JP6945042B2 - Method for producing sesaminol disaccharide or trisaccharide glycoside, and novel sesaminol glycoside - Google Patents

Description

The present invention is a method for producing a sesaminol disaccharide or trisaccharide glycoside, sesaminol 2'-O-β-D-glucosyl (1 → 2) -O- [β-D-glucosyl (1 → 2)]. The present invention relates to a method for selecting -β-D-glucosides, proteins, polynucleotides, expression vectors, transformants and plants.

Sesamum sesame seeds are rich in lignans, a secondary metabolite of the phenylpropanoid family. Its representative, sesamin, has low polarity and various biological activities have been reported, and it is sold as a health food in Japan. Sesaminol has a hydroxyl group in which sesamin is oxidized, glucose is added to this hydroxyl group, and glucose is added to the 2nd and 6th positions of the glucose residue, respectively. Accumulated as -β-D-glucosyl (1 → 2) -O- [β-D-glucosyl (1 → 6)]-β-D-glucoside (hereinafter referred to as SG3 (2,6)) (Non-Patent Document 1). These are more water-soluble than sesamin aglycone, and are expected to have different activities due to differences in physical characteristics, and can be added to beverages and the like due to their high water solubility.

So far, sesame-derived UDP-glucosyltransferase (UDP) has been used as a protein that catalyzes the reaction of glucosylating sesaminol to produce sesaminol 2'-O-β-D-glucoside (hereinafter referred to as SG1). -Sugar dependent protein glycosyltransferase; UGT) UGT71A9 and SG1 glucose 6-position are glucosylated, and sesaminol 2'-O-β-D-glucosyl (1 → 6) -β-D-glucoside (hereinafter SG2 (6) ) Has been reported (Patent Document 1, Non-Patent Document 1). A protein that specifically transfers glucose to the sugar of a sesaminol glycoside, such as UGT94D1, is referred to as a glycoside-specific glycosyltransferase (hereinafter, also referred to as "GGT"). There is. Also, various GTTs have a common amino acid sequence that is well conserved across species. However, it has been difficult to estimate the position where the sugar acceptor compound or sugar is added from the amino acid sequence information.

In addition, the 2-position of the glucose residue of SG1 or the 2-position of the first glucose residue bound to sesaminol of SG2 (6) is actually glucosylated to sesaminol 2'-O-β. No protein producing -D-glucosyl (1 → 2) -β-D-glucoside (hereinafter referred to as SG2 (2)) or SG3 (2,6) was found.

Japanese Unexamined Patent Publication No. 2006-129728

Noguchi, A. , Et al (2008) Sequantial glucosylation of a furofuran lignan, (+)-sesaminol, by Sesamum indicum UGT71A9 and UGT94D1 glucosyltrans. Plant J. 54, 415-427.

An object of the present invention is to provide a method for producing a sesaminol disaccharide or trisaccharide glycoside by imparting a sugar to a predetermined position of a sugar residue of a sesaminol disaccharide or a disaccharide glycoside. ..

As a result of diligent research to solve the above problems, the present inventor has the second position of the glucose residue of SG1, the second position of the first glucose residue of SG2 (6), and the 2nd position of SG2 (2). Glycoside the 2nd position of the second glucose residue, SG2 (2), SG3 (2,6), and sesaminol 2'-O-β-D-glucosyl (1 → 2) -O- [ β-D-glucosyl (1 → 2)]-β-D-glucoside (hereinafter referred to as SG3 (2,2)) is produced by a sesaminol glycoside-specific glycoside enzyme (hereinafter, "sesaminol GGT"). By discovering (also described as) and using this protein, sugar is added to a predetermined position of the sugar residue of the sesaminol disaccharide or disaccharide glycoside, and the sesaminol disaccharide or trisaccharide glycoside is added. The present invention was completed by finding that it can be produced.

That is, the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention is a method for producing a sesaminol disaccharide or trisaccharide glycoside, which is represented by the following general formula (1). Alternatively, the step (i) of allowing at least one protein (P1) selected from the group consisting of the following proteins (P1-1) to (P1-4) to act on the disaccharide glycoside and the UDP-sugar is included. It is characterized by.
(P1-1) Protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2 (P1-2) Amino acid sequence in which one or several amino acids are deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 1 or 2. A protein (P1-3) SEQ ID NO: consisting of and having a β-1 → 2 binding glycosyltransferase activity to a hexose residue of a sesaminol monosaccharide or disaccharide glycoside represented by the following general formula (1). A sesaminol monosaccharide or disaccharide glycoside consisting of an amino acid sequence having 90% or more identity with respect to the amino acid sequence of 1 or 2 and represented by the following general formula (1) to a hex source residue. Encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 4 of a protein (P1-4) having β-1 → 2 binding glycosyl transfer activity. A protein consisting of the amino acid sequence to be used and having a β-1 → 2 binding glycosyltransferase activity to the hexose residue of the sesaminol monosaccharide or disaccharide glycoside represented by the following general formula (1).

