Patent Application
20200283782
The present invention relates to a method for creating a transformed plant having a blue-hued flower color, characterized in that delphinidin-type anthocyanin and a flavone C-glycoside are made to coexist within the plant cells, and a transformed plant characterized in that delphinidin-type anthocyanin and a flavone C-glycoside coexist within the plant cells, or its inbred or outbred progenies, or their propagules, partial plant bodies (especially cut flowers), processed forms (especially processed cut flowers), tissues or cells.
Roses, chrysanthemums and carnations are industrially important plants around the world. In particular, roses are the most popular flowering plant with records of their cultivation from before the Christian Era and artificial breeding has been carried out thereon over several hundred years. However, due to the problem that there are no wild species with a blue-hued flower color among related species that can be crossed, it has been difficult to create a rose variety with a blue-hued flower color by conventional cross breeding or mutation breeding. The creation of a completely new blue-hued flower color will stimulate a new demand for the flower as the areas of use thereof expands, leading to expansion of production and consumption. Therefore, attempts have been made to produce roses having a blue-hued flower color by genetic engineering techniques.
For example, purple to blue flowers are known to contain large amounts of delphinidin-type anthocyanins such as delphinidin, petunidin and malvidin. However, flowers such as roses cannot produce such delphinidin-type anthocyanins and so studies have been conducted in which delphinidin is artificially produced by expression of the flavonoid 3′,5′-hydroxylase gene which is necessary for the synthesis thereof (NPL1). However, in order to express the enzyme gene for producing the target substance in a recombinant plant, even if the metabolism of the plant is artificially altered, competition with endogenous enzymes of the plant itself frequently occurs and in most cases accumulation of the target product does not occur at all or occurs to a limited extent.
Furthermore, in addition to the structure of the anthocyanin itself, the color of the flower changes depending on coexisting flavonoids (referred to as copigments), metal ions, the pH of the vacuole, etc. Flavones and flavonols are typical copigments which, by being stacked so as to sandwiched anthocyanin, have an effect of making the anthocyanin appear blue, and dark (NPL2). This is known as the copigment effect. Flavones in particular are known to exhibit a strong copigment effect. For example, it has been reported that analysis of genetically modified carnations found that flavones exhibited a significant copigment effect (NPL3). Further, it has been reported that in the Dutch Iris, the higher the ratio of total flavone content to total delphinidin content, the greater the copigment effect and the color becomes blue (NPL4).
However, not all plants are able to produce flavone, and roses, petunias, and the like do not accumulate flavone. Thus, attempts have been made to express, in such plants, genes encoding proteins that act to synthesize flavone from flavanone in order to modify the color of the flowers (PTL1).
Furthermore, in addition to the free form, flavone is also distributed as a glycoside, and although flavone O-glycoside and flavone C-glycoside are produced, it is known that flavone C-glycoside in particular exhibits a strong copigment effect. For example, it has been reported that isovitexin, which is a type of flavone C-glycoside, exhibits a copigment effect with anthocyanin in Iris ensata Thunb and by stabilizing anthocyanin, a blue flower color is also stabilized (NPL5). With regard to flavone C-glycoside, two biosynthetic pathways have thus far been reported. In one of the pathways, flavone C-glycoside is synthesized from flavanone by reactions catalyzed by flavanone 2-hydroxylase and C-glucosyltransferase, and dehydratase. In the other pathway, flavone C-glycoside is synthesized from flavanone by reactions catalyzed by flavone synthase and flavone C-glucosyltransferase (NPL6).
However, there have been no reports so far of examples of these genes being introduced into plants that do not produce flavone C-glycoside. Further, the copigment effect is considered to be influenced by the quantitative ratio of anthocyanin and flavone, modification by sugar or acyl groups on the anthocyanin and flavone, etc., and it is not always possible to make the flower color blue by merely expressing the flavone synthase gene and accumulating flavone. When the torenia flavone synthase gene is expressed in petunias, the bluish-purple color of the flower fades (NPL7). Further, when a Gentiana scabra-derived flavone synthase gene is expressed in tobacco, flavone was synthesized (NPL8) but the flower color still faded. Furthermore, there have been attempts to modify the flower color of roses by artificially incorporating flavone and malvidin (PTL2), but the creation of roses having a blue-hued flower color has been unsuccessful.
In fact, up until now, in flower color modification of roses aiming for a blue-hued flower color, roses with a purple color (RHS color chart hue group: Purple group) or a bluish-purple color (Purple-Violet group, Violet group) were the limits and roses with purplish-blue color (Violet-Blue group) or blue color (Blue group) flowers have not been created. Thus, there is still a need for the development of a blue color expression control technique to enable the creation of roses with a true, blue flower color.
The object of the present invention is to provide a transformed plant having a blue-hued flower color or its inbred or outbred progenies, or their propagules, partial plant bodies (especially cut flowers), processed forms (especially processed cut flowers), tissues or cells.
The present inventors carried out extensive research in order to solve the problem above and as a result of repeated experimentation, discovered that a transformed plant having a blue-hued flower color (RHS color chart 5th edition: Violet-Blue group/Blue group and/or a hue angle of 339.7° to 270.0°) that could not be previously obtained could be created by making a delphinidin-type anthocyanin and a flavone C-glycoside coexist in the petal of plants such as roses, and thereby the present invention was completed.
Specifically, the present invention relates to the following:
[1] A method for creating a transformed plant characterized in that a delphinidin-type anthocyanin and a flavone C-glycoside are made to coexist in a cell of a plant.
[2] The method according to 1, wherein the flavone C-glycoside is selected from the group consisting of flavone 6-C-glucoside, flavone 8-C-glucoside, and a combination thereof.
[3] The method according to 2, wherein the flavone C-glycoside is apigenin 6-C-glucoside and/or luteolin 6-C-glucoside.
[4] The method according to any one of 1 to 3, wherein the delphinidin-type anthocyanin is selected from the group consisting of malvidin 3,5-diglucoside, delphinidin 3,5-diglucoside, petunidin 3,5-diglucoside, acylated anthocyanins (e.g., delphinidin 3-(6″-p-coumaroyl-β-glucosyl)-5-β-glucoside or delphinidin 3-(6″-p-malonyl-β-glucosyl)-3′,5′-β-diglucoside), and combinations thereof.
[5] The method according to any one of 1 to 4 comprising transforming a host plant with a vector comprising a flavone synthase (FNS) gene or a homologue thereof and a flavone C-glucosyltransferase (CGT) gene or a homologue thereof.
[6] The method according to 5, wherein the vector further comprises a flavonoid 3′,5′-hydroxylase (F3′S′H) gene or a homologue thereof and a methyltransferase (MT) gene or a homologue thereof.
