panels B and D).
  • Figure 28 depicts the plasmid map of pCW109-IFS.
  • the following sequence descriptions and Sequences Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1.821-1.825.
  • the Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUB standards described in Nucleic Acids Research 75:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2j:345-373 (1984) which are herein inco ⁇ orated by reference.
  • the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1.822.
  • SEQ ID NOJ is the nucleotide sequence comprising the soybean cDNA insert in clone sgslc.pk006.o20 encoding an enzymatically active isoflavone synthase.
  • SEQ ID NO:2 is the deduced amino acid sequence of an enzymatically active soybean isoflavone synthase derived from the nucleotide sequence of SEQ ID NO: 1.
  • SEQ ID NO: 3 is the nucleotide sequence of an oligonucleotide primer used in the construction of yeast strain WHT1.
  • SEQ ID NO:4 is the nucleotide sequence of an oligonucleotide primer used in the construction of the yeast strain WHT 1.
  • SEQ ID NO: 5 is the nucleotide sequence of an oligonucleotide primer used to amplify the cDNA insert from clone sgslc.pk006.o20.
  • SEQ ID NO:6 is the nucleotide sequence of an oligonucleotide primer used to amplify the cDNA insert from clone sgslc.pk006.o20.
  • SEQ ID NO: 7 is the nucleotide sequence of an oligonucleotide primer used for PCR amplification of the soybean clone with sequence corresponding to the one found in NCBI General Identifier No. 2739005. This oligonucleotide sequence corresponds to nucleotides 3 to 26 of the NCBI sequence.
  • SEQ ID NO: 8 is the nucleotide sequence of an oligonucleotide primer used for PCR amplification of the soybean clone with sequence corresponding to the one found in NCBI General Identifier No. 2739005. This oligonucleotide sequence corresponds to the complement of nucleotides 1798 to 1824 of the NCBI sequence.
  • SEQ ID NO: 9 is the nucleotide sequence of an enzymatically active soybean isoflavone synthase having an NCBI General Identifier No. 2739005.
  • SEQ ID NO: 10 is the deduced amino acid sequence of an enzymatically active soybean isoflavone synthase derived from of SEQ ID NO: 9 and having an NCBI General Identifier No. 2739006.
  • SEQ ID NOJ 1 is the nucleotide sequence of an oligonucleotide primer used for PCR amplification of the isoflavone synthase genes from mung bean, red clover, white clover, lentil, hairy vetch, alfalfa, lupine and snow pea.
  • SEQ ID NO: 12 is the nucleotide sequence of an oligonucleotide primer used for PCR amplification of the isoflavone synthase genes from mung bean, red clover, white clover, lentil, hairy vetch, alfalfa, lupine and snow pea.
  • SEQ ID NO: 13 is the nucleotide sequence of an oligonucleotide primer used in the second round of PCR amplification of the white clover, lentil, hairy vetch, alfalfa and lupine isoflavone synthase genes.
  • SEQ ID NO: 14 is the nucleotide sequence of an oligonucleotide primer used in the second round of PCR amplification of the white clover, lentil, hairy vetch, alfalfa and lupine isoflavone synthase genes.
  • SEQ ID NO: 15 is the nucleotide sequence comprising the alfalfa cDNA insert in clone alfalfal encoding an almost entire alfalfa isoflavone synthase.
  • SEQ ID NO: 16 is the deduced amino acid sequence of an almost entire alfalfa isoflavone synthase derived from the nucleotide sequence of SEQ ID NO: 15.
  • SEQ ID NO: 17 is the nucleotide sequence comprising the hairy vetch cDNA insert in clone hairy vetch 1 encoding an almost entire hairy vetch isoflavone synthase.
  • SEQ ID NO: 18 is the deduced amino acid sequence of an almost entire hairy vetch isoflavone synthase derived from the nucleotide sequence of SEQ ID NO: 17.
  • SEQ ID NO: 19 is the nucleotide sequence comprising the lentil cDNA insert in clone lentil 1 encoding an almost entire lentil isoflavone synthase.
  • SEQ ID NO:20 is the deduced amino acid sequence of an almost entire lentil isoflavone synthase derived from the nucleotide sequence of SEQ ID NOJ 9.
  • SEQ ID NO:21 is the nucleotide sequence comprising the lentil cDNA insert in clone lentil2 encoding an almost entire lentil isoflavone synthase.
  • SEQ ID NO:22 is the deduced amino acid sequence of an almost entire lentil isoflavone synthase derived from the nucleotide sequence of SEQ ID NO:21.
  • SEQ ID NO:23 is the nucleotide sequence comprising the mung bean cDNA insert in clone mung beanl encoding an entire mung bean isoflavone synthase.
  • SEQ ID NO:24 is the deduced amino acid sequence of an entire mung bean isoflavone synthase derived from SEQ ID NO:23.
  • SEQ ID NO:25 is the nucleotide sequence comprising the mung bean cDNA insert in clone mung bean2 encoding an entire mung bean isoflavone synthase.
  • SEQ ID NO:26 is the deduced amino acid sequence of an entire mung bean isoflavone synthase derived from SEQ ID NO:25.
  • SEQ ID NO:27 is the nucleotide sequence comprising the mung bean cDNA insert in clone mung bean3 encoding an entire mung bean isoflavone synthase.
  • SEQ ID NO:28 is the deduced amino acid sequence of an entire mung bean isoflavone synthase derived from SEQ ID NO:27.
  • SEQ ID NO:29 is the nucleotide sequence comprising the mung bean cDNA insert in clone mung bean4 encoding an entire mung bean isoflavone synthase.
  • SEQ ID NO:30 is the deduced amino acid sequence of an entire mung bean isoflavone synthase derived from SEQ ID NO:30.
  • SEQ ID NO:31 is the nucleotide sequence comprising the red clover cDNA insert in clone red cloverl encoding an entire red clover isoflavone synthase.
  • SEQ ID NO:32 is the deduced amino acid sequence of an entire red clover isoflavone synthase derived from SEQ ID NO: 31.
  • SEQ ID NO:33 is the nucleotide sequence comprising the red clover cDNA insert in clone red clover2 encoding an entire red clover isoflavone synthase.
  • SEQ ID NO:34 is the deduced amino acid sequence of an entire red clover isoflavone synthase derived from SEQ ID NO:33.
  • SEQ ID NO:35 is the nucleotide sequence comprising the snow pea cDNA insert in clone snow peal encoding an entire snow pea isoflavone synthase.
  • SEQ ID NO:36 is the deduced amino acid sequence of an entire snow pea isoflavone synthase derived from SEQ ID NO: 37.
  • SEQ ID NO:37 is the nucleotide sequence comprising the white clover cDNA insert in clone white cloverl encoding an almost entire white clover isoflavone synthase.
  • SEQ ID NO:38 is the deduced amino acid sequence of an almost entire white clover isoflavone synthase derived from SEQ ID NO:37.
  • SEQ ID NO:39 is the nucleotide sequence comprising the white clover cDNA insert in clone white clover2 encoding an almost entire white clover isoflavone synthase.
  • SEQ ID NO:40 is the deduced amino acid sequence of an almost entire white clover isoflavone synthase derived from SEQ ID NO: 39.
  • SEQ ID NO:41 is the nucleotide sequence of an oligonucleotide primer used for PCR amplification of the isoflavone synthase coding region in clone sgslc.pk006.o20.
  • SEQ ID NO:42 is the nucleotide sequence of an oligonucleotide primer used for PCR amplification of the isoflavone synthase coding region in clone sgslc.pk006.o20.
  • SEQ ID NO:43 is the nucleotide sequence of an oligonucleotide primer used to determine the transcription of the soybean isoflavone synthase in transgenic tobacco.
  • SEQ ID NO:44 is the nucleotide sequence of an oligonucleotide primer used to determine the transcription of the soybean isoflavone synthase in transgenic tobacco.
  • SEQ ID NO:45 is the nucleotide sequence of an oligonucleotide primer to the maize R coding region used to amplify genomic DNA to determine the presence of a chimera containing the maize R region between the region encoding the C 1 DNA binding domain and the CI activation domain (CRC) in transgenic corn cells.
  • CRC CI activation domain
  • SEQ ID NO:46 is the nucleotide sequence of an oligonucleotide primer to the 3' untranslated region from potato protease inhibitor II gene used to amplify genomic DNA to determine the presence of CRC in transgenic corn cells.
  • SEQ ID NO:47 is the nucleotide sequence comprising the sugarbeet cDNA insert in clone sugarbeetl, encoding an almost entire sugarbeet isoflavone synthase.
  • SEQ ID NO:48 is the deduced amino acid sequence of an almost entire sugarbeet isoflavone synthase derived from SEQ ID NO:47.