[In the general formula (1), (S1) represents a hexose residue, and n represents a number of 1 or 2. The first hexose residue (S1) bound to sesaminol is bound to sesaminol via the carbon at position 1. When n is 2, the two hexose residues (S1) may be the same or different, and these are β-1 → 2 bonds or β-1 → 6 bonds. ]
In the present specification, β-1 → 2 bond and β-1 → 6 bond are O-glycosidic bonds.

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the UDP-sugar is preferably UDP-hexose, more preferably UDP-glucose.

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the above (S1) is preferably a D-glucose residue.

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, β-1 → 2 binding to a hexose residue of the sesaminol 1 sugar or disaccharide glycoside represented by the above general formula (1) The glycosyl transfer activity is the glucose transfer activity (I) of β-1 → 2 bond to the glucose residue of SG1, and the glucose transfer activity of β-1 → 2 bond to the first glucose residue of SG2 (6). It is preferable that any one or more of (II) and the glucose transfer activity (III) of β-1 → 2 binding to the second glucose residue of SG2 (2).

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the sesaminol 1 sugar or disaccharide glycoside represented by the above general formula (1) is SG1 represented by the following formula (2). The UDP-sugar is UDP-glucose, and it is preferable that SG2 (2) represented by the following formula (3) is produced by the glucose transfer of β-1 → 2 bond to the glucose residue of SG1.

In the method for producing a sesaminol disaccharide or a trisaccharide glycoside of the present invention, SG2 (2) and UDP-glucose represented by the above formula (3) are composed of the following proteins (P2-11) to (P2-18). It is preferable to include a step (ii) in which at least one protein (P2) selected from the group is allowed to act to produce SG3 (2,6) represented by the following formula (4).
(P2-11) Protein consisting of the amino acid sequence of SEQ ID NO: 5 (P2-12) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 5 and , Sesaminol 2'-O-β-D-glucosyl (1 → 12) -β-D-glucoside protein with β-1 → 6 binding glucose transfer activity to the first glucose residue (P2- 13) Glucose transfer of β-1 → 6 bond to the first glucose residue of SG2 (2), which consists of an amino acid sequence having 90% or more identity with respect to the amino acid sequence of SEQ ID NO: 5. An amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the active protein (P2-14) SEQ ID NO: 6 and SG2 ( 2) Amino acid sequence of protein (P2-16) SEQ ID NO: 7 consisting of the amino acid sequence of protein (P2-15) SEQ ID NO: 7 having β-1 → 6 binding glucose transfer activity to the first glucose residue Consists of an amino acid sequence in which one or several amino acids have been deleted, substituted and / or added, and the β-1 → 6 binding glucose transfer activity of SG2 (2) to the first glucose residue. Protein (P2-17) consisting of an amino acid sequence having 90% or more identity with respect to the amino acid sequence of SEQ ID NO: 7 and β-1 to the first glucose residue of SG2 (2). → Protein with 6-binding glucose transfer activity (P2-18) From the amino acid sequence encoded by the polynucleotide that hybridizes under stringent conditions with the polynucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 8. A protein that has β-1 → 6 binding glucose transfer activity to the first glucose residue of SG2 (2).

In the method for producing a sesaminol disaccharide or a trisaccharide glycoside of the present invention, the above protein (P1) is allowed to act on SG2 (2) and UDP-glucose represented by the above formula (3), and the following formula (5) is obtained. It is preferable to further include a step of producing the indicated SG3 (2,2).

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the sesaminol disaccharide or disaccharide glycoside represented by the above general formula (1) is SG2 (6) represented by the following formula (6). ), And the UDP-sugar is UDP-glucose, which is represented by the above formula (4) by the glucose transfer of β-1 → 2 bond to the first glucose residue of SG2 (6). It is preferable to generate SG3 (2,6).

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the sesaminol 1 sugar or disaccharide glycoside represented by the above general formula (1) is SG2 (2) represented by the above formula (3). ), And the UDP-sugar is UDP-glucose, which is represented by the above formula (5) by the glucose transfer of β-1 → 2 bond to the second glucose residue of SG2 (2). It is preferable to generate SG3 (2,2).

SG3 (2,2) represented by the above formula (5) is also one of the present inventions.

