WO2008140334A1 - Compositions and methods for regulating plant gene expression - Google Patents

Compositions and methods for regulating plant gene expression Download PDF

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Publication number
WO2008140334A1
WO2008140334A1 PCT/NZ2008/000104 NZ2008000104W WO2008140334A1 WO 2008140334 A1 WO2008140334 A1 WO 2008140334A1 NZ 2008000104 W NZ2008000104 W NZ 2008000104W WO 2008140334 A1 WO2008140334 A1 WO 2008140334A1
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Prior art keywords
sequence
polynucleotide
promoter
plant
promoter polynucleotide
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PCT/NZ2008/000104
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French (fr)
Inventor
Richard Espley
Roger P Hellens
Andrew C Allan
David Chagne
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Richard Espley
Roger P Hellens
Andrew C Allan
David Chagne
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Publication of WO2008140334A1 publication Critical patent/WO2008140334A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation

Definitions

  • the present invention relates to promoter polynucleotides for regulating gene expression in plants, and uses thereof.
  • polynucleotide of interest it is often desirable to control expression of a polynucleotide of interest, in a particular tissue, at a particular developmental stage, or under particular conditions, in which the polynucleotide is not normally expressed.
  • the polynucleotide of interest may encode a protein or alternatively may be intended to effect silencing of a corresponding target gene.
  • Plant promoter sequences are useful in genetic manipulation for directing expression of polynucleotides in transgenic plants.
  • a genetic construct is often introduced into a plant cell or plant.
  • Such constructs include a plant promoter operably linked to the polynucleotide sequence of interest.
  • Such a promoter need not normally be associated with the gene of interest.
  • the promoter controls expression of the operably linked polynucleotide of interest thus leading to the desired transgene expression and resulting desired phenotypic characteristics in the plant.
  • Promoters used in genetic manipulation are typically derived from the 5' un-transcribed region of genes and contain regulatory elements that are necessary to control expression of the operably linked polynucleotide. Promoters useful for plant biotechnology can be classified depending on when and where they direct expression. For example promoters may be tissue specific or constitutive (capable of transcribing sequences in multiple tissues). Other classes of promoters include inducible promoters that can be triggered by external stimuli such as environmental, and chemical stimuli.
  • a relatively high level of expression of the transformed sequence of interest is desirable. This is often achieved through use of viral promoter sequences such as the Cauliflower Mosaic Virus 35S promoter. In some circumstances it may be more preferable to use a plant derived promoter rather than a promoter derived from a microorganism. It may also be preferable in some circumstances to use a promoter derived from the species to be transformed.
  • viral promoter sequences such as the Cauliflower Mosaic Virus 35S promoter.
  • the apple (Malus species) is a major fruit species grown in New Zealand and other temperate climates throughout the world. Valuable traits that may be improved by genetic manipulation of apple include: fruit flavour, fruit colour, content of health promoting components (such as anthocyanins and flavanoids) in fruit, stress tolerance/resistance, pest tolerance/resistance and disease tolerance/resistance. Genetic manipulation of such traits in apple, and other plant species, is limited by the availability of promoters capable of appropriately controlling the expression of genes of interest.
  • the invention provides an isolated promoter polynucleotide comprising at least two sequence motifs with at least 70% identity to the sequence SEQ ID NO: 1, wherein the promoter polynucleotide is capable of controlling transcription of an operably linked polynucleotide in a plant.
  • the promoter polynucleotide comprises at least three of the sequence motifs. In a further embodient the promoter polynucleotide comprises at least four of the sequence motifs.
  • the promoter polynucleotide comprises at least five of the sequence motifs.
  • the promoter polynucleotide comprises six of the sequence motifs.
  • At least one of the motifs is interupted by at least one of the other sequence motifs.
  • At least one of the motifs is interupted by at least two other sequence motifs.
  • the promoter polynucleotide comprises six of the sequence motifs and one of the motifs is interupted by two of the other sequence motifs.
  • sequence motif has 80% identity with the sequence of SEQ ID NO: 1.
  • sequence motif has 90% identity with the sequence of SEQ ID NO: 1.
  • sequence motif has 91% identity with the sequence of SEQ ID NO: 1.
  • sequence motif has 95% identity with the sequence of SEQ ID NO: 1.
  • sequence motif has the sequence of SEQ ID NO: 1.
  • the promoter polynucleotide comprises a sequence element with the at least 70% identity to the sequence of SEQ ID NO: 2. In a further embodiment the promoter polynucleotide comprises a sequence element with the sequence of SEQ ID NO: 2.
  • the promoter polynucleotide also comprises a microsatellite sequence element with at least 70% identity to the sequence of SEQ ID NO: 3.
  • the promoter polynucleotide also comprises a microsatellite sequence element with the sequence of SEQ ID NO: 3.
  • the promoter polynucleotide also comprises a region with at least 70% identity to the sequence of SEQ ID NO: 4.
  • the promoter polynucleotide comprises a region with the sequence of SEQ ID NO: 4.
  • the promoter polynucleotide also comprises a region with at least 70% identity to the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide comprises a region with the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide also comprises at least 66 contiguous polynucleotides of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide is a naturally occuring sequence found in a solanaceous species.
  • solanaceous species is from the genus Malus.
  • solanaceous species is Malus domestica.
  • the promoter polynucleotide comprises at least 140 bases of the sequence of SEQ ID NO: 5. In a further embodiment the promoter polynucleotide comprises at least 200 bases of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide comprises at least about 462 bases of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide comprises at least 500 bases of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide comprises at least 505 bases of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide comprises at least 750 bases of the sequence of SEQ ID NO: 5. . .. .
  • the promoter polynucleotide comprises at least 934 bases of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide comprises at least 1000 bases of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide comprises at least 1500 bases of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide comprises at least 1801 bases of the sequence of SEQ ID NO: 5.
  • the promoter polynucleotide is modulated by a MYB transcription factor.
  • the promoter polynucleotide is positively modulated, activated, or up- regulated, by the MYB transcription factor.
  • the MYB transcription factor comprises an R2R3 DNA binding domain.
  • the MYB transcription factor comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6.
  • the MYB transcription factor comprises the sequence of SEQ ID NO: 6.
  • the MYB transcription factor is encoded by a polynucleotide with at least 70% identity to the sequence of SEQ ID NO: 7.
  • the MYB transcription factor is encoded by a polynucleotide with the sequence of SEQ ID NO: 7.
  • the promoter polynucleotide is up-regulated by by the gene product of the gene with which the promoter polynucleotide is endogenously associated.
  • the promoter polynucleotide is a promoter of an autoregulated gene, where expression of the gene product up-regulates the promoter leading to further gene product expression.
  • the promoter polynucleotide is endogenously associated with the MYB transcription factor in naturally occuring plants, and the promoter is autoregulated by the MYB transcription factor.
  • the promoter polynucleotide is capable of controlling transcription of an operably linked polynucleotide sequence constitutively in substantially all tissues of a plant.
  • the promoter polynucleotide is capable of controlling transcription of an operably linked polynucleotide sequence in any plant, plant cell, or plant tissue in which the MYB transcription factor is expressed.
  • the MYB transcription factor may be naturally expressed in the plant or may be expressed in the plant through genetic manipulation of the plant.
  • the invention provides a genetic construct comprising a promoter polynucleotide of the invention.
  • the promoter polynucleotide is operably linked to a polynucleotide sequence to be expressed.
  • the invention provides a vector comprising a genetic construct of the invention.
  • the invention provides a host cell transformed with the promoter polynucleotide of the invention.
  • the invention provides a plant cell or plant transformed with the promoter polynucleotide of the invention.
  • the invention provides a plant cell or plant transformed with a genetic 15 construct of the invention.
  • the plant cell or plant is also transformed with a polynucleotide or genetic construct for expresssing a MYB transcription factor that modulates expression of the promoter polynucleotide of the invention. >0
  • the plant cell or plant naturally expresses the MYB transcription factor.
  • the MYB transcription factor comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6. >5
  • the MYB transcription factor comprises the sequence of SEQ ID NO: 6.
  • the invention provides a method for producing a plant cell or plant with modifed expression of at least one polynucleotide, the method comprising transformation of the 50 plant cell or plant with a promoter polynucleotide of the invention
  • the plant cell or plant is transformed with a genetic construct of the invention.
  • the plant cell or plant is also transformed with a polynucleotide or genetic construct capable of expresssing a MYB transcription factor that modulates expression of the promoter polynucleotide of the invention.
  • the plant cell or plant naturally expresses the MYB transcription factor.
  • the MYB transcription factor comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6.
  • the MYB transcription factor comprises the sequence of SEQ ID NO: 6.
  • the promoter polynucleotide of the invention may be transformed into the plant to control expression of a polynucleotide that is operably linked to the promoter prior to transformation.
  • the promoter polynucleotide may be transformed into the genome of the plant without an operably linked polynucleotide, but the promoter may control expression of an endogenous polynucleotide, typically adjacent to the insert site, and typically, to the 3' end of the inserted promoter polynucleotide.
  • a further aspect of the invention provides a method for producing a plant cell or plant with a modified phenotype, the method comprising the stable incorporation into the genome of the plant, a promoter polynucleotide of the invention
  • the plant cell or plant is transformed within a genetic construct of the invention.
  • the plant cell or plant is also transformed with a genetic construct for expresssing a MYB transcription factor that modulates expression of the promoter )0 polynucleotide of the invention.
  • the plant cell or plant naturally expresses the MYB transcription factor.
  • the MYB transcription factor comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6.
  • the MYB transcription factor comprises the sequence of SEQ ID NO: 6.
  • the invention provides a plant cell or plant produced by a method of the invention.
  • the invention provides a seed, propagule, progeny or part of a plant, of the invention.
  • the seed, propagule, progeny or part of a plant comprises the transformed promoter polynucleotide.
  • the promoter polynucleotide of the invention may be derived from any species and/of may be produced synthetically or recombinantly.
  • the promoter polynucleotide is derived from a plant species.
  • the promoter polynucleotide is derived from a gymnosperm plant species.
  • the promoter polynucleotide is derived from an angiosperm plant species.
  • the promoter polynucleotide is derived from a from dicotyledonuous plant species.
  • the promoter polynucleotide is derived from a monocotyledonous plant species.
  • polypeptide encoded by the polynucleotide to be expressed in a construct of the invention may be derived from any species and/or may be produced synthetically or recombinantly. In one embodiment the polypeptide is derived from a plant species.
  • polypeptide is derived from a gymnosperm plant species.
  • polypeptide is derived from an angiosperm plant species.
  • polypeptide is derived from a from dicotyledonous plant species.
  • polypeptide is derived from a monocotyledonous plant species.
  • the MYB transcription factor that regulates the promoter of the invention may be derived from any species and/or may be produced synthetically or recombinantly.
  • the MYB transcription factor is derived from a plant species.
  • the MYB transcription factor is derived from a gymnosperm plant species.
  • the MYB transcription factor is derived from an angiosperm plant species.
  • the MYB transcription factor is derived from a from dicotyledonuous plant species.
  • the MYB transcription factor is derived from a monocotyledonous plant species.
  • the plant cells and plants, of the invention, or produced by the methods of the invention may be derived from any species.
  • the plant cell or plant is derived from a gymnosperm plant species.
  • the plant cell or plant is derived from an angiosperm plant species. In a further embodiment the plant cell or plant, is derived from a from dicotyledonous plant species.
  • the plant cell or plant is derived from a monocotyledonous plant 5 species.
  • Preferred plant species include fruit plant species selected from a group comprising but not limited to the following
  • Particularly preferred fruit plant species are: Malus domestica, Actidinia deliciosa, A. chinensis, A. eriantha, A. arguta and hybrids of the four Actinidia species and Prunis persica.
  • 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. >0
  • Preferred plants also include crop plant species selected from a group comprising but not limited to the following genera: Glycine, Zea, Hordeum and Ory ⁇ a.
  • Particularly preferred crop plant species include Glycine max, Zea mays and Oryza sativa. 15
  • Preferred plants also include those of the Rosaceae family.
  • Preferred Rosaceae genera include Exochorda, Maddenia, Oemleria, Osmaroriia, Prinsepia, Prunus, Maloideae, Amelanchier, Aria, Aronia, Chaenomeles, Chamaemespilus, Cormus, 50 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, Peraphyllum, Photinia, Pseudocydonia,
  • 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, Primus 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
  • BSP-2004-1 Prunus sp. BSP -2004-2, Prunus sp. EB-2002, Amelanchier alnifolia, Amelanchier arbor ea, 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 Amelanchier sp. 'dentata', Amelanchier sp. ' dent at a' , Amelanchier sp. 'erecta', Amelanchier sp. 'erecta' x Amelanchier laevis, Amelanchier sp.
  • CFRA 538 Fr agar ia 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.
  • Tetraglochin cristatum Waldsteinia fragarioides, Waldsteinia geoides, Adenostoma fasciculatum, Adenostoma sparsifolium, Aruncus dioicus, Cercocarpus betuloides, Cercocarpus ledifolius, Chamaebatiaria millefolium, Chamaerhodos erecta, Gillenia stipulata, Gillenia trifoliata, Holodiscus discolor, Holodiscus microphyllus, Lyonothamnus ⁇ oribundus, Neillia affinis, Neillia gracilis, Neillia sinensis, Neillia sparsiflora, Neillia thibetica, Neillia thyrsiflora, Neillia uekii, Neviusia alabamensis, Physocarpus alternans, Physocarpus amnrensis, Physocarpus capitatus, Phys
  • R ⁇ saceae genera include: Malus, Pyrus, Cydonia, Prunus, Eriobotrya, and Mespilus.
  • Rosaceae species include: Malus domestica, Malus sylvestris, Pyrus communis, Pyrus pyrifolia, Pyrus bretschneideri, Cydonia oblonga, Prunus salicina, Prunus cerasifera, Primus persica, Eriobotrya japonica, Prunus dulcis, Prunus avium, Mespilus germanica and Prunus domestica.
  • a particularly preferred Rosaceae genus is Malus.
  • a particularly preferred Malus species is Malus domestica.
  • Malus sieversii 93.051 GO 1-048 Malus aldenhamii, Malus pumila Niedzwetzkyana, Malus x domestica cv. 'Prairiefire', Malus x domestica cv. 'Geneva', Malus sieversii 92.103 30-312.
  • a particularly preferred Malus cultivar is Malus x domestica niedwetzkyana.
  • MYB transcription factor is a term well understood by those skilled in the art to refer to a class of transcription factors characterised by a structurally conserved DNA binding domain consisting of single or multiple imperfect repeats.
  • a MYB transcription with an R2R3 DNA binding domain is a term well understood by those skilled in the art to refer to MYB transcription factors of the two-repeat class.
  • 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 preferably at least 15 nucleotides in length.
  • the fragments of the invention preferably comprises at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 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.
  • fragment in relation to promoter polynucleotide sequences is intended to include sequences comprising cis-elements and regions of the promoter polynucleotide sequence capable of regulating expression of a polynucleotide sequence to which the fragment is operably linked.
  • fragments of promoter polynucleotide sequences of the invention comprise at least 46, more preferably at least 69, more preferably at least 92, more preferably at least 1 15, more preferably at least 138, more preferably at least 150, more preferably at least 200, more preferably at least 300, more preferably at least 400, more preferably at least 500, more preferably at least 600; more preferably at least 700, more preferably at least 800, more
  • 10 preferably at least 900, more preferably at least 1000, more preferably at least 1 100, more preferably at least 1200, more preferably at least 1300, more preferably at least 1400, more preferably at least 1500, more preferably at least 1600 and most preferably at least 1700 contiguous nucleotides of the specified polynucleotide. Fragments of the promoter polynucleotide sequences can be used to control expression of an operably linked polynucleotide
  • 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 template.
  • a primer is preferably at least 5, more preferably at least 6,
  • ZO more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 1 1, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.
  • 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.
  • a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more
  • polynucleotides of the invention being "derived from” a particular genera or species, means that the polynucleotide has the same sequence as a polynucleotide found naturally in that genera or species. The polynucleotide which is derived from a genera or species may therefore be produced synthetically or recombinantly.
  • 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.
  • the polypeptides 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.
  • polypeptides disclosed being derived from a particular genera or species, means that the polypeptide has the same sequence as a polypeptide found naturally in that genera or species.
  • the 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 polynucleotides and polypeptides possess biological activities that are the same or similar to those of the inventive polynucleotides or polypeptides.
  • variants of the inventive polynucleotides and polypeptides possess biological activities that are the same or similar to those of the inventive polynucleotides or polypeptides.
  • variant with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides 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, more preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, more preferably at least 200 nucleotide positions, more preferably at least 300 nucleotide positions, more preferably at least 400 nucleotide positions, more preferably at least 500 nucleotide positions, more preferably at least 600 nucleotide positions, more preferably at least 700 nucleotide positions, more preferably at least 800 nucleotide positions, more preferably at least 900 nucleotide positions, more preferably at least 1000 nucleotide positions, more preferably at least 1 100 nucleotide positions, more preferably at least 1200 nucleotide positions, more preferably at least 1300 nucleotide positions, more preferably at least 1400 nucleotide positions, more preferably at least 1500 nucleotide positions, more preferably at least 1600 nucleotide positions, more preferably at least 1700 nucle
  • Polynucleotide sequence identity can be determined in the following manner. - The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN
  • 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. Longden,I. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276- 277) which can be obtained from the world wide web at http://www.hgmp.mrc.ac.uk/Software/EMBOSS/.
