WO2009061215A1 - Compositions et méthodes de modification de la production de pigment dans des plantes - Google Patents

Compositions et méthodes de modification de la production de pigment dans des plantes Download PDF

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Publication number
WO2009061215A1
WO2009061215A1 PCT/NZ2008/000287 NZ2008000287W WO2009061215A1 WO 2009061215 A1 WO2009061215 A1 WO 2009061215A1 NZ 2008000287 W NZ2008000287 W NZ 2008000287W WO 2009061215 A1 WO2009061215 A1 WO 2009061215A1
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Prior art keywords
polynucleotide
seq
sequence
plant
polypeptide
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PCT/NZ2008/000287
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English (en)
Inventor
Andrew Charles Allan
Richard Victor Espley
Roger Paul Hellens
Kui Lin-Wang
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The New Zealand Institute For Plant And Food Research Limited
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Publication of WO2009061215A1 publication Critical patent/WO2009061215A1/fr

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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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis

Definitions

  • the present invention is in the field of pigment development in plants.
  • anthocyanin pigments in fruit is an important determinant of fruit quality. These pigments provide essential cultivar differentiation for consumers and are implicated in the health attributes.
  • Anthocyanins belong to the diverse group of ubiquitous secondary metabolites, collectively known as flavonoids. In plants, flavonoids are believed to have a variety of functions, including defence and protection against light stress, and the pigmented anthocyanin compounds play an important physiological role as attractants in plant/animal interactions (Harborne and Grayer, 1994; Koes et al. , 1994).
  • cyanidin which, in the form of cyanidin 3-0- galactoside, is the pigment primarily responsible for red colouration in apple skin (Lancaster, 1992; Tsao et al, 2003).
  • major anthocyanins are pelargonidin 3- glucoside (purple) and pelargonidin 3-rutinoside (Hong and Wrolstad, 1990).
  • the control of anthocyanin accumulation in strawberry is a key question in understanding and manipulating fruit colour.
  • Transcription factors may regulate expression of more than one gene in any given biosynthetic pathway and therefore can be useful tools for regulating production from such biosynthetic pathways.
  • the Arabidopsis gene PAPl when overexpressed in transgenic Arabidopsis led to up-regulation of a number of genes in the anthocyanin biosynthesis pathway from PAL to CHS and DFR (Borevitz et al. , 2000, Tohge et al., 2005).
  • sequences derived from strawberry species may be useful to alleviate public concerns about cross-species transformation in the genetic manipulation of anthocyanin production.
  • down-regulation of such a strawberry sequence it may be necessary to transform the plant with a sequence that is identical, or at least highly similar, to the endogenous strawberry sequence.
  • Strawberry sequences may also be useful to provide probes or primers for assessing expression of corresponding endogenous sequences in strawberry species during marker-assisted breeding.
  • FaMYBl gene from strawberry encodes an R2R3 MYB protein which is predominantly expressed in the red ripe strawberry fruit.
  • Heterologous expression in tobacco showed that FaMYBl is a repressor of anthocyanin production, and suggests a role for FaMYBl as a transcriptional regulator of late flavonoid biosynthesis genes (Aharoni et al., 2001 28(3) 319- 332.), rather than anthocyanin generating enzymes.
  • the invention provides an isolated polynucleotide comprising a sequence encoding a polypeptide with the amino acid sequences of SEQ ID NO: 1 or 2 or a variant thereof, wherein the polypeptide or variant thereof is an R2R3 MYB transcription factor that positively regulates anthocyanin production in a plant.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 1 or 2.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 1.
  • the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 2.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
  • the invention provides an isolated polynucleotide comprising a sequence encoding a polypeptide with the amino acid sequences of SEQ ID NO:1 or 2 or a variant thereof, wherein the polypeptide or variant thereof is an R2R3 MYB transcription factor that positively regulates the promoter of a gene in the anthocyanin biosynthetic pathway in a plant.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 1 or 2.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 1.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 2.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
  • the gene in the anthocyanin biosynthetic pathway encodes dihydroflavonol 4-reductase (DFR).
  • the promoter has at least 70% identity to the sequence of SEQ ID NO: 33.
  • the promoter has the sequence of SEQ ID NO: 33.
  • the invention provides an isolated polynucleotide comprising the sequence of any one of the sequences of SEQ ID NO: 3 to 6 or a variant thereof, wherein the polynucleotide or variant thereof encodes an R2R3 MYB transcription factor that positively regulates anthocyanin production in a plant.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 3 to 6.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 3.
  • polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 3.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 4.
  • polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 4.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 5.
  • polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 5.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 6.
  • polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 6.
  • the invention provides an isolated polynucleotide comprising the sequence of any one of the sequences of SEQ ID NO: 3 to 6 or a variant thereof, wherein the polynucleotide or variant thereof encodes an R2R3 MYB transcription factor that positively regulates the promoter of a gene in the anthocyanin biosynthetic pathway in a plant.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 3 to 6.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 3.
  • polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 3.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 4.
  • polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 4.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 5.
  • polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 5.
  • the variant comprises a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 6.
  • polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 6.
  • the gene in the anthocyanin biosynthetic pathway encodes dihydroflavonol 4-reductase (DFR).
  • the invention provides an isolated polypeptide comprising: a) the amino acid sequences of SEQ ID NO: 1 or 2 or a variant thereof, wherein the polypeptide or variant thereof is an R2R3 MYB transcription factor that positively regulates anthocyanin production in a plant; or b) a fragment, of at least 5 amino acids in length, of the sequence of a), capable of performing the same function as the polypeptide in a).
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 1 or 2.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 1.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 2.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
  • the invention provides an isolated polypeptide comprising: a) the amino acid sequences of SEQ ID NO: 1 or 2 or a variant thereof, wherein the polypeptide or variant thereof is an R2R3 MYB transcription factor that positively regulates the promoter of a gene in the anthocyanin biosynthetic pathway in a plant. b) a fragment, of at least 5 amino acids in length, of the sequence of a), capable of performing the same function as the polypeptide in a).
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 1 or 2.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO : 1.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the variant comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 2.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
  • gene in the anthocyanin biosynthetic pathway encodes dihydroflavonol 4-reductase (DFR).
