AU667392B2 - Genetic sequences encoding flavonol synthase enzymes and uses therefor - Google Patents

Description

OPI DATE 03/03/94 APPLN. ID 46901/93 11|1 |i |1 1 1 AOJP DATE 26/05/94 PCT NUMBER PCT/AU93/00400 1111111111111111111 11111 AU9346901 (51) International Patent Classification 5 (11) International Publication Number: WO 94/03606 C12N 15/52, 15/53, 9/02 Al C12N 9/04, AO1H 5/00, 5/02 (43) International Publication Date: 17 February 1994 (17.02.94) (21) International Application Number: PCT/AU93/00400 (81) Designated States: AU, CA, JP, NZ, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, (22) International Filing Date: 5 August 1993 (05.08.93) MC, NL, PT, SE).

Priority data: Published PL 3944 5 August 1992 (05.08.92) AU With international search report.

(71) Applicant (for all designated States except US): INTERNA- TIONAL FLOWER DEVELOPMENTS PTY. LTD.

[AU/AU]; 16 Gipps Street, Collingwood, VIC 3066

(AU).

(72) Inventors; and Inventors/Applicants (for US only): HOLTON, Timothy, Albert [AU/AU]; Unit 1, 19 Oldis Street, Northcote, VIC 3070 KEAM, Lisa, Ann [AU/AU]; 33 Freeman Street, North Fitzroy, VIC 3068 (AU).

(74)Agents: SLATTERY, John, M. et al.; Davies Collison Cave, 1 Little Collins Street, Melbourne, VIC 3000 (AU).

66739 2 (54)Title: GENETIC SEQUENCES ENCODING FLAVONOL SYNTHASE ENZYMES AND USES THEREFOR (57) Abstract The present invention relates generally to genetic sequences encoding flavonoid pathway metabolising enzymes and in particular enzymes having flavonol synthase activity and their use such as in manipulating production of pigmentory molecules in plants. More particularly, the present invention provides genetic sequences encoding flavonol synthase (FLS).

,r i;*p WO 94/03606 PCT/AU93/00400 1 GENETIC SEQUENCES ENCODING FLAVONOL SYNTHASE ENZYMES AND USES THEREFOR The present invention relates generally to genetic sequences encoding flavonoid metabolising enzymes and in particular enzymes having flavonol synthase activity and their use such as in manipulating production of pigmentory molecules in plants.

Bibliographic details of the publications referred to hereinafter in the specification are collected at the end of the description. SEQ ID No's referred to herein in relation to 1 0 nucleotide and amino acid sequences are defined after the Bibliography.

The flower industry strives to develop new and different varieties of flowering plants.

An effective way to create such novel varieties is through the manipulation of flower colour and classical breeding techniques have been used with some success to produce a wide range of colours for most of the commercial varieties of flowers. This approach has been limited, however, by the constraints of a particular species' gene pool and for this reason it is rare for a single species to have a full spectrum of coloured varieties. For example, the development of blue varieties of the major cut flower species such as rose, chrysanthemum, carnation, lily, tulip and gerbera would offer a significant opportunity in both the cut flower and ornamental markets.

The colours of flowers and other plant parts are predominantly due to two types of pigments: flavonoids and carotenoids. Flavonoids are the most common and the most important of the flower pigments. The most important classes of flavonoids with 2 5 respect to flower colour are anthocyanins, flavonols and flavones. Anthocyanins are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole.

One important factor for flower colour is co-pigmentation of anthocyanins with tannins 3 0 and certain flavone and flavonol glycosides (Scott-Moncrieff, 1936). When compared over a range of pH values, co-pigmented anthocyanins are always found to be bluer than the normal pigment. Co-pigmentation of anthocyanins with flavonol glycosides can also be important for the development of colour in fruit (Yoshitama et al., 1992).

The molar ratio of anthocyanin to co-pigment can also exert a strong influence on 3 5 colour. It has recently been demonstrated that flavonol aglycones are essential for pollen germination and pollen tube growth (Mo et al., 1992). The ability to control the production of co-pigments, such as flavonols, in plants could therefore have useful ii li. WO 94/03606 PCT/AU93/00400 2 applications in altering flower colour and manipulating plant fertility.

The biosynthetic pathway for the anthocyanin pigments is well established (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and de Vlaming, 1984; Schram et al., 1984; Stafford, 1990). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA with one molecule ofpcoumaroyl-CoA. This reaction is catalysed by the enzyme chalcone synthase. The product of this reaction, 2',4,4',6'-tetrahydroxychalcone, is normally rapidly isomerised to produce naringenin by the enzyme chalcone-flavanone isomerase.

1 0 Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3-hydroxylase to produce dihydrokaempferol (DHK). The B-ring of dihydrokaempferol can be hydroxylated at either the or both the 3' and 5' positions, to produce dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively.

DHK, DHQ and DHM may be converted to coloured anthocyanins (pelargonidin 3- 1 5 glucoside, cyanidin 3-glucoside and delphinidin 3-glucoside) by the action of at least two enzymes (dihydroflavonol-4-reductase and flavonoid-3-glucosyltransferase).

Flavonols such as kaempferol quercetin and myricetin are formed from dihydroflavonols by the introduction of a double bond between C-2 and C-3 (Forkmann, 1991), as illustrated in Figure 1. Flavonols often accumulate in glycosylated forms and may also be methylated. Methylation can occur either before or after glycosylation. In vitro conversion of dihydroflavonols to flavonols was first observed in enzyme preparations from parsley cell cultures (Britsch et al., 1981).

Flavonol synthase activity has also been detected in flower extracts from Matthiola 2 5 (Spribille and Forkmann, 1984), Petunia (Forkmann et al., 1986) and Dianthus (Forkmann, 1991). Flavonol synthase enzyme activity requires 2-oxoglutarate, ascorbate and ferrous ions as cofactors. In flowers of Petunia hybrida, the genetic locus Fl controls the formation of flavonols: flavonol synthesis is greatly reduced in mutants homozygous recessive for this gene (Wiering et al., 1979; Forkmann et al., 3 0 1986). In vitro enzyme assays with the flavonol synthase from petunia showed that DHK and DHQ were readily converted to the respective flavonols, whereas DHM was a poor substrate. The ability to control flavonol synthase activity in flowering plants would provide a means to manipulate petal colour by altering flavonol production, thereby enabling a single species to express a broader spectrum of flower colours. As 3 5 stated above, the ability to control flavonol production also has implications in respect of male fertility. Such control may be by modulating the level of production of an indigenous enzyme or by introducing a non-indigenous enzyme k II- pr WO 94/03606 PCT/AU93/00400 3 As used herein an "indigenous" enzyme is one which is native to or naturally expressed in a particular cell. A "non-indigenous" enzyme is an enzyme not native to the cell but expressed through the introduction of genetic material into a plant cell; for example, through a transgene. An "endogenous" enzyme is an enzyme produced by a cell but which may or may not be indigenous to that cell.

In accordance with the present invention, the genetic sequences encoding flavonol synthase (hereinafter referred to as "FLS") have been identified and cloned from a 1 0 number of sources and used to generate transgenic plants. These recombinant sequences permit the modulation of levels of flavonol production thereby providing a means to manipulate petal colour and male fertility. The recombinant sequences also permit the modulation of DHK metabolism as well as the metabolism of other substrates, such as DHQ and DHM. Since DHK, DHQ and DHM are precursors of 1 5 the coloured anthocyanins modulation of their concentrations by expression of FLS sequences provides another means of manipulating flower colour.

Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to a 2 0 sequence encoding a plant FLS or a functional mutant, derivative, part, fragment, homologue or analogue of said FLS. The expression "FLS" includes reference to polypeptides and proteins having FLS activity as well as any mutants, derivatives, parts, fragments, homologues or analogues of such polypeptides or proteins and which have FLS activity. A molecule having FLS activity may also be a fusion polypeptide or protein between a polypeptide or protein having FLS activity and an extraneous peptide, polypeptide or protein.

As used herein, the term "isolated nucleic acid molecule" is meant to include a genetic sequence in a non-naturally-occurring condition. Generally, this means isolated away 3 0 from its natural state or formed by procedures not necessarily encountered in its natural environment. More specifically, it includes nucleic acid molecules formed or maintained in vitro, including genomic DNA fragments, recombinant or synthetic molecules and nucleic acids in combination with heterolcgous nucleic acids such as heterologous nucleic acids fused or operably-linked to the genetic sequences of the 3 5 present invention. The term "isolated nucleic acid molecule" also extends to the genomic DNA or cDNA, or part thereof encoding FLS or a functional mutant, derivative, part, fragment, homologue or analogue of FLS, in reverse orientation I WO 94/03606 PCT/AU93/00400 4 relative to its or another promoter. It further extends to naturally-occurring sequences following at least a partial purification relative to other nucleic acid sequences. The term "isolated nucleic acid molecule" as used herein is understood to have the same meaning as a "nucleic acid isolate".

The expression "genetic sequences" is used herein in its most general sense and encompasses any contiguous series of nucleotide bases specifying directly, or via a complementary series of bases, a sequence of amino acids comprising a FLS molecule including a polypeptide or protein having FLS activity. Such a sequence of amino 1 0 acids may constitute a full-length FLS such as is set forth in, for example, SEQ ID No:l or SEQ ID No:4 or SEQ ID No:5 or an active truncated form thereof or a functional mutant, derivative, part, fragment, homologue or analogue thereof.

Alternatively, the amino acid sequence may comprise part of, for example, these sequences or all or part of the sequences set forth in SEQ ID No:2 or SEQ ID No:3.

Another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which: encodes a FLS; and (ii) has at least 50% nucleotide sequence similarity to the nucleotide sequence set forth in at least one of SEQ ID No: 1 or SEQ ID No:2 or SEQ ID No:3 or SEQ ID No:4 or SEQ ID More particularly, the present invention is directed to an isolated DNA molecule comprising a sequence of nucleotides which: 2 5 encodes a FLS; and (ii) has at least 65-75% nucleotide sequence similarity to the nucleotide sequence set forth in at least one of SEQ ID No: 1 or SEQ ID No:2 or SEQ ID No:3 or SEQ ID No:4 or SEQ ID 3 0 Preferred percentage similarities include 80%, 85%, 90%, 92-95%, 96-98% and 99- 100%. Although the pecentage similarities referred to above assume an overall comparison between the sequences set forth in at least one of SEQ ID No:l or SEQ ID No:2 or SEQ ID No:3 or SEQ ID No:4 or SEQ ID No:5 and another genetic sequence, it is clear that there may be specific regions within the molecules being compared 3 5 having less than 50% similarity. In this respect, the present invention is further defined as a nucleic acid molecule, and in particular a DNA molecule, comprising a sequence of nucleotides which: WO 94/03606 PCT/AU93/00400 encodes a FLS; and (ii) has at least 50-75% nucleotide sequence similarity to one or more regions of the sequence set forth in at least one of SEQ ID No:l or SEQ ID No:2 or SEQ ID No:3 or SEQ ID No:4 or SEQ ID The nucleic acid sequences contemplated herein also encompass oligonucleotides useful as genetic probes or as "antisense" molecules capable of regulating expression of the corresponding gene in a plant. An "antisense molecule" as used herein may also encompass a gene construct comprising the structural genomic or cDNA gene or part 1 0 thereof in reverse orientation relative to its or another promoter.

With respect to this aspect of the invention there is provided an oligonucleotide of 5-50 nucleotides having substantial similarity or complementarity to a part or region of a molecule with a nucleotide sequence set forth in at least one of SEQ ID No: or SEQ 1 5 ID No:2 or SEQ ID No:3 or SEQ ID No:4 or SEQ ID No:5. By "substantial similarity or complementarity" in this context is meant a hybridizable similarity under low, alternatively and preferably medium and alternatively and most preferably high stringency conditions, as defined below. Such an oligonucleotide is useful, for example, in screening FLS genetic sequences from various sources or for monitoring an introduced genetic sequence in a transgenic plant. Such an oligonucleotide is generally in the form of a primer or a probe. Preferably, the oligonucleotide is directed to a conserved FLS genetic sequence or a sequence conserved within a plant genus, plant species and/or plant strain or variety.

2 5 In one aspect of the present invention, the oligonucleotide corresponds to the 5' or the 3' end of the FLS genetic sequence. For convenience, the 5' end is considered herein to define a region substantially between the start codon of the structural gene to a centre portion of the gene, and the 3' end is considered herein to define a region substantially between the centre portion of the gene and the terminating codon of the structural gene.

It is clear, therefore, that oligonucleotides may hybridize to the 5' end or the 3' end or to a region common to both the 5' and the 3' ends. The present invention extends to all such oligonucleotides.

