Patent Grant
8859848
The present invention relates to compositions and methods for producing plants with altered phloretin glycosyltransferase activity and/or altered phlorizin content.
The dihydrochalcone phlorizin (phloretin 2′-glucoside, see
Until recently phlorizin was believed to exist only in Malus species. However phloretin glycosides have been reported in the leaves of Australian native sarsaparilla (Smilax glyciphylla, Cox et al. 2005), sweet tea (Lithocarpus polystachyus, Dong et al. 2007) and at very low levels in strawberry fruit (Hilt et al. 2003). In apple trees, phlorizin is found primarily in the young shoots, roots, leaves and bark. In fruit, phlorizin is most abundant in the seeds, with intermediate levels in both the core and the skin, and the lowest level in the cortex. Variation has been assessed within apple trees, between orchards, between different cultivars and among mutants (Hunter and Hull 1993, Awad et al. 2000). Despite this information, little is known of the in planta function of phlorizin in apple tree physiology, although it has been suggested that it might act in apple tree growth and development (Zhang et al. 2007) or be an inhibitor of bacterial (MacDonald and Bishop 1952) or fungal growth (Gessler et al. 2006).
The molecular basis for production of phlorizin in planta has not been described. Phloretin is a product of the phenylpropanoid pathway (Watts et al. 2004), with conversion to its glucoside, phlorizin, likely to be catalysed by the action of a uridine diphosphate (UDP) glycosyltransferase (UGT). UGTs mediate the transfer of a sugar residue from an activated nucleotide sugar to acceptor molecules (aglycones). Plants contain large families of UGTs with over 100 genes being described in Arabidopsis. These genes have a common signature motif of 44 amino acids thought to be involved in binding of the UDP moiety of the activated sugar (Li et al. 2001). A phylogenetic analysis established the presence of distinct Groups (A-N) and Families (UGT71-92) of UGT genes in Arabidopsis (Ross et al. 2001) and this facilitated the characterisation of many new activities (Jackson et al. 2001; Lim et al. 2002; Jones et al. 2003; Messner et al. 2003; Lim et al. 2004; Lim et al. 2005). Although initially thought to be promiscuous enzymes, recent evidence suggests that their broad substrate specificity is limited by regio-specificity (Hansen et al. 2003; Lim et al. 2003), and in some cases UGTS have been shown to be highly specific (Fukuchi-Mizutani et al. 2003). Using a functional genomics approach we have identified and characterised a UGT from apple belonging to the previously uncharacterised UGT Family 88. We establish that MpUGT88A1 mediates the glycosylation of the dihydrochalcone phloretin to phlorizin which may indicate that other members of UGT Family 88 utilise similar substrates.
It would be beneficial to have a means to increase phlorizin levels in plants.
It is an object of the invention to provide improved compositions and methods for modulating activity and/or phlorizin content in plants or at least to provide the public with a useful choice.
In a first aspect the invention provides a method for producing a plant cell or plant with increased phlorizin content, the method comprising transformation of a plant cell or plant with a polynucleotide encoding a polypeptide with the amino acid sequence of any one of SEQ ID NO: 1 to 5 or a variant of the polypeptide.
Preferably the variant has phloretin glycosyltransferase activity.
In a further aspect the invention provides a method for producing a plant cell or plant with increased phloretin glycosyltransferase activity, the method comprising transformation of a plant cell or plant with a polynucleotide encoding a polypeptide with the amino acid sequence of any one of SEQ ID NO: 1 to 5, or a variant of the polypeptide.
Preferably the variant has the activity of phloretin glycosyltransferase activity.
Preferably the variant comprises the sequence of SEQ ID NO: 16.
In one embodiment the variant has at least 70% sequence identity to a polypeptide with the amino acid sequence of any one of SEQ ID NO: 1 to 5.
In a further embodiment the polynucleotide encodes a polypeptide with an amino acid sequence that has at least 70% identity to the sequence of SEQ ID NO: 1.
In a further embodiment the polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO: 1.
In a further embodiment the polynucleotide encodes a polypeptide with an amino acid sequence that has at least 70% identity to the sequence of SEQ ID NO: 2.
In a further embodiment the polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO: 2.
In a further embodiment the polynucleotide encodes a polypeptide with an amino acid sequence that has at least 70% identity to the sequence of SEQ ID NO: 3.
In a further embodiment the polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO: 3.
In a further embodiment the polynucleotide encodes a polypeptide with an amino acid sequence that has at least 70% identity to the sequence of SEQ ID NO: 4.
In a further embodiment the polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO: 4.
In a further embodiment the polynucleotide encodes a polypeptide with an amino acid sequence that has at least 70% identity to the sequence of SEQ ID NO: 5.
In a further embodiment the polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO: 5.
In a preferred embodiment of the method for producing a plant cell or plant with increased phlorizin content, the plant cell or plant is also transformed with a polynucleotide encoding a phloretin synthase.
Transformation with the phloretin glycosyltransferase and synthase may be sequential, in either order. Alternatively transformation with the phloretin glycosyltransferase and synthase may be simultaneous. When simultaneous, sequences encoding the phloretin glycosyltransferase and synthase may be on the same or separate constructs or vectors.
In a further aspect the invention provides a method of producing a plant cell or plant with increased phlorizin content, the method comprising transformation of a plant cell or plant with a polynucleotide comprising a nucleotide sequence selected from any one the sequences of SEQ ID NO: 7 to 18, or a variant thereof.
Preferably the variant encodes a polypeptide with phloretin glycosyltransferase activity.
Preferably the variant encodes a polypeptide comprising the sequence of SEQ ID NO: 16.
In a further aspect the invention provides a method of producing a plant cell or plant with increased phloretin glycosyltransferase activity, the method comprising transformation of a plant cell or plant with a polynucleotide comprising a nucleotide sequence selected from any one the sequences of SEQ ID NO: 6 to 15, or a variant thereof.
Preferably the variant encodes a polypeptide which has phloretin glycosyltransferase activity.
