WO2013004281A1 - Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses - Google Patents

Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses Download PDF

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Publication number
WO2013004281A1
WO2013004281A1 PCT/EP2011/061165 EP2011061165W WO2013004281A1 WO 2013004281 A1 WO2013004281 A1 WO 2013004281A1 EP 2011061165 W EP2011061165 W EP 2011061165W WO 2013004281 A1 WO2013004281 A1 WO 2013004281A1
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Prior art keywords
seed
sunflower
seq
plant
oleate desaturase
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PCT/EP2011/061165
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French (fr)
Inventor
Alberto Javier Leon
Andrés Daniel ZAMBELLI
Roberto Juan REID
Monica Mariel Morata
Marcos KASPAR
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Advanta International Bv
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Priority to UAA201400941A priority Critical patent/UA119026C2/en
Priority to EP23199371.8A priority patent/EP4303306A3/en
Priority to PCT/EP2011/061165 priority patent/WO2013004281A1/en
Priority to BR112013033907A priority patent/BR112013033907A8/en
Priority to CA2837760A priority patent/CA2837760A1/en
Priority to EP11729620.2A priority patent/EP2726616A1/en
Priority to RU2014103477A priority patent/RU2630641C2/en
Priority to US13/822,279 priority patent/US10143149B2/en
Application filed by Advanta International Bv filed Critical Advanta International Bv
Priority to CN201180071926.4A priority patent/CN103842510A/en
Priority to AU2011372738A priority patent/AU2011372738A1/en
Priority to ARP120102374A priority patent/AR086832A1/en
Priority to UY0001034184A priority patent/UY34184A/en
Publication of WO2013004281A1 publication Critical patent/WO2013004281A1/en
Priority to ZA2013/09107A priority patent/ZA201309107B/en
Priority to HK14112239.0A priority patent/HK1198772A1/en
Priority to US16/164,696 priority patent/US11234389B2/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
    • 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)
    • 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
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits
    • A01H1/101—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine
    • A01H1/104—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine involving modified lipid metabolism, e.g. seed oil composition
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • 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

Definitions

  • NUCLEOTIDE SEQUENCES MUTATED BY INSERTION THAT ENCODE A TRUNCATED OLEATE DESATURASE PROTEIN, PROTEINS, METHODS AND
  • the present invention relates to isolated nucleotide sequences that encode a mutated oleate desturase sunflower protein, to the mutated protein, to plants
  • the oleate desaturase acid enzyme (OLD) is involved in the enzymatic conversion of oleic acid to linoleic acid.
  • the microsomal OLD have been cloned and characterized using the marker technology by T-DNA (T-DNA tagging, Okuley, et al. (1994) Plant Cell 6:147-158). Nucleotide sequences of higher plants encoding microsomal OLD have been described in document W094/11516 PCT of Lightner et al .
  • Sunflower is generally cultivated to obtain oils that contain saturated fatty acids (palmitic and stearic) and unsaturated fatty acids (oleic and linoleic) .
  • the stearic acid content is always lower than 10% (Guston et al . (1986) The lipid handbook, Chapman and Hall Great Britain), usually between 3 and 7%.
  • sunflower seeds In relation 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% to 70% (Knowles (1988) Recent advances in oil crops breeding, AOCS Proceedings) and sunflower with high oleic content, which has a linoleic acid content of 2% to 10% and oleic acid content from 75% to 90% (Soldatov
  • the Pervenets population was distributed worldwide and used in many breeding programs in order to convert 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 in seeds is dependent on two enzymatic reactions: the desaturation of 18:0 (stearic acid) to 18:1 and the subsequent desaturation of 18:1 to 18:2 (linoleic acid).
  • the oleate desaturase enzyme (OLD) catalyzes the desaturation of 18:1 to 18:2 (Ohlrogge and Browse (1995) The Plant Cell, 7:957-970, Somerville and
  • Sunflower oil is naturally rich in 18:2 (55-70%) and consequently poor in 18:1 (20-25%).
  • LO low oleic
  • the Pervenets mutation has been shown to be associated with gene duplications within the OLD gene, leading to gene silencing. This decrease in transcription of OLD explains the decrease in the amount of enzyme and therefore the low OLD activity demonstrated (Hongtrakul et al. (1998) Crop Sci 38:1245-1249) consistently to the 18:1 accumulation in sunflower seeds. This finding led to the development of molecular markers characteristic of the mutation that can be used in breeding programs to facilitate the selection of HO genotypes (WO2005/106022 ; Lacombe et al . (2001) Life Sci 324:839-845).
  • polyunsaturated fatty acids It is used as a salad oil, cooking oil, or for production of margarine.
  • This oil should at least contain a level of 18:1 of 55% to 65% in relation to the total fatty acids.
  • the benefit of this oil is its high oxidative stability after the extraction process and the stability of the flavor of fried products. Sunflower oil with a high concentration of 18:1 does not need to be
  • This lower enzyme activity when expressed in a plant, in particular a sunflower plant, causes an increase in the amount of oleic acid in the plant oil or seed oil as compared to existing plants.
  • the invention thus relates to an isolated nucleotide sequence comprising an insertion that changes the reading frame, which nucleotide sequence encodes a truncated sunflower OLD protein.
  • the inserted nucleotide sequence comprises a premature stop codon and encodes a truncated sunflower oleate desaturase protein that comprises the amino acid sequence of SEQ ID No : 1 or SEQ ID No : 2.
  • the coding sequence of the truncated oleate desaturase protein can be the nucleotide sequence shown in SEQ ID No : 3 or SEQ ID No : .
  • the invention provides a truncated sunflower oleate desaturase protein comprising no more than 110 amino acids of the N- terminal end.
  • the amino acid sequence consists of SEQ ID NO:l or SEQ ID No : 2.
  • this invention relates to a sunflower plant comprising two alleles of the OLD with an insertion encoding an OLD truncated protein.
  • Each allele comprises a nucleotide sequence that has an insertion comprising a premature stop codon and wherein said sequence encodes an OLD truncated protein.
  • the nucleotide sequence is SEQ ID No : 3 or SEQ ID No : 4.
  • the mutated plant produces seeds with an oleic acid content between 80% and 95% with respect to the total percentage of fatty acids of the seed.