[7] The method according to 6, wherein:
the FNS gene or homologue thereof is selected from the group consisting of
(1-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 19,
(1-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 19 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (1-a),
(1-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 20,
(1-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 20 and having the same activity as a protein encoded by the polynucleotide set forth in (1-c), and
(1-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20 and having the same activity as a protein encoded by the polynucleotide set forth in (1-c);
the CGT gene or homologue thereof is selected from the group consisting of
(2-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 21,
(2-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 21 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (2-a),
(2-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 22,
(2-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 22 and having the same activity as a protein encoded by the polynucleotide set forth in (2-c), and
(2-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 22 and having the same activity as a protein encoded by the polynucleotide set forth in (2-c);
the F3′S′H gene or homologue thereof is selected from the group consisting of
(3-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9,
(3-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 9 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (3-a),
(3-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10,
(3-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (3-c), and
(3-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (3-c);
the MT gene or homologue thereof is selected from the group consisting of
(4-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 17,
(4-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 17 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (4-a),
(4-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 18,
(4-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (4-c), and
(4-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (4-c).
[8] The method according to 7, wherein the CGT gene or a homologue thereof has added thereto an Arabidopsis alcohol dehydrogenase (ADH) gene-derived 5′ untranslated region (5′-UTR) (SEQ ID NO: 23).
[9] The method according to any one of 1 to 4 comprising transforming a host plant with a vector comprising a flavanone 2-hydroxylase (F2H) gene or a homologue thereof, a flavone C-glucosyltransferase (CGT) gene or a homologue thereof, and a dehydratase (FDH) gene or a homologue thereof.
[10] The method according to 9, wherein the vector further comprises a flavonoid 3′,5′-hydroxylase (F3′S′H) gene or a homologue thereof, and a methyltransferase (MT) gene or a homologue thereof.
[11] The method according to 10, wherein:
the F2H gene or homologue thereof is selected from the group consisting of
(5-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 3,
(5-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 3 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (5-a),
(5-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4,
(5-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 4 and having the same activity as a protein encoded by the polynucleotide set forth in (5-c), and
(5-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 and having the same activity as a protein encoded by the polynucleotide set forth in (5-c);
the CGT gene or homologue thereof is selected from the group consisting of
(6-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 13,
(6-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 13 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (6-a),
(6-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14,
(6-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded by the polynucleotide set forth in (6-c), and
(6-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded by the polynucleotide set forth in (6-c);
the FDH gene or homologue thereof is selected from the group consisting of
(7-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 15,
(7-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 15 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (7-a),
(7-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 16,
(7-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 16 and having the same activity as a protein encoded by the polynucleotide set forth in (7-c), and
(7-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and having the same activity as a protein encoded by the polynucleotide set forth in (7-c);
the F3′5′H gene or homologue thereof is selected from the group consisting of
(8-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9,
(8-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 9 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (8-a),
(8-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10,
(8-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded the polynucleotide set forth in (8-c), and
(8-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and the same activity as a protein encoded by the polynucleotide set forth in (8-c); and the MT gene or homologue thereof is selected from the group consisting of
(9-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 17,
(9-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 17 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (9-a),
(9-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 18,
(9-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (9-c), and
(9-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (9-c).
[12] A transformed plant or an inbred or outbred progeny thereof characterized in that delphinidin-type anthocyanin and flavone C-glycoside coexist within a cell thereof.
[13] The transformed plant or inbred or outbred progeny thereof according to 12, wherein the flavone C-glycoside is selected from the group consisting of flavone 6-C-glucoside, flavone 8-C glucoside and a combination thereof.
[14] The transformed plant or inbred or outbred progeny thereof according to 13, wherein the flavone C-glycoside is apigenin 6-C-glucoside.
[15] The transformed plant or inbred or outbred progeny thereof according to any one of 12 to 14, wherein the delphinidin-type anthocyanin is selected from the group consisting of malvidin 3,5-diglucoside, delphinidin 3,5-diglucoside, petunidin 3,5-diglucoside, and acylated anthocyanins (for example, delphinidin 3-(6″-p-coumaroyl-β-glucosyl)-5-β-glucoside or delphinidin 3-(6-p-malonyl-β-glucosyl)-3′,5′-β-diglucoside) and combinations thereof.
[16] The transformed plant or inbred or outbred progeny thereof according to any one of 12 to 15 comprising a flavone synthase (FNS) gene or a homologue thereof, and a flavone C-glucosyltransferase (CGT) gene or a homologue thereof.
[17] The transformed plant or inbred or outbred progeny thereof according to 16 further comprising a flavonoid 3′,5′-hydroxylase (F3′5′H) gene or a homologue thereof and a methyltransferase (MT) gene or a homologue thereof.
[18] The transformed plant or inbred or outbred progeny thereof according to 17, wherein: the FNS gene or homologue thereof is selected from the group consisting of
(1-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 19,
(1-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 19 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (1-a),
(1-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 20,
(1-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 20 and having the same activity as a protein encoded by the polynucleotide set forth in (1-c), and
(1-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20 and having the same activity as a protein encoded by the polynucleotide set forth in (1-c);
the CGT gene or homologue thereof is selected from the group consisting of
(2-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 21,
(2-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 21 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (2-a),
(2-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 22,
(2-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 22 and having the same activity as a protein encoded by the polynucleotide set forth in (2-c), and
(2-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 22 and having the same activity as a protein encoded by the polynucleotide set forth in (2-c);
the F3′S′H gene or homologue thereof is selected from the group consisting of
(3-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9,
(3-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 9 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (3-a),
(3-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10,
(3-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (3-c), and
(3-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (3-c); and
the MT gene or homologue thereof is selected from the group consisting of
(4-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 17,
(4-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 17 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (4-a),
(4-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 18,
(4-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (4-c), and
(4-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (4-c).
[19] The transformed plant or inbred or outbred progeny thereof according to 18, wherein the CGT gene or a homologue thereof has added thereto an Arabidopsis alcohol dehydrogenase (ADH) gene-derived 5′ untranslated region (5′-UTR) (SEQ ID NO: 23).
[20] The transformed plant or inbred or outbred progeny thereof according to any one of 12 to 15 comprising a flavanone 2-hydroxylase (F2H) gene or a homologue thereof, a flavone C-glucosyltransferase (CGT) gene or a homologue thereof, and a dehydratase (FDH) gene or a homologue thereof.
[21] The transformed plant or inbred or outbred progeny thereof according to 20 further comprising a flavonoid 3′,5′-hydroxylase (F3′S′H) gene or a homologue thereof, and a methyltransferase (MT) gene or a homologue thereof.