  • the promoter has at least 70% identity to the sequence of SEQ ID NO: 33.
  • the promoter has the sequence of SEQ ID NO: 33.
  • the invention provides a polynucleotide encoding a polypeptide of the invention.
  • the invention provides an antibody raised against a polypeptide of the invention.
  • the invention provides a genetic construct comprising a polynucleotide of any one of the invention.
  • the invention provides a vector comprising a polynucleotide of the invention.
  • the invention provides a vector comprising a genetic construct of the invention.
  • the invention provides a host cell genetically modified to express a polynucleotide of any one of the invention.
  • the invention provides a host cell comprising a genetic construct of the invention.
  • the invention provides a host cell comprising a vector of the invention.
  • the invention provides a plant cell genetically modified to express a polynucleotide of the invention.
  • the invention provides a plant cell or comprising the genetic construct of the invention.
  • the invention provides a plant which comprises the plant cell of the invention.
  • the invention provides a method for producing a polypeptide of the invention, the method comprising the step of culturing a host cell genetically modified to express a polynucleotide of the invention
  • the host cell comprises a genetic construct of the invention.
  • the provides a method for producing a plant cell or plant with altered anthocyanin production, the method comprising the step of transformation of a plant cell or plant with a genetic construct including: a) at least one polynucleotide encoding of a MYB polypeptide of the invention; b) at least one gene encoding of a MYB polypeptide of the invention c) at least one polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a) or b); d) at least one polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of c); or e) at least one polynucleotide capable of hybridising under stringent conditions to the polynucleotide of a) or a gene of b).
  • the method includes the additionall step of transforming the plant with a construct designed to express a bHLH transcription factor, such that the bHLH transcription factor is co-expressed with the MYB polypeptide of the invention
  • the bHLH transcription factor comprises an amino acid sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 7 to 9. In a further embodiment the bHLH transcription factor comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 7.
  • the bHLH transcription factor comprises an amino acid sequence with the sequence of SEQ ID NO: 7.
  • the bHLH transcription factor comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 8.
  • the bHLH transcription factor comprises an amino acid sequence with the sequence of SEQ ID NO: 8.
  • the bHLH transcription factor comprises an amino acid sequence with at least 70% identity to the sequence of SEQ ID NO: 9.
  • the bHLH transcription factor comprises an amino acid sequence with the sequence of SEQ ID NO: 9.
  • Preferred combinations of transcription factors to be co-expressed in the method of the invention include FaMYBlO (SEQ ID NO: 1) with AtBHLHl (SEQ ID NO: 8); FaMYBlO (SEQ ID NO: 1) with AtBHLH2 (SEQ ID NO: 9); FvMYBlO (SEQ ID NO: 2) with MdBHLH33 (SEQ ID NO: 7); FvMYBlO (SEQ ID NO: 2) with AtBHLHl (SEQ ID NO: 8); and FvMYBlO (SEQ ID NO: 2) with AtBHLH2 (SEQ ID NO: 9).
  • Use of variants of each co-expressed sequence is also included in the method of the invention.
  • the invention provides a plant produced by the method of the invention.
  • the invention provides a method for selecting a plant altered in anthocyanin production, the method comprising testing of a plant for altered expression of a polynucleotide of the invention. In a further aspect the invention provides a method for selecting a plant altered in anthocyanin production, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
  • the invention provides a plant selected by the method of the invention.
  • the invention provides a method for selecting a plant cell or plant that has been transformed, the method comprising the steps a) transforming a plant cell or plant with a polynucleotide or polypeptide of the invention capable of regulating anthocyanin production in a plant; b) expressing the polynucleotide or polypeptide in the plant cell or plant; and c) selecting a plant cell or plant with increased anthocyanin pigmentation relative to other plant cells or plants, the increased anthocyanin pigmentation indicating that the plant cell or plant has been transformed.
  • the invention provides a transformed plant selected by the method.
  • the transcription factors and variants of the invention that are capable of regulating anthocyanin production in plants, are capable of regulating the production of the anthocyanins selected from the group including but not limited to: cyanidin-3-glucoside, cyanidin-3-0- rutinoside, cyanadin-3-glucoside and cyanadin-3-pentoside; pelargonidin 3-glucoside and pelargonidin 3-rutinoside.
  • anthocyanins are pelargonidin 3-glucoside and pelargonidin 3-rutinoside.
  • the plants or plant cells with altered production of anthocyanins are altered in production of anthocyanins selected from the group including but not limited to: cyanidin-3-glucoside, cyanidin-3-O-rutinoside, cyanadin- 3-glucoside and cyanadin-3-pentoside; pelargonidin 3-glucoside and pelargonidin 3-rutinoside.
  • anthocyanins are pelargonidin 3-glucoside and pelargonidin 3-rutinoside.
  • the polynucleotides and polynucleotide variants, of the invention may be derived from any species or may be produced by recombinant or synthetic means.
  • polynucleotide or variant is derived from a plant species.
  • polynucleotide or variant is derived from a gymnosperm plant species.
  • polynucleotide or variant is derived from an angiosperm plant species.
  • polynucleotide or variant is derived from a from dicotyledonous plant species.
  • polypeptides and polypeptide variants of the invention may be derived from any species, or may be produced by recombinant or synthetic means.
  • polypeptides or variants of the invention are derived from plant species.
  • polypeptides or variants of the invention are derived from gymnosperm plant species.
  • polypeptides or variants of the invention are derived from angiosperm plant species.
  • polypeptides or variants of the invention are derived from dicotyledonous plant species.
  • polypeptide or variant is derived from a monocotyledonous plant species.
  • the plant cells and plants of the invention may be from any species. In one embodiment the plants cells and plants of the invention are from gymnosperm species.
  • plants cells and plants of the invention are from angiosperm species.
  • plants cells and plants of the invention are from dicotyledonous species.
  • plants cells and plants of the invention are from monocotyledonous species.
  • Preferred plant species include fruit plant species selected from a group comprising but not limited to the following genera: Malus, Pyrus Prunis, Rubus, Rosa, Fragaria, Actinidia, Cydonia, Citrus, and Vaccinium.