In one embodiment, the nucleic acid sequence encoding a FLS or a functional mutant, 3 5 derivative, part, fragment, homologue or analogue thereof is used to reduce the activity of an indigenous FLS, such as by using co-suppression (US Patent Number 5,034,323). Alternatively, the nuci.ic acid sequence encoding this enzyme or various i ~I~ WO 94/03606 PCT/AU93/00400 6 functional mutants, derivatives, parts, fragments, homologues or analogues thereof, is used in the antisense orientation to reduce activity of the indigenous FLS. Although not wishing to limit the present invention to any one theory, it is possible that an antisense FLS transcript or fragment or part thereof (for example, an oligonucleotide nmlecule) would form a duplex with all or part of the naturally-occurring mRNA specified for the enzyme thus preventing accumulation of or translation from the mRNA into active enzyme.

In another alternative, ribozymes could be used to inactivate target nucleic acid 1 0 sequences. Ribozymes are well described by Haseloff and Gerlach (1988). With respect to this embodiment, the ribozyme would preferably comprise a hybridizing portion and a catalytic portion wherein the hybridizing portion comprises one and preferably two nucleotide arms capable of hybridizing to a mRNA transcript from a gene having a nucleotide sequence substantially as set forth in at least one of SEQ ID 1 5 No: 1 or SEQ ID No:2 or SEQ ID No:3 or SEQ ID No:4 or SEQ ID In a further embodiment, the nucleic acid sequence encoding a FLS or a functional mutant, derivative, part, fragment, homologue or analogue thereof is used to elevate the activity of an indigenous FLS above the normal endogenous or existing level, or 2 0 alternatively to provide FLS activity where the normal endogenous or existing level of activity is negligible or zero.

Reference herein to the altering of FLS activity relates to an elevation or reduction in activity of 30% or more, or more preferably of 30-50%, or even more preferably 2 5 75% or still more preferably 75% or greater above or below the normal endogenous or existing levels of activity. Such elevation or reduction may be referred to as "modulation" of FLS enzyme activity. Generally, modulation is at the level of transcription or translation of FLS genetic sequences. The level of activity can be assayed using a modified method of Forkmann et al. (1986).

The nucleic acids of the present invention may be ribonucleic acids or deoxyribonucleic acids, single stranded or covalently closed circular molecules. Preferably, the nucleic acid molecule is, or originates from, cDNA. The present invention also extends to other nucleic acid molecules which hybridize to the genetic sequences herein disclosed.

i II WO 94/03606 PCT/AU93/00400 7 According to this aspect of the present invention there is provided an isolated nucleic acid molecule comprising a sequence of nucleoides which: encodes a FLS; and (ii) hybridizes to the nucleotide sequence set forth in at least one of SEQ ID No: 1 or SEQ ID No:2 or SEQ ID No:3 or SEQ ID No:4 or SEQ ID No:5 or a complementary respective form thereof under low stringency conditions.

For the purpose of defining the level of stringency, reference can conveniently be made to Maniatis et al. (1982) at pages 387-389, and especially paragraph 11, which is 1 0 herein incorporated by reference. A low stringency is defined herein as being in 4-6 x SSC 1% SDS at 37-450C for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridization, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 1-4 x SSC 0.5-1% SDS at greater than or equal to 45 0

C

1 5 for 2-3 hours or high stringent conditions considered herein to be 0.1-1 x SSC 0.1- SDS at greater than or equal to 60 0 C for 1-3 hours.

In its most preferred embodiment, the present invention extends to a nucleic acid molecule having or comprising a nucleotide sequence set forth in at least one of SEQ ID No:l or SEQ ID No:2 or SEQ ID No:3 or SEQ ID No:4 or SEQ ID No:5 or to a molecule having at least 50%, more preferably at least 55%, even more preferably at least 60%, still more preferably at least 65-70%, and yet even more preferably greater than 85% similarity at the level of nucleotide or amino acid sequence to at least one or more regions of the nucleotide or amino acid sequence set forth, respectively, in the 2 5 above-referenced sequences and wherein the nucleic acid encodes or is complementary to a sequence which encodes an enzyme having FLS activity. It should be noted, however, that nucleotide or amino acid sequences may have similarities below the above given percentages and yet still encode a FLS-like molecule and such molecules may still be considered within the scope of the present invention where they have 3 0 regions of sequence conservation.

The nucleic acid molecules contemplated herein may exist, in either orientation, alone or in combination with a vector molecule and preferably an expression-vector. The term "vector molecule" is used in its broadest sense to include any intermediate vehicle 3 5 for the nucleic acid molecule, capable of facilitating transfer of the nucleic acid into the l plant cell and/or facilitating integration into the plant genome. An intermediate vehicle may, for example, be adapted for use in electroporation, microprojectile bombardment, Agrobacterium-mediated transfer or insertion via DNA or RNA viruses. The WO 94/03606 PCT/AU93/00400 8 intermediate vehicle and/or the nucleic acid molecule contained therein may or may not need to be stably integrated into the plant genome. Such vector molecules may also replicate and/or express in prokaryotic cells. Preferably, the vector molecules or parts thereof are capable of integration into the plant genome. The nucleic acid molecule may additionally contain a promoter sequence capable of directing expression of the nucleic acid molecule in a plant cell. The nucleic acid molecule and promoter may also be introduced into the cell by any number of means such as those described above. The vector molecule may also comprise a genetic sequence encoding a ribozyme as hereinbefore defined capable of cleaving a FLS mRNA transcript The nucleic acid or its complementary form may encode the full-length enzyme or a derivative thereof. By "derivative" is meant any single or multiple amino acid substitutions, deletions, and/or additions relative to the naturally-occurring enzyme and which retains FLS activity. In this regard, the nucleic acid includes the naturally- 1 5 occurring nucleotide sequence encoding FLS or may contain single or multiple nucleotide substitutions, deletions and/or additions to said naturally-occurring sequence. The nucleic acid sequences of the present invention or its complementary form may also encode a "part" of a FLS, whether active or inactive, and such a nucleic acid molecule may be useful as an oligonucleotide probe, primer for polymerase chain 2 0 reactions or in various mutagenic techniques, or for the generation of antisense molecules or ribozyme molecules capable of regulating expression of the corresponding gene in a plant.

Amino acid insertional derivatives of the FLS of the present invention include amino 2 5 and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino 3 0 acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with Table 1, overleaf.

Where FLS is derivatised by amino acid substitution, the amino acids are generally 3 5 replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like. Amino acid Ssubstitutions are typically of single residues. Amino acid insertions will usually be in l l -s~n=rrr~FDI=Pg~i--~rr--~-LU b WO 94/03606 PCT/AU93/00400 9 the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.

TABLE 1 for amino acid substitutions Suitable residues Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gin; His Asp Glu Cys Ser Gin Asn; Glu Glu Asp Gly Pro His Asn; Gin Be Leu; Val Leu Ile; Val Lys Arg; Gin; Glu Met Leu; le; Val Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu; Met 3 5 The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield, 1964) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The WO 94/03606 PCT/AU93/00400 manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently described, for example, in Sambrook et al. (1989).

Other examples of recombinant or synthetic mutants and derivatives of the FLS enzyme of the present invention include single or multiple substitutions, deletions and/or additions of any molecule associated with the enzyme such Ls carbohydrates, lipids and/or proteins or polypeptides.

The terms "analogues" and "derivatives" also extend to any functional chemical equivalent of FLS and also to any amino acid derivative described above. For convenience, reference to "FLS" herein and in particular hereinafter includes reference to any functional mutant, derivative, part, fragment, homologue or analogue thereof.

The present invention is exemplified using nucleic acid sequences derived from petunia, tobacco, carnation and chrysanthemum, since these represent the most convenient and preferred sources of material to date. However, one skilled in the art will immediately appreciate that similar sequences can be isolated from any number of sources such as other plants. All such nucleic acid sequences encoding directly or indirectly FLS are encompassed by the present invention regardless of their source.

Examples of other suitable sources of genes encoding FLS enzymes include, but are not limited to rose, snapdragon, lisianthus, cyclamen, grape and parsley.

In accordance with the present invention, a nucleic acid sequence encoding a FLS may be introduced into and expressed in a transgenic plant in either orientation thereby providing a means to convert DHK and/or other suitable substrates, if synthesised in the plant cell, ultimately into flavonols or derivatives of same or alternatively to inhibit such conversion of metabolites by reducing or eliminating endogenous or existing FLS activity. The production of these flavonols will modify petal colour and may contribute to the production of bluer colours via co-pigmentation with anthocyanins. Expression of the nucleic acid sequence in the plant may be constitutive, inducible or developmental and may also be tissue-specific. The term "expression" is used in its broadest sense to include production of RNA or of both RNA and protein. It also extends to partial expression of a nucleic acid molecule.

According to this aspect of the present invention there is provided a method for producing a transgenic flowering plant capable of synthesizing FLS, said method ~as nser~r~~ WO 94/03606 PCT/AU93/00400 11 comprising stably transforming a cell of a suitable plant with a nucleic acid sequence which comprises a sequence of nucleotides encoding said FLS under conditions permitting the eventual expression of said nucleic acid sequence, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid sequence. The transgenic plant may thereby produce non-indigenous FLS at elevated levels relative to the amount expressed in a comparable non-transgenic plant.

Another aspect of the present invention contemplates a method for producing a 1 0 transgenic plant with reduced indigenous or existing FLS activity, said method comprising stably transformin a cell of a suitable plant with a nucleic acid molecule which comprises a sequence of nucleotides encoding or complementary to a sequence encoding a FLS activity, regenerating a transgenic plant from the cell and where necessary growing said transgenic plant under conditions sufficient to permit the 1 5 expression of the nucleic acid.

Yet another aspect of the present invention contemplates a method for producing a genetically modified plant with reduced indigenous or existing FLS activity, said method comprising altering the Fl gene through modification of the indigenous 2 0 sequences via homologous recombination from an appropriately altered Fl gene or derivative or part thereof introduced into the plant cell, and regenerating the genetically modified plant from the cell.

In a preferred embodiment, the present invention contemplates a method for producing a transgenic flowering plant exhibiting altered inflorescence properties, said method comprising stably transforming a cell of a suitable plant with a nucleic acid sequence of the present invention, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid sequence into a FLS. Alternatively, said method may comprise stably 3 0 transforming a cell of a suitable plant with a nucleic acid sequence of the present invention or its complementary sequence, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to alter the level of activity of the indigenous or existing FLS. Preferably the altered level would be less than the indigenous or existing level of FLS activity in a comparable non- 3 5 transgenic plant. Without wishing to limit the present invention, one theory of mode of action is that reduction of the indigenous FLS activity requires the expression of the introduced nucleic acid sequence or its complementary sequence. However, -C ~r=n;iCI~31^---~TI WO 94/03606 4CT/AU93/00400 12 expression of the introduced genetic sequence or its complement may not be required to achieve the desired effect: namely, a flowering plant exhibiting altered inflorescence properties.

In a related embodiment, the present invention contemplates a method for producing a flowering plant exhibiting altered inflorescence properties, said method comprising alteration of the Fl gene through modification of the indigenous sequences via homologous recombination from an appropriately altered Fl gene or derivative or part thereof introduced into the plant cell, and regenerating the genetically modified plant 1 0 from the cell.

Preferably, the altered inflorescence includes the production of white, yellow, pink, violet or blue flowers or other colour shades depending on the genotype and physiological conditions of the recipient plant.

Accordingly, the present invention extends to a method for producing a transgenic plant capable of expressing a recombinant gene encoding a FLS or which carries a nucleic acid sequence which is substantially complementary to all or a part of a mRNA molecule optionally transcribable where required to effect regulation of a FLS, said method comprising stably transforming a cell of a suitable plant with the isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to a sequence encoding, a FLS, where necessary under conditions permitting the eventual expression of said isolated nucleic acid molecule, and regenerating a transgenic plant from the cell. By "suitable plant" is meant a plant capable of producing DHK, or other substrates of FLS, and possessing the appropriate physiological properties required for the development of the colour desired.

The present invention is exemplified by generation of transgenic petunia and tobacco plants containing introduced FLS genetic sequences. The use of petunia and tobacco plants represents a particularly convenient and useful model for the generation of transgenic plants carrying genetic sequences and the results obtained from such transgenic plants are generally applicable to other plants. One skilled in the art will immediately recognise the variations applicable to this method snch as increasing or decreasing the expression of the enzyme naturally present in a target plant. This would lead to differing shades of colours. Other suitable target plants, in addition to petunia lisianthus, lily, iris and pelargonium.

i WO 94/03606 PCT/AU93/00400 13 The present invention, therefore, extends to all transgenic plants containing all or part of the nucleic acid sequence of the present invention, or antisense forms thereof and/or any homologues or related forms thereof and in particular those transgenic plants which exhibit altered inflorescence properties. The transgenic plants may contain an introduced nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding a FLS. Generally the nucleic acid would be stably introduced into the plant genome, although the present invention also extends to the introduction of a FLS nucleotide sequence within an autonomously-replicating 1 0 nucleic acid sequence such as a DNA or RNA virus capable of replicating within the plant cell. The invention also extends to seeds from such transgenic plants. Such seeds, especially if coloured, will be useful, inter alia, as proprietary tags for plants.

