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WO2013004280A1 - Isolated mutated nucleotide sequences that encode a modified oleate destaurase sunflower protein, modified protein, methods and uses - Google Patents

Isolated mutated nucleotide sequences that encode a modified oleate destaurase sunflower protein, modified protein, methods and uses Download PDF

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Publication number
WO2013004280A1
WO2013004280A1 PCT/EP2011/061164 EP2011061164W WO2013004280A1 WO 2013004280 A1 WO2013004280 A1 WO 2013004280A1 EP 2011061164 W EP2011061164 W EP 2011061164W WO 2013004280 A1 WO2013004280 A1 WO 2013004280A1
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ncimb
seq
sequence
protein
sunflower
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Inventor
Alberto Javier Leon
Andrés Daniel ZAMBELLI
Roberto Juan REID
Monica Mariel Morata
Marcos KASPAR
Enrique Martinez-Force
Rafael Garcés
Joaquin J. SALAS
Monica VENEGAS-CALERON
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Consejo Superior de Investigaciones Cientificas CSIC
Advanta International BV
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Consejo Superior de Investigaciones Cientificas CSIC
Advanta International BV
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Priority to PCT/EP2011/061164 priority Critical patent/WO2013004280A1/en
Priority to ARP120102375A priority patent/AR087014A1/en
Publication of WO2013004280A1 publication Critical patent/WO2013004280A1/en
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Priority to ARP240100392A priority patent/AR131904A2/en
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    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
    • A01H6/1464—Helianthus annuus [sunflower]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • 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
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C12Y114/19006—DELTA12-fatty-acid desaturase (1.14.19.6), i.e. oleoyl-CoA DELTA12 desaturase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • ISOLATED MUTATED NUCLEOTIDE SEQUENCES THAT ENCODE A MODIFIED OLEATE DESTAURASE SUNFLOWER PROTEIN, MODIFIED PROTEIN,
  • the present invention relates to isolated mutated nucleotide sequences that encode a modified oleate
  • OLD desaturase sunflower protein
  • Oleate desaturase enzyme is involved in the enzymatic conversion of oleic acid to linoleic acid.
  • the microsomal OLD has been cloned and characterized by using the marker technology by T-DNA ("T-DNA tagging") (Okuley, et al., (1994) Plant Cell 6:147-158). Higher plants nucleotide sequences that encode microsomal OLD have been described in the document PCT W094/11516 of Lightner et al .
  • Sunflower is generally cultivated to obtain oils containing saturated fatty acids (palmitic and stearic) and unsaturated fatty acids (oleic and linoleic) .
  • Stearic acid content is always lower than 10% (Guston, FD et al . "The lipid handbook", Chapman and Hall 1986), usually between 3 and 7%.
  • unsaturated fatty acids there are two types of sunflower seeds: the normal sunflower which has a linoleic acid content between 50% and 70%
  • oilseeds focused on changing the composition of their fatty acids, for example by means of conventional breeding programs, mutagenesis or transgenesis .
  • Mutations are typically induced with extremely high doses of radiation and/or chemical mutagens (Gaul, H. Radiation Botany (1964) 4:155-232). High doses exceed the lethal dose of 50% (LD50), and generally the 90% lethal dose (LD90), which maximizes the percentage of possible
  • genotypes with low content of 18:1 into genotypes with a high content of 18:1 in their seed.
  • the accumulation of 18:1 fatty acids in seeds depends on two enzymatic reactions: the desaturation of 18:0 to 18:1 and the subsequent desaturation of 18:1 to 18:2.
  • the oleate desaturase enzyme (OLD) catalyzes the desaturation of oleic acid (18:1) to linoleic acid (18:2) (Ohlrogge and Browse (1995) The Plant Cell, 7:957-970, Somerville and Browse (1996) Trends Cell Biol 6:148-153; Schwartzbeck
  • Sunflower oil is naturally rich in linoleic acid (55-70%) and consequently low in oleic acid (20-25%) .
  • Traditional sunflower oil high in linoleic acid is considered a healthful vegetable oil that has a proper taste, and has been considered first quality in the world market due to the high percentage of polyunsaturated fatty acids. It is used as a dressing for salads, cooking oils, or for the production of margarine.
  • This oil should at least contain a concentration of oleic acid from 55 to 65% in relation to total fatty acids. The benefit of this oil is its high oxidative stability after the extraction process and the stability of the flavor in fried products.
  • Sunflower oil with a high oleic acid concentration does not require to be hydrogenated to improve its thermal stability, therefore does not include trans fatty acids.
  • Monounsaturated fatty acids display a good combination of low melting points, stability and viscosity (Noureddini et al . (1992) J Am Oil Chem Soc 12:1189-1191) .
  • the evolution of the oxidation of high oleic versus standard sunflower oils shows that a higher content of oleic acid gives a higher termooxidative resistance, with low peroxide value and polymer formation (Marmesat et al . (2009) Grasas y Aceites 60:155-160).
  • oils with high proportions of oleic acid would be much more resistant to oxidation than oils containing similar proportions of linoleic acid.
  • the invention thus relates to isolated nucleotide sequences encoding a modified oleate desaturase sunflower protein, wherein the nucleotide sequences comprise a point mutation.
  • nucleotide sequence encodes a modified oleate desaturase sunflower protein and comprises a point mutation.
  • the modified oleate desaturase sunflower protein comprises at least one of the following amino acid substitutions in the wild type
  • nucleotide sequence comprises one of the following amino acid sequences: SEQ ID No: 1; SEQ ID No: 2; SEQ ID No: 3; SEQ ID No: 4; SEQ ID No: 5; SEQ ID No: 6; SEQ ID No: 7; SEQ ID No: 8 or SEQ ID No: 9.
  • the nucleotide sequence is one of the following sequences: SEQ ID No: 10, SEQ ID No: 11; SEQ ID No: 12; SEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15; SEQ ID No: 16; SEQ ID No: 17 or SEQ ID No: 18; or sequences that are at least 90% homologous, wherein said sequences at least 90% homologous maintain the point
  • the percentage homology refers in particular to the percentage of identical residues in the sequence.
  • the invention further relates to a protein, in particular a sunflower protein, that has a reduced oleate desaturase activity, wherein said protein comprises at least one of the following amino acid substitutions with respect to the wild type sequence: a) Ser by Leu at position 131, b) Gly by Asp at position 103; c) Ser by Asn at position 135; d) Gly by Glu at position 144; e) Gly by Glu at position 226; f) Pro by Ser at position 253; g) Leu by Phe at
  • the protein has one of the following sequences: a) the sequence shown in SEQ ID No: 1; b) the sequence shown in SEQ ID No: 2; c) the sequence shown in SEQ ID No: 3; d) the sequence shown in SEQ ID No: 4; e) the sequence shown in SEQ ID No: 5; f) the sequence shown in SEQ ID No: 6; g) the sequence shown in SEQ ID No: 7; h) the sequence shown in SEQ ID No: 8; or SEQ ID No: 9.
  • the invention further relates to sunflower plants comprising a gene encoding an oleate desaturase protein having a point mutation modifying the amino acid sequence, and thereby affecting the activity of the oleate desaturase protein.
  • the nucleotide sequence of the gene comprises a point mutation with respect to the wild type sequence, as for example one or more of the following: a) G by A at position 308; b) C by T at position 392; c) G by A at position 404; d) G by A at position 431; e) G by A at position 677; f) C by T at position 757; g) C by T at position 799; h) C by T at position 823; o i) G by A at position 243.
  • the plant can produce seeds with an oleic content of between 80% and 95% with respect to the total percentage of the fatty acids of said seed.
  • the invention further relates to sunflower plants that produce an oleate desaturase protein that has a lower enzymatic activity as compared to the wildtype oleate desaturase protein without point mutations.
  • Such plants are obtainable by introgression of a mutation as found in a plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 into a wildtype plant not carrying the mutation.
  • Introgression can for example take place by crossing a plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 with another plant and selecting in the F2 for plants that produce a sunflower oil that has a high oleic acid content between 80 and 95% as compared to the total fatty acid content in the oil.
  • the invention further relates to plants producing an oleate desaturase protein having a combination of two of more of the following amino acid substitutions as compared to the wild type amino acid sequence: a) Gly by Asp at position 103, b) Ser by Leu at position 131; c) Ser by Asn at position 135; d) Gly by Glu at position 144; e) Gly by Glu at position 226; f) Pro by Ser at position 253; g) Leu by Phe at position 267; h) Pro by Ser at position 275, which plants are obtainable by introgression of the mutation as found in two or more of the plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 into a wildtype sunflower or into a sunflower that already
  • Sunflower seeds comprising an oleate desaturase gene having a point mutation and that encodes a modified oleate desaturase protein are also provided.
  • the nucleotide sequence comprises at least one point mutation selected from: a) G by A at position 308; b) C by T at position 392; c) G by A at position 404; d) G by A at position 431; e) G by A at position 677; f) C by T at position 757; g) C by T at position 799; h) C by T at position 823; and i) G by A at position 243.
  • the seeds are seeds of which a representative sample was deposited at NCIMB and under one of the following accession numbers: NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742, NCIMB 41743.
  • the seed may have an oleic acid content of between 80% and 95% with respect to the total percentage of fatty acids of said seed.
  • At least one of the one or more point mutations present in homozygous state.
  • the genome of the plant or seed comprises more than one of the above listed point mutations, they are all present in homozygous state.
  • an oil is provided that is high in oleic acid, containing between 80% to 95% with respect to the total fatty acid percentage of the oil.
  • the oil can be obtained from seeds of which a representative sample was deposited under one of the accession numbers listed above.
  • the invention further relates to use of the sunflower seeds to obtain oil with high oleic acid content. Oil can suitably be obtained by extracting the seeds.