The protein of the present invention is characterized by comprising the amino acid sequence of SEQ ID NO: 2.
The polynucleotide of the present invention is characterized by comprising the nucleotide sequence of SEQ ID NO: 4.
The expression vector of the present invention is characterized by containing the above-mentioned polynucleotide of the present invention.

The transformant of the present invention is a transformant which is a non-human host into which an expression vector has been introduced, and the expression vector is the following polynucleotides (N1-1) to (N1-4) and a polynucleotide (N2). It is characterized by containing at least one polynucleotide selected from the group consisting of -1) to (N2-4).
(N1-1) Nucleotides consisting of the nucleotide sequence of SEQ ID NO: 3 or 4 (N1-2) Nucleotides in which one or several nucleotides are deleted, substituted and / or added to the nucleotide sequence of SEQ ID NO: 3 or 4. It is a polynucleotide having a sequence, and the transglycosylation activity of β-1 → 2 binding to the first hexose residue of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1). Nucleotide encoding the protein having (N1-3) A nucleotide having a nucleotide sequence having 90% or more identity with respect to the nucleotide sequence of SEQ ID NO: 3 or 4, and in the above general formula (1). Nucleotide (N1-4) SEQ ID NO: 3 or 4 encoding a protein having β-1 → 2 binding glycotransfer activity to the first hexose residue of the sesaminol monosaccharide or disaccharide glycoside shown. A sesaminol monosaccharide or disaccharide glycoside consisting of a nucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of No. 1 and a polynucleotide that hybridizes under stringent conditions and represented by the above general formula (1). Polynucleotide (N2-2) SEQ ID NO: 6 consisting of the nucleotide sequence of nucleotide (N2-1) SEQ ID NO: 6 encoding a protein having β-1 → 2 binding glycosyl transfer activity to the first hexsource residue. A polynucleotide having a nucleotide sequence in which one or several nucleotides are deleted, substituted, and / or added to the nucleotide sequence of the above, and sesaminol 1 sugar or 2 sugars represented by the above general formula (1). 90% or more identical to the nucleotide sequence of nucleotide (N2-3) SEQ ID NO: 6 encoding a protein having β-1 → 6 binding glycosyl transfer activity to the first hex source residue of the sugar body A polynucleotide having a nucleotide sequence having sex, and β-1 → 6 binding to the first hexsource residue of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1). Nucleotide encoding a protein having transglycosylation activity (N2-4) Consists of a nucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6, and a polynucleotide that hybridizes under stringent conditions, and described above. A polynucleotide encoding a protein having a β-1 → 6-binding glycosyltransferase activity to the first hexose residue of a sesaminol monosaccharide or disaccharide glycoside represented by the general formula (1).

In the transformant of the present invention, the non-human host is preferably a plant or a portion thereof, and more preferably a sesame plant or a portion thereof.

The method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention is characterized by comprising a step of cultivating or culturing the transformant of the present invention.

The method for selecting plants of the present invention uses a polynucleotide containing at least a part of the nucleotide sequence of SEQ ID NO: 3, 4 or 6, and plants having different sesaminol disaccharide or trisaccharide glycoside synthesizing ability. It is characterized by sorting the body.

According to the present invention, by using a protein (sesaminol GGT) that transfers a sugar from β-1 to 2 to a hexose residue of a sesaminol glycoside, a predetermined position of the sesaminol monosaccharide or disaccharide glycoside It is possible to produce a sesaminol disaccharide or trisaccharide glycoside having a predetermined structure by transferring the sugar to.
Further, according to the present invention, a novel sesaminol trisaccharide glycoside can be provided.