  • Particularly preferred fruit plant species are: Malus domestica, Actinidia deliciosa, A. chinensis, A. eriantha, A. arguta and hybrids of the four Actinidia species, Prunis persica, Pyrus communis., Rubus, Rosa, and Fragaria.
  • Preferred plants also include vegetable plant species selected from a group comprising but not limited to the following genera: Brassica, Lycopersicon and Solanum,
  • Particularly preferred vegetable plant species are: Lycopersicon esculentum and Solanum tuberosum
  • Preferred plants also include crop plant species selected from a group comprising but not limited to the following genera: Glycine, Zea, Hordeum and Oryza.
  • Particularly preferred crop plant species include Glycine max, Zea mays and Oryza sativa.
  • Preferred plants also include those of the Rosaceae family.
  • Preferred Rosaceae genera include Exochorda, Maddenia, Oemleria, Osmaronia, Prinsepia, Prunus, Maloideae, Amelanchier, Aria, Aronia, Chaenomeles, Chamaemespilus, Cormus, Cotoneaster, CrataegusOsmaronia, Prinsepia, Prunus, Maloideae, Amelanchier, Aria, Aronia, Chaenomeles, Chamaemespilus, Cormus, Cotoneaster, Crataegu, Cydonia, Dichotomanthes, Docynia, Docyniopsis, Eriobotrya, Eriolobus, Heteromeles, Kageneckia, Lindleya, Malacomeles, Malus, Mespilus, Osteomeles, Peraphy
  • Preferred Rosaceae species include Exochorda giraldii, Exochorda racemosa, Exochorda, Exochorda giraldii, Exochorda racemosa, Exochorda serratifolia, Maddenia hypoleuca, Oemleria cerasiformis, Osmaronia cerasiformis, Prinsepia sinensis, Prinsepia uniflora, Prunus alleghaniensis, Prunus americana, Prunus andersonii, Prunus angustifolia, Prunus apetala, Prunus argentea, Prunus armeniaca, Prunus avium, Prunus bifrons, Prunus brigantina, Prunus bucharica, Prunus buergeriana, Prunus campanulata, Prunus caroliniana, Prunus cerasifera, Prunus cerasus, Prunus choreiana, Prunus cocomili
  • BSP-2004-1 Prunus sp. BSP -2004-2, Prunus sp, EB-2002, Amelanchier alnifolia, Amelanchier arborea, Amelanchier asiatica, Amelanchier bartramiana, Amelanchier canadensis, Amelanchier cusickii, Amelanchier fernaldii, Amelanchier florida, Amelanchier humilis, Amelanchier intermedia, Amelanchier laevis, Amelanchier lucida, Amelanchier nantucketensis, Amelanchier pumila, Amelanchier quinti-martii, Amelanchier sanguinea, Amelanchier stolonifera, Amelanchier utahensis, Amelanchier wiegandii, Amelanchier x neglecta, Amelanchier bartramiana x
  • CFRA 538 Fragaria sp.,Geum andicola, Geum borisi, Geum bulgaricum, Geum calthifolium, Geum chiloense, Geum geniculatum, Geum heterocarpum, Geum macrophyllum, Geum montanum, Geum reptans, Geum rivale, Geum schof ⁇ eldii,Geum speciosum, Geum urbanum, Geum vernum, Geum sp.
  • Rosaceae genera include: Fragaria, Malus, Pyrus, Cydonia, Prunus, Eriobotrya, and Mespilus.
  • Rosaceae species include: Fragaria x ananassa, Fragaria vesca, Malus domestica, Malus sylvestris, Pyrus communis, Pyrus pyrifolia, Pyrus bretschneideri, Cydonia oblonga, Prunus salicina, Prunus cerasifera, Prunus persica, Eriobotrya japonica, Prunus dulcis, Prunus avium, Mespilus germanica and Prunus domestica.
  • a more particularly preferred Rosaceae genera is Fragaria.
  • Preferred Fragaria species include Fragaria daltoniana, Fragaria gracilis, Fragaria grandiflora, Fragaria iinumae, Fragaria moschata, Fragaria nilgerrensis, Fragaria nipponica, Fragaria nubicola, Fragaria orientalis, Fragaria pentaphylla, Fragaria vesca, Fragaria virginiana, Fragaria vi ⁇ dis, Fragaria x ananassa, Fragaria sp. CFRA 538.
  • Fragaria species are Fragaria x ananassa, Fragaria chiloensis and Fragaria vesca.
  • plant is intended to include a whole plant, any part of a plant, propagules and progeny of a plant.
  • 'propagule' means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.
  • the term "positiviely regulates anthocyanin production” means that when a plant expresses, or expresses increased levels of the transciption factor, the result is an increase in anthocyanin production in the plant to a relative suitable control plant.
  • the increased level of expression of the transcription factor may be brought about by genetic manipulation such as transformation with a polynucleotide or genetic construct of the invention. Alternatively the increased expression may be naturally occuring in selected plants from a population.
  • Suitable control plants include plants of the same species or variety that are not genetically modified to increase expression of the transcription factor, such as plants transformed with a control construct, for example an empty vector construct. Other control plants may include other members of the population from which plants with naturally occuring high expression of the transcription factor are selected.
  • polynucleotide(s), means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.
  • a "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, e.g., a sequence that is at least 15 nucleotides in length.
  • the fragments of the invention comprise 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide of the invention.
  • a fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.
  • primer refers to a short polynucleotide, usually having a free 3 'OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.
  • probe refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay.
  • the probe may consist of a "fragment" of a polynucleotide as defined herein.
  • polypeptide encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds.
  • Polypeptides of the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques.
  • the term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.
  • a "fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide.
  • the term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above enzymatic activity.
  • isolated as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment.
  • An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.
  • recombinant refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and/or is recombined with sequences that are not present in its natural context.
  • a "recombinant" polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.
  • polynucleotides or polypeptides of the invention being derived from a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species.
  • the polynucleotide or polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.
  • variant refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides.
  • variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides.
  • variant with reference to polypeptides and polypeptides encompasses all forms of polypeptides and polypeptides as defined herein.
  • Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least
  • Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the invention.
  • Polynucleotide sequence identity can be determined in the following manner.
  • the subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • the default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences.
  • Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. MoI. Biol. 48, 443-453).
  • Needleman- Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. LongdenJ. and Bleasby,A. EMBOSS: The European
  • European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi. ac.uk/emboss/align/.
  • the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
  • Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.
  • variant polynucleotides of the present invention hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions.
  • hybridize under stringent conditions refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration.
  • a target polynucleotide molecule such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot
  • the ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
  • Tm melting temperature
  • Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65 0 C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65 0 C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65 0 C.
  • exemplary stringent hybridization conditions are 5 to 10° C below Tm.
  • Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) 0 C.
  • Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l;26(21):5004-6.
  • Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C below the Tm.
  • Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention.
  • a sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.
  • Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention.
  • a skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al, 1990, Science 247, 1306).
  • Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.
  • variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more
  • Polypeptide sequence identity can be determined in the following manner.
  • the subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/ ' ).
  • BLASTP from the BLAST suite of programs, version 2.2.5 [Nov 2002]
  • bl2seq which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/ ' ).
  • NCBI ftp://ftp.ncbi.nih.gov/blast/ '
  • the default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.
  • Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs.
  • EMBOSS-needle available at http:/www.ebi. ac.uk/emboss/align/
  • GAP Human, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227- 235.
  • suitable global sequence alignment programs for calculating polypeptide sequence identity.
  • a preferred method for calculating polypeptide sequence identity is based on aligning sequences to be compared using Clustal W (Thompson et al 1994, Nucleic Acid Res 11 (22)4673-4680)
  • Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.Rov/blast/).
  • the similarity of polypeptide sequences may be examined using the following unix command line parameters:
  • Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 "6 more preferably less than 1 x 10 "9 , more preferably less than 1 x 10 ⁇ 12 , more preferably less than 1 x 10 "15 , more preferably less than 1 x 10 ⁇ 18 , more preferably less than 1 x 10 "2I , more preferably less than 1 x 10 "30 , more preferably less than 1 x 10 "40 , more preferably less than 1 x 10 ⁇ 0 , more preferably less than 1 x 10 "60 , more preferably less than 1 x 10 "70 , more preferably less than 1 x 10 "80 , more preferably less than 1 x 10 "90 and most preferably 1x10 " 10 ° when compared with any one of the specifically identified sequences.
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
  • the term "genetic construct” refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule.
  • a genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
  • the insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA.
  • the genetic construct may be linked to a vector.
  • vector refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell.
  • the vector may be capable of replication in at least one additional host system, such as E. coli.
  • expression construct refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
  • An expression construct typically comprises in a 5' to 3' direction: a) a promoter functional in the host cell into which the construct will be transformed, b) the polynucleotide to be expressed, and c) a terminator functional in the host cell into which the construct will be transformed.
  • coding region or "open reading frame” (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences.
  • the coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon.
  • a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences.
  • “Operably-linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
  • noncoding region refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency. Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
  • promoter refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
  • transgene is a polynucleotide that is taken from one organism and introduced into a different organism by transformation.
  • the transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
  • a "transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation.
  • the new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.
  • An "inverted repeat” is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,
  • Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
  • regulating anthocyanin production is intended to include both increasing and decreasing anthocyanin production. Preferably the term refers to increasing anthocyanin production.
  • Anthocyanins that may be regulated include but are not limited to cyanindin-3- glucoside, cyaniding-3-O-rutinoside, cyanadin-3-galactoside and cyanadin-3-pentoside.
  • the terms "to alter expression of and “altered expression” of a polynucleotide or polypeptide of the invention are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the invention is modified thus leading to altered expression of a polynucleotide or polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations.
  • the "altered expression” can be related to an increase or decrease in the amount of messenger RNA and/or polypeptide produced and may also result in altered activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.
  • polypeptides SEQ ID NO: 1 and 2 from the strawberry species Fr agar ia x ananassa and Fr agar ia vesca respectively.
  • the polypeptides are both MYB R2R3 transcription factors that positively regulate anthocyanin production in plants.
  • the invention provides fragments and variants of the sequences.
  • the polypeptides share 93% seqeuence identity and are thus variants of each other.
  • the transcription factors also positively regulate the promoters of genes encoding enzymes in the anthocyanin biosynthetic pathway in plants.
  • Table 1 Summary of the relationship between the polynucleotides and polypeptides is found in Table 1 (Summary of Sequences).
  • the invention provides genetic constructs, vectors and plants comprising the polynucleotide sequences.
  • the invention also provides plants comprising the genetic constructs and vectors of the invention.
  • the invention provides plants altered, relative to suitable control plants, in production of anthocyanin pigments.
  • the invention provides both plants with increased and decreased production of anthocyanin pigments.
  • the invention also provides methods for the production of such plants and methods for the selection of such plants.
  • Suitable control plants may include non-transformed plants of the same species and variety, or plants of the same species or variety transformed with a control construct, such as an empty vector construct.
  • compositions of the invention include the production of fruit, or other plant parts, with increased levels of anthocyanin pigmentation, for example production of apples with red skin and or red flesh.
  • the invention also provides methods for selecting transformed plant cells and plants by selecting plant cells and plants which have increased anthocyanin pigment, the increased anthocyanic pigment indicating that the plants are transformed to express a polynucleotide or polypeptide of the invention.
  • polypeptides of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art.
  • such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al, Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference.
  • PCR polymerase chain reaction
  • the polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
  • hybridization probes include use of all, or portions of, the polypeptides having the sequence set forth herein as hybridization probes.
  • Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65°C in 5. 0 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution; washing (three washes of twenty minutes each at 55°C) in 1.
  • An optional further wash (for twenty minutes) can be conducted under conditions of 0. 1 X SSC, 1% (w/v) sodium dodecyl sulfate, at 60°C.
  • polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR amplification.
  • a partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5'RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56) and hybridization- based method, computer/database -based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al, 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene.
  • the fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Divergent primers are designed from the known region.
