Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-10-16
2004-08-03
Fredman, Jeffrey (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S410000, C435S252300
Reexamination Certificate
active
06770747
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a gene encoding a protein having a glycosyl transferase activity to aurones, said protein, and the uses thereof.
BACKGROUND ART
The color of flowers are mainly based on three pigments: flavonoids, carotenoids, and betalains. Yellow colors are mostly derived from carotenoids and betalains, but in some plants they are derived from flavonoids. Among the flavonoid pigments, major pigments that are thought to be associated with the development of yellow flowers are divided into three groups: chalcones, aurones, and yellow flavonols (Saito, Biohorti 1, pp. 49-57, 1990)
Aurones are substances in which two phenyl groups are joined together through three carbon atoms of dihydrofuran. As aurones, there are known 4,6,4'-trihydroxy aurone, aureusidin, sulfuretin, bracteatin, and the like. For example, aureusidin and bracteatin are contained in snapdragons, aureusidin is contained in limoniums, aureusidin is contained in morning glories, sulfuretin is contained in dahlias, bracteatin is contained in
Helichrysum bracteatum
, and sulfuretin is contained in
Helianthus tuberosus.
Flavonoids have generally been modified by acylation, glycosilation, methylation and the like, and carotenoids and betalains have also been glycosilated in many cases. Among various modifications, glycosilation plays an important role in the color of flowers such as (1) contribution to enhancing the stability and solubility of pigments, (2) the presence as a step preceding acylation that greatly affects the hue of colors, and (3) copigmentation effects by the glycosilated flavonoids, and the like.
It has been reported that, in snapdragon, a yellow pigment aurones (aureusidin, bracteatin), a kind of flavonoid, is present in a glycosilated at its position 6 corresponding to position 7 of flavonoids, and since aurones are present as glycosides in other aurone-containing plants as well, it has been considered that glycosilation is essential for the stability of aurones.
There are many reports on the genes for glycosyl transferases derived from plants that transfer a glycosyl group to flavonoids and on the activities of those enzymes.
By way of example, genes encoding UDP-glucose: flavonoid 3-glucosyl transferases (3GT) that transfer a glycosyl group to the position 3 of flavonoids have been obtained from many plants including corn, barley, and snapdragon, and has been analyzed in detail (The Flavonoids: Advanced in Research Since 1986. Published by Chapman & Hall, 1993).
Also, genes encoding UDP-glucose: flavonoid 5-glucosyl transferases (5GT) that transfer a glycosyl group to the position 5 of flavonoids have been cloned from perillas, torenias, and verbenas (International Patent Publication No. WO 99/05287).
However, as to the gene encoding UDP-glucose: flavonoid 7-glucosyl transferase (7GT) that transfers a glycosyl group to the position 7 of flavonoids, there is only one report on the purification of flavanone-specific 7-glucosyl transferase in grapefruits (Archives of Biochemistry and Biophysics 282, 1: 50-57, 1990).
With regard to enzymes that transfer a glycosyl group to the position 6 of aurones, there is a report on the measurement of a reaction that transfers a glycosyl group to the position 6 of sulfuretin, a kind of aurone (Plant Science 122: 125-131, 1997), but this only studied the enzymatic property using a partially purified product, and has not been purified in a pure form.
On the other hand, there is a report on the isolation of a glycosyl transferase, pS.b UFGT1, that has an activity of transferring glucose to the position 7 of baicaleins, a kind of flavone, from the hairy roots of a Labuatae,
Scutellaria baicalensis
(1997, presented at the Fifteenth annual meeting of Japanese Society of Plant Cell and Molecular Biology). The gene product is also reported to be capable of transferring a glycosyl group to the position 7 of anthocyanidins and flavonols, but not reported on aurones (presented at the Fifteenth annual meeting of Japanese Society of Plant Cell and Molecular Biology).
As genes having a high homology to pS.b UFGT1, tabacco-derived IS10a and IS5a have been reported (Plant Molecular Biology, 31: 1061-1072, 1996), but its activity of transferring a glycosyl group to position 7 (7GT activity) has not been studied.
Reports to date teach that the glycosyl transferases that use flavonoids as substrates have a great variation in substrate specificity even among flavonoids. For example, when the gene of flavonoid-3-glycosyl transferase derived from gentians were cloned, expressed in
E coli
, and the activity was determined, it was found to exhibit a 61% activity to cyanidins, a 38% activity to pelargonidins, and a good activity to anthocyanins relative to a 100% glycosyl transferase activity to delphinidins. On the other hand, it only shows an activity of 7.0%, 6.5%, and 4.4% to kaempferol, quercetin, and myricetin, respectively. Furthermore, it does not transfer a glycosyl group to dihydroflavonols (Tanaka et al., Plant Cell Physiol. 37: 711, 1996).
Also, when the gene of flavonoid-3-glycosyl transferase derived from grapes was cloned and the activity was determined in
E. coli
, its Km was 30 &mgr;M and Vmax was 905 nkatals/mg to cyanidins, whereas to quercetins the Km was 15 &mgr;M and Vmax was 18.9 nkatals/mg, exhibiting a great difference in reaction rates (Ford et al., J. Biol. Chem. 273: 9224, 1998).
These reports indicate that glycosyl transferases can distinguish the kinds of flavonoids and that the glycosyl transferase activity to a flavonoid does not readily permit the estimation of the glycosyl transferase activity to another flavonoids.
DISCLOSURE OF THE INVENTION
As hereinabove described, glycosyl transferases using flavonoids as substrates have a great variation in substrate specificity and the estimation of a glycosyl transferase activity to a specific flavonoid cannot be easily made based on known glycosyl transferases.
Thus, the present inventors have attempted to obtain a gene encoding a protein having a glycosyl transferase activity to aurones among the flavonoid pigments, and thereby have completed the present invention.
The present inventors have demonstrated that a gene product of the pS.b UFGT1 gene derived from
Scutellaria baicalensis
has an activity of transferring a glycosyl group to aurones, and, using this gene as a probe, have obtained a gene encoding a protein having an activity of transferring a glycosyl group to aurones from snapdragons (
Antirrhinum majus
).
Also, using said gene obtained from snapdragons (
Antirrhinum majus
) as a probe, the present inventors have further obtained two genes encoding a protein having an activity of transferring a glycosyl group to aurones from a petunia (
Petunia hybrida
).
Thus, the present invention provides a gene encoding a protein having an activity of transferring a glycosyl group to aurones. Furthermore, the present invention provides a gene encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 2, 8, or 10 and having an activity of transferring a glycosyl group to aurones.
The present invention further provides a gene encoding a protein that has an amino acid sequence modified by the addition, deletion and/or substitution with other amino acids of one or more amino acids in the amino acid sequence as set forth in SEQ ID NO: 2, 8, or 10, and that has an activity of transferring a glycosyl group to aurones.
The present invention further provides a gene encoding a protein that hybridizes to a nucleic acid having a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 2, 8, or 10 or a portion thereof under a stringent condition, and that has an activity of transferring a glycosyl group to aurones.
The present invention also provides a vector comprising said gene.
The present invention further provides a host transformed with said vector. The host may be a microorganism, plant cells, animal cells, or plants.
The present invention also provides a method of producing a protein having an activity of transferring a glycosyl grou
Fukui Yuko
Kusumi Takaaki
Sakakibara Keiko
Tanaka Yoshikazu
Yoshikawa Takafumi
Fredman Jeffrey
Suntory Limited
Switzer Juliet C.
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