FIG. 1 is a reaction diagram schematically showing a route for synthesizing SG3 (2,6) or SG3 (2,2). FIG. 2A is a photograph of agarose gel electrophoresis of a PCR product obtained using a sesame seed cDNA as a template and a forward primer for protein SiGGT21842 and a reverse primer for protein SiGGT21842, and FIG. 2B is a sesame gel electrophoresis. It is a photograph of agarose gel electrophoresis of a PCR product obtained by using a forward primer for protein SiGGT21646 and a reverse primer for protein SiGGT21646 using seed cDNA as a template. FIG. 3A is a chart of reverse phase LC-MS analysis of the reaction solution using SG1 and the purified protein SiGGT21842 solution, and FIG. 3B is a standard reverse phase LC of SG2 (2). -MS analysis chart, FIG. 3 (c) is a chart of reverse phase LC-MS analysis of the reaction solution using SG1 and the negative control fraction. FIG. 4A is a chart of reverse phase LC-MS analysis of the reaction solution using SG2 (2) and the purified protein SiGGT21842 solution, and FIG. 4B is a chart of SG2 (2) and a negative control image. It is a chart of the reverse phase LC-MS analysis of the reaction solution using a minute. FIG. 5 (a) is a chart of reverse phase LC-MS analysis of the reaction solution using SG2 (6) and the purified protein SiGGT21842 solution, and FIG. 5 (b) is SG3 (2, 6) and negative. It is a chart of the reverse phase LC-MS analysis of the reaction solution using the control fraction, and FIG. 5 (c) shows the reverse phase LC-MS analysis of the reaction solution using SG2 (6) and the negative control fraction. It is a chart. FIG. 6A is a chart of reverse phase LC-MS analysis of the negative control reaction solution of Example 4, and FIG. 6B is a reverse phase of the reaction solution of the first reaction of Example 4. It is a chart of LC-MS analysis, and FIG. 6C is a chart of reverse phase LC-MS analysis of the reaction solution of the second reaction of Example 4. FIG. 7 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 1. FIG. 8 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 2. FIG. 9 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 3. FIG. 10 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 4. FIG. 11 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 5. FIG. 12 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 6. FIG. 13 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 7.

Hereinafter, the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention will be described while showing specific embodiments. However, the present invention is not limited to the following embodiments, and can be appropriately modified and applied without changing the gist of the present invention.

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, a protein (P1) described later is added to the sesaminol disaccharide or disaccharide glycoside represented by the following general formula (1) and UDP-sugar. The step (i) of acting is included. In the present specification, the sesaminol monosaccharide or disaccharide glycoside represented by the following general formula (1) is also referred to as "sesaminol glycoside (1)". The method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention may include one or more steps other than the above step (i) as long as the effects of the present invention are not impaired. For example, the step of preparing the sesaminol glycoside (1) which is the substrate of the step (i) and the step of purifying the obtained sesaminol disaccharide or trisaccharide glycoside may be included. Further, for example, when the sesaminol 2 or trisaccharide glycoside obtained in step (i) is a sesaminol disaccharide glycoside, the glycoside and UDP-sugar are further added to the protein (P1) or described later. It may include a step of allowing the protein (P2) to act.

[In the general formula (1), (S1) represents a hexose residue, and n represents a number of 1 or 2. The first hexose residue (S1) bound to sesaminol is bound to sesaminol via the carbon at position 1. When n is 2, the two hexose residues (S1) may be the same or different, and these are β-1 → 2 bonds or β-1 → 6 bonds. ]

(S1) in the above general formula (1) is a hexose residue, and examples of the hexose residue include residues such as glucose, allose, altrose, mannose, gulose, idose, galactose, and talose. May be D-type or L-type, but is preferably D-type. Of these, it is more preferably a glucose residue, and even more preferably a D-glucose residue.
n is the number of hexose residues (S1), which is 1 or 2.

In the present specification, the "first hexose residue (S1)" in the sesaminol glycoside (1) means the hexose residue (S1) bound to the hydroxyl group of sesaminol. Further, the hexose residue (S1) bound to the "first hexose residue (S1)" is referred to as "second hexose residue (S1)" or the like. When n is 1, the sesaminol glycoside (1) has one hexose residue. In other words, when n is 1, the first hexose residue of the sesaminol glycoside (1) is the hexose residue of the glycoside. When n is 2, that is, when two hexose residues (S1) are bound, the first (S1) and the second (S1) are β-1 → 2 bonds or β. -1 → 6 are combined.

When one hexose residue (S1) is bound to sesaminol as the sesaminol glycoside (1) (when n is 1), SG1 of the above general formula (2) is preferable.
The sesaminol monosaccharide or disaccharide glycoside represented by the general formula (1), which is the substrate of the step (i), can be used alone or in combination of two or more.

When two hexose residues (S1) are bound to sesaminol as the sesaminol glycoside (1) (when n is 2), SG2 (2) of the above formula (3) or the above SG2 (6) of the formula (6) is preferable.
As the sesaminol glycoside (1), it is preferable to use at least one selected from the group consisting of SG1, SG2 (2) and SG2 (6).