  • the invention also relates to progeny of the seeds and plants as described herein, wherein the progeny
  • a point mutation in the gene that encodes an oleate desaturase protein wherein said mutation leads to the synthesis of a modified oleate desaturase protein, in particular a oleate desaturase protein that has a reduced enzymatic activity as compared to a wildtype protein not carrying the point mutation.
  • step b) identifying and selecting at least one plant obtained in step b) comprising a nucleotide sequence having at least one point mutation and wherein said sequence encodes a protein that has modified oleate desaturase activity.
  • the point mutation comprises at least one
  • nucleotide substitution with respect to the wild type sequence such as : a) G by A at position 308; b) C by T at position 392; c) G by A at position 404; d) G by A at position 431; e) G by A at position 677; f) C by T at position 757; g) C by T at position 799; h) C by T at position 823; or i) G by A at position 243, wherein said sequence encodes a protein that has modified oleate
  • a method to identify sunflower plants or seeds with a high content of oleic acid which comprises:
  • the part of a plant is a seed.
  • the point mutation comprises at least one change in the nucleotide sequence encoding oleate desaturase, such as: a) G by A at position 308; b) C by T at position 392; c) G by A at position 404; d) G by A at position 431; e) G by A at position 677; f) C by T at position 757; g) C by T at position 799; h) C by T at position 823; or i) G by A at position 243, wherein said sequence encodes a protein that has modified oleate
  • the method to detect the presence of a point mutation in the nucleotide sequences of the invention may be any known technique, such as ASA (Soleimani et al . (2003) Plant Mol Biol Rep 21: 281-288), PAMSA (Gaudet et al . (2007) Plant Mol Biol Rep 25:1-9), SSCP (Germano and Klein (1999) Theor Appl Genet 99:37-49) or TaqMan® (Jones et al . (2008) Pest Management Science 64:12-15).
  • ASA Soleimani et al . (2003) Plant Mol Biol Rep 21: 281-288
  • PAMSA Gadet et al . (2007) Plant Mol Biol Rep 25:1-9
  • SSCP Germano and Klein (1999) Theor Appl Genet 99:37-49
  • TaqMan® Japanese Management Science 64:12-15.
  • the invention further relates to the use of the nucleotides sequence of the invention to prepare a probe to detect, in a sunflower plant, a molecular marker associated with the high oleic phenotype.
  • the nucleotide sequence can furthermore be used in a cisgenic or transgenic approach to make plants and seeds which produce a modified oleate desaturase resulting in a high oleic acid in the seed oil of the plant.
  • a procedure to obtain high oleic sunflower oil comprises extracting the oil from seeds of which a representative sample was deposited at NCIBM under accession number NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 or NCIMB 41743.
  • Figure 1 shows the secondary structure of the native oleate desaturase protein (A) and point mutations (B) , a-h indicates the location of the different amino acid substitutions produced by the point mutations.
  • the oleate desaturase enzyme (OLD, EC 1.3.1.35) is also known as: oleic acid desaturase; linoleate synthase; oleoyl-CoA desaturase; oleoylphosphatidylcholine desaturase, oleoyl-PC desaturase, delta-12 oleate desaturase, FAD2.
  • the present invention is thus related to mutated nucleotides sequences that encode sunflower proteins having modified oleate desaturase activity, wherein the modified oleate desaturase encoded by said mutated polynucleotides has a lower enzymatic activity that leads the plant or parts of the plant, such as the seed, to contain an elevated percentage of oleic acid.
  • the changes or modifications in the amino acid sequence of the oleate desaturase protein may be one or more of the following substitutions :
  • the amino acid sequence of the modified oleate desaturase protein may be one of the sequences shown in SEQ ID Nos. 1 to 9. Any amino acid sequence of an oleate
  • desaturase protein comprising amino acid substitutions shown above are within the scope of the present invention.
  • the amino acid sequences shown as SEQ ID Nos. 1 to 9 can be modified also in other places than the sites of the substitutions shown here, being all of them within the scope of the present invention.
  • such reference is made by comparing the modified amino acid sequences with the sequence of the oleate desaturase from sunflower variety HA89, RHA266 and line 29002 (SEQ ID No:
  • oleate desaturase lines HA89, RHA266 and 29002 are the same and they all correspond to SEQ ID No: 19.
  • the point mutation can also generate a premature stop codon and then the nucleotide sequence will encode a truncated protein.
  • the truncated oleate desaturase protein is shown in SEQ ID No. 9.
  • the mutated nucleotide sequences have a point mutation that leads to the change of one amino acid for another in the encoded polypeptide. For example, the
  • nucleotide sequence can have any of the following changes: a) G by A at position 308;
  • the invention thus relates to any nucleotide substitution that leads to one or more of the following amino acid substitutions:
  • sequence of the mutated nucleotides of the invention is one of the
  • SEQ ID No: 10 SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17 or SEQ ID No: 18.
  • the position of each of the point mutations indicated in the nucleotides sequence was made based on the nucleotides sequence that encodes the oleate desaturase of wild type of the sunflower variety HA89 (SEQ ID No: 20) .
  • mutated such as lines HA89, 29002 and RHA266 to obtain plants that produce seeds with high oleic acid content.
  • Selected mutations in the coding sequence of the sunflower oleate desaturase were point mutations that generated the substitution of an amino acid for another one in the amino acid sequence of the oleate desaturase.
  • the mutated seeds were deposited under the
  • Mutant plants and seeds of the invention can be obtained using different mutagenesis schemes.
  • a mutagenic agent is injected, for example EMS at a concentration between 5 and 15% to the heads of the plants.
  • the fatty acid profile of mutated M2 seeds and seed parentals were analyzed and only those were selected that showed a high content of oleic acid, for example over 80% with respect to the fatty acid content of the seed,
  • mutated plants and seeds were obtained by immersing the parental seeds in a mutagenic agent, such as EMS, at a concentration between 0.3 and 0.7%. Subsequently, the fatty acid profile of the M2 seeds was compared with the fatty acid profile of the
  • parental seeds and those that showed a high content of oleic acid for example over 80% with respect to the fatty acid content of seed, preferably about 90% or higher compared to the total fatty acid content of the seed were selected.
  • Table 1 shows the mutagenesis method used for each mutated line of the invention, the mutation obtained in the nucleotides sequence and the amino acid substitution in the sequence of the oleate desaturase enzyme.
  • P palmitic acid
  • S stearic acid
  • O oleic acid
  • L
  • Sunflower line 29074 presented a point mutation that exchanges the nucleotide C by T at position 392 of the coding region of the oleate desaturase (SEQ ID No: 11) . This change leads to the synthesis of a modified oleate
  • Sunflower line 29075 presented a point mutation that exchanges the nucleotide G by A at position 243 of the coding region of the oleate desaturase (SEQ ID No: 18)
  • the nucleotide exchange leads to the development of a stop codon that replaces tryptophan, generating a truncated modified oleate desaturase, and its amino acid sequence is shown in SEQ ID No: 9.
  • Sunflower line 29076 presented a point mutation exchanging nucleotide C by T at position 757 of the coding region of the oleate desaturase (SEQ ID NO: 15) . This change leads to the synthesis of an oleate desaturase modified wherein the amino acid proline is replaced by serine at position 253 (SEQ ID No. 6) corresponding to domain VIII, more particularly to the transmembrane helix (TM4) (f in
  • Sunflower line 29077 presented a point mutation exchanging nucleotide C by T at position 823 of the coding region of the oleate desaturase (SEQ ID No: 17) . This change leads to the synthesis of a modified oleate desaturase wherein the amino acid proline is replaced by serine at position 275 (SEQ ID No: 8) corresponding to domain IX formed by the C-terminal region (h in Figure 1) .
  • Sunflower line 29078 presented a point mutation exchanging the nucleotide G by A at position 404 of the coding region of the oleate desaturase (SEQ ID No: 12) . This change leads to the synthesis of a modified oleate
  • Sunflower line 29079 presented a point mutation exchanging the nucleotide G by A at position 431 of the coding region of the oleate desaturase (SEQ ID No: 13) . This change leads to the synthesis of a modified oleate
  • amino acid glycine is replaced by the amino acid glutamic acid at position 144 (SEQ ID No: 4) which corresponds to a site near the histidine domain II (d in Figure 1) .
  • Sunflower line 29082 presented a point mutation exchanging the nucleotide G by A at position 308 of the coding region of the oleate desaturase (SEQ ID No: 10) . This change leads to the synthesis of a modified oleate
  • amino acid glycine is replaced by the amino acid aspartic acid at position 103 (SEQ ID No. 1) which corresponds to a site near the histidine domain I (a in Figure 1) .
  • Sunflower line 29081 presented a point mutation exchanging nucleotide C by T at position 799 of the coding region of the oleate desaturase (SEQ ID No: 16) . This change leads to the synthesis of a modified oleate desaturase wherein the amino acid leucine is substituted by the amino acid phenylalanine at position 267 (SEQ ID No. 7) located in domain IX (g in Figure 1) .
  • Sunflower line 39096 presented a point mutation exchanging the nucleotide G by A at position 677 of the coding region of the oleate desaturase (SEQ ID No. 14) . This change leads to the synthesis of a modified oleate
  • the point mutations of the invention can be introduced into an agronomically desirable background. This can be done analogous to prior art describing how different point mutations associated to agronomical traits of interest were introgressed into elite material. For instance, herbicide resistant trait was introgressed into elite inbred lines of sunflower by
  • RHA 447 is an F7-derived F8 restorer sunflower line selected from the cross RHA 377/RHA 348.
  • RHA 377 PI 560145
  • RHA 348 PI 509058
  • a high-oleic oilseed germplasm are restorer lines released by USDA-ARS and the North Dakota Agricultural Experiment Station in 1990 and 1986,
  • the pedigree breeding method was used to develop RHA 447. Analyses for oleic acid concentration were conducted on seed harvested from F3 to F7 plants by gas chromatography (Miller et al . (2006) Crop Sci 46:484-485).