  • SEQ ID NO:49 is the nucleotide sequence of an oligonucleotide primer used for the PCR amplification of the soybean isoflavone synthase coding region in clone sgslc.pk006.o20.
  • SEQ ID NO:50 is the nucleotide sequence of an oligonucleotide primer used for the PCR amplification of the soybean isoflavone synthase coding region in clone sgslc.pk006.o20.
  • SEQ ID NO:51 is the nucleotide sequence of an oligonucleotide primer used to amplify the genomic sequence comprising the isoflavone synthase in clone sgslc.pk006.o20.
  • SEQ ID NO:52 is the nucleotide sequence of a genomic fragment encoding the isoflavone synthase in clone sgslc.pk006.o20.
  • SEQ ID NO:53 is the nucleotide sequence of a genomic fragment encoding the CYP93C1 isoflavone synthase.
  • SEQ ID NO:54 is the nucleotide sequence comprising the lupine cDNA insert in clone lupine 1 encoding an entire lupine isoflavone synthase.
  • SEQ ID NO:55 is the deduced amino acid sequence of an entire lupine isoflavone synthase derived from SEQ ID NO:54.
  • SEQ ID NO:56 is the nucleotide sequence comprising the alfalfa cDNA insert in clone alfalfa2 encoding an almost entire alfalfa isoflavone synthase.
  • SEQ ID NO:57 is the amino acid sequence of an almost entire alfalfa isoflavone synthase derived from SEQ ID NO:56.
  • SEQ ID NO:58 is the nucleotide sequence comprising the alfalfa cDNA insert in clone alfalfa3 encoding an almost entire alfalfa isoflavone synthase.
  • SEQ ID NO:59 is the amino acid sequence of an almost entire alfalfa isoflavone synthase derived from SEQ ID NO:58.
  • SEQ ID NO: 60 is the amino acid sequence comprising the sugarbeet cDNA insert in clone sugarbeet2, encoding an almost entire sugarbeet isoflavone synthase.
  • SEQ ID NO:61 is the deduced amino acid sequence of an almost entire sugarbeet isoflavone synthase derived from SEQ ID NO:60.
  • SEQ ID NO: 62 is the nucleotide sequence of an oligonucleotide primer used for the PCR amplification of the soybean chalcone reductase coding region in clone src3c.pk009.e4.
  • SEQ ID NO: 63 is the nucleotide sequence of an oligonucleotide primer used for the PCR amplification of the soybean chalcone reductase coding region in clone src3c.pk009.e4.
  • SEQ ID NO: 64 is the nucleotide sequence of an oligonucleotide primer used for the PCR amplification of the soybean chalcone reductase present in monocot cells.
  • SEQ ID NO:65 is the nucleotide sequence of an oligonucleotide primer used for the PCR amplification of the soybean chalcone reductase present in monocot cells.
  • SEQ ID NO: 66 is the amino acid sequence of the consensus sequence produced by the
  • the instant invention discloses nucleotide and amino acid sequences for isoflavone synthases from legumes such as soybean, alfalfa, lupine, hairy vetch, lentil, mung bean, red clover, snow pea, and white clover and non-legumes such as sugarbeet.
  • Plant P450 enzymes catalyze a diverse range of reactions, including molecular transformations in primary metabolism, and in the metabolism and detoxification of xenobiotics. Although tentative identification of any given gene or conceptual translation product as a P450 is relatively simple based on its similarity to other known P450s, the assignment of actual catalytic function cannot necessarily be inferred from nucleic acid or protein sequence information alone.
  • the instant disclosure demonstrates and teaches the identification of a cDNA from soybean that encodes isoflavone synthase based on the ability of the encoded polypeptide to convert the normal substrate for the reaction, 2S-flavanone, to genistein. Demonstration of activity has been accomplished in subcellular fractions of a yeast strain, WHT1, which has been specifically altered to also express a P450 reductase from Helianthus tuber osum. In this manner, and using the materials identified and described herein, other nucleic acid sequences from soybean and from other plants that are predicted to encode P450s may be tested to determine whether any of those P450's possess isoflavone synthase activity.
  • the isoflavonoids are biogeneticaly related to the flavonoids but constitute a distinctly separate class in that they contain a rearranged C15 skeleton and may be regarded as derivatives of 3-phenylchroman.
  • Isoflavones are the most abundant of the natural isoflavonoid derivatives, with over 160 isoflavone aglycones being recognized.
  • nucleic acid sequence is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • a nucleic acid sequence in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
  • substantially similar refers to nucleic acid sequences wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid sequences wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid sequence to mediate alteration of gene expression by gene silencing through for example antisense or co- suppression technology.
  • Substantially similar also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-a-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.
  • antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed.
  • alterations in a nucleic acid sequence which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide are well known in the art.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product.
  • Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide.
  • substantially similar nucleic acid sequences may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar sequences, such as homologous sequences from distantly related organisms, to highly similar sequences, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
  • One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min.
  • a more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60°C.
  • Another preferred set of highly stringent conditions uses two final washes in 0JX SSC, 0J% SDS at 65°C.
  • nucleic acid sequences of the instant invention may also be characterized by their percent identity to the nucleic acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art.
  • Preferred are those nucleic acid sequences whose sequences are at least about 85% identical and more preferably at least about 90% identical to the nucleotide sequences reported herein. More preferred are nucleic acid sequences that are at least about 90% identical and more preferably at least about 95% identical to the nucleotide sequences reported herein. More preferred are nucleic acid sequences that are 95% identical to the nucleotide sequences reported herein.
  • Substantially similar nucleic acid sequences of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Preferred are those nucleic acid sequences whose nucleotide sequences encode amino acid sequences that are at least about 95% identical and even more preferably at least about 98% identical to the amino acid sequences reported herein. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sha ⁇ (1989) CABIOS.
  • a "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises.
  • Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer- based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).
  • a sequence often or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
  • gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
  • oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid sequence comprising the primers.
  • a "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid sequence comprising the sequence.
  • the instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for pu ⁇ oses known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
  • Codon degeneracy refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid sequence comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein.
  • the skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid sequence for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
  • Synthetic nucleic acid fragments can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid sequences which may then be enzymatically assembled to construct the entire desired nucleic acid sequence. "Chemically synthesized”, as related to nucleic acid sequence, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid sequences may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines.
  • nucleic acid sequences can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell.
  • the skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
  • Gene refers to a nucleic acid sequence that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • Native gene refers to a gene as found in nature with its own regulatory sequences.
  • Chimeric gene refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • Coding sequence refers to a nucleotide sequence that codes for a specific amino acid sequence.
  • Regulatory sequences refer to nucleotide sequences located upstream (5' non- coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an "enhancer” is a nucleotide sequence which can stimulate promoter activity. It may be an innate element of the promoter or a heterologous element inserted to enhance the level and/or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
  • Promoters which cause a nucleic acid sequence to be expressed in most cell types at most times are commonly referred to as “constitutive promoters".
  • “Organ-specific” or “development-specific” promoters are those that direct gene expression almost exclusively in specific organs, such as leaves or seeds, or at specific development stages in an organ, such as in early or late embryogenesis, respectively.
  • New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 75:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid sequences of different lengths may have identical promoter activity.
  • the origin of the promoter chosen to drive the expression of the coding sequence is not critical as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA for the desired protein genes in the desired host tissue.
  • the "translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence.
  • the translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence.
  • the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Molecular Biotechnology 5:225-236).
  • the "3' non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • the use of different 3' non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 7:671-680.
  • RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into polypeptide by the cell.
  • cDNA refers to a double-stranded DNA that is complementary to and derived from mRNA.
  • Sense RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell.
  • Antisense RNA refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Patent No. 5,107,065, inco ⁇ orated herein by reference).
  • the complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
  • operably linked refers to the association of two or more nucleic acid sequences on a single nucleic acid sequence so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid sequence of the invention. Expression may also refer to translation of mRNA into a polypeptide.
  • Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.
  • Overexpression refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
  • Co-suppression refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Patent No. 5,231,020, inco ⁇ orated herein by reference).
  • Altered levels refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
  • Transformation refers to the transfer of a nucleic acid sequence into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:211) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Patent No. 4,945,050, inco ⁇ orated herein by reference).
  • Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook”).
  • a nucleic acid sequence encoding a soybean isoflavone synthase was isolated and identified from a cDNA library. Nucleic acid sequences encoding three alfalfa, one hairy vetch, one snow pea, one lupine, two lentil, two red clover, two white clover, two sugarbeet, and four mung bean isoflavone synthases have been isolated-using RT-PCR. Nucleic acid sequences encoding two soybean isoflavone synthases have been isolated from genomic DNA. The nucleic acid sequences of the instant invention may be used to isolate cDNAs and genes encoding homologous enzymes from the same or other plant species.
  • sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
  • genes encoding other isoflavone synthase proteins could be isolated directly by using all or a portion of the instant nucleic acid sequence as aDNA hybridization probe to screen libraries from any desired plant employing methodology well known to those skilled in the art.
  • Specific oligonucleotide probes based upon the instant nucleic acid sequence can be designed and synthesized by methods known in the art (Sambrook). Moreover, the entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
  • primers can be designed and used to amplify a part of or full-length of the instant sequences.
  • the resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length cDNA or genomic fragments under conditions of appropriate stringency.
  • two short segments of the instant nucleic acid sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid sequences encoding homologous genes from DNA or RNA.
  • the polymerase chain reaction may also be performed on a library of cloned nucleic acid sequences wherein the sequence of one primer is derived from the instant nucleic acid sequences, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes.
  • the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl.
  • Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36 A; Sambrook).
  • the nucleic acid sequence of the instant invention may be used to create transgenic plants and transgenic seeds in which expression of nucleic acid sequences (or their complements) encoding the disclosed enzyme result in levels of the corresponding endogenous enzyme that are higher or lower than normal.
  • expression of the instant nucleic acid sequence may result in the production of the encoded enzyme in cell types or developmental stages in which they are not normally found. Either strategy would have the effect of altering the level of isoflavonoids.
  • Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development.
  • the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals may also be provided.
  • the instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.
  • Plasmid vectors comprising the isolated polynucleotide (or chimeric gene) may be constructed.
  • the choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBOJ. ⁇ :2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 275:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.
  • the nucleic acid sequence of the instant invention may be used to create transgenic plants that have increased expression of the disclosed enzyme and that are additionally transformed with a chimeric gene encoding a transcription factor that regulates expression of one or more genes in the phenylpropanoid pathway.
  • the chimeric transcription factor gene has regulatory sequences such that its expression is coordinated with that of the isoflavone synthase gene developmentally and preferably within the same cell type. This combination of expression of isoflavone synthase and transcription factor regulating phenylpropanoid pathway genes has the effect of enhancing the level of isoflavonoid synthesis due to increased levels of substrates for isoflavone synthase.
  • the chimeric transcription factor gene regulates expression of at least one gene in the phenylpropanoid pathway. While not intending to be bound by any theory or theories of operation it is believed to regulate as many as two, three or four genes in the phenylpropanoid pathway.
  • a plant cell that does not naturally produce isoflavonoids and does not have an active phenylpropanoid pathway would not produce the substrates for isoflavone synthase to convert to isoflavonoids.
  • Activation of the phenylpropanoid pathway in the desired cells or at the desired developmental stage would provide these substrates allowing the synthesis of isoflavonoids.
  • the present invention is also directed to a method of altering the level of isoflavonoids in a cell comprising exposing said cell to a phenylpropanoid pathway altering agent.
  • the cell may be a plant cell such as a monocot, including and not limited to corn, or a dicot, such as soybean, for example.
  • a phenylpropanoid pathway altering agent may be any agent that results in an increase or decrease in the level of expression of an enzyme in the phenylpropanoid pathway, such as isoflavone synthase, phenylalanine ammonia lyase, chalcone synthase, among others.
  • Such phenylpropanoid pathway altering agents include and are not limited to a transcription factor and stress.
  • Transcription factors include and are not limited to chimeric transcription factors, a chimera containing the maize R region between the region encoding the CI DNA binding domain and the CI activation domain (CRC) for example.
  • Stresses to a plant cell include ultraviolet light, temperature, pressure, chemicals including and not limited to herbicides, and phosphate level. Phosphate levels may be increased or decreased such that decreasing phosphate levels may result in phosphate starvation.
  • a chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene sequence encoding that polypeptide to plant promoter sequences.
  • a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid sequence can be constructed by linking the gene or gene sequence in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.
  • Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U. S. Patent Nos. 5,190,931, 5,107,065 and
  • a preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.
  • the instant isoflavone synthases (or portions of the enzymes) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the enzymes by methods well known to those skilled in the art.
  • the antibodies are useful for detecting the enzymes in situ in cells or in vitro in cell extracts.
  • Preferred heterologous host cells for production of isoflavone synthase are yeast hosts. Yeast expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art.
  • chimeric genes for production of the instant isoflavone synthase.
  • These chimeric genes could then be introduced into appropriate hosts via transformation to provide high level expression of the enzymes.
  • An example of a vector for high level expression of the instant isoflavone synthase in a yeast host is provided (Example 5).
  • nucleic acid sequences of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes.
  • the instant nucleic acid sequences may be used as restriction sequence length polymo ⁇ hism (RFLP) markers.
  • RFLP restriction sequence length polymo ⁇ hism
  • Southern blots Mantonis
  • the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 7:174-181) in order to construct a genetic map.
  • nucleic acid sequences of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymo ⁇ hisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 52:314-331). The production and use of plant gene-derived probes for use in genetic mapping is described in Beraatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4(1):31-A ⁇ .
  • Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
  • nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).
  • FISH direct fluorescence in situ hybridization
  • nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 114(2):95-96), polymo ⁇ hism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 7(5:325-332), allele-specific ligation (Landegren et al. (1988) Science 2 ⁇ 7:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 75:3671), Radiation Hybrid Mapping (Walter et al.
  • isoflavonoids The physiological activities associated with isoflavonoids in both plants and humans makes the manipulation of their contents in crop plants highly desirable. For example, increasing levels of isoflavonoids in soybean seeds would increase the efficiency of extraction and lower the cost of isoflavonoid-related products sold. Decreasing levels of isoflavonoids in soybean seeds would be beneficial for production of soy-based infant formulas where the estrogenic effects of isoflavonoids are undesirable. Decreasing levels of isoflavonoids may also increase palatability of soy foods. Raising levels of isoflavonoid phytoalexins in vegetative plant tissue could increase plant defenses to pathogen attack, thereby improving resistance and lowering the need for pesticide use. Manipulation of isoflavonoid levels in roots could lead to improved nodulation and increased efficiencies of nitrogen fixation. To date, however, it has proven difficult to develop soybean or other plant lines with consistently high levels of isoflavonoids.
  • Identification of the functional isoflavone synthase gene is extremely important because isoflavone synthase catalyzes the central reaction in pathways producing isoflavonoids.
  • Manipulation of the isoflavone synthase gene via molecular techniques is expected to allow production of soybeans and other plants with high, stable levels of isoflavonoids.
  • Introduction of the isoflavone synthase gene in non-legume crop species including, but not limited to, corn, wheat, rice, sunflower, and canola could lead to synthesis of isoflavonoids in these species. Synthesis of isoflavonoids would 1) confer disease resistance to the crops and/or 2) produce crops which would benefit human and/or livestock health.
  • 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 ⁇ Em ⁇ s" 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. Elicitor treatment was conducted as follows. The epidermal surfaces of the hypocotyls were opened using a razor blade. The incisions were approximately 2 cm long and 1 to 2 mm deep; one was made on each hypocotyl. Fungal-derived elictors were prepared by the method of Sharp et al. (Sharp, J. K. et al. (1984) J. Biol. Chem. 259:11312-11320).
  • Soybean suspension cell cultures were grown at 25°C in 250 mL flasks that were tightly covered with two layers of aluminum foil to prevent illumination. Cells were grown in 35 mL of Murashige and Skoog medium (Gibco BRL) supplemented with 0.75 mg/L 2, 4-dichlorophenoxy acetic acid and 0.55 mg/mL 6-benzyl aminopurine. Cells were diluted (1 :3 ratio) into fresh medium every 7 days and elicitor treatment was conducted 3 days after cell dilution. One hundred fifty milligrams of the same fungal elicitor used to treat the hypocotyls was dissolved in 15 mL of 10 mM KH2PO4 and was filter sterilized.
  • the supernate was carefully transferred into 13 mL polyallomer tubes which fit into a Sorvall TH641 rotor and centrifuged at 160,000 g for 40 minutes to 2 h at 4°C.
  • the precipitated microsomes were washed twice with the storage buffer (buffer B: 80 mM KH 2 PO 4 , pH 8.5, 14 mM ⁇ -mercaptoethanol, 30% (v/v) glycerol) and resuspended with storage buffer.
  • the microsomal pellet was gently homogenized by hand using a disposable plastic pestle, and the suspension was divided into several aliquots which were frozen on dry-ice.
  • the reaction mixture was prepared at room temperature and consisted of 100 ⁇ M naringenin or liquiritigenin, 80 mM K 2 HPO , 0.5 mM glutathione (Sigma, G-4251), 20% w/v sucrose, and 30 to 150 ⁇ g 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 ⁇ L.
  • the reaction was started by the addition of 40 nmol of NADPH per each 100 ⁇ L of final reaction volume.