The sesaminol glycoside (1) is contained in sesame seeds and can be obtained by purification by a known method (for example, "J Agricult Food Chem. 2006 Feb 8; 54 (3): 633". -8. HPLC analysis of sesame minerals in sesame seeds. Moazzami AA1, Anderson RE, Kamal-Eldin A. ”). Further, for example, SG1 is UGT71A9 described in Patent Document 1 (Japanese Patent Laid-Open No. 2006-129728) or Non-Patent Document 1 (Noguchi, A., et al (2008) Plant J. 54, 415-427.). Can be obtained by acting on sesaminol and UDP-glucose. SG2 (2) can be obtained by further acting UGT94D1 described in Non-Patent Document 1. In addition, the sesaminol glycoside (1) can also be chemically synthesized.
Further, as such a sesaminol monosaccharide or disaccharide glycoside, a commercially available product can also be used. Examples of such commercially available products include product number: NS185201, manufacturer: Nagara Science Co., Ltd. (commercially available product of SG2 (2)), product number: NS185301, and manufacturer: Nagara Science Co., Ltd. (commercially available product of SG2 (6)). ) Etc.

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the above-mentioned UDP (uridine diphosphate) -sugar is not particularly limited, and examples thereof include UDP-pentose, UDP-hexose, and UDP-heptose. One or two or more types of UDP-sugar can be used.
Examples of UDP-pentose include UDP-arabinose and UDP-xylose.
Examples of the UDP-hexose include UDP-glucose, UDP-allose, UDP-altrose, UDP-mannose, UDP-gulose, UDP-idose, UDP-galactose, and UDP-talose.
Examples of UDP-heptulose include UDP-sedoheptulose and the like.
Of these, UDP-hexose is preferred, and UDP-glucose is even more preferred.
When the UDP-sugar is UDP-glucose, the substrate specificity with the protein (P1) in the present invention is high, the synthesis rate of the sesaminol disaccharide or the trisaccharide glycoside by the protein (P1) is increased, and the efficiency is increased. Sesaminol disaccharide or trisaccharide glycoside can be produced.

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the protein (P1) has a β-1 → 2 binding glycosyl transfer activity to the hexose residue of the sesaminol glycoside (1). Sesaminol GGT. This transglycosylation activity is usually UDP-sugar-specific transglycosylation activity.
Specifically, the protein (P1) in the present invention is at least one selected from the group consisting of the following proteins (P1-1) to (P1-4). These may be used alone or in combination of two or more.

(P1-1) Protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2 (P1-2) Amino acid sequence in which one or several amino acids are deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 1 or 2. The protein (P1-3) SEQ ID NO: consisting of and having the glycosyltransferase activity of β-1 → 2 binding to the hexose residue of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1). A sesaminol monosaccharide or disaccharide glycoside having an amino acid sequence having 90% or more identity with respect to the amino acid sequence of 1 or 2 and represented by the above general formula (1) to a hex source residue. Encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 4 of a protein (P1-4) having β-1 → 2 binding glycosyl transfer activity. A protein consisting of the amino acid sequence to be used and having a β-1 → 2 binding glycosyltransferase activity to the hexose residue of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1).

Further, in the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, β-1 → 2 to the hexose residue of the sesaminol disaccharide or disaccharide glycoside represented by the above general formula (1). The binding glycosyl activity is a glycosyl transfer activity in which a sugar residue is β-1 → 2 bound to the 2-position of the hexose residue. More specifically, when the substrate sesaminol glycoside (1) is (A) n is 1, or n is 2, and two hexose residues (S1) are β-1 → 6 bonded. If so, it is the glycosyltransferase activity of β-1 → 2 binding to the first hexose residue, (B) n is 2, and the two hexose residues (S1) are β-1 → 2 binding. If so, it is the glycosyl transfer activity of β-1 → 2 binding to the second hexose residue (S1). This activity produces a sesaminol disaccharide or trisaccharide glycoside containing one or two β-1 → 2 bonds in the molecule (more specifically, its sugar portion).
In the present invention, the sugar transferred to the sesaminol glycoside is the same as the sugar in the UDP-sugar described above, and examples thereof include pentose, hexose, and heptose, preferably hexose, and more preferably glucose.
The transglycosylation activity of β-1 → 2 bond to the hexose residue of the sesaminol monosaccharide or disaccharide glycoside represented by the general formula (1) is that of β-1 → 2 bond to the glucose residue of SG1. Glucose transfer activity (I), glucose transfer activity (II) of β-1 → 2 binding to the first glucose residue of SG2 (6), and glucose transfer activity (II) of SG2 (2) to the second glucose residue It is preferable that the activity is any one or more of the β-1 → 2 bond glucose transfer activities (III), and it is more preferable that all the activities (I) to (III) are active.