  • the oil obtained from any of the sunflower seeds of the invention has an oleic acid content higher than 80% with respect to the total fatty acid content of seed, preferably greater than 85% and more preferably greater than 90%.
  • sequences of the disclosed nucleic acids can be used as molecular markers or probes provided that part of the sequence
  • the exchange of C by T at nucleotide position 392 (corresponding to codon 131 that encodes for Leucine) shown as SEQ ID No: 11 can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques such as ASA (Soleimani et al . (2003) Plant Mol Biol Rep 21: 281-288), PAMSA (Gaudet et al . (2007) Plant Mol Biol Rep 25:1-9) SSCP (Germano and Klein (1999) Theor Appl Genet 99:37-49) or TaqMan® (Jones et al . (2008) Pest Management Science 64:12-15).
  • ASA Soleimani et al . (2003) Plant Mol Biol Rep 21: 281-288
  • PAMSA Gaudet et al . (2007) Plant Mol Biol Rep 25:1-9
  • SSCP Germano and Klein (1999) Theor Appl Genet 99:37-49
  • TaqMan® Japanese Management Science
  • a portion of the sequence shown as SEQ ID No. 12 which comprises the exchange of G by A at nucleotide position 404 can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
  • a portion of the sequence shown as SEQ ID No. 13 which includes the exchange of G by A at nucleotide position 431 can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
  • a portion of the sequence shown as SEQ ID NO: 14 comprising the exchange of G by A at nucleotide position 677 can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
  • a portion of the sequence shown as SEQ ID NO: 15 comprising the exchange of T by C at nucleotide position 757 can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
  • a portion of the sequence shown as SEQ ID NO: 16 comprising the exchange of T by C at nucleotide position 799 can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
  • a portion of the sequence shown as SEQ ID No. 17 which includes the exchange of C by T at nucleotide position 823 (corresponding to codon 275 that encodes serine) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
  • a portion of the sequence shown as SEQ ID No. 18 which includes the exchange of G by A at nucleotide position 243 (corresponding to codon 81 that encodes Stop) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any know techniques described above. It is apparent to those skilled in the art that shown mutations can be useful to identify sunflower plants with high oleic acid content in any scheme of plant breeding and using any known technique.
  • the mutated nucleotide sequences can also be used to be introduced through an appropriate vector to a plant, where the genetically modified plant obtained is a plant with a high oleic acid content phenotype.
  • batch CA04-3 and 40 rows identified as batch CA04-3 and 40 rows as a batch CA04-1501. Each row was 6 meters long.
  • the EMS is a mutagenic agent that induces transitions G/C to A/T (Jander et al . (2003. Plant Physiol. 131:139-146).
  • Ml self-pollinated seeds each M0 plant was bagged before flowering. The flower heads of the plants of each EMS treatment were harvested, threshed and stored.
  • Ml seeds HA89-10% EMS (seeds mutagenized with 10% EMS) were sown in batches ca05, ca05-6077 and CA05- 6079. In this way, a plot of 1 ha with origin CA04-3 was obtained and a plot of 0.6 ha of origin CA04-1501 was obtained. An additional lot of 0.6 ha was planted with HA89 mutated with 15% of EMS (originally CA04-1501) known as Lot ca05-6080.
  • a total of 12,000 Ml plants from each batch was bagged for self-pollination and the flower heads of the rows were harvested and threshed individually.
  • M2 seeds were taken from each plant and analyzed by infrared spectroscopy (NIR) according to the protocol described in Fassio and Cozzolino, Industrial Crops and Products (2004) 20:321-329.
  • NIR infrared spectroscopy
  • composition of the selected seeds by NIR was analyzed by gas chromatography (GC) , evaluating 30 individual seeds of each M2 flower head, according to the protocol described in
  • the mutant plant CA06- 497-827 showed a phenotype of high-oleic acid content. It was identified and referred to as 29081.
  • RHA266 line was sown in Balcarce (wholesome Aires, Argentina) in the season 2004/5 and 103 rows were identified under the lot number CA04-2. Each row was six meters long.
  • WO2008/071715 Each M0 plant was bagged before flowering in order to produce the Ml self-pollinated seeds. The flower heads of the plants of each EMS treatment were harvested, threshed and stored. In the next field planting season, Ml seeds RHA266-15% EMS were planted in one hectare on December 1, 2005 under the lot number CA05-6073. A total of 12,000 Ml plants were bagged and the 10,400 flower heads resulting that reached maturity were harvested and threshed
  • Sunflower seeds are cut by the sagittal axis and placed in a 2ml glass vial containing 0.25 ml of methylation solution consisting of methanol, toluene, dimethoxypropane and sulfuric acid in the ratio 66:28:4:2.
  • the seeds were covered and incubated for one hour at 80 °C. They were allowed to cool at room temperature and then 1 ml of heptane was added (Garces and Mancha (2003) Anal Biochem 317:247- 254) .
  • the methyl esters of present fatty acids in the superior phase (heptane) were separated on a gas
  • tissue samples were taken from each high-oleic mutant. Genomic DNA was isolated and diluted to a stock concentration of 100 ng/ ⁇ . The coding sequence of the oleate desaturase of the high oleic mutants and the coding sequence of the oleate desaturase of line HA89 (wild type) were amplified in two overlapping segments. The specific primers used for each amplificon were: ler amplicon (705 bp)
  • OLD1-F2 GAAAAGTCTGGTCAAACAGTCAACAT SEQ ID No: 21
  • OLD1-R2 CCGATGTCGGACATGACTATC SEQ ID No: 22
  • PCR polymerase chain reaction
  • IX buffer Invitrogen
  • 0.2 mm dNTPs GE Healthcare
  • 2.5 mm MgC ⁇ 2 Invitrogen
  • 0.2 ⁇ of each primer 0.5 ⁇ 1 of Platinum Taq DNA polymerase (5 ⁇ / ⁇ 1) (Invitrogen) and lOOng of genomic DNA.
  • the PCR reaction was carried out on a GeneAmp PCR
  • Terminator kit v3.1 Cycle Sequencing (Applied Biosystems) following the manufacturer's instructions.
  • the files of the sequencing of the oleate desaturase obtained for each amplicon were assembled using the program Vector NTI Suite-Contig Express, version 7.0 (Informax) .
  • the resulting DNA sequences of the oleate desaturase were aligned with the sequences of line HA89 (GenBank Accession Number AY802989) .

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Abstract

The invention relates to isolated nucleotide sequences, comprising a point mutation and wherein the sequences encode a modified oleate desaturase sunflower protein. The modified oleate desaturase sunflower protein comprises at least one of the following amino acid substitutions: a) Gly by Asp at position 103, b) Ser by Leu at position 131, c) Ser by Asn at position 135, d) Gly by Glu at position 144,e) Gly by Glu at position 226, f) Pro by Ser at position 253, g) Leu by Phe at position 267, h) Pro by Ser at position 275, or it is a truncated oleate desaturase.

Description

ISOLATED MUTATED NUCLEOTIDE SEQUENCES THAT ENCODE A MODIFIED OLEATE DESTAURASE SUNFLOWER PROTEIN, MODIFIED PROTEIN,
METHODS AND USES The present invention relates to isolated mutated nucleotide sequences that encode a modified oleate
desaturase sunflower protein (OLD) , to the modified protein, to methods for making the protein, to plants and seeds producing the protein and uses of the nucleotide sequences, the protein and the plants and seeds.
BACKGROUND
Oleate desaturase enzyme (OLD) is involved in the enzymatic conversion of oleic acid to linoleic acid. The microsomal OLD has been cloned and characterized by using the marker technology by T-DNA ("T-DNA tagging") (Okuley, et al., (1994) Plant Cell 6:147-158). Higher plants nucleotide sequences that encode microsomal OLD have been described in the document PCT W094/11516 of Lightner et al .
Sunflower is generally cultivated to obtain oils containing saturated fatty acids (palmitic and stearic) and unsaturated fatty acids (oleic and linoleic) . Stearic acid content is always lower than 10% (Guston, FD et al . "The lipid handbook", Chapman and Hall 1986), usually between 3 and 7%. According to the content of unsaturated fatty acids there are two types of sunflower seeds: the normal sunflower which has a linoleic acid content between 50% and 70%
(Knowles, PF "Recent advances in oil crops breeding" AOCS Proceedings 1988) and sunflower with high oleic acid content which has a linoleic acid content of 2% to 10% and an oleic acid content of 75% to 90% (Soldatov, KI "Chemical
mutagenesis in sunflower breeding", Proc . 7th International Sunflower Conference, 352-357, 1976). In order to give a solution to the need to obtain vegetable oils of interest to the industry as well as for food applications, efforts are being made to improve
varieties of oilseeds focused on changing the composition of their fatty acids, for example by means of conventional breeding programs, mutagenesis or transgenesis .
Mutations are typically induced with extremely high doses of radiation and/or chemical mutagens (Gaul, H. Radiation Botany (1964) 4:155-232). High doses exceed the lethal dose of 50% (LD50), and generally the 90% lethal dose (LD90), which maximizes the percentage of possible
mutations .
Mutagenesis conducted by Soldatov in 1976 in a population of sunflower allowed to obtain the population of Pervenets mutants. The average content of 18:1 fatty acids of seed for this cultivar is higher than 65%, the individual content is between 60% and 80% while in normal varieties (LO, low oleic) this content is approximately 20%. The
Pervenets population was distributed worldwide and used in many breeding programs in order to transform certain
genotypes with low content of 18:1 into genotypes with a high content of 18:1 in their seed.
The accumulation of 18:1 fatty acids in seeds depends on two enzymatic reactions: the desaturation of 18:0 to 18:1 and the subsequent desaturation of 18:1 to 18:2. The oleate desaturase enzyme (OLD) catalyzes the desaturation of oleic acid (18:1) to linoleic acid (18:2) (Ohlrogge and Browse (1995) The Plant Cell, 7:957-970, Somerville and Browse (1996) Trends Cell Biol 6:148-153; Schwartzbeck
(2001) Phytochemistry, 57:643-652).