Preferably the variant encodes a polypeptide comprising the sequence of SEQ ID NO: 16.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to any one of the sequences of SEQ ID NO: 6 to 15.
In a further embodiment the polynucleotide comprises a sequence with at least 70% identity to any one the sequences of SEQ ID NO: 6 to 15.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 6.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 7.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 7.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 7.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 8.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 8.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 9.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 9.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 10.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 10.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 11.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 11.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 12.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 12.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 13.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 13.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 14.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 14.
In a further embodiment the polynucleotide comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 15.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 15.
In a preferred embodiment of the method of producing a plant cell or plant with increased phlorizin content, the plant cell or plant is also transformed with a polynucleotide encoding a phloretin synthase.
Transformation with the phloretin glycosyltransferase and synthase may be sequential, in either order. Alternatively transformation with the phloretin glycosyltransferase and synthase may be simultaneous. When simultaneous, sequences encoding the glycosyltransferase and synthase may be on the same or separate constructs or vectors.
In a further aspect the invention provides an isolated polynucleotide encoding a polypeptide comprising a sequence selected any one of SEQ ID NO: 2 to 5 or a variant thereof.
Preferably the variant has phloretin glycosyltransferase activity.
Preferably the variant comprises the sequence of SEQ ID NO: 16.
In one embodiment the variant comprises a sequence with at least 80% identity to SEQ ID NO: 2:
In a further embodiment the polypeptide comprises the sequence of SEQ ID NO: 2.
In one embodiment the variant comprises a sequence with at least 80% identity to SEQ ID NO: 3:
In a further embodiment the polypeptide comprises the sequence of SEQ ID NO: 3.
In one embodiment the variant comprises a sequence with at least 80% identity to SEQ ID NO: 4:
In a further embodiment the polypeptide comprises the sequence of SEQ ID NO: 4.
In one embodiment the variant comprises a sequence with at least 80% identity to SEQ ID NO: 5:
In a further embodiment the polypeptide comprises the sequence of SEQ ID NO: 5.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 8 to 15, or a variant thereof.
Preferably the variant encodes a phloretin glycosyltransferase.
Preferably the variant encodes a polypeptide comprising the sequence of SEQ ID NO: 16.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 8.
In one embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 8.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 9.
In one embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 9.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 10.
In one embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 10.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 11.
In one embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 11.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 12.
In one embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 12.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 13.
In one embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 13.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 14.
In one embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 14.
In one embodiment the variant comprises a sequence with at least 70% sequence identity to the sequence of SEQ ID NO: 15.
In one embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 15.
In a further aspect the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 to 5, or a variant thereof.
Preferably the variant has phloretin glycosyltransferase activity.
Preferably the variant comprises the sequence of SEQ ID NO: 16.
In one embodiment the variant polypeptide has at least 80% sequence identity to an amino acid sequence selected from any one of SEQ ID NO: 2 to 5.
In a further embodiment the isolated polypeptide has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2.
In a further embodiment the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
In a further embodiment the isolated polypeptide has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3.
In a further embodiment the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 3.
In a further embodiment the isolated polypeptide has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4.
In a further embodiment the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 4.
In a further embodiment the isolated polypeptide has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5.
In a further aspect the invention provides an isolated polynucleotide encoding a polypeptide of the invention.
In a further aspect the invention provides an isolated polynucleotide comprising:
In a further aspect the invention provides a genetic construct which comprises a polynucleotide of the invention.
In a further aspect the invention provides an expression construct which comprises a polynucleotide of the invention.
In a further aspect the invention provides an RNAi construct which comprises a polynucleotide of the invention.
In a further aspect the invention provides a vector comprising an expression construct, genetic construct or RNAi construct of the invention.
In a further aspect the invention provides a host cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.
Preferably the host cell is also genetically modified to express a polynucleotide encoding phloretin synthase.
In a further aspect the invention provides a host cell comprising an expression construct or genetic construct of the invention.
Preferably the host cell is also comprises an expression construct or genetic construct including a polynucleotide encoding phloretin synthase.
In a further aspect the invention provides a method for producing a phloretin glycosyltransferase polypeptide, the method comprising culturing a host cell comprising an expression construct of the invention or a genetic construct of the invention, capable of expressing a phloretin glycosyltransferase polypeptide.
In a further aspect the invention provides a method for producing the enzymic product of a phloretin glycosyltransferase, the method comprising culturing a host cell including an expression construct of the invention or an genetic construct of the invention, capable of expressing a phloretin glycosyltransferase polypeptide, in the presence of enzymic substrate which may be supplied to, or may be naturally present within the host cell.
In a further aspect the invention provides a method for the biosynthesis of phlorizin comprising the steps of culturing a host cell comprising an expression construct of the invention or a genetic construct of the invention, capable of expressing a phloretin glycosyltransferase, in the presence of a phloretin which may be supplied to, or may be naturally present within the host cell.
Preferably the host cell is also genetically modified to express a polynucleotide encoding phloretin synthase.
Preferably the host cell is a plant cell. Preferably the plant cell is part of a plant.
In a further aspect the invention provides a plant cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.
In a further aspect the invention provides a plant cell which comprises an expression construct of the invention or the genetic construct of the invention.
In a further aspect the invention provides a plant which comprises a plant cell of the invention.
Preferably the plant cells or plants of the invention are also genetically modified to express a polynucleotide encoding a phloretin synthase.
Preferably the host cell is also genetically modified to express a polynucleotide encoding a phloretin synthase.
It may be desirable, in some cases, to reduce phloretin glycosyltransferase activity and/or phlorizin content in a plant. This can be achieved by down-regulating expression of endogenous phloretin glycosyltransferase genes using the phloretin glycosyltransferase sequences, or fragments thereof, disclosed herein. Methods for down-regulation or silencing endogenous genes in plants are well known to those skilled in the the art and described herein.
In a further aspect the invention provides a method for selecting a plant altered in phloretin glycosyltransferase activity, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant altered in phloretin glycosyltransferase activity, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a method for selecting a plant with altered phlorizin content; the method comprising testing of a plant for altered expression of a polynucleotide or polypeptide of the invention.