  • the 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/.
  • GAP Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
  • Sequence identity may also be calculated by aligning sequences to be compared using Vector NTI version 9.0, which uses a Clustal W algorithm (Thompson et al., 1994, Nucleic Acids Research 24, 4876-4882), then calculating the percentage sequence identity between the aligned sequences using Vector NTI version 9.0 (Sept 02, 2003 ⁇ 1994-2003 InforMax, licenced to Invitrogen).
  • 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 polynucleotides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI on the world wide web at 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 polynucleotide sequences preferably exhibit an E value of less than ⁇ x 10 "l0 more preferably less than 1 x 10 "20 , more preferably less than 1 x 10 "30 , more preferably less than 1 x 10 "40 , more preferably less than 1 x 10 "5 ⁇ more preferably less than 1 x 10 "60 more preferably less than 1 x 10 "70 more preferably less than 1 x 10 "8 ⁇ more preferably less than 1 x 10 "90 and most preferably less than 1 x 10 "l0 ° when compared with any one of the specifically identified sequences.
  • variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, 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 I X 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. On average, the 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 such as those in constructs of the invention encoding proteins to be expressed, also encompasses polynucleotides that differ from the specified sequences 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.
  • 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 on the world wide web at 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 on the world wide web at 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 on the world wide web at 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.
  • Sequence identity may also be calculated by aligning sequences to be compared using Vector NTI version 9.0, which uses a Clustal W algorithm (Thompson et al., 1994, Nucleic Acids Research 24, 4876-4882), then calculating the percentage sequence identity between the aligned polypeptide sequences using Vector NTI version 9.0 (Sept 02, 2003 ⁇ 1994-2003 InforMax, licenced to Invitrogen).
  • 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 on the world wide web at ftp://ftp.ncbi.nih.gov/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 "l 2 , more preferably less than 1 x 10 ⁇ 15 , more preferably less than 1 x 10 ⁇ 18 , more preferably less than 1 x 10 "2 I , more preferably less than 1 x 10 "30 , more preferably less than 1 x 10 "4 ⁇ more preferably less than 1 x 10.
  • 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 a promoter polynucleotide such as a promoter polynucleotide of the invention including 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 synthetic or 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, such as a promoter polynucleotide sequence of the invention, 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 includes 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 sequences may include elements required for transcription initiation and termination and for regulation of translation efficiency.
  • noncoding also includes intronic sequences within genomic clones.
  • 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 a polynucleotide sequence capable of regulating the expression of a polynucleotide sequence to which the promoter is operably linked. Promoters may 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.
  • the applicants have isolated a promoter polynucleotide sequence from apple and demonstrated that a sequence motif in the promoter, when present in more than one copy, such as when included as part of a minisatellite repeat unit, strongly effects transcription of an operably linked polynucleotide in plants.
  • a promoter sequence comprising more than one copy of the motif is positively regulated by a MYB transcription factor resulting in a significant increase in expression driven by the promoter.
  • the invention also provides fragments and variants of the promoter polynucleotide capable of such regulation of expression.
  • the invention provides genetic constructs and vectors comprising the promoter polynucleotide sequences, and transgenic plant cells and transgenic plants comprising the promoter polynucleotide sequence, genetic constructs, or vectors of the invention. . .
  • the invention also provides methods for producing plants with modified gene expression and modified phenotype.
  • the invention further provides plants produced by the methods of the invention.
  • polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art.
  • such polynucleotides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et ah, Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference.
  • PCR polymerase chain reaction
  • the polynucleotides 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 polynucleotides 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 0 C) in 1.
  • 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 polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence, untranslated sequences, and promoter sequences. Such methods include PCR-based methods, 5'RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56), genome walking using a Genome WalkerTM kit (Clontech, Mountain View, California), and hybridization- based method, computer/database -based methods.
  • 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 polynucleotide. 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.
  • the promoter sequences disclosed may be further characterized to identify other fragments, such as cis-elements and regions, capable of regulating to expression of operably linked sequences, using techniques well-known to those skilled in the art. Such techniques include 5' and/or 3' deletion analysis, linker scanning analysis and various DNA footprinting techniques (Degenhardt et al., 1994 Plant Cell:6(8) 1123-34; Directed Mutagenesis: A Practical Approach IRL Press (1991)). Fragments include truncated versions of longer promoter sequences which may terminate (at the 3' end) at or close to the transcriptional start site. Methods for identifying the transcription start site of a promoter are well-known to those skilled in the art (discussed in Hashimoto et al., 2004, Nature Biotechnology 22, 1 146-1 149).
  • the techniques described above may be used to identify a fragment that defines essential region of the promoter that is able to confer the desired expression profile.
  • the essential region may function by itself or may be fused to a core promoter to drive expression of an operably linked polynucleotide.
  • the core promoter can be any one of known core promoters such as the Cauliflower Mosaic
  • Virus 35S or 19S promoter U.S. Pat. No. 5,352,605
  • ubiquitin promoter U.S. Pat. No. 5,510,474
  • the IN2 core promoter U.S. Pat. No. 5,364,780
  • a Figwort Mosaic Virus promoter Gruber, et al. "Vectors for Plant Transformation” Methods in Plant Molecular Biology and Biotechnology) et al. eds, CRC Press pp.89-1 19 (1993)).
  • Promoter fragments can be tested for their utility in driving expression in any particular cell or tissue type, or at any particular developmental stage, or in response to any particular stimulus by techniques well-known to those skilled in the art. Techniques include operably-linking the promoter fragment to a reporter or other polynucleotide and measuring reporter activity or polynucleotide expressions in plants. Some of such techniques are described in the Examples section of this specification.
  • Variant polynucleotides may be identified using PCR-based methods (Mullis et al, Eds. 1994 The Polymerase Chain Reaction, Birkhauser).
  • Polynucleotide and polypeptide variants 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 1 1-16, 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.
  • BLAST suite of programs including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from the world wide web at ftp://ftp.ncbi.nih.gov/blast/, or from the National Center for Biotechnology Information (NCBI),
  • 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
  • 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, is described in the publication of Altschul et al.. Nucleic Acids Res. 25: 3389-3402, 1997.
  • the "hits" to one or more database sequences by a queried sequence produced by BLASTN, !0 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
  • ⁇ 0 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.
  • sequences having an E value of 0.01 or less over aligned and matched portions the probability of finding a match by chance in that database is 1% or less using the BLASTN,
  • 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 (Bairoch 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.
  • the PROSITE database www.expasy.org/prosite
  • Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.
  • the genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides disclosed, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or particularly plant organisms.
  • the genetic constructs of the invention are intended to include expression constructs as herein defined. Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987).
  • 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 al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides. 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).
  • 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. Plants comprising such cells also form an aspect of the invention.
  • strategies for genetically manipulating plants 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.
  • Strategies may also be designed to increase expression of a polynucleotide/polypeptide in response to an external stimuli, such as an environmental stimuli.
  • Environmental stimuli may include environmental stresses such as mechanical (such as herbivore activity), dehydration, salinity and temperature stresses.
  • 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 or to reduce expression of a polynucleotide/polypeptide in response to an external stimuli. Such strategies are known as gene silencing strategies.
  • Genetic constructs for expression of genes in transgenic plants typically include promoters, such as promoter polynucleotides of the invention, for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.
  • promoters such as promoter polynucleotides of the invention, for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.
  • 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 zin gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solatium tuberosum PI-II terminator.
  • CaMV cauliflower mosaic virus
  • Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators the Zea mays zin gene terminator
  • the Oryza sativa ADP-glucose pyrophosphorylase terminator the Solatium 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., Spangenbert. 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 may include an antisense copy of a polynucleotide. 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. Usually 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 polynucleotides useful for 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
  • 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).
  • UTR untranslated region
  • 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 sequence operably-linked to promoter 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.
  • plant is intended to include a whole plant or 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.
  • a "transgenic” or transformed” 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 or transformed plant or from a different species.
  • a transformed plant includes a plant which is either stably or transiently transformed with new genetic material.
  • 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. Plants resulting from such standard breeding approaches also form part of the present invention.
  • Figure 1 shows the promoter polynucleotide sequence of SEQ ID NO: 5, showing the position of the repeat motifs (1 , 2, 3A, 3B, 4, 5 and 6), the microsatellite (microsat) and several restriction enzyme sites.
  • Figure 2 shows a schematic representation of the MdMYBlO R
  • the figure also shows schematic representation of the structure and location of the additional repeat unit composed of repeat units 2, 3a, 3b, 4, 5 and 6 found in the promoter of the red-fleshed cultivar R 6 , relative to the promoter from the white-fleshed cultivar.
  • Example phenotypes for the MdMYBlO Ri and R 6 promoter versions are shown to the left, Malus x domestica Royal Gala (i) and Mctlus x domestica niedzwetzkyana (ii).
  • Figure 3 shows the portion of the sequence of the promoter from the red-fleshed apple cultivar including repeat motifs 1 , 2, 3a. 3b, 4, 5 and 6 and the microsatellite region.
  • Figure 4 shows /nms-activation of the promoters from white-fleshed (Ri) and red-fleshed (R6) cultivars by the MdMYBlO gene in transient tobacco transformation assays. Both promoters were infiltrated with and without MdMYBlO. Error bars shown are ⁇ S. E. of the means of 6 replicate experiments.
  • Figure 5 shows that amplification of a PCR product comprising the minsatellite motif serves as a marker that distinguishes white-fleshed and red-fleshed apple cultivars.
  • a total of 87 cultivars were screened using the PCR primer pair described in Example 3.
  • PCR products were separated on 0.9% agarose gels and stained with ethidium bromide.
  • the figure shows the PCR amplification obtained over a subset of 10 apple varieties. Two alleles were found: a 496 bp fragment corresponding to the promoter of SEQ ID NO: 5, which was only present in red flesh varieties (lanes 1-6) and was absent in white- fleshed varieties (lanes 7-10), and a 392 bp allele present in both types of fruit.
  • Red-fleshed varieties . 1 : open-pollinated (OP) Malus 'Mildew Immune Seedling' 93.051 G01-048; 2: M. - ⁇ purpurea 'Aldenhamensis'; 3: M. pumila var. niedzwet ⁇ kyana; 4: M. 'Prairifire'; 5: OP M. pumila var. niedzwetzkyana 'Geneva'; 6: OP M. x domestica 'Pomme Grise' 92.103 30-312; 7: M. x domestica 'Granny Smith'; 8: M. x domestica 'Royal Gala'; 9: M. x domestica 'Fuji'; 10: M. x domestica 'Braeburn'.
  • OP open-pollinated
  • 2 M. - ⁇ purpurea '
  • Figure 6 shows the native apple promoter containing the minisatellite induces ectopic anthocyanin accumulation
  • Red colouration has developed around the infiltration site in the leaves of Nicotiana tabacum 8 days after transient transformation with R 6 MdMYBlO (i) and 35S:MdMYB10 (ii) but not with R
  • native promoter from Malus domestica 'Royal Gala'.
  • R 6 native promoter from Malus x pumila var. niedzwetzkyana.
  • Figure 7 shows the interaction of the native apple promoters and MdMYBlO in the dual luciferase transient tobacco assays.
  • Ri native promoter from Malus domestica 'Royal Gala'.
  • R 6 native promoter from Malus x pumila var. niedzwetzkyana.
  • Figure 8 shows the number of repeat units affects the transactivation rate
  • Figure 9 shows identification of areas of the promoter critical to transactivation by deletion study, (a) Cartoon (not drawn to scale) of the different promoter deletions of Ri 5 (i) and R 6 , (ii), denoted as ⁇ a - ⁇ d.
  • Deleted areas are shown in white with dotted lines and the relative positions of the repeat unit Ri to the microsatellite and minisatellite are displayed, (b) Corresponding data from promoter deletion studies with luciferase fusions of Ri, (i) and (ii), and R 6 , (iii) and (iv), co- infiltrated with MdMYBlO, (i) and (iii) respectively (pale grey bars) and with MdMYBlO and MdbHLH3, (ii) and (iv) respectively (dark grey bars). Error bars shown are means ⁇ S. E. of 6 replicate reactions.
  • Figure 10 shows a schematic representation of the cloning of the minsatellite repeat unit (copies 1-6) from the apple MdMYBlO R 6 promoter (MdMYBlO long) into the MYBlO promoter from pear (PcMYBlO(GP)) to produce the chimeric promoter PcMYB 10R6(GP-R6).
  • the MdMYBlO promoter from white-fleshed apple (MdMYBlO short) is included in the figure for reference.
  • restriction sites Dral and Bsgl
  • PCR priming sites CB02 and REl 61
  • Figure 1 1 shows the effect of MdMYBlO genomic and 35S:PcMYB10 constructs on luciferase reporter gene driven by PcMYBlO promoter containing or not the MdMYB 10-promoter R6 repeats.
  • Activity is expressed as a ratio of the Luciferase (LUC) to the CaMV35s-Renilla (REN) activities. Error bars represent the standard error (SE) for 4 replicates.
  • the transcription factor constructs are all driven by the CaMV35S promoter and are as follows: MdMlO, MdMYBlO; PcMlO, PcMYBlO; b33, MdbHLH33 and b2, Arabidopsis thaliana bHLH2.
  • Figure 12 shows an alignment between the sequences of the MYBlO promoters from white- fleshed apple and pear and highlights with, underligning, the conserved 23 bp repeat motif.
  • Example 1 Isolation of the full length MdMYBlO promoter polynucleotides from white- fleshed and red-fleshed apple cultivars, and identification of additional elements within the promoter from the red-fleshed cultivar.
  • Genomic DNA was isolated from the leaves of a white-fleshed apple cultivar (Malm domestica Royal Gala) and from the leaves of a red-fleshed apple cultivar (Mains x piimila niedwetzkyana) using a Qiagen DNeasy Plant Mini Kit, according to the manufacturers instructions (Qiagen, Valencia, California).
  • a 1.7-1.8 Kb region of the upstream regulatory region of the MdMYBlO gene was isolated from the DNA of both the white-fleshed and the red-fleshed cultivar by PCR genome walking using a GenomeWalkerTM kit (Clontech, Mountain View, California), following .the manufacturers instructions.
  • the isolated promoters were sequenced by standard techniques. The sequence of the promoter from the red-fleshed cultivar is shown in SEQ ID NO: 5. The sequence of the promoter from the white-fleshed cultivar is shown in SEQ ID NO: 8.
  • the sequence of the MdMYBlO polypeptide is shown in SEQ ID NO: 6.
  • the polynucleotide sequence (cDNA) encoding the MdMYBlO polypeptide is shown in SEQ ID NO: 7.
  • the applicants identified a 23-base pair sequence motif found in both promoters.
  • the motif is present as a single copy (with a lbp difference versus the motif in the promoter from the red-fleshed cultivar).
  • the motif is present at a corresponding position, but in addition, the motif is duplicated in five tandem repeats to form a minisatellite repeat unit.
  • the sequence of the repeat motif is shown in SEQ ID NO: 1.
  • the sequence of the minisatellite unit comprising five copies of the repeat motif is shown in SEQ ID NO: 2.
  • Figure 1 shows the sequence of the promoter from the red-fleshed variety as shows the position of the repeated motifs.
  • the minisatellite unit precedes a di-nucleotide microsatellite found in both promoters.
  • the sequence of the microsatellite is shown in SEQ ID NO: 3.
  • Figure 2 shows a schematic representation of promoter from the white-fleshed cultivar and shows the relative position and structure of the additional minisatellite repeat unit found in the promoter of the red-fleshed cultivar.
  • Minisatellites similar to these, have been shown to have an effect on transcriptional regulation in humans ( Kominato et al, (1997). J. Biol. Chem. 272, 25890, Lew et al, (2000). Proc. Natl. Acad. Sci. U. S. A. 97, 12508 and to produce phenotypic alteration in Saccharomyces cerevisiae fVerstrepen et al, (2005f Nat. Genet. 37, 986/
  • Example 2 Demonstration of regulation of expression of operably linked polynucleotide sequences by the promoter polynucleotides of the invention.
  • the promoter sequences for MdMYBlO from the red-fleshed and white-fleshed cultivars were separately inserted into the cloning site of pGreen 0800-LUC (Hellens et al., 2005, R. P. Hellens, A. C. Allan, E. N. Friel EN, K. Bolitho, K. Grafton, M. D. Templeton, S. Karunairetnam, W. A. Laing, Plant Methods 1:13).
  • 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.