[22] The transformed plant or inbred or outbred progeny thereof according to 21, wherein: the F2H gene or homologue thereof is selected from the group consisting of
(5-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 3,
(5-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 3 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (5-a),
(5-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4,
(5-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 4 and having the same activity as a protein encoded by the polynucleotide set forth in (5-c), and
(5-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 and having the same activity as a protein encoded by the polynucleotide set forth in (5-c);
the CGT gene or homologue thereof is selected from the group consisting of
(6-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 13,
(6-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 13 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (6-a),
(6-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14,
(6-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded by the polynucleotide set forth in (6-c), and
(6-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded by the polynucleotide set forth in (6-c);
the FDH gene or homologue thereof is selected from the group consisting of
(7-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 15,
(7-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 15 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (7-a),
(7-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 16,
(7-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 16 and having the same activity as a protein encoded by the polynucleotide set forth in (7-c), and
(7-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and having the same activity as a protein encoded by the polynucleotide set forth in (7-c);
the F3′5′H gene or homologue thereof is selected from the group consisting of
(8-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9,
(8-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 9 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (8-a),
(8-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10,
(8-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (8-c), and
(8-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and the same activity as a protein encoded by the polynucleotide set forth in (8-c); and
the MT gene or homologue thereof is selected from the group consisting of
(9-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 17,
(9-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 17 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (9-a),
(9-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 18,
(9-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (9-c), and
(9-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (9-c).
[23] The transformed plant or inbred or outbred progeny thereof according to any one of 12 to 22 having a flower color in the Blue group or Violet-Blue group of the RHS color chart and/or a hue angle of 339.7° to 270° in the CIE L*a*b* color system.
[24] A propagule, part of a plant, tissue or cell of the transformed plant or inbred or outbred progeny thereof according to any one of claims 12 to 23.
[25] A cut flower of the transformed plant or inbred or outbred progeny thereof according to any one of claims 12 to 23, or a processed product created from the cut flower.
[26] A vector comprising a flavone synthase (FNS) gene or a homologue thereof and a flavone C-glucosyltransferase (CGT) gene or a homologue thereof.
[27] The vector according to 26 further comprising a flavonoid 3′,5′-hydroxylase (F3′5′H) gene or a homologue thereof, and a methyltransferase (MT) gene or a homologue thereof.
[28] The vector according to 27, wherein:
the FNS gene or homologue thereof is selected from the group consisting of
(1-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 19,
(1-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO:19 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (1-a),
(1-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 20,
(1-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 20 and having the same activity as a protein encoded by the polynucleotide set forth in (1-c), and
(1-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20 and the same activity as a protein encoded by the polynucleotide set forth in (1-c);
the CGT gene or homologue thereof is selected from the group consisting of
(2-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 21,
(2-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO:21 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (2-a),
(2-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 22,
(2-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 22 and having the same activity as a protein encoded by the polynucleotide set forth in (2-c), and
(2-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 22 and having the same activity as a protein encoded by the polynucleotide set forth in (2-c);
the F3′5′ gene or homologue thereof is selected from the group consisting of
(3-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9,
(3-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 9 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (3-a),
(3-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10,
(3-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (3-c), and
(3-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (3-c);
the MT gene or homologue thereof is selected from the group consisting of
(4-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 17,
(4-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 17 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (4-a),
(4-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 18,
(4-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (4-c), and
(4-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (4-c).
[29] The vector according to 28, wherein the CGT gene or a homologue thereof has added thereto an Arabidopsis alcohol dehydrogenase (ADH) gene-derived 5′ untranslated region (5′-UTR) (SEQ ID NO: 23).
[30] A vector comprising a flavanone 2-hydroxylase (F2H) gene or a homologue thereof, a flavone C-glycoside (CGT) gene or a homologue thereof, and a dehydratase (FDH) gene or a homologue thereof.
[31] The vector according to 30 further comprising a flavonoid 3′,5′-hydroxylase (F3′S′H) gene or a homologue thereof, and a methyltransferase (MT) gene or a homologue thereof.
[32] The vector according to 31, wherein:
the F2H gene or homologue thereof is selected from the group consisting of
(5-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 3,
(5-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 3 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (5-a),
(5-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4,
(5-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 4 and having the same activity as a protein encoded by the polynucleotide set forth in (5-c), and
(5-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 and the same activity as a protein encoded by the polynucleotide set forth in (5-c);
the CGT gene or homologue thereof is selected from the group consisting of
(6-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 13,
(6-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 13 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (6-a),
(6-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14,
(6-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded the polynucleotide set forth in (6-c), and
(6-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded by the polynucleotide set forth in (6-c);
the FDH gene or homologue thereof is selected from the group consisting of
(7-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 15,
(7-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 15 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (7-a),
(7-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 16,
(7-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 16 and having the same activity as a protein encoded by the polynucleotide set forth in (7-c), and
(7-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and having the same activity as a protein encoded by the polynucleotide set forth in (7-c);
the F3′5′H gene or homologue thereof is selected from the group consisting of
(8-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9,
(8-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 9 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (8-a),
(8-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10,
(8-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (8-c), and
(8-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (8-c); and the MT gene or homologue thereof is selected from the group consisting of
(9-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 17,
(9-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 17 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (9-a),
(9-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 18,
(9-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (9-c), and
(9-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (9-c).
[33] An isolated polynucleotide selected from the group consisting of:
(6-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 13;
(6-b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 13 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (6-a);
(6-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14;
(6-d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded by the polynucleotide set forth in (6-c), and
(6-e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and the same activity as a protein encoded by the polynucleotide set forth in (6-c).
According to the present invention, a plant variety having a blue-hued flower color (RHS color chart 5th edition: Violet-Blue group/Blue group and/or a hue angle of 339.7° to 270.0°) that could not be previously obtained could be created.
The present invention is a method for creating a transformed plant having a blue-hued flower color and relates to a method characterized in that delphinidin-type anthocyanin and flavone C-glycoside are made to coexist within the plant cells.
Anthocyanins are a group of pigments that exist widely in plants which are known to exhibit red, blue, and purple flower colors. Depending on the number of hydroxyl groups in the B-ring of the anthocyanidin site, which is an aglycone, the anthocyanidins are classified into the three groups pelargonidin, cyanidin, and delphinidin. The chromophore is the aglycone moiety and pelargonidin-based anthocyanins exhibit a bright red color, cyanidin-based anthocyanins exhibit a purple-red color, and delphinidin-based anthocyanins exhibit a purple-red color. Herein, for example, “delphinidin-based anthocyanin” can refer to delphinidin, malvidin, petunidin, or derivatives thereof, but is preferably malvidin.