In a further aspect the invention provides a method for selecting a plant with altered phlorizin content; the method comprising testing of a plant for altered expression of a polypeptide or polypeptide of the invention.
In a further aspect the invention provides a plant cell or plant produced by the method of the invention. Preferably the plant is genetically modified to include a polynucleotide or polypeptide of the invention.
In a preferred embodiment of the method the plant cell or plant is also transformed with a polynucleotide encoding a phloretin synthase.
In a further aspect the invention provides a group or population of plants selected by the method of the invention.
In a further aspect the invention provides a method of producing phlorizin, the method comprising extracting phlorizin from a plant cell or plant of the invention.
In a further aspect the invention provides an antibody raised against a polypeptide of the invention.
In a further aspect the invention provides method of producing phlorizin, the method comprising contacting phloretin with the expression product of an expression construct comprising a polynucleotide of the invention or a polypeptide of the invention to obtain phlorizin.
The polynucleotides and variants of polynucleotides, of the invention may be derived from any species. The polynucleotides and variants may also be recombinantly produced and also may be the products of “gene shuffling’ approaches.
In one embodiment the polynucleotide or variant, is derived from a plant species.
In a further embodiment the polynucleotide or variant, is derived from a gymnosperm plant species.
In a further embodiment the polynucleotide or variant, is derived from an angiosperm plant species.
In a further embodiment the polynucleotide or variant, is derived from a from dicotyledonuous plant species.
The polypeptides and variants of polypeptides of the invention may be derived from any species. The polypeptides and variants may also be recombinantly produced and also may also be expressed from the products of “gene shuffling’ approaches.
In one embodiment the polypeptides or variants of the invention are derived from plant species.
In a further embodiment the polypeptides or variants of the invention are derived from gymnosperm plant species.
In a further embodiment the polypeptides or variants of the invention are derived from angiosperm plant species.
In a further embodiment the polypeptides or variants of the invention are derived from dicotyledonous plant species.
The plant cells and plants of the invention, including those from which the polynucleotides, variant polynucleotides, polypeptide and variant polypeptides are derived, may be from any species.
In one embodiment the plants cells and plants are from gymnosperm species.
In a further embodiment the plants cells and plants are from angiosperm species.
In a further embodiment the plants cells and plants are from dicotyledonuous species.
In a further embodiment the plants cells and plants of the invention are from monocotyledonous species.
In a preferred embodiment the plants cells and plants are from a species selected from a group comprising but not limited to the following genera: Smilax, Lithocarpus, Fragaria and Malus.
Preferred plant species are: Smilax glyciphylla, Lithocarpus polystachyus, Fragaria, Malus domestica and Malus sieboldii.
A particularly preferred genus is Malus.
Preferred Malus species include: Malus aldenhamii Malus angustifolia, Malus asiatica, Malus baccata, Malus coronaria, Malus domestica, Malus doumeri, Malus florentina, Malus floribunda, 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 sylvestris, Malus toringoides, Malus transitoria, Malus trilobata, Malus tschonoskii, Malus×domestica, Malus×domestica×Malus sieversii, Malus sylvestris, Malus×domestica×Pyrus communis, Malus xiaojinensis, Malus yunnanensis, Malus sp., Mespilus germanica,
A particularly preferred plant species is Malus domestica.
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 “comprising” as used in this specification means “consisting at least in part of”. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.
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 capable of specific hybridization to a target of interest, e.g., a sequence that is at least 15 nucleotides in length. The fragments of the invention comprise 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.
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 target.
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.
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. Polypeptides of the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof
A “fragment” of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above enzymatic activity.
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 polynucleotides or polypeptides of the invention being derived from a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species. The polynucleotide or polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.
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 occuring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides. The term “variant” with reference to polypeptides and polypeptides encompasses all forms of polypeptides 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 sequence of the present invention. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the invention.
Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
The identity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq -i nucleotideseq1 -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. Mol. 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 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 may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
A preferred method for calculating polynucleotide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)
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 polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
The similarity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq -i nucleotideseq1 -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 1×10−6 more preferably less than 1×10−9, more preferably less than 1×10−12, more preferably less than 1×10−15, more preferably less than 1×10−18, more preferably less than 1×10−21, more preferably less than 1×10−30, more preferably less than 1×10−40, more preferably less than 1×10−50, more preferably less than 1×10−60, more preferably less than 1×10−70, more preferably less than 1×10−80, more preferably less than 1×10−90 and most preferably less than 1×10−100 when compared with any one of the specifically identified sequences.
Alternatively, variant polynucleotides of the present invention hybridize to the specified polynucleotide sequences, 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 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
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)° 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. 1; 26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C. below the Tm.
Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a “silent variation”. Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.
Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.
The function of a variant polynucleotide of the invention as a phloretin glycosyltransferase may be assessed for example by expressing such a sequence in bacteria and testing activity of the encoded protein as described in the Example section. Function of a variant may also be tested for it ability to alter phloretin glycosyltransferase activity or phloretin content in plants, also as described in the Examples section herein.
Polypeptide Variants
The term “variant” with reference to polypeptides encompasses naturally occurring, recombinantly 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 [November 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity 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.
A preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)
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 [November 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters:
bl2seq -i peptideseq1 -j peptideseq2 -F F -p blastp
Variant polypeptide sequences preferably exhibit an E value of less than 1×10−6 more preferably less than 1×10−9, more preferably less than 1×10−12, more preferably less than 1×10−15, more preferably less than 1×10−18, more preferably less than 1×10−21, more preferably less than 1×10−30, more preferably less than 1×10−40, more preferably less than 1×10−50, more preferably less than 1×10−60, more preferably less than 1×10−70, more preferably less than 1×10−80, more preferably less than 1×10−90 and most preferably 1×10−100 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).
The function of a polypeptide variant as a phloretin glycosyltransferase may be assessed by the methods described in the Example section herein.
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 the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.
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:
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.