  • standard molecular biology approaches can be utilized (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
  • transgenic plant from a particular species, it may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species.
  • the benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms.
  • down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species. Variants (including orthologues) may be identified by the methods described.
  • Variant polypeptides may be identified using PCR-based methods (Mullis et al, Eds. 1994 The Polymerase Chain Reaction, Birkhauser).
  • the polynucleotide sequence of a primer useful to amplify variants of polynucleotide molecules of the invention by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.
  • polypeptide variants may also be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.
  • variant sequences of the invention may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.
  • An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast ⁇ or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38 A, Room 8N805, Bethesda, MD 20894 USA.
  • NCBI National Center for Biotechnology Information
  • the NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases.
  • BLASTN compares a nucleotide query sequence against a nucleotide sequence database.
  • BLASTP compares an amino acid query sequence against a protein sequence database.
  • BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database.
  • tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames.
  • tBLASTX compares the six- frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
  • BLAST family of algorithms including BLASTN, BLASTP, and BLASTX
  • BLASTN, BLASTP, and BLASTX The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences.
  • the hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
  • the BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments.
  • the Expect value (E) indicates the number of hits one can "expect” to see by chance when searching a database of the same size containing random contiguous sequences.
  • the Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
  • the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
  • Pattern recognition software applications are available for finding motifs or signature sequences.
  • MEME Multiple Em for Motif Elicitation
  • MAST Motif Alignment and Search Tool
  • the MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found.
  • MEME and MAST were developed at the University of California, San Diego.
  • PROSITE Boiroch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences.
  • PROSITE database www.expasy.org/prosite
  • the PROSITE database contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235).
  • Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.
  • a variant polynucleotide of the invention as encoding a transcription factor capable of regulating pigment production in a plant transcription factors can be tested for this ability to regulate expression of known anthocyanin biosynthesis genes (e.g. Example 4) or can be tested for their capability to regulate pigment production (e.g. Examples 5 and 6).
  • polypeptides of the invention may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 43 IA Peptide Synthesizer (Foster City, California). Mutated forms of the polypeptides may also be produced during such syntheses.
  • peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 43 IA Peptide Synthesizer (Foster City, California). Mutated forms of the polypeptides may also be produced during such syntheses.
  • polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification,).
  • polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
  • the genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynycleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms.
  • the genetic constructs of the invention are intended to include expression constructs as herein defined.
  • the invention provides a host cell which comprises a genetic construct or vector of the invention.
  • Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.
  • Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et ah, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et ah, Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention.
  • Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention.
  • the expressed recombinant polypeptide which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, VoI 182, Guide to Protein Purification).
  • Methods for producing plant cells and plants comprising constructs and vectors are useful in methods well known in the art (e.g. Sambrook et ah,
  • the invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention. Plants comprising such cells also form an aspect of the invention.
  • Production of plants altered in pigment production may be achieved through methods of the invention.
  • Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide capable of regulating pigment production in such plant cells and plants.
  • Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polypeptides or polypeptides capable of regulating pigment production in such plant cells and plants.
  • a number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant MoI Biol, 48, 297).
  • strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed.
  • the expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species. Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.
  • Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detest presence of the genetic construct in the transformed plant.
  • the promoters suitable for use in the constructs of this invention are functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired.
  • the promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi.
  • promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention.
  • constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is herein incorporated by reference.
  • Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.
  • CaMV cauliflower mosaic virus
  • Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators the Zea mays zein gene terminator
  • the Oryza sativa ADP-glucose pyrophosphorylase terminator the Solarium tuberosum PI-II terminator.
  • NPT II neomycin phophotransferase II gene
  • aadA gene which confers spectinomycin and streptomycin resistance
  • phosphinothricin acetyl transferase ⁇ bar gene for Ignite (AgrEvo) and Basta (Hoechst) resistance
  • hpt hygromycin phosphotransferase gene
  • reporter genes coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated.
  • a visible signal e.g., luciferase, GUS, GFP
  • the reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.
  • Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. "Regulatory elements” is used here in the widest possible sense and includes other genes which interact with the gene of interest.
  • Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide of the invention may include an antisense copy of a polynucleotide of the invention. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.
  • an “antisense” polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,
  • Genetic constructs designed for gene silencing may also include an inverted repeat.
  • An 'inverted repeat' is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,
  • the transcript formed may undergo complementary base pairing to form a hairpin structure.
  • a spacer of at least 3-5 bp between the repeated region is required to allow hairpin formation.
  • Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al, 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.
  • genetic construct as used herein also includes small antisense RNAs and other such polypeptides effecting gene silencing.
  • Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347).
  • sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5' or 3' untranslated region (UTR).
  • Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al, 2002, Plant Physiol. 128(3): 844-53; Jones et al, 1998, Planta 204: 499-505).
  • the use of such sense suppression strategies to silence the expression of a polynucleotide of the invention is also contemplated.
  • the polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5' or 3' UTR sequence, or the corresponding gene.
  • Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.
  • soybean US Patent Nos. 5, 416, 011 ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830); pineapple (US Patent Serial No. 5, 952, 543); poplar (US Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871); cereals (US Patent No. 6, 074, 877); pear (Matsuda et al., 2005, Plant Cell Rep.
  • Prunus Prunus (Ramesh et al., 2006, Plant Cell Rep. 25(8):821-8; Song and Sink 2005, Plant Cell Rep. 2006; 25(2):117-23; Gonzalez Padilla et al., 2003, Plant Cell Rep. 22(l):38-45); strawberry (Oosumi et al., 2006, Planta.; 223(6):1219-30; Folta et al., 2006, Planta. 2006 Apr 14; PMID: 16614818), rose (Li et al., 2003, Planta. 218(2):226-32), and Rubus (Graham et al., 1995, Methods MoI Biol. 1995;44: 129-33). Transformation of other species is also contemplated by the invention. Suitable methods and protocols for transformation of other species are available in the scientific literature.
  • nucleotide and/or polypeptide of the invention may be employed to alter expression of a nucleotide and/or polypeptide of the invention. Such methods include but are not limited to Tilling (Till et al, 2003, Methods MoI Biol, 2%, 205), so called “Deletagene” technology (Li et al, 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors, (e.g. Jouvenot et al, 2003, Gene Therapy 10, 513). Additionally antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al, 2003, Nat. Biotechnol., 21(1), 35).