The invention further extends to fruit and to vegetable plants and leaves of, for example, ornamental plants.

Another aspect of the present invention is directed tc -mbinant forms of FLS. The recombinant forms of the enzymes will provide a source of material for research to develop, for example, more active enzymes and may be useful in developing in vitro systems for production of flavonols and/or coloured compounds.

Still a further aspect of the present invention contemplates the use of the genetic sequences described herein in the manufacture of a genetic construct capable of expressing a FLS or down-regulating an indigenous FLS enzyme in a plant.

2 5 Another aspect of the present invention is directed to a prokaryotic or eukaryotic organism carrying a genetic sequence encoding a FLS extrachromasomally in plasmid form. In one embodiment, the plasmid is pCGP481 in Escherichia coli. The microorganism Escherichia coli strain DH5a containing the plasmid pCGP481 was deposited with the Australian Government Analytical Laboratories, 1 Suakin Street, 3 0 Pymble, New South Wales, 2037, Australia on August 5, 1993 under Accession Number N93/33236.

The present invention is further described by reference to the following non-limiting Figures and Examples.

The amino acid abbreviations used throughout the specification, including in the Examples, are shown overleaf in Table 2.

WO 94/03606 PTA9/00 PCr/AU93/00400 14 TABLE 2 Amino acid 3-letter single-letter L-alanine Ala A L-arginine Arg R L-asparagine Asn N L-aspartic acid Asp D L-cysteine Cys C L-glutamnine Gin Q L-glutamic acid Glu E L-glycine Gly G L-histidine His H L-isoleucine Ile I L-leucine Leu L L-lysine Lys K L-meUtionine Met M L-phenylalanine Phe F L-proline Pro P L-serine Ser S L-threonine Thr T L-tryptophan Trp, W L-tyrosine Tyr Y L-valine Val V

I

WO 94/03606 PCr/AU93/00400 Table 3 provides a summary of the SEQ ID No's assigned to genetic sequences referred to herein: TABLE 3 Sequence SEQ ID No cDNA insert of pCGP481 SEQ ID No: 1 cDNA insert of pCGP489 SEQ ID No:2 cDNA insert of pCGP49O SEQ ID No:3 cDNA insert of pCGP777 SEQ ID No:4 cDNA insert of pCGP874 SEQ ID Oligo #1 SEQ ID No:6 Oligo #2 SEQ ID No:7 Oligo #3 SEQ ID No:8 Oligo #4 SEQ IOD No:9 Oligo #5 SEQ ID No: Oligo #6 SEQ ID No: 11 Oligo #7 SEQ ID No: 12 Oligo #8 SEQ ID No: 13 Oligo #9 SEQ ID No: 14 Oligo #10 SEQ ID No: Oligo #11 SEQ ID No: 16 In the Figures: 3 0 Figure I is a schematic representation of the conversion of dihydroflavonols *to flavonols in Petunia hybrida. Enzymes involved in each step of the pathway are indicated as follows: F3'H Flavonoid 3'-hydroxylase; F3'5'H Flavonoid hydroxylase; FLS flavonol synthase. DHK dihydrokaempferol, DHQ dihydroquercetin, DHM dihydromyricetin, K kaempferol, Q quercetin, M 3 5 myricetin.

V WO 94/03606 PCT/AU93/00400 16 Figure 2 is an autoradiograph of an RNA gel blot probed with 32 P-labelled pDIOXC3 cDNA insert. Each lane contained 20 gg of total RNA isolated from the following 1- OGB limb tissue of flowers at the five stages of development; T: OGB tube tissue from stage 3-4 flowers; L: leaf tissue from six week old OGB seedlings.

Figure 3 shows the sequencing strategy used to obtain the complete nucleotide sequence of the cDNA insert of pCGP481. Arrows indicate the direction and length of sequences read from individual sequencing reactions. Sequencing reactions using custom-made oligonucleotide primers (Oligos 7-9; SEQ ID No's 12-14) are also shown.

Figure 4 is a diagrammatic representation of the construction of pCGP631.

pCGP631 was constructed by cloning the pCGP481 cDNA insert in a sense orientation behind the yeast glyceraldehyde-3-phosphate dehydrogenase promoter in the 1 5 expression vector pYGA22m. The cDNA insert from pCGP481 was ligated as a Eco.RI/ZXhI fragment with the large fragment that resulted from the EcoRI/SalI digestion of pYGA22m. IR inverted repeat of 2 im plasmid, TRP1 TRP1 gene, Ap ampicillin resistance marker.

Figure 5 shows a FLS assa3 of yeast extracts using DHQ as substrate. The autoradiograph shows conversion of 14 C]-DHQ to quercetin by enzyme extracts of yeast transformed with the plasmid pCGP631. No FLS activity was detected in untransformed yeast. C unlabelled quercetin without yeast extract. The position of migration of unlabelled quercetin is circled.

1 .~1 genomic DNA or cDNA, or part thereof encoding FLS or a functional mutant, derivative, part, fragment, homologue or analogue of FLS, in reverse orientation U _,OMI rC*4~irCq~p 1PVc ~Y irl~i~ii~~ =~51 rr rn ii--i WO 94/03606 PC'r/AU93/00400 EXAMPLE 1 MATERIALS Enzymes All enzymes were obtained from commercial sources and used according to the manufacturer's recommendations.

Bacterial Strains The following Escherichia coli strains were used: 1 0 PLK-F' and SURE, both obtained from Stratagene; XL1-Blue (Bullock et al.,1987), and DH5a (Hanahan, 1983 and BRL, 1986). The Agrobacterium tumefaciens strain used was the disarmed AGLO (Lazo et al., 1991).

Plant Material 1 5 The Petunia hybrida varieties used are indicated in Table 4, overleaf.

Flowers of Dianthus caryophyllus cv. Laguna were obtained from Van Wyk and Son Flower Supply, Victoria.

Chrysanthemum morifolium cultivars were obtained from Baguley Flower and Plant Growers, Victoria.

EXAMPLE 2 PLANT GROWING CONDITIONS STAGES Growth of plants Petunia hybrida plants were grown in specialised growth rooms with a 14 hour day length at a light intensity of 10,000 lux and a temperature of 22 to 26 0 C. OGB flowers were harvested at developmental stages defined as follows: Stage 1: Unpigmented, closed bud (<25 mm in length).

Stage 2: Pigmented, closed bud (25-35 mm in length).

Stage 3: Dark purple bud with emerging corolla (>35 mm in length).

Stage 4: Dark purple opened flower pre-anther dehiscence (>50 mm in length).

3 5 Stage 5: Fully opened flower with all anthers dehisced.

i WO 94/03606 WO 9403606PCr/AU93/00400 18 TABLE 4 Plant variety Genotype Source/Reference Old Glory Blue Ani, An2, An3, An4, An6, Ball Seed, USA (F1 Hybrid) An8, An9, AnlO, AMi1, Hfl, Htl, Rt, Po, BI, Fl (inferred from phenotype) V23 Ani1, An2, An3, An4, An6 An8, An9, Anl10, phi1, Hfl1, Hf2, htl, Rt, po, BI, Fl Wallroth et al. 1986 Doodeman et al. 1984 R51 Ani1, An2, An3, an4, An6, Wallroth et al. 1986 An8, An9, An10, Anl11, van Tunen et al. 1990 Phi1, hf 1, hf2, Ht 1, rt, Doodeman et al. 1984 Po, bi, fl Ba2O ani1, An2 an4, An6, hfl1, hf2, E. Farcy 2 0 Htl, Phi, Ph2, Ph5, Gf, mfl, INRA, Dijon mf2, Rt, fl V26 Ani1, An2, An3, an4, An6, A. Gerats An 8, An9, Anl10, Ani11, Htl1, Free University 2 5 Hfl, hf2, Phi1, ph2, Ph5, mf I, Amsterdam mf2, Mtl, Mt2, po, Gf, Rt, Fl Flowers of the other petunia varieties were harvested prior to anther dehiscence, at the 3 0 stage of maximum pigment accumulation.

Stages of Dianthus caryophyllus flower development were defined as follows: Stage 1: No visible flower bud.

3 5 Stage 2: Flower buds opening: tips of florets visible.

Stage 3: Tips of nearly all florets exposed; outer florets opening, none horizontal.

Stage 4: Outer florets horizontal.

j derivative, part, fragment, homol.ogue or analogue thereof is used to reduce the activity of an indigenous FLS, such as by using co-suppression (US Patent Number 5,034,323). Alternatively, the nucl-lic acid sequence encoding this enzyme or various 4 WO 94/03606 PCTr/AU93/00400 Stages of Chrysanthzemum flower development were defined as follows: Stage 0: Stage 1: Stage 2: Stage 3: Stage 4: Stage 5: Stage 6: No visible flower bud.

Flower bud visible: florets completely covered by the bracts.

Rlower buds opening: tips of florets visible.

Florets tightly overlapped.

Tips of nearly all florets exposed; outer florets opening but none horizontal.

Outer florets horizontal.

Flower approaching maturity.

EXAMPLE 3 1 5 Synthesis of Oligonucleotides Oligonucleotides were synthesised on an Applied Biosystems PCR-Mate DNA synthesiser using methods recommended by the manufacturer. The oligonucleo tides synthesised were, Oligo 1: GAGAGAGAGAGAGAGAGAGATCTCGAGi'I 1111111111111 SEQID No:6 Oligo 2: Oligo 3: Oligo 4: TGGGGHI'1(T,C)(C,G)AIIT(A,G)T 1(A,G)AICA SEQ ID No:7 TI(A,G)TIAA(T,C)CA(T,C)GGI(A,T)TICC SEQ ID No:8

GGI(T,C)TF=(T,C)(C,G)A(A,G)MT(A,G)TIAA(T,C)CA(T,C)GG

SEQ ID No:9 Oligo 5: GYG(T,C)TTIGG(A,G)CAIGGIGG(A,G)TA Oligo 6: GG(A,G)CAIGGIGG(A,G)TA(A,G)TA(A,GTT Oligo 7: ATCAGAGTACA'ITAGGTC Oligo 8: GTCCCAAATGAAGTCCAAG Oligo 9: TTCTrCGTTIGCTFCCCT Oligo 10: CTAGGiTACC'GGGCCCAAAGGATCCTCTAGAGTAC Oligo 11: TCTAGAGGATCCTTTGGGCCCGGTAC SEQ ID SEQ ID No: 11 SEQ ID No: 12 SEQ ID No: 13 SEQ ID No: 14 SEQ ID No: SEQ ID No: 16 3 5 Where two nucleotides are given in parentheses, this indicates a choice of one or other of the nucleotides; the abbreviation represents deoxyinosine.

rra~r WO 94/03606 PCr/AU93/00400 EXAMPLE 4 CLONING OF A DIOXYGENASE FROM PETUNIA Construction of a petunia cDNA library Total RNA was isolated from the petal tissue of P. hybrida cv. OGB stage 3 to 4 flowers using the method of Turpen and Griffith (1986). Poly (A) RNA was selected from the total RNA by three cycles of oligo-dT cellulose chromatography (Aviv and Leder, 1972).

Two micrograms of poly(A)+ RNA were reverse transcribed in a 20 pL volume 1 0 containing 1 x Superscript" reaction buffer, 10 mM dithiothreitol, 500 M dATP, 500 gM dGTP, 500 M dTTP, 500 gM 5-methyl-dCTP, 0.75 gig Oligo 1 (SEQ ID No:6) and 2 p.L Superscript T M reverse transcriptase (BRL). The reaction mix was incubated at 37 0 C for 50 minutes, 44 0 C for 10 minutes, then placed on ice.

1 5 Second strand reaction mix (140 L) was added to the first strand reaction. The second strand reaction mix consisted of 21 mM Tris-HC1, 104 mM KC1, 5.3 mM MgCl2, 171 gM 3-NAD, 11.4 mM (NH 4 2

SO

4 214 jM dATP, 642 4M dCTP, 214 mM dGTP, 214 gM dTTP, 4 mM DTT, 10 iCi 32 P-dCTP (3000 Ci/mmole), 15 units E. coli DNA ligase, 40 units E. coli DNA polymerase I (Boehringer) and 0.8 units RNAse H. The final mixture was incubated for 150 minutes at 16 0 C. To make the double-stranded cDNA blunt-ended, 10 units of T4 DNA polymerase was added, and the reaction was continued for a further 15 minutes at 16 0 C. The reaction was stopped and the cDNA purified by phenol/chloroform extraction, followed by chloroform extraction and ethanol precipitation.