  • the promoter-LUC fusion was used in transient transformation by mixing 100 ⁇ l of Agrobacterium strain GV3101 (MP90) transformed with the reporter cassette with or without another Agrobacterium culture (900 ⁇ l) transformed with a cassette containing MdMYBlO fused to the 35S promoter. Nicotiana tabacum 'Samsun' plants were grown until at least 6 leaves were available for infiltration with Agrobacterium.
  • a 10 ⁇ l loop of confluent bacterium were re- suspended in 10 ml of infiltration media (10 mM MgCb, 0.5 ⁇ M acetosyringone), to an OD 6 Q 0 of 0.2, and incubated at room temperature without shaking for 2 h before infiltration.
  • 10 ml of infiltration media (10 mM MgCb, 0.5 ⁇ M acetosyringone), to an OD 6 Q 0 of 0.2
  • Approximately 150 ⁇ l of this Agrobacterium mixture was infiltrated at six points into a young leaf of N. tabacum and transient expression was analysed 3 days after inoculation.
  • Six technical replicates of 3 mm 0 leaf discs were excised from each plant using a leaf hole-punch and buffered in Phosphate Buffer Saline (PBS).
  • PBS Phosphate Buffer Saline
  • Plate-based assays were conducted using a Berthold Orion Microplate Luminometer (Berthold Detection Systems, Oak Ridge, TN, USA) according to the manufacturer's specifications for the dual luciferase assay, using the Dual Glow assay reagents (Promega, Madison, WI) for firefly luciferase and Renilla luciferase. Luminescence was calculated using Simplicity version 4.02 software (Berthold Detection Systems).
  • the results show that co-expression of the MdMYBlO transcription factor results in a 10- fold increase in expression of the luciferase sequence that is operably linked to the promoter (R 6 ) from the red-fleshed cultivar.
  • the effect of MdMYBlO from the white-fleshed cultivar is much smaller.
  • This result shows that the promoter polynucleotide of the invention is positively regulated by the MYB transcription factor MdMYBlO.
  • Example 3 The presence of the minisatellite unit in the promoter of the invention is consistently associated with red-flesh in naturally occurring red-fleshed apple varieties.
  • the fruit flesh (cortex) of most apple cultivars is white or off-white in colour.
  • the skin is usually green or red, the skin reddening in response to developmental, hormonal and light signals (Ubi et a!., 2006, Plant Sci. 170, 571 ).
  • Apple genomic DNA from 19 cultivars was amplified using a pair of PCR primers located in the
  • R ⁇ refers to the absence of the minisatellite unit as found in the promoter from the white-fleshed Royal Gala cultivar.
  • R 6 refers to the presence of the minisatellite unit as found in the promoter from the red-fleshed Malus x piimila niedwetzkyana cultivar.
  • the presence of additional repeat units in the promoter from the red-fleshed cultivar are likely to account for the known increased expression level of MdMYlO and resulting anthocyanin accumulation red- fleshed apple cultivars.
  • Example 4 Expression of the MdMYBlO transcription factor driven by the promoter of the invention results in anthocyanin production in transiently transformed tobacco.
  • Example 5 Expression of the MdMYBlO transcription factor driven by the promoter of the invention can transactivate reporter gene expression at a level similar or higher than CaMV35S promoter driven expression of the MdMYBlO transcription factor.
  • Example 6 The number of copies of the 23 bp repeat unit influences transcription.
  • Example 15 relationship between the anthocyanin-regulating MYB and bHLH co-factors and it has previously been shown the dependency of MdMYBlO on a co-factor bHLH in transient assays (Espley et al, 2007). In this assay, activation for both the Ri and R 6 promoters is enhanced with the addition of 35S:MdbHLH3 for all the constructs tested.
  • Example 7 Deletion analysis of the promoter of the invention emphasises the importance of the minisatellite region, containing multiple copies of the 23 bp repeat unit, in enhancing transcription.
  • the R 6 )LUC deletions were less affected than R
  • the R 6 ILUC promoters appeared to show a lesser dependence on the bHLH for increased activity although this may be due to saturation or depletion of one or other of the co- infiltrated transcription factors.
  • Ri Ad and R 6 Ad there was barely detectable activity, with or without the bHLH, confirming the requirement of the 3' region for transactivation.
  • the data suggests that the R 6 promoter can still activate luciferase transcription in truncated form (500 bp) whereas the corresponding version of Ri (R]Ab) cannot.
  • genomic DNA was extracted from Malus x domestica 'Sciros' (Pacific RoseTM, derived from a cross between 'Gala' and 'Splendour').
  • Nested primers were designed to the coding region of MdMYBlO; primary 5'- CACTTTCCCTCTCCATGAATCTCAAC-3 (SEQ ID NO: 18), and secondary 5'- CAGGTTTTCGTTATATCCCTCCATCTC-3 (SEQ ID NO: 19).
  • a 1.7 Kb region of upstream DNA, immediately adjacent to the transcription start site was isolated from the genomic DNA by PCR genome walking using a Genome WalkerTM kit (Clontech, Mountain View, California,
  • Genomic DNA was subsequently isolated from Malus x domestica 'Granny Smith', Malus x domes tica 'Royal Gala' and Malus x pumila var. niedzwetzkyana using forward and reverse primers 5'-ACCCTGAACACGTGGGAACCG-3 (SEQ ID NO: 20) and 5'-GCTAAGCTTAGCTGCTAGCAGATAAGAG-S (SEQ ID NO: 21) respectively.
  • the PCR products were cloned using the TOPO TA cloning® kit (Invitrogen,
  • Apple genomic DNA from 19 cultivars was amplified using a pair of PCR primers located in the 15 MYBlO promoter (forward: 5'-GGAGGGGAATGAAGAAGAGG-S ' [SEQ ID NO: 22]; reverse: 5'-TCCACAGAAGCAAACACTGAC-S ' [SEQ ID NO: 23]).
  • PCR reactions were carried out in 16.5 ⁇ l volume containing Ix PCR buffer mix (Invitrogen), 1.3 mMMgC12, 100 ⁇ M of each dNTP, 0.72 % formamide, 10 ⁇ M of each primer, 0.5 U of Platinum Taq DNA polymerase (Invitrogen) and 2 ng of genomic DNA.
  • PCR amplifications were performed in a Hybaid PCR JO Express Thermal Cycler (Thermo Electron Corporation, Waltham, MA, USA) with conditions as follows: 94°C for 2 min 45 sec followed by 40 cycles at 94 0 C for 55 sec, 55 0 C for 55 sec and 72 0 C for 1 min 39 sec, and a final elongation at 72 0 C for 10 min.
  • the PCR products obtained were cloned using the TOPO TA cloning® kit (Invitrogen). Four clones were sequenced for each PCR product. The sequences were aligned using Vector NTI (Invitrogen). >5
  • Luciferase reporter constructs were derivatives of pGreen 0800-LUC (Hellens et al. 2005) in which the promoter sequence for the native MdMYBlO promoter or the deletion fragments were ?0 inserted.
  • Native promoter sequences were PCR amplified using the primers 5' - ACCCTGAACACGTGGGAACCG - 3' (SEQ ID NO: 24) and 5' - GCTAAGCTTAGCTGCTAGCAGATAAGAG - 3' (SEQ ID NO: 25) and cloned into the multi-cloning region of pGreen 0800-LUC.
  • Ri and R 6 promoter fragments were cloned in as native promoter sequences whilst changes to the repeat frequency for the R 2 , R 3 and R 4 promoter fragments were synthesised (Geneart AG, Regensburg, Germany) and cloned into Ri using the restriction enzymes Spel and Dr a ⁇ .
  • An inverse PCR approach was used for the R
  • GAGCTCATGTTAGCTTTTCTATATATCGA - 3' (SEQ ID NO: 27).
  • the pSAK construct for 35S:MdMYB10 and 35S:MdbHLH3 was as previously described (Espley et al, 2007) whilst the promoter sequences were substituted for the R
  • the promoter-LUC fusions were used in transient transformation by mixing 100 ⁇ l of Agrobacterium strain GV3101 (MP90) transformed with the reporter cassette with or without another Agrobacterium culture(s) (900 ⁇ l) transformed with a cassette containing MYBJO fused to the 35S, Rl or R6 promoters and MdbHLH3 fused to the 35S promoter. Nicotiana tabacum 'Samsun' plants were grown until at least 6 leaves were available for infiltration with Agrobacterium.
  • a 10 ⁇ l loop of confluent bacterium were re- suspended in 10 ml of infiltration media (10 mM MgC12, 0.5 ⁇ M acetosyringone), to an OD 600 of 0.2, and incubated at room temperature without shaking for 2 h before infiltration. Approximately 150 ⁇ l of this Agrobacterium mixture was infiltrated at six points into a young leaf of N. tabacum. Transient expression was analysed three days after inoculation. Six technical replicates of 3 mm 0 leaf discs were excised from each plant using a leaf hole-punch and buffered in Phosphate Buffer Saline (PBS).
  • PBS Phosphate Buffer Saline
  • Plate-based assays were conducted using a Berthold Orion Microplate Luminometer (Berthold Detection Systems, Oak Ridge, TN, USA) according to the manufacturer's specifications for the dual luciferase assay, using the Dual Glow assay reagents (Promega) for firefly luciferase and renilla luciferase. Luminescence was calculated using Simplicity version 4.02 software (Berthold Detection Systems). Induction of anthocyanin pigmentation in tobacco
  • N. tabacum were grown as previously mentioned and maintained in the glasshouse for the duration of the experiment. Agrobacterium cultures were incubated as for the dual luciferase assay and separate strains containing the MdMYBlO gene fused to either the 35S, Ri or R 6 promoter sequences and the MdbHLH3 gene fused to the 35S promoter were mixed (500 ⁇ l each) and infiltrated into the abaxial leaf surface. Six separate infiltrations were performed into N. tabacum leaves (two plants per treatment) and changes in colour were observed over an eight day period.
  • each leaf included positive ⁇ Agrobacterium cultures containing 33S:MdMYB10 + 35S:MdbHLH3) and negative ⁇ Agrobacterium with empty vector) controls.
  • the binary vector pSAK277 containing the MdMYBlO cDNA driven by the R 6 or Ri promoters was transferred into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method.
  • Transgenic Mains domestica 'Royal Gala” plants were generated by Agrobacterium-mediated transformation of leaf pieces, using a method previously reported (Yao et ⁇ l. 1995).
  • Example 8 Isolation of the PcMYBlO promoter from pear and identification of a sequence motif analogous to the repeat motif found in apple MdMYBlO promoters.
  • Genomic DNA was isolated from the leaves of a pear cultivar ⁇ Pyrus communis 'William's Bon Chretien') using a Qiagen DNeasy Plant Mini Kit, according to the manufacturers instructions (Qiagen, Valencia, California). Promoter sequences were isolated by PCR using the primers REl 58 (5'-ACCCTGAACACGTGGGAACCG-S', SEQ ID NO: 28) and REl 59 (5'- CTCTT ATCTGCTAGCAGCT AAGCTTAGC-3', SEQ ID NO: 29).
  • SEQ ID NO: 12 100% The high degree of conservation between these three sequences, and their conserved position within the promoters, from three different sources, strongly suggest that each of the three sequences perform the same function. 5
  • Example 9 Production of a chimeric promoter with altered activity by insertion of copies of a repeated motif from the MdMYBlO promoter from red-fleshed apple into the MdMYBlO promoter from pear.
  • MdMYBlO controls the accumulation of anthocyanin in apple.
  • Transient experiments described in the Examples above have shown that the MYBlO protein is able to auto-regulate its own promoter leading to a high level of expression of a Luciferase reporter gene driven by the long 15 version of MdMYBlO promoter (which includes the 6 repeats of a putative transcription factor binding site), when co-infiltrated with bHLH33 transcription factor.
  • the applicants have now introduced the 6 repeats into the green pear MYBlO promoter controlling luciferase reporter gene and assessed the reporter activity in presence of PcMYBlO and MdMYBlO TFs.
  • the green pear MYBlO promoter (SEQ ID NO: 13) was cloned in the pGreen0800LUC vector.
  • the R6 region (SEQ ID NO: 14) of the MdMYBlO promoter was amplified using primers CB02F/RE161, digested by Dral and cloned in the PcMYBlO promoter at the blunted Bsgl site .5 (see Figure 10) to produce the recombinant chimeric promoter of SEQ ID NO: 15.
  • Apple and pear MYBlO constructs in presence of bHLH33 and bHLH2 respectively, strongly activate the DFR, MdMYB 10R6 and PcMYB 10R6 promoters, and only slightly activate MdMYBlORl and PcMYBI ORl promoters.
  • the introduction of the apple R6 repeats in the pear promoter leads to a 6-fold increase in the luciferase activity in presence of the 35S:PcMYB10 construct and an 8-fold increase in presence of the MdMYBlO genomic construct.
  • the pear MYBlO promoter was cloned into the pGreen0800-LUC vector (Hellens et al. 2005).
  • the R6 region of the MdMYBlO promoter was amplified using primers CB02F (5'- CAGAAATGTTAGACTGGTAGCTATTAAC-3', SEQ ID NO: 30) and RE161 (5'- CCAGTGACGTGCATGTCTGATATCC-3', SEQ ID NO: 31), digested by Dral and cloned in the PcMYBlO promoter at the blunted Bsgl site (see Figure 10).
  • PcMYB 10R6-LUC were transformed into GV3101 by electroporation and used to infiltrate Nicotiana benthaniama leaves as described previously (Hellens et al. 2006). After 4 days, leaf discs were collected and Firefly luciferase (LUC) and renillia luciferase (REN) activities were measured on a luminometer using the Dual GlowTM reagents (PROMEGA).
  • Apple and pear MYBlO constructs in presence of MdbHLH33 and AtbHLH2 respectively, strongly activated the DFR, MdMYB 10R6 and PcMYB 10R6 promoters, and only slightly activated MdMYBlORl and PcMYBIORl promoters.
  • the introduction of the apple R6 repeats in the pear promoter leads to a 6-fold increase in the luciferase activity in presence of the 35S:PcMYB10 construct and an 8-fold increase in presence of the MdMYBlO genomic construct. . .. . .
  • minisatellite repeat unit from MdMYBlO promoter from red-
  • apple minisatellite sequence inserted Malus domestica polynucleotide into pear PcMYBlO promoter niedwetzkyana

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Abstract

The invention provides an isolated promoter polynucleotide comprising at least two sequence motifs with at least 70 % identity to the sequence SEQ ID No: 1, wherein the promoter polynucleotide is capable of controlling transcription of an operably linked polynucleotide in a plant and is capable of being modulated by a MYB transcription factor, specifically MdMYB 10. The invention also provides genetic constructs and vectors comprising the promoter polynucleotide sequence, and transgenic plant cells and transgenic plants comprising the promoter polynucleotide sequence, genetic constructs or vectors of the invention. The invention also provides methods for producing plants with modified gene expression and modified phenotype. The invention further provides plants produced by the methods of the invention.

Description

COMPOSITIONS AND METHODS FOR REGULATING PLANT GENE EXPRESSION
TECHNICAL FIELD
The present invention relates to promoter polynucleotides for regulating gene expression in plants, and uses thereof.
BACKGROUND ART
An important for goal for agriculture is to produce plants with beneficial agronomic traits. Recent advances in genetic manipulation provide the tools to transform plants with polynucleotide sequences of interest and to express such sequences within the transformed plants. This has led to the development of plants capable of expressing pharmaceuticals and other chemicals, plants with increased pest resistance, increased stress tolerance and many other beneficial traits.
It is often desirable to control expression of a polynucleotide of interest, in a particular tissue, at a particular developmental stage, or under particular conditions, in which the polynucleotide is not normally expressed. The polynucleotide of interest may encode a protein or alternatively may be intended to effect silencing of a corresponding target gene.
Plant promoter sequences are useful in genetic manipulation for directing expression of polynucleotides in transgenic plants. To achieve this, a genetic construct is often introduced into a plant cell or plant. Typically such constructs include a plant promoter operably linked to the polynucleotide sequence of interest. Such a promoter need not normally be associated with the gene of interest. Once transformed, the promoter controls expression of the operably linked polynucleotide of interest thus leading to the desired transgene expression and resulting desired phenotypic characteristics in the plant.
Promoters used in genetic manipulation are typically derived from the 5' un-transcribed region of genes and contain regulatory elements that are necessary to control expression of the operably linked polynucleotide. Promoters useful for plant biotechnology can be classified depending on when and where they direct expression. For example promoters may be tissue specific or constitutive (capable of transcribing sequences in multiple tissues). Other classes of promoters include inducible promoters that can be triggered by external stimuli such as environmental, and chemical stimuli.
Often a relatively high level of expression of the transformed sequence of interest is desirable. This is often achieved through use of viral promoter sequences such as the Cauliflower Mosaic Virus 35S promoter. In some circumstances it may be more preferable to use a plant derived promoter rather than a promoter derived from a microorganism. It may also be preferable in some circumstances to use a promoter derived from the species to be transformed.
It would be beneficial to have a variety of promoters available in order to ensure that transgenes are transcribed at an appropriate level in the right tissues, and at an appropriate stage of growth or development.