Delphinidin-type anthocyanins, when made to coexist with substances such as flavones, flavanols, organic esters and tannins, may develop a bluish color due to intermolecular interactions therewith. This phenomenon is called copigmentation and substances that induce such phenomena are called copigments. Copigmentation not only has the effect of deepening color to induce the appearance of a blue color but also has the effect of darkening colors and improving color stability. The present inventors, at this time, confirmed that rose petals developed a blue color due to the effect of copigmentation of delphinidin-type anthocyanin and flavone C-glycoside.
Flavone, which is a type of organic compound, is a cyclic ketone of a flavan derivative that exists in plants mainly as a glycoside. Flavone in a narrow sense is 2,3-didehydroflavan-4-one and is a compound with the chemical formula C15H10O2 and a molecular weight of 222.24. However, flavone in the broader sense (flavone group) is just one category of flavonoids and flavonoids that have a flavone structure as the basic skeleton and do not have a hydroxyl group at position 3 are categorized as ‘flavones’. Herein ‘flavone C-glycoside’ refers to flavones in the broad sense of the term, and from among glycosides of flavone derivatives, specifically refers to glycosides with aglycone bonded directly to the anomeric carbon of aldose. Examples of flavone C-glycoside include luteolin C-glycoside, tricetin C-glycoside, apigenin C-glycoside, and acacetin C-glycoside, but are not limited thereto. The flavone C-glycoside also include glycosides of apigenin, luteolin, tricetin, and acacetin derivatives. Two biosynthetic pathways of flavone C-glycoside are known in plants (
Accumulation of flavone C-glycoside within plant cells can be achieved by transforming a host plant with a vector containing genes that are essential for pathway 1 (namely, a flavanone 2-hydroxylase (F2H) gene, flavone C-glucosyltransferase (CGT) gene, and a dehydratase (FDH) gene) or a homologues thereof, or a vector containing genes that are essential for pathway 2 (namely, a flavone synthase (FNS) gene and a flavone C-glucosyltransferase (CGT) gene) or homologues thereof.
The source of the F2H gene, which is an essential gene in pathway 1, or a homologue thereof, is not particularly limited provided the desired function is exhibited, but is preferably a Glycyrrhiza-derived F2H gene or a homologue thereof comprising a polynucleotide selected from the following group consisting of:
(a) a polynucleotide consisting of the base sequence of SEQ ID NO: 3;
(b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 3 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (a);
(c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4;
(d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 4 and having the same activity as a protein encoded by the polynucleotide set forth in (c); and
(e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 and the same activity as a protein encoded by the polynucleotide set forth in (c).
The source of the CGT gene, which is an essential gene in pathway 1, or a homologue thereof, is not particularly limited provided the desired function is exhibited, but is preferably an Oryza sativa-derived codon-usage modified CGT gene or a homologue thereof comprising a polynucleotide selected from the following group consisting of:
(a) a polynucleotide consisting of the base sequence of SEQ ID NO: 13;
(b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 13 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (a);
(c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14;
(d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded by the polynucleotide set forth in (c); and
(e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and having the same activity as a protein encoded by the polynucleotide set forth in (c).
The source of the FDH gene, which is an essential gene in pathway 1, or a homologue thereof, is not particularly limited provided the desired function is exhibited, but is preferably a Lotus japonicus-derived FDH gene or a homologue thereof comprising a polynucleotide selected from the following group consisting of:
(a) a polynucleotide consisting of the base sequence of SEQ ID NO: 15;
(b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 15 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (a);
(c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 16;
(d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 16 and having the same activity as a protein encoded by the polynucleotide set forth in (c); and
(e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and having the same activity as a protein encoded by the polynucleotide set forth in (c).
The source of the FNS gene, which is an essential gene in pathway 2, or a homologue thereof, is not particularly limited provided the desired function is exhibited, but is preferably a Torenia-derived FNS gene or a homologue thereof comprising a polynucleotide selected from the following group consisting of:
(a) a polynucleotide consisting of the base sequence of SEQ ID NO: 19;
(b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 19 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (a);
(c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 20;
(d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 20 and having the same activity as a protein encoded by the polynucleotide set forth in (c); and
(e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20 and having the same activity as a protein encoded by the polynucleotide set forth in (c).
The source of the CGT gene, which is an essential gene in pathway 2, or a homologue thereof, is not particularly limited provided the desired function is exhibited, but is preferably a Gentiana scabra-derived CGT gene or a homologue thereof comprising a polynucleotide selected from the following group consisting of:
(a) a polynucleotide consisting of the base sequence of SEQ ID NO: 21;
(b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 21 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (2-a);
(c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 22;
(d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 22 and having the same activity as a protein encoded by the polynucleotide set forth in (2-c); and
(e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 22 and the same activity as a protein encoded by the polynucleotide set forth in (c).
It is preferable for the CGT gene, which is an essential gene in pathway 2, or a homologue thereof to have added thereto an Arabidopsis alcohol dehydrogenase (ADH) gene-derived 5′ untranslated region (5′-UTR) (SEQ ID NO: 23).
Accumulation of a delphinidin-type anthocyanin within plant cells can be achieved by incorporating a flavonoid 3′,5′-hydroxylase (F3′S′H) gene or a homologue thereof and a methyltransferase (MT) gene or a homologue thereof into a host plant (PTL2). Thus, delphinidin-type anthocyanin and flavone C-glycoside can be made to coexist with cells of a host plant by transforming the host plant with a vector that further contains an F3′S′H gene or a homologue thereof and an MT gene or a homologue thereof in addition to the essential genes for pathways 1 and 2 and the homologues thereof.
The source of the F3′S′H gene or a homologue thereof, is not particularly limited provided the desired function is exhibited, but is preferably a Campanula-derived F3′S′H gene or a homologue thereof comprising a polynucleotide selected from the following group consisting of:
(a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9;
(b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 9 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (a);
(c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10;
(d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 10 and having the same activity as a protein encoded by the polynucleotide set forth in (c); and
(e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and the same activity as a protein encoded by the polynucleotide set forth in (c).
The source of the MT gene or a homologue thereof, is not particularly limited provided the desired function is exhibited, but is preferably a Torenia-derived MT gene or a homologue thereof comprising a polynucleotide selected from the following group consisting of:
(a) a polynucleotide consisting of the base sequence of SEQ ID NO: 17;
(b) a polynucleotide hybridizing to a polynucleotide consisting of a base sequence complimentary to the base sequence of SEQ ID NO: 17 under stringent conditions and encoding a protein having the same activity as a protein encoded by the polynucleotide set forth in (a);
(c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 18;
(d) a polynucleotide encoding a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (c);
(e) a polynucleotide encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 and having the same activity as a protein encoded by the polynucleotide set forth in (c).