“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” refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5′ UTR and the 3′ UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.
Terminators are sequences, which terminate transcription, and are found in the 3′ untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
The term “promoter” refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
A “transgene” is a polynucleotide that is taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
An “inverted repeat” is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,
Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
Host Cells
Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.
A “transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.
The applicants have identified novel polynucleotides (SEQ ID NO: 6 to 15) that encode novel polypeptides (SEQ ID NO: 2 to 5) that have phloretin glycosyltransferase activity, as summarised in Table 1.
The applicants have shown that all of the phloretin glycosyltransferase polypeptides sequences disclosed (SEQ ID NO: 1 to 5) show significant sequence conservation and are variants of one another.
Similarly the applicants have shown that all of the disclosed phloretin glycosyltransferase polynucleotides sequences (SEQ ID NO: 6 to 15) show significant sequence conservation and are variants of one another.
The invention provides genetic constructs, vectors and plants containing the novel polynucleotide sequences (SEQ ID NO: 6 to 15) or sequences encoding the novel polypeptide sequences (SEQ ID NO: 2 to 5). The invention also provides plants comprising the genetic construct and vectors of the invention.
The invention provides plants altered in phloretin glycosyltransferase activity, relative to suitable control plants, and plants altered in phlorizin content relative to suitable control plants. The invention provides plants with increased phloretin glycosyltransferase activity and increased phlorizin.
The invention also provides methods for the production of such plants and methods of selection of such plants.
Suitable control plants include non-transformed plants of the same species or variety or plants transformed with control constructs.
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 polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polypeptides 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 include use of all, or portions of, the polypeptides having the sequence set forth herein as hybridization probes. The technique of hybridizing labelled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65° C. in 5.0×SSC, 0.5% sodium dodecyl sulfate, 1×Denhardt's solution; washing (three washes of twenty minutes each at 55° C.) in 1.0×SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0.5×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C. An optional further wash (for twenty minutes) can be conducted under conditions of 0.1×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 partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5′RACE (Frohman Mass., 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer/database—based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. 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.
Methods for Identifying Variants
Physical Methods
Variant polypeptides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer, useful to amplify variants of polynucleotide molecules of the invention by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.
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.
Polypeptide variants may also be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.
Computer Based Methods
The variant sequences of the invention, including both 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 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.
An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [November 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 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 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.
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, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce “Expect” values for alignments. The Expect value (E) indicates the number of hits one can “expect” to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. 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, 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. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. 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.
The function of a variant polynucleotide of the invention as encoding phloretin glycosyltransferases can be tested for the activity, or can be tested for their capability to alter phlorizin content in plants by methods described in the examples section herein.
Methods for Isolating Polypeptides
The polypeptides of the invention, including variant polypeptides, may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco Calif., or automated synthesis, for example using an Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.). Mutated forms of the polypeptides may also be produced during such syntheses.
The polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification).
Alternatively the polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
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 of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.
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 Polynucleotides, Constructs or Vectors
The invention provides a host cell which comprises a genetic construct or vector of the invention.
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 of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 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 of the invention. Plants comprising such cells also form an aspect of the invention.
Alteration of phloretin glycosyltransferase activity may be altered in a plant through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct designed to alter expression of a polynucleotide or polypeptide which modulates phloretin glycosyltransferase activity, or phlorizin content in such plant cells and plants. Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polynucleotides or polypeptides which modulate phloretin glycosyltransferase activity and/or phlorizin content in such plant cells and plants.
Methods for transforming plant cells, plants and portions thereof with polypeptides 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.
Methods for Genetic Manipulation of Plants
A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297, Hellens R P, et al (2000) Plant Mol Biol 42: 819-32, Hellens R et al Plant Meth 1: 13). 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. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.
Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.
Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detest presence of the genetic construct in the transformed plant.
The promoters suitable for use in the constructs of this invention are functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is herein incorporated by reference.
Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum 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., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.
Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. “Regulatory elements” is used here in the widest possible sense and includes other genes which interact with the gene of interest.
Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide of the invention may include an antisense copy of a polynucleotide of the invention. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.
An “antisense” polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,
Genetic constructs designed for gene silencing may also include an inverted repeat. An ‘inverted repeat’ is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,
The transcript formed may undergo complementary base pairing to form a hairpin structure. 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 polypeptides effecting gene silencing.
Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). 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 polynucleotide of the invention is also contemplated.
The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5′ or 3′ UTR sequence, or the corresponding gene.
Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (McIntyre, 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.
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); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (U.S. Pat. Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. 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 al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073 and 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 (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); cereals (U.S. Pat. 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):117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep. 22(1):38-45); strawberry (Oosumi et al., 2006 Planta. 223(6):1219-30; Folta et al., 2006 Planta Apr. 14; PMID: 16614818), rose (Li et al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol. 1995; 44:129-33), tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple (Yao et al., 1995, Plant Cell Rep. 14, 407-412) and Actinidia eriantha (Wang et al., 2006, Plant Cell Rep. 25, 5: 425-31). Transformation of other species is also contemplated by the invention. Suitable methods and protocols are available in the scientific literature.
Several further methods known in the art may be employed to alter expression of a nucleotide and/or polypeptide of the invention. Such methods include but are not limited to Tilling (Till et al., 2003, Methods Mol Biol, 2%, 205), so called “Deletagene” technology (Li et al., 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors. (e.g. Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al., 2003, Nat. Biotechnol., 21(1), 35). Transposon tagging approaches may also be applied. Additionally peptides interacting with a polypeptide of the invention may be identified through technologies such as phase-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a polypeptide of the invention. Use of each of the above approaches in alteration of expression of a nucleotide and/or polypeptide of the invention is specifically contemplated.
The terms “to alter expression of” and “altered expression” of a polynucleotide or polypeptide of the invention, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the invention is modified thus leading to altered expression of a polynucleotide or polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The “altered expression” can be related to an increase or decrease in the amount of messenger RNA and/or polypeptide produced and may also result in altered activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.
