Genetic sequences encoding flavonoid pathway enzymes and uses therefor

Claim 1

A nucleic acid isolate comprising a sequence of nucleotides encoding a dihydrokaempferol (DHK) hydroxylating enzyme or derivative thereof which hydroxylates one or more of DHK, DHQ, naringenin and eriodicytol, or the sequence complementary thereto, said sequence comprising: the nucleotide sequence set forth in Figure 9 or 10, or the sequence complementary thereto, or a nucleotide sequence capable of hybridizing to the nucleotide sequence set forth in Figure 9 or 10, or to the sequence complementary thereto, under stringency conditions of 20% formamide, 6xSSC, 1% w/v SDS at 42°C for 16 hours followed by washing in 6xSSC, 1% w/v SDS at 65°C for 1 hour.

Claim 23

A method for producing a transgenic plant capable of expressing a recombinant dihydrokaempferol (DHK) hydroxylating enzyme or which directs transcription of a nucleic acid sequence which is substantially complementary to all or a part of a mRNA molecule translatable to a DHK hydroxylating enzyme, said method comprising introducing into a cell of a suitable plant a nucleic acid isolate comprising a sequence of nucleotides encoding a dihydrokaempferol (DHK) hydroxylating enzyme or derivative thereof which hydroxylates one or more of DHK, DHQ, naringenin and eriodicytol, or the sequence complementary thereto, said sequence comprising: the nucleotide sequence set forth in Figure 9 or 10, or the sequence complementary thereto, or a nucleotide sequence capable of hybridizing to the nucleotide sequence set forth in Figure 9 or 10, or to the sequence complementary thereto, under stringency conditions of 20% formamide, 6xSSC, 1% w/v SDS at 42°C for 16 hours followed by washing in 6xSSC, 1% w/v SDS at 65°C for 1 hour, under conditions permitting the eventual expression of said nucleic acid isolate, 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 isolate, wherein said transgenic plant exhibits altered inflorescence properties as a result of the introduction of said nucleic acid isolate.

Claim 26

The method according to any one of claims 23 to 25 wherein the recombinant enzyme is flavonoid 3',5'-hydroxylase.

Claim 27

The method according to any one of claims 23 to 26 wherein the expression of nucleic acid isolate is developmentally regulated.

Claim 28

The method according to any one of claims 23 to 27 wherein the recombinant enzyme is of petunia, verbena, delphinium, grape, iris, freesia, hydrangea, cyclamen, potato, pansy or eggplant origin.

Claim 30

The method according to any one of claims 23 to 29 wherein the transgenic plant is a rose, petunia, chrysanthemum, carnation, gerbera, geranium, alstroemeria or tobacco.

Claim 32

The method of any one of claims 23 to 31 wherein the nucleic acid isolate is contained in a vector molecule capable of transferring said nucleic acid isolate into a plant cell or tissue.

Claim 35

A method for cloning a nucleic acid molecule comprising a sequence of nucleotides encoding a dihydrokaempferol (DHK) hydroxylating enzyme or derivative thereof which hydroxylates one or more of DHK, DHQ, naringenin and eriodicytol, or the sequence complementary thereto, said sequence comprising: the nucleotide sequence set forth in Figure 9 or 10, or the sequence complementary thereto, or a nucleotide sequence capable of hybridizing to the nucleotide sequence set forth in Figure 9 or 10, or to the sequence complementary thereto, under stringency conditions of 20% formamide, 6xSSC, 1% w/v SDS at 42°C for 16 hours followed by washing in 6xSSC, 1% w/v SDS at 65°C for 1 hour, said method comprising amplification of DHK hydroxylating enzyme nucleotide sequences or complementary sequences from a suitable preparation of nucleic acid molecules from cells of a plant by one or more polymerase chain reactions using one or more oligonucleotide primers based on the amino acid sequence FGA/SGRRICAG.

Claim 37

The method according to any one of claims 35 to 36 wherein the DHK hydroxylating enzyme is a flavonoid 3',5'-hydroxylase.

Claim 40

The method according to any one of claims 23 to 34 wherein said method further comprises the step of obtaining from said transgenic plant a transgenic flower, transgenic reproductive parts, or transgenic vegetative parts.

Claim 41

The method according to any one of claims 35 to 38 wherein said method further comprises the step of isolation of said nucleic acid molecule.