  • the invention relates to a sunflower plant capable of producing seed having an oleic acid content between 80 and 95% with respect to the total fatty acid content of the seed, which plant is obtainable by crossing a plant of line 29065 that has the accession number NCIMB 41733 or of line 29066 that has the accession number NCIMB 41734 with another plant and
  • the invention also provides sunflower seed which comprises two alleles of the OLD with an insertion encoding a truncated oleate desaturase protein.
  • Each allele comprises a nucleotide sequence that has an insert comprising a premature stop codon and wherein the sequence encodes a truncated protein desaturase oleate.
  • the nucleotide sequence may be the sequence shown in SEQ ID No : 3 or SEQ ID No : 4.
  • the amino acid sequence of the oleate desaturase protein may be the sequence shown in SEQ ID No : 1 or SEQ ID No : 2.
  • the seed is of line 29065, a
  • a sunflower oil which has an oleic acid content between 80% and 95% with respect to the total fatty acid content of the oil, which oil is obtainable from or obtained from sunflower seed that comprises two OLD alleles which have an insertion and encode a truncated oleate desaturase protein.
  • the oil is obtained from the seeds of line 29065, a representative seed sample of which was deposited under the accession number NCIMB 41733 or line 29066, a representative seed sample of which was deposited under the accession number NCIMB 41734.
  • the present invention further provides the use of sunflower seed for oil extraction.
  • the seed is of line 29065, a representative seed sample of which was deposited under the accession number NCIMB 41733 or of line 29066, a representative seed sample of which was deposited under the accession number NCIMB 41734.
  • the invention further relates to progeny of the claimed seed, wherein said progeny comprises an insertion in the gene that encodes an oleate desaturase protein, in which such insertion leads to the synthesis of a truncated oleate desaturase protein.
  • the invention further relates to a method for obtaining a sunflower plant with high oleic acid content which comprises the following steps:
  • truncated oleate desaturase protein comprises a sequence of no more than 110 amino acids of the N-terminal end of the sunflower oleate desaturase, for example, the protein shown in SEQ ID No : 1 or SEQ ID No:2.
  • the invention provides a method for obtaining high oleic sunflower oil comprising extracting oil from seeds, a representative seed sample of which was deposited under accession number NCIMB 41733 or NCIMB 41734. DESCRIPTION OF THE FIGURES
  • Figure 1 displays a graph with the percentage of oleic acid in relation to all major fatty acids for
  • segments within the boxes represent the medians.
  • the present invention relates to mutated nucleotide sequences encoding an oleate desaturase that have an
  • the inserted sequence comprises a premature stop codon and therefore the sunflower oleate desaturase protein is truncated.
  • the truncated oleate desaturase protein encoded has a lower enzyme activity leading to the plant or plant parts, such as seeds, to accumulate a large amount of oleic acid.
  • the truncated amino acid sequence can be any sequence that comprises no more than the first 110 amino acids of the N-terminal end of the sunflower oleate
  • the truncated amino acid sequence may be the sequence shown in SEQ ID No : 1 or the sequence shown in SEQ ID No : 2.
  • nucleotide sequences mutated by an insertion comprise a premature stop codon that lead to the synthesis of a sunflower truncated OLD protein. It should be
  • nucleotide sequence that encodes an oleate desaturase protein that comprises not more than the first 110 amino acids of the N-terminal end of the wild type sunflower OLD, for example, the one from line HA89, are within the scope of this invention.
  • nucleotide sequences that comprise an insertion that has a premature stop codon for example the sequences shown in SEQ ID No : 3 and SEQ ID No : 4 are within the scope of the present invention.
  • Different sunflower lines have been mutated, for example lines HA89 and 29010 to obtain plants that produce seeds with high oleic acid content.
  • Selected mutations in the coding sequence of the sunflower oleate desaturase gene were mutations by insertion of sequences that comprised a premature stop codon, said nucleotide sequences encode truncated oleate desaturase proteins.
  • NCIMB Food and Marine Bacteria
  • the present invention relates to all seeds and plants, in particular sunflower seeds and plants that carry in their genome a mutation that leads to production of a truncated oleate desaturase protein having an enzyme activity that is lowered as compared to existing oleate desaturase enzymes leading to a higher oleic acid content in the seed oil.
  • the mutation is an
  • Such plants and seeds that have a higher oleic acid content in the oil can be obtained by crossing with plants of the deposited lines 29065 (NCIMB 41733) or 29066
  • NCIMB 41734 selecting for progeny plants that have the higher oleic acid content. Selection is suitably made in the F2 since the mutation leading to the truncation must
  • the mutated gene can be brought into a sunflower plant by means of genetic engineering, in
  • Mutated plants and seeds of the invention can be obtained using different mutagenesis schemes.
  • a mutagenic agent such as EMS in a concentration between 5 and 15% is injected into the flower heads of the plants, the seeds are harvested and then X-ray radiation is applied.
  • the fatty acid profile of mutated M2 seeds and parental seeds was analyzed, and those that showed a high content of oleic acid were selected, for example over 80% compared to the fatty acid content of seed, preferably about 90% compared to the total fatty acid content of the seed.
  • mutated plants and seeds were obtained by X-ray application.
  • the fatty acid profile of M2 seeds was compared to the fatty acid profile of the parental seeds and those that showed a high content of oleic acid, for example having over 80% with respect to the total fatty acid content of the seeds, preferably about 90% compared to the total fatty acid content of the seed were selected.
  • the oil produced 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 the seed,
  • Fl plants obtained in each cross were self-pollinated and their seeds (F2) were harvested. F2 seeds from each cross were planted in the field at Balcarce during the 2008/2009 season .
  • homozygous plants were identified for the Pervenets mutation and homozygous for mutations 29065 and 29066, obtained in each of the crosses. Said plants were self-pollinated and seeds were harvested individually. A group of 30 seeds was taken from each plant, grounded in a block and the fatty acid composition of oil was determined by gas
  • nucleotide and amino acids sequences were compared with the corresponding sequences of the sunflower wild type line HA89 (SEQ ID No : 5 and SEQ ID No:6). These sequences were identical to the corresponding ones of 29010 wild-type sunflower line.
  • the device for X-ray emission was a Philips MG 160, 4KW with a maximum output of 160KV, 19mA and power use of 120 KV, 15 mA.
  • the exposure time and dose used were 7 min 55 sec and 144 Grays, respectively, with a distance between the focus of the source and the object (seeds) of 40 centimeters.