EcoRI adaptors (Promega) were ligated with the cDNA and then kinased with polynucleotide kinase (Amersham) using conditions recommended by the manufacturer. The enzymes were denatured by heat (70 0 C for 20 minutes) and the DNA was purified by phenol/chloroform extraction and ethanol precipitation. The cDNA was digested with 50 units XhQI (Boehringer) in a reaction volume of 100 L, using conditions recommended by the manufacturer. The enzyme was heat killed 0 C for 20 minutes) and the cDNA digest passed through a Sephacryl S400 spun column (Pharmacia) which had been equilibrated in STE buffer (Sambrook et al., 1989). The eluate was phenol/chloroform extracted and ethanol precipitated. After 3 5 microcentrifugation at 4 0 C for 30 minutes the cDNA pellet was rinsed with 70% (v/v) ethanol, air dried and resuspended in 10 (iL of TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH I C"a io3rrrrlsll WO 94/03606 PCT/AU93/00400 21 One-quarter of the cDNA (2.5 giL) was ligated with 1 gg of XZAPII EcoRI/XhoI/ CIAP treated vector (Stratagene) in 5 piL reaction buffer consisting of 50 mM Tris-HCl (pH 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP and 2 units of T4 DNA ligase. The reaction was incubated at 4 0 C for 4 days.

After incubating at room temperature for two hours, the ligation reaction mixture was packaged using the Packagene system (Promega). The total number of recombinants was 1 x 106. An amount of 1 x 106 plaque forming units (pfu) of the packaged cDNA was plated at 50,000 pfu per 15 cm diameter plate after transfecting E. coli PLK-F' 1 0 cells. The plates were incubated at 37 0 C for eight hours, then stored overnight at 4 0

C.

Phage were eluted from the plates into phage storage buffer (8 mM MgSO4, 100 mM NaC1, 0.01% gelatin, 50 mM Tris-HC1, pH 8.0) to form an amplified cDNA library stock.

1 5 Design of dioxygenase oligonucleotide primers A number of dioxygenases have been sequenced, from organisms as diverse as plants (Matsuda et al., 1991; Martin et al., 1991), fungi and bacteria (Cohen et al., 1990). A sequence conservation. Amino acid sequences of a number of different plant 2 0 dioxygenases were aligned using the CLUSTAL programs of Higgins and Sharp (1988). The sequences used were Candi (Martin et al., 1991), hyoscyamine 6(hydroxylase (H6H) (Matsuda et al., 1991), flavanone 3-hydroxylase (F3H), E8 (Deikman and Fischer, 1988), A2 (Menssen et al., 1990) and Toml3 (Holdsworth et al., 1987). This analysis revealed two well-conserved regions, shown in Table TABLE Candi (snapdragon) WGVMHLINHGVP NYYPKCPQP H6H (Hyoscyamus niger) FGLFQVINHGFP NYYPPCPDP F3H (barley) WGIFQVIDHGVD NFYPRCPQP E8 (tomato) WGFFQVVNHGIP NYYPPCPQP A2 (maize) WGVMHIAGHGIP NYYPRCPQP Toml3 (tomato) WGFFELVNHGIP SNYPPCPKP Consensus wGffqvinHGip nyYPpCPqP f imhlvd vd sf r d v eiag f n k k WO 94/03606 PCrAU93/00400 22 Oligonucleotides were designed to hybridise to genes encoding sequences similar to the consensus sequences. The sequences of each of these oligonucleotides, designated Oligo 2-6 (SEQ ID No's 7 11), are shown above. The inclusion of deoxyinosine (I) covered the different possibilities for codon usage where more than two codons could encode the same amino acid. Deoxyinosine base-pairs with similar efficiency to A, T, G and C (Martin et al., 1985; Ohtsuka et al., 1985).

PCR amplification of petunia dioxygenase gene fragments 1 0 Total RNA was isolated from stages 3-4 flowers of Ba20 (flfl) and V26 (FlFl). 25 jig of total ANA was ethanol precipitated, pelleted and resuspended in 10.5 tgL water.

One tiL (0.5 fig) of Oligo 1 (SEQ ID No:6) was added and the mixture was heated at 0 C for 10 minutes then placed on ice. The following were then added: 4 iL Superscript' reaction buffer (5x stock), 2 giL of 100 mM dithiothreitol, 0.5 giL of 1 5 mM dATP, 0.5 gL of 5 mM dCTP, 0.5 gL of 5 mM dGTP, 0.5 [L of 5 mM dTTP, and 0.5 jiL of [a- 32 p]-dCTP. The mixture was incubated at 37 0 C for 2 minutes.

Following the addition of 1 IiL (200 units) of Superscript M reverse transcriptase, the reaction was incubated at 37 0 C for 60 minutes and then terminated by the addition of iL of STE. The cDNA was purified by Sephacryl S200 spun-column 2 0 chromatography, followed by ethanol precipitation and resuspension in 100 giL TE buffer. This cDNA was used as the template for PCR.

PCR reactions for amplification of petunia dioxygenase gene fragments contained 4 gL of cDNA, 10 mM Tris-HC1 (pH 50 mM KCI, 2 mM MgCl2, 0.01% (w/v) gelatin, 0.2 mM each dNTP, 0.4 gtM each primer and 1.25 units Taq polymerase (Cetus). Reaction mixes (50 gtL) were cycled 40 times: 94°C for 50 seconds; 42°C for 1 minute; 72 0 C for 1 minute. Fifteen microlitres of each PCR was electrophoresed on a 1.25% agarose gel. DNA fragments in the size range 300-500 bp were collected onto NA-45 membrane. The DNA was eluted from the membrane and then ethanol 3 0 precipitated, pelleted by centrifugation and resuspended in 25 giL of TE buffer. One giL of DNA fragments from each of the V26 PCRs were pooled and 32 P-labelled using an oligo-labelling kit (BRESATEC). DNA fragments from the Ba20 PCRs were labelled in a similar manner.

WO 94/03606 PCT/AU93/00400 23 Isolation of dioxygenase homologues from a petunia petal cDNA library Duplicate lifts of 16,000 plaques were hybridised with 5 x 105 cpm/gL of either the V26 probe or the Ba20 probe, and washed as follows: High stringency conditions (hybridisation: 50% formamide, 6 x SSC, 1% SDS at 42 0 C for 16 hours and washing: 2 x SSC, 1% SDS at 65 0 C for 2 x 15 minutes followed by 0.2 x SSC, 1% SDS at 65 0 C for 2 x 15 minutes) were used to detect sibling clones.

Fourteen clones hybridised to the V26 probe but not the Ba20 probe. A further 12 clones hybridised more strongly to the V26 probe than the Ba20 probe.

1 0 Plasmid cDNA clones in pBluescript were rescued from XZAPII clones using the helper phage R408 (Stratagene).

DNA sequencing of this and other clones was performed essentially by the method of Sanger et al. (1977) using the Sequenase enzyme (USB, version 2.1).

EXAMPLE 5 NORTHERN ANALYSIS Total RNA was isolated from tissue that had been frozen in liquid nitrogen and ground 2 0 to a fine powder using a mortar and pestle. An extraction buffer of 4 M guanidium isothiocyanate, 50 mM Tris-HC1 (pH 20 mM EDTA, 0.1% Sakosyl, was added to the tissue and the mixture was homogenised for 1 minute using a polytron at maximum speed. The suspension was filtered through Miracloth (Calbiochem) and centrifuged in a JA20 rotor for 10 minutes at 10,000 rpm. The supernatant was 2 5 collected and made to 0.2 g/ mL CsCI Samples were then layered over a 10 mL cushion of 5.7 M CsCI, 50 mM EDTA (pH 7.0) in 38.5 mL Quick- seal centrifuge tubes (Beckman) and centrifuged at 42,000 rpm for 16 hours at 25 0 C in a 70Ti rotor.

Pellets were resuspended in TE/SDS (10 mM Tris-HC1 (pH 7.5),1 mM EDTA, 0.1% SDS) and extracted with phenol:chloroform:isoamyl alcohol (25:24:1) saturated in 10 mM EDTA (pH Following ethanol precipitation, the RNA pellets were resuspended in TE/SDS.

RNA samples (20 4g) were electrophoresed through a 2.2 M formaldehyde 1.2% agarose gel using running buffer containing 20 mM 3 5 morpholinopropanesulphonic. acid (pH 5 mM sodium acetate, 0.1 mM EDTA (pH The RNA was transferred to Hybond-N membrane (Amersham) as recommended by the manufacturer and probed with 32 P-labelled 0.9 kb EcoRI-XhoI 1 According to this aspect of the present invention there is provided a method for producing a transgenic flowering plant capable of synthesizing FLS, said method WO 94/03606 PCT/AU93/00400 24 pDIOXC3 cDNA fragment (108 cpm/tig, 2 x 106 cpm/L). Prehybridisation (one hour at 42 0 C) and hybridisation (16 hours at 42 0 C) were carried out in 50% (v/v) formamide, 1 M NaCI, 1% SDS, 10% dextran sulphate. Degraded salmon sperm DNA (100 pg/mL) was added with the 32 P-labelled probe for the hybridisation step. Filters were washed in 2 x SSC/ 1% SDS at 65C for 1 to 2 hours and then 0.2 x SSC/ 1% SDS at 65 0 C for 30 to 60 minutes. Filters were exposed to Kodak XAR film with an intensifying screen at -70 0 C for 48 hours (Figure 2).

RNA gel blot analysis revealed that the gene corresponding to the cDNA clone 1 0 pDIOXC3 was expressed at the highest level during Stage 1 of flower development and then declined. The expression pattern is similar to that of FLS enzyme ai-dvity in petunia flowers (Forkmann et al., 1986).

1 5 EXAMPLE 6 RFLP MAPPING OF pDIOXC3 There is one genetic locus in P. hybrida, Fl, that controls FLS activity. It was therefore expected that a cDNA clone encoding a P. hybrida FLS would map to the Fl locus, p;rvded that the Fl locus encodes the structural gene for FLS. Fl has been 2 0 mapped to chromosome II of the P. hybrida genome and is linked to within 2% recombination of the PAcl gene (Cornu et al., 1990). RFLP analysis of DNA isolated from an F 2 population of plants derived from a cross between the inbred lines V23 (FIFl) and R51 (flfl) was used to obtain linkage data for the various dioxygenase homologues.

Isolation of Genomic DNA DNA was isolated from leaf tissue of V23 x R51 F2 plants essentially as described iby Dellaporta et al., (1983). The DNA preparations were further purified by CsCl buoyant density centrifugation (Sambrook et al., 1989).

Southern blots The genomic DNA (10 pg) was digested for 16 hours with 60 units of Xhba and electrophoresed through a 0.7% agarose gel in a running buffer of TAE (40 mM Tris-acetate, 50 mM EDTA). The DNA was then denatured in denaturing solution 3 5 M NaCO/.5 M NaOH) for 1 to 1.5 hours, neutralised in 0.5 M Tris-HCl (pH 7.5)/1.5 M NaCl for 2 hours and the DNA was then transferred to a Hybond-N (Amersham) filter in 20 x SSC.

I

WO 94/03606 PCT/AU93/00400 DNA fragments (50 to 100 ng) were radioactively labelled with 50 pICi of [a- 32 p]dCTP using an oligolabelling kit (Bresatec). Unincorporated [ca- 32 P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column. A PAc1 probe was synthesised from a 2.7 kb HindgIIIamHI fragment of oPAcl (Baird and Meagher, 1987). A pDIOXC3 cDNA probe was synthesised from a 0.9 kb EoQRI-Xho.

fragment of pDIOXC3.

Duplicate Southern blots of genomic DNA digested with XhIa were hybridised with 1 0 either the pDIOXC3 probe or the PAcl probe, to detect RFLP patterns. For 40 out of the 42 plants analysed there was co-segregation of the V23, VR and R51 RFLP patterns for PAl with the corresponding RFLP patterns of pDIOXC3, demonstrating the corresponding genes are closely linked recombination).

1 5 These data provided strong evidence that the gene corresponding to pDIOXC3 is linked to the Fl locus. This linkage, as well as the Northern analysis, provided circumstantial evidence that the pDIOXC3 cDNA might encode FLS.

EXAMPLE 7 ISOLATION OF FULL-LENGTH SIBLING cDNA CLONES OF pDIOXC3 From preliminary sequence analysis it was shown that pDIOXC3 did not represent a full-length clone of the corresponding transcript. To obtain a full-length version of 2 5 pDIOXC3, approximately 20,000 recombinants from the cDNA library were screened for clones that hybridised to the 0.9 kb EgcRI-Xhol fragment from pDIOXC3. Six clones produced strong hybridisation signals and were chosen for further analysis. A number of clones appeared to he full-length based on agreement between the size of the cDNA insert and the mRNA. The complete sequence of the cDNA insert from one of 3 0 these clones, designated pCGP481, was determined by compilation of sequence from different pBluescript subclones obtained using standard cloning procedures (Sambrook et al., 1989). For some regions it was necessary to synthesise specific oligonucleotide primers (Oligos 7-9; SEQ ID No's 12 14) to obtain overlapping sequence data. The complete nucleotide sequence and deduced amino acid sequence of pCGP481 is shown as SEQ ID No:l.