Herein, the term “nucleotide” refers to DNA or RNA. Herein, the term “stringent conditions” refers to conditions that allow selective and detectable specific binding of polynucleotides or oligonucleotides to genomic DNA. Stringent conditions are defined according to an appropriate combination of salt concentration, organic solvent (e.g. formamide) temperature, and other publicly known conditions. Namely, stringency is increased depending on whether the salt concentration is decreased, the organic solvent concentration is increased, or the hybridization temperature is increased. Furthermore, washing conditions after hybridization are also affected by stringency. These washing conditions are defined according to salt concentration and temperature, and washing stringency is increased depending on whether the salt concentration is decreased or temperature is increased. Thus, the term “stringent conditions” means conditions under which specific hybridization occurs only when the base sequences have a high sequence identity where the degree of “sequence identity” between each base sequence is, for example, as an average for the whole sequence, at least about 80%, preferably at least about 90%, more preferably at least about 95%, even more preferably at least 97% and most preferably 98%. An example of “stringent conditions” are a temperature of 60° C. to 68° C., a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, a pH of 6 to 8, and as a specific example, hybridization can be performed under conditions that include 5×SSC (750 mN NaCl, 75 mM trisodium citrate), 1% SDS, 5×Denhardt's solution 50% formaldehyde at 42° C. and washing can be performed under conditions that include 0.1×SSC (15 mM NaCl. 1.5 mM trisodium citrate), 0.1% SDS, and 55° C.
Hybridization can be performed according to publicly known methods in the art such as the method disclosed in Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987) or methods based thereon. Further, when using commercially available libraries, hybridization can be performed by following the method described in the attached instruction manual. Genes selected by such hybridization methods may be derived from nature and may be a gene derived from a plant or a gene that is not derived from a plant. Further, genes selected by hybridization may be cDNA, genomic DNA, or chemically synthesized DNA.
“An amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids” means an amino acid sequence, wherein, for example, any number of amino acids from 1 to 20, preferably 1 to 5, and more preferably 1 to 3, have been deleted, substituted, inserted and/or added. Site-directed mutagenesis, which is a genetic engineering method, is useful for introducing specific mutations at specific sites and can be performed by following the method disclosed in, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. By expressing this mutant DNA using a suitable expression system, a protein consisting of an amino acid sequence including deletion, substitution, insertion, and/or addition of one or more amino acids can be obtained.
Further, the polynucleotides can be obtained by methods publicly known to a person skilled in the art. For example, chemical synthesis methods such as the phosphoramidite method or nucleic acid amplification methods using primers designed based on the nucleotide sequence of the target gene using a plant nucleic acid sample as a template can be used to obtain the polynucleotide.
Herein, the term “sequence identity” refers to the quantity (number) of amino acid residues or nucleotides composing polypeptide sequences (or amino acid sequences) or polynucleotide sequences (or nucleotide sequences), that can be determined to be identical between the two chains, and refers to the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. The “sequence identity” can be easily calculated. Many methods for measuring sequence identity between two polynucleotide sequences or polypeptide sequences are known and the term “sequence identity” is well known to a person skilled in the art (for example, refer to Lesk, A. M. (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, D. W. (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin, A. M. & Grifin, H. G. (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991)).
Furthermore, the value of “sequence identity” herein may be a value calculated using a sequence identity search program publicly known to a person skilled in the art, unless otherwise specified, and is preferably a value calculated using the Clustal W program of the MacVector application (version 9.5 Oxford Molecular Ltd., Oxford, England). In the present invention, the degree of sequence identity between each amino acid sequence is, for example, at least about 90%, preferably at least about 95%, more preferably at least about 97%, and most preferably at least about 98%.
The polynucleotide of the present invention (nucleic acid, gene) ‘encodes’ a protein of interest. Herein “encodes” means expressing the protein of interest so as to be in an active state. ‘Encode’ can also refer to both the encoding of a protein by a continuous structural sequence (exons) or through intervening sequences (introns).
The sequence of a gene comprising a native base sequence can be obtained by analysis using a DNA sequencer. Further, DNA encoding an enzyme having a modified amino acid sequence can be synthesized by conventional site-directed mutagenesis or PCR based on DNA having the native base sequence. For example, a modified DNA fragment is obtained by treating native cDNA or genomic DNA with a restriction enzyme, this fragment is used as a template and site-directed mutagenesis or PCR is carried out thereon using a primer into which a desired mutation has been introduced and the desired modified DNA fragment is obtained. Thereafter, the DNA fragment into which this mutation has been introduced can be joined to a DNA fragment encoding a different portion of the target enzyme.
Alternatively, in order to obtain DNA that encodes an enzyme comprising a shortened amino acid sequence, for example, an amino acid sequence longer than the target amino acid sequence, for example, the DNA encoding the full-length amino acid sequence is cut with a desired restriction enzyme, and if as a result thereof, the DNA fragment does not encode the entire amino acid sequence, a DNA fragment that encodes the lacking sequence can be synthesized and joined.
Furthermore, the obtained polynucleotide can be expressed in E. coli or yeast gene expression systems, and by measuring the enzymatic activity it can be confirmed whether the obtained polynucleotide encodes a protein having the desired activity.
The present invention also relates to (recombinant) vectors comprising the polynucleotide above, especially expression vectors, as well as plants transformed by the vectors.
Furthermore, the vectors of the present invention contain an expression control region, for example, a promoter, a terminator, or a replication origin that is dependent on the type of host plant into which the vector is introduced. Examples of promoters that will constitutively express polynucleotides within plant cells include the cauliflower mosaic virus 35S promoter, the El235S promoter that links two enhancer domains of the 35S promoter, the rd29A gene promoter, the rbcS promoter, the mac-1 promoter, etc. Furthermore, for tissue specific gene expression, promoters for the genes that are specifically expressed in the tissue can be used.
The vectors can be produced according to conventional methods such as using restriction enzymes and ligases. Transformation of the host plant with a vector can also be carried out according to conventional methods.
Under the present state of the art, techniques can be used for introducing a polynucleotide into a plant and for expressing this polynucleotide constitutively or tissue-specifically. The introduction of DNA into a plant can be performed by a method that is publicly known to a person skilled in the art, for example, the agrobacterium method, binary vector method, electroporation method, PEG method, or particle gun method.
Plants that can be used as hosts in the present invention are not particularly limited but plants of the genus Rosa in the family Rosaceae, the genus Chrysanthemum in the family Asteraceae, and the genus Dianthus in the family Caryophyllaceae (e.g. carnation) can be used, and particularly preferable is the cultivated rose (scientific name: Rosa hybrida) of the genus Rosa in the family Rosaceae. Note that the term “rose plant” used herein refers to the cultivated rose (scientific name: Rosa hybrida) of the genus Rosa in the family Rosaceae. Roses are mainly divided into hybrid tea, floribunda, polyantha, etc., but there are only two types of the main pigment (anthocyanin) contained in the petals of each of the strains, namely cyanidin and pelargonidin. The variety of rose plant used as the host in the present invention is not particularly limited and these varieties and strains can be used as appropriate. For example, rose varieties that can be used as the host can include Ocean Song, Noblesse, Rita Perfumera, Cool Water, Fame, Topless, Peach Avalanche, etc.