  • the flower heads of the plants were mutagenized by EMS injection (methanesulfonic acid ethyl ester) in doses of 5, 10 and 15% using a suitable mutagenesis protocol. Each M0 plant was bagged before flowering in order to produce self- pollinated Ml seeds. The flower heads of the plants of each EMS treatment were harvested, threshed and stored. The flower heads of the plants were mutagenized by injection with EMS (methanesulfonic acid ethyl ester) in doses of 5, 10 and 15% using the protocol described in O2006/024351 , and WO2008/071715.
  • EMS is a mutagenic agent that induces transitions G/C to A/T (Jander et al .
  • each M0 plant was covered with a nylon mesh bag before flowering.
  • the flower heads of the plants of each 5% EMS treatment were harvested, threshed and identified (lot CA04- 3) .
  • Said Ml seeds were subsequently mutagenized by X- ray application. Irradiation was carried out at the
  • the X-ray emission device was a Philips MG 160, 4KW with a maximum output of 160KV, 19mA and power use of 120 KV, 15 mA.
  • the exposure time and dose used were 7 min 55 sec and 144 Grays, respectively, with a distance between the source focus and the object (seeds) of 40 centimeters .
  • the irradiated seeds were sown on December 12, 2005, in 16 rows of 100 meters long in Balcarce under the lot number CA05-6362.
  • the flower heads of the 555 plants obtained were bagged before flowering in order to produce seeds by self-pollination, M2.
  • the flower heads in each row were harvested and threshed individually.
  • M2 seeds from 555 plants were analyzed by gas chromatography (GC) to determine the fatty acid composition in 30 individual M2 seeds per plant.
  • GC gas chromatography
  • the mutant plant CA05-6362-263 showed a phenotype with a high content of oleic and was called 29066.
  • Table 1 shows the mutagenesis method used for each mutant line of the invention, the nucleotide seguence and amino acid seguence of the truncated oleate desaturase protein .
  • 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 vials were capped 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) Analytical Biochemistry 317:247-254).
  • the methyl esters of fatty acids present in the upper phase (heptane) were separated on a gas
  • Table 2 are shown the fatty acid composition analyses done by gas chromatography on seed samples from mutant plants from the invention.
  • P palmitic acid
  • S stearic acid
  • 0 oleic acid
  • L
  • KLA reaction by PCR was performed on a GeneAmp PCR System 9700 computer (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
  • genomic libraries were generated for each of the mutants (29065 and 29066) in order to perform chromosome walking by PCR using the system GenomeWalkerTM
  • ACCCGTTCGTTCTCCTACGT (SEQ ID 0LD1-Walk3'-F2 No:12) AGTGGCAGGCGAAACGGTCA (SEQ ID NO:
  • GCCCACGACACTTACCAGA SEQ ID MUTHA89HO-W32-2 No:24
  • Terminator v3.1 Cycle Sequencing System (Applied Biosystems) following the manufacturer's instructions.

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Abstract

Isolated nucleotide sequences mutated by insertion encoding a truncated sunflower oleate desaturase protein, truncated protein, methods, procedures and uses. The isolated nucleotide sequences comprise an insertion that includes a stop codon, and wherein the sequences encode a truncated sunflower oleate desaturase protein. The truncated sunflower oleate desaturase protein may be for example the sequence shown in SEQ ID No: 1 or SEQ ID No: 2.

Description

NUCLEOTIDE SEQUENCES MUTATED BY INSERTION THAT ENCODE A TRUNCATED OLEATE DESATURASE PROTEIN, PROTEINS, METHODS AND
USES The present invention relates to isolated nucleotide sequences that encode a mutated oleate desturase sunflower protein, to the mutated protein, to plants
producing the protein, to seeds and oils produced by the plant, to methods for obtaining the mutated sequences and the plant, and to uses of the protein and the plant,
desaturasedesaturase
BACKGROUND
The oleate desaturase acid enzyme (OLD) is involved in the enzymatic conversion of oleic acid to linoleic acid. The microsomal OLD have been cloned and characterized using the marker technology by T-DNA (T-DNA tagging, Okuley, et al. (1994) Plant Cell 6:147-158). Nucleotide sequences of higher plants encoding microsomal OLD have been described in document W094/11516 PCT of Lightner et al .
Sunflower is generally cultivated to obtain oils that contain saturated fatty acids (palmitic and stearic) and unsaturated fatty acids (oleic and linoleic) . The stearic acid content is always lower than 10% (Guston et al . (1986) The lipid handbook, Chapman and Hall Great Britain), usually between 3 and 7%. In relation 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% to 70% (Knowles (1988) Recent advances in oil crops breeding, AOCS Proceedings) and sunflower with high oleic content, which has a linoleic acid content of 2% to 10% and oleic acid content from 75% to 90% (Soldatov
(1976) Chemical mutagenesis in sunflower breeding,
Proceedings of the 7th International Sunflower Conference, 352-357) . There is also a sunflower line that has a high content of palmitic acid between 22 and 40% (Ivanov et al. (1988) Sunflower Breeding for High Palmitic Acid Content in the Oil, Proceedings of the 12th Interntional Sunflower Conference, Vol II, 453-465) and another line of sunflower with low content of saturated fatty acids (less than 6%) (EP-A-0496504) .
In order to respond to the need for vegetable oils of interest to both the industry and to human food
consumption, efforts are made in improving varieties of oilseeds focused on modifying the fatty acid composition of seeds, for example, by means of conventional breeding programs, mutagenesis or transgenesis .
Mutations are typically induced with extremely high doses of radiation or chemical mutagens (Gaul (1964)
Radiation Botany 4:155-232). High doses exceed the lethal dose 50% (LD50) , and generally lethal doses of 90% (LD90) maximize the percentage of possible mutations.
Mutagenesis conducted by Soldatov in 1976 in a population of sunflower allowed obtaining the population of so-called Pervenets mutants. The average content of oleic fatty acid (18:1) of the seeds of this cultivar is higher than 65%, the individual content is between 60 and 80% while in normal varieties (low oleic, LO) this content is
approximately 20%. The Pervenets population was distributed worldwide and used in many breeding programs in order to convert 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 in seeds is dependent on two enzymatic reactions: the desaturation of 18:0 (stearic acid) to 18:1 and the subsequent desaturation of 18:1 to 18:2 (linoleic acid). The oleate desaturase enzyme (OLD) catalyzes the desaturation of 18:1 to 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 18:2 (55-70%) and consequently poor in 18:1 (20-25%). Traditional
varieties are classified as low oleic (LO) . As there exists a high interest among consumers of eating more healthy oils with high oleic acid content, there is also an interest in developing high oleic sunflower plants, which preferably also lead to similar crop yields as the conventional
sunflower varieties.