I~nta cfl ~CI WO 94/03606 PCr/AU93/00400 26 EXAMPLE 8 EXPRESSION OF PCGP481 cDNA IN YEAST Construction of the yeast expression vector pYGA22m M13-mpl8 was digested with EcoRI and ]gII to produce a 700 bp fragment that contained a multicloning site. This fragment was ligated with the 9 kb EcoRI-]gII fragment from pYGA2269 (Ashikari et al., 1989). The resulting construct, designated pYGA22m, contained the multicloning site inserted downstream of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter.

1 0 Construction of pCGP631 A 1.3 kb EcoRI-Xhl fragment that included the entire cDNA insert from pCGP481 was ligated with the 9 kb EcoRI/SalI fragment from pYGA22m. The resulting plasmid, designated pCGP631 (Figure contained the pCGP481 cDNA fragment ligated in a sense orientation behind the glyceraldehyde-3-phosphate dehydrogenase promoter.

Yeast transformation The yeast strain G-1315 (Mata, trpl) (Ashikara et al., 1989) was transformed with pCGP631 according to Ito et al., (1983). The transformants were selected by their 2 0 ability to restore G-1315 to tryptophan prototrophy.

Preparation of yeast extracts for assay of FLS activity Single isolates of G-1315/pCGP631 and a G-1315 revertant that grew on media lacking tryptophan were used to inoculate 10 mL of YNBC 1.2% yeast nitrogen base without amino acids (Difco) and 0.3% Casamino acid (Difco)} and incubated with shaking for 2 days at 30 0

C.

Yeast cells were harvested by centrifugation, washed once with TE buffer and resuspended in 100 L of buffer B (10 mM Tris-HCl (pH 1.2 M sorbitol, 0.1 mM DTT, 0.1 mM EDTA) containing zymolyase (0.1 mg/mL) (Seikagakukogyo, Japan) and kept at 30 0 C for 1 hour. Spheroplasts were collected by centrifugation and resuspended in 500 [tL of 0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM 2-mercaptoethanol and 1 mM PMSF. The suspension was then vortexed with glass beads (diameter 0.4 mm) for 2 minutes. The supernatant after centrifugation 3 5 was used as a crude extract.

L e WO 94/03606 PCT/AU93/00400 27 FLS assay of yeast enzyme extracts FLS activity was measured by the method of Forkmann et al. (1986) with modifications. The reaction mixture contained in a total of 200 pL: 0.1 M potassium phosphate (pH 1.4 mM 2-mercaptoethanol, 250 gM 2-oxoglutarate, 5 mM ascorbic acid, 50 gIM ferrous sulphate, 5000 cpm of 14 C-DHQ and 40 iL of crude extract Incubation was carried out for 10 minutes or 1 hour at 30 0 C. The mixture was immediately extracted with 500 jiL of ethyl acetate and chromatographed on a cellulose plate (Merck Art 5577, Germany) with Forestal (acetic acid: HC1: water 30: 3: 1 0 along with un,,belled DHQ and quercetin. Radioactivity was localised by autoradiography. An enzyme extract prepared from G-1315/ pCGP631 was shown to have FLS activity while an equivalent fraction prepared from non-transformed yeast had no activity (Figure 1 5 The yeast expression results confirmed that the cDNA insert from pCGP481 encoded an FLS enzyme. Forkmann et al. (1986) suggested that two enzymes, a 2-hydroxylase and a dehydratase, are necessary for the conversion of dihydroflavonols to flavonols.

However, the results show that expression the enzyme encoded by the petunia FLS cDNA clone in yeast is sufficient for this conversion, suggesting that only one enzyme is required for the conversion of dihydroflavonols to flavonols.

EXAMPLE 9 MANIPULATION OF FLAVONOL AND ANTHOCYANIN SYNTHESIS IN TRANSGENIC PLANTS Binary constructs The binary expression vector pCGP478 was constructed by replacing the 2XhI-K=nI fragment of the multiple cloning site from pCGP293 (Brugliera et al., 1993) with a synthetic polylinker containing sites for XhAl, BamHI, A.al and Asp718. The 3 0 synthetic polylinker was made by annealing the two oligonucleotides Oligo 10 and Oligo 11 (SEQ ID No's 15 and 16). The order of the restriction enzyme sites between the MAC promoter and thte mas terminator of pCGP478 facilitates direct subcloning of cDNA inserts from directional XZAPII clones in an antisense orientation.

A 1.2 kb Xbha/Aan718 fragment containing the complete cDNA frem pCGP481 was cloned in an antisense orientation between the MAC promoter and mas terminator of pCGP478 to create pCGP479. The plasmid pCGP479 was introduced into 1 WO 94/03606 PCT/AU93/00400 28 Agrobacterium tumefaciens strain AGLO (Lazo et al., 1991) using the method of Gynheung et al. (1988). Cells of A. tumefaciens carrying pCGP479 were selected on MG/L agar plates containing 100 gg/mL gentamycin.

A 1.2 kb XKaI/Ag718 fragment containing the cDNA from pCGP481 was cloned in a sense orientation between the MAC promoter and mas terminator of pCGP293 to create.

pCGP482. The plasmid pCGP482 was introduced into A. tumefaciens strain AGLO (Lazo et al., 1991) using the method of Gynheung et al. (1988). Cells of A.

tumefaciens carrying pCGP482 were selected on MG/L agar plates containing 100 1 0 .tg/mL gentamycin.

Production of transgenic plants Petunia cv. VR (Flfl) plants were transformed by co-cultivation of leaf discs with AGLO/pCGP479 using the method of Horsch et al. (1985). Transgenic plants were 1 5 grown to flowering and scored for altered flower colour compared with nontransformed VR flowers. Four out of 12 transgenic plants produced redder flowers than non-transgenic controls. Apart from the change in flower colour in the transgenic petunias no other effects of antisense expression of the FLS cDNA were observed.

2 0 Petunia cv. Old Glory Blue plants were transformed by co-cultivation of leaf discs with AGLO/pCGP482 using the method of Horsch et al. (1985). Transgenic plants were grown to flowering and scored for altered flower colour compared with nontransformed Old Glory Blue flowers. Three out of 15 transgenic plants produced redder flowers than non-transgenic controls. Non-transgenic Old Glory Blue flowers are generally blue-violet while the colours of the Old Glory Blue flowers transformed with pCGP482 ranged in colour from blue-violet to purple.

Tobacco plants (Nicotiana tabacum cv. Xanthi) were transformed by co-cultivation of leaf discs with AGLO/pCGP479 using the method of Horsch et al. (1985). Tobacco 3 0 flowers are normally light pink and produce low levels of cyanidin derivatives in the limb of the corolla. Transformation of tobacco with the antisense FLS gene construct caused a reduction in flavonol production and lead to the production of red flowers.

Red pigmentation was also increased in the filaments. The red flower colour was due to a three-fold increase in anthocyanin production in the corolla limb.

The colour changes observed may be described in terms of the numbers from the Royal Horticultural Society's Colour Chart as shown in Table 6, overleaf: WO 94/03606 PCT/AU93/00400 29 TABLE 6 MODIFICATION OF FLOWER COLOUR IN TRANSGENIC PLANTS Control: Petunia cv.VR Purple-violet Transgenic: VR/pCGP479 Red-purple 74A Control: Petunia cv. Old Glory Blue Violet-blue 89A 89C Transgenic: OGB/pCGP482 Violet 83A 83B Control: Nicotiana tabacum cv. Xanthi Red-purple Transgenic: Xanthi/pCGP479 Red 51B It should be noted, however, that other biochemical and physiological conditions will affect the individual outcome and the citing of the specific colour change achieved by expression of the FLS sense and antisense constructs in transgenic plants should not be interpreted as limiting the possible range of colour changes which may be observed.

Extraction and analysis of flavonoids from flowers Flavonol aglycones were isolated from petunia flowers by boiling a single corolla in 1 mL of 2 M HCI for 30 minutes and extracting the flavonoids with 150 p.L ethyl acetate.

For thin layer chromatography (TLC), 4 gtL of ethyl acetate extracts and 4 p.L flavonol 2 5 standards (kaempferol, quercetin and myricetin) were applied to a TLC plate and developed as described above. Flavonols were visualised under UV light after fuming with ammonia.

Flavonols extracted from petuniaVR and petunia VR/pCGP479 flowers were analysed by thin layer chromatography. The red VR/pCGP479 flowers produced markedly less flavonols than non-transgenic VR flowers. Flowers from tobacco plants transformed with pCGP479 were similarly analysed and found to have reduced flavonol content Anthocyanins were extracted from petunia and tobacco flowers with 0.5% HCI in 3 5 methanol. Anthocyanin concentrations were estimated from A 530 measurements of the extracts as described by Gerats (1985).

i i -ll-l i~l~l~a~a4nra~i~l WO 94/03606 PCT/AU93/00400 EXAMPLE 10 ISOLATION OF cDNA HOMOLOGUES FROM NICOTIANA Construction and screening of a Nicotiana alata cDNA library A cDNA library in the vector XZAP was made from RNA isolated from styles of S6S6 Nicotiana alata as described by Chen et al. (1992).

Approximately 36,000 cDNA clones were hybridised with 32 P-labelled pCGP481 cDNA fragment in 6 x SSC, 35% formamide, 1% SDS for 16 hours at 1 0 42 0 C. The filters were washed under medium stringency conditions in 2 x SSC, 1% SDS at 65 0 C and then autoradiographed. Hybridising plaques were picked off into PSB to allow the phage to elute. Eight plasmid clones were rescued using the singlestranded helper phage VCSM13 (Stratagene). The clone containing the largest cDNA insert, pCGP489, was sequenced (SEQ ID No:2) and showed 88% similarity to the 1 5 Petunia FLS genetic sequence at the nucleotide level, and 91% similarity over 241 amino acids to the Petunia FLS sequence encoded by pCGP481.

Construction and screening of a Nicotiana sylvestris cDNA library A cDNA library in the vector XZAPII was made from RNA isolated from styles of 2 0 Nicotiana sylvestris using methods described by Chen et al. (1992).

Approximately 120,000 cDNA clones were hybridised with 32 P-labelled pCGP481 cDNA fragment in low stringency hybridisation buffer (6xSSC, 35% formamide, 1% SDS) for 16 hours at 42 0 C. The filters were washed in 2xSSC, 1% (w/v) 2 5 SDS at 65 0 C and then autoradiographed. Hybridising plaques were picked off into PSB to allow the phage to elute. Three plasmid clones were rescued using the singlestranded helper phage VCSM13. The clone containing the largest cDNA insert, pCGP490, was sequenced (SEQ ID No:3) and showed 84% similarity to the Detunia FLS genetic sequence at the nucleotide level, and 93% similarity over the 45 amino acid sequence to the Petunia FLS sequence encoded by pCGP481.

A comparison of the amino acid sequences of the two Nicotiana cDNA clones pCGP489 (SEQ ID No:2) and pCGP490 (SEQ ID No:3) with the amino acid sequence of the petunia FLS cDNA clone (SEQ ID No:l) is shown in Table 7, overleaf.

LI II I I ii El WO 94/03606 PCr/AU93/00400 31 TABLE 7 COMPARISON OF SEQUENCES FROM NICOTIANA WITH pCGP481 PetFLSI RDPDENKMVKLiAD)ASKEWGIFQLINHG.IPDEAIADLQKVGKEFFEHVPQEEKELIAKTP pCGP489 2 VPQEEK~mIAKs P PetFLS GSNDIEGYGTSLQKEVEGKKGWVDHLFH-KIWPPSAVNYRYWPKNPPSYREANEEYGKRMR pCGP4 89 GSqnIEGYGTSLQKEVEGKrGWVDHLFHKIWPPSAiNYRYWPKNPPSYREANEEYaKRlR PetFLS EVVDRIFKSLSLGLGLEGHEMIEAAGGDEIVYLLKINYYPPCPRPDLALGVVA{TDMSYI pCGP4 89 EVaekmFKSLSLGLGLEaHEMmiEAAGGedIVYLLKINYYPPC PRPDLALGVVAHTDMShI 3 0 PetFLS TILVPNEVQGLQVFKDGHWYDVKYIPNALIVHIGDQVEILSNGKYKSVYHRTVNKDKTR pCGP489 .TILVPNEVQGLQVFKDG1{WYDVnYIPNALIVHIGDQlEILSNGKYKSVYHRTTIVtKDKTR PetFLS pCGP489.

pCGP49O 3 MSWPVFLEPPSEHEVGPI PKLLSEANPPKFKTKKYKDYVYCKLNKLPQ MSWPVFLEP PSEHEVGP IPKLvnEANPPKFKTKKYKDYVYCKLNKLPQ PVFLEPPSEHEVGPI sKLvnEANPPKFKTKKYKDYVYCKLNKLPQ 1 Petunia FLS amino acid sequence (SEQ lID No:l1) 2 Nicotana~ alata sequence (SEQ ID No:2) 4 5 3 Nicotiana sylvestris sequence (SEQ ID No:3) Amino acid mismatches in the compared sequences; all other amino acids are identical over the sequence lengths compared.