According to the present invention a transformed plant having blue-hued flower color, wherein delphinidin-type anthocyanin and a flavone C-glycoside coexist within the cells can be obtained, the transformed plant preferably being of the genus Rosa in the family Rosaceae, the genus Chrysanthemum in the family Asteraceae, and the genus Dianthus in the family Caryophyllaceae (e.g. carnation), and particularly preferably the rose plant. The obtained transformed plant has a flower color belonging to the Blue group or Violet-Blue group of the RHS color chart and/or a hue angle of 339.7° to 270.0° in the CIE L*a*b* color system.
The present invention also relates: to a cut flower of the transformed plant obtained above, or an inbred or outbred progeny thereof; a propagule thereof; a part of the plant; tissue; or cell; or a processed product produced from the cut flower (particularly processed cut flowers).
The processed cut flowers include pressed flowers, preserved flowers, dried flowers, resin-sealed products, etc. but are not limited thereto.
The present invention is specifically described in the following examples.
Anthocyanin and flavone C-glycoside were prepared so as to perform a simulation of the copigment effect between anthocyanin and flavone C-glycoside. The malvin (malvidin 3,5-diglucoside) and isovitexin (apigenin 6-C-glucoside) used in this experiment were purchased from Funakoshi Corporation.
With respect to the anthocyanin (malvin) obtained thereby, flavone C-glycoside (isovitexin) was added at molar concentration ratios of 0, 2 and 4 equivalents in a pH 4.5 buffer and the absorption spectrum was measured. The concentration of the anthocyanin was made to be 0.5 mM.
By adding the flavone C-glycoside, the absorbance of the aqueous anthocyanin solution increased and the maximum absorbance (λ max) shifted to a longer wavelength with the addition of flavone C-glycoside. This confirms that isovitexin exhibits a copigment effect when combined with malvin.
pSPB4743 has the basic skeleton of pBINPLUS and contains the following 4 expression cassettes.
(1) El235S promoter, pansy-derived F3′S′H, full length cDNA (SEQ ID NO:1) and D8 terminator
(2) 35S promoter, Glycyrrhiza-derived F2H, full length cDNA (SEQ ID NO: 3), and perilla-derived AT terminator
(3) 35S promoter, Oryza sativa-derived CGT, full length cDNA (SEQ ID NO: 5), and perilla-derived AT terminator
(4) 35S promoter, Glycyrrhiza-derived FDH, full length cDNA (SEQ ID NO. 7), and perilla-derived AT terminator
This plasmid constitutively expresses the pansy F3′5H #40 gene, the Glycyrrhiza F2H gene, the Oryza sativa CGT gene, and the Glycyrrhiza FDH gene in the plant.
The pSPB4743 constructed in this manner was introduced into an orange rose variety “Rita Perfumera” and a total of 16 transformants were obtained. Pigment analysis was carried out thereon and 15 were confirmed to accumulate delphinidin, and the maximum delphinidin content thereof was 94% (average 89.5%). Furthermore, in 10 of the transformants the flavone C-glycoside isovitexin was confirmed and the maximum amount produced per gram of fresh petals was 0.55 mg.
Analyzed values of the transformants are shown below in Table 2
pSPB4743, which was constructed in the same manner as for Example 2, was introduced into a pink rose variety “Noblesse” and a total of 20 transformants were obtained. Pigment analysis was carried out thereon and all were confirmed to accumulate delphinidin, and the maximum delphinidin content thereof was 88% (average 83.5%). Furthermore, in 18 of the transformants the flavone C-glycoside isovitexin was confirmed and the maximum amount produced per gram of fresh petals was 0.06 mg.
Representative analysis values of the transformants are shown below in Table 3.
pSPB6188 has the basic skeleton of pBINPLUS and contains the following 4 expression cassettes.
(1) El235S promoter, Campanula-derived F3′5′H, full length cDNA (SEQ ID NO:9) and D8 terminator
(2) 35S promoter, Glycyrrhiza-derived F2H, full length cDNA (SEQ ID NO: 3), and perilla-derived AT terminator
(3) El235S promoter, Oryza sativa-derived CGT, full length cDNA (SEQ ID NO:5) and a perilla-derived AT terminator
(4) El235S promoter, Oryza sativa-derived FDH, full length cDNA (SEQ ID NO:11) and Arabidopsis-derived HSP terminator
This plasmid constitutively expresses the Campanula F3′5′H gene, the Glycyrrhiza F2H gene, the Oryza sativa CGT gene, and the Oryza sativa FDH gene in the plant.
The pSPB6188 constructed in this manner was introduced into an orange rose variety “Rita Perfumera” and a total of 77 transformants were obtained. Pigment analysis was carried out thereon and 68 were confirmed to accumulate delphinidin, and the maximum delphinidin content thereof was 99.6% (average 93.3%). Furthermore, in 57 of the transformants, the flavone C-glycoside isovitexin was confirmed and the maximum amount constructed per gram of fresh petals was 0.72 mg.
Representative analysis values of the transformants are shown below in Table 4.
pSPB6188, which was constructed in the same manner as for Example 4, was introduced into a pink rose variety “Noblesse” and a total of 51 transformants were obtained. Pigment analysis was carried out thereon and all were confirmed to accumulate delphinidin, and the maximum delphinidin content thereof was 99.7% (average 66.9%). Furthermore, in 48 of the transformants the flavone C-glycoside isovitexin was confirmed and the maximum amount produced per gram of fresh petals was 0.58 mg.
Representative analysis values of the transformants are shown below in Table 5.
pSPB5588 has the basic skeleton of pBINPLUS and contains the following 4 expression cassettes.
(1) El235S promoter, pansy-derived F3′S′H, full length cDNA (SEQ ID NO:1) and D8 terminator
(2) 35S promoter, Campanula-derived F2H, full length cDNA (SEQ ID NO: 3), and perilla-derived AT terminator
(3) 35S promoter, Oryza sativa-derived codon-usage modified CGT, full length cDNA (SEQ ID NO:13) and an Arabidopsis-derived HSP terminator
(4) 35S promoter, Lotus japonicus-derived FDH, full length cDNA (SEQ ID NO:15) and Arabidopsis-derived HSP terminator
This plasmid constitutively expresses the pansy F3′5′H #40 gene, the Glycyrrhiza F2H gene, the Oryza sativa codon-usage modified CGT gene, and the Lotus japonicus FDH gene in the plant.