Studies carried out by Garces et al. in 1989 and 1991 (Garces et al . (1989) Phytochemistry 28:2597-2600;
Garces and Mancha (1991) Phytochemistry 30:2127-2130) showed that the high oleic phenotype (high oleic, HO) is associated with a marked decrease in the activity of the OLD enzyme, which catalyzes the desaturation of 18:1 to 18:2 in HO seeds during critical stages of the synthesis of the lipidic reserves, which explains the accumulation of 18:1.
The Pervenets mutation has been shown to be associated with gene duplications within the OLD gene, leading to gene silencing. This decrease in transcription of OLD explains the decrease in the amount of enzyme and therefore the low OLD activity demonstrated (Hongtrakul et al. (1998) Crop Sci 38:1245-1249) consistently to the 18:1 accumulation in sunflower seeds. This finding led to the development of molecular markers characteristic of the mutation that can be used in breeding programs to facilitate the selection of HO genotypes (WO2005/106022 ; Lacombe et al . (2001) Life Sci 324:839-845).
Traditional sunflower oil with high content of 18:2 is considered a healthy vegetable oil that has a proper taste, and it has been considered first class quality oil in the world market due to its high percentage of
polyunsaturated fatty acids. It is used as a salad oil, cooking oil, or for production of margarine.
By modifying the fatty acid profile of sunflower oil, new oil can be developed having higher oxidative
stability compared to conventional oil. This oil should at least contain a level of 18:1 of 55% to 65% in relation to the total fatty acids. The benefit of this oil is its high oxidative stability after the extraction process and the stability of the flavor of fried products. Sunflower oil with a high concentration of 18:1 does not need to be
hydrogenated to improve stability and does not form trans fatty acids. BRIEF DESCRIPTION OF THE INVENTION
It is an object of this invention to provide an isolated nucleotide sequence encoding a mutated oleate desaturase protein (OLD) having a lower enzyme activity. This lower enzyme activity when expressed in a plant, in particular a sunflower plant, causes an increase in the amount of oleic acid in the plant oil or seed oil as compared to existing plants.
The invention thus relates to an isolated nucleotide sequence comprising an insertion that changes the reading frame, which nucleotide sequence encodes a truncated sunflower OLD protein. In a preferred embodiment, the inserted nucleotide sequence comprises a premature stop codon and encodes a truncated sunflower oleate desaturase protein that comprises the amino acid sequence of SEQ ID No : 1 or SEQ ID No : 2. The coding sequence of the truncated oleate desaturase protein can be the nucleotide sequence shown in SEQ ID No : 3 or SEQ ID No : .
According to a further aspect thereof this
invention provides a truncated sunflower oleate desaturase protein comprising no more than 110 amino acids of the N- terminal end. In a preferred embodiment the amino acid sequence consists of SEQ ID NO:l or SEQ ID No : 2.
According to another aspect this invention relates to a sunflower plant comprising two alleles of the OLD with an insertion encoding an OLD truncated protein. Each allele comprises a nucleotide sequence that has an insertion comprising a premature stop codon and wherein said sequence encodes an OLD truncated protein. In a preferred embodiment the nucleotide sequence is SEQ ID No : 3 or SEQ ID No : 4. The mutated plant produces seeds with an oleic acid content between 80% and 95% with respect to the total percentage of fatty acids of the seed.
In a further embodiment thereof, the invention relates to a sunflower plant capable of producing seed having an oleic acid content between 80 and 95% with respect to the total fatty acid content of the seed, which plant is obtainable by crossing a plant of line 29065 that has the accession number NCIMB 41733 or of line 29066 that has the accession number NCIMB 41734 with another plant and
selecting in the F2 for plants that produce seed having an oleic acid content between 80 and 95% with respect to the total percentage of fatty acids of the seed.
The invention also provides sunflower seed which comprises two alleles of the OLD with an insertion encoding a truncated oleate desaturase protein. Each allele comprises a nucleotide sequence that has an insert comprising a premature stop codon and wherein the sequence encodes a truncated protein desaturase oleate. The nucleotide sequence may be the sequence shown in SEQ ID No : 3 or SEQ ID No : 4. The amino acid sequence of the oleate desaturase protein may be the sequence shown in SEQ ID No : 1 or SEQ ID No : 2. In a preferred embodiment the seed is of line 29065, a
representative seed sample of which was deposited under the accession number NCIMB 41733 or of line 29066, a
representative seed sample of which was deposited under the accession number NCIMB 41734.
According to another aspect of this invention a sunflower oil is provided, which has an oleic acid content between 80% and 95% with respect to the total fatty acid content of the oil, which oil is obtainable from or obtained from sunflower seed that comprises two OLD alleles which have an insertion and encode a truncated oleate desaturase protein. In a preferred embodiment the oil is obtained from the seeds of line 29065, a representative seed sample of which was deposited under the accession number NCIMB 41733 or line 29066, a representative seed sample of which was deposited under the accession number NCIMB 41734.
The present invention further provides the use of sunflower seed for oil extraction. In a preferred embodiment the seed is of line 29065, a representative seed sample of which was deposited under the accession number NCIMB 41733 or of line 29066, a representative seed sample of which was deposited under the accession number NCIMB 41734.
The invention further relates to progeny of the claimed seed, wherein said progeny comprises an insertion in the gene that encodes an oleate desaturase protein, in which such insertion leads to the synthesis of a truncated oleate desaturase protein.
The invention further relates to a method for obtaining a sunflower plant with high oleic acid content which comprises the following steps:
a) mutagenesis of 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 that has an insertion that comprises a premature stop codon, and wherein said nucleotide sequence encodes a truncated oleate desaturase protein. In one embodiment the truncated oleate desaturase protein comprises a sequence of no more than 110 amino acids of the N-terminal end of the sunflower oleate desaturase, for example, the protein shown in SEQ ID No : 1 or SEQ ID No:2.