  • Transposon tagging approaches may also be applied.
  • peptides interacting with a polypeptide of the invention may be identified through technologies such as phase-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a polypeptide of the invention.
  • Use of each of the above approaches in alteration of expression of a nucleotide and/or polypeptide of the invention is specifically contemplated.
  • Methods are also provided for selecting plants with altered pigment production. Such methods involve testing of plants for altered for the expression of a polynucleotide or polypeptide of the invention. Such methods may be applied at a young age or early developmental stage when the altered pigment production may not necessarily be visible, to accelerate breeding programs directed toward improving anthocyanin content.
  • a polynucleotide such as a messenger RNA
  • exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
  • Polynucleotides or portions of the polynucleotides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with altered levels of anthocyanin.
  • the polypeptides of the invention may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.
  • antibodies may be raised against polypeptides of the invention.
  • Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998).
  • Such antibodies may be used in methods to detect altered expression of polypeptides which modulate flower size in plants.
  • Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).
  • the plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.
  • Figure 1 shows an alignment of the strawberry MYBlO protein sequences with other MYB sequences involved in anthocyanin production.
  • Figure 2 shows a bootstrap phylogenetic analysis of the strawberry MYBlO protein sequences with other MYB sequences involved in anthocyanin production.
  • Figure 3 shows % identity of strawberry MYBlO proteins with other anthocyanin related MYB transcription factors.
  • FIG. 4 shows Strawberry and other Rosaceous MYB genes in phylogeny with MYB genes from other species.
  • Strawberry MYBlO (arrowed: FaMYBlO and FvMYBlO) clusters with other Rosaceous MYBlOs, and genes from other species involved in regulating anthocyanin biosynthesis (red dots).
  • Subgroup numbers are those described by Stracke et al, (2001) and are shown as a suffix after most MYB descriptors.
  • Arabidopsis genes identified by Arabidopsis unique identifiers.
  • the accession numbers for other species of genes, or translated products, in the GenBank database are as follows: AmROSEAl [ABB83826], AmROSEA2 [ABB83827],
  • AmVENOSA [ABB83828], MdMYBlO [DQ267896], VvMYBAl [AB242302], VvMYBA2
  • Figure 5 shows qPCR analysis of the expression of FvMYBlO and FaMYBlO gene transcripts in the developmental series from both wild and cultivated strawberry, reveals large increases in the relative transcript levels of this transcription factor in the fruit tissues of both species (A). In Fragaria x ananassa transcript levels were barely detectable, until fruit were full size (B), and upon ripening and colour change there was an almost 40,000 fold increase in relative transcript level. Expression levels of FvMYBlO in Fragaria vesca are less dramatic and follow colour change; wild strawberry has an earlier colour change, which occurs only in the skin (C). Under stressful conditions (high light) the petals of Fragaria vesca became pigmented (D). Transcript of FvMYBlO, from this tissue, shows the largest increase over non-pigmented petals. This is further evidence of MYBlO's involvement in driving strawberry colour.
  • FIG. 6 shows trans-activation assays where the strawberry MYB (FaMYBlO and FvMYBlO) genes were infiltrated into N. benthamiana leaves either with or without the apple or Arabidopsis BHLH genes. Trans-activation was measured using the AtDRF-LUC reporter cassette.
  • Figure 7 shows that transformation of strawberry with 35S:FaMYB10 elevates anthocyanin synthesis. Visible reddening' is seen in leaves (A; WT on right) and roots (B; WT in right). The extracted pigment is anthocyanin, and is increased from 80 mg per g leaf to 160 mg per g in some lines (C).
  • Example 1 Isolation and characterisation of R2R3 MYB transcription factors of the invention.
  • Messenger RNA mRNA was isolated from this tissue by standard techniques, and subjected to 3' and 5' RACE (GeneRacer, Invitrogen).
  • Degenerate primers (SEQ ID NO 17) were used as shown in Table 2 below (with a 32 fold degeneracy) designed to the consensus DNA sequence of the R2R3 DNA binding domain based on the sequence of anthocyanin regulators in diverse species. Numerous cDNAs encoding R2R3 MYB domains were obtained. The applicants identified two sequences that they designated FaMYBlO (from Fragaria x ananassa) and FvMYBlO (from Fragaria vesca) as candidate sequences encoding transcription factors that modulate anthocyanin production. The complete sequences for FvMYBlO and FaMYBlO cDNA were compiled from overlapping fragments. Then full length clones (both genomic and cDNA) isolated using gene specific primers (SEQ ID NO 24 & 25) designed to the 5' and 3' UTR regions.
  • FvMYBlO and FaMYBlO are related to the Arabidopsis subgroup 10 MYBs, including PAPl.
  • the sequences show only 40% identity to the entire PAPl protein but show 68% amino acid identity with the R2R3 DNA binding domain of PAPl but.
  • the two strawberry sequences were used to search the NCBI EST data base using TBLASTX (Nucleotide query - Translated db [tblastx]" is useful for identifying novel genes in error prone query sequence) (http://www.ncbi.nlm.nih.gov/blast/ ) on HortResearch's proprietory database.
  • a bootstrapped circular phylogenic tree shown in Figure 4 was generated using MEGA version 3.1 (Kumar et al, 2004) and shows that FaMYBlO FvMYBlO in the same clade as Arabidopsis PAPl, PAP2, AtMYBl 13 and AtMYBlU.
  • Example 2 In vivo expression of the R2R3 MYB transcription factors of the invention correlates strongly with anthocyanin levels in strawberry fruit and flowers.
  • Strawberry tissue was collected at 6 time points during fruit development: stage 1, pre-opened bud; stage 2, fully open flower; stage 3, petal drop; stage 4, expanding fruitlet; stage 5, expanded fruit and stage 6, red-ripe fruit, from plants grown under glasshouse conditions in full potting mix, using natural light with daylight extension to 16 h.
  • Fragaria vesca was grown under constant lighting, a treatment that induced red pigmented petals.