The pSPB5588 constructed in this manner was introduced into an orange rose variety “Rita Perfumera” and a total of 92 transformants were obtained. Pigment analysis was carried out on 65 thereof and 44 were confirmed to accumulate delphinidin, and the maximum delphinidin content thereof was 100% (average 62.3%). Furthermore, in 37 of the transformants the flavone C-glycoside isovitexin was confirmed and the maximum amount produced per gram of fresh petals was a high content of 2.02 mg.
Representative analysis values of the transformants are shown below in Table 6.
pSPB5588, which was constructed in the same manner as for Example 4, was introduced into an orange rose variety “Noblesse” and a total of 60 transformants were obtained. Pigment analysis was carried out thereon and 42 were confirmed to accumulate delphinidin, and the maximum delphinidin content thereof was 96.9% (average 54.4%). Furthermore, in 29 of the transformants, the flavone C-glycoside isovitexin was confirmed and the maximum amount produced per gram of fresh petals was a high content of 1.60 mg.
Representative analysis values of the transformants are shown below in Table 7.
pSPB6486 has the basic skeleton of pBINPLUS and contains the following 5 expression cassettes.
(1) El235S promoter, Campanula-derived F3′5′H, full length cDNA (SEQ ID NO:9) and D8 terminator
(2) El235S promoter, Torenia-derived MT, full length cDNA (SEQ ID NO: 17), and NOS terminator
(3) 35S promoter, Glycyrrhiza-derived F2H, full length cDNA (SEQ ID NO: 3) and a perilla-derived AT terminator
(4) 35S promoter, Oryza sativa-derived codon-usage modified CGT, full length cDNA (SEQ ID NO:13) and Arabidopsis-derived HSP terminator
(5) 35S promoter, Lotus japonicus-derived FDH, full length cDNA (SEQ ID NO:15) and Arabidopsis-derived HSP terminator
This plasmid constitutively expresses the Campanula F3′5′H gene, the Torenia MT gene, the Glycyrrhiza F2H gene, the Oryza sativa codon-usage modified CGT gene, and the Lotus japonicus FDH gene in the plant.
The pSPB6486 constructed in this manner was introduced into a blue-hued rose variety “Ocean song” and a total of 27 transformants were obtained. Pigment analysis was carried out thereon and 26 were confirmed to accumulate malvidin, and the maximum malvidin content thereof was 74.5% (average 57.0%). Furthermore, in this system, in addition to the isovitexin analyzed so far as the flavone C-glycoside, vitexin (apigenin 8-C-glucoside), vicenin-2 (apigenin 6,8-C diglucoside), isoorientin (luteolin 6-C-glucoside) and orientin (luteolin 8-C-glucoside) were also identified and quantified. Flavone C-glycoside could be detected in all of the transformants in which malvidin was detected and from the total amount thereof, the maximum amount produced per gram of fresh petals was a high content of 1.563 mg. Furthermore, in most transformants, the total amount of flavone C-glycoside was a high content of 1 mg or more per gram of fresh petals which was approximately 10 times or more than the amount of malvidin produced.
Representative analysis values of the transformants are shown below in Table 8.
pSPB6438 has the basic skeleton of pBINPLUS and contains the following 4 expression cassettes.
(1) El235S promoter, pansy-derived F3′5′H, full length cDNA (SEQ ID NO: 1) and NOS terminator
(2) El235S promoter, Torenia-derived MT gene, full length cDNA (SEQ ID NO: 17) and NOS terminator
(3) El235S promoter, Torenia-derived FNS gene, full length cDNA (SEQ ID NO: 19) and D8 terminator
(4) El235S promoter, Gentiana scabra-derived CGT, full length cDNA (SEQ ID NO: 21) and Arabidopsis-derived HSP terminator
This plasmid constitutively expresses the pansy F3′5′H #40 gene, the Torenia MT gene, the Torenia FNS gene, and the Gentiana scabra CGT gene.
The pSPB6438 constructed in this manner was introduced into an orange rose variety “Rita Perfumera” and a total of 122 transformants were obtained. Pigment analysis was carried out thereon and 71 transformants were confirmed to accumulate malvidin, and the maximum malvidin content thereof was 69.6% (average 25.9%). Furthermore, in this system, in addition to the isovitexin analyzed so far as the flavone C-glycoside, vitexin (apigenin 8-C-glucoside) and vicenin-2 (apigenin 6,8-C diglucoside) were also identified and quantified. Of the transformants in which malvidin could be detected, flavone C-glycoside could be confirmed in 16, and from the total amount thereof, the maximum amount produced per gram of fresh petals was 0.02 mg. In contrast thereto, the total amount of flavones (apigenin, luteolin, and tricetin) was a high content of 2.07 mg per gram of fresh petals.
Representative analysis values of the transformants are shown below in Table 9.
pSPB7013 has the basic skeleton of pBINPLUS and contains the following 4 expression cassettes.
(1) El235S promoter, Campanula-derived F3′5′H, full length cDNA (SEQ ID NO:9) and D8 terminator
(2) El235S promoter, Torenia-derived MT, full length cDNA (SEQ ID NO: 17) and NOS terminator
(3) El235S promoter, Torenia-derived FNS, full length cDNA (SEQ ID NO: 19) and a D8 terminator
(4) El235S promoter, Gentiana scabra-derived CGT, full length cDNA (SEQ ID NO:21) (Arabidopsis ADH gene-derived 5′-UTR (SEQ ID NO: 23) added to 5′ end) and Arabidopsis-derived HSP terminator
This plasmid constitutively expresses the Campanula F3′5′H gene, the Torenia MT gene, the Torenia FNS gene, and the Gentiana scabra CGT gene in plants.
The pSPB7013 constructed in this manner was introduced into a blue-hued rose variety “Ocean Song” and a total of 15 transformants were obtained. Pigment analysis was carried out thereon and all transformants were confirmed to accumulate malvidin, and the maximum malvidin content thereof was 67.2% (average 40.9%). Furthermore, in this system, in addition to the isovitexin, vitexin, and vicenin-2 analyzed so far as the flavone C-glycoside, isoorientin (luteolin 6-C-glucoside) and orientin (luteolin 8-C-glucoside) were also identified and quantified. Flavone C-glycoside could be detected in all of the transformants in which malvidin was detected, and from the total amount thereof, the maximum amount produced per gram of fresh petals was a high content of 1.410 mg.
Representative analysis values of the transformants are shown below in Table 10.