_U

WO 94/03606 PCT/AU93/00400 32 EXAMPLE 11 ISOLATION OF cDNA HOMOLOGUE FROM DIANTHUS Construction of a Dianthus cDNA library Total RNA was isolated from the petal tissue of D. caryophyllus cv. Laguna stage 3 flowers, using the method of Turpen and Griffith (1986). Poly(A)+ RNA was selected from the total RNA by Oligotex dT-30 (Takana, Japan) following the manufacturer's protocol.

cDNA was made and a library constructed in ,ZAPII according to the protocol used for the petunia library.

The primary library, which contained 150,000 pfu, was plated at 37,500 pfu per 15 cm 1 5 diameter plate after transfecting E. coli SURE cells. The plates were incubated at 37 0

C

for eight hours, then stored overnight at 4 0 C. Phage were eluted from the plates into phage storage buffer (8 mM MgSO4, 100 mM NaCI, 0.01% gelatin, 50 mM Tris-HC1, pH 8.0) to form an amplified cDNA library stock.

2 0 Isolation of a FLS homologue from a Dianthus cDNA library A total of 100,000 plaques were screened, in duplicate, with 32 P-labelled 1.1 kb EcoRI-HindII pDIOXC3 cDNA fragment (5 x 105 cpm/p.L). Hybridisation was carried out in a low stringency buffer (6 x SSC, 0.5% SDS, 5 x Denhardt's solution, 0.01 M EDTA, 100 gg/ml) for 16 hours at 420C. The filters were washed in 2 x SSC/1% SDS at 65 0 C and then autoradiographed. Hybridised cDNA clones were rescued from XZAPII using the single-stranded helper phage Exassist (Stratagene) according to the manufacturer's instructions.

When sequenced, one of the isolated clones, pCGP777 (SEQ ID No:4), revealed 3 0 similarity at both the nucleotide level and the amino acid level to the Petunia FLS sequence encoded by pCGP481.

WO 94/03606 PCT/AU93/00400 33 EXAMPLE 12 ISOLATION OF cDNA HOMOLOGUE FROM CHRYSANTHEMUM Construction of a Chrysanthemum cDNA library Total RNA w, isolated from the petal tissue of Chrysanthemum morifolium cv. Dark Pink Pompom (Reference Number 5999), stages 2 and 3 flowers, again using the method of Turpen and Griffith (1986). An amount of 30 plg of the total RNA was used as template for cDNA synthesis.

1 0 Following fractionation and ligation, the cDNA reaction mixture was packaged using the Packagene system (Promega). The titre of the unamplified library was 3.7 x 104 pfu/ml.

Isolation of a FLS homologue from a Chrysanthemum cDNA library 1 5 An amount of 90,000 pfu (of amplified library; 2.6 x 107 pfu/ml) of the packaged cDNA was plated at 10,000 pfu per 15 cm diameter plate after transfecting XL1-Blue cells. The plates were incubated at 37 0 C overnight, then stored at 4°C. Duplicate lifts were taken onto Colony/Plaque Screen T M filters (DuPont), treated as recommended by the manufacturer, and screened with the 32 P-labelled EcoRI-XhoI pCGP481 cDNA 2 0 fragment. Hybridisation was carried out in a low stringency buffer (6 x SSC, SDS, 20% for;namide) for 16 hours at 420C. The filters were washed twice for 30 minutes in 2 x SSC/1% SDS at 65 0 C, and then autoradiographed.

The isolated clone pCGP874 (SEQ ID No:5), when sequenced, revealed 2 5 similarity at the nucleotide level and 72% similarity at the amino acid level to the Petunia FLS sequence encoded by pCGP481.

EXAMPLE 13 EXPRESSION OF PCGP874 cDNA IN YEAST 3 0 Construction of the yeast expression vector pYGA22m and pCGP492 The yeast expression vector pYGA22m was contructed as described in Example 8 above. A 1.3 kb EcoRI-XhoI fragment that included the entire cDNA insert from pCGP874 was ligated with the 9 kb EcoRI/SalI fragment from pYGA22m, in the same manner as for the construction of pCGP 631, described above and shown in Figure 4.

3 5 The resulting plasmid, designated pCGP492, contained the pCGP874 cDNA fragment ligated in a sense orientation behind the glyceraldehyde-3-phosphate dehydrogenase promoter.

WO 94/03606 PCT/AU93/00400 34 Yeast transformation and assay of FLS activity Transformation of yeast and preparation of extracts for assay of FLS activity were carried out as described above in Example 8. FLS activity was again measured by the method of Forkmann et al. (1986) with modifications, using unlabelled DHK and DHQ as substrates. The results confirmed that the pCGP874 cDNA encodes a functional chrysanthemum FLS enzyme.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

~cr~-rcp~ efc~-~3fi -r~~lp 13 LIs~ WO 94/03606 PCT/AU93/00400

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ft- -EI- ~-rrr^a~ Ica~a x re WO 94/03606 PCT/AU93/00400 39 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT (other than US): INTERNATIONAL FLOWER DEVELOPME.rS PTY

LTD

(US only): HOLTON, KEAM, L.A.

(ii) TITLE OF INVENTION: GENETIC SEQUENCES ENCODING FLAVONOL SYNTHASE ENZYMES AND USES THEREFOR (iii) NUMBER OF SEQUENCES: 16 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: AU INTERNATIONAL FILING DATE: 05-AUG-1993

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: SLATTERY, JOHN M.

REFERENCE/DOCKET NUMBER: EJH/JMS/EK (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 61 3 254 2777 TELEFAX: 61 3 254 2770 TELEX: AA 31787 WO 94/03606 PCT/AU93/00400 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1211 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 59..1101 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GTTTTTCTAA AAACAGAGGG CCTAACTTCT GTATAGACAA AGAAAAAAAG AAAAGAAA ATG AAA ACA GCT CAA GGT GTC AGT GCA ACC CTA ACA ATG GAA GTG GCA Met Lys Thr Ala Gln Gly Val Ser Ala Thr Leu Thr Met Glu Val Ala AGA GTA CAA Arg Val Gln TCA GAG TAC Ser Glu Tyr ATA GCA TCG TTA AGC AAG TGC ATGGAC Ile Ala Ser Leu Ser Lys Cys Met Asp ACA ATT CCA Thr Ile Pro ATT AGG TCC GAG Ile Arg Ser Glu GAG CAA CCA GCA GCC ACA ACC CTG Glu Gln Pro Ala Ala Thr Thr Leu CAT GGG His Gly GTA GTT CTT CAA Val Val Leu Gln CCA GTG ATT GAC Pro Val Ile Asp CGT GAC CCT GAT Arg Asp Pro Asp

GAG

Glu AAC AAG ATG GTG Asn Lys Met Val CTC ATA GCT GAT Leu Ile Ala Asp AGC AAA GAG TGG Ser Lys Glu Trp ATA TTC CAA CTG ATC AAC CAT GGC ATT Ile Phe Gln Leu Ile Asn His Gly Ile GAT GAG GCT ATC Asp Glu Ala Ile GCG GAT Ala Asp TTA CAG AAA GTA GGG AAA GAG Leu Gln Lys Va.l Gly Lys Glu 100 TTC TTT GAA Phe Phe Glu 105 CAT GTT CCA CAG GAG GAG His Val Pro Gln Glu Glu 110 -L IUB air c WO 94/03606 PCI/AU93/00400 -41- AAA GAG CTG ATT GCC AAG ACT CCA GGA TCA Lys Giu Leu Ile Ala Lys Thr Pro Giy Ser AAC GAC ATT GAA GGC TAT Asn Asp Ile 125 Glu Giy Tyr GGA ACT Gly Thr 130 TCT CTG CAG AAG GAA GTG GAA GOC AAG Ser Leu Gin Lys Giu Vai Giu Giy Lys 135

AAA

Lys 140 GGT TGG GTG GAT Gly Trp Val Asp TTG TTC CAT AAG Leu Phe His Lys TGG CCT CCT TCT Trp Pro Pro Ser GTC AAC TAT CGT Vai Asn Tyr Arg TGG CCT AAA AAC Trp Pro Lys Asn CCT TCA TAC AGG Pro Ser Tyr Arg

GAA

Glu 170 GCA AAC GAA GAA Ala Asn Giu Glu TAT GGA Tyr Gly 175 AAG AGG ATG Lys Arg Met GAA GTT GTA GAC Glu Val Val Asp ATT TTT AAG AGC Ile Phe Lys Ser GGG CTT GGG CTT GAA GGC CAT Gly Leu Gly Leu Giu Gly His 195

GAA

Glu 200 ATG ATA GAG GCA GCT Met Ile Giu Ala Ala TTG TCT CTT Leu Ser Leu 190 GGT GGT GAT Gly Gly Asp TGC CCA AGG Cys Pro Ara GAG ATA Glu Ile 210 GTT TAC TTG TTG Val Tyr Leu Leu ATC AAC TAT TAC Ile Asn Tyr Tyr CCA CCA Pro Pro 220 CCC GAT TTG GCT Pro Asp Leu Ala 225 ACC ATT CTT GTC Thr Ile Leu Val CTT GGT Leu Gly 230 GTT GTG GCC CAT Val Val Ala His GAC ATG TCA TAT Asp Met Ser Tyr AAT GAA GTC CAA GGC CTC CAA GTG TTC Asn Giu Val Gin Gly Leu Gin Val Phe 250 AAG GAT Lys Asp 255 GTC CAT Val His GGC CAT TGG Gly His Trp ATT GGT GAC Ile Gly Asp 275

TAT

Tyr 260 GAT GTC AAG TAC Asp Val Lys Tyr CCA AAT GCC TTA ATT Pro Asn Ala Leu Ile 270 CAA GTT GAC, ATT CTT AGC AAT GGC AAA Gin Val Giu ie Leu Ser Asn Gly Lys 280 AAG AGT GTA Lys Ser Val TAC CAT AGG Tyr His Arg 290 ACA ACG GTG AAC Thr Thr Val Asn 295 AAG GAC AAG ACA AGA ATG TCA TGG CCG Lys Asp Lys Thr Arg Met Ser Trp Pro 300 970 WO 94/03606 WO 9403606PCr/AU93/00400 -42-

GTT

Val 305 TTC TTG GAG CCC Phe Leu GJlu Pro TCA GAG CAT GAA Ser Giu His Giu GTT GGG Val. Gly 315 CCA AIT CCT Pro Ile Pro AAG 1018 Lys 320 CTG C'IT AGT GAG Leu Leu Ser Giu

GCC

Ala 325 AAC CCA CCC Asn Pro Pro AAA TITC Lys Phe 330 AAG ACC AAG AAG TAC AAG 1066 Lys Thr Lys Lys Tyr Lys 335 GAT TAC GTC TAT TGT AAG CIT AAC AAG CIT CCT CAG TGAAGAAGC Asp TEyr Val Tyr Cys Lys Leu Asn Lys Leu Pro Gin 340 345 ACCTCTATGT ATGGAGCGAT TAGCTATATC TTCGCGAGTG TTATGG'FITT ATTTGTACTG TCCTAATTAA 'PTACACAAAA AAAAAAAAAA AA.AAAAAAAA ilil 1161 1211 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 890 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 3. .725 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GC GTA CCA CA.A GAA GAG AAA GAG ATG Val. Pro Gin Glu Glu Lys Glu Met 1 ATT GCA AAG ACT Ile Ala Lys Ser CCA GCC TCG Pro Gly Ser GAG AAT ATT GAA GCG TAT GGT ACA TCT TTG CAC AAG CAA GTT GAA CCC Gin Asn Ile Gli Gly T'yr Cly Thr Ser Leu Gin Lys Glu Val Glu Gly 25 WO 94/03606 WO 9403606PCFP/AU93/00400 -43- AAA AGA GGT TOG GTT GAT CAT TTG T CAT AAG ATT TOG CCT CCT TCT Lys Arg Gly Trp Val Asp His Leu Phe His Lys Ile Trp Pro Pro Ser 0CC ATC AAT TAT CGT TAT TOO Ala Ile Asn Tyr Arg Tyr Trp AAA AAC CCT CCT Lys Asn Pro Pro TAC AGO GAA Tyr Arg Glu GCA AAT Ala Asn GAG GAA TAT GCA Giu Giu Tyr Ala AGO, CTG CGA GAA Arg Leu Arg Giu GCG GAG AAG ATG Ala Giu Lys Met AAG AGC TTA TCA CTT GOG CTT GOG TTA Lys Ser Leu Ser Leu Gly Leu Gly Leu GCC CAT GAA ATO Ala His Giu Met GAG GCA GCA GGT Giu Ala Ala Giy GAA OAT ATA GTT Giu Asp Ile Val TTG TTO AAG ATC Leu Leu Lys Ile AAT TAT Asn Tyr 110 TAC CCA CCA Tyr Pro Pro ACA GAC ATO r Asp Met 130 CCA AGG CCT GAT Pro Arg Pro Asp OCA CTT GOA GTT Ala Leu Gly Val GTG GCC CAT Val Ala His 125 TCC CAT ATA ACC Ser His Ile Thr CTT GTC CCA AAT GAA GTC CAA GOC Leu Val Pro Asn Giu Val Gin Gly 140 CTC CAA Leu Gin 145 GTC TTC AAG OAT Val Phe Lys Asp CAT TOG TAT OAT His Trp Tyr Asp AAC TAC ATA CCA Asn Tyr Ile Pro 0CC CTA ATT OTC Ala Leu Ile Val ATT GOT GAC CAA Ile Gly Asp Gin GAO ATC CTT AGC Olu Ile Leu Ser 000 AAA TAC AAG, Gly Lys Tyr Lys