Sunflower oil is naturally rich in linoleic acid (55-70%) and consequently low in oleic acid (20-25%) .
Traditional varieties are classified as low oleic (LO) . Due to the fact that the population is highly interested in eating healthier oils with a high oleic acid content, high oleic sunflower plants are being developed. They also show similar crop yields when compared to traditional low-oleic varieties.
Research carried out by Garces and Mancha in 1989 and 1991 (Garces R, Garcia J, Mancha M. (1989) "Lipid characterization in seeds of a high oleic acid sunflower mutant" Phytochemistry 28:2597-2600, Garces R, Mancha M (1991) "In vitro oleate desaturase in sunflower seeds developping" Phytochemistry 30:2127-2130) showed that the "High Oleic" phenotype is associated with a marked decrease in the activity of the OLD enzyme, which catalyzes the desaturation of 18:1 to 18:2 during the critical stages of the construction of lipid stock, explaining thus the
accumulation of 18:1.
It has been proven that the Pervenets mutation is associated with OLD gene duplication leading to its genetic silencing. This decrease in transcription of OLD explains the decrease in the amount of enzyme and thus the lower OLD activity that produces a greater accumulation of oleic acid in sunflower seeds (Hongtrakul et al . (1998) Crop Sci
38:1245-1249) . This finding led to the development of molecular markers that are characteristic of the mutation and that can be used in breeding programs to facilitate the selection of HO genotypes (WO 2005/106022; Lacombe et al . (2001) Life Sci 324:839-845).
Traditional sunflower oil high in linoleic acid is considered a healthful vegetable oil that has a proper taste, and has been considered first quality in the world market due to the high percentage of polyunsaturated fatty acids. It is used as a dressing for salads, cooking oils, or for the production of margarine. By altering the fatty acid content of sunflower oil, a new oil that has oxidative stability and improved taste can be developed. This oil should at least contain a concentration of oleic acid from 55 to 65% in relation to total fatty acids. The benefit of this oil is its high oxidative stability after the extraction process and the stability of the flavor in fried products. Sunflower oil with a high oleic acid concentration does not require to be hydrogenated to improve its thermal stability, therefore does not include trans fatty acids. Monounsaturated fatty acids display a good combination of low melting points, stability and viscosity (Noureddini et al . (1992) J Am Oil Chem Soc 12:1189-1191) . The evolution of the oxidation of high oleic versus standard sunflower oils shows that a higher content of oleic acid gives a higher termooxidative resistance, with low peroxide value and polymer formation (Marmesat et al . (2009) Grasas y Aceites 60:155-160). The relative rates of autoxidation for oleic and linoleic acids are well known and they give good notice of their stability. Thus, if we give the value 1 to the oleic acid autooxidation rate, linoleic fatty acid will display a value of 27
(Gunstone (2004) In Gunstone FD (Ed.) The Chemistry of Fats and Oils. Blackwell Publishing, Oxford, pp. 150-168) which means they are much more unstable and easier to oxidize. Therefore, oils with high proportions of oleic acid would be much more resistant to oxidation than oils containing similar proportions of linoleic acid.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide the means to produce sunflower seeds and plants having an oil with a high oleic acid concentration. The invention thus relates to isolated nucleotide sequences encoding a modified oleate desaturase sunflower protein, wherein the nucleotide sequences comprise a point mutation.
According to the invention, isolated mutant nucleotide sequences are thus provided, wherein said
nucleotide sequence encodes a modified oleate desaturase sunflower protein and comprises a point mutation.
In a preferred embodiment, the modified oleate desaturase sunflower protein comprises at least one of the following amino acid substitutions in the wild type
sequence: a) Gly by Asp at position 103, b) Ser by Leu at position 131; c) Ser by Asn at position 135; d) Gly by Glu at position 144; e) Gly by Glu at position 226; f) Pro by Ser at position 253; g) Leu by Phe at position 267; h) Pro by Ser at position 275; or it is a truncated desaturase oleate.
In another preferred embodiment the oleate
desaturase sunflower protein that is encoded by the
nucleotide sequence comprises one of the following amino acid sequences: SEQ ID No: 1; SEQ ID No: 2; SEQ ID No: 3; SEQ ID No: 4; SEQ ID No: 5; SEQ ID No: 6; SEQ ID No: 7; SEQ ID No: 8 or SEQ ID No: 9.
In a preferred embodiment, the nucleotide sequence is one of the following sequences: SEQ ID No: 10, SEQ ID No: 11; SEQ ID No: 12; SEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15; SEQ ID No: 16; SEQ ID No: 17 or SEQ ID No: 18; or sequences that are at least 90% homologous, wherein said sequences at least 90% homologous maintain the point
mutation. Homology in this context means sequence
similarity. The percentage homology refers in particular to the percentage of identical residues in the sequence.
The invention further relates to a protein, in particular a sunflower protein, that has a reduced oleate desaturase activity, wherein said protein comprises at least one of the following amino acid substitutions with respect to the wild type sequence: a) Ser by Leu at position 131, b) Gly by Asp at position 103; c) Ser by Asn at position 135; d) Gly by Glu at position 144; e) Gly by Glu at position 226; f) Pro by Ser at position 253; g) Leu by Phe at
position 267; h) Pro by Ser at position 275 or it is an truncated desaturase oleate.
In a preferred embodiment, the protein has one of the following sequences: a) the sequence shown in SEQ ID No: 1; b) the sequence shown in SEQ ID No: 2; c) the sequence shown in SEQ ID No: 3; d) the sequence shown in SEQ ID No: 4; e) the sequence shown in SEQ ID No: 5; f) the sequence shown in SEQ ID No: 6; g) the sequence shown in SEQ ID No: 7; h) the sequence shown in SEQ ID No: 8; or SEQ ID No: 9.
The invention further relates to sunflower plants comprising a gene encoding an oleate desaturase protein having a point mutation modifying the amino acid sequence, and thereby affecting the activity of the oleate desaturase protein. The nucleotide sequence of the gene comprises a point mutation with respect to the wild type sequence, as for example one or more of the following: a) G by A at position 308; b) C by T at position 392; c) G by A at position 404; d) G by A at position 431; e) G by A at position 677; f) C by T at position 757; g) C by T at position 799; h) C by T at position 823; o i) G by A at position 243. The plant can produce seeds with an oleic content of between 80% and 95% with respect to the total percentage of the fatty acids of said seed.
The invention further relates to sunflower plants that produce an oleate desaturase protein that has a lower enzymatic activity as compared to the wildtype oleate desaturase protein without point mutations. Such plants are obtainable by introgression of a mutation as found in a plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 into a wildtype plant not carrying the mutation.
Introgression can for example take place by crossing a plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 with another plant and selecting in the F2 for plants that produce a sunflower oil that has a high oleic acid content between 80 and 95% as compared to the total fatty acid content in the oil.
The invention further relates to plants producing an oleate desaturase protein having a combination of two of more of the following amino acid substitutions as compared to the wild type amino acid sequence: a) Gly by Asp at position 103, b) Ser by Leu at position 131; c) Ser by Asn at position 135; d) Gly by Glu at position 144; e) Gly by Glu at position 226; f) Pro by Ser at position 253; g) Leu by Phe at position 267; h) Pro by Ser at position 275, which plants are obtainable by introgression of the mutation as found in two or more of the plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 into a wildtype sunflower or into a sunflower that already contains one or more of the other point mutations.
Sunflower seeds comprising an oleate desaturase gene having a point mutation and that encodes a modified oleate desaturase protein are also provided. The nucleotide sequence comprises at least one point mutation selected from: a) G by A at position 308; b) C by T at position 392; c) G by A at position 404; d) G by A at position 431; e) G by A at position 677; f) C by T at position 757; g) C by T at position 799; h) C by T at position 823; and i) G by A at position 243. In a preferred embodiment, the seeds are seeds of which a representative sample was deposited at NCIMB and under one of the following accession numbers: NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742, NCIMB 41743. The seed may have an oleic acid content of between 80% and 95% with respect to the total percentage of fatty acids of said seed.
In one embodiment, at least one of the one or more point mutations present in homozygous state. Preferably, in case the genome of the plant or seed comprises more than one of the above listed point mutations, they are all present in homozygous state.
According to another aspect of the invention, an oil is provided that is high in oleic acid, containing between 80% to 95% with respect to the total fatty acid percentage of the oil. The oil can be obtained from seeds of which a representative sample was deposited under one of the accession numbers listed above.
The invention further relates to use of the sunflower seeds to obtain oil with high oleic acid content. Oil can suitably be obtained by extracting the seeds.
The invention also relates to progeny of the seeds and plants as described herein, wherein the progeny
comprises a point mutation in the gene that encodes an oleate desaturase protein, wherein said mutation leads to the synthesis of a modified oleate desaturase protein, in particular a oleate desaturase protein that has a reduced enzymatic activity as compared to a wildtype protein not carrying the point mutation.
Also provided is a method for obtaining a sunflower plant with high oleic acid content, comprising the following steps:
a) mutagenesis of a part of a sunflower plant, in particular of the seed;
b) obtaining at least one progeny of the mutant plant or seed, and
c) identifying and selecting at least one plant obtained in step b) comprising a nucleotide sequence having at least one point mutation and wherein said sequence encodes a protein that has modified oleate desaturase activity. The point mutation comprises at least one
nucleotide substitution with respect to the wild type sequence such as : a) G by A at position 308; b) C by T at position 392; c) G by A at position 404; d) G by A at position 431; e) G by A at position 677; f) C by T at position 757; g) C by T at position 799; h) C by T at position 823; or i) G by A at position 243, wherein said sequence encodes a protein that has modified oleate
desaturase activity.