In the protein (P1-2) and the protein (P2) described later, one or several amino acids are deleted, substituted and / or added to the amino acid sequence, which means that any one or several amino acids in the same sequence are deleted. It means that there is a deletion, substitution and / or addition of one or several amino acids at a position in the amino acid sequence of, and two or more of the deletion, substitution and addition may occur at the same time.
As the number of such deleted, substituted and / or added amino acids, 1 or several is preferably 1 to 30, more preferably 1 to 20, and 1 to 10. It is more preferably present, and particularly preferably 1 to 5.

The identity (sequence identity) of the protein (P1-3) with respect to the amino acid sequence of SEQ ID NO: 1 or 2 is the glycosyltransferase activity of β-1 → 2 binding to the hexose residue of the sesaminol glycoside (1). For example, it is preferably 92% or more, more preferably 95% or more, 96% or more, 97% or more, still more preferably 98% or more, and particularly preferably 99% or more. ..

The identity of the amino acid sequence and the nucleotide sequence can be calculated by default parameters using, for example, analysis software such as BLAST.

Further, in the present invention, the polynucleotide that hybridizes with the polynucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 4 under stringent conditions is composed of the nucleotide sequence of SEQ ID NO: 3 or 4. It is preferable that the polynucleotide has 90% or more identity (sequence identity). More preferably, it has 92% or more, 95% or more, 96% or more, 97% or more, more preferably 98% or more, and particularly preferably 99% or more identity with the nucleotide sequence of SEQ ID NO: 3 or 4. ..

As used herein, the term "polynucleotide" means DNA or RNA.

In the present specification, "a polynucleotide that hybridizes with a polynucleotide having a nucleotide sequence complementary to a nucleotide sequence under stringent conditions" is a part or all of a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence. Refers to a polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like, using the above as a probe. Hybridization methods include, for example, "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Biology Press 2001" and "Ausube, Mobile, Carlent". Etc. can be used.

As used herein, the "stringent condition" may be, for example, any of a low stringent condition, a medium stringent condition, and a high stringent condition.
“Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 32 ° C. “Medium stringent conditions” include, for example, 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 42 ° C. or 5 × SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5). , 50% formamide, 42 ° C. “High stringent conditions” are, for example, under the conditions of 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 50 ° C. or 0.2 × SSC, 0.1% SDS, 65 ° C. be. Under these conditions, it can be expected that a polynucleotide having a higher identity can be efficiently obtained as the temperature is raised. However, multiple factors such as temperature, salt concentration, probe concentration and length, ionic strength, and time can be considered as factors that affect stringency, and those skilled in the art should appropriately select these factors. It is possible to achieve similar stringency in.
“Stringent conditions” include, for example, Sambrook & Russel, Molecular Cloning: A Laboratory Manual Vol. 3. The conditions described in Cold Spring Harbor, Laboratory Press 2001 and the like can also be adopted.

In the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the protein (P1) is preferably a protein (P1-1). That is, the protein (P1) is preferably a protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2. The protein consisting of the amino acid sequence of SEQ ID NO: 1 and the protein consisting of the amino acid sequence of SEQ ID NO: 2 have a β-1 → 2 bond of a sugar residue at the 2-position of the hexose residue of the sesaminol glycoside (1) described above. It is a sesaminol GGT having a glycosyltransferase activity.
In the present specification, the protein consisting of the amino acid sequence of SEQ ID NO: 1 is also referred to as "protein SiGGT21842", and the protein consisting of the amino acid sequence of SEQ ID NO: 2 is also referred to as "protein SiGGT21842R229Q".

By performing the step (i) using the protein (P1), the hexose is transferred from β-1 to 2 to the 2-position of the hexose residue bound to the sesaminol glycoside (1), and the hexose has a predetermined structure. A sesaminol disaccharide or trisaccharide glycoside can be produced. More specifically, when the sesaminol glycoside (1) is a sesaminol 1 glycoside (for example, SG1 when the sugar residue is a glucose residue), the first hexose residue. The sugar transfer of β-1 → 2 bond to to produces a sesaminol disaccharide glycoside containing one β-1 → 2 bond in the molecule. The sesaminol glycoside (1) is a sesaminol disaccharide glycoside (for example, the sugar residue is a glucose residue) in which n is 2 and two hexose residues (S1) are β-1 → 6 bound. In the case of SG2 (6), etc.), a sesame containing one β-1 → 2 bond in the molecule due to the glycoside transfer of β-1 → 2 bond to the first hexose residue. Nool trisaccharide glycosides (triglucosides) are produced. The sesaminol glycoside (1) is a sesaminol disaccharide glycoside in which n is 2 and two hexose residues (S1) are β-1 → 2 bound (for example, the sugar residue is a glucose residue). In the case of SG2 (2), etc.), a sesame containing two β-1 → 2 bonds in the molecule due to the glycoside transfer of β-1 → 2 bonds to the second hexose residue. Nool trisaccharide glycosides are produced. By performing step (i) in this way, a sesaminol disaccharide or trisaccharide glycoside containing 1 or 2 β-1 → 2 bonds in the molecule can be produced.