The apple (Malus species) is a major fruit species grown in New Zealand and other temperate climates throughout the world. Valuable traits that may be improved by genetic manipulation of apple include: fruit flavour, fruit colour, content of health promoting components (such as anthocyanins and flavanoids) in fruit, stress tolerance/resistance, pest tolerance/resistance and disease tolerance/resistance. Genetic manipulation of such traits in apple, and other plant species, is limited by the availability of promoters capable of appropriately controlling the expression of genes of interest.
It is therefore an object of the present invention to provide a promoter polynucleotide useful for controlling gene expression in apple and other plants and/or at least to provide a useful choice.
SUMMARY OF THE INVENTION
In one aspect the invention provides an isolated promoter polynucleotide comprising at least two sequence motifs with at least 70% identity to the sequence SEQ ID NO: 1, wherein the promoter polynucleotide is capable of controlling transcription of an operably linked polynucleotide in a plant.
In one embodient the promoter polynucleotide comprises at least three of the sequence motifs. In a further embodient the promoter polynucleotide comprises at least four of the sequence motifs.
In a further embodient the promoter polynucleotide comprises at least five of the sequence motifs.
In a further embodient the promoter polynucleotide comprises six of the sequence motifs.
In a further embodiment at least one of the motifs is interupted by at least one of the other sequence motifs.
In a further embodiment at least one of the motifs is interupted by at least two other sequence motifs.
In a further embodiment the promoter polynucleotide comprises six of the sequence motifs and one of the motifs is interupted by two of the other sequence motifs.
In a further embodiment the sequence motif has 80% identity with the sequence of SEQ ID NO: 1.
In a further embodiment the sequence motif has 90% identity with the sequence of SEQ ID NO: 1.
In a further embodiment the sequence motif has 91% identity with the sequence of SEQ ID NO: 1.
In a further embodiment the sequence motif has 95% identity with the sequence of SEQ ID NO: 1.
In a further embodiment the sequence motif has the sequence of SEQ ID NO: 1.
In a further embodiment the promoter polynucleotide comprises a sequence element with the at least 70% identity to the sequence of SEQ ID NO: 2. In a further embodiment the promoter polynucleotide comprises a sequence element with the sequence of SEQ ID NO: 2.
In a further embodiment the promoter polynucleotide also comprises a microsatellite sequence element with at least 70% identity to the sequence of SEQ ID NO: 3.
In a further embodiment the promoter polynucleotide also comprises a microsatellite sequence element with the sequence of SEQ ID NO: 3.
In a further embodiment the promoter polynucleotide also comprises a region with at least 70% identity to the sequence of SEQ ID NO: 4.
In a further embodiment the promoter polynucleotide comprises a region with the sequence of SEQ ID NO: 4.
In a further embodiment the promoter polynucleotide also comprises a region with at least 70% identity to the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide comprises a region with the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide also comprises at least 66 contiguous polynucleotides of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide is a naturally occuring sequence found in a solanaceous species.
In a further embodiment the solanaceous species is from the genus Malus.
In a further embodiment the solanaceous species is Malus domestica.
In a further embodiment the promoter polynucleotide comprises at least 140 bases of the sequence of SEQ ID NO: 5. In a further embodiment the promoter polynucleotide comprises at least 200 bases of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide comprises at least about 462 bases of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide comprises at least 500 bases of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide comprises at least 505 bases of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide comprises at least 750 bases of the sequence of SEQ ID NO: 5. . .. .
In a further embodiment the promoter polynucleotide comprises at least 934 bases of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide comprises at least 1000 bases of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide comprises at least 1500 bases of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide comprises at least 1801 bases of the sequence of SEQ ID NO: 5.
In a further embodiment the promoter polynucleotide is modulated by a MYB transcription factor.
In a further embodiment the promoter polynucleotide is positively modulated, activated, or up- regulated, by the MYB transcription factor.
Preferably the MYB transcription factor comprises an R2R3 DNA binding domain. Preferably the MYB transcription factor comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6.
Preferably the MYB transcription factor comprises the sequence of SEQ ID NO: 6.
Preferably the MYB transcription factor is encoded by a polynucleotide with at least 70% identity to the sequence of SEQ ID NO: 7.
Preferably the MYB transcription factor is encoded by a polynucleotide with the sequence of SEQ ID NO: 7.
Preferably the promoter polynucleotide is up-regulated by by the gene product of the gene with which the promoter polynucleotide is endogenously associated.
Preferably the promoter polynucleotide is a promoter of an autoregulated gene, where expression of the gene product up-regulates the promoter leading to further gene product expression.
Preferably the promoter polynucleotide is endogenously associated with the MYB transcription factor in naturally occuring plants, and the promoter is autoregulated by the MYB transcription factor.
Preferably the promoter polynucleotide is capable of controlling transcription of an operably linked polynucleotide sequence constitutively in substantially all tissues of a plant.
More preferably the promoter polynucleotide is capable of controlling transcription of an operably linked polynucleotide sequence in any plant, plant cell, or plant tissue in which the MYB transcription factor is expressed.
The MYB transcription factor may be naturally expressed in the plant or may be expressed in the plant through genetic manipulation of the plant.
In a further aspect the invention provides a genetic construct comprising a promoter polynucleotide of the invention. In one embodiment the promoter polynucleotide is operably linked to a polynucleotide sequence to be expressed.
5 In a further aspect the invention provides a vector comprising a genetic construct of the invention.
In a further aspect the invention provides a host cell transformed with the promoter polynucleotide of the invention. IO
In a further aspect the invention provides a plant cell or plant transformed with the promoter polynucleotide of the invention.
In a further aspect the invention provides a plant cell or plant transformed with a genetic 15 construct of the invention.
In one embodiment the plant cell or plant is also transformed with a polynucleotide or genetic construct for expresssing a MYB transcription factor that modulates expression of the promoter polynucleotide of the invention. >0
In a further embodiment the plant cell or plant naturally expresses the MYB transcription factor.
In a further embodiment the MYB transcription factor comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6. >5
Preferably the MYB transcription factor comprises the sequence of SEQ ID NO: 6.
In a further aspect the invention provides a method for producing a plant cell or plant with modifed expression of at least one polynucleotide, the method comprising transformation of the 50 plant cell or plant with a promoter polynucleotide of the invention
In one embodiment the plant cell or plant is transformed with a genetic construct of the invention. In a further embodiment the plant cell or plant is also transformed with a polynucleotide or genetic construct capable of expresssing a MYB transcription factor that modulates expression of the promoter polynucleotide of the invention.
5 In a further embodiment the plant cell or plant naturally expresses the MYB transcription factor.
In a further embodiment the MYB transcription factor comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6.
IO Preferably the MYB transcription factor comprises the sequence of SEQ ID NO: 6.
It will be appreciated by those skilled in the art that, the promoter polynucleotide of the invention may be transformed into the plant to control expression of a polynucleotide that is operably linked to the promoter prior to transformation.
15
Alternatively the promoter polynucleotide may be transformed into the genome of the plant without an operably linked polynucleotide, but the promoter may control expression of an endogenous polynucleotide, typically adjacent to the insert site, and typically, to the 3' end of the inserted promoter polynucleotide.
>0
In a further aspect of the invention provides a method for producing a plant cell or plant with a modified phenotype, the method comprising the stable incorporation into the genome of the plant, a promoter polynucleotide of the invention
.5 In one embodiment the plant cell or plant is transformed within a genetic construct of the invention.
In a further embodiment the plant cell or plant is also transformed with a genetic construct for expresssing a MYB transcription factor that modulates expression of the promoter )0 polynucleotide of the invention.
In a further embodiment the plant cell or plant naturally expresses the MYB transcription factor. In a further embodiment the MYB transcription factor comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6.
Preferably the MYB transcription factor comprises the sequence of SEQ ID NO: 6.
In a further aspect the invention provides a plant cell or plant produced by a method of the invention.
In a further aspect the invention provides a seed, propagule, progeny or part of a plant, of the invention.
Preferably the seed, propagule, progeny or part of a plant comprises the transformed promoter polynucleotide.
The promoter polynucleotide of the invention may be derived from any species and/of may be produced synthetically or recombinantly.
In one embodiment the promoter polynucleotide, is derived from a plant species.
In a further embodiment the promoter polynucleotide, is derived from a gymnosperm plant species.
In a further embodiment the promoter polynucleotide, is derived from an angiosperm plant species.
In a further embodiment the promoter polynucleotide, is derived from a from dicotyledonuous plant species.
In a further embodiment the promoter polynucleotide, is derived from a monocotyledonous plant species.
The polypeptide encoded by the polynucleotide to be expressed in a construct of the invention, may be derived from any species and/or may be produced synthetically or recombinantly. In one embodiment the polypeptide is derived from a plant species.
In a further embodiment the polypeptide is derived from a gymnosperm plant species.
In a further embodiment the polypeptide is derived from an angiosperm plant species.
In a further embodiment the polypeptide is derived from a from dicotyledonous plant species.
In a further embodiment the polypeptide is derived from a monocotyledonous plant species.
The MYB transcription factor that regulates the promoter of the invention may be derived from any species and/or may be produced synthetically or recombinantly.
In one embodiment the MYB transcription factor, is derived from a plant species.
In a further embodiment the MYB transcription factor, is derived from a gymnosperm plant species.
In a further embodiment the MYB transcription factor, is derived from an angiosperm plant species.
In a further embodiment the MYB transcription factor, is derived from a from dicotyledonuous plant species.
In a further embodiment the MYB transcription factor, is derived from a monocotyledonous plant species.
The plant cells and plants, of the invention, or produced by the methods of the invention, may be derived from any species.
In one embodiment the plant cell or plant, is derived from a gymnosperm plant species.
In a further embodiment the plant cell or plant, is derived from an angiosperm plant species. In a further embodiment the plant cell or plant, is derived from a from dicotyledonous plant species.
In a further embodiment the plant cell or plant, is derived from a monocotyledonous plant 5 species.
Preferred plant species (from which the promoter polynucleotides and variants, polypeptides and variants, MYB transcription factor and variants, and plant cells and plants may be derived) include fruit plant species selected from a group comprising but not limited to the following
10 genera: Malus, Pyrus Prunis, Rubus, Rosa, Fragaria, Actinidia, Cydonia, Citrus, and Vaccinium.
Particularly preferred fruit plant species are: Malus domestica, Actidinia deliciosa, A. chinensis, A. eriantha, A. arguta and hybrids of the four Actinidia species and Prunis persica.
15 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. >0
Preferred plants also include crop plant species selected from a group comprising but not limited to the following genera: Glycine, Zea, Hordeum and Ory∑a.
Particularly preferred crop plant species include Glycine max, Zea mays and Oryza sativa. 15
Preferred plants also include those of the Rosaceae family.
Preferred Rosaceae genera include Exochorda, Maddenia, Oemleria, Osmaroriia, Prinsepia, Prunus, Maloideae, Amelanchier, Aria, Aronia, Chaenomeles, Chamaemespilus, Cormus, 50 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, Peraphyllum, Photinia, Pseudocydonia,
Pyracantha, Pyrus, Rhaphiolepis, Sorbus, Stranvaesia, Torminalis, Vauquelinia, Rosoideae, Acaena, Acomastylis, Agήmonia, Alchemilla, Aphanes, Aremonia, Bencomia, Chamaebatia, Cliffortia, Coluήa, Cowania, Dalibarda, Dendriopoterium, Dryas, Duchesnea, Erythrocoma, Fallugia, Filipendula, Fragaria, Geum, Hagenia, Horkelia, Ivesia, Kerria, Leucosidea, Marcetella, Margyricarpus, Novosieversia,Oncostylus, Polylepis, Potent ilia, Rosa, Rubus, Sanguisorba, Sarcopoterium, Sibbaldia, Sieversia, Taihangia, Tetraglochin, Waldsteinia, Rosaceae incertae sedis, Adenostoma, Aruncus, Cercocarpus, Chamaebatiaria, Chamaerhodos, Gillenia, Holodisciis, Lyonothamnus, Neillia, Neviusia, Physocarpus, Purshia, Rhodotypos, Sorbaria, Spiraea and Stephanandra.