According to another aspect thereof the invention provides a method for obtaining high oleic sunflower oil comprising extracting oil from seeds, a representative seed sample of which was deposited under accession number NCIMB 41733 or NCIMB 41734. DESCRIPTION OF THE FIGURES
Figure 1 displays a graph with the percentage of oleic acid in relation to all major fatty acids for
homozygous plants for the high oleic Pervenets mutation, for lines 29065 and 29066 obtained in crosses 20342x29065;
20342x29066; 20340x29065 and 20340x29066. In all cases the percentages of oleic acid in F2 plants carrying mutations of the present invention in homozygous state (mut 29065 Hm and mut 29066 Hm, respectively) are compared with F2 plants carrying Pervenets mutation in homozygous state (mut
Pervenets Hm) . The points represent the mean and the
segments within the boxes represent the medians.
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to mutated nucleotide sequences encoding an oleate desaturase that have an
insertion, where the inserted sequence comprises a premature stop codon and therefore the sunflower oleate desaturase protein is truncated. The truncated oleate desaturase protein encoded has a lower enzyme activity leading to the plant or plant parts, such as seeds, to accumulate a large amount of oleic acid.
The truncated amino acid sequence can be any sequence that comprises no more than the first 110 amino acids of the N-terminal end of the sunflower oleate
desaturase protein. In a preferred embodiment the truncated amino acid sequence may be the sequence shown in SEQ ID No : 1 or the sequence shown in SEQ ID No : 2.
The nucleotide sequences mutated by an insertion comprise a premature stop codon that lead to the synthesis of a sunflower truncated OLD protein. It should be
understood that any nucleotide sequence that encodes an oleate desaturase protein that comprises not more than the first 110 amino acids of the N-terminal end of the wild type sunflower OLD, for example, the one from line HA89, are within the scope of this invention.
The nucleotide sequences that comprise an insertion that has a premature stop codon, for example the sequences shown in SEQ ID No : 3 and SEQ ID No : 4 are within the scope of the present invention.
Different sunflower lines have been mutated, for example lines HA89 and 29010 to obtain plants that produce seeds with high oleic acid content. Selected mutations in the coding sequence of the sunflower oleate desaturase gene were mutations by insertion of sequences that comprised a premature stop codon, said nucleotide sequences encode truncated oleate desaturase proteins.
Mutant seeds of the invention were deposited under the Budapest Treaty in the National Library of Industry,
Food and Marine Bacteria (NCIMB) collection on July 20, 2010 with the following accession numbers: line 29065 is accession number NCIMB 41733 and line 29066 is accession number NCIMB 41734.
The present invention relates to all seeds and plants, in particular sunflower seeds and plants that carry in their genome a mutation that leads to production of a truncated oleate desaturase protein having an enzyme activity that is lowered as compared to existing oleate desaturase enzymes leading to a higher oleic acid content in the seed oil. In one embodiment, the mutation is an
insertion that carries a stop codon. Other mutations that lead to a truncation of the oleate desaturase to a maximum of 110 amino acids are also within the scope of this invention. Such plants and seeds that have a higher oleic acid content in the oil can be obtained by crossing with plants of the deposited lines 29065 (NCIMB 41733) or 29066
(NCIMB 41734) and selecting for progeny plants that have the higher oleic acid content. Selection is suitably made in the F2 since the mutation leading to the truncation must
preferably be present homozygously for the lower enzyme activity to lead to the higher oleic acid content.
Alternatively, the mutated gene can be brought into a sunflower plant by means of genetic engineering, in
particular cisgenesis, with the mutant oleic desaturase gene described herein or any other gene leading to the same truncated expression product of not more than 110 amino acids .
Mutated plants and seeds of the invention can be obtained using different mutagenesis schemes. In a preferred embodiment a mutagenic agent such as EMS in a concentration between 5 and 15% is injected into the flower heads of the plants, the seeds are harvested and then X-ray radiation is applied.
Following mutagenesis, the fatty acid profile of mutated M2 seeds and parental seeds was analyzed, and those that showed a high content of oleic acid were selected, for example over 80% compared to the fatty acid content of seed, preferably about 90% compared to the total fatty acid content of the seed. In another preferred embodiment, mutated plants and seeds were obtained by X-ray application. Subsequently, the fatty acid profile of M2 seeds was compared to the fatty acid profile of the parental seeds and those that showed a high content of oleic acid, for example having over 80% with respect to the total fatty acid content of the seeds, preferably about 90% compared to the total fatty acid content of the seed were selected.
The oil produced 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 the seed,
preferably higher than 85% and more preferably higher than 90%.
Directed crosses were performed between lines that belong to Advanta Semillas with high oleic phenotype, which are carriers of Pervenets mutation (20342 and 20340), and the high oleic mutants 29065 and 29066 of the invention:
20342x29065; 2042x29066; 20340x29065 and 20340x29066. Fl plants obtained in each cross were self-pollinated and their seeds (F2) were harvested. F2 seeds from each cross were planted in the field at Balcarce during the 2008/2009 season .
By means of using specific molecular markers, homozygous plants were identified for the Pervenets mutation and homozygous for mutations 29065 and 29066, obtained in each of the crosses. Said plants were self-pollinated and seeds were harvested individually. A group of 30 seeds was taken from each plant, grounded in a block and the fatty acid composition of oil was determined by gas
chromatography.
In all cases, homozygous plants for mutations 29065 and 29066 showed average percentages of oleic acid higher than their corresponding counterparts with Pervenets
mutation (see Table 3 and Figure 1) .
The nucleotide and amino acids sequences were compared with the corresponding sequences of the sunflower wild type line HA89 (SEQ ID No : 5 and SEQ ID No:6). These sequences were identical to the corresponding ones of 29010 wild-type sunflower line.
The invention is illustrated by the following examples which should not be interpreted as limiting the scope thereof. On the contrary, it should be clearly understood that it is possible for those skilled in the field after reading the present disclosure to resort to other embodiments, modifications and equivalents of the invention, without deviating from the spirit of the present invention and/or scope of appended claims.
EXAMPLES EXAMPLE 1
1.1 Mutagenesis by X-ray irradiation (generation of line 29065)
M0 seeds of line 29010, proprietary of Advanta Semillas SAIC, were mutagenised by application of X-ray. Irradiation was carried out at the Institute of Genetics "Edward A. Favret", CICVyA-CNIA, INTA-Castelar, Argentina. The device for X-ray emission was a Philips MG 160, 4KW with a maximum output of 160KV, 19mA and power use of 120 KV, 15 mA. The exposure time and dose used were 7 min 55 sec and 144 Grays, respectively, with a distance between the focus of the source and the object (seeds) of 40 centimeters.