Methods of Selecting Plants
Methods are also provided for selecting plants with altered phloretin glycosyltransferase or phlorizin content. Such methods involve testing of plants for altered for the expression of a polynucleotide or polypeptide of the invention. Such methods may be applied at a young age or early developmental stage when the altered phloretin glycosyltransferase activity or phlorizin content may not necessarily be easily measurable.
The expression of a polynucleotide, such as a messenger RNA, is often used as an indicator of expression of a corresponding polypeptide. Exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). Polynucleotides or portions of the polynucleotides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with altered levels of phloretin glycosyltransferase or phlorizin. The polynucleotides of the invention may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.
Alternatively antibodies may be raised against polypeptides of the invention. Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998). Such antibodies may be used in methods to detect altered expression of polypeptides which modulate flower size in plants. Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).
These approaches for analysis of polynucleotide or polypeptide expression and the selection of plants with altered phloretin glycosyltransferase or altered phlorizin content are useful in conventional breeding programs designed to produce varieties with altered phloretin glycosyltransferase activity or phlorizin content.
Plants
The term “plant” is intended to include a whole plant, 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.
The plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.
The function of a variant polynucleotide of the invention as encoding a phloretin glycosyltransferase may be assessed for example by expressing such a sequence in bacteria and testing activity of the encoded protein as described in the example sections herein.
Alteration of phloretin glycosyltransferase activity and/or phlorizin content may also be altered in a plant through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide which modulates phloretin glycosyltransferase activity and/or phlorizin content in such plant cells and plants. Such methods preferably also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more other polynucleotides or polypeptides which modulate ascorbic acid content in such plant cells and plants. Preferably a combination of phloretin glycosyltransferase and a phloretin synthase is expressed in the plant cells or plants.
Methods for Extracting Phlorizin from Plants
Methods are also provided for the production of phlorizin by extraction of phlorizin from a plant of the invention. Phlorizin may be extracted from plants by many different methods known to those skilled in the art.
Ridgway and Tucker 1997 extract phlorizin from young apple leaves and twigs (containing up to 10% dry weight of phlorizin). They showed that the most important determinant in a given tissue's phlorizin content is its period of development, and that cultivar, including rootstock type, had a much less pronounced effect. They recommend that production of phlorizin is best carried out by the coppicing of fast growing rootstocks, such as M25 or MM106, this being akin to the production of willow or poplar for biomass; for which mechanical harvesting has already been developed. Yields of 250 kg of phlorizin per hectare should then be readily obtained. Yields could be improved by genetic engineering. (Ridgway T & Tucker G (1997) Apple: a new agrochemical crop. Biochemical Society Transactions 25, 110S)
Hunter and Hull (1993) also provide a method for apple. Dong, H. et al (2007) provide a preparative separation of phlorhizin from the crude extract of Lithocarpus polystachyus. Cox et al., provide a method of extraction from Smilax.
These methods may be up-scaled for larger scale phlorizin extraction using approaches well-known to those skilled in the art.
This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
The present invention will be better understood with reference to the accompanying drawings in which:
The invention will now be illustrated with reference to the following non-limiting example.
Sequence Identification and Phylogenetics
GenBank apple EST sequences were BLAST searched (expect value of exp−05) using previously published UGT genes from GenBank. EST sequences were automatically parsed through two rounds of contig building and a set of “non-redundant contig” sequences was derived. Amino acid alignments of predicted proteins were constructed using ClustaIX (version 1.8). All proteins were checked for the presence of the common signature motif of 44 amino acids found in plant UGTs (Li et al. 2001). A full-length sequence in each contig was selected for complete sequencing.
For phylogenetic analysis all amino acid sequences were initially aligned using ClustalX then manually edited. Arabidopsis UGT sequences were obtained from the following website http://www.p450.kvl.dk/UGT.shtml. Confidence values for groupings in phylogenetic trees were obtained using BOOTSTRAP N-J TREE using 1,000 bootstrap trials. Trees were visualised in TREEVIEW (v.1.6.6).
The MpUGT88A Gene and its Predicted Protein
Gene mining identified over 60 UDP-glycosyltransferase-encoding genes amongst the ˜270,000 apple EST sequences in GenBank. In silico expression profiling of the apple UGT ESTs identified the gene ‘MpUGT88A1’ that was highly expressed (represented by 255 ESTs) in all tissues known to produce phlorizin including leaf, seed and fruit (
A framework phylogenetic tree was constructed using MpUGT88A1 and representative members of the Arabidopsis UGT tree published by Ross et al. (2001). This framework tree indicated that MpUGT88A1 clustered with the sole Arabidopsis Family 88 UGT sequence AtUGT88A1 (data not shown). The apple clone was therefore designated MpUGT88A1 according the nomenclature described in Ross et al. (2001). MpUGT88A1 showed 42.5% amino acid identity with AtUGT88A1, and significant homology with two full length UGTs sequences from rice (Oryza sativa, BAC10743) and sweetleaf (Stevia rebaudiana, AAR06919) and a partial sequence from black gram (Vigna mungo, BAA36412, 52% amino acid identity in the region of overlap). MpUGT88A1 and these four sequences are shown aligned in
Plant Material, RNA Extraction and qPCR
Total RNA was isolated from apple tissues by a modified silica RNA extraction method (Nieuwenhuizen et al. 2007). The RNA concentration of each sample was measured using a NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Del., USA). RNA samples (˜3 μg in 30 μl reactions) were treated for 30 min at 37° C. with DNase I (Ambion, Inc., Austin, Tex., USA) to remove any minor genomic DNA contamination. The DNase was heat inactivated for 30 min at 50° C. Reverse transcription was performed in 20 μl reactions as per manufacturer's instructions using SuperScript III™ RNase H-reverse transcriptase (Invitrogen, Auckland, NZ), 500 ng of RNA and the primer. NotI-PA 5′-GACTAGTTCT AGATCGCGAG CGGCCGCCCT(15)-3′ (SEQ ID NO:17).