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, Primus 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 cocomilia, Prunus cyclamina, Prunus davidiana, Prunus debilis, Prunus domestica, Prunus dulcis, Prunus emarginata, Prunus fasciculata, Prunus ferganensis, Prunus fordiana, Prunus fremontii, Prunus fruticosa, Prunus geniculata, Prunus glandulosa, Prunus gracilis, Prunus grayana, Prunus hortulana, Prunus ilicifolia, Prunus incisa, Prunus jacquemontii, Prunus japonica, Prunus kuramica, Prunus laurocerasus, Prunus leveilleana, Prunus lusitanica, Prunus maackii, Prunus mahaleb, Prunus mandshurica, Prunus maritima, Prunus maximowiczii, Prunus mexicana, Prunus microcarpa, Prunus mira, Prunus mume, Prunus munsoniana, Prunus nigra, Prunus nipponica, Prunus padus, Prunus pensylvanica, Prunus persica, Prunus petunnikowii, Prunus prostrata, Prunus pseudocerasus, Prunus pumila, Prunus rivularis, Prunus salicina, Prunus sargentii, Prunus sellowii, Prunus serotina, Prunus serrulata, Prunus sibirica, Prunus simonii, Prunus spinosa, Prunus spinulosa, Prunus subcordata, Prunus subhirtella, Prunus takesimensis, Prunus tenella,Prunus texana, Prunus tomentosa, Prunus tschonoskii, Prunus umbellata, Prunus verecunda, Prunus virginiana, Prunus webbii, Prunus x yedoensis, Prunus zippeliana, Prunus sp. BSP-2004-1, Prunus sp. BSP -2004-2, Prunus sp. EB-2002, Amelanchier alnifolia, Amelanchier arbor ea, 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 Amelanchier sp. 'dentata', Amelanchier sp. ' dent at a' , Amelanchier sp. 'erecta', Amelanchier sp. 'erecta' x Amelanchier laevis, Amelanchier sp. 'serotina', Aria alnifolia, Aronia prunifolia, Chaenomeles cathayensis, Chaenomeles speciosa, Chamaemespilus alpina, Cormus domestica, Cotoneaster apiculatus, Cotoneaster lacteus, Cotoneaster pannosus, Crataegus azarolus, Crataegus columbiana, Crataegus crus-galli, Crataegus curvisepala, Crataegus laevigata, Crataegus mollis, Crataegus monogyna, Crataegus nigra, Crataegus rivularis, Crataegus sinaica, Cydonia oblonga, Dichotomanthes tristaniicarpa, Docynia delavayi, Docyniopsis tschonoskii, Eriobotrya japonica, Eriobotrya prinoides, Eriolobus trilobatus, Heteromeles arbutifolia, Kageneckia angustifolia, Kageneckia oblonga, Lindleya mespiloides, Malacomeles denticulata, Malus angustifolia, Malus asiatica, Malus baccata, Malus coronaria, Malus doumeri, Malus βorentina, Malus βoribunda, Malus fusca, Malus halliana, Malus honanensis, Malus hupehensis, Malus ioensis, Malus kansuensis, Malus mandshurica, Malus micromalus, Malus niedzwetzkyana, Malus ombrophilia, Malus orientalis, Malus prattii, Malus prunifolia, Malus pumila, Malus sargentii, Malus sieboldii, Malus sieversii, Malus sylvestr is, Malus toringoides, Malus transitoria, Malus trilobata, Malus tschonoskii, Malus x domestica, Malus x domestica x Malus sieversii, Malus x domestica x Pyrus communis, Malus xiaojinensis, Malus yunnanensis, Malus sp., Mespilus germanica, Osteomeles anthyllidifolia, Osteomeles schwerinae, Peraphyllum ramosissimum, Photinia fraseri, Photinia pyrifolia, Photinia serrulata, Photinia villosa, Pseudocydonia sinensis, Pyracantha coccinea, Pyracantha fortuneana, Pyrus calleryana, Pyrus caucasica, Pyrus communis, Pyrus elaeagrifolia, Pyrus hybrid cultivar, Pyrus pyrifolia, Pyrus salicifolia, Pyrus ussuriensis, Pyrus x bretschneideri, Rhaphiolepis indica, Sorbus americana, Sorbus aria, Sorbus aucuparia, Sorbus californica, Sorbus commixta, Sorbus hupehensis, Sorbus scopulina, Sorbus sibirica, Sorbus torminalis, Stranvaesia davidiana, Torminalis clusii, Vauquelinia californica, Vauquelinia corymbosa, Acaena anserinifolia, Acaena argentea, Acaena caesiiglauca, Acaena cylindristachya, Acaena digitata, Acaena echinata, Acaena elongata, Acaena eupatoria, Acaena fissistipula, Acaena inermis, Acaena laevigata, Acaena latebrosa, Acaena lucida, Acaena macrocephala, Acaena magellanica, Acaena masafuerana, Acaena montana, Acaena multifida, Acaena novaezelandiae, Acaena ovalifolia, Acaena pinnatifida, Acaena splendens, Acaena subincisa, Acaena x anserovina, Acomastylis elata, Acomastylis rossii, Acomastylis sikkimensis, Agrimonia eupatoria, Agrimonia nipponica, Agrimonia parviflora, Agrimonia pilosa, Alchemilla alpina, Alchemilla erythropoda, Alchemilla japonica,
Alchemilla mollis, Alchemilla vulgaris, Aphanes arvensis, Aremonia agrimonioides, Bencomia brachystachya, Bencomia caudata, Bencomia exstipulata, Bencomia sphaerocarpa, Chamaebatia foliolosa, Cliffortia burmeana, Cliffortia cuneata, Cliffortia dentata, Cliffortia graminea, Cliffortia heterophylla, Cliffortia nitidula, Cliffortia odorata, Cliffortia ruscifolia, Cliffortia sericea, Coluria elegans, Coluria geoides, Cowania stansburiana, Dalibarda repens, Dendriopoteriwn menendezii, Dendriopoterium pulidoi, Dryas drummondii, Dryas octopetala, Duchesnea chrysantha, Duchesnea indica, Erythrocoma triflora, Fallugia paradoxa, Filipendula multijuga Filipendula purpurea, Filipendula ulmaria, Filipβndula vulgaris, Fr agar ia chiloensis ,Fr agar ia daltoniana ,Fr agar ia gracilis ,Fr agar Aa grandiflora, Fr agar ia iinumae ,Fr agar ia moschata, Fr agar ia nilgerrensis, Fr agar ia nipponica ,Fr agar ia nubicola ,Fr agar ia oriental is ,Fragaria pentaphylla, Fr agar ia vesca ,Fr agar ia virginiana ,Fr agar ia viridis ,Fr agar ia x ananassa , Fr agar ia sp. CFRA 538 ,Fr agar ia sp.,Geum andicola ,Geum borisi ,Geum bulgaricum, Geum calthifolium, Geum chiloense ,Geum geniculatum, Geum heterocarpum, Geum macrophyllum ,Geum montanum ,Geum reptans ,Geum rivale ,Geum schofιeldii,Geum speciosum ,Geum urbanum ,Geum vernum ,Geum sp. 'Chase 2507 K',Hagenia abyssinica,Horkelia cuneata ,Horkelia fusca, Ivesia gordoni,Kerria japonica,Leucosidea sericea,Marcetella maderensis ,Marcetella moquiniana,Margyricarpus pinnatus, Margyricarpus setosus.Novosieversia glacialis,Oncostylus cockaynei ,Oncostylus leiospermus,Polylepis australis,Polylepis besseri ,Polylepis crista-galli, Polylepis hieronymi ,Polylepis incana , Polylepis lanuginosa, Polylepis multijuga , Polylepis neglect a, Polylepis pauta .Polylepis pepei .Polylepis quadrijuga, Polylepis racemosa .Polylepis reticulata .Polylepis rugulosa , Polylepis sericea , Polylepis subsericans, Polylepis tarapacana.Polylepis tomentella , Polylepis weberbaueri, Potentilla anserina ,Potentilla arguta ,Po tent ilia bifurca, Potentilla chinensis, Potentilla dickinsii ,Potentilla erecta ,Potentilla fragarioides, Potentilla fruticosa , Potentilla indica , Potentilla micrantha , Potentilla multifida , Potentilla nivea , Potentilla norvegica, Potentilla palustris , Potentilla peduncular is, Potentilla reptans .Potentilla salesoviana, Potentilla stenophylla, Potentilla tridentata.Rosa abietina,Rosa abyssinica, Rosa acicularis,Rosa agrestis, Rosa alba, Rosa alba x Rosa corymbifera, Rosa altaica, Rosa arkansana, Rosa arvensis, Rosa banksiae.Rosa beggeriana, Rosa blanda, Rosa bracteata, Rosa brunonii, Rosa caesia, Rosa californica, Rosa canina.Rosa Carolina, Rosa chinensis, Rosa cinnamomea, Rosa columnifera, Rosa cotymbifera,Rosa cymosa,Rosa davurica, Rosa dumalis, Rosa ecae, Rosa eglanteria, Rosa elliptica, Rosa fedtschenkoana, Rosa foetida, Rosa foliolosa, Rosa gallica, Rosa gallica x Rosa dumetorum, Rosa gigantea, Rosa glauca, Rosa helenae, Rosa henryi, Rosa hugonis, Rosa hybrid cultivar, Rosa inodora, Rosa jundzillii, Rosa laevigata, Rosa laxa, Rosa luciae, Rosa majalis, Rosa marretii, Rosa maximowicziana, Rosa micrantha, Rosa mollis, Rosa montana, Rosa moschata, Rosa moyesii, Rosa multibracteata, Rosa multiflora, Rosa nitida, Rosa odorata, Rosa palustris, Rosa pendulina, Rosa persica, Rosa phoenicia, Rosa platyacantha, Rosa primula, Rosa pseudoscabriuscula, Rosa roxburghii, Rosa rubiginosa, Rosa rugosa, Rosa sambucina, Rosa sempervirens, Rosa sericea, Rosa sertata, Rosa setigera, Rosa sherardii, Rosa sicula, Rosa spinosissima, Rosa stellata, Rosa stylosa, Rosa subcanina, Rosa subcollina, Rosa suffulta, Rosa tomentella, Rosa tomentosa, Rosa tunquinensis, Rosa villosa, Rosa virginiana, Rosa wichurana, Rosa willmottiae, Rosa woodsii; Rosa x damascena, Rosa x fortuniana, Rosa x macrantha, Rosa xanthina, Rosa sp., Rubus alceifolius, Rubus allegheniensis, Rubus alpinus, Rubus amphidasys, Rubus arcticus, Rubus argutus, Rubus assamensis, Rubus australis, Rubus bifrons, Rubus caesius, Rubus caesius x Rubus idaeus, Rubus canadensis, Rubus canescens, Rubus caucasicus, Rubus chamaemorus, Rubus corchorifolius, Rubus crataegifolius, Rubus cuneifolius, Rubus deliciosus, Rubus divaricatus, Rubus ellipticus, Rubus flagellaήs, Rubus fruticosus, Rubus geoides, Rubus glabratus, Rubus glaucus, Rubus gunnianus, Rubus hawaiensis, Rubus hawaiensis x Rubus rosifolius, Rubus hispidus, Rubus hochstetterorum, Rubus humulifolius, Rubus idaeus, Rubus lambertianus, Rubus lasiococcus, Rubus leucodermis, Rubus lineatus, Rubus macraei, Rubus maximiformis, Rubus minusculus, Rubus moorei, Rubus multibracteatus, Rubus neomexicanus, Rubus nepalensis, Rubus nessensis, Rubus nivalis, Rubus niveus, Rubus nubigenus, Rubus occidentalis, Rubus odoratus, Rubus palmatus, Rubus parviβorus, Rubus parvifolius, Rubus parvus, Rubus pectinellus, Rubus pedatus, Rubus pedemontanus, Rubus pensilvanicus, Rubus phoenicolasius, Rubus picticaulis, Rubus pubescens, Rubus rigidus, Rubus robustus, Rubus roseus, Rubus rosifolius, Rubus sanctus, Rubus sapidus, Rubus saxatilis, Rubus setosus, Rubus spectabilis, Rubus sulcatus, Rubus tephrodes, Rubus trianthus, Rubus tricolor, Rubus triβdus, Rubus trilobus, Rubus trivialis, Rubus ulmifolius, Rubus ursinus, Rubus urticifolius, Rubus vigorosus, Rubus sp. JPM-2004, Sanguisorba albiflora, Sanguisorba alpina, Sanguisorba ancistroides, Sanguisorba annua, Sanguisorba canadensis, Sanguisorba filiformis, Sanguisorba hakusanensis, Sanguisorba japonensis, Sanguisorba minor, Sanguisorba obtusa, Sanguisorba officinalis, Sanguisorba parviflora, Sanguisorba stipulata, Sanguisorba tenuifolia, Sarcopoterium spinosum, Sibbaldia procumbens, Sieversia pentapetala, Sieversia pusilla, Taihangia rupestris,
Tetraglochin cristatum, Waldsteinia fragarioides, Waldsteinia geoides, Adenostoma fasciculatum, Adenostoma sparsifolium, Aruncus dioicus, Cercocarpus betuloides, Cercocarpus ledifolius, Chamaebatiaria millefolium, Chamaerhodos erecta, Gillenia stipulata, Gillenia trifoliata, Holodiscus discolor, Holodiscus microphyllus, Lyonothamnus βoribundus, Neillia affinis, Neillia gracilis, Neillia sinensis, Neillia sparsiflora, Neillia thibetica, Neillia thyrsiflora, Neillia uekii, Neviusia alabamensis, Physocarpus alternans, Physocarpus amnrensis, Physocarpus capitatus, Physocarpus malvaceus, Physocarpus monogynus, Physocarpus opulifolius, Purshia tridentata, Rhodotypos scandens, Sorbaria arborea, Sorbaria sorbifolia, Spiraea betulifolia, Spiraea cantoniensis, Spiraea densiβora, Spiraea japonica, Spiraea nipponica, Spiraea x vanhouttei, Spiraea sp., Stephanandra chinensis, Stephanandra incisa and Stephanandra tanakae.
Particularly preferred Rόsaceae genera include: Malus, Pyrus, Cydonia, Prunus, Eriobotrya, and Mespilus.
Particularly preferred Rosaceae species include: Malus domestica, Malus sylvestris, Pyrus communis, Pyrus pyrifolia, Pyrus bretschneideri, Cydonia oblonga, Prunus salicina, Prunus cerasifera, Primus persica, Eriobotrya japonica, Prunus dulcis, Prunus avium, Mespilus germanica and Prunus domestica.
A particularly preferred Rosaceae genus is Malus.
A particularly preferred Malus species is Malus domestica.
Particularly preferred Malus species/cultivars include Malus sieversii 93.051 GO 1-048, Malus aldenhamii, Malus pumila Niedzwetzkyana, Malus x domestica cv. 'Prairiefire', Malus x domestica cv. 'Geneva', Malus sieversii 92.103 30-312.
A particularly preferred Malus cultivar is Malus x domestica niedwetzkyana.
DETAILED DESCRIPTION
The term "comprising" as used in this specification and claims means "consisting at least in part of; that is to say when interpreting statements in this specification and claims which include "comprising", the features prefaced by this term in each statement all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner. In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
The term "MYB transcription factor" is a term well understood by those skilled in the art to refer to a class of transcription factors characterised by a structurally conserved DNA binding domain consisting of single or multiple imperfect repeats.
The term "A MYB transcription with an R2R3 DNA binding domain" is a term well understood by those skilled in the art to refer to MYB transcription factors of the two-repeat class.
Polynucleotides and fragments
The term "polynucleotide(s)," as used herein, 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 preferably at least 15 nucleotides in length. The fragments of the invention preferably comprises at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 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. The term "fragment" in relation to promoter polynucleotide sequences is intended to include sequences comprising cis-elements and regions of the promoter polynucleotide sequence capable of regulating expression of a polynucleotide sequence to which the fragment is operably linked.
5 Preferably fragments of promoter polynucleotide sequences of the invention comprise at least 46, more preferably at least 69, more preferably at least 92, more preferably at least 1 15, more preferably at least 138, more preferably at least 150, more preferably at least 200, more preferably at least 300, more preferably at least 400, more preferably at least 500, more preferably at least 600; more preferably at least 700, more preferably at least 800, more
10 preferably at least 900, more preferably at least 1000, more preferably at least 1 100, more preferably at least 1200, more preferably at least 1300, more preferably at least 1400, more preferably at least 1500, more preferably at least 1600 and most preferably at least 1700 contiguous nucleotides of the specified polynucleotide. Fragments of the promoter polynucleotide sequences can be used to control expression of an operably linked polynucleotide
15 in a transgenic plant cells or plants.
The term "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 template. Such a primer is preferably at least 5, more preferably at least 6,
ZO more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 1 1, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.
>5
The term "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. Preferably such a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more
30 preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length. The term "derived from" with respect to polynucleotides of the invention being "derived from" a particular genera or species, means that the polynucleotide has the same sequence as a polynucleotide found naturally in that genera or species. The polynucleotide which is derived from a genera or species may therefore be produced synthetically or recombinantly.
Polypeptides and fragments
The term "polypeptide", as used herein, 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. The polypeptides 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.
The term "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.
The term "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.
The term "derived from" with respect to polypeptides disclosed being derived from a particular genera or species, means that the polypeptide has the same sequence as a polypeptide found naturally in that genera or species. The polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.
Variants
As used herein, the term "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 polynucleotides and polypeptides possess biological activities that are the same or similar to those of the inventive polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.
Polynucleotide variants
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 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, more preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, more preferably at least 200 nucleotide positions, more preferably at least 300 nucleotide positions, more preferably at least 400 nucleotide positions, more preferably at least 500 nucleotide positions, more preferably at least 600 nucleotide positions, more preferably at least 700 nucleotide positions, more preferably at least 800 nucleotide positions, more preferably at least 900 nucleotide positions, more preferably at least 1000 nucleotide positions, more preferably at least 1 100 nucleotide positions, more preferably at least 1200 nucleotide positions, more preferably at least 1300 nucleotide positions, more preferably at least 1400 nucleotide positions, more preferably at least 1500 nucleotide positions, more preferably at least 1600 nucleotide positions, more preferably at least 1700 nucleotide positions and most preferably over the entire length of the specified polynucleotide sequence.
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 on the world wide web at 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 identity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn
The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities = ".
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). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,I. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276- 277) which can be obtained from the world wide web at http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The 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/.
Alternatively the GAP program, which computes an optimal global alignment of two sequences without penalizing terminal gaps, may be used to calculate sequence identity. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
Sequence identity may also be calculated by aligning sequences to be compared using Vector NTI version 9.0, which uses a Clustal W algorithm (Thompson et al., 1994, Nucleic Acids Research 24, 4876-4882), then calculating the percentage sequence identity between the aligned sequences using Vector NTI version 9.0 (Sept 02, 2003 ©1994-2003 InforMax, licenced to Invitrogen).
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. Such sequence similarity with respect to polynucleotides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI on the world wide web at ftp://ftp.ncbi.nih.gov/blast/.
The similarity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx
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 polynucleotide sequences preferably exhibit an E value of less than ϊ x 10 "l0 more preferably less than 1 x 10 "20, more preferably less than 1 x 10 "30, more preferably less than 1 x 10 "40, more preferably less than 1 x 10 "5^ more preferably less than 1 x 10 "60 more preferably less than 1 x 10 "70 more preferably less than 1 x 10 "8^ more preferably less than 1 x 10 "90 and most preferably less than 1 x 10 "l0° when compared with any one of the specifically identified sequences.
Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions.
The term "hybridize under stringent conditions", and grammatical equivalents thereof, 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. 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.
With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al, Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al, 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al, Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84: 1390). 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 650C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in I X SSC, 0.1% SDS at 650 C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 650C. With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)0 C.
With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec 6;254(5037): 1497-500) 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 such as those in constructs of the invention encoding proteins to be expressed, also encompasses polynucleotides that differ from the specified sequences 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 contemplated. 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 on the world wide web at ftp://ftp.ncbi.nih.gov/blast/, via the tblastx algorithm as previously described.
Polypeptide variants
The term "variant" with reference to polypeptides encompasses naturally occurring, recombinant^ and synthetically produced polypeptides. 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 preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.
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 on the world wide web at 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/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227- 235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. Sequence identity may also be calculated by aligning sequences to be compared using Vector NTI version 9.0, which uses a Clustal W algorithm (Thompson et al., 1994, Nucleic Acids Research 24, 4876-4882), then calculating the percentage sequence identity between the aligned polypeptide sequences using Vector NTI version 9.0 (Sept 02, 2003 ©1994-2003 InforMax, licenced to Invitrogen).
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. Such 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 on the world wide web at ftp://ftp.ncbi.nih.gov/blast/. The similarity of polypeptide sequences may be examined using the following unix command line parameters:
bl2seq -i peptideseql -j peptideseq2 -F F -p blastp
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 "l 2, more preferably less than 1 x 10 ~ 15, more preferably less than 1 x 10 ~18, more preferably less than 1 x 10 "2 I, more preferably less than 1 x 10 "30, more preferably less than 1 x 10 "4^ more preferably less than 1 x 10.'50, 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 100 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.
Conservative substitutions of one or several amino acids of a described 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).
Constructs, vectors and components thereof
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 a promoter polynucleotide such as a promoter polynucleotide of the invention including 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 synthetic or 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.
The term "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.
The term "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, such as a promoter polynucleotide sequence of the invention, 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.
The term "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. When inserted into a genetic construct, a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences. The term "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.
The term "noncoding region" includes 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 sequences may include elements required for transcription initiation and termination and for regulation of translation efficiency. The term "noncoding" also includes intronic sequences within genomic clones.
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.
The term "promoter" refers to a polynucleotide sequence capable of regulating the expression of a polynucleotide sequence to which the promoter is operably linked. Promoters may 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.