Further is provided a method to identify sunflower plants or seeds with a high content of oleic acid, which comprises:
a) providing one part of a sunflower plant, and b) detecting in said part the presence of a point mutation in the sequence that encodes a protein that has oleate desaturase activity. In a preferred embodiment, the part of a plant is a seed. The point mutation comprises at least one change in the nucleotide sequence encoding oleate desaturase, such as: a) G by A at position 308; b) C by T at position 392; c) G by A at position 404; d) G by A at position 431; e) G by A at position 677; f) C by T at position 757; g) C by T at position 799; h) C by T at position 823; or i) G by A at position 243, wherein said sequence encodes a protein that has modified oleate
desaturase activity.
The method to detect the presence of a point mutation in the nucleotide sequences of the invention may be any known technique, such as ASA (Soleimani et al . (2003) Plant Mol Biol Rep 21: 281-288), PAMSA (Gaudet et al . (2007) Plant Mol Biol Rep 25:1-9), SSCP (Germano and Klein (1999) Theor Appl Genet 99:37-49) or TaqMan® (Jones et al . (2008) Pest Management Science 64:12-15).
The invention further relates to the use of the nucleotides sequence of the invention to prepare a probe to detect, in a sunflower plant, a molecular marker associated with the high oleic phenotype.
The nucleotide sequence can furthermore be used in a cisgenic or transgenic approach to make plants and seeds which produce a modified oleate desaturase resulting in a high oleic acid in the seed oil of the plant.
A procedure to obtain high oleic sunflower oil is provided. The procedure comprises extracting the oil from seeds of which a representative sample was deposited at NCIBM under accession number NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 or NCIMB 41743.
DESCRIPTION OF THE FIGURES
Figure 1 shows the secondary structure of the native oleate desaturase protein (A) and point mutations (B) , a-h indicates the location of the different amino acid substitutions produced by the point mutations. DETAILED DESCRIPTION OF THE INVENTION
The oleate desaturase enzyme (OLD, EC 1.3.1.35) is also known as: oleic acid desaturase; linoleate synthase; oleoyl-CoA desaturase; oleoylphosphatidylcholine desaturase, oleoyl-PC desaturase, delta-12 oleate desaturase, FAD2.
The present invention is thus related to mutated nucleotides sequences that encode sunflower proteins having modified oleate desaturase activity, wherein the modified oleate desaturase encoded by said mutated polynucleotides has a lower enzymatic activity that leads the plant or parts of the plant, such as the seed, to contain an elevated percentage of oleic acid.
In a preferred embodiment, the changes or modifications in the amino acid sequence of the oleate desaturase protein may be one or more of the following substitutions :
a) Gly by Asp at position 103
b) Ser by Leu at position 131
c) Ser by Asn at position 135
d) Gly by Glu at position 144
e) Gly by Glu at position 226
f) Pro by Ser at position 253
g) Leu by Phe at position 267
h) Pro by Ser at position 275
The amino acid sequence of the modified oleate desaturase protein may be one of the sequences shown in SEQ ID Nos. 1 to 9. Any amino acid sequence of an oleate
desaturase protein comprising amino acid substitutions shown above are within the scope of the present invention. For example, the amino acid sequences shown as SEQ ID Nos. 1 to 9 can be modified also in other places than the sites of the substitutions shown here, being all of them within the scope of the present invention. In all cases when referring to the substituted sites of the sunflower oleate desaturase protein, such reference is made by comparing the modified amino acid sequences with the sequence of the oleate desaturase from sunflower variety HA89, RHA266 and line 29002 (SEQ ID No:
19) considered as the wild type. It has to be clarified that the sequence of the oleate desaturase lines HA89, RHA266 and 29002 is the same and they all correspond to SEQ ID No: 19. The point mutation can also generate a premature stop codon and then the nucleotide sequence will encode a truncated protein. In a preferred embodiment, the truncated oleate desaturase protein is shown in SEQ ID No. 9.
The mutated nucleotide sequences have a point mutation that leads to the change of one amino acid for another in the encoded polypeptide. For example, the
nucleotide sequence can have any of the following changes: a) G by A at position 308;
b) C by T at position 392;
c) G by A at position 404;
d) G by A at position 431;
e) G by A at position 677;
f) C by T at position 757;
g) C by T at position 799;
h) C by T at position 823;
i) G by A at position 243.
All of them are analyzed with respect to the sequence encoding the sunflower oleate desaturase, more particularly regarding the sequence encoding the oleate desaturase HA89 line, line RHA266 or line 29002 (SEQ ID No. 20) or other wild-type sequence. It has to be clarified that the nucleotide sequence encoding the oleate desaturase lines HA89, RHA266 and 29002 is the same and they all correspond to SEQ ID No : 20. It should also be understood that changes or mutations in a nucleotide shown above can be found in any sequence that encodes a sunflower oleate desaturase, particularly of any line or known sunflower variety, where the sequence of the sunflower line or variety can be also found with other modifications such as deletions, additions or others. Any nucleotide sequence that encodes a sunflower polypeptide with oleate desaturase activity comprising at least one of the point mutations or changes shown above falls within the scope of the present invention
It is to be understood that due to the degeneration of the genetic code, other point mutations or changes in the nucleotides different from those shown above can be obtained, which encode the same amino acid
substitutions in the sequence of the sunflower oleate desaturase protein.
The invention thus relates to any nucleotide substitution that leads to one or more of the following amino acid substitutions:
a) Gly by Asp at position 103
b) Ser by Leu at position 131
c) Ser by Asn at position 135
d) Gly by Glu at position 144
e) Gly by Glu at position 226
f) Pro by Ser at position 253
g) Leu by Phe at position 267
h) Pro by Ser at position 275.
In a preferred embodiment, the sequence of the mutated nucleotides of the invention is one of the
following: SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17 or SEQ ID No: 18. The position of each of the point mutations indicated in the nucleotides sequence was made based on the nucleotides sequence that encodes the oleate desaturase of wild type of the sunflower variety HA89 (SEQ ID No: 20) .
Different mutant sunflower lines have been
mutated, such as lines HA89, 29002 and RHA266 to obtain plants that produce seeds with high oleic acid content.
Selected mutations in the coding sequence of the sunflower oleate desaturase were point mutations that generated the substitution of an amino acid for another one in the amino acid sequence of the oleate desaturase.
The mutated seeds were deposited under the
Budapest Treaty at NCIMB Ltd. on July 20th, 2010 with the following accession numbers:
line 29074 accession number NCIMB 41735;
line 29075 accession number NCIMB 41736;
line 29076 accession number NCIMB 41737;
line 29077 accession number NCIMB 41738;
line 29078 accession number NCIMB 41739;
line 29079 accession number NCIMB 41740;
line 29081 accession number NCIMB 41741;
line 29082 accession number NCIMB 41742;
line 39096 accession number NCIMB 41743.
Mutant plants and seeds of the invention can be obtained using different mutagenesis schemes. In a preferred embodiment a mutagenic agent is injected, for example EMS at a concentration between 5 and 15% to the heads of the plants. The fatty acid profile of mutated M2 seeds and seed parentals were analyzed and only those were selected that showed a high content of oleic acid, for example over 80% with respect to the fatty acid content of the seed,
preferably about 90% with respect to the total fatty acid content of the seed. In another preferred embodiment, mutated plants and seeds were obtained by immersing the parental seeds in a mutagenic agent, such as EMS, at a concentration between 0.3 and 0.7%. Subsequently, the fatty acid profile of the M2 seeds was compared with the fatty acid profile of the
parental seeds and those that showed a high content of oleic acid, for example over 80% with respect to the fatty acid content of seed, preferably about 90% or higher compared to the total fatty acid content of the seed were selected.
Table 1 shows the mutagenesis method used for each mutated line of the invention, the mutation obtained in the nucleotides sequence and the amino acid substitution in the sequence of the oleate desaturase enzyme. Table 1
Line Mutagenesis Type of Sequence of Amino acid Sequence of Accession Method Nucleotide mutated substitution the encoded Number
Mutation nucleotyde of the protein
invention
29074 Injection of C by T at SEQ ID No: 11 Ser by Leu at SEQ ID No: 2 NCIMB 41735 EMS at 10% in position position 131
the head 392
29075 Injection of G by A at SEQ ID No: 18 Trp by Stop SEQ ID No: 9 NCIMB 41736 EMS at 10% in position at position
the head 243 81
29076 Injection of C by T at SEQ ID No: 15 Pro by Ser at SEQ ID No: 6 NCIMB 41737 EMS at 10% in position position 253
the head 757
29077 Injection of C by T at SEQ ID No: 17 Pro by Ser at SEQ ID No: 8 NCIMB 41738 EMS at 10% in position position 275
the head 823
29078 Injection of G by A at SEQ ID No: 12 Ser by Asn at SEQ ID No: 3 NCIMB 41739 EMS at 10% in position position 135 the head 404
29079 Injection of G by A at SEQ ID No: 13 Gly by Glu at SEQ ID No: 4 NCIMB 41740 EMS at 15% in position position 144
the head 431
29082 Immersion of G by A at SEQ ID No: 10 Gly by Asp at SEQ ID No: 1 NCIMB 41742 the seeds in position position 103
EMS at 0.7% 308
29081 Immersion of C by T at SEQ ID No: 16 Leu by Phe at SEQ ID No: 7 NCIMB 41741 the seeds in position position 267
EMS at 0.3% 799
39096 Injection of G by A at SEQ ID No: 14 Gly by Glu at SEQ ID No: 5 NCIMB 41743 EMS at 15% in position position 226
the head 677
The results of the analysis of the fatty acids content in the mutated seeds can be seen in Table 2. Table 2
Figure imgf000017_0001
P: palmitic acid; S: stearic acid; O: oleic acid; L:
linoleic acid. Sunflower line 29074 presented a point mutation that exchanges the nucleotide C by T at position 392 of the coding region of the oleate desaturase (SEQ ID No: 11) . This change leads to the synthesis of a modified oleate
desaturase wherein the amino acid serine is substituted by the amino acid leucine at position 131 (SEQ ID No: 2) which corresponds to domain IV of the first peripheral segment associated to a PMS1 membrane (b in Figure 1) .