Quantitative PCR reactions (20 μL) were repeated four times on an ABI Prism 7900HT (Applied Biosystems, Foster City, Calif., USA) using 3 μL of diluted cDNA, 0.5 μM MpUGT88A1-specific primers (Fwd 5′-GAAGGGTGTG TTGCCAGAAG GGT-3′ (SEQ ID NO:18); Rev 5′-GTCACGAACC CACCAACCGA CT-3′ [SEQ ID NO:19]), and 5 μL of Lightcycler 480 SYBR Green Master (Roche Diagnostics) following the manufacturer's instructions. Cycling conditions included an initial hot start at 95° C. for 5 min, followed by 45 cycles of 95° C. for 10 s, 60° C. for 10 s and 72° C. for 12 s. Fluorescence was measured at the end of each annealing step. Each PCR reaction was followed by a melting curve program to check that only single products were amplified, starting with denaturation at 95° C. for 5 s before cooling to 65° C. for 1 min then increasing at 0.1° C.s−1 with continuous fluorescence measurement until 97° C. was reached. Negative controls consisted of water in place of cDNA and were run with all reactions. Data were analysed using LightCycler 480 software version 1.2.0.169. For each gene, a standard curve was generated using a cDNA serial dilution, and the resultant efficiencies were used to calculate expression relative to apple actin (CN938023), to minimise variation in cDNA template levels.
Expression of MpUGT88A1
In silico expression profiling of MpUGT88A1 indicated that ESTs were represented in libraries made from all apple tissues except senescing leaf and developing fruit harvested 24-87 DAFB (
Expression of MpUGT88A1 in E. coli
The open reading frame of EST111441 (MpUGT88A1) was amplified using primers RA335 5′-ACGGGATCCA TGGGAGACGT CATTGTACTG-3′ (SEQ ID NO:20) and RA336 5′-CCCAAGCTTT TATGTAATGC TACTAACAAA GTTGAC-3′(SEQ ID NO:21). Amplified bands were purified using Qiaquick® PCR cleanup columns (Qiagen GmbH, Hilden, Germany), digested with BamHI and HindIII (underlined in the primers above), and ligated into the corresponding sites of the pET30a(+) vector (Novagen, Madison, Wis., USA). The clone was sequence verified against the original EST. Recombinant N-terminal His6-tagged protein was expressed from pET-30a(+) plasmids in E. coli BL21-Codon-Plus™-RIL cells. Cultures were grown in a ZYM-5052 autoinducible media (Studier 2005) at 37° C. for 4 h at 300 rpm. The temperature was then lowered to 16° C. and incubation continued for a further 60 h. Recombinant proteins were purified on 5 ml His-Trap chelating HP columns (Amersham Biosciences, Buckinghamshire, UK) and eluted using a continuous 0-250 mM imidazole gradient as described in Green et al (2007). The concentrate was then applied to a 1.6 times 40 cm G200 Superdex gel filtration column (Pharmacia Biotech, Auckland, NZ) pre-equilibrated with 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5 mM DTT at a flow rate of 1 ml·min−1. Highest purity fractions were pooled, adjusted to 15% glycerol and stored at −80° C. A pET-30(+) vector-only control was expressed and purified as above.
Recombinant protein was analysed on 12% (w/v) SDS-Tris-Tricine gels, electroblotted onto polyvinyldifluoride membrane, and blocked as described in (Nieuwenhuizen et al. 2007). Proteins were immunolocalised with a His6 monoclonal antibody (Roche, Mannheim, Germany), 1:1000, (w/v), diluted in TBS buffer containing milk powder. Membranes were incubated with an anti-rabbit alkaline phosphatase conjugated secondary antibody (Sigma-Aldrich, St. Louis, Mo., USA) and binding visualised using BCIP®NBT (nitro-blue tetrazolium) Liquid Substrate System (Sigma-Aldrich).
UGT Activity Assays
UGT activity assays were performed in triplicate in 50 μl reactions using ˜1-2 μg of recombinant protein purified on His-Trap and G200 Superdex columns. Reactions were performed in glycosyltransferase assay buffer (50 mM TrisHCl, pH 7.5, 2 mM DTT) with 2 mM substrate and 1 μl [3H]-UDP-glucose (uridine diphospho-D-[6-3H] glucose, 13.6 Ci/mmol, GE Heathcare Buckinghamshire UK). Reactions were performed at 30° C. for 30 min and terminated by addition of 10 μL of 2 M HCl. The reaction mixtures were extracted twice with 100 μL of ethyl acetate and 20 μl of the organic phase were combined with 1 ml of non-aqueous scintillation fluid and analysed by liquid scintillation counting (Tri-Carb 2900TR, PerkinElmer, Boston, Mass., USA). Boiled enzyme and pET30a(+) vector controls were run in parallel with all enzyme reactions.
The effect on UGT activity of different pH (5-10), temperature (15-50° C.) and ionic strength of monovalent (Na+, 0-100 mM; K+, 0-10 mM) and divalent ions (Mg+ or Mn2+, 0-25 mM) was determined as described above using 2 μM phloretin. Reactions were shown to be linear over 1 h under standard conditions. The apparent Km value for phloretin was determined by varying the phloretin concentration from 4 μM to 0.01 μM with a fixed [3H]-UDP-glucose concentration of 27 uM. The Km value for UDP-glucose was determined by varying ([3H]-UDP-glucose concentration from 27 uM to 0.135 Um and by mixing ([3H]-UDP-glucose (13.5 uM) with cold UDP-glucose (10-100 uM) at a fixed phloretin concentration of 2 μM.
For LC-MS analysis, reactions contained ˜10 μg enzyme, 10 μM phloretin and UDP-glucose (Sigma Aldrich), UDP-galactose or UDP-xylose (CarboSource Services, University of Georgia, Ga., USA) at a final concentration of 250 μM. Reactions were performed for 16 h, and stopped by addition of 10 μl of 10% glacial acetic acid. The products of 3×100 μL reactions were combined for LC-MS analysis.