The applicants have isolated a promoter polynucleotide sequence from apple and demonstrated that a sequence motif in the promoter, when present in more than one copy, such as when included as part of a minisatellite repeat unit, strongly effects transcription of an operably linked polynucleotide in plants. The applicants have also shown that a promoter sequence comprising more than one copy of the motif, such as when included as part of a minisatellite repeat unit, is positively regulated by a MYB transcription factor resulting in a significant increase in expression driven by the promoter.
The invention also provides fragments and variants of the promoter polynucleotide capable of such regulation of expression. The invention provides genetic constructs and vectors comprising the promoter polynucleotide sequences, and transgenic plant cells and transgenic plants comprising the promoter polynucleotide sequence, genetic constructs, or vectors of the invention. . . The invention also provides methods for producing plants with modified gene expression and modified phenotype. The invention further provides plants produced by the methods of the invention.
Methods for isolating or producing polynucleotides
The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polynucleotides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et ah, Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polynucleotides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
Further methods for isolating polynucleotides of the invention, or useful in the methods of the invention, include use of all or portions, of the polynucleotides set forth herein as hybridization probes. The technique of hybridizing labeled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic. 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 550C) in 1. 0 X SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0. 5 X SSC, 1% (w/v) sodium dodecyl sulfate, at 600C. 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.
The 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 polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence, untranslated sequences, and promoter sequences. Such methods include PCR-based methods, 5'RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56), genome walking using a Genome Walker™ kit (Clontech, Mountain View, California), 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 polynucleotide. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
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. Additionally when 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.
The promoter sequences disclosed may be further characterized to identify other fragments, such as cis-elements and regions, capable of regulating to expression of operably linked sequences, using techniques well-known to those skilled in the art. Such techniques include 5' and/or 3' deletion analysis, linker scanning analysis and various DNA footprinting techniques (Degenhardt et al., 1994 Plant Cell:6(8) 1123-34; Directed Mutagenesis: A Practical Approach IRL Press (1991)). Fragments include truncated versions of longer promoter sequences which may terminate (at the 3' end) at or close to the transcriptional start site. Methods for identifying the transcription start site of a promoter are well-known to those skilled in the art (discussed in Hashimoto et al., 2004, Nature Biotechnology 22, 1 146-1 149).
The techniques described above may be used to identify a fragment that defines essential region of the promoter that is able to confer the desired expression profile. The essential region may function by itself or may be fused to a core promoter to drive expression of an operably linked polynucleotide.
The core promoter can be any one of known core promoters such as the Cauliflower Mosaic
Virus 35S or 19S promoter (U.S. Pat. No. 5,352,605), ubiquitin promoter (U.S. Pat. No. 5,510,474) the IN2 core promoter (U.S. Pat. No. 5,364,780) or a Figwort Mosaic Virus promoter (Gruber, et al. "Vectors for Plant Transformation" Methods in Plant Molecular Biology and Biotechnology) et al. eds, CRC Press pp.89-1 19 (1993)).
Promoter fragments can be tested for their utility in driving expression in any particular cell or tissue type, or at any particular developmental stage, or in response to any particular stimulus by techniques well-known to those skilled in the art. Techniques include operably-linking the promoter fragment to a reporter or other polynucleotide and measuring reporter activity or polynucleotide expressions in plants. Some of such techniques are described in the Examples section of this specification.
Methods for identifying variants
Physical methods
Variant polynucleotides may be identified using PCR-based methods (Mullis et al, Eds. 1994 The Polymerase Chain Reaction, Birkhauser).
Alternatively library screening methods, well known to those skilled in the art, may be employed (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). When identifying variants of the probe sequence, hybridization and/or wash stringency will typically be reduced relatively to when exact sequence matches are sought.
Computer-based methods
Polynucleotide and polypeptide variants, 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 1 1-16, 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 the world wide web at ftp://ftp.ncbi.nih.gov/blast/, or from the National Center for Biotechnology Information (NCBI),
5 National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA. 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
0 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.
5
The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al.. Nucleic Acids Res. 25: 3389-3402, 1997.
The "hits" to one or more database sequences by a queried sequence produced by BLASTN, !0 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.
>5 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
\0 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. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN,
BLASTP, BLASTX, tBLASTN or tBLASTX algorithm. Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673- 4680, at the world wide web at http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or T- COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. MoI. Biol. (2000) 302: 205-217) or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. MoI. Evol. 25, 351).
Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. 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 (Bairoch 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. The PROSITE database (www.expasy.org/prosite) 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.
Methods for producing constructs and vectors
The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides disclosed, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or particularly plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined. Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987).
Methods for producing host cells comprising constructs and vectors
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 al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides. 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
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. Plants comprising such cells also form an aspect of the invention.
Methods for transforming plant cells, plants and portions thereof with polynucleotides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual. Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.
The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al, 1999, Plant Cell Rep. 18, 572); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al, 1996, Plant Cell Rep. 15, 1996, 877); tomato (US Patent Serial No. 5, 159, 135); potato (Kumar et al, 1996 Plant J. 9, : 821); cassava (Li et al, 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et άl, 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al, 1985, Science 227, 1229); cotton (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); perennial ryegrass (Bajaj et al, 2006, Plant Cell Rep. 25, 651); grasses (US Patent Nos. 5, 187, 073, 6. 020, 539); peppermint (Niu et al, 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al, 1995, Plant Sci.104, 183); caraway (Krens et al, 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US Patent Nos. 5, 416, 01 1 ; 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); and cereals (US Patent No. 6, 074, 877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(1 ):45-51 ); Prunus (Ramesh et al., 2006, Plant Cell Rep. 25(8):821-8; Song and Sink 2005, Plant Cell Rep. 2006; 25(2): 1 17-23; 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.
Methods for genetic manipulation of plants
A number of strategies for genetically manipulating plants are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant MoI Biol, 48, 297). For example, 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. Strategies may also be designed to increase expression of a polynucleotide/polypeptide in response to an external stimuli, such as an environmental stimuli. Environmental stimuli may include environmental stresses such as mechanical (such as herbivore activity), dehydration, salinity and temperature stresses. 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 or to reduce expression of a polynucleotide/polypeptide in response to an external stimuli. Such strategies are known as gene silencing strategies.
Genetic constructs for expression of genes in transgenic plants typically include promoters, such as promoter polynucleotides of the invention, for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.
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 zin gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solatium tuberosum PI-II terminator.
Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase {bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene ( hpt) for hygromycin resistance. Use of genetic constructs comprising 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. 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., Spangenbert. 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 may include an antisense copy of a polynucleotide. 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.,
5'GATCTA 3' (coding strand) 3'CTAGAT 5' (antisense strand)
3'CUAGAU 5' mRNA 5'GAUCUCG 3' antisense RNA
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.,
5'-GATCTA TAGATC-3' 3'-CTAGAT ATCTAG-5'
The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually 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.
The term genetic construct as used herein also includes small antisense RNAs and other such polynucleotides useful for 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). In some cases 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 sequence operably-linked to promoter 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.
Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (Mclntyre, 1996, Transgenic Res, 5, 257)
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.
Plants
The term "plant" is intended to include a whole plant or any part of a plant, propagules and progeny of a plant. The term 'propagule' means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.
A "transgenic" or transformed" 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 or transformed plant or from a different species. A transformed plant includes a plant which is either stably or transiently transformed with new genetic material.
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. Plants resulting from such standard breeding approaches also form part of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the promoter polynucleotide sequence of SEQ ID NO: 5, showing the position of the repeat motifs (1 , 2, 3A, 3B, 4, 5 and 6), the microsatellite (microsat) and several restriction enzyme sites.
Figure 2 shows a schematic representation of the MdMYBlO R| promoter from the white-fleshed cultivar with a single repeat motif (1) and the microsatelite (MS). The figure also shows schematic representation of the structure and location of the additional repeat unit composed of repeat units 2, 3a, 3b, 4, 5 and 6 found in the promoter of the red-fleshed cultivar R6, relative to the promoter from the white-fleshed cultivar. Example phenotypes for the MdMYBlO Ri and R6 promoter versions are shown to the left, Malus x domestica Royal Gala (i) and Mctlus x domestica niedzwetzkyana (ii).
Figure 3 shows the portion of the sequence of the promoter from the red-fleshed apple cultivar including repeat motifs 1 , 2, 3a. 3b, 4, 5 and 6 and the microsatellite region.
Figure 4 shows /nms-activation of the promoters from white-fleshed (Ri) and red-fleshed (R6) cultivars by the MdMYBlO gene in transient tobacco transformation assays. Both promoters were infiltrated with and without MdMYBlO. Error bars shown are ± S. E. of the means of 6 replicate experiments.
Figure 5 shows that amplification of a PCR product comprising the minsatellite motif serves as a marker that distinguishes white-fleshed and red-fleshed apple cultivars. A total of 87 cultivars were screened using the PCR primer pair described in Example 3. PCR products were separated on 0.9% agarose gels and stained with ethidium bromide. The figure shows the PCR amplification obtained over a subset of 10 apple varieties. Two alleles were found: a 496 bp fragment corresponding to the promoter of SEQ ID NO: 5, which was only present in red flesh varieties (lanes 1-6) and was absent in white- fleshed varieties (lanes 7-10), and a 392 bp allele present in both types of fruit. Red-fleshed varieties: . 1 : open-pollinated (OP) Malus 'Mildew Immune Seedling' 93.051 G01-048; 2: M. -^purpurea 'Aldenhamensis'; 3: M. pumila var. niedzwet∑kyana; 4: M. 'Prairifire'; 5: OP M. pumila var. niedzwetzkyana 'Geneva'; 6: OP M. x domestica 'Pomme Grise' 92.103 30-312; 7: M. x domestica 'Granny Smith'; 8: M. x domestica 'Royal Gala'; 9: M. x domestica 'Fuji'; 10: M. x domestica 'Braeburn'.
Figure 6 shows the native apple promoter containing the minisatellite induces ectopic anthocyanin accumulation, (a) Red colouration has developed around the infiltration site in the leaves of Nicotiana tabacum 8 days after transient transformation with R6MdMYBlO (i) and 35S:MdMYB10 (ii) but not with R|. All three were co-infiltrated with 35S:MdbHLH3. (b) Regenerating Royal Gala apple callus transformed with R6:MdMYB10. R| = native promoter from Malus domestica 'Royal Gala'. R6 = native promoter from Malus x pumila var. niedzwetzkyana.
Figure 7 shows the interaction of the native apple promoters and MdMYBlO in the dual luciferase transient tobacco assays. To compare the transactivation activity of the apple promoters to 35S, these were co-infiltrated with the R| and R6 promoter-luciferase fusions. The results provide a measure for the potential activity of the apple promoters and they show a significant increase in the case of the Rό-driven MdMYBlO. Ri = native promoter from Malus domestica 'Royal Gala'. R6 = native promoter from Malus x pumila var. niedzwetzkyana.
Figure 8 shows the number of repeat units affects the transactivation rate, (a) Cartoon (not drawn to scale) of the different promoters with repeat units ranging from zero (R0) to six (R6).
The two native promoters from apple are marked, Ri as from Malus x domestica 'Royal gala' and R6 from Mains x pumila var. niedzwetzkyana. The position of the repeat units (in black) relative to the microsatellite (grey diagonal box) is shown. R)+ is differentiated with grey vertical shading to represent the substituted sequence replacing the spatial effect of the minisatellite. (b) Results of R0 to R6 promoters co-infiltrated with 35S:MdMYB10 alone (light grey bars) and with 35S:MdMYB10 and 35SMdbHLH3 (dark grey bars). Error bars shown are means ± S. E. of 6 replicate reactions.
Figure 9 shows identification of areas of the promoter critical to transactivation by deletion study, (a) Cartoon (not drawn to scale) of the different promoter deletions of Ri5 (i) and R6, (ii), denoted as Δa - Δd. Deleted areas are shown in white with dotted lines and the relative positions of the repeat unit Ri to the microsatellite and minisatellite are displayed, (b) Corresponding data from promoter deletion studies with luciferase fusions of Ri, (i) and (ii), and R6, (iii) and (iv), co- infiltrated with MdMYBlO, (i) and (iii) respectively (pale grey bars) and with MdMYBlO and MdbHLH3, (ii) and (iv) respectively (dark grey bars). Error bars shown are means ± S. E. of 6 replicate reactions.
Figure 10 shows a schematic representation of the cloning of the minsatellite repeat unit (copies 1-6) from the apple MdMYBlO R6 promoter (MdMYBlO long) into the MYBlO promoter from pear (PcMYBlO(GP)) to produce the chimeric promoter PcMYB 10R6(GP-R6). The MdMYBlO promoter from white-fleshed apple (MdMYBlO short) is included in the figure for reference.
The position of the restriction sites (Dral and Bsgl) and PCR priming sites (CB02 and REl 61) is also shown.
Figure 1 1 shows the effect of MdMYBlO genomic and 35S:PcMYB10 constructs on luciferase reporter gene driven by PcMYBlO promoter containing or not the MdMYB 10-promoter R6 repeats. Activity is expressed as a ratio of the Luciferase (LUC) to the CaMV35s-Renilla (REN) activities. Error bars represent the standard error (SE) for 4 replicates. All the promoter sequences were fused to the luciferase reporter and are abbreviated as follows: DFR, Arabidopsis DFR promoter; MdIOs, MdMYBlO Rl promoter; MdlOR6, MdMYBlO R6 promoter; PcIOS, PcMYBIORl promoter and PclOR6, PcMYB 10R6 promoter. The transcription factor constructs are all driven by the CaMV35S promoter and are as follows: MdMlO, MdMYBlO; PcMlO, PcMYBlO; b33, MdbHLH33 and b2, Arabidopsis thaliana bHLH2. Figure 12 shows an alignment between the sequences of the MYBlO promoters from white- fleshed apple and pear and highlights with, underligning, the conserved 23 bp repeat motif.
EXAMPLES
The invention will now be illustrated with reference to the following non-limiting examples.
Example 1: Isolation of the full length MdMYBlO promoter polynucleotides from white- fleshed and red-fleshed apple cultivars, and identification of additional elements within the promoter from the red-fleshed cultivar.
Isolation of genomic DNA
Genomic DNA was isolated from the leaves of a white-fleshed apple cultivar (Malm domestica Royal Gala) and from the leaves of a red-fleshed apple cultivar (Mains x piimila niedwetzkyana) using a Qiagen DNeasy Plant Mini Kit, according to the manufacturers instructions (Qiagen, Valencia, California).
Promoter isolation
A 1.7-1.8 Kb region of the upstream regulatory region of the MdMYBlO gene was isolated from the DNA of both the white-fleshed and the red-fleshed cultivar by PCR genome walking using a GenomeWalker™ kit (Clontech, Mountain View, California), following .the manufacturers instructions.
The isolated promoters were sequenced by standard techniques. The sequence of the promoter from the red-fleshed cultivar is shown in SEQ ID NO: 5. The sequence of the promoter from the white-fleshed cultivar is shown in SEQ ID NO: 8.
The sequence of the MdMYBlO polypeptide is shown in SEQ ID NO: 6. The polynucleotide sequence (cDNA) encoding the MdMYBlO polypeptide is shown in SEQ ID NO: 7.
By comparing the sequences of the promoters from white-fleshed and red-fleshed apple cultivars the applicants identified a 23-base pair sequence motif found in both promoters. In the promoter from the white-fleshed cultivar, the motif is present as a single copy (with a lbp difference versus the motif in the promoter from the red-fleshed cultivar). In the promoter from the red- fleshed cultivar the motif is present at a corresponding position, but in addition, the motif is duplicated in five tandem repeats to form a minisatellite repeat unit.
The sequence of the repeat motif is shown in SEQ ID NO: 1.
The sequence of the minisatellite unit comprising five copies of the repeat motif is shown in SEQ ID NO: 2.
Figure 1 shows the sequence of the promoter from the red-fleshed variety as shows the position of the repeated motifs. The minisatellite unit precedes a di-nucleotide microsatellite found in both promoters.
The sequence of the microsatellite is shown in SEQ ID NO: 3.
Figure 2 shows a schematic representation of promoter from the white-fleshed cultivar and shows the relative position and structure of the additional minisatellite repeat unit found in the promoter of the red-fleshed cultivar. Minisatellites, similar to these, have been shown to have an effect on transcriptional regulation in humans (Kominato et al, (1997). J. Biol. Chem. 272, 25890, Lew et al, (2000). Proc. Natl. Acad. Sci. U. S. A. 97, 12508 and to produce phenotypic alteration in Saccharomyces cerevisiae fVerstrepen et al, (2005f Nat. Genet. 37, 986/
Example 2: Demonstration of regulation of expression of operably linked polynucleotide sequences by the promoter polynucleotides of the invention.
