Genetic sequences having methyltransferase activity and uses therefor

Abstract

The present invention relates generally to a genetic sequence encoding a polypeptide having methyltransferase activity and the use of the genetic sequence and/or the polypeptide to modify one or more phenotypic characteristics of a plant. More particularly, the methyltransferase of the present invention acts on flavonoids, preferably wherein the flavonoid is an anthocyanin. Even more particularly, the present invention relates to a polypeptide having S-adenosyl-L-methionine: anthocyanin 3′-O-methyltransferase or S-adenosyl-L-methionine: anthocyanin 3′, 5′-O-methyltransferase activity. The present invention still further provides a genetic sequence encoding a polypeptide having methyltransferase activity derived from Petunia, Torenia Fuchsia or Plumbago or botanically related plants. The instant invention further relates to antisense and sense molecules corresponding to all or part of the subject genetic sequence as well as genetically modified plants as well as cut flowers, parts, extracts and reproductive tissue from such plants.

1 . An isolated nucleic acid comprising a sequence of nucleotides encoding or complementary to a sequence encoding a flavonoid methyltransferase (FMT) or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1  wherein the FMT modulates or otherwise facilitates the methylation of an anthocyanin. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1  wherein the FMT is a Class I S-adenosyl-L-methionine O-methyltransferase (SAM-OMTs). 
     
     
         4 . The isolated nucleic acid molecule of  claim 3  wherein the FMT is 3′FMT or 3′5′FMT. 
     
     
         5 . The isolated nucleic acid molecule of  claim 2  wherein the anthocyanin molecule is derivative of delphinidin. 
     
     
         6 . The isolated nucleic acid molecule of  claim 2  wherein the anthocyanin molecule is a derivative of petunidin or cyanidin. 
     
     
         7 . The isolated nucleic acid molecule of  claim 2  wherein the anthocyanin molecule is delphinidin 3-glucoside, delphinidin 3,5-diglucoside or delphinidin 3-rutinoside. 
     
     
         8 . The isolated nucleic acid molecule of  claim 2  wherein methylation of an anthocyanin molecule results in the production of a petunidin, malvidin or peonidin derivative. 
     
     
         9 . The isolated nucleic acid molecule of  claim 1  wherein the molecule is of  Petunia, Torenia, Fuchsia, Plumbago  or botanically related plant origin. 
     
     
         10 . An isolated nucleic acid molecule according to  claim 1  having the nucleotide sequence comprising:
 (i) a nucleotide sequence set forth in SEQ ID NO:1; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:1; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO: 1 or its complementary form; 
 (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:2; 
 (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:2; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent or homolog thereof. 
     
     
         11 . The isolated nucleic acid molecule of  claim 1  having the nucleotide sequence comprising:—
 (i) a nucleotide sequence set forth in SEQ ID NO:4; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:4; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO: 4 or its complementary form; 
 (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:5; 
 (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:5; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         12 . The isolated nucleic acid molecule of  claim 1  having the nucleotide sequence comprising:
 (i) a nucleotide sequence set forth in SEQ ID NO:6; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:6; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO:6 or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         13 . An isolated nucleic acid molecule of  claim 1  having the nucleotide sequence comprising:
 (i) a nucleotide sequence set forth in SEQ ID NO:26; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:26; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO: 26 or its complementary form; 
 (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:7; 
 (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:7; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         14 . An isolated nucleic acid molecule of  claim 1  having the nucleotide sequence comprising:
 (i) a nucleotide sequence set forth in SEQ ID NO:11; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:11; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO: 11 or its complementary form; 
 (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:12; 
 (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:12; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         15 . An isolated nucleic acid molecule of  claim 1  having the nucleotide sequence comprising:—
 (i) a nucleotide sequence set forth in SEQ ID NO:21; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:21; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO: 21 or its complementary form; 
 (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:22; 
 (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:22; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         16 . An isolated nucleic acid molecule of  claim 1  having the nucleotide sequence comprising:
 (i) a nucleotide sequence set forth in SEQ ID NO:41; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:41; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO: 41 or its complementary form; 
 (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:42; 
 (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:42; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         17 . An isolated nucleic acid molecule according to  claim 1  having the nucleotide sequence comprising:
 (i) a nucleotide sequence set forth in SEQ ID NO:43; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:43; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to SEQ ID NO: 43 or its complementary form; 
 (iv) a nucleotide sequence capable of encoding the amino acid sequence set forth in SEQ ID NO:44; 
 (v) a nucleotide sequence capable of encoding an amino acid sequence having at least about 50% similarity after optimal alignment to SEQ ID NO:44; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         18 . A genetic construct comprising a nucleic acid molecule encoding or complementary to a sequence encoding an FMT of  claim 1  or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent or homolog thereof. 
     