AGT

Ser 180 OTO TAT CAT AGG Val 7Lyr His Arg ACA OTG ACA AAG Thr Val Thr Lys OAT AAG Asp Lys 190 ACA AGA ATO TCA TOG CCA OTT TTC Thr Arg Met Ser Trp Pro Val Phe 195 GAO CCA CCA TCA Giu Pro Pro Cer GAO CAT GAA Oiu His Glu 205 CCC AAA TTC Pro Lys Phe OTT 000 CCA Val Gly Pro 210 ATT CCT AAG CTO OTT AAT GAG 0CC AAT Ile Pro Lys'Leu Val Asn Glu Ala Asn 215 671 WO 94/03606 PCT/AU93/00400 -44- AAG ACC AAG AAG TAC AAG GAT TAT GTC TAT TGT AAG CTT AAC AAG CTT 719 Lys Thr Lys Lys Tyr Lys Asp Tyr Val Tyr Cys Lys Leu Asn Lys Leu 225 230 235 CCT CAG TGAAGAAACT CCTCTATATA TGTTTGGCAG CGATTAGCTA CTATATGTTC 775 Pro Gin 240 GTCAGTATTA TATTATGGTT TGTACTATCC TTACCAACAG ATGTCTTATT ATGATTAAGG 835 ACTATATATT TACACTTAAA ACTTTTTGAT ACTAGCTAAT AACTGACTTA TTAAG 890 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 236 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..135 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCA GTA TTC TTG GAG CCA CCA TCA GAG CAT GAA GTA GGG CCA ATT TCT 48 Pro Val Phe Leu Glu Pro Pro Ser Glu His Glu Val Gly Pro Ile Ser 1 5 10 AAG CTG GTT AAT GAG GCC AAT CCA CCC AAA TTC AAG ACC AAG AAG TAC 96 Lys Leu Val Asn Glu Ala Asn Pro Pro Lys Phe Lys Thr Lys Lys Tyr 25 AAG GAT TAT GTT TAT TGT AAG CTT AAC AAG CTT CCT CAG TGAAGAAACC 145 Lys Asp Tyr Val Tyr Cys Lys Leu Asn Lys Leu Pro Gin 40 CCTCTATATA TGTTTGGCAG CGAATAGCTA GTATATTCGT GAGTACTAAA TTATGGTTTG 205 TACTATCCTT ACCAAGAGAT GTCTTATTAT G 236 9i i L WO 94/03606 PCT/AU93/00400 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 1236 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 70..1068 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAATTCGGCA CGAGGTTAAA TCAAAAATAT AAAACAAACA AATAAATAAA TAAATAAATA AATAAAAGT ATG GGC ATA TCT TCA GAA AGA GTA CAA AAC ATA GCC TTA Met Gly Ile Ser Ser Glu Arg Val Gin Asn Ile Ala Leu AAA TCC Lys Ser CAG AAC ATA GAC GAC ATT CCA CCC GAA TAC ATA AGA TTA GAG Gin Asn Ile Asp Asp Ile Pro Pro Glu Tyr Ile Arg Leu Glu GAT GAA CAA CCA GCA Asp Glu Gin Pro Ala ATC ACA Ile Thr ACA GTC CTC GAC ACG GTT CTC GAG GTC Thr Val Leu Asp Thr Val Leu Glu Val CCT GCG ATC GAC Pro Ala Ile Asp AGC CTC GAG GAA GAC GAC GTT GTA AAA Ser Leu Glu Glu Asp Asp Val Val Lys CTC GTC Leu Val TTG AGT GCA AGC AAA GAG TGG GGA Leu Ser Ala Ser Lys Glu Trp Gly TTT CAG GTC ACC AAC CAC GGA Phe Gin Val Thr Asn His Gly ATT CCG ACT GAA GTC ATT Ile Pro Thr Glu Val Ile GAA AAA Glu Lys 85 TTG CAA AAA Leu Gin Lys GTC GGT AAG ATG TTT Val Gly Lys Met Phe

V

WO 94/03606 PCr/AU93/00400 -46- TTT CGA Phe Arg GCT CCC GCA GAG GAG AAG GAG ACG ATT Ala Pro Ala Giu Giu Lys Giu Thr Ile 100 AAA CCC GAG GGT Lys Pro Giu Gly GGT GTT GAA GGG TAT GGG ACC ATG ITrG CAA AAG GAG ATT CAA GGG Gly Val Glu Gly Tyr Gly Thr Met Leu Gin Lys Giu Ile Gin Gly AAA GGT TGG GTT GAT CAT TTG TT'T CAC Lys Gly Trp Val Asp His Leu Phe His 130 A.AG GTT Lys Val 135 TGG CCT CCT Trp, Pro Pro AGT GTT Ser Vai 140 ATT AAC TAC Ile Asn Tyr GAA GAG TAC Glu Glu Tyr 160 TGG TGG CCT AAG ACT CCT TCT TAT AGO Trp Trp Pro Lys Thr Pro Ser Tyr Arg GAG GCG AAC Glu Aia Asn 155 ACA AAG TAC CTA Thr Lys Tyr Leu AGA ATA GTA GCC GAC AAG CTC TTC AAG Arg Ile Val Ala Asp Lys Leu Phe Lys 165 170 TTA GAA GAA GAT GAA GTC AAA AAA TCA Leu Glu Glu Asp Glu Val Lys Lys Ser 185 TGT ATG Cys Met 175 TCA AAG GGA CTT Ser Lys Gly Leu GGC AAT GAA GAC ATA GTG TAC CI'T CTC Gly Asn Glu Asp Ile Val Tyr Leu Leu 195 ATC AAC TAC TAC Ile Asn T[yr T[yr CCT TGT CCT CGA Pro Cys Pro Arg GAC TTG GCT CTA Asp Leu Ala Leu

GGG

Gly 215 GTG GCC GCT CAC Val Ala Ala His ACT GAC Thr Asp 220 TTG AGC GTC Leu Ser Val GTC TCT AGA Val Ser Arg 240 ACC ATT CTT GTT Thr Ile Leu Val AAT GAT GTT GCC Asn Asp Val Ala GGT CTT CAG Gly Leu Gin 235 CCT AAT GCA Pro Asn Ala GAC GGA CGT TGG Asp Gly Arg Trp GAT GTC AAG TAC Asp Val Lys T[yr

ATT

Ile 250 CTC ATC Leu Ile 255 ATC CAC GTT GGT Ile His Val Gly CAA ATG GAG ATA Gin Met Giu Ile AGC AAT GGA GAG Ser Asn Gly Glu

TAC

Tyr 270 AAG GCG GTG CTT Lys Ala Val Leu AGG TCG ACA GTG Arg Ser Thr Val AAA GAA AGA ACA Lys Glu Arg Thr C WO 94/03606 PCT/AU93/00400 -47- ATA TCG TGG CCC GTG TTC CTG GAA CCG CCA TCA GAC TTT Ile Ser Trp Pro Val Phe Leu Glu Pro Pro Ser Asp Phe 290 295 CCT ATT CCA AAG CTC ATT AGT GAT GAA AAG CCA GCC AAG Pro Ile Pro Lys Leu Ile Ser Asp Glu Lys Pro Ala Lys 305 310 AAG GTG TTT TCC GAG TAC AAG TAT TGT AAG CTG AAC AAG Lys Val Phe Ser Glu Tyr Lys Tyr Cys Lys Leu Asn Lys 320 325 330 TGAAGACTGA AGATGTTTAA TATTAGTCTT ATATGTTTAA TAAAGGCT.

CAGTTATCAC CTTGTTATTT GAATGTGCCA CTAAAATCAC TGTTAATT ATATATGATT TGTTCTCATT CATGTATGTT AAAAAAAAAA AAAAAAAA GCA GTC GGG Ala Val Gly 300 TAT AAG ACG Tyr Lys Thr 315 CTA CCT ATG Leu Pro Me'.

AT TGTTGGTTAT AA GGTGATATGG 972 1020 1068 1128 1188 1236 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1250 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 11..1065 (xi) SEQUENCE DESCRIPTION: SEQ ID AGCACACACA ATG GAG GTG GAA AGA GTT CAA GAA ATA GCA ACA CTC TCA Met Glu Val Glu Arg Val Gln Glu Ile Ala Thr Leu Ser 1 5 AAC CTA AAT GGC ACA ATC CCA AGT GAG TTC ATA AGA CTG GAA AAC GAA Asn Leu Asn Gly Thr Ile Pro Ser Glu Phe Ile Arg Leu Glu Asn Glu 20 WO 94/03606 WO 9403606PCT/AU93/00400 -48- CAA CCA Gin Pro ATC GAT Ile Asp AAA GCG Lys Ala CCA AGT Pro Ser GAG CTA Giu Leu AAA GGT Lys Gly 110 AAG AAA Lys Ly3 TTT ATT Phe Ile ACA AAT Thr Asn CTT GGG Leu Gly 175 CAA GCG Gin Ala 190

GCA

Ala

CTT

Lei-

AGC

Ser

GAA

Glu

CCA

Pro Val

GGA

Gly

AAC

Asn

GAG

Giu 160

CTG

Leu

TTG

Leu ACG ACC Thr Thr AGC CAG Scr Gin s0 AAG CAT Lys Asp CTC ATT Leu Ile CAA GA.A Gin Giu GAA GGG Giu Gly TGG GTG Trp Val 130 TAT CAA Tyr Gin 145 GAA TAC Giu Tyr TTG TCA Leu Ser GGT GGC Gly Gly

ACT

Thr

GCG

Ala

TGG

Trp

AGC

Ser

GAA

Giu

TAT

Tyr 115

GAT

Asp Phe

ACA

Thr

AAA

Lys

GAA

Giu 195 CTC CAT Leu His CAT AAC Asp Asn GGT AT Gly Ile AAG TTA Lys Leu 85 AAA GAA Lys Glu 100 GGA ACA Gly Thr CAT TTG His Leu TGG CCA Trp Pro CAA AGC Gin Ser 165 GGG CTT Gly Leu 180 GAC TTG; Asp Leu GGC GTC Gly Val GAA TCC Giu Ser 55 TTr CAA Phe Gin CAA AAT Gin Asn GTC ATT Val Ile AAG, CTT Lys Leu TTT CAT Phe His 135 AAG AAC Lys Asn 150 TTG ATA Leu Ile GGA CTG Gly Leu ATC TAC Ile Tyr CTT GCT TTG TTG GAG GTT CCA GTG Leu Leu Glu Val Pro Val Leu