  • the pH of the reaction mixture was 8.0 before the addition of the substrate, NADPH and microsomes. Microsomes were thawed, an aliquot removed and the remaining sample was immediately frozen on dry ice and stored in the freezer. The reactions using microsomes prepared from soybean elicitor-treated hypocotyls were run for incubation periods of up to 24 h, while the reactions using the yeast microsomes were allowed to run for incubation periods of up to 14 h. Following incubation, 200 ⁇ L of ethyl acetate was added directly to the mixture and the mixture was shaken for 1 minute using a vortex mixer. Separation of the organic phase was accelerated by centrifugation for 2 minutes at 4°C. The organic phase was removed and analyzed.
  • the second column was used for plant samples where the ethyl acetate was evaporated and the samples resuspended in 80% methanol. In these cases separation used a 10 minutes linear gradient from 20% methanol/80% 10 mM ammonium acetate, pH 8.3 to 100% methanol using a flow rate of 0.8 ml per minute. Genistein and daidzein were monitored by the absorbance at 260 nm and naringenin and liquiritigenin were monitored by the absorbance at 280 nm.
  • Peak areas were converted to nanograms using, as standards for calibration, authentic naringenin, liquiritigenin, genistein, and daidzein (Indofine Chemical Company, Inc., Somerville, NJ) dissolved in ethanol.
  • Analyses using LC/MS employed 10 ⁇ L of the ethyl acetate phase that had been first evaporated with nitrogen gas and resuspended in 100 ⁇ L of 25% acetonitrile in water. These samples were analyzed by a Hewlett-Packard/Micromass LC/MS instrument.
  • a twenty-five microliter sample was run on a Zorbax Eclipse XDB-C8 reverse-phase column (3 X 150 mm, 3.5 micron) isocratically with 25% of solvent B in solvent A.
  • Solvent A was 0.1% formic acid in water
  • solvent B was 0.1% formic acid in acetonitrile.
  • Mass spectrometry was carried out by electro-spray scanning from 200-400 m/e, using +60 volt cone voltage. The diode array signals were monitored between 200-400 nm in both instruments.
  • Positive control samples consisting of soybean microsomes which were prepared from elicitor-treated hypocotyls and suspension culture cells were used to establish the in vitro assay system. Optimization of this in vitro assay system was critical for validation of the yeast expression system for functional cloning. We observed positive results (i.e., the synthesis of genistein) in assays that used either the microsomes of elicitor-treated soybean hypocotyls ( Figure 4) or those obtained from elicitor-treated cell suspension cultures ( Figure 6).
  • EXAMPLE 3 Composition of Soybean cDNA Library. Isolation and Sequencing of cDNA Clone 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-ZAPTM XR vector according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA).
  • 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. (1991) Science 252:1651-1656). The resulting ESTs were analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
  • Example 3 The cDNA sequences obtained in Example 3 were analyzed for similarity to all publicly available DNA sequences contained in the "nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr” database using the BLASTX algorithm (Gish, W. and States, D. J. (1993) Nature Genetics 5:266-272) provided by the NCBI.
  • NCBI National Center for Biotechnology Information
  • the insert in cDNA clone sgslc.pk006.o20 was identified as a candidate isoflavone synthase gene by a BLAST search against the NCBI database.
  • the 5' sequence of this insert was determined to be related to Glycine max cytochrome P450 monooxygenase CYP93Clp (CYP93C1) mRNA, the complete coding sequence of which may be found as NCBI General Identifier No. 2739005.
  • the CYP93Clp cDNA sequence was obtained using random isolation and screening to identify soybean P450s involved in herbicide metabolism (Siminszky B., et al. (1999) Proc. Natl. Acad. Sci. U.S.A.
  • Isoflavone synthase catalyzes in soybeans the oxidation of 7,4'dihyroxyflavanone (liquiritigenein) or 5,7,4'trihydroxyflananone (naringenin) to daidzein or genistein respectively.
  • Previously published work Karls and Griesbach (1986) Eur. J. Biochem 755:311-318; Hashim et al. (1990) FEBS 277:219-222) suggested that the enzyme that catalyzes this reaction is a cytochrome P450.
  • the polypeptide encoded by this insert was evaluated for its ability to catalyze the formation of genistein from naringenin.
  • the ability of the cDNA insert in clone sgslc.pk006.o20 to encode an isoflavone synthase was evaluated by expression of the encoded polypeptide in an engineered yeast (Saccharomyces cerivisae) strain. Microsomes prepared from the engineered yeast strain transformed with a plasmid encoding the putative isoflavone synthase were assayed for their ability to mediate the synthesis of genistein in the presence of substrate (naringenin).
  • Yeast strain W303-1B was used as the starting material and modified by homologous recombination.
  • the coding sequence of the P450 reductase HTl isolated from Helianthus tuberosus was inserted into the integrative plasmid pYeDPl 10 (Pompon, D. et al. (1996) Meth. Enz. 272:51-64). Insertion was achieved after PCR amplification for addition of Bam HI and ⁇ co Rl restriction sites 5' and 3' of the coding region, respectively, using the primers listed as S ⁇ Q ID NO:3 and S ⁇ Q ID NO:4.
  • Plasmid DNA 200 ng was used as template for PCR with primers that are homologous to the vector sequences flanking the cDNA cloning site (S ⁇ Q ID NO:5 and S ⁇ Q ID NO:6).
  • Amplification was performed using the GC melt kit (Clontech) with a 1 M final concentration of GC melt reagent. Amplification took place in a Perkin Elmer 9700 thermocycler for 30 cycles as follows: 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. The amplified insert was then incubated with a modified pRS315 plasmid (NCBI General Identifier No. 984798; Sikorski, R. S. and Hieter, P. (1989) Genetics 122: 19-27) that had been digested with Not I and Spe I.
  • NCBI General Identifier No. 984798 NCBI General Identifier No. 984798; Sikorski, R. S. and Hieter, P. (1989) Genetics 122: 19-27
  • Plasmid pRS315 had been previously modified by the insertion of a bidirectional gal 1/10 promoter between the Xho I and Hind III sites.
  • the plasmid was then transformed into the WHTl yeast strain using standard procedures.
  • the insert recombines though gap repair to form the desired plasmid (Hua, S. B., et al. (1997) Plasmid 55:91-96.).
  • the resulting transformed yeast strain is named Isoflavone Synthase GM1 (hereinafter referred to as "GM1”), and bears ATCC Accession No. 203606.
  • Yeast microsomes were prepared according to the methods of Pompon et al. (Pompon, D., et al. (1996) Meth. Enz. 272:51-64). Briefly, a yeast colony was grown overnight (to saturation) in SG (-Leucine) medium at 30°C with good aeration. A 1 :50 dilution of this culture was made into 500 mL of YPGE medium with adenine supplementation and allowed to grow at 30°C with good aeration to an OD600 of 1.6 (24-30 h). Fifty mL of 20% galactose was added, and the culture was allowed to grow overnight at 30°C.
  • the cells were recovered by centrifugation at 5,500 ⁇ m for five minutes in a Sorvall GS-3 rotor.
  • the cell pellet was resuspended in 80 mL of TEK buffer (0J M KCl in TE) and left at room temperature for five minutes.
  • the cells were recovered by centrifugation as described above.
  • the cell pellet was resuspended in 5 mL of TES-B (0.6M sorbitol in TE), and glass beads (0.5 mm diameter) were gently added until they reached the surface of the suspension.
  • the cells were disrupted by shaking up and down for five minutes, with an agitation frequency of at least once every 0.5 second.
  • TES-B Five mL of TES-B were added to the crude extract, and the beads were washed with some agitation. The supernatant was withdrawn and saved. The wash was repeated twice and the liquid fractions were pooled. The combined fractions were clarified by spinning at 11,000 ⁇ m in a Sorvall SS34 rotor. The pellet was discarded and the microsomes were precipitated by the addition of NaCl to a final concentration of 0J5 M. PEG 4000 was added to a final concentration of 0J g/mL. The mixture was incubated on ice for at least 15 minutes, and the microsomal fraction was recovered by at 8,500 ⁇ m for 10 minutes in an SS34 rotor.
  • the molecular weights of the materials corresponding to the expected genistein and daidzein peaks from the yeast microsome assay samples were 270.32 and 255.2, respectively.
  • the molecular weights of authentic genistein and daidzein are 270.23 and 255.2, respectively.
  • Genistein synthesis corresponds quantitatively with the amount of input GM1 microsomes ( Figure 14 and Figure 15).
  • the genistein peak in the assay using GM1 as a source was about 10 times higher than the peak observed from soybean microsome prepared from elicitor-treated hypocotyls (compare Figure 4 and Figure 13).
  • Genistein synthesis by yeast microsomes using GM1 also demonstrated an absolute requirement for NADPH. Without the cofactor, the reaction mixture did not synthesize any detectable genistein over a 4-h incubation ( Figure 16).