     
         19 . The genetic construct according to  claim 18 , wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
 (i) the nucleotide sequence set forth in any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43;   (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43;   (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43, or the complementary form thereof;   (iv) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 42, or SEQ ID NO: 44;   (v) a nucleotide sequence encoding an amino acid sequence having at least about 50% similarity after optimal alignment to any one of SEQ ID NO:2 SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 42, or SEQ ID NO: 44;   (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form;   
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         20 . A genetically modified plant or part thereof or cells therefrom comprising the nucleic acid of  claim 1 . 
     
     
         21 . The genetically modified plant or part thereof or cells therefrom of  claim 20  said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
 (i) the nucleotide sequence set forth in any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43, or the complementary form thereof; 
 (iv) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 42, or SEQ ID NO: 44; 
 (v) a nucleotide sequence encoding an amino acid sequence having at least about 50% similarity after optimal alignment to any one of SEQ ID NO:2 SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 42, or SEQ ID NO: 44; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         22 . The genetically modified plant or part thereof or cells therefrom of  claim 20  wherein saidplant or part thereof or cells therefrom is from a cut-flower species. 
     
     
         23 . The genetically modified plant or part thereof or cells therefrom of  claim 20  wherein said plant or part thereof or cells therefrom is a horticultural plant species. 
     
     
         24 . The genetically modified plant or part thereof or cells therefrom of  claim 20  wherein said plant or part thereof or cells therefrom is an agricultural plant species. 
     
     
         25 . The genetically modified plant or part thereof or cells thereof of  claim 23  or  24  wherein the plant exhibits altered flowers or inflorescence. 
     
     
         26 . The genetically modified plant or part thereof or cells therefrom of  claim 20  wherein said altered part is a sepal, bract, petiole, peduncle, ovary or anther stem. 
     
     
         27 . The genetically modified plant or part thereof or cells therefrom of  claim 20  wherein said altered part is a leaf, root, flower, seed, fruit, nut, berry or vegetable. 
     
     
         28 . The genetically modified plant or part thereof or cells therefrom of  claim 22  wherein the plant is selected from a rose, carnation, lisianthus, petunia, lily, pansy, gerbera, chrysanthemum,  Torenia, Begonia, Cyclamen, Nierembergia, Catharanthus, Pelargonium, Orchid , grape,  Euphorbia  or  Fuchsia.    
     
     
         29 . Flowers cut or severed from a plant of  claim 20 . 
     
     
         30 . Progeny, offspring of progeny or vegetation propagates lines of the genetically modified plant of  claim 20 . 
     
     
         31 . An extract from a plant or plant part of  claim 20 . 
     
     
         32 . The extract of  claim 31  wherein the extract is a flavoring or food additive or health product or beverage or juice or coloring. 
     
     
         33 . A method for producing a genetically modified plant capable of synthesizing FMT, said method comprising stably transforming a cell of a suitable plant with the nucleic acid molecule of  claim 1  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. 
     
     
         34 . A method for producing a genetically modified plant with reduced indigenous or existing FMT activity, said method comprising stably transforming a cell of a suitable plant with the nucleic acid molecule of  claim 1 , regenerating a transgenic plant from the cell and where necessary growing said transgenic plant under conditions sufficient to permit the expression of the nucleic acid. 
     