Dual Lucifer ase Assay of Transiently Transformed Tobacco Leaves
The promoter sequences for MdMYBlO from the red-fleshed and white-fleshed cultivars (SEQ ID NOs: 4 and 5 respectively) were separately inserted into the cloning site of pGreen 0800-LUC (Hellens et al., 2005, R. P. Hellens, A. C. Allan, E. N. Friel EN, K. Bolitho, K. Grafton, M. D. Templeton, S. Karunairetnam, W. A. Laing, Plant Methods 1:13). In the same construct, 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. The promoter-LUC fusion was used in transient transformation by mixing 100 μl of Agrobacterium strain GV3101 (MP90) transformed with the reporter cassette with or without another Agrobacterium culture (900 μl) transformed with a cassette containing MdMYBlO fused to the 35S promoter. Nicotiana tabacum 'Samsun' plants were grown until at least 6 leaves were available for infiltration with Agrobacterium. A 10 μl loop of confluent bacterium were re- suspended in 10 ml of infiltration media (10 mM MgCb, 0.5 μM acetosyringone), to an OD6Q0 of 0.2, and incubated at room temperature without shaking for 2 h before infiltration. Approximately 150 μl of this Agrobacterium mixture was infiltrated at six points into a young leaf of N. tabacum and transient expression was analysed 3 days after inoculation. Six technical replicates of 3 mm 0 leaf discs were excised from each plant using a leaf hole-punch and buffered in Phosphate Buffer Saline (PBS). Plate-based assays were conducted using a Berthold Orion Microplate Luminometer (Berthold Detection Systems, Oak Ridge, TN, USA) according to the manufacturer's specifications for the dual luciferase assay, using the Dual Glow assay reagents (Promega, Madison, WI) for firefly luciferase and Renilla luciferase. Luminescence was calculated using Simplicity version 4.02 software (Berthold Detection Systems).
The results, as shown in Figure 4, show that the promoter (R6) from the red-fleshed cultivar containing the minisatellite repeat unit drives expression of the operably linked sequence encoding luciferase at 7 times the level of expression driven by the promoter (R|) from the white-fleshed cultivar (from which the minisatellite repeat unit is absent) when the MdMYBlO protein is also expressed. This result demonstrates the significance of the extra sequence present in R6 promoter (including additional copies of the repeat motif) from the red-fleshed variety.
The results also show that co-expression of the MdMYBlO transcription factor results in a 10- fold increase in expression of the luciferase sequence that is operably linked to the promoter (R6) from the red-fleshed cultivar. The effect of MdMYBlO from the white-fleshed cultivar is much smaller. This result shows that the promoter polynucleotide of the invention is positively regulated by the MYB transcription factor MdMYBlO. Example 3: The presence of the minisatellite unit in the promoter of the invention is consistently associated with red-flesh in naturally occurring red-fleshed apple varieties.
Minisatellite region PCR amplification and sequencing
5
The fruit flesh (cortex) of most apple cultivars is white or off-white in colour. The skin is usually green or red, the skin reddening in response to developmental, hormonal and light signals (Ubi et a!., 2006, Plant Sci. 170, 571 ). There are, however, a number of high anthocyanin, red- fleshed apples, including Malus x pumila niedzwetzkyana, originating from the wild-apple
0 forests of Khazakhstan.
In apple, anthocyanin accumulation is specifically regulated by MdMYBlO, with MdMYBlO transcript levels greatly elevated in red-fleshed varieties (Espley et al., 2007, Plant J 49, 414).
5 Genomic DNA samples from several red-fleshed and white-fleshed apple cultivars listed in the Table 1 below were supplied by Charles J Simon and Philip Forsline, Agricultural Research Services USDA.
Apple genomic DNA from 19 cultivars was amplified using a pair of PCR primers located in the
»0 MdMYBlO promoter (forward: 5 '-GGAGGGGAATGAAGAAGAGG-S ' - SEQ ID NO: 9; reverse: 5'-TCCACAGAAGCAAACACTGAC-S' - SEQ ID NO: 10). PCR reactions were carried out in 16.5 μl volume containing Ix PCR buffer mix (Invitrogen, Carlsbad, California),
1.3 mM MgCl2, 100 μM of each dNTP, 0.72 % formamide, 10 μM of each primer, 0.5 U of
Platinum® Taq DNA polymerase (Invitrogen) and 2 ng of genomic DNA. PCR amplifications
15 were performed in a Hybaid PCR Express Thermal Cycler (Thermo Electron Corporation,
Waltham, Massachusetts) with conditions as follows: 940C for 2 min 45 sec followed by 40 cycles at 940C for 55 sec, 55°C for 55 and 720C for 1 min 39 sec, and a final elongation at 720C for 10 min. The PCR products obtained were cloned using the TOPO TA cloning® kit
(Invitrogen). Four clones were sequenced for each PCR product. The sequences were aligned
)0 using Vector NTI (Invitrogen). Association of the minisatellite with the red-fleshed phenotype
Previously we have shown that MdMYBlO is linked to the red flesh and red foliage phenotype in apple (Chagne et al, 2007, BMC Genomics, 8, 212). Further, PCR amplification of the promoter region from red and white-fleshed varieties consistently showed that the R6 minisatellite was amplified in all the red phenotypes (Figure 3). We determined the association of the repeat motif with the red-fleshed phenotype by sequencing the region encompassing the minisatellite motif over 19 diverse apple varieties (11 red and 8 white flesh; Table 1). A number of sequence variations were found in the upstream region, but only the minisatellite polymorphism is perfectly associated with the elevated accumulation of anthocyanins that causes red flesh and red foliage. The same region was PCR-amplified from a further set of 68 white-fleshed apple cultivars and wild accessions taken from two collections of Malus species, and in each case the product corresponding to the minisatellite motif was absent (data not shown). All the white- fleshed versions tested contained only the Ri version whilst the red-fleshed versions contained both Ri and R6Or R6OnIy.
Table 1
Accession Flesh colour G/T SNP Pos 81 Minisatellite motif A/T SNP Pos 448
Malus x domestica 'Babine'* Red GG R1R6 AT
Malus x domestica 'Okanagan'* Red GG R1R6 AT
Malus x domestica 'Simcoe'* Red GG R6R6 TT
Malus x domestica 'Slocan'* Red GT R1R6 AT
Malus maηorensis ' Formosa '* Red GT R1R6 AT
Malus sieversii 629319* Red GG R6R6 TT
Malus sieversu FORM 35 (33-01)* Red GT R1R6 AT
Malus sieversu 01 P22* Red GG R6R6 TT
Malus sieversu 3563 q* Red GG R6R6 TT
Malus Aldenhamii Red TT R1R6 AT
Malus x domestica 91136 B6-77 Red GT R1R6 AT
Malus x domestica 'Close' White GT R1R1 AT
Malus x domestica 'Mr Fitch' White TT R1R1 AA
Malus x domestica 'Guldborg' White GT R1R1 AT
Malus x domestica 'Alkmene' White TT R1R1 AA
Malus x domestica 'Red Melba' White TT R1R1 AA
Malus x domestica 'Rae Ime' White GG R1R1 TT
Malus x domestica 'Lady Williams' White TT R1R1 AA
Malus x domestica 'Granny Smith' White GT R1R, AA
Association test (r2) 0185 1 0491
"Rι" refers to the absence of the minisatellite unit as found in the promoter from the white-fleshed Royal Gala cultivar. "R6" refers to the presence of the minisatellite unit as found in the promoter from the red-fleshed Malus x piimila niedwetzkyana cultivar.
Given that the single repeat unit is present in the promoter from the white-fleshed, the presence of additional repeat units in the promoter from the red-fleshed cultivar are likely to account for the known increased expression level of MdMYlO and resulting anthocyanin accumulation red- fleshed apple cultivars.
Example 4: Expression of the MdMYBlO transcription factor driven by the promoter of the invention results in anthocyanin production in transiently transformed tobacco.
Previous studies have shown that when MdMYBlO was fused to 35S and co-infiltrated into N tabacum with a 35S driven co-factor bHLH, a high level of anthocyanin pigmentation could be detected at the infiltration site (Espley et al, 2007). The applicants therefore infiltrated Nicotiana tabacum with Agrobacterium suspensions of MdMYBlO driven by the Ri and R6 promoter sequences. Ri is the native promoter from Malus domestica 'Royal Gala'. R6 is the native promoter from Malus x pumila var. niedzwetzkyana. When R6IMdMYBlO was co-infiltrated with 35S:MdbHLH3 a similar level of colouration was achieved as with 35S:MdMYB10 (Figure. 6A). The applicants were unable to detect anthocyanin accumulation with the Rι :MdMYB10 infiltration, with or without 35S:MdbHLH3.
To investigate the properties of the R6 promoter in apple, the applicants transformed Royal Gala with MdMYBlO cDNA driven by either the R6 or Ri promoters. Whilst the Rl promoter is found in Royal Gala R6 is not. It has prevously been shown that when Royal Gala is transformed with 35S:MdMYB10, red callus is produced which regenerates to produce red plants (Espley et al, 2007). The applicants observed a similar callus phenotype when Royal Gala is transformed with R6IMdMYBlO, with bright red areas on regenerating callus (Figure 6B). However, whilst 35S:MdMYB10 was capable of driving anthocyanin accumulation in the transformed callus in the absence of light, we noted that the R6IMdMYBl O transformants required light for the induction of pigmentation. No sustained pigmentation was seen on regenerating apple callus transformed with Ri iMdMYBlO. Similarly, callus transformed with an empty vector cassette showed no pigmentation. Example 5: Expression of the MdMYBlO transcription factor driven by the promoter of the invention can transactivate reporter gene expression at a level similar or higher than CaMV35S promoter driven expression of the MdMYBlO transcription factor.
5 To further investigate the effect of the promoter on MdMYB 10 transcript and predicted protein levels, the applicants repeated the assay from Example 2, replacing the 35S promoter with either the Ri or R6 promoters. Results indicated that the high transcript abundance of MdMYBlO driven by the R6 promoter enables transactivation of the reporter, particularly when the reporter is fused to R^ (Figure 7); The results show a similar level of activity to the 35S promoter. With 10 the R| luciferase fusion, R6:MdMYB10 appears to exert stronger transactivation than
35S:MdMYB10. The R| :MdMYB10 fusion did not influence transactivation to the same extent.
Example 6: The number of copies of the 23 bp repeat unit influences transcription.
[ 5 A series of constructs were built, using standard molecular biology techniques, to test the effect on transcription of the number of 23 bp repeat units present in the upstream region. These constructs were based on the native promoter sequences but with repeat units ranging from one (R i ) to six (R^) and were fused to the luciferase reporter as above (Figure 8a). To test the spatial effect that the presence of the minisatellite sequence might exert on other non-identified motifs, a
>0 further construct (Rι+) was built where the minisatellite sequence from R6 was replaced with non-specific DNA of the same length from a cloning vector (Promega, Madison, WI, USA). The results indicate a correlation between the number of repeat units and the activation of the promoter (Figure 8b). When co-infiltrated with 35S:MdMYB10 there is basal activity from both Rι and R|+ and an increasing activation from R2 to R6. There are numerous examples of the
15 relationship between the anthocyanin-regulating MYB and bHLH co-factors and it has previously been shown the dependency of MdMYBlO on a co-factor bHLH in transient assays (Espley et al, 2007). In this assay, activation for both the Ri and R6 promoters is enhanced with the addition of 35S:MdbHLH3 for all the constructs tested. Example 7: Deletion analysis of the promoter of the invention emphasises the importance of the minisatellite region, containing multiple copies of the 23 bp repeat unit, in enhancing transcription.
To define the upstream region directly responsible for transcriptional enhancement, both versions of the native promoter (Riand R^) were subjected to various sequence deletion treatments (Figure 9a). The five versions for each native promoter were fused to luciferase and co-infiltrated into tobacco with 35S:MdMYB10, +/- 35S:MdbHLH3.
When the deletion versions of R|:LUC were infiltrated with just 35S:MdMYB10, luciferase activity was barely detectable and significantly lower than the native non-deleted version (Figure 9b). Only when 35S:MdbHLH3 was co-infiltrated with 35S:MdMYB10 did luminescence rise above background. Although there is a putative bHLH binding domain at the 5' end of the isolated promoter region, when this was deleted (Ri Δa) there was still a significant increase in LUC:REN ratio with co-infiltration of the bHLH, suggesting that there may be an alternative site for bHLH binding. The R6)LUC deletions were less affected than R| with activity halved for R6Δa and R6Δb and a lesser reduction with R6Ac. With the restoration of the putative bHLH binding domain on both R|Δc and R6Ac, there is an increase in activity when 35S:MdbHLH3 is co-infiltrated.
In this assay, the R6ILUC promoters appeared to show a lesser dependence on the bHLH for increased activity although this may be due to saturation or depletion of one or other of the co- infiltrated transcription factors. For both Ri Ad and R6Ad there was barely detectable activity, with or without the bHLH, confirming the requirement of the 3' region for transactivation. The data suggests that the R6 promoter can still activate luciferase transcription in truncated form (500 bp) whereas the corresponding version of Ri (R]Ab) cannot.
Experimental procedures
Isolation of MdMYBlO upstream promoter region
For isolation of the upstream promoter region, genomic DNA was extracted from Malus x domestica 'Sciros' (Pacific Rose™, derived from a cross between 'Gala' and 'Splendour').
Nested primers were designed to the coding region of MdMYBlO; primary 5'- CACTTTCCCTCTCCATGAATCTCAAC-3 (SEQ ID NO: 18), and secondary 5'- CAGGTTTTCGTTATATCCCTCCATCTC-3 (SEQ ID NO: 19). A 1.7 Kb region of upstream DNA, immediately adjacent to the transcription start site was isolated from the genomic DNA by PCR genome walking using a Genome Walker™ kit (Clontech, Mountain View, California,
5 USA), following the manufacturers instructions. Genomic DNA was subsequently isolated from Malus x domestica 'Granny Smith', Malus x domes tica 'Royal Gala' and Malus x pumila var. niedzwetzkyana using forward and reverse primers 5'-ACCCTGAACACGTGGGAACCG-3 (SEQ ID NO: 20) and 5'-GCTAAGCTTAGCTGCTAGCAGATAAGAG-S (SEQ ID NO: 21) respectively. The PCR products were cloned using the TOPO TA cloning® kit (Invitrogen,
10 Carlsbad, California, USA) and the sequences aligned using Vector NTI (Invitrogen).
Minisatellite region PCR amplification and sequencing
Apple genomic DNA from 19 cultivars was amplified using a pair of PCR primers located in the 15 MYBlO promoter (forward: 5'-GGAGGGGAATGAAGAAGAGG-S ' [SEQ ID NO: 22]; reverse: 5'-TCCACAGAAGCAAACACTGAC-S ' [SEQ ID NO: 23]). PCR reactions were carried out in 16.5 μl volume containing Ix PCR buffer mix (Invitrogen), 1.3 mMMgC12, 100 μM of each dNTP, 0.72 % formamide, 10 μM of each primer, 0.5 U of Platinum Taq DNA polymerase (Invitrogen) and 2 ng of genomic DNA. PCR amplifications were performed in a Hybaid PCR JO Express Thermal Cycler (Thermo Electron Corporation, Waltham, MA, USA) with conditions as follows: 94°C for 2 min 45 sec followed by 40 cycles at 940C for 55 sec, 550C for 55 sec and 720C for 1 min 39 sec, and a final elongation at 720C for 10 min. The PCR products obtained were cloned using the TOPO TA cloning® kit (Invitrogen). Four clones were sequenced for each PCR product. The sequences were aligned using Vector NTI (Invitrogen). >5
Plasmid construction
Luciferase reporter constructs were derivatives of pGreen 0800-LUC (Hellens et al. 2005) in which the promoter sequence for the native MdMYBlO promoter or the deletion fragments were ?0 inserted. Native promoter sequences were PCR amplified using the primers 5' - ACCCTGAACACGTGGGAACCG - 3' (SEQ ID NO: 24) and 5' - GCTAAGCTTAGCTGCTAGCAGATAAGAG - 3' (SEQ ID NO: 25) and cloned into the multi-cloning region of pGreen 0800-LUC. Ri and R6 promoter fragments were cloned in as native promoter sequences whilst changes to the repeat frequency for the R2, R3 and R4 promoter fragments were synthesised (Geneart AG, Regensburg, Germany) and cloned into Ri using the restriction enzymes Spel and Dr a\. An inverse PCR approach was used for the R|+ construct with the inclusion of unique restriction sites (BamHl and Sad) for the cloning of non-specific DNA (from pGEM T Easy, Promega, Madison, WI, USA) using the primers 5' - GGATCCTTCTGCACGACAACATTGACAA - 3' (SEQ ID NO: 26) and 5' -
GAGCTCATGTTAGCTTTTCTATATATCGA - 3' (SEQ ID NO: 27). The pSAK construct for 35S:MdMYB10 and 35S:MdbHLH3 was as previously described (Espley et al, 2007) whilst the promoter sequences were substituted for the R| and R^MdMYBlO versions. All constructs were verified by DNA sequencing.