  • EXAMPLE 6 Identification of CYP93C1 as a Soybean Isoflavone Synthase
  • the function of the protein encoded by this mRNA has yet to be identified.
  • the cDNA insert in clone sgslc.pk006.o20 encodes an isoflavone synthase and has sequence similarities with CYP93C1.
  • CYP93C1 encodes a functional isoflavone synthase
  • cDNA was prepared and cloned into the yeast vector pRS315-gal and transformed into yeast strain WHT1 to assay for its ability to produce genistein.
  • the CYP93C1 mRNA was amplified from RNA isolated from soybean tissue (cv. S 1990) infected with the fungal pathogen Sclerotinia slerotiorum using RT-PCR. Fungal infection causes an increase in the amount of isoflavonoid produced and thus the amount of isoflavone synthase transcript was increased in the infected tissue. Soybean plants were infected 45 days after planting seeds and were harvested two days later.
  • RNA was prepared using the TRIzol Reagent following the manufacturer's instructions (Gibco BRL) and 1 ⁇ g of the resulting total RNA was converted into a first strand cDNA using the SuperscriptTM Preamplification system and using oligodT as the reverse transcription primer.
  • One microliter of first strand cDNA was amplified by PCR using the primers listed as SEQ ID NO:7 and SEQ ID NO:8:
  • nucleotide sequence in SEQ ID NO: 7 corresponds to nucleotides 3 to 26 of the sequence found in NCBI General Identifier No. 2739005.
  • nucleotide sequence in SEQ ID NO:8 corresponds to the complement of nucleotides 1798 to 1824 of the sequence found in NCBI General Identifier No. 2739005.
  • Amplification was performed on a Perkin Elmer Applied Biosystems GeneAmp PCR System using the Advantage-GC cDNA polymerase mix (Clontech), following the manufacturer's instructions, with a 1 M final concentration of GC melt reagent. Previous to amplification, the mixture was incubated at 94°C for 5 minutes.
  • Amplification was performed using 30 cycles of: 94°C for 30 seconds, 53°C for 30 seconds and 72°C for 2 minutes. Following amplification, the mixture was incubated at 72°C for 7 minutes. The amplified product was then cloned into pCR2J using "The Original TA Cloning Kit” (Invitrogen). Plasmid DNA was purified using QIAFilter cartridges (Qiagen Inc) according to the manufacturer's instructions. Sequence was generated on an ABI Automatic sequencer using dye terminator technology and using a combination of vector and insert-specific primers. Sequence editing was performed using DNAStar (DNASTAR, Inc.). The sequence generated represents coverage at least two times in each direction.
  • the above plasmid was then cloned into the yeast vector pRS315-gal using gap repair as described in Example 4. Standard procedures were used to transform the resulting plasmid into the WHT1 yeast strain. Microsomes were prepared from the WHT1 yeast strain containing the soybean CYP93C1 sequence and assayed for the production of genistein and daidzein as described in Example 5. The resulting microsomes exhibited isoflavone synthase activities. To compare the rates of genistein and daidzein synthesis by microsomes of the yeast transformant containing the soybean CYP93C1 sequence, samples representing incubation periods of 2, 4, 6, 8 and 10 h were analyzed.
  • Amplification was performed on a Perkin-Elmer Applied Biosystems GeneAmp PCR System 9700PCR using Advantage-GC cDNA polymerase mix (Clontech) according to the manufacturer's instructions and with a final concentration of GC melt reagent equal to 1 M. Amplification was preceded in all cases by incubation at 94°C for 5 minutes and was followed by incubation at 72°C for 7 minutes. Two sets of primers were used for PCR amplification. Primer set one is composed of SEQ ID NO: 11 and SEQ ID NO: 12 and primer set two is composed of SEQ ID NO: 13 and SEQ ID NO: 14:
  • the initial amplification of all samples was done using 1 ⁇ L of first strand cDNA and primer set one (SEQ ID NO: 11 and SEQ ID NO: 12).
  • Amplification of mung bean was performed using 30 cycles of 94°C for 30 seconds, 48°C for 30 seconds and 72°C for 2 minutes.
  • Amplification of red clover was performed using 30 cycles of 94°C for 30 seconds, 50°C for 30 seconds and 72°C for 1 minute.
  • Amplification of white clover, lentil, hairy vetch, alfalfa and lupine was carried out in two steps.
  • the first amplification reaction was performed using 30 cycles of 94°C for 30 seconds, 50°C for 30 seconds and 72°C for one minute.
  • a second amplification reaction was done with 1 ⁇ L of the resulting product and primer set two (SEQ ID NO: 13 and SEQ ID NOJ 4) using 30 cycles of 94°C for 30 seconds, 50.5°C for 30 seconds and 72°C for one minute.
  • Amplification of snow pea was performed in three different PCR reactions. The first reaction was performed using 30 cycles of 94°C 30 seconds, 50.5°C for 30 seconds and 72°C for one minute. One microliter from the resulting product was used for a second amplification reaction using primer set one and 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for one minute.
  • the resulting reaction was analyzed on a 1% agarose gel and the band at the expected size was gel purified using the QIAquick Gel Extraction Kit (Qiagen).
  • the purified DNA was resuspended in 30 ⁇ L of water and 1 ⁇ L was used as a template for a third PCR reaction using primer set one with 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 90 seconds.
  • the resulting mung bean, red clover and snow pea PCR sequences were cloned into pCR2J using "The Original TA Cloning Kit” (Invitrogen).
  • the resulting white clover, lentil, hairy vetch, alfalfa and lupine PCR sequences were cloned into pCR-2.1 using TOPOTM TA Cloning Kit (Invitrogen).
  • Plasmid DNA was purified using QIAFilter cartridges (Qiagen Inc) or Wizard Plus Minipreps DNA Purification System (Promega) following the manufacturer's instructions. Sequence was generated on an ABI Automatic sequencer using dye terminator technology and using a combination of vector and insert-specific primers. Sequence editing was performed using DNAStar (DNASTAR, Inc.). All sequences represent coverage at least two times in both directions.
  • the nucleotide sequence of comprising the cDNA insert in clone alfalfa 1 is shown in SEQ ID NO: 15; the deduced amino acid sequence of this DNA is shown in SEQ ID NO: 16.
  • the nucleotide sequence comprising the cDNA insert in clone alfalfa 2 is shown in SEQ ID NO:57; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:58.
  • the nucleotide sequence comprising the cDNA insert in clone alfalfa 3 is shown in SEQ ID NO:59; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:60.
  • the nucleotide sequence comprising the cDNA insert in clone hairy vetch 1 is shown in SEQ ID NO: 17; the deduced amino acid sequence of this DNA is shown in SEQ ID NOJ 8.
  • the nucleotide sequence comprising the cDNA insert in clone lentil 1 is shown in SEQ ID NO: 19; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:20.
  • the nucleotide sequence comprising the cDNA insert in clone lentil 2 is shown in SEQ ID NO:21; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:22.
  • the nucleotide sequence comprising the cDNA insert in clone mung bean 1 is shown in SEQ ID NO:23; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:24.
  • the nucleotide sequence comprising the cDNA insert in clone mung bean 2 is shown in SEQ ID NO:25; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:26.
  • the nucleotide sequence comprising the cDNA insert in clone mung bean 3 is shown in SEQ ID NO:27; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:28.
  • the nucleotide sequence comprising the cDNA insert in clone mung bean 4 is shown in SEQ ID NO:29; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:30.
  • the nucleotide sequence comprising the cDNA insert in clone red clover 1 is shown in SEQ ID NO: 31 ; the deduced amino acid sequence of this DNA is shown in SEQ ID NO: 32.
  • the nucleotide sequence comprising the cDNA insert in clone red clover 2 is shown in SEQ ID NO:33; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:34.
  • the nucleotide sequence comprising the cDNA insert in clone snow pea 1 is shown in SEQ ID NO:35; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:36.
  • the nucleotide sequence comprising the cDNA insert in clone white clover 1 is shown in SEQ ID NO:37; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:38.
  • the nucleotide sequence comprising the cDNA insert in clone white clover 2 is shown in SEQ ID NO:39; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:40.
  • the nucleotide sequence comprising the cDNA insert in clone lupine 1 is shown in SEQ ID NO:54; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:55.
  • Plasmids corresponding to mung bean 2, red clover 2 and snow pea 1 were amplified and the plant-specific DNA (corresponding to SEQ ID NO:25, SEQ ID NO:33 and SEQ ID NO:35) were transferred to the yeast vector pRS315-gal following the gap repair method explained in Example 4 to produce the yeast expression strains isoflavone synthase VR2, isoflavone synthase TP2, and isoflavone synthase PSI, respectively.