     
         35 . A method for producing a genetically modified plant with reduced indigenous or existing FMT activity, said method comprising altering an FMT encoding gene through modification of the indigenous sequences via homologous recombination from an appropriately altered nucleic acid of  claim 1  introduced into the plant cell, and regenerating the genetically modified plant from the cell. 
     
     
         36 . A method for producing a genetically modified plant with altered levels of FMT encoded in the nucleic acid molecule of  claim 1 , said method comprising introducing into a cell or cells of said plant a genetic sequence selected from:
 (i) an antisense sequence to FMT mRNA;   (ii) a sense sequence to FMT DNA; and/or   (iii) an RNAi-inducing sequence specific for FMT mRNA;   
       and regenerating a genetically modified plant from said cell. 
     
     
         37 . A method for producing a transgenic plant capable of expressing a recombinant gene encoding an FMT or part thereof 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 an FMT, said method comprising stably transforming a cell of a suitable plant with the isolated nucleic acid molecule of  claim 1 , where necessary under conditions permitting the eventual expression of said isolated nucleic acid molecule, and regenerating a transgenic plant from the cell. 
     
     
         38 . A method for producing a transgenic plant of any one of  claims 33  to  37 , wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
 (i) the nucleotide sequence set forth in any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43; 
 (ii) a nucleotide sequence having at least about 50% similarity after optimal alignment to any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43; 
 (iii) a nucleotide sequence capable of hybridizing under low stringency conditions to any one of SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 41 or SEQ ID NO: 43, or the complementary form thereof; 
 (iv) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 42, or SEQ ID NO: 44; 
 (v) a nucleotide sequence encoding an amino acid sequence having at least about 50% similarity after optimal alignment to any one of SEQ ID NO:2 SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 42, or SEQ ID NO: 44; 
 (vi) a nucleotide sequence capable of hybridizing under low stringency conditions to the nucleotide sequence in (iv) or (v) or its complementary form; 
 
       wherein said nucleotide sequence encodes a FMT molecule or a mutant, part, fragment or portion thereof or a functional and/or structural equivalent, homolog, analogue or derivative thereof. 
     
     
         39 . An isolated oligonucleotide of at least 5 nucleotides having substantial similarity or complementarily to a part or region of a molecules with a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:11 or SEQ ID NO:21 or SEQ ID NO:26 or SEQ ID NO:41 or SEQ ID NO:43 or having at least about 50% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:11 or SEQ ID NO:21 or SEQ ID NO:26 or SEQ ID NO:41 or SEQ ID NO:43 under low stringency conditions, wherein said nucleotide sequence encodes a polypeptide having FMT activity. 
     
     
         40 . An isolated oligonucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:13 or SEQ ID NO:14 or SEQ ID NO:15 or SEQ ID NO:16 or SEQ ID NO:17 or SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:20 or SEQ ID NO:23 or SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:28 or SEQ ID NO:29 or SEQ ID NO:30 or SEQ ID NO:31 or SEQ ID NO:32 or SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35 or SEQ ID NO:36 or SEQ ID NO:37 or SEQ ID NO:38 or SEQ ID NO:39 or SEQ ID NO:40. 
     
     
         41 . An isolated recombinant FMT encoded by a nucleic acid molecule of  claim 1 . 
     
     
         42 . The recombinant FMT of  claim 41  wherein the recombinant FMT is a fusion molecule comprising two or more heterologous amino acid sequences. 
     
     
         43 . An isolated recombinant FMT nucleic acid molecule of  claim 1  comprising a fusion of two or more heterologous nucleotide sequences. 
     
     
         44 . A prokaryotic organism carrying a genetic sequence encoding an FMT molecule according to  claim 1  extrachromasomally in plasmid form. 
     
     
         45 . A eukaryotic organism carrying a genetic sequence encoding an FMT molecule according to  claim 1  extrachromasomally in plasmid form.