The irradiated seeds were sown on December 12,
2005, in 12 rows 100 meters long in Balcarce with lot number CA05-6347. The flower heads of the 1606 plants obtained were bagged before flowering in order to produce seeds by self- pollination, M2. The flower heads in each row were harvested and threshed individually. The M2 seeds of the 1606 plants were analyzed by gas chromatography (GC) to determine the fatty acid composition in 30 individual M2 seeds per plant. The mutant plant CA05-6347-725 showed a phenotype with a high content of oleic and was called 29065. 1.2 Mutagenesis by EMS injection and subsequent irradiation with X rays (generation of line 29066)
Seventy-five rows of seeds of line HA89 were sown in the Biotechnology Research Center of Advanta Semillas SAIC in Balcarce (Buenos Aires, Argentina) in the season
2004/5 and were identified under the lot number CA04-3, and 40 rows under the batch number CA04-1501. Each row was 6 meters long.
The flower heads of the plants were mutagenized by EMS injection (methanesulfonic acid ethyl ester) in doses of 5, 10 and 15% using a suitable mutagenesis protocol. Each M0 plant was bagged before flowering in order to produce self- pollinated Ml seeds. The flower heads of the plants of each EMS treatment were harvested, threshed and stored. The flower heads of the plants were mutagenized by injection with EMS (methanesulfonic acid ethyl ester) in doses of 5, 10 and 15% using the protocol described in O2006/024351 , and WO2008/071715. 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 self-pollinated Ml seeds, each M0 plant was covered with a nylon mesh bag before flowering. The flower heads of the plants of each 5% EMS treatment were harvested, threshed and identified (lot CA04- 3) .
Said Ml seeds were subsequently mutagenized by X- ray application. Irradiation was carried out at the
Institute of Genetics "Edward A. Favret", CICVyA-CNIA, INTA- Castelar, Argentina. The X-ray emission device was a Philips MG 160, 4KW with a maximum output of 160KV, 19mA and power use of 120 KV, 15 mA. The exposure time and dose used were 7 min 55 sec and 144 Grays, respectively, with a distance between the source focus and the object (seeds) of 40 centimeters .
The irradiated seeds were sown on December 12, 2005, in 16 rows of 100 meters long in Balcarce under the lot number CA05-6362. The flower heads of the 555 plants obtained were bagged before flowering in order to produce seeds by self-pollination, M2. The flower heads in each row were harvested and threshed individually. M2 seeds from 555 plants were analyzed by gas chromatography (GC) to determine the fatty acid composition in 30 individual M2 seeds per plant. The mutant plant CA05-6362-263 showed a phenotype with a high content of oleic and was called 29066.
Table 1 shows the mutagenesis method used for each mutant line of the invention, the nucleotide seguence and amino acid seguence of the truncated oleate desaturase protein .
Table 1
Figure imgf000013_0001
EXAMPLE 2
Analysis of the fatty acid composition of the seeds oil by gas chromatography
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 vials were capped 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) Analytical Biochemistry 317:247-254). The methyl esters of fatty acids present in the upper phase (heptane) were separated on a gas
chromatograph Agilent 6890 with autosampler Model 7683B. The injector temperature was 240°C and a capillary
chromatographic column Durabond 15 meters in length, inner diameter of 250 μΜ and 250 μΜ film was used (J & W
Scientific) at 200°C. H2 gas was used as running gas at a pressure of 9.56 psi, flow 1.7 ml/min, and an average speed of 69 cm/sec. The methyl esters were detected with a flame ionization detector (FID) at 300°C. The results were
integrated and analyzed using Chem32 Agilent Technologies software. The relative amounts of each fatty acid were measured in relation to a standard solution of methylated fatty acids (Alltech) .
In Table 2 are shown the fatty acid composition analyses done by gas chromatography on seed samples from mutant plants from the invention.
Table 2
Figure imgf000014_0001
P: palmitic acid; S: stearic acid; 0: oleic acid; L:
linoleic acid
Table 3
Comparison of the levels of oleic acid (%) of sunflower seeds from F2 plants homozygous (Hm) for mutations 29065 and 29066 compared with F2 plants homozygous (Hm) for Pervenets mutation. Percentage oleic acid content for both Hm 29065 and 29066 was significantly higher than its respective Hm Pervenets counterpart. Homozygous
Average oleic Standard
Population high oleic n acid % deviation mutation
29065 87.50 1.08 12
20340x29065
Pervenets 82.86 5.38 18
29066 87.91 2.56 114
20340x29066
Pervenets 86.26 2.64 90
29065 89.08 3.56 56
20342x29065
Pervenets 75.81 11.15 57
29066 88.90 2.17 37
20342x29066
Pervenets 87.17 2.08 41 n: amount of F2 plants whose fatty acid composition was analyzed by gas chromatography. EXAMPLE 3
Sequencing of the sunflower genes that encode a polypeptide that has oleate desaturase activity
For sequencing studies, tissue samples were taken from each mutant with high oleic content, the genomic DNA was isolated and diluted to a stock concentration of 100 ng/μΐ . In order to obtain the coding sequence of the full oleate desaturase of high oleic mutants 29065 and 29066 and the original lines 29010 and HA89 (wild type) specific primers were designed for the gene. These were used to amplify by PCR (polymerase chain reaction) and generate two overlapping DNA segments (amplicons) that covered the entire OLD gene. The sequence of designed primers follows:
1st OLD1-F2 GAAAAGTCTGGTCAAACAGTCAACAT (SEQ ID No : 7 ) amplicon OLD1-R2 CCGATGTCGGACATGACTATC (SEQ ID No: 8)
(705 bp)
2nd OLDladv-F2 AAATACTTTAACAACACAGTGGGC (SEQ ID No: 9) amplicon 0LD1-R3 CCAGAACCAGGACAACAGCCATTGTC (SEQ ID No: 10) (733 bp) All primers are specific for the desaturase oleate gene. The following conditions were used for polymerase chain reaction (PCR) in a final volume of 25μ1: IX buffer ( Invitrogen) , 0.2 mm dNTPs (GE Healthcare), 2.5 mm MgCl2 (Invitrogen), 0.2 μΜ of each primer, 0.5μ1 of Platinum Taq (5ϋ/μ1) (Invitrogen) and 100 ng of genomic DNA. KLA reaction by PCR was performed on a GeneAmp PCR System 9700 computer (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 72°C for 10 minutes .