Substrate Preference of MpUGT88A1
The entire coding region of the MpUGT88A1 cDNA was cloned in frame into the expression vector pET30a(+) for the production of His6-tagged recombinant protein in Escherichia coli. The recombinant protein was isolated by Ni2+ affinity and gel filtration chromatography. Successful expression of the fusion protein was indicated in SDS-PAGE by the appearance of a protein of the expected 58 kDa size (53.5 kDa for MpUGT88A1+4.5 kDa for the His6 tag and associated residues) in MpUGT88A1 extracts but not in equivalent vector control extracts (
For functional characterisation of MpUGT88A1 aliquots of the purified recombinant protein were assayed for glycosyltransferase activity using [3H]-UDP-glucose as the sugar donor. Fifteen substrates (caffeic acid, catechin, chlorogenic acid, 2-coumaric acid, 3-coumaric acid, 4-coumaric acid, cyanidin, 3-(3,4 dihydroxyphenyl) propionic acid, epicatechin, 3-hydroxybenzoic acid, naringenin, phloretin, protocatechuic acid, quercetin and rutin) that are natural constituents of apple fruit or commonly available phenolic compounds were screened for activity. Of these substrates, only phloretin was utilised by MpUGT88A1 as an acceptor. The vector control showed no activity towards phloretin, indicating the glycosylation reaction was specific to the MpUGT88A1 enzyme.
Kinetic parameters were determined for MpUGT88A1 with respect to phloretin and UDP-glucose. The enzyme showed a Km of 0.62 uM±0.1 μM for phloretin with a turnover rate of 9.72×10−4 mol/sec/mol. The observed Km for UDP-glucose was ˜13 uM which is consistent with that observed for other UGT enzymes.
MpUGT88A1 protein was tested over a pH range of 5-10. The enzyme showed significant activity from pH 7-8 with maximum activity at pH 7.7 (
LC-MS Analysis of in vitro Reaction Mixtures
LC-MS employed an LTQ linear ion trap mass spectrometer fitted with an ESI interface (ThermoQuest, Finnigan, San Jose, Calif., USA) coupled to an Ettan™ MDLC (GE Healthcare Bio-Sciences, Uppsala, Sweden).
Phenolic compound separation was achieved using a Prodigy 5 μm ODS(3) 100 Å (Phenomenex, Torrance, Calif., USA), 150×2 mm analytical column maintained at 35° C. A 0.2 μm in-line filter (Alltech, Deerfield, Ill., USA) was installed before the column. Solvents were (A) acetonitrile+0.1% formic acid and (B) water+0.1% formic acid and the flow rate was 200 μL·min−1. The initial mobile phase, 5% A/95% B, was held for 5 min then ramped linearly to 10% A at 10 min, 17% A at 25 min, 23% A at 30 min, 30% A at 40 min, 97% A between 48-53 min before resetting to the original conditions. Sample injection volume was 50 μL.
MS data was acquired in the negative mode using a data-dependent LC-MS3 method. This method isolates and fragments the most intense parent ion to give MS2 data, then isolates and fragments the most intense daughter ion (MS3 data). To maximise sensitivity, the full scan range was set to m/z 420-490 from 0-42.9 min for the detection of phloretin glycosides and then to m/z 270-280 from 43 min for the detection of unreacted phloretin. The ESI voltage, capillary temperature, sheath gas pressure and sweep gas were set at −10 V, 275° C., 40 psi, and 5 psi, respectively.
LC-MS Analysis
Products of the reaction between MpUGT88A1, phloretin and UDP-glucose were analysed by HPLC (
The MpUGT88A1 enzyme was tested with phloretin in the presence of two additional activated sugar donors—UDP-xylose and UDP-galactose. HPLC chromatograms indicated that a single glycosylated product was formed with UDP-xylose with a retention time of 40.5 min (
Mass spectrometry was used to further characterise the products of the MpUGT88A1 reactions. Phloretin (
Expression of Md—138221 in E. coli
The Md—138221 sequence (SEQ ID NO: 14) was also amplified and cloned into pET30a(+) by standard techniques as described above for MpUGT88A1.
The cloned sequence was confirmed by sequencing before transfer into RIL cells as described above. Assays for phloretin glycosyl transferase activity were then completed as described above. Md—138221 also showed phloretin glycosyltransferase activity at approximately 3% of that shown for MpUGT88A1.
Discussion
A genomics approach was used to identify the gene MpUGT88A1 in apple (cv. Royal Gala) with homology to Family 88 UGTs of unknown function from Arabidopsis, rice and sweetleaf (S. rebaudiana). Glycosyltransferase assays and LC-MS revealed that the recombinant MpUGT88A1 enzyme could specifically glycosylate phloretin in the presence of UDP-glucose, UDP-xylose and UDP-galactose. This is the first report of functional characterisation of a UGT that utilises a dihydrochalcone as its primary substrate.
The glycosylated product of phloretin and UDP-glucose co-migrated with a known phlorizin standard, indicating that MpUGT88A1 is likely to be the enzyme that glycosylates phloretin to phlorizin in planta. This assertion is supported by other lines of evidence. Expression data indicated that the MpUGT88A1 gene was highly expressed in leaf, root, flower and fruit, which is consistent with the presence of phlorizin in these apple tissues. The recombinant MpUGT88A1 protein also produced glycosides with UDP-xylose and UDP-galactose (with one of the products glycosylated with UDP-galactose having the same retention time as phlorizin). Apples have been reported to produce two phloretin glycosides in addition to phloretin 2′-glucoside (phlorizin), namely phloretin 2′-galactoside (Barth et al. 2005) and phloretin 2′-xyloglucoside (Lommen et al. 2000). Although the exact nature of the glycosylation in these reactions has not been determined (as no authentic apple compounds were available for comparison), the MS spectra clearly indicated that the sugar molecules were attached to phloretin. It seems likely the regio-specificity of MpUGT88A1 is to the 2′-hydroxyl position, as substrates (e.g. naringenin and quercetin) with hydroxyl groups in alternative positions were not utilised as substrates.