In the present invention, if desired, sesaminol GGT having a β-1 → 6 bond glycosyl transfer activity to the first hexose residue of the sesaminol monosaccharide or disaccharide glycoside may be used. Such an enzyme is also referred to as a protein (P2). The sugar transfer activity of β-1 → 6 bond to the first hexose residue of the sesaminol glycoside (1) is the sugar transfer of β-1 to 6 bond of sugar to the 6-position of the hexose residue. It is active. Preferred embodiments of sugar are as described above, with glucose being particularly preferred. This activity produces a sesaminol disaccharide or trisaccharide glycoside containing one β-1 → 6 bond in the molecule (more specifically, its sugar portion). This transglycosylation activity is usually UDP-sugar-specific transglycosylation activity.
The β-1 → 6 bond glycosyl transfer activity to the first hexose residue of the sesaminol glycoside (1) is the β-1 → 6 bond glucose transfer activity (IV) of SG1 to the glucose residue. , And any one or two of the glucose transfer activity (V) of β-1 → 6 bond to the first glucose residue of SG2 (2), preferably (IV) and ( It is more preferable that both activities of V) are active.

As the protein (P2) in the present invention, at least one protein selected from the group consisting of the following proteins (P2-1) to (P2-8) is preferable.
(P2-1) Protein consisting of the amino acid sequence of SEQ ID NO: 5 (P2-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 5 and , For the amino acid sequence of protein (P2-3) SEQ ID NO: 5 having glycosyl transfer activity that binds sugar β-1 → 6 to the 6-position of the first hex source residue of the sesaminol glycoside (1). A glycosyl transfer activity that comprises an amino acid sequence having 90% or more identity and that binds a sugar β-1 to 6 at the 6-position of the first hex source residue of the sesaminol glycoside (1). Protein (P2-4) consisting of a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6 and an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions, and has a sesaminol arrangement. A protein (P2-5) having a transglycosylation activity that binds a sugar β-1 to 6 at the 6-position of the first hex source residue of the sugar body (1). A protein (P2-) consisting of the amino acid sequence of SEQ ID NO: 7. 6) The first hex source residue of the sesaminol glycoside (1), which consists of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 7. At the 6-position of, it consists of an amino acid sequence having 90% or more identity with respect to the amino acid sequence of protein (P2-7) SEQ ID NO: 7 having a transglycosylation activity that binds a sugar from β-1 to 6 and has an amino acid sequence of 90% or more. Complementary to the nucleotide sequence of SEQ ID NO: 8 of a protein (P2-8) having a transglycosylation activity that binds a sugar from β-1 to 6 at the 6-position of the first hexose residue of the sesaminol glycoside (1). Consists of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a similar nucleotide sequence, and at the 6-position of the first hexose residue of the sesaminol glycoside (1). , Protein with glycosyl transfer activity that binds sugar β-1 → 6

The number of deleted, substituted and / or added amino acids in (P2-2) and (P2-6) is preferably 1 to 30, preferably 1 to 20. It is more preferably 1 to 10 pieces, and particularly preferably 1 to 5 pieces.

The identity of SEQ ID NO: 5 or 7 with respect to the amino acid sequence in (P2-3) and (P2-7) is preferably 92% or more, more preferably 95% or more, 96% or more, 97% or more, More preferably, it is 98% or more, and particularly preferably 99% or more.

The polynucleotides (P2-4) and (P2-8) are preferably those having 90% or more identity with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 or 8. More preferably, it has 92% or more, 95% or more, 96% or more, 97% or more, more preferably 98% or more, and particularly preferably 99% or more identity with the nucleotide sequence of SEQ ID NO: 6 or 8. ..

Among them, the protein (P2) is preferably a protein (P2-1) or (P2-5). That is, the protein (P2) is preferably a protein consisting of the amino acid sequence of SEQ ID NO: 5 or 7. The protein consisting of the amino acid sequence of SEQ ID NO: 5 and the protein consisting of the amino acid sequence of SEQ ID NO: 7 have β-1 → 6 sugar residues at the 6-position of the first hexose residue of the sesaminol glycoside (1). It is a sesaminol GGT having a glycosyl transfer activity to be bound. These proteins have β-1 → 6-linked glucose transfer activity (IV) to the glucose residue of SG1 and β-1 → 6-linked glucose to the first glucose residue of SG2 (2). Has metastatic activity (V).
In the present specification, the protein consisting of the amino acid sequence of SEQ ID NO: 5 is also referred to as "protein SiGGT21646", and the protein consisting of the amino acid sequence of SEQ ID NO: 7 is also referred to as "UGT94D1".
Sesaminol disaccharide or trisaccharide containing one β-1 → 6 bond in the molecule by performing a step of allowing protein (P2) to act on sesaminol disaccharide or disaccharide glycoside and UDP-sugar. Glycosides can be obtained. UDP-sugar and preferred embodiments thereof are as described above.