GTG

Val

GTT

Val1

GCT

Ala

CAA

Gin 120

ATA

Ile

CCT

Pro

GGG

G ly

GAA

Giu

ATG

Met 200

CTT

GTC

Val

GTG

Val

OCA

G ly

AAA

Lys 105

AAA

Lys

GTT

Val1

CCT

Pro

GTG

Val1

GAA

Giu 185

TTG

Leu

GCT

Ala

AAC

Asn

AAA

Lys

CCA

Pro

GAA

G lu

TGG

Trp

TCT

Ser

GCA

Ala 170

GAT

Asp

AAA

Lys AT A Ile

CCC

Cly

?ITC

Phe

OCT

Gly

CAA

Gin

CCT

Pro 140

AGA

Arg

AAG

Lys

GTG

Val1

AAC

As n 193 241 289 337 385 433 481 529 577 625 673 TAC CCA CCA TGT CCA TGC CCC GAG COG OTA GCC CCA CAT Tyr Pro Pro Cys Pro Cys Pro Glu Leu Ala Leu Gly Val Ala Pro His 210 215 220 WO 94/03606 P~r/AU93/00400 -49- ACO GAO ATG Thr Asp Met OTA CAA GTO Leu Gin Val 240 TOA ATC ACC ATA Ser Ile Thr Ile GTC COG AAT GAA Vai Pro Asn Giu GTT CAA GGT Val Gin Gly 235 TTr AAA GAT GGT Phe Lys Asp Giy CAA TGG TAT GAT GTT GOT TAO ATT OCT Gin Trp Tyr Asp Vai Aia Tyr Ile Pro 245 250 GOT GAO CAG ATT GAG ATA OTG AGO AAT Giy Asp Gin Ile Giu Ile Leu Ser Asn 265 AAT GOT Asn Aia 255 OTO API' ATT CAC Leu Ile Ile His

GGA

G iy 270 AAA TAT AAG AGT Lys Tyr Lys Ser

GTG

Vai 275 TAT CAC AGA TOA Tyr His Arg Ser GTG AAT AAG GAG Vai Asn Lys Giu ACA AGA ATG TOG Thr Arg Met Ser OCA GOA PIT 'PTG Pro Aia Phe Leu

GAG

Giu 295 OCA COG OCA GAG Pro Pro Pro Giu 'PIT GAG Phe Giu 300 GTT GOT OCA Val Giy Pro AAG ACC AAG Lys Thr Lys 320 OCA AAG OTO GTO AAT Pro Lys Leu Vai Asn AAA GAO GAT Lys Asp Asp OCA OCA AAA TAO Pro Pro Lys Tyr 315 AAG TAO AAA GAO Lys Tyr Lys Asp TAT GTO Tyr Vai 325 TAT TGC AAG OTA AAT AAG OTT Tyr Cys Lys Leu Asn Lys Leu 330 1009 COG CAG Pro Gin TGA GGG API' 'TA GAT ATT ATO TOO AAA CAT ATA TOT ATT TOT 1057 335 GAA OGO AT GOGTOTOTAA. GOTOTTOAGT TXITTTGAATO TOOGAPIOTA ATATOAGACT OTOATOGAAT 'ITAGTTICAAA GOTATATOAA OATAOAATAA GAGOAGTAAO TOA'PI'ATOO 1i75 AOTTTOGOAT 'PITOATCIT TATATTOGAT AITAGOTATO TATOATOOTT TAOAAGGTAA 1235 AAAA~AAA 7 i250 1250 L .anriPirnar_~i-rrrau~l-ra~--rm=~-~i-i WO 94/03606 PCT/AU93/00400 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GAGAGAGAGA GAGAGAGAGA TCTCGAGTTT TTTTTTTTTT TTTTT INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (ix) FEATURE: either X or Y I inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGGGGIITTT T(T,C)(C,G)AIITI(A,G)T I(A,G)AICA 26 FE WO 94/03606 PCT/AU93/00400 -51- INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (ix) FEATURE: either X or I inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TI(A,G)TIAA(T,C)CA (T,C)GGI(A,T)TICC INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (ix) FEATURE: either X or Y I inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGI(T,C)TITT(T,C)(C,G) A(A,G)ITI(A,G)TIAA (T,C)CA(T,C)GG WO 94/03606 PCT/AU93/00400 -52- INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (ix) FEATURE: either X or Y I inosine (xi) SEQUENCE DESCRIPTION: SEQ ID ,3(T,C)TTIGG(A,G)C AIGGIGG(A,G)TA INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (ix) FEATURE: either X or Y I inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GG(A,G)CAIGGIG G(A,G)TA(A,G)TA(A,G)TT WO 94/03606 PCT/AU93/00400 -53- INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGACAAGGAG GATAATAATT INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATCAGAGTAC ATTAGGTC 18 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GTCCCAAATG AAGTCCAAG 19 L -Cnrsa3lra~~ WO 94/03606 PCT/AU93/00400 -54- INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TTCTTCGTTT GCTTCCCT 18 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTAGGTACCG GGCCCAAAGG ATCCTCTAGA GTAC 34 k

Claims (17)

1. An isolated nucleic acid molecule comprising a sequence ofnucleotides encoding or complementary to a sequence encoding a flavonol synthase (FLS) as hereinbefore defined.

2. An isolated nucleic acid molecule according to claim 1 wherein said nucleic acid is DNA.

3. An isolated nucleic acid molecule according to claim 1 or 2 wherein the plant is selected from the group consisting of petunia, snapdragon, tobacco, rose, carnation, chrysanthemum, lisianthus, cyclamen, parsely and grape.

4. An isolated nucleic acid molecule according to claim 3 wherein the plant is selected from the group consisting of petunia, tobacco, carnation and chrysanthemum. An isolated nucleic acid molecule according to claim 4 having a nucleotide sequence or complementary nucleotide sequence which comprises the sequence as set forth in SEQ ID No:l or having at least 50% similarity thereto.

6. An isolated nucleic acid molecule according to claim 4 having a nucleotide sequence or complementary nucleotide sequence which comprises the sequence as set forth in SEQ ID No:2 or having at least 50% similarity thereto. *0 4

7. An isolated nucleic acid molecule according to claim 4 having a nucleotide sequence or complementary nucleotide sequence which comprises the sequence as set forth in SEQ ID No:3 or having at least 50% similarity thereto.

8. An isolated nucleic acid molecule according to claim 4 having a nucleotide sequence or complementary nucleotide sequence which comprises the sequence as set forth in SEQ ID No:4 or having at least 50% similarity thereto. 960110,p:\operejh,46901-93.004,55 0 9601 10,p:\oper\ejh,46901-93.004,55 il- 56

9. An isolated nucleic acid molecule according to claim 4 having a nucleotide sequence or complementary nucleotide sequence which comprises the sequence as set forth in SEQ ID No:5 or having at least 50% similarity thereto.

10. An isolated DNA molecule comprising a sequence of nucleotides which encodes a FLS of plant origin; and (ii) has at least 50% nucleotide sequence similarity to the sequence set forth in SEQ ID No:l.

11. An isolated DNA molecule according to claim 10 having a nucleotide sequence as set forth in SEQ ID No:l.

12. An isolated DNA molecule according to claim 10 having a nucelotide sequence as set forth in SEQ ID No:2.

13. An isolated DNA molecule according to claim 10 having a nucelotide sequence as set forth in SEQ ID No:3.

14. An isolated DNA molecule according to claim 10 having a nucleotide sequence as set forth in SEQ ID No:4. 6 i An isolated DNA molecule according to claim 10 having a nucleotide sequence 20 as set forth in SEQ ID

16. An isolated nucleic acid molecule which: encodes a FLS of plant origin; and (ii) hybridises under low stringency conditions to the nucleotide sequence set forth in SEQ ID No:1 or to a complementary strand thereof.

17. An isolated nucleic acid molecule according to claim 16 having a nucleotide sequence as set forth in SEQ ID No:1. a O Io 0 25 16. An isolated nu50110,pcleic oper\jhacid molecul46901-93004,56e which:

57- 18. An isolated nucleic acid molecule according to claim 16 having a nucleotide sequence as set forth in SEQ ID No:2. 19. An isolated nucleic acid molecule according to claim 16 having a nucleotide sequence as set forth in SEQ ID No:3. An isolated nucleic acid molecule according to claim 16 having a nucleotide sequence as set forth in SEQ ID No:4. 21. An isolated nucleic acid molecule according to claim 16 having a nucleotide sequence as set forth in SEQ ID 22. A vector comprising the nucleic acid molecule according to claim 1 or 10 or 16. 23. A vector according to claim 22 wherein the nucleic acid molecule is operably linked to a promoter. 24. A vector according to claim 23 capable of replication and expression in a eukaryotic cell. 0 4 t 441$ 25. A vector according to claim 23 capable of replication and expression in a prokaryotic cell. 9. 0 4 4. 26. An oligonucleotide capable of hybridising under low stringency conditions to part a, L of the nucleotide sequence or its complementary form set forth in at least one of the *,,;sequences selected from the list consisting of SEQ ID No:l 1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID 4 t 27. A transgenic plant carrying a non-indigenous genetic sequence encoding a FLS. 28. A transgenic plant according to claim 27 wherein the genetic sequence is capable Ri of expression and said expression is optionally regulatable. 9 t 6 A7 960110,p:\oper\ejh,46901-93.004,57 r Lf__L

58- 29. A transgenic plant according to claim 28 wherein the expression is developmentally regulated. A transgenic plant according to claim 27 or 28 wherein the FLS is from a plant selected from the group consisting of petunia, snapdragon, tobacco, rose, carnation, chrysanthemum, lisianthus, cyclamen, parsely and grape. 31. A transgenic plant according to claim 27 or 28 wherein the FLS is from a plant selected from the group consisting of petunia, tobacco, carnation and chrysanthemum. 32. A transgenic plant according to claim 27 or 28 wherein said plant is selected from the group consisting of petunia, rose, carnation, chrysanthemum, gerbera, tobacco, lisianthus, lily, iris and pelargonium. 33. A transgenic plant according to claim 27 or 28 wherein the FLS is encoded by a nucleotide sequence as set forth in SEQ ID No:l or having at least 50% similarity thereto. 34. A transgenic plant according to claim 27 or 28 wherein the FLS is encoded by 20 a nucleotide sequence as set forth in SEQ ID No:2 or having at least 50% similarity thereto. A transgenic plant according to claim 27 or 28 wherein the FLS is encoded by a nucleotide sequence as set forth in SEQ ID No:3 or having at least 50% similarity 25 thereto. 36. A transgenic plant according to claim 27 or 28 wherein the FLS is encoded by a nucleotide sequence as set forth in SEQ ID No:4 or having at least 50% similarity thereto. 44.444 o 44 4 C 4 4 4 0444 4444p eta o a @444 a t 44 4( 444' 4at. 37. A transgenic plant according to R a nucleotide sequence as set forth in SI O 3 L claim 27 or 28 wherein the FLS is encoded by EQ ID No:5 or having at least 50% similarity 960110,p:\oper\ejh,46901-93.004,58 -59 thereto. 38. A transgenic plant selected from the group consisting of petunia, rose, carnation, chrysanthemum, gerbera, tobacco, lisianthus, lily, iris and pelargonium carrying a non- indigenous genetic sequence encoding a FLS, said genetic sequence optionally capable of being expressed and wherein said FLS is encoded by a DNA molecule comprising a DNA strand capable of hybridising under low stringency conditions to a nucleic acid molecule comprising all or part of the sequence of nucleotides set forth in at least one of SEQ ID No:l, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID 39. A cut flower from a transgenic plant according to any one of claims 27 to 38. A method for producing a transgenic flowering plant capable of exhibiting altered inflorescence properties, said method comprising introducing into a cell of a suitable plant the nucleic acid molecule according to claim 1 or 10 or 16, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit expression of the nucleic acid sequence into FLS. 41. A method according to claim 40 wherein the transgenic plant is selected from the 20 group consisting of petunia, rose, carnation, chrysanthemum, gerbera, tobacco, lisianthus, o .0 lily, iris and pelargonium. 42. A method according to claim 41 wherein the introduced nucleic acid is DNA and encodes FLS having the nucleotide sequence substantially as set forth in at least one of the sequences selected from the list consisting of SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID 43. A method for producing a transgenic flowering plant capable of exhibiting altered inflorescence properties, said method comprising introducing into a cell of a plant carrying an indigenous FLS, the nucleic acid according to any one of claims 1 or 10 or 16 under conditions to induce co-suppression of said indigenous FLS. 9601 10,pAoperjh,46901-93.004,59 r L I. 1, 44. A method according to claim 43 wherein the transgenic plant is selected from the list consisting of petunia, rose, carnation, chrysanthemum, gerbera, tobacco, lisianthus, lily, iris and pelargonium. 45. A method according to claim 44 wherein the introduced nucleic acid is DNA and encodes FLS and has the nucleotide sequence substantially as set forth in at least one of the sequences selected from the list consisting of SEQ ID No:l 1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID 46. An isolated nucleic acid molecule according to any one of claims 1 to 21 or a vector according to any one of claims 22 to 25 or an oligonucleotide according to claim 26 or a transgenic plant according to any one of claims 27 to 38 or a cut flower according to claim 39 or a method according to any one of claims 39 to 42 substantially as described herein with reference to the Figures and/or Examples. a 0- *0 '9 00 0 0« 0 0 j 0 a a0 Dated this 10th day of January, 1995 INTERNATIONAL FLOWER DEVELOPMENTS PTY LTD by DAVIES COLLISON CAVE Patent Attorneys for the Applicant. 960110,p:\opcr\ejh,46901-93.004,60 I I