  • RNA was isolated (adapted from Chang et ai, 1993) from the tissue or fruit (six samples from the same plant, skin and cortex combined). First strand cDNA synthesis was carried out using oligo dT according to the manufacturers instructions (Transcriptor, Roche Diagnostics, Mannheim, Germany).
  • Reactions were performed in triplicate using 2 ⁇ l 5x Master Mix, 0.5 ⁇ M each primer, 1 ⁇ l diluted cDNA and nuclease-free water (Roche Diagnostics) to a final volume of 10 ⁇ l. A negative water control was included in each run. Fluorescence was measured at the end of each annealing step. Amplification was followed by a melting curve analysis with continual fluorescence data acquisition during the 65°C to 95°C melt.
  • the raw data was analysed with the LightCycler software version 4 and expression was normalised to strawberry actin Fragaria x ananassa (genebank number; BAC81670) to minimise variation in cDNA template levels, with the pre-opened bud (stage 1) sample acting as calibrator with a nominal value of 1.
  • stage 1 For each gene a standard curve was generated with a cDNA serial dilution and the resultant PCR efficiency calculations (ranging between 1.700 and 1.945) were imported into relative expression data analysis.
  • qPCR amplicons were sequenced and confirmed as the expected plant DNA sequences. Error bars shown in qPCR data are technical replicates, the means ⁇ S.E. of 3 replicate qPCR reactions.
  • Example 3 Induction of the promoter of an anthocyanin biosynthetic gene by over- expression of the R2R3 MYB transcription factors of the invention in plants.
  • the promoter sequence for Arabidopsis DFR was inserted into the cloning site of pGreen 0800-LUC (Hellens et al, 2005) and modified to introduce an JVC ⁇ I site at the 3' end of the sequence, * allowing the promoter to be cloned as a transcriptional fusion with the firefly luciferase gene (LUC).
  • TFs that bind the promoter and increase the rate of transcription could be identified as an increase in luminescence activity.
  • Arabidopsis DFR (7Ti, AT5g42800) was isolated from genomic Arabidopsis DNA.
  • a luciferase gene from Renilla (REN) under the control of a 35S promoter, provided an estimate of the extent of transient expression.
  • Activity is expressed as a ratio of LUC to REN activity so that where the interaction between a TF (+/- bHLH) and the promoter occurred, a significant increase in the LUC activity relative to REN would be observed.
  • the promoter-LUC fusion in pGreenll 0800-LUC was used in transient transformation by mixing 100 ⁇ l of Agrobacterium strain GV3101 (MP90) transformed with the reporter cassette with two other Agrobacterium cultures (450 ⁇ l each) transformed with cassettes containing a MYB TF gene fused to the 35S promoter and a bHLH TF gene in either pART27 (Gleave, 1992) or pGreenll 62-SK binary vectors (Hellens et al, 2000). Nicotiana benthamiana 'plants were grown under glasshouse conditions in full potting mix, using natural light with daylight extension to 16 h, until at least 6 leaves were available for infiltration with Agrobacterium.
  • Plants were maintained in the glasshouse for the duration of the experiment.
  • Agrobacterium was cultured on Lennox agar (Invitrogen) supplemented with selection antibiotics and incubated at 28 0 C.
  • a 10 ⁇ l loop of confluent bacterium were re- suspended in 10 ml of infiltration media (10 mM MgCl 2 , 0.5 ⁇ M acetosyringone), to an OD 6O0 of 0.2, and incubated at room temperature without shaking for 2 h before infiltration.
  • Infiltrations were performed according to the methods of Voinnet et al, (2003).
  • Approximately 150 ⁇ l of this Agrobacterium mixture was infiltrated at six points into a young leaf of N. benthamiana and transient expression was assayed 3 days after inoculation.
  • Firefly luciferase and renilla luciferase were assayed using the dual luciferase assay reagents (Promega, Madison, WI). Three days after inoculation, 2 cm leaf discs (6 technical replicates from each plant) were removed and ground in 500 ⁇ l of passive lysis buffer (PLB). Ten ⁇ l of a 1/100 dilution of this crude extract was assayed in 40 ⁇ l of luciferase assay buffer, and the chemiluminescence measured. 40 ⁇ l of Stop and GlowTM buffer was then added and a second chemiluminescence measurement made. Absolute relative luminescence units (RLU) were measured in a Turner 20/20 luminometer (Turner BioSystems, Sunnyvale, CA), with a 5 s delay and 15 s integrated measurement.
  • RLU Absolute relative luminescence units
  • the dual luciferase system has been demonstrated to provide a rapid method of transient gene expression analysis (Matsuo et al, 2001, Hellens et al, 2005). It requires no selectable marker and results can be quantified with a simple enzymatic assay.
  • Nicotiana benthamiana was used to test the interaction of our candidate TFs with an Arabidopsis anthocyanin biosynthesis gene promoter AtDFR (7TJ, AT5g42800). This is known to be regulated by Arabidopsis PAPl and PAP2 MYB TFs (Zimmermann et al, 2004, Tohge et al, 2005).
  • FvMYBlO and FaMYBlO have the amino acid residues that specify interaction with bHLHs (Grotewold et al., 2000, Zimmermann et al, 2004), transient assays were performed in the presence or absence of known bHLH cofactors. These were of the IHf clade of bHLH (Heim et al, 2003) that has been shown to be involved in the regulation of anthocyanin biosynthesis, from apple MdbHLH3 (CN934367), and MdbHLH33 (DQ266451) and Arabidopsis AtbHLHl (At5g41315) and AtbHLH2 (Atlg63650).
  • FaMYBlO promoter activity with FaMYBlO was enhanced by both AtbHLHl and A ⁇ HLH2.
  • Example 4 Induction of anthocyanin biosynthesis by over-expression of the R2R3 MYB transcription factors of the invention in plants.
  • Seeds of Fragaria ananassa were surface sterilized in 20 %bleach (0.8% sodium hypochlorite) with 1 drop of Tween-20 for 20 min followed by three rinses in sterile water. Seeds were germinated on 1/2 MS basal salt and vitamins (Duchefa) + 3% sucrose + 0.7% agar (Germantown) (pH5.7) medium. Seedlings were subcultured onto fresh medium every four weeks.