  • the eight amino acids at the amino- and carboxy-terminus correspond to those translated from the primers used in PCR amplification and not necessarily belong to the endogenous genes.
  • Microsomes were isolated from the resulting yeast WHT1 strains containing the mung bean, red clover or snow pea genes, and assayed for isoflavone synthase activity as described in Example 5, with minor modifications. After incubation for 16 hours, 200 ⁇ L of ethyl acetate was added to recover the isoflavonoids from the assay solution, the ethyl acetate was evaporated under nitrogen using a heating module evaporation system and the sample resuspended in 200 ⁇ L of 80% methanol. A 10 ⁇ L sample of this solution was injected into a Phenomenex Luna 3 ⁇ CI 8 (2) column (size: 150 x 4.6 mm.
  • the samples were eluted over 10 minutes using an increasing methanol gradient (from 20% methanol/80% 100 mM ammonium acetate buffer (pH 5.9) to 100% methanol (v/v)) at a flow rate of 1 mL per minute.
  • the levels of genistein and naringenin in the eluted samples were monitored through the abso ⁇ tion spectrum at 260 and 290 nm.
  • the genistein signal was verified by comparisons of retention time, diode array detected abso ⁇ tion spectra. As seen in Table 1 , microsomes from all three strains produced genistein and therefore exhibited isoflavone synthase activity.
  • Sugarbeet a member of the family Chenopodiaceae, is one of the few non-legume species to have been shown to have isoflavonoids present (Geigert, et al. (1973) Tetrahedron.29:2103-2106).
  • a second amplification reaction was done with 1 ⁇ L of the resulting product with primer set two (SEQ ID NO: 13 and SEQ ID NO: 14) and using 30 cycles of 94°C for 30 seconds, 50.5°C for 30 seconds and 72°C for one minute.
  • the resulting PCR sequence was cloned into pCR2.1 using TOPOTM TA Cloning Kit (Invitrogen). Plasmid DNA was purified using QIAFilter cartridges (Qiagen Inc) or Wizard Plus Minipreps DNA Purification System (Promega) following the manufacturer's instructions. Sequence was generated on an ABI Automatic sequencer using dye terminator technology and using a combination of vector and insert-specific primers. Sequence editing was performed using DNAStar (DNASTAR, Inc.).
  • nucleotide sequence comprising the cDNA insert in clone sugarbeet 1 is shown in SEQ ID NO:47; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:48.
  • the nucleotide sequence comprising the cDNA insert in clone sugarbeet 2 is shown in SEQ ID NO:61; the deduced amino acid sequence of this DNA is shown in SEQ ID NO:61.
  • Table 2 summarizes the relationship of the isoflavone synthase nucleotide and amino acid sequences disclosed herein. Reported are the percent identity of the nucleotide sequences set forth in SEQ ID NOs:9, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 47 and 54 to instant soybean isoflavone synthase sequence set forth in SEQ ID NOJ. In addition, the percent identity of the amino acid sequences deduced from the instant nucleotide sequences as set forth in SEQ ID NOs:10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
  • a consensus sequence was determined by aligning the amino acid sequences of the present invention using the Clustal method of alignment and this sequence is shown in SEQ ID NO:66. Amino acids not conserved are indicated by Xaa. These are: Xaajo Phe or Leu Xaai6 Ser or Leu Xaa 2 3 Ser or Thr Xaa 2 5 He or Lys Xaa39 Lys or Arg
  • Xaa 85 Asp or Gly To verify that the similarity between the isoflavone synthase nucleotide sequences from soybean and from sugarbeet were not due to artifacts of PCR, a nucleic acid sequence containing the soybean isoflavone synthase set forth in SEQ ID NO: 1 was used as a probe for Southern blot analysis against sugarbeet genomic DNA. Hybridization was done overnight at 65°C in 6X SSC, 5X Denhardts. Filters were washed 2 times in 2X SSC, 1% SDS at room temperature and 2 times in 0.2X SSC, 0.5% SDS at 65°C. Hybridizing bands were detected indicating that sugarbeet does contain genes with high homology to the soybean isoflavone synthase sequence. EXAMPLE 9
  • the resulting DNA sequence contains from the second codon to the stop codon of the soybean isoflavone synthase gene sequence followed by a Kpn I site.
  • the following three sequences (in 5' to 3' order) were assembled in pUC18 vector (New England Biolabs) to yield plasmid pOY160 (depicted in Figure 20):
  • 35S/cabL a promoter sequence comprising 1.3 Kb from the cauliflower mosaic virus (CaMV) 35S promoter extending to 8 bp downstream from the transcription start site followed by a 60 bp leader sequence derived from the chlorophyll a/b binding protein gene 22L (Ha ⁇ ster M. H. et al. (1988) Mol. Gen. Genet. 272:182-190);
  • CaMV cauliflower mosaic virus
  • IFS the isoflavone synthase gene fragment generated by PCR amplification using the primers from SEQ ID NOJ 1 and SEQ ID NO:42.
  • IFS The 5' end of IFS was ligated to Nco I-digested, filled-in, 35S/cabL.
  • the 3' end of IFS was digested with Kpn I and ligated to Kpn I-digested Nos3'.
  • the vector pPZP211 contains an npt II gene fragment under the control of the 35S
  • CaMV promoter conferring kanamycin resistance as the plant selectable marker (Hajdukiewicz P. et al. (1994) Plant Mol. Biol. 25:989-994).
  • the plasmid pOY204 was transformed into the Agrobacterium tumefaciens strain LBA4404 and was subsequently introduced into Nicotiana tobaccum by leaf disc co-cultivation following standard procedures (De Blaere et al. 1987 Meth. Enzymol.
  • Verification of the presence of the isoflavone synthase coding region in the genome of the tested tobacco shoots was done by separating the reaction product using a 1% agarose gel and staining with ethidium bromide. The expected 1.6 Kb fragment was obtained as the reaction product in all the transgenic tobacco shoots and not in the untransformed tobacco controls.
  • RT-PCR Transcription of the isoflavone synthase gene in the transgenic tobacco shoots was confirmed using RT-PCR.
  • Total steady-state plant RNA was extracted from four randomly-selected tobacco shoots resulting from transformation with pOY204 using the RNeasy Plant Mini Kit (Qiagen) following standard protocols.
  • RT-PCR amplification was performed using "The Superscript One Step RT-PCR Kit” (Gibco BRL) with the primers: 5'-GACGCCTCACTTACGACAACTCTGTG-3' [SEQ ID NO:43]
  • the activity of the soybean isoflavone synthase in the transgenic tobacco was determined by analyzing shoots for the presence of genistein. Approximately one gram of tissue from shoots of five-week-old rooting transformants and from untransformed tobacco plants were ground in liquid nitrogen and extracted for 20 minutes at room temperature using 10 mL of 80% ethanol. After filtration through Acrodisc CR-PTFE syringe filters (Gelman Sciences), 3 mL from each extraction solution were concentrated to 1 mL by evaporation under nitrogen gas flow using a 50°C heating block.
  • the hydroxyl groups in the samples were derivatized to trimethylsilylate by the addition of 100 ⁇ L of BSTFA (N, O-bis(trimethylsilyl)-trifluoroacetamide; Supelco) and incubation at 37°C for 1 h.
  • BSTFA N, O-bis(trimethylsilyl)-trifluoroacetamide; Supelco
  • the samples were dried under nitrogen gas and re-dissolved in 20 ⁇ L chloroform immediately before manual injection into the gas chromatograph.
  • Two ⁇ L of sample were manually injected onto a 15 meter dry bed GC capillary column ( J&W, Jones Chromatography, Mid Glamorgan, UK) through an injector port operated in the split mode (5:1).
  • the initial oven temperature was set at 200°C and the column was set at a linear temperature gradient from 200°C to 300°C in 20 minutes with a helium gas flow rate of 1.5 mL/minute.
  • the mass spectrum was monitored using a Hewlett Packard 5973 mass-selective detector at an ionization potential of 70 eV.
  • the mass ions identified from the cracking pattern of pure genistein treated as mentioned above are 414 and 399 m/z. These peaks represent the products of partially derivatized genistein, the form obtained following the above procedure. Twenty nine of thirty three tobacco transformants analyzed by gas chromatography had an identifiable genistein peak at 8.7 minutes.
  • the prevalence of genistein in the flowers relates to the expression of the anthocyanin biosynthetic pathway, which is active in the flowers as indicated by the pink flower color.
  • An active anthocyanin pathway produces the naringenin substrate for isoflavone synthase.
  • Arabidopsis thaliana was transformed with the plasmid pOY204 via inplanta vacuum infiltration following standard protocols (Bechtold et al. (1993) CR Life Sciences 57(5:1194-1199). Briefly, three-week-old Arabidopsis thaliana ectotype WS plants were submerged in 500 mL of Agrobacterium, strain GV3101 harboring pOY204, suspended in basic MS media (Gibco BRL) and vacuum was applied repeatedly for 10 minutes. The infiltrated plants were allowed to set seeds for another three weeks.