The transformants (rose variety “Ocean Song” used as host) created in Examples 8 and 10 were categorized into groups, wherein: (1) delphinidin had accumulated as the main pigment but flavone was not present; (2) malvidin had accumulated as the main pigment and flavone C-glycoside created through pathway 1 was present; (3) malvidin had accumulated as the main pigment and flavone C-glycoside created through pathway 2 was present; and the host (wherein cyanidin had accumulated as the main pigment). The color of each of the petals thereof were measured using a spectrophotometer CM-2022 (Minolta Co., Ltd) with a 10-degree field of view and a D65 light source and analyzed using the color management software SpectraMagic™ (Minolta Co., Ltd.) (n=5).
Even in rose types in which the main pigment was delphinidin, the hue angle of the petals shifted towards blue. Furthermore, when flavone C-glycoside was made to coexist in rose types in which the main pigment was malvidin, this tendency was even more remarkable and the hue angle also greatly shifted to the blue side. The system of Example 10 was found to exhibit such a remarkable tendency. From the results above, it was confirmed that the color of petals turned more blue due to the coexistence of malvidin and flavone C-glycoside.
The results are shown in Table 11.
Torenia MT + Glycyrrhiza
Lotus japonicus FDH
Gentiana scabra CGT
pSPB6495 has the basic skeleton of pBINPLUS and contains the following 5 expression cassettes.
(1) El235S promoter, Campanula-derived F3′5′H, full length cDNA (SEQ ID NO: 9) and D8 terminator
(2) El235S promoter, lavender-derived 3AT, full length cDNA (SEQ ID NO: 24) and Arabidopsis-derived HSP terminator
(3) 35S promoter, Glycyrrhiza-derived F2H gene, full length cDNA (SEQ ID NO: 3) and Perilla-derived AT terminator
(4) 35S promoter, Oryza sativa-derived codon-usage modified CGT, full length cDNA (SEQ ID NO: 13) and Arabidopsis-derived HSP terminator
(5) 35S promoter, Lotus japonicus-derived FDH, full length cDNA (SEQ ID NO: 15) and Arabidopsis-derived HSP terminator
This plasmid constitutively expresses the Campanula F3′,5′H gene, the Lavender 3AT gene, the Glycyrrhiza F2H gene, the Oryza sativa codon-usage modified CGT gene, and the Lotus japonicus FDH gene in the plant.
The pSPB6495 constructed in this manner was introduced into a blue rose variety “Ocean song” and a total of 228 transformants were obtained. Pigment analysis was carried out thereon and 59 transformants were confirmed to accumulate acylated delphinidin. Furthermore, in this system, in addition to the isovitexin analyzed so far as the flavone C-glycoside, vitexin (apigenin 8-C-glucoside), vicenin-2 (apigenin 6,8-C-diglucoside), isoorientin (luteolin 6-C-glucoside), and orientin (luteolin 8-C-glucoside) were also identified and quantified. Flavone C-glycoside could be detected in all of the transformants in which acylated delphinidin was detected. There was a transformant having a high maximum amount thereof as high as from the total amount thereof, the maximum amount produced per gram of fresh petals included a transformant with a high content of 1.720 mg, but the average value was 0.833 mg.
Representative analysis values of the transformants are shown below in Table 12.
pSPB7189 has the basic skeleton of pBINPLUS and contains the following 5 expression cassettes.
(1) El235S promoter, Campanula-derived F3′S′H, full length cDNA (SEQ ID NO:9) and NOS terminator
(2) El235S promoter, Clitoria ternatea-derived A3'S′GT, full length cDNA (SEQ ID NO: 26) and Arabidopsis-derived HSP terminator
(3) El235S promoter, Rose-derived 53GT, full length cDNA (SEQ ID NO: 28) (RNAi), and Arabidopsis-derived HSP terminator
(4) SAT promoter and Perilla-derived 3GT, full length cDNA (SEQ ID NO: 30), and Arabidopsis-derived HSP terminator
(5) El235S promoter, Dahlia-derived 3MaT, full length cDNA (SEQ ID NO: 32), and Arabidopsis-derived HSP terminator
(6) 35S promoter, Glycyrrhiza-derived F2H, full length cDNA (SEQ ID NO: 3), and Perilla-derived AT terminator
(7) 35S promoter, Oryza sativa-derived codon-usage modified CGT, full length cDNA (SEQ ID NO: 13), and Arabidopsis-derived HSP terminator
(8) 35S promoter, Lotus japonicus-derived FDH, full length cDNA (SEQ ID NO: 15), and Arabidopsis-derived HSP terminator.
This plasmid constitutively expresses the Campanula F3′5′H gene, the Clitoria ternatea A3′,5′GT gene, the Perilla 3GT gene, the Dahlia 3MaT gene, the Glycyrrhiza F2H gene, the Oryza sativa codon-usage modified CGT gene, and the Lotus japonicus FDH gene, and inhibits expression of the endogenous Rose 5,3GT gene in plants.
The pSPB7189 constructed in this manner was introduced into a blue-hued rose variety “Ocean Song” and a total of 101 transformants were obtained. Pigment analysis was carried out thereon and only 1 transformant was confirmed to accumulate delphinidin, and acylation thereof was not confirmed. Furthermore, in this system, in addition to the isovitexin used so far as the flavone C-glycoside, vitexin (apigenin 8-C-glucoside), vicenin-2 (apigenin 6,8-C-diglucoside), isoorientin (luteolin 6-C-glucoside), and orientin (luteolin 8-C-glucoside) were also identified and quantified. Flavone C-glycoside could be detected in this transformant, and from the total amount thereof, the amount produced per gram of fresh petals was a high content of 1.024 mg. Analyzed values of the transformants are shown below in Table 13.
The transformants (rose variety “Ocean Song” used as host) created in Examples 12 and 13 were categorized into groups, wherein: (1) delphinidin (a portion of which was acylated) had accumulated as the main pigment and flavone C-glycoside had been created through pathway 1; and (2) delphinidin had accumulated as the main pigment and flavone C-glycoside had been created through pathway 1. The color of each petal was measured using a spectrophotometer CM-2022 (Minolta Co., Ltd) with a 10-degree field of view and a D65 light source and analyzed using the color management software SpectraMagic™ (Minolta Co., Ltd.) (n=5).
For rose types in which the main pigment was delphinidin, even if a portion thereof was acylated, the hue angle of the petals was not confirmed to shift towards blue compared to transformants created in Examples 8 and 10 (however, there was a greater blue shift than for acylated anthocyanin alone). From the results above, it was confirmed that the color of petals turned more blue due to the coexistence of malvidin and flavone C-glycoside.
The results are shown in Table 14.
Oryza sativa CGT (codon-usage
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-193480 | Oct 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/036935 | 10/2/2018 | WO | 00 |