  • Agrobacterim tumefaciens strain EHAl 05 (Hood et al. 1993), harbouring the binary plasmd pGreen II 0029 62-sk (Hellens et al. 2000), was used in transformation experiments.
  • the pGreen II 0029 62-sk vector contains the NOS/NPT II for kanmycin resistance, and a CaMV 35s promoter-driven full length FaMYBlO cDNA.
  • Agrobacterium culture was grown in 30 ml of LB (Invitrogen) + 50mg/L Kanamycin + 25mg/L Rifampicin broth overnight at 28°C in an incubator-shaker. Overnight culture was centrifuged at 4500 rpm for 10 min and discarded supernatant, wash with liquid MS + 2% sucrose medium once, and then the pellet was resuspended in 20ml of liquid MS + 2% sucrose medium. Transformation of Fragaria ananassa
  • Inoculated leaf strips were transferred onto co-cultivation medium MS basal salts and vitamins (Duchefa) + 3% sucrose + 0.7% phytagel (Sigma) + 3mg/L BAP (6- benzylaminopurine) + 0.2mg/L IBA (3-indolebutyric acid) + 100 ⁇ M Acetosyringone, and incubated at 25°C for 2 days with 16h photoperiod.
  • the leaf strips were transferred onto regeneration and selection medium MS basal salts and vitamins (Duchefa) + 3% sucrose + 0.7% agar (Germantown) + 3mg/L BAP + 0.2mg/L IBA + 300mg/L Timentin + 150mg/L Kanamycin.
  • the leaf explants were subcultured onto fresh regeneration and selection medium every three weeks.
  • Adventitious buds were initiated from the calli formed on the leaf strips at two to three months post the inoculation.
  • Transformation of strawberry with 35S:FaMYB10 elevates anthocyanin synthesis. Visible reddening was seen in leaves (A; WT on right) and roots (B; WT in right). Extracted pigment was anthocyanin and increased to from 80 mg per g leaf to 160 mg per g in some lines (C).
  • Flavonoids Advances in Research Since 1986. Chapman & Hall, London, p 589-618 Heim, M.A., Jakoby, M., mostr, M., Martin, C, Bailey, P.C. and Weisshaar, B. (2003) The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity.
  • the TT8 gene encodes a Basic Helix-Loop-Helix domain protein required for expression of
  • TESTA GLABRAl locus which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell, 11, 1337-1350

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Abstract

La présente invention concerne des polynucléotides qui codent de nouveaux facteurs de transcription et les facteurs de transcription codés qui sont capables de réguler la production d'anthocyanine dans des plantes. Cette invention porte également sur des constructions comprenant les polynucléotides, et sur les cellules hôtes, les cellules de plante et les plantes transformées avec les polynucléotides, les constructions et les vecteurs. Cette invention concerne également des méthodes de production de plantes dans lesquelles la production d'anthocyanine est modifiée et des plantes produites au moyen desdites méthodes.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104059920A (zh) * 2014-07-11 2014-09-24 西南大学 桑树二氢黄酮醇还原酶启动子及其重组表达载体和应用
CN105463128A (zh) * 2014-10-30 2016-04-06 四川省农业科学院园艺研究所 一种草莓病毒的定量检测方法
CN106939038A (zh) * 2016-01-04 2017-07-11 深圳市农科集团有限公司 一种玉米发育调控蛋白、编码基因及应用
CN107164391A (zh) * 2017-06-30 2017-09-15 沈阳农业大学 一种草莓开花基因FvbHLH78及其应用
CN108588092A (zh) * 2018-07-13 2018-09-28 四川农业大学 一种梨花色素苷合成转录因子PbMYB109及其应用
CN110106189A (zh) * 2019-05-31 2019-08-09 福建省农业科学院果树研究所 VbMYB基因及其编码蛋白与应用
CN112626084A (zh) * 2020-12-31 2021-04-09 安徽农业大学 草莓MYB转录因子FvMYB24基因、表达蛋白及应用
IT202100000785A1 (it) 2021-01-18 2022-07-18 Consiglio Nazionale Ricerche Metodo di editazione genetica per incrementare il contenuto di antocianine in cellule di patata

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WO2007027105A2 (fr) * 2005-08-30 2007-03-08 The Horticulture And Food Research Institute Of New Zealand Limited Compositions et procedes servant a moduler la production de pigments dans les vegetaux

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104059920A (zh) * 2014-07-11 2014-09-24 西南大学 桑树二氢黄酮醇还原酶启动子及其重组表达载体和应用
CN105463128A (zh) * 2014-10-30 2016-04-06 四川省农业科学院园艺研究所 一种草莓病毒的定量检测方法
CN106939038A (zh) * 2016-01-04 2017-07-11 深圳市农科集团有限公司 一种玉米发育调控蛋白、编码基因及应用
CN106939038B (zh) * 2016-01-04 2020-09-08 深圳市农科集团有限公司 一种玉米发育调控蛋白、编码基因及应用
CN107164391A (zh) * 2017-06-30 2017-09-15 沈阳农业大学 一种草莓开花基因FvbHLH78及其应用
CN108588092A (zh) * 2018-07-13 2018-09-28 四川农业大学 一种梨花色素苷合成转录因子PbMYB109及其应用
CN108588092B (zh) * 2018-07-13 2021-06-29 四川农业大学 一种梨花色素苷合成转录因子PbMYB109及其应用
CN110106189A (zh) * 2019-05-31 2019-08-09 福建省农业科学院果树研究所 VbMYB基因及其编码蛋白与应用
CN112626084A (zh) * 2020-12-31 2021-04-09 安徽农业大学 草莓MYB转录因子FvMYB24基因、表达蛋白及应用
CN112626084B (zh) * 2020-12-31 2022-03-29 安徽农业大学 草莓MYB转录因子FvMYB24基因、表达蛋白及应用
IT202100000785A1 (it) 2021-01-18 2022-07-18 Consiglio Nazionale Ricerche Metodo di editazione genetica per incrementare il contenuto di antocianine in cellule di patata

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