  • Extracts were prepared and analyzed by HPLC and GC/MS as described in Example 2, except that after hydrolysis, the dried ethyl acetate extracts were resuspended in 1 mL of 80% methanol. Five of twelve randomly-selected Arabidopsis transformants analyzed by HPLC had an identifiable genistein peak at 8.7 minutes.
  • EXAMPLE 12 Enhancing Isoflavonoid Levels in Transgenic Arabidopsis To determine whether activation of the phenylpropanoid pathway results in increased accumulation of isoflavonoids in IFS-transformed Arabidopsis, the pathway was activated by UV light treatments. Homozygous Arabidopsis transformants of line A109-4, which synthesize genistein, were identified through germination on kanamycin-containing medium by first selecting a transformant that segregated kanamycin resistance in a 3.J ratio. A resistant progeny from this generation that then produced 100% resistant progeny was identified as a homozygote.
  • Plants from this population and wild type Arabidopsis plants were transferred to 2-inch pots 10 days after germination and grown for 10 more days. Plants were placed directly under 366 nm UV light for 16 h (46 mWatt/cm 2 , using an UVL-56 BLAK-Ray Lamp from UV Products, Inc., San Gabriel, CA). Control plants were placed under the same described environment except for the UV illumination. The above ground parts oi Arabidopsis plants were pulverized in liquid nitrogen to fine powder immediately after UV treatment. The tissues were extracted with 10 mL 80% methanol per 1 gram of fresh weight.
  • the genistein content from tissue extracts of UV-treated and untreated plants was determined by HPLC using a Phenomenex Luna 3u (2) column (150 X 4.6 mm) and a mobil phase linear gradient which goes in 15 minutes from 20% methanol, 80% 10 mM ammonium acetate, pH 8.3 to 100% methanol followed by 100% methanol for 5 minutes as described in Example 2. Aliquots from the same extracts were also assayed for anthocyanin accumulation using photospectrometry as described by Bariola, P. A., et. al. ((1999) Plant Physiol. 779:331-342).
  • Anthocyanins are products of one branch of the phenylpropanoid pathway, and the level of their accumulation is an indication of the activity of this pathway. As seen in the table above, genistein was not detectable and the anthocyanin levels increased by about 28% after UV treatment in the control plants. In plants expressing IFS the anthocyanin levels were not significantly increased while the genistein levels more than doubled.
  • the ability to obtain isoflavone synthase activity in monocot cells was tested by transforming the soybean gene from clone sgslc.pk006.o20 into corn suspension cells and assaying for genistein production.
  • the soybean isoflavone synthase gene was cloned in a vector for expression in monocot cells and its activity determined by the expression of genistein in corn.
  • a chimeric isoflavone synthase gene plasmid was prepared (pOY206) using the pGEM9Zf cloning vector (Promega) for expression of the instant isoflavone synthase in monocots.
  • Plasmid pDETRIC contains the bar gene from Streptomyces hygroscopicus that confers resistance to the herbicide glufosinate (Thompson et al. (1987) EMBO J. 6:25X9).
  • the bar gene is under the control of the CaMV 35S promoter, its translation-initiation codon has been changed from GTG to ATG for proper translation initiation in plants (De Block et al. ( 1987) EMBO J. 6:2513), and uses the Agrobacterium tumefaciens octopine synthase polyadenylation signal.
  • pDP7951 contains in the 5'-3' orientation:
  • TMV tobacco mosaic virus
  • CaMV 35S promoter was prepared and used in corn cell transformations.
  • the Sma I fragment of DP7951 containing CRC was ligated to Nco I and Kpn I ends that had been blunt ended with Mung bean nuclease (New England Biolabs) to create the chimeric gene:
  • This plasmid is called pOY162, and its restriction enzyme map is shown in Figure 23.
  • Black Mexican Sweet (BMS) suspension culture is a commonly used, corn-derived, monocot cell line. Cultures were maintained in MS2D medium (MS salts with vitamins
  • Transformations were performed by microprojectile bombardment using a DuPont Biolistic PDS 1000/He system (Klein T. M. et al. (1987) Nature 327:10-13). Gold particles (0.6 microns) were coated with mixtures of plasmid DNAs as indicated in Table 4:
  • Tissue from 25 lines transformed with Group 1 , 5 white lines resulting from transformation with Group 2, 7 red lines transformed with Group 2, 6 white lines transformed with Group 3, and 6 red lines transformed with Group 3 was harvested and analyzed for the presence of genistein using HPLC and GC-MS. Extracts were prepared and analyzed as described in Example 2. The genistein HPLC peak and the identifying 414 and 399 m/z MS peaks were detected in the extracts from all seven red lines transformed with Group 2 while no genistein was detected in any of the white lines transformed with the same plasmids. Lines transformed with Group 3 did not have genistein whether they were red or white. Sixteen lines transformed with Group 4 also produced genistein. A summary of these results is shown in Table 5.
  • EXAMPLE 14 Synthesis of Daidzein in Monocot Cells
  • the activity of chalcone reductase determines the relative levels of substrates available for isoflavone synthase to produce genistein or daidzein (see Figure 1).
  • Chalcone reductase reduces 4,2',4',6'-tetrahydroxychalcone to 4,2',4'-trihydroxychalcone, thus producing liquiritigenin as the substrate for isoflavone synthase to produce daidzein.
  • Chalcone reductases are present in legumes, but have not been found in most non-legume plants including Arabidopsis, tobacco, and corn.
  • a plasmid DNA containing a soybean chalcone reductase gene was introduced into corn suspension cells by microprojectile bombardment, together with a selection marker, CRC, and IFS constructs as described in Example 13.
  • a soybean cDNA clone encoding chalcone reductase was identified by homology to known chalcone reductase genes of alfalfa (Ballance and Dixon (1995) Plant Phys. 707:1027-1028).
  • the cDNA library was prepared using mRNAs from eight-day-old soybean roots inoculated with cyst Nematode for four days, and sequenced as described in Example 3. BLAST analysis was performed as described in Example 4.
  • the DNA containing the entire coding region from the identified clone, src3c.pk009.e4 was amplified using PCR with the primers shown in SEQ ID NO:62 and SEQ ID NO:63
  • the 5' primer had an Nco I site at the start of the coding region.
  • the 1.3 kb PCR product was subcloned into the pTOPO2J vector (Invitrogen Inc., Carlsbad, CA).
  • the 1.3 kb coding region fragment was excised as a Nco I/Kpn I fragment, using the Nco I site and the Kpn I site from the vector.
  • This fragment was isolated and ligated between the 35S/CabL promoter and Nos 3' polyadenylation signal sequence in the pUC18 vector as described in Example 9, to produce plasmid pCHR40, which was used in the BMS transformation experiments. '
  • Transformation of corn suspension cells was done as described in Example 13, using pDETRIC, pCHR40, pOY206 and pOYl 62. Selection and culturing were as described in
  • Example 13 Each selected line was assayed for the presence of the IFS and CRC genes using PCR as in Example 13. The presence of the CHR gene was determined by the appearance of a 0.6 kb fragment when performing PCR on the tissues using the primers shown in SEQ ID NO:64 and SEQ ID NO:65:
  • the resulting fragment is bound by Not I sites in the primer sequences and contains a 5' leader sequence, the coding region for isoflavone synthase, the untranslated 3' region from SEQ ID NOJ, and a stretch of 18 A residues at the 3' end.
  • This fragment was digested with Not I and ligated to Not I-digested and phosphatase-treated pKS67.
  • the plasmid pKS67 was prepared by replacing in pRB20 (described in U.S. 5,846,784) the 800 bp Nos 3' fragment, described in Example 9, with the 285 bp Nos 3' fragment, described in Example 12.
  • Plasmid pKS93s contains a T7 promoter/HPT/T7 terminator cassette for expression of the HPT enzyme in certain strains of E. coli, such as NovaBlue (DE3) (from Novagen), that are lysogenic for lambda DE3 (which carries the T7 RNA Polymerase gene under lacV5 control).
  • Plasmid pK93s also contains the 35S/HPT/NOS 3' cassette for constitutive expression of the HPT enzyme in plants. These two expression systems allow selection for growth in the presence of hygromycin to be used as a means of identifying cells that contain plasmid DNA sequences in both bacterial and plant systems. Transformation of Soybean Somatic Embryo Cultures
  • Soybean embryonic suspension cultures were maintained in 35 mL liquid media (SB55) on a rotary shaker (150 ⁇ m) at 28°C with a mix of fluorescent and incandescent lights providing a 16 h day 8 h night cycle. Cultures were subcultured every 2 to 3 weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid media.