Sunflower line 29075 presented a point mutation that exchanges the nucleotide G by A at position 243 of the coding region of the oleate desaturase (SEQ ID No: 18) The nucleotide exchange leads to the development of a stop codon that replaces tryptophan, generating a truncated modified oleate desaturase, and its amino acid sequence is shown in SEQ ID No: 9.
Sunflower line 29076 presented a point mutation exchanging nucleotide C by T at position 757 of the coding region of the oleate desaturase (SEQ ID NO: 15) . This change leads to the synthesis of an oleate desaturase modified wherein the amino acid proline is replaced by serine at position 253 (SEQ ID No. 6) corresponding to domain VIII, more particularly to the transmembrane helix (TM4) (f in
Figure 1) .
Sunflower line 29077 presented a point mutation exchanging nucleotide C by T at position 823 of the coding region of the oleate desaturase (SEQ ID No: 17) . This change leads to the synthesis of a modified oleate desaturase wherein the amino acid proline is replaced by serine at position 275 (SEQ ID No: 8) corresponding to domain IX formed by the C-terminal region (h in Figure 1) .
Sunflower line 29078 presented a point mutation exchanging the nucleotide G by A at position 404 of the coding region of the oleate desaturase (SEQ ID No: 12) . This change leads to the synthesis of a modified oleate
desaturase wherein the amino acid serine is replaced by the amino acid asparagine at position 135 (SEQ ID No. 3) corresponding to domain IV of the first peripheral segment associated to PMS1 a membrane (c in Figure 1) .
Sunflower line 29079 presented a point mutation exchanging the nucleotide G by A at position 431 of the coding region of the oleate desaturase (SEQ ID No: 13) . This change leads to the synthesis of a modified oleate
desaturase wherein the amino acid glycine is replaced by the amino acid glutamic acid at position 144 (SEQ ID No: 4) which corresponds to a site near the histidine domain II (d in Figure 1) .
Sunflower line 29082 presented a point mutation exchanging the nucleotide G by A at position 308 of the coding region of the oleate desaturase (SEQ ID No: 10) . This change leads to the synthesis of a modified oleate
desaturase wherein the amino acid glycine is replaced by the amino acid aspartic acid at position 103 (SEQ ID No. 1) which corresponds to a site near the histidine domain I (a in Figure 1) .
Sunflower line 29081 presented a point mutation exchanging nucleotide C by T at position 799 of the coding region of the oleate desaturase (SEQ ID No: 16) . This change leads to the synthesis of a modified oleate desaturase wherein the amino acid leucine is substituted by the amino acid phenylalanine at position 267 (SEQ ID No. 7) located in domain IX (g in Figure 1) .
Sunflower line 39096 presented a point mutation exchanging the nucleotide G by A at position 677 of the coding region of the oleate desaturase (SEQ ID No. 14) . This change leads to the synthesis of a modified oleate
desaturase wherein the amino acid leucine is substituted for the amino acid phenylalanine at position 267 (SEQ ID No: 5) corresponding to the domain VIII, more particularly to the transmembrane helix (TM3) (e in Figure 1) .
According to the invention the point mutations of the invention can be introduced into an agronomically desirable background. This can be done analogous to prior art describing how different point mutations associated to agronomical traits of interest were introgressed into elite material. For instance, herbicide resistant trait was introgressed into elite inbred lines of sunflower by
conventional breeding methods for the purpose of developing and deploying imidazolinone-resistant and sulfonylurea- resistant cultivars (Al-Khatib and Miller (2000) Crop Sci 40:869-870; Miller and Al-Khatib (2002) Crop Sci 42:988-989; Miller and Al-Khatib (2004) Crop Sci 44:1037-1038). An important development in sunflower breeding was the
introduction of a high oleic acid mutant from the open- pollinated mutant cultivar Pervenets. After 1980, the
Pervenets germplasm became available to sunflower breeders in the USA and Europe, where it has been used widely as the primary source of breeding hybrid cultivars with high oleic acid (Skoric (1988) Sunflower breeding, Uijarstvo 25:3-90). Oil with high oleic acid content has steadily gained market acceptance, especially for food and industrial purposes where high oxidative stability is required (Fick and Miller (1997) Sunflower breeding. In: A. A. Schneiter (Ed.),
Sunflower Technology and Production, pp.395-439. American Society of Agronomy, Madison) . Thus, the sunflower high oleic mutation Pervenets was also introgressed into
different sunflower varieties. RHA 447 is an F7-derived F8 restorer sunflower line selected from the cross RHA 377/RHA 348. RHA 377 (PI 560145), a high linoleic oilseed germplasm, and RHA 348 (PI 509058), a high-oleic oilseed germplasm, are restorer lines released by USDA-ARS and the North Dakota Agricultural Experiment Station in 1990 and 1986,
respectively. The pedigree breeding method was used to develop RHA 447. Analyses for oleic acid concentration were conducted on seed harvested from F3 to F7 plants by gas chromatography (Miller et al . (2006) Crop Sci 46:484-485).
The oil obtained from any of the sunflower seeds of the invention has an oleic acid content higher than 80% with respect to the total fatty acid content of seed, preferably greater than 85% and more preferably greater than 90%.
A portion of the sequences of the disclosed nucleic acids (SEQ ID No: 10 to 18) can be used as molecular markers or probes provided that part of the sequence
comprises the corresponding point mutation. For example, the exchange of C by T at nucleotide position 392 (corresponding to codon 131 that encodes for Leucine) shown as SEQ ID No: 11 can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques such as ASA (Soleimani et al . (2003) Plant Mol Biol Rep 21: 281-288), PAMSA (Gaudet et al . (2007) Plant Mol Biol Rep 25:1-9) SSCP (Germano and Klein (1999) Theor Appl Genet 99:37-49) or TaqMan® (Jones et al . (2008) Pest Management Science 64:12-15).
In other preferred embodiments a portion of the sequence shown as SEQ ID No. 12 which comprises the exchange of G by A at nucleotide position 404 (corresponding to codon 135 that encodes asparagine) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
In other preferred embodiments a portion of the sequence shown as SEQ ID No. 13 which includes the exchange of G by A at nucleotide position 431 (corresponding to codon 144 which encodes glutamic) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
In other preferred embodiments a portion of the sequence shown as SEQ ID NO: 14 comprising the exchange of G by A at nucleotide position 677 (corresponding to codon 226 which encodes glutamic) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
In other preferred embodiments a portion of the sequence shown as SEQ ID NO: 15 comprising the exchange of T by C at nucleotide position 757 (corresponding to codon 253 which encodes serine) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
In other preferred embodiments a portion of the sequence shown as SEQ ID NO: 16 comprising the exchange of T by C at nucleotide position 799 (corresponding to codon 267 that encodes phenylalanine) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
In other preferred embodiments a portion of the sequence shown as SEQ ID No. 17 which includes the exchange of C by T at nucleotide position 823 (corresponding to codon 275 that encodes serine) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any known techniques described above.
In other preferred embodiments a portion of the sequence shown as SEQ ID No. 18 which includes the exchange of G by A at nucleotide position 243 (corresponding to codon 81 that encodes Stop) can be used as a molecular marker to detect, or select sunflower plants with high oleic acid content, using any know techniques described above. It is apparent to those skilled in the art that shown mutations can be useful to identify sunflower plants with high oleic acid content in any scheme of plant breeding and using any known technique.
The mutated nucleotide sequences can also be used to be introduced through an appropriate vector to a plant, where the genetically modified plant obtained is a plant with a high oleic acid content phenotype.
This invention is best illustrated by the following example which should not be interpreted as a restriction on the scope thereof. On the contrary, it should be clearly understood that other embodiments, modifications and equivalents can be analyzed, which, after reading the present disclosure, may be found by those knowledgeable in the field without departing from the spirit of the present invention and/or scope of appended claims.
EXAMPLES EXAMPLE 1
a) Mutagenesis of sunflower line HA89 by injection in the head with EMS and selection of mutant plants 29074, 29075, 29076, 29077, 29078 and 29079
Seeds from line HA89 were sown in Biotechnology Seeds Research Station Advanta at Balcarce (Buenos Aires, Argentina) in season 2004/5. Seventy-five rows were
identified as batch CA04-3 and 40 rows as a batch CA04-1501. Each row was 6 meters long.
Flower buds (reproductive stage Rl or R2 according to the scale of development of Schneiter and Miller (1981. Crop Sci 21: 901-903) of the plants were mutagenized by injection with EMS (methanesulfonic acid ethyl ester) at doses of 5, 10 and 15% using the protocol described in
WO2006/024351 and WO2008/071715. The EMS is a mutagenic agent that induces transitions G/C to A/T (Jander et al . (2003. Plant Physiol. 131:139-146). In order to produce Ml self-pollinated seeds each M0 plant was bagged before flowering. The flower heads of the plants of each EMS treatment were harvested, threshed and stored. In the next planting season Ml seeds HA89-10% EMS (seeds mutagenized with 10% EMS) were sown in batches ca05, ca05-6077 and CA05- 6079. In this way, a plot of 1 ha with origin CA04-3 was obtained and a plot of 0.6 ha of origin CA04-1501 was obtained. An additional lot of 0.6 ha was planted with HA89 mutated with 15% of EMS (originally CA04-1501) known as Lot ca05-6080.
A total of 12,000 Ml plants from each batch was bagged for self-pollination and the flower heads of the rows were harvested and threshed individually.