In the case of the two mutants found with high content of oleic acid (29065 and 29066) amplification product could not be obtained when OLD1 -F2 and OLD1-R2 primers were used. This led to postulate that these mutants possessed large segments of DNA inserted into the region comprised between these primers as a result of the mutagenic treatment with X-rays. This would mean that due to the large segment flanked by OLD1-F2 and OLD1-R2 primers, no
amplification product could be obtained.
In order to establish the nucleotide sequence of the inserted segments, genomic libraries were generated for each of the mutants (29065 and 29066) in order to perform chromosome walking by PCR using the system GenomeWalker™
Universal Kit Protocol-at-a-Glance (Clontech) following the manufacturer's instructions.
Taking the nucleotide sequence of the oleate desaturase gene of lines HA89 and 29010 as a reference, genome walking was carried out both in a 5 ' -and 3' -sense for each mutant. The following specific primers were used for each of the mutants:
Mutant 29065
First AACCACCCTTCACCATCGGCG (SEQ ID time 0LD1-Walk3 ' -Fl No:ll)
ACCCGTTCGTTCTCCTACGT (SEQ ID 0LD1-Walk3'-F2 No:12) AGTGGCAGGCGAAACGGTCA (SEQ ID
0LD1-Walk5 ' -Rl No: 13)
AGCCGAGAGTGAGAGTGACG (SEQ ID 0LD1-Walk5 ' -R2 No: 14)
Second ATGATCGCAGTCCCCAAAAG (SEQ ID time MUT29010HO-W2-5 ' -1 No:15)
CCAATCAGCCTACAATAACAA (SEQ ID MUT29010HO-W2-5 ' -2 No: 16)
AAGGGACGAGTAAAGACGAG (SEQ ID MUT29010HO-W2-3 * -1 No: 17)
TCGTCGACCCATTGATAATC (SEQ ID MUT29010HO-W2-3 ' -2 No: 18)
Mutant 29066
First AACCACCCTTCACCATCGGCG (SEQ ID time OLDl- alk3 ' -Fl No: 11)
ACCCGTTCGTTCTCCTACGT (SEQ ID
OLDl-Walk3 ' -F2 No: 12)
AGTGGCAGGCGAAACGGTCA (SEQ ID OLDl-Walk5 ' -Rl No: 13)
AGCCGAGAGTGAGAGTGACG (SEQ ID OLDl- alk5 ' -R2 No: 14)
Second GGAAAACAGGGTTATTGGCA (SEQ ID time MUTHA89HO-W2-51 No: 19)
CAACAAAGCACGCACCCACA (SEQ ID
MUTHA89HO-W2-5 ' No: 20)
Third TCATGGTAGTCAACTCCCTC (SEQ ID time MUTHA89HO-W52-1 No: 21)
CGCTTTCTTTCCTTCGGGCAA (SEQ ID MUTHA89HO-W52-2 No:22)
GCTAATCGTGATCCACAGGC (SEQ ID MUTHA89HO-W32-1 No: 23)
GCCCACGACACTTACCAGA (SEQ ID MUTHA89HO-W32-2 No:24)
Fourth CCCAACTCTATATTTTCAAG (SEQ ID time MUTHA89HO-W53-1 No:25)
ACTTGAAAGTTTGGTTTCGG (SEQ ID
MUTHA89HO-W53-2 No: 26) AGGGAACGGGGCAACATTTG (SEQ I D
MUTHA89HO-W33-1 No:27)
GCCGCCTAACGAGAGACTT (SEQ I D MUTHA89HO-W33-2 No : 28 )
Two microliters of each product that resulted from each PCR was analyzed by agarose gel electrophoresis and DNA concentration was estimated by comparison with the molecular weight marker Low DNA Mass Ladder (Invitrogen) . The
remainder product of the PCR was purified using a Gel
Wizard® SV (Promega) and PCR Clean-Up System (Promega) . The purified PCR products were sequenced using the BigDye®
Terminator v3.1 Cycle Sequencing System (Applied Biosystems) following the manufacturer's instructions.
The files of the sequencing of the oleate desaturase obtained for each amplicon were assembled using CAP3 Sequence Assembly Program (http://pbil.univ- lyonl.fr/cap3.php). The resulting DNA sequences of the oleate desaturase were aligned with the sequences of line
HA89 deposited in GenBank (accession number AY802989) using Clustal W Program version 2.1 (http; // ww.clustal .org) identifying nucleotide insertions present in the mutants 29065 and 29066, which showed to have a size of 785 base pairs (bp) and 4872 bp, respectively. In addition, analysis of the mutated OLD sequences and their corresponding wild type showed that in the case of mutant line 29065 a 785-bp insertion was located at nucleotide position 310 of the coding region of the OLD gene while in the mutant line 29066 insertion of 4872 bp was located at nucleotide position 201. L72K087/16
1/1
PCT
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Figure imgf000019_0001
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Figure imgf000019_0002
Cajide, Maria
FOR INTERNATIONAL BUREAU USE ONLY -5 This form was received by the
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Claims

31 CLAIMS
1. An isolated nucleotide sequence, comprising an insertion and encoding a truncated sunflower oleate
desaturase protein.
2. The sequence according to claim 1, wherein the insertion comprises a premature stop codon.
3. The sequence according to claim 1 or 2, wherein the sequence encodes a truncated sunflower oleate desaturase protein comprising an amino acid sequence selected from the group consisting of SEQ ID No : 1 and SEQ ID No : 2.
4. The sequence according to any one of the claims
1-3, wherein said nucleotide sequence is selected from the group consisting of SEQ ID No : 3 and SEQ ID No : 4.
5. Truncated sunflower oleate desaturase protein comprising at least 110 amino acids of the N-terminal end of wild type oleate desaturase.
6. The protein according to claim 5, comprising an amino acid sequence selected from the group consisting of SEQ ID No:l and SEQ ID No : 2.
7. A sunflower plant, which comprises an oleate desaturase gene that has a nucleotide sequence comprising an insertion and encoding a truncated oleate desaturase
protein.