  • Soybean embryonic suspension cultures were transformed with pKS93s by the method of particle gun bombardment (see Klein et al. (1987) Nature 327:10-13) using a DuPont Biolistic PDSIOOO/He instrument.
  • Five ⁇ L of pKS93s plasmid DNA (1 g/L), 50 ⁇ L CaCl 2 (2.5 M), and 20 ⁇ L spermidine (0J M) were added to 50 ⁇ L of a 60 mg/mL 1 mm gold particle suspension.
  • the particle preparation was agitated for 3 minutes, spun in a microfuge for 10 seconds and the supernate removed.
  • the DNA-coated particles were then washed once with 400 ⁇ L of 70% ethanol and resuspended in 40 ⁇ L of anhydrous ethanol.
  • the DNA/particle suspension was sonicated three times for 1 second each. Five ⁇ L of the DNA-coated gold particles were then loaded on each macro carrier disk.
  • Transformed embryonic clusters were removed from liquid culture media and placed on a solid agar media, SB103, containing 0.5% charcoal to begin maturation. After 1 week, embryos were transferred to SB 103 media minus charcoal. After 5 weeks on SB 103 media, maturing embryos were separated and placed onto SB 148 media. During maturation embryos were kept at 26°C with a mix of fluorescent and incandescent lights providing a 16 h day 8 h night cycle. After 3 weeks on SB 148 media, embryos were analyzed for the expression of the isoflavonoids. Each embryonic cluster gave rise to 5 to 20 somatic embryos. Non-transformed somatic embryos were cultured by the same method as used for the transformed somatic embryos. Analysis of Transformed Somatic Embryos
  • somatic embryos were harvested from 12 independently transformed lines. Somatic embryos were collected individually and stored in 96-well plates at — 80° until lyophilized. Somatic embryos were lyophilized for 24 hours. Three to five lyophilized somatic embryos were pooled in a micro centrifuge tube and the dry weight was measured three times. Three samples of dried embryos were assayed for each transformed line. An 80% methanol solution was added to the lyophilized somatic embryos and the samples incubated for 24 h in the dark at room temperature to extract isoflavonoids. The 80% methanol solution was filtered through a Costar nylon membrane microcentrifuge filter with 0.22 ⁇ m pore size (Sigma).
  • the peaks and spectra corresponding to daidzein, glycitin and genistein conjugated with malonylated glucosides were determined by LC/MS. Isoflaovonoids were monitored through the abso ⁇ tion spectra at 260 and 280 nm. The isoflavonoid signals observed in the soybean somatic embryo samples were verified by comparisons of the retention times and diode array detected abso ⁇ tion spectra with those of the standards. The areas of all peaks corresponding to the isoflaovones in a sample were added and divided by the dry weight of that sample. These dry weight based normalized area sums were used for statistical analysis. An analysis of variance test (ANOVA; Steel, R. G. D. and Torrie, J. H. (1996)
  • Figure 25 shows a graph depicting the distribution of the sum of isoflavone area per mg of dry weight of soybean somatic embryos transgenic for the isoflavone synthase gene and a control line. The results are depicted in the graph in ascending order of the amount of total isoflavones produced. Some lines, such as the ones represented in bars 7 through 14, contained approximately the same levels of isoflavones as the control line. While most of the lines showed intermediate increases or decreases in the amounts of isoflavones produced, there are clear examples of lines having markedly increased or decreased amounts of isoflavones.
  • bar 25 represents a line which expresses 208% as much isoflavones as the control line
  • bar 24 represents a line which expresses 184% as much isoflavones as the control line
  • bar 1 represents a line which produces only 25% of the isoflavones as the control line.
  • transgenic expression of isoflavone synthase affords the ability to manipulate isoflavonoid levels as desired for a particular application; i.e., transformants may be chosen for advancement that have large changes in isoflavonoid levels (i.e., very high as in IS 19 or very low as in IS6) or more subtle changes in the content of isoflavonoids.
  • Genomic sequences encoding isoflavone synthase may be used to express isoflavone synthase as well as the cDNA sequences. Therefore the genomic sequences containing the coding regions for the soybean isoflavone synthase genes were isolated.
  • Soybean genomic DNA was prepared from Glycine max cv. Wye following standard protocols (DNeasy Plant Maxi Kit, Qiagen, Valencia, CA). Using this DNA as template, a genomic DNA fragment including the sequence corresponding to the soybean insert in sgslc.pk006.o20 was produced by PCR with the primers listed as SEQ ID NO:41 and SEQ ID NO:42. A genomic DNA fragment including the sequence of CYP93C1 was produced with the primers listed as SEQ ID NO:7 and SEQ ID NO:51 :
  • the intron sequence in SEQ ID NO:52 corresponds to nucleotides 895 to 1112 (217 nucleotides), while the intron sequence in SEQ ID NO:53 corresponds to nucleotides 947 to 1082 (135 nucleotides) in SEQ ID NO:53.
  • Alignment of the intron nucleotide sequences using the Clustal method of alignment and the default parameters shows that the intron sequences are 46.3% identical.
  • soybean IFS gene was transformed in conjunction with the CRC gene.
  • a vector containing a chimeric isoflavone synthase gene was constructed as follows.
  • the 1.6 Kb isoflavone synthase coding region from clone sgslc.pk006.o20 (SEQ ID NO: 1 ) was amplified using a standard PCR reaction in a GeneAmp PCR System using Pfu polymerase (Stratagene) with the primers shown in SEQ ID NO:41 and SEQ ID NO:42 as in Example 9.
  • the plasmid pCW109 (World Patent Publication No. WO94/11516) was digested with Nco I.
  • the 3.2 Kb fragment containing the beta-conglycinin/P-IFS-phaseolin 3' chimeric gene was purified from pCW109-IFS as a Hind III fragment and ligated with Hind Ill-digested and phosphatase-treated pZBL102.
  • pZBL102 is derived from pKS18HH (described in US Patent No. 5,846,784) by replacing the long Nos 3' fragment in pKS18HH with the short Nos 3' fragment described in Example 13.
  • the Sal I site between the two hygromycin phosphotransferase coding regions was deleted, and a Not I site was added between the Hind III and Sal I sites 5' to the 35S promoter of the 35S-HPT gene.
  • the resulting plasmid has a T7 promoter/HPT/T7 terminator cassette for expression of the HPT enzyme in certain strains of E. coli that are lysogenic for lambda DE3.
  • the lambda DE3 carries the T7 RNA Polymerase gene under lacV5 control and is found in commercially available E. coli strains such as NovaBlue (DE3) (from Novagen).
  • Plasmid pWSJOOl also contains the 35S/HPT/NOS 3' cassette for constitutive expression of the HPT enzyme in plants.
  • a vector containing a chimeric CRC gene was constructed as follows.
  • the plasmid pDP7951 of Example 13, Figure 22, was digested with Smal and the fragment containing the CRC coding region was purified.
  • This CRC fragment was ligated to a modified vector containing the sequences of pCW109 (World Patent Publication No. WO94/11516) with the substitution of a phaseolin promoter fragment extending to -410 and including leader sequences to +77 (Slightom et al., 1991 Plant Mol Biol Man B16J) instead of the beta- conglycinin promoter.
  • Modification included digestion with Ncol and S 1 nuclease treatment followed by religation to remove the ATG sequence of the Ncol site that follows the promoter fragment.
  • the vector was then digested with Kpnl and the ends filled in so that the Smal CRC fragment was inserted in a blunt-end ligation.
  • the Hindlll fragment containing the phaseolin promoter-CRC-phaseolin 3' chimeric gene was isolated and ligated with Hindlll digested pZBL 102 (described above).
  • the resulting plasmid was called pOY203. Transformation Of Somatic Soybean Embryo Cultures and Regeneration Of Soybean Plants
  • Soybean embryogenic suspension cultures were transformed with pWSJOOl or pWSJOOl in conjunction with pOY203 by the method of particle gun bombardment as in Example 15. Besides the media used for the soybean somatic embryo cultures described in Example 15, the following media were used:
  • Transformed embryogenic clusters were removed from liquid culture and placed on a solid agar media (SB 103) containing no hormones or antibiotics. Embryos were cultured for eight weeks at 26°C with mixed florescent and incandescent lights on a 16:8 h day/night schedule. During this period, individual embryos were removed from the clusters and analyzed at various stages of embryo development. Selected lines were assayed by PCR for the presence of the an additional IFS gene using the primers shown in SEQ ID NO:43 and SEQ ID NO:44.
  • Extracts are prepared and analyzed by HPLC as described in Example 15 except that a 150 to 200 mg chip of soybean seed is used for the analysis. Seeds with statistically significant variation in the level of isoflavonoid concentration are further analyzed.
  • Tne indications made below relate to the microorganism rcterred to in the description

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