Transactivation analysis using transformed tobacco leaves
The promoter sequences for MdMYBlO were inserted into the cloning site of pGreen 0800-LUC (Hellens et al, 2005). In the same construct, 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. The promoter-LUC fusions were used in transient transformation by mixing 100 μl of Agrobacterium strain GV3101 (MP90) transformed with the reporter cassette with or without another Agrobacterium culture(s) (900 μl) transformed with a cassette containing MYBJO fused to the 35S, Rl or R6 promoters and MdbHLH3 fused to the 35S promoter. Nicotiana tabacum 'Samsun' plants were grown until at least 6 leaves were available for infiltration with Agrobacterium. A 10 μl loop of confluent bacterium were re- suspended in 10 ml of infiltration media (10 mM MgC12, 0.5 μM acetosyringone), to an OD600 of 0.2, and incubated at room temperature without shaking for 2 h before infiltration. Approximately 150 μl of this Agrobacterium mixture was infiltrated at six points into a young leaf of N. tabacum. Transient expression was analysed three days after inoculation. Six technical replicates of 3 mm 0 leaf discs were excised from each plant using a leaf hole-punch and buffered in Phosphate Buffer Saline (PBS). Plate-based assays were conducted using a Berthold Orion Microplate Luminometer (Berthold Detection Systems, Oak Ridge, TN, USA) according to the manufacturer's specifications for the dual luciferase assay, using the Dual Glow assay reagents (Promega) for firefly luciferase and renilla luciferase. Luminescence was calculated using Simplicity version 4.02 software (Berthold Detection Systems). Induction of anthocyanin pigmentation in tobacco
N. tabacum were grown as previously mentioned and maintained in the glasshouse for the duration of the experiment. Agrobacterium cultures were incubated as for the dual luciferase assay and separate strains containing the MdMYBlO gene fused to either the 35S, Ri or R6 promoter sequences and the MdbHLH3 gene fused to the 35S promoter were mixed (500 μl each) and infiltrated into the abaxial leaf surface. Six separate infiltrations were performed into N. tabacum leaves (two plants per treatment) and changes in colour were observed over an eight day period. To control for leaf-to-leaf variability, at least 2 leaves were infiltrated, and each leaf included positive {Agrobacterium cultures containing 33S:MdMYB10 + 35S:MdbHLH3) and negative {Agrobacterium with empty vector) controls.
Transformation of apple
The binary vector pSAK277 containing the MdMYBlO cDNA driven by the R6 or Ri promoters was transferred into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method. Transgenic Mains domestica 'Royal Gala" plants were generated by Agrobacterium-mediated transformation of leaf pieces, using a method previously reported (Yao et αl. 1995).
Example 8: Isolation of the PcMYBlO promoter from pear and identification of a sequence motif analogous to the repeat motif found in apple MdMYBlO promoters.
Isolation of the MYBlO promoter from pear
Genomic DNA was isolated from the leaves of a pear cultivar {Pyrus communis 'William's Bon Chretien') using a Qiagen DNeasy Plant Mini Kit, according to the manufacturers instructions (Qiagen, Valencia, California). Promoter sequences were isolated by PCR using the primers REl 58 (5'-ACCCTGAACACGTGGGAACCG-S', SEQ ID NO: 28) and REl 59 (5'- CTCTT ATCTGCTAGCAGCT AAGCTTAGC-3', SEQ ID NO: 29).
By comparing the sequences of the MYBlO promoter from apple (Example 1) and pear, the applicants identified presence of a 23 bp motif, in the pear promoter, very similar to that found in apple MYBlO proteins. An alignment of the MYBlO promoter from the white-fleshed apple and from pear, highlighting the 23bp motif with underligning, is shown in Figure 12.
Both the apple and pear promoters showed some positional conservation with the Rl repeat being at position -220 (from the ATG site) in apple and position -227 in pear. Similarly, the position of the microsatelite appeared to be conserved with the microsatelite in apple starting at postion -253 and in pear at -259.
The applicants identified three versions of the 23 bp element, from white-fleshed apple, red- fleshed apple and pear, as summarized in Table 2 below.
Table 2. Comparison of 23bp motifs from apple and pear, highlighting variable positions
SEQ ID NO Sequence Species found in
1 gttagactggtagctattaacaa white-feshed apple, red-fleshed apple
11 gttagactggtagctaataacaa white-fleshed apple
12 gttagaccggtagctaataacaa pear
Percent identity between the sequences is shown in Table 3 below.
Table 3. Percent identity between 23 bp motifs from apple and pear
SEQ ID NO: 1 SEQ ID NO: 11 SEQ ID NO: 12
SEQ ID NO: 1 100% 96% 91%
SEQ ID NO: 11 100% 96%
SEQ ID NO: 12 100% The high degree of conservation between these three sequences, and their conserved position within the promoters, from three different sources, strongly suggest that each of the three sequences perform the same function. 5
Example 9: Production of a chimeric promoter with altered activity by insertion of copies of a repeated motif from the MdMYBlO promoter from red-fleshed apple into the MdMYBlO promoter from pear.
0 Introduction
MdMYBlO controls the accumulation of anthocyanin in apple. Transient experiments described in the Examples above have shown that the MYBlO protein is able to auto-regulate its own promoter leading to a high level of expression of a Luciferase reporter gene driven by the long 15 version of MdMYBlO promoter (which includes the 6 repeats of a putative transcription factor binding site), when co-infiltrated with bHLH33 transcription factor. The applicants have now introduced the 6 repeats into the green pear MYBlO promoter controlling luciferase reporter gene and assessed the reporter activity in presence of PcMYBlO and MdMYBlO TFs.
>0 Materials
The green pear MYBlO promoter (SEQ ID NO: 13) was cloned in the pGreen0800LUC vector. The R6 region (SEQ ID NO: 14) of the MdMYBlO promoter was amplified using primers CB02F/RE161, digested by Dral and cloned in the PcMYBlO promoter at the blunted Bsgl site .5 (see Figure 10) to produce the recombinant chimeric promoter of SEQ ID NO: 15.
All the constructs (including MdMYBlO genomic, 35S:PcMYB10, AtbHLH2 and bHLH33 and the different LUC reporter constructs: DFR-LUC, MdMYB lOshort-LUC, MdMYB 101ong-LUC, PcMYB lOshort-LUC, PcMYB 10R6-LUC) were transformed into GV3101 by electroporation 0 and used to infiltrate Nicotiana benthaniama leaves as described previously (Hellens at al. 2006). After 5 days, leaf discs were collected and Firefly luciferase (LUC) and renillia luciferase (REN) activities were measured on a luminometer using the Dual Glow™ reagents (PROMEGA). Results
Apple and pear MYBlO constructs, in presence of bHLH33 and bHLH2 respectively, strongly activate the DFR, MdMYB 10R6 and PcMYB 10R6 promoters, and only slightly activate MdMYBlORl and PcMYBI ORl promoters. The introduction of the apple R6 repeats in the pear promoter leads to a 6-fold increase in the luciferase activity in presence of the 35S:PcMYB10 construct and an 8-fold increase in presence of the MdMYBlO genomic construct.
The pear MYBlO promoter was cloned into the pGreen0800-LUC vector (Hellens et al. 2005). The R6 region of the MdMYBlO promoter was amplified using primers CB02F (5'- CAGAAATGTTAGACTGGTAGCTATTAAC-3', SEQ ID NO: 30) and RE161 (5'- CCAGTGACGTGCATGTCTGATATCC-3', SEQ ID NO: 31), digested by Dral and cloned in the PcMYBlO promoter at the blunted Bsgl site (see Figure 10). All the constructs (including MdMYBlO genomic, 35S:PcMYB10, AtbHLH2 and MdbHLH33 and the different LUC reporter constructs: DFR-LUC, MdMYBlORl-LUC, MdMYB 10R6-LUC, PcMYB10Rl-LUC,
PcMYB 10R6-LUC) were transformed into GV3101 by electroporation and used to infiltrate Nicotiana benthaniama leaves as described previously (Hellens et al. 2006). After 4 days, leaf discs were collected and Firefly luciferase (LUC) and renillia luciferase (REN) activities were measured on a luminometer using the Dual Glow™ reagents (PROMEGA). Apple and pear MYBlO constructs, in presence of MdbHLH33 and AtbHLH2 respectively, strongly activated the DFR, MdMYB 10R6 and PcMYB 10R6 promoters, and only slightly activated MdMYBlORl and PcMYBIORl promoters. The introduction of the apple R6 repeats in the pear promoter leads to a 6-fold increase in the luciferase activity in presence of the 35S:PcMYB10 construct and an 8-fold increase in presence of the MdMYBlO genomic construct. . .. . .
The above Examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention. Summary of Sequences
SEQ ID
Sequence type Information Species NO:
Malus domestica and polynucleotide 23 bp sequence motif, version 1 Mains domestica niedwetzkyana
minisatellite repeat unit, from MdMYBlO promoter from red-
Malus domestica polynucleotide fleshed cultivar Malus x domestica niedwetzkyana niedwetzkyana, including repeat motifs 2, 3A, 3B, 4, 5 and 6
Malus domestica polynucleotide microsatellite niedwetzkyana
region of MdMYBlO promoter from red-fleshed cultivar Malus x
Malus domestica polynucleotide domestica niedwetzkyana including niedwetzkyana minisatellite repeat unit, microsatellite and repeat unit 1
whole MdMYBlO promoter from
Malus domestica polynucleotide red-fleshed cultivar Malus x niedwetzkyana domestica niedwetzkyana
polypeptide MdMYBlO Malus domestica
polynucleotide MdMYBlO coding region Malus domestica
whole MdMYBlO promoter from polynucleotide white-fleshed cultivar Malus Malus domestica domestica Royal Gala polynucleotide forward primer artificial
polynucleotide reverse primer artifical
polynucleotide 23 bp sequence motif, version 2 Malus domestica
polynucleotide 23 bp sequence motif, version 3 Pyrus communis
polynucleotide Whole pear PcMYBlO promoter Pyrus communis
apple minisatellite sequence inserted Malus domestica polynucleotide into pear PcMYBlO promoter niedwetzkyana
polynucleotide Chimeric apple/pear promoter Artificial
polypeptide Pear PcMYBlO Pyrus communis
polynucleotide Pear PcMYBlO coding sequence Pyrus communis
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial
polynucleotide primer artificial polynucleotide primer artificial polynucleotide primer artificial polynucleotide primer artificial

Claims

Claims:
1. An isolated promoter polynucleotide comprising at least two sequence motifs with at least 5 70% identity to the sequence of SEQ ID NO: 1, 12 or 13 wherein the promoter polynucleotide is capable of controlling transcription of an operably linked polynucleotide in a plant.
2. The promoter polynucleotide of claim 1 in which the sequence motifs each have at least 70% identity to the sequence .of SEQ ID NO: 1.
[0
3. The promoter polynucleotide of claim 1 or 2, in which at least one sequence motif has the sequence of SEQ ID NO: 1.
4. The promoter polynucleotide of any one of claims 1 to 3, in which at least one sequence motif 15 has the sequence of SEQ ID NO: 12.
5. The promoter polynucleotide of any one of claims 1 to 4, in which at least one sequence motif has the sequence of SEQ ID NO: 13. O
6. The promoter polynucleotide of any one of claims 1 to 5, in which at least one of the sequence motifs is interupted by at least one of the other sequence motifs.
7. The promoter polynucleotide of any one of claims 1 to 6, comprising a sequence with at least 70% identity to the sequence of SEQ ID NO: 2. 5 . .. . .
8. The promoter polynucleotide of any one of claims 1 to 7, comprising the sequence of SEQ ID NO: 2.
9. The promoter polynucleotide of any one of claims 1 to 8, comprising a microsatellite 0 sequence with at least 70% identity to the sequence of SEQ ID NO: 3.
10. The promoter polynucleotide of any one of claims 1 to 9, comprising a microsatellite sequence with the sequence of SEQ ID NO: 3.
1 1. The promoter polynucleotide of any one of claims 1 to 10, comprising a sequence with at least 70% identity to the sequence of SEQ ID NO: 4.
12. The promoter polynucleotide of any one of claims 1 to 1 1, comprising the sequence of SEQ ID NO: 4.
13. The promoter polynucleotide of any one of claims 1 to 12, comprising a sequence with at least 70% identity to the sequence of SEQ ID NO: 5.
14. The promoter polynucleotide of any one of claims 1 to 13, comprising a sequence with the sequence of SEQ ID NO: 5.
15. The promoter polynucleotide of any one of claims 1 to 12, comprising at least 66 contiguous polynucleotides of the sequence of SEQ ID NO: 5.
16. The promoter polynucleotide of any one of claims 1 to 15, that is modulated by a MYB transcription factor.
17. The promoter polynucleotide of any one of claims 1 to 16, that is modulated by a MYB transcription factor comprising an R2R3 DNA binding domain.
18. The promoter polynucleotide of any one of claims 1 to 17, that is modulated by a MYB transcription factor that comprises a sequence with at least 70% identity to the sequence of SEQ ID NO: 6 or 16.
19. The promoter polynucleotide of any one of claims 1 to 18, that is modulated by a MYB transcription factor that comprises the sequence of SEQ ID NO: 6 or 16.
20. The promoter polynucleotide of any one of claims 16 to 19, wherein the promoter polynucleotide is endogenously associated with the MYB transcription factor in a naturally occuring plant, and the promoter is autoregulated by the MYB transcription factor.
21. The promoter polynucleotide of any one of claims 1 to 20, that is capable of controlling transcription of an operably linked polynucleotide sequence constitutively, in substantially all tissues of a plant.
22. The promoter polynucleotide of any one of claims 16 to 20, that is capable of controlling transcription of an operably linked polynucleotide sequence in any plant in which the MYB transcription factor is expressed.
23. A genetic construct comprising a promoter polynucleotide of any one of claims 1 to 22.
24. A genetic construct comprising a promoter polynucleotide of any one of claims 1 to 22, operably linked to a polynucleotide sequence to be expressed.
25. A host cell transformed with the promoter polynucleotide of any one of claims 1 to 22.
26. A plant cell or plant transformed with the promoter polynucleotide of any one of claims 1 to
22.
27. The plant cell or plant of claim 26 further transformed with a polynucleotide encoding a MYB transcription factor that modulates expression of the promoter polynucleotide.
28. The plant cell or plant of claim 26 that naturally expresses a MYB transcription factor that modulates expression of the promoter polynucleotide.
29. A method for producing a plant cell or plant with modifed expression of at least one polynucleotide, the method comprising transformation of the plant cell or plant with a promoter polynucleotide of any one of claims 1 to 22.
30. The method of claim 29 in which the plant cell or plant is also transformed with a polynucleotide capable of expresssing a MYB transcription factor that modulates expression of the promoter polynucleotide.
31. The method of claim 29 in which the plant cell or plant naturally expresses the MYB transcription factor that modulates expression of the promoter polynucleotide.
32. A method for producing a plant cell or plant with a modifed phenotype, the method comprising transformation of the plant cell or plant with a promoter polynucleotide of any one of claims 1 to 22.
33. The method of claim 32 in which the plant cell or plant is also transformed with a polynucleotide capable of expresssing a MYB transcription factor that modulates expression of the promoter polynucleotide.
34. The method of claim 32 in which the plant cell or plant naturally expresses the MYB transcription factor that modulates expression of the promoter polynucleotide.
35. A plant cell or plant produced by a method of any on of claims 29 to 34.
36. A seed, propagule, progeny or part of a plant, of any one of claims 26 to 28 and 35, wherein the seed, propagule, progeny or part of a plant comprises the transformed promoter polynucleotide.
PCT/NZ2008/000104 2007-05-11 2008-05-12 Compositions and methods for regulating plant gene expression WO2008140334A1 (en)

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CN116640775A (en) * 2023-06-14 2023-08-25 中国热带农业科学院热带生物技术研究所 Longan DlMYB15 gene capable of enhancing anthocyanin synthesis regulation capability of MBW complex

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Title
CHAGNE D. ET AL.: "Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple", BMC GENOMICS, vol. 8, no. 212, 2007, pages 1 - 11, XP021028026 *
DATABASE GENBANK [online] ESPLEY R.V. ET AL.: "Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10", Database accession no. (DQ267896) *
DATABASE GENBANK [online] TAKOS A.M. ET AL.: "Light-Induced Expression of a MYB Gene Regulates Anthocyanin Biosynthesis in Red Apples", Database accession no. (DQ886414) *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2285968A1 (en) * 2008-05-12 2011-02-23 The New Zealand Institute for Plant and Food Research Limited Chimeric compositions and methods for regulating plant gene expression
EP2285968A4 (en) * 2008-05-12 2011-08-10 Nz Inst Plant & Food Res Ltd Chimeric compositions and methods for regulating plant gene expression
US8686125B2 (en) 2008-05-12 2014-04-01 The New Zealand Institute For Plant And Food Research Limited Chimeric promoters comprising MYB10 repeat element and methods for regulating plant gene expression
CN116640775A (en) * 2023-06-14 2023-08-25 中国热带农业科学院热带生物技术研究所 Longan DlMYB15 gene capable of enhancing anthocyanin synthesis regulation capability of MBW complex
CN116640775B (en) * 2023-06-14 2024-01-26 中国热带农业科学院热带生物技术研究所 Longan DlMYB15 gene capable of enhancing anthocyanin synthesis regulation capability of MBW complex

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