8. The sunflower plant according to claim 7, wherein the insertion comprises a stop codon.
9. The plant according to claim 7, wherein the nucleotide sequence is selected from the group consisting of SEQ ID No: 3 and SEQ ID No : 4. 32
10. The plant according to claim 7, wherein said plant produces seeds with an oleic acid content between 80% and 95% with respect to the total percentage of fatty acids of the seed.
11. Sunflower plant capable of producing seed having an oleic acid content between 80 and 95% with respect to the total percentage of fatty acids of the seed, which plant is obtainable by crossing a plant of line 29065 that has the accession number NCIMB 41733 or of line 29066 that has the accession number NCIMB 41734 with another plant and selecting in the F2 for plants that produce seed having an oleic acid content between 80 and 95% with respect to the total percentage of fatty acids of the seed.
12. A sunflower seed comprising an oleate desaturase gene comprising a nucleotide sequence that comprises an insertion and encoding a truncated oleate desaturase protein.
13. The seed according to claim 12, wherein the nucleotide sequence is selected from the group consisting of SEQ ID No: 3 and SEQ ID No : 4.
14. The seed according to claim 12, wherein the truncated oleate desaturase protein comprises an amino acid sequence selected from the group consisting of SEQ ID No : 1 and SEQ ID No : 2.
15. The seed according to claim 12, wherein said seed is selected from the group consisting of:
a) line 29065 that has the accession number NCIMB
41733 and
b) line 29066 that has the accession number NCIMB 41734. 33
16. The seed according to claim 12, wherein said seed has an oleic acid content between 80% and 95% with respect to the total fatty acid content of the seed.
17. Sunflower seed having an oleic acid content between 80% and 95% with respect to the total fatty acid content in the seed and obtainable from plants as claimed in any one of the claims 7-11.
18. Sunflower oil, comprising an oleic acid content between 80% and 95% from the total fatty acid content in the oil, which oil is obtainable from sunflower seed as claimed in any one of the claims 12-17.
19. Sunflower oil, characterized in that it has an oleic acid content between 80% and 95% with respect to the total percentage of fatty acids and the oil is obtained from sunflower seed of claim 15.
20. The use of sunflower seed of any one of the claims 12-17 to obtain oil.
21. Use according to claim 20, wherein said seed is selected from the group consisting of:
a) line 29065 that has the accession number NCIMB
41733 and
b) line 29066 that has the accession number NCIMB
41734 22. Progeny of seed as claimed in any one of the claims 12-17, characterized in that it comprises an
insertion in the gene that encodes an oleate desaturase protein, wherein said insertion leads to the synthesis of a truncated oleate desaturase protein.
23. A method for obtaining a sunflower plant with a high oleic acid content, which comprises the following steps : 34 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 that has an insertion comprising a premature stop codon, and wherein said nucleotide sequence encodes a truncated oleate desaturase protein.
24. The method according to claim 23, wherein the step of mutagenesis is performed by injecting a mutagenizing agent in the flower head of the plant.
25. The method according to claim 24, characterized in that it further comprises a step of irradiation of seeds with X-rays.
26. The method according to claim 24, wherein the mutagenizing agent is methanesulfonic acid ethyl ester at a concentration between 5% and 15%.
27. The method according to claim 24, wherein the step of mutagenesis is carried out by irradiating seeds with X-rays .
28. The method according to claim 24, wherein the truncated oleate desaturase protein comprises a sequence of no more than 110 amino acids of the N-terminal end.
29. The method according to claim 28, characterized in that the sequence of the truncated oleate desaturase protein is selected from the group consisting of SEQ ID No : 1 and SEQ ID No : 2.
30. Method for obtaining high oleic sunflower oil, comprising extracting oil from sunflower seeds selected from the group consisting of line 29065 (NCIMB 41733) and line 29066 (NCIMB 41734) .
PCT/EP2011/061165 2011-07-01 2011-07-01 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses WO2013004281A1 (en)

Priority Applications (15)

Application Number Priority Date Filing Date Title
UAA201400941A UA119026C2 (en) 2011-07-01 2011-01-07 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
US13/822,279 US10143149B2 (en) 2011-07-01 2011-07-01 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
BR112013033907A BR112013033907A8 (en) 2011-07-01 2011-07-01 ISOLATED NUCLEOTIDE SEQUENCE, SUNFLOWER TRUNCATED PROTEIN OLEATE DESATURASE, SUNFLOWER PLANT, SUNFLOWER PLANT CAPABLE OF PRODUCING SEEDS, SUNFLOWER SEED, SUNFLOWER OIL, USE OF SUNFLOWER SEED, SEED PROGENIE, METHOD FOR OBTAINING A SUNFLOWER PLANT WITH A HIGH OLEIC ACID CONTENT, AND METHOD FOR OBTAINING HIGH OLEIC SUNFLOWER OIL
CA2837760A CA2837760A1 (en) 2011-07-01 2011-07-01 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
CN201180071926.4A CN103842510A (en) 2011-07-01 2011-07-01 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
RU2014103477A RU2630641C2 (en) 2011-07-01 2011-07-01 Nucleotide sequences, subject to mutation by insert coding truncated protein of oleate-desaturase, proteins, methods and applications
PCT/EP2011/061165 WO2013004281A1 (en) 2011-07-01 2011-07-01 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
EP23199371.8A EP4303306A3 (en) 2011-07-01 2011-07-01 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
EP11729620.2A EP2726616A1 (en) 2011-07-01 2011-07-01 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
AU2011372738A AU2011372738A1 (en) 2011-07-01 2011-07-01 Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
ARP120102374A AR086832A1 (en) 2011-07-01 2012-06-29 SEQUENCES OF NUCLEOTID MUTED BY INSERTION CODING A TRUNCATED OLEATO DESATURASA PROTEIN, PROTEINS, METHODS AND USES
UY0001034184A UY34184A (en) 2011-07-01 2012-07-02 SEQUENCES OF NUCLEOTIDE MUTATES BY INSERTION THAT CODIFY A TRUNCATED OLEATO DESATURASA PROTEIN, PROTEINS, 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
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RU2717642C1 (en) * 2018-12-26 2020-03-24 Автономная некоммерческая образовательная организация высшего образования Сколковский институт науки и технологий Snp-panel for genotyping and genomic selection of sunflower by content of fatty acids in seed oil

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CN103525841A (en) * 2013-10-21 2014-01-22 昆明理工大学 Delta<12>-fatty acid desaturases gene and recombinant expression vector thereof
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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