In order to determine the seeds that showed a modified fatty acid profile with respect to the fatty acid profile of the parental line HA89, M2 seeds were taken from each plant and analyzed by infrared spectroscopy (NIR) according to the protocol described in Fassio and Cozzolino, Industrial Crops and Products (2004) 20:321-329. The
composition of the selected seeds by NIR was analyzed by gas chromatography (GC) , evaluating 30 individual seeds of each M2 flower head, according to the protocol described in
Garces and Mancha (2003) Anal Biochem 317:247-254. Following this procedure 8 mutant plants with modified fatty acid composition were identified and selected, which were called 29074, 29075.29076, 29077, 29078 and 29079. b) Mutagenesis of line 29002 and selection of 29081 mutant plant
Ten thousand seeds of line 29002 were treated with 0.3% of EMS according to the protocol described in U.S. 2004/0083502 and sown at Balcarce, on November 16, 2006 (lot No. CA06-497) . 2,851 Ml plants were obtained and bagged before flowering in order to produce M2 seeds self- pollinated. The flower heads were harvested and threshed individually and analyzed by NIR. From the 2,759 flower heads analyzed by NIR, 102 were selected and their fatty acid composition was confirmed by GC, evaluating 30
individual M2 seeds per flower head. The mutant plant CA06- 497-827 showed a phenotype of high-oleic acid content. It was identified and referred to as 29081. c) Sunflower line RHA266 Mutagenesis and selection of the 39096 mutant plant
RHA266 line was sown in Balcarce (Buenos Aires, Argentina) in the season 2004/5 and 103 rows were identified under the lot number CA04-2. Each row was six meters long.
Flower buds (reproductive stage Rl or R2 according to the scale of development of Schneiter and Miller (1981. Crop Sci 21:901-903) of the plants were mutagenized by injection with EMS at doses of 5, 10 and 15% using
mutagenesis protocol described in WO2006/024351, and
WO2008/071715. Each M0 plant was bagged before flowering in order to produce the Ml self-pollinated seeds. The flower heads of the plants of each EMS treatment were harvested, threshed and stored. In the next field planting season, Ml seeds RHA266-15% EMS were planted in one hectare on December 1, 2005 under the lot number CA05-6073. A total of 12,000 Ml plants were bagged and the 10,400 flower heads resulting that reached maturity were harvested and threshed
individually. The seeds of each plant were analyzed by NIR to detect the seeds with an altered fatty acid composition with respect to the original RHA266 seed. The composition of 227 selected plants by NIR was individually studied by GC, analyzing 30 M2 seeds by flower head. The CA05-6073-4133 plant showed a high oleic phenotype and was referred to as 39096. d) Sunflower line HA89 Mutagenesis by EMS immersion at 0.7% and selection of the mutant plant 29082
Ten thousand seeds of line HA89 were treated with 0.7% EMS with respect to the protocol described in U.S.
2004/0083502 and sown at Balcarce on November 14, 2006 (lot No. CA06-495) . 3,421 plants were obtained and bagged before flowering period to produce self-pollinated M2 seeds. The flower heads were harvested and threshed individually and analyzed by near infrared spectroscopy (NIR) . From the 3,442 flower heads analyzed by NIR, 154 were selected and their fatty acid composition was confirmed by GC, after evaluating 30 M2 seeds by flower head. The mutant plant-CA06 495-2100 showed a phenotype with high content of oleic acid. It was identified and referred to as 29082. EXAMPLE 2
Analysis of fatty acid composition of seed oil
Sunflower seeds are cut by the sagittal axis and placed in a 2ml glass vial containing 0.25 ml of methylation solution consisting of methanol, toluene, dimethoxypropane and sulfuric acid in the ratio 66:28:4:2. The seeds were covered and incubated for one hour at 80 °C. They were allowed to cool at room temperature and then 1 ml of heptane was added (Garces and Mancha (2003) Anal Biochem 317:247- 254) . The methyl esters of present fatty acids in the superior phase (heptane) were separated on a gas
chromatograph Agilent 6890 with automatic injector Model 7683B. The injector temperature was 240°C and a Durabond capillary chromatographic column of 15 meters long was used, with an inner diameter of 0.25 mm and film of 0.25 microns (J & W Scientific) at 200°C. H2 was used as running gas at a pressure of 9.56 psi, flow 1.7 ml / min, and at an average speed of 69 cm/sec.
The methyl esters were detected with an ionized flame detector Type FID at 300°C. The results were
integrated and analyzed using Agilent Technologies Chem32 software. The relative amounts of each fatty acid were measured in relation to a standard solution of methylated fatty acids (Alltech) .
EXAMPLE 3
Sequencing of the sunflower genes that encode a polypeptide having desaturase oleate activity
For sequencing studies, tissue samples were taken from each high-oleic mutant. Genomic DNA was isolated and diluted to a stock concentration of 100 ng/μΐ. The coding sequence of the oleate desaturase of the high oleic mutants and the coding sequence of the oleate desaturase of line HA89 (wild type) were amplified in two overlapping segments. The specific primers used for each amplificon were: ler amplicon (705 bp)
OLD1-F2 GAAAAGTCTGGTCAAACAGTCAACAT (SEQ ID No: 21) OLD1-R2 CCGATGTCGGACATGACTATC (SEQ ID No: 22)
2nd amplicon (733 bp)
OLDladv-F2 AAATACTTTAACAACACAGTGGGC (SEQ ID No: 23)
OLD1-R3 CCAGAACCAGGACAACAGCCATTGTC (SEQ ID No: 24) All primers are specific for the gene of the oleate
desaturase. The following conditions were used for the polymerase chain reaction (PCR) in a final volume of 25μ1: IX buffer ( Invitrogen) , 0.2 mm dNTPs (GE Healthcare), 2.5 mm MgC±2 ( Invitrogen) , 0.2μΜ of each primer , 0.5μ1 of Platinum Taq DNA polymerase (5υ/μ1) (Invitrogen) and lOOng of genomic DNA. The PCR reaction was carried out on a GeneAmp PCR
System 9700 ( Perkin-Elmer) . The cycling conditions were: an initial denaturation step at 94°C for 1 minute followed by 35 cycles consisting of 94°C for 45 seconds, 57°C for 45 seconds and 72°C for 70 seconds, and a final elongation step of 72°C for 10 minutes.
Two microliters of each resulting product of the PCR was analyzed by electrophoresis in agarose gel and the DNA concentration was estimated in comparison with the molecular weight marker Low DNA Mass Ladder (Invitrogen) . The remainder of the product of the PCR was purified using a Wizard® SV Gel and the PCR Clean-Up System (Promega) . The purified PCR products were sequenced using the BigDye®
Terminator kit v3.1 Cycle Sequencing (Applied Biosystems) following the manufacturer's instructions.
The files of the sequencing of the oleate desaturase obtained for each amplicon were assembled using the program Vector NTI Suite-Contig Express, version 7.0 (Informax) . The resulting DNA sequences of the oleate desaturase were aligned with the sequences of line HA89 (GenBank Accession Number AY802989) .
Translations of the nucleotide sequences obtained from the new mutants into amino acid sequence were aligned with the amino acid sequence of the oleate desaturase HA89. In both cases the Vector NTI Suite program-AlignX, version 7.0 (Informax) was used and polymorphisms identified in a single nucleotide and changes in the amino acids of the mutants (29074, 29075, 29076, 29077, 29078, 29079, 29081, 29082 and 39096) . Print Out (Original in Electronic Form)
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Claims

1. An isolated mutated nucleotide sequence, wherein said nucleotide sequence comprises at least one point mutation and wherein said sequence encodes a modified oleate desaturase sunflower protein.
2. The sequence according to claim 1 wherein it encodes a modified oleate desaturase sunflower protein comprising at least one amino acid substitution selected from the group consisting of:
a) Gly by Asp at position 103,
b) Ser by Leu at position 131,
c) Ser by Asn at position 135,
d) Gly by Glu at position 144,
e) Gly by Glu at position 226,
f) Pro by Ser at position 253,
g) Leu by Phe at position 267; and
h) Pro by Ser at position 275
with respect to the wild type oleate desaturase of SEQ ID No: 19.
3. The sequence according to claim 1 or 2, wherein the sequence encodes a truncated oleate desaturase sunflower enzyme .
4. The sequence according to any one of the claims 1-3, wherein the sequence encodes a modified oleate
desaturase sunflower protein comprising an amino acids sequence selected from the group consisting of:
a) the sequence shown in SEQ ID No: 1,
b) the sequence shown in SEQ ID No: 2,
c) the sequence shown in SEQ ID No: 3,
d) the sequence shown in SEQ ID No: 4,
e) the sequence shown in SEQ ID No: 5,
f) the sequence shown in SEQ ID No: 6, g) the sequence shown in SEQ ID No: 7 ; h) the sequence shown in SEQ ID No: 8; and
i) the sequence shown in SEQ ID No: 9
5. The sequence according to any one of the claims 1-4, wherein that nucleic acids sequence is selected from the group consisting of:
a) the sequence shown in SEQ ID No: 10
b) the sequence shown in SEQ ID No: 11; c) the sequence shown in SEQ ID No: 12; d) the sequence shown in SEQ ID No: 13; e) the sequence shown in SEQ ID No: 14; f) the sequence shown in SEQ ID No: 15; g) the sequence shown in SEQ ID No: 16; h) the sequence shown in SEQ ID No: 17; and i) the sequence shown in SEQ ID No: 18. j) sequences at least 90 ¾ homologous to the sequences of a) to h) , wherein said sequences at least 90% homologous maintain the point mutation introduced in the sequences a) to h) .
6. A protein having reduced oleate desaturase activity, wherein said protein is a sunflower protein and comprises at least one amino acid substitution in a domain selected from the group consisting of the domains:
a) IV of the first peripheral segment associated to a PMS1;
b) VIII, more particularly the transmembrane helix
( TM4 ) ;
c) IX formed by the C-terminal region;
d) II of histidine;
e) I of histidine;
f) IX; and
g) VIII, more particularly the transmembrane helix
(TM3) .