No sequences within UGT Family 88 have been ascribed a definitive function to date. SvUGT88B1 from S. rebaudiana is reported to show trace activity with kaempferol (Richman et al. 2005), whilst VmUFGT4, a partial clone isolated from V. mungo seedling tissue (Mato et al. 1998) and OsBAC10743 from rice, are completely uncharacterised. Although UGT sequences from Arabidopsis have been extensively characterised in vitro (e.g. Lim et al. 2002), no function has been ascribed to AtUGT88A1. AtUGT88A1 is moderately expressed in a number of Arabidopsis tissues but especially in cauline leaves (Arabidopsis eFP browser; http://www.bar.utoronto.ca).
Family 88 UGTs are classified as belonging to the larger Group E UGTs, which also contains UGT Families 71 and 72. Some enzymes in these families have been functionally characterised and show activity towards a wide range of substrates including: phenylpropanoids, benzoates chlorinated phenols and anilines (AtUGT71C1, Lim and Bowles 2004; AtUGT72E2, Lim and Bowles 2004; AtUGT72B, Loutre et al. 2003); coumarins, flavonoids and naphthols (NtGT1a, NtGT3, Nicotiana tabacum, Taguchi et al. 2003); quercetin, isoflavones and triterpene saponins (UGT71G1, Medicago truncatula, Achnine et al. 2005) and hydroxyquinone (arbutin synthase, Rauvolfia serpentina, Hefner and Stockigt 2003). It is possible that some of these enzymes also accept dihydrochalcones as substrates, but this has not been reported. Other Family 88 UGTs may be specific for dihydrochalcones, but this is unlikely given that dihydrochalcone glycosides are found almost exclusively in Malus (and have been used as markers for the detection of apple admixtures in other fruit juices and purees, Dragovic-Uzelac et al. 2005). Family 88 UGTs are most likely to show as yet undetermined stereo-specificity represented by the 2′ hydroxyl position in phloretin.
In conclusion, our results have identified a key enzyme in the biochemical pathway to phlorizin production in apples. The ability to manipulate phlorizin levels in fruit is an important target in apple breeding programmes as “the use of phlorizin may provide the molecular basis for the clinical observation that an apple a day keeps the doctor away” (Ehrenkranz et al. 2005).
Transient Transformation of Tobacco Leaves.
Tobacco (Nicotiana benthamiana) is transiently transformed with Agrobacterium cultures containing a gene/polynucleotide (e.g. SEQ ID NO:6-15) encoding phloretin glycosyltransferase (e.g. SEQ ID NO: 1-5) cloned in pGreen (Hellens et al., 2000) mixed with Agrobacterium containing the gene for the silencing suppressor P19 as previously described (Hellens et al., 2005). Controls are run using Agrobacterium containing P19 in pGreen alone. Tobacco leaves were harvested 9 days after transformation and frozen in liquid nitrogen.
Stable Transformation of Apple Plants
Two constructs containing MdPGT1 (EST111441) were produced, in pHEX2 (Hellens et al., 2005) by standard techniques, to characterise the role of the MdPGT1 gene in transgenic apple plants.
An RNAi knockout construct was produced. This construct contained 455 nucleotides of the EST111441 gene (nucleotides 473-928 of SEQ ID NO: 6) in sense orientation and the same 455 nucleotides oriented in the antisense direction to form an inverted repeat. Constitutive expression of the hairpin dsRNA was driven by a cauliflower mosaic virus (CaMV) 35S promoter.
An over-expression construct was produced. This construct contained the full length EST111441 (SEQ ID NO: 6) driven by a CaMV 35S promoter.
Transgenic apple plants were regenerated as described in Yao et al., (1995) Plant cell Reports 14: 407-412.
Fourteen apple lines containing the EST111441 knockout construct were regenerated on kanamycin-containing medium. Eleven of the lines were confirmed by PCR to contain the EST111441 knockout construct. Five lines that showed the most significant decrease in expression of EST111441 RNA (compared to EST111441 expression in wild type leaves) were transferred to the containment greenhouse for further analysis (including analysis of phloretin and phlorizin levels).
Eight apple lines containing the EST111441 over-expression construct were regenerated on kanamycin-containing medium. These lines will be characterised in the same way as the knockout lines to choose the best plants for transfer into the containment greenhouse.
Phlorizin Measurement
Collected leaves are immediately flash-frozen in liquid N2, in the field, stored at −40° C., lyophilized and ground to a fine powder. Extracts are prepared by pre-washing 200 mg of powder in diethylether and extracting at 40° C. for 3 hr in 70% acetone-8.6 mM ascorbic acid. Acetone is removed by evaporation under pressure and distilled H2O added to a constant 5 ml vol. Concentrations of phloridzin and phloretin are estimated by reverse phase HPLC against commercial standards, with a 45:55 MeOH:H2O solvent, buffered with PIC-A reagent (Supelco, column 25 cm×4.6 mm containing a 5 pm C18 bonded phase, preceded by a 2 cm guard column packed with 37-75 um Porasil B, flow rate 0.9 ml min−1) and detected at 254 nm with a UV detector. Hunter and Hull 1993
These experiments can show that over-expression of the phloretin glycosyltransferase sequence of the invention can increase phloretin glycosyltransferase activity or phlorizin production in plants.
The Md—111441 sequence was used to identify orthologous phloretin glycosyltransferase genes from HortResearch proprietary sequence data bases.
Four variant sequences were identified as summarised in Table 1 below.
Malus
sieboldii
An ClustalX (Version 1.8) alignment (Hall, 1999) of the polypeptide sequences of Md—111441 and all of the four variants is shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 564691 | Dec 2007 | NZ | national |
This application is a Continuation of International Application No. PCT/NZ2008/000341, filed Dec. 19, 2008, which claims the benefit of New Zealand Patent Application No. 564691, filed Dec. 21, 2007, which is hereby incorporated by reference to the extent there is no inconsistency with the present disclosure.
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| Number | Date | Country | |
|---|---|---|---|
| 20110030098 A1 | Feb 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/NZ2008/000341 | Dec 2008 | US |
| Child | 12818725 | US |