7. The protein according to claim 6 wherein it comprises at least one amino acids substitution selected from the group consisting of:
a) Gly by Asp at position 103;
b) Ser by Leu at position 131;
c) Ser by Asn at position 135;
d) Gly by Glu at position 144;
e) Gly by Glu at position 226;
f) Pro by Ser at position 253;
g) Leu by Phe at position 267; and
h) Pro by Ser at position 275
with respect to the wild type oleate desaturase of SEQ ID No: 19.
8. The protein according to claim 6, characterized in that it comprises an amino acid sequence selected from the group consisting of:
a) the sequence shown in SEQ ID No: 1;
b) the sequence shown in SEQ ID No: 2;
c) the sequence shown in SEQ ID No: 3;
d) the sequence shown in SEQ ID No: 4;
e) the sequence shown in SEQ ID No: 5;
f) the sequence shown in SEQ ID No: 6;
g) the sequence shown in SEQ ID No: 7 ; and
h) the sequence shown in SEQ ID No: 8
9. The protein according to claim 6, wherein the protein is sunflower and it is truncated.
10. The protein according to claim 9,
characterized in that it comprises the amino acid sequence shown in SEQ ID No. 9.
11. A sunflower plant, characterized in that it comprises a gene that encodes an oleate desaturase enzyme, wherein the nucleotide sequence of said gene has a point mutation and encodes a modified oleate desaturase protein.
12. The plant according to claim 11, wherein the nucleotide sequence comprises a point mutation selected from the group consisting of:
a) G by A at position 308;
b) C by T at position 392;
c) G by A at position 404;
d) G by A at position 431;
e) G by A at position 677;
f) C by T at position 757;
g) C by T at position 799;
h) C by T at position 823; and
i) G by A at position 243;
all of them with respect to the wild type nucleotides sequence of SEQ ID No: 19.
13. The plant according to claim 11, wherein the plant produces seeds with an oleic acid content between 80% and 95% with respect to the total percentage of fatty acids of the said seed.
14. A sunflower seed, which comprises an oleate desaturase gene having a point mutation in its nucleotide sequence and the sequence encodes a modified oleate
desaturase protein.
15. The seed according to claim 14, wherein the nucleotide sequence comprises a mutation selected from the group consisting of:
a) G by A at position 308;
b) C by T at position 392;
c) G by A at position 404;
d) G by A at position 431;
e) G by A at position 677;
f) C by T at position 757;
g) C by T at position 799;
h) C by T at position 823; i) G by A at position 243,
with respect to the wild type nucleotides sequence of SEQ No: 20.
16. The seed according to claim 14-15, wherein a representative sample of said seed was deposited and is selected from the group consisting of:
a) line named 29074 access number NCIMB 41735; b) line named 29075 access number NCIMB 41736; c) line named 29076 access number NCIMB 41737; d) line named 29077 access number NCIMB 41738; e) line named 29078 access number NCIMB 41739; f) line named 29079 access number NCIMB 41740; g) line named 29082 access number NCIMB 41742; h) line named 29081 access number NCIMB 41741; and
i) line named 39096 access number NCIMB 41743.
17. The seed according to any one of the claims 14-16, wherein said seed has an oleic acid content between 80% and 95% with respect to the total percentage of fatty acids of the said seed.
18. Vegetable oil, characterized in that it has oleic acid content between 80% and 95% with respect to the total percentage of fatty acids and it is obtained from the sunflower seed of any one of the claims 14-17.
19. The use of the sunflower seed of any one of the claims 14-17 to obtain oil.
20. Progeny of the seed of any one of the claims
14-17, characterized in that comprises a point mutation in the gene that encodes an oleate desaturase protein, wherein said mutation leads to the synthesis of a modified oleate desaturase protein.
21. A method for obtaining a sunflower plant with high oleic acid content, characterized because it comprises the following steps:
a) mutagenesis of a part of a sunflower plant; b) obtaining at least one progeny of the mutant plant, and
c) identifying and selecting at least one plant obtained in step b) comprising a nucleotide sequence having at least one point mutation and wherein said sequence encodes a protein that has modified oleate desaturase activity .
22. The method according to claim 21, wherein the step of mutagenesis was performed by injecting a
mutagenizing agent in the flower head of the plant.
23. The method according to claim 21, wherein the step of mutagenesis is performed by contacting a seed with a mutagenizing agent.
24. The method according to claim 22 or 23, wherein the mutagenizing agent is methanesulfonic acid ethyl ester at a concentration between 5% and 15%.
25. The method according to claim 22 or 23, wherein the mutagenizing agent is methanesulfonic acid ethyl ester at a concentration between 0.3% and 0.7%.
26. The method according to claim 21, wherein the nucleotide sequence that has a point mutation comprises at least one nucleotide substitution selected from the group consisting of:
a) G by A at position 308;
b) C by T at position 392;
c) G by A at position 404;
d) G by A at position 431;
e) G by A at position 677;
f) C by T at position 757; g) C by T at position 799;
h) C by T at position 823; and
i) G by A at position 243;
with respect to the wild type nucleotide sequence of SEQ ID No: 20 and where said sequence encodes a protein that has modified oleate desaturase activity.
27. Method for identifying plants or sunflower seeds with high oleic acid content, which comprises:
a) providing a portion of a sunflower plant, and b) detecting in that part the presence of at least one point mutation in the gene that encodes a protein that has oleate desaturase activity.
28. The method according to claim 27, wherein the part of the plant is a seed.
29. The method according to claim 27 or 28, wherein the point mutation comprises at least one nucleotide substitution selected from the group consisting of:
a) G by A at position 30<
b) C by T at position 392,
c) G by A at position 404,
d) G by A at position 431,
e) G by A at position 677,
f) C by T at position 757,
g) C by T at position 799,
h) C by T at position 823; and
i) G by A at position 243,
with respect to the wild type sequence of SEQ ID No: 20 and wherein said sequence encodes a protein that has modified oleate desaturase activity.
30. The method according to any one of the claims
27-29, characterized in that the encoded protein comprises at least one amino acid substitution selected from the group consisting of: a) Gly by Asp at position 103;
b) Ser by Leu at position 131;
c) Ser by Asn at position 135;
d) Gly by Glu at position 144;
e) Gly by Glu at position 226;
f) Pro by Ser at position 253;
g) Leu by Phe at position 267;
h) Pro by Ser at position 275,
with respect to the amino acid sequence of the wild type protein of SEQ ID No: 19.
31. The method according to any one of the claims 27-30, characterized in that the detection of the presence of the point mutation in the gene comprises techniques selected from the group consisting of ASA, PAMSA, SSCP and TaqMan®.
32. The use of the nucleotide sequence of claim 5 for preparing a probe to detect a molecular marker
associated with high oleic phenotype in a sunflower plant, wherein the probe comprises a sequence of at least 8 nucleotides from a sequence selected from the sequence listed in claim 5 that include the point mutation.
33. A method for obtaining high oleic sunflower oil, characterized in that it comprises extracting oil from seeds of which a representative sample was deposited under an NCIMB accession number selected from the group consisting of NCIMB 41735, NCIMB 41736; NCIMB 41737, NCIMB 41738; NCIMB 41739, NCIMB 41740, NCIMB 41742; NCIMB 41741 and NCIMB
41743.
34. Plant that produces an oleate desaturase protein that has a lower enzymatic activity as compared to a wildtype oleate desaturase protein, obtainable by
introgression of a point mutation as found in a plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 into a wildtype plant not carrying the mutation.
35. Plant as claimed in claim 34, wherein introgression takes place by crossing a plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 with another plant and selecting in the F2 for plants that produce a sunflower oil that has a high oleic acid content between 80 and 95% as compared to the total fatty acid content in the oil.
36. Plant producing an oleate desaturase protein having a combination of two of more of the following amino acid substitutions as compared to the wild type amino acid sequence: a) Gly by Asp at position 103, b) Ser by Leu at position 131; c) Ser by Asn at position 135; d) Gly by Glu at position 144; e) Gly by Glu at position 226; f) Pro by Ser at position 253; g) Leu by Phe at position 267; h) Pro by Ser at position 275, which plants are obtainable by introgression of the mutation as found in two or more of the plant grown from seeds of which a representative sample was deposited under one of the accession numbers NCIMB 41735, NCIMB 41736, NCIMB 41737, NCIMB 41738, NCIMB 41739, NCIMB 41740, NCIMB 41741, NCIMB 41742 and NCIMB 41743 into a wildtype sunflower or into a sunflower that already contains one or more of the other point mutations.
PCT/EP2011/061164 2011-07-01 2011-07-01 Isolated mutated nucleotide sequences that encode a modified oleate destaurase sunflower protein, modified protein, methods and uses Ceased WO2013004280A1 (en)

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ARP120102375A AR087014A1 (en) 2011-07-01 2012-06-29 SEQUENCES OF ISOLATED MUTED NUCLEOTIDS THAT CODIFY A MODIFIED SUNFLOWER OLEATO PROTEIN, MODIFIED PROTEIN, METHODS AND USES
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WO2021014010A1 (en) 2019-07-24 2021-01-28 Soltis Sunflower with high oleic acid content and method for obtaining same
WO2024094771A1 (en) 2022-11-02 2024-05-10 KWS SAAT SE & Co. KGaA Oleic acid sunflower mutants and methods for detection

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WO2021014010A1 (en) 2019-07-24 2021-01-28 Soltis Sunflower with high oleic acid content and method for obtaining same
FR3099178A1 (en) 2019-07-24 2021-01-29 Soltis High oleic acid sunflower and process for obtaining it
WO2024094771A1 (en) 2022-11-02 2024-05-10 KWS SAAT SE & Co. KGaA Oleic acid sunflower mutants and methods for detection

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