In the step (i) in the production method of the present invention, a sesaminol glycoside (1) and a UDP-sugar may be used as substrates, and a protein (P1) may be allowed to act on these substrates, and the conditions and the like are particularly limited. Not done.

Step (i) can be carried out, for example, by adding the protein (P1) to a reaction system containing a sesaminol monosaccharide or disaccharide glycoside represented by the general formula (1) and UDP-sugar. The reaction is preferably carried out in an aqueous solution. The reaction conditions in step (i) are preferably, for example, 10 to 50 ° C. and pH 5 to 8. The reaction temperature is more preferably 30 to 40 ° C. The ratio of the substrate and the protein (P1) may be appropriately set.

In addition, a polynucleotide encoding a protein (P1) is introduced into a non-human host, the protein (P1) is expressed in the non-human host (transformant of the present invention described later), and the transformant is used in a step (step). i) can also be done. The transformant can be prepared by the method described later.
In this case, as the host of the transformant, it is preferable to use a plant, preferably a sesame plant (more preferably sesame); a microorganism such as Escherichia coli, yeast, or aspergillus.

When a sesaminol disaccharide glycoside is produced by the method for producing a sesaminol disaccharide or a trisaccharide glycoside of the present invention, the sesaminol disaccharide glycoside is preferably SG2 (2).
When the sesaminol trisaccharide glycoside is produced by the method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention, the sesaminol trisaccharide glycoside is SG3 (2) represented by the above formula (5). , 2), or SG3 (2,6) represented by the above formula (4) is preferable.
The method for producing a sesaminol disaccharide or trisaccharide glycoside of the present invention is suitable as a method for producing these sesaminol disaccharide or trisaccharide glycoside.

Next, an example of a method for producing SG2 (2) as a sesaminol disaccharide or a trisaccharide glycoside will be described in more detail.
The method for producing SG2 (2) includes the step (i) of allowing the protein (P1) to act on SG1 and UDP-glucose. A step of preparing SG1 may be performed before performing this step.

(Step to prepare SG1)
The method for preparing SG1 is not particularly limited. For example, as described above, UGT71A9 described in Patent Document 1 or Non-Patent Document 1 can be obtained by acting on sesaminol and UDP-glucose.
Further, SG1 may be prepared by extracting and purifying from sesame seeds and the like.

(Step (i) of allowing protein (P1) to act on SG1 and UDP-glucose)
By allowing protein (P1) to act on SG1 and UDP-glucose, SG2 (2) is synthesized by the glucose transfer of β-1 → 2 bond to the glucose residue of SG1.
In the above, the protein (P1) functions as sesaminol GGT having the glucose transfer activity (I) of β-1 → 2 binding to the glucose residue of SG1.

Next, a method for producing SG3 (2,6) as a sesaminol disaccharide or a trisaccharide glycoside will be described in more detail with two examples.

The first method for producing SG3 (2,6) is the step (i) of allowing the protein (P1) to act on SG1 and UDP-glucose, and the action of the protein (P2) on SG2 (2) and UDP-glucose. Includes step (ii). Further, the step of preparing the SG1 may be included.

As described above, the protein (P2) is sesaminol GGT having β-1 → 6 binding glycosyl transfer activity to the hexose residue of the sesaminol glycoside (1).
In the first method, the protein (P2) preferably has a glucose transfer activity (IV) of β-1 → 6 bond to the glucose residue of SG1 as the glucose transfer activity. As such a protein (P2), it is preferable that it is at least one selected from the group consisting of the following proteins (P2-11) to (P2-18). These may be used alone or in combination of two or more.

(P2-11) Protein consisting of the amino acid sequence of SEQ ID NO: 5 (P2-12) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 5 and , 90% or more identity to the amino acid sequence of protein (P2-13) SEQ ID NO: 5, which has β-1 → 6 binding glucose transfer activity to the first glucose residue of SG2 (2). Complementary to the nucleotide sequence of protein (P2-14) SEQ ID NO: 6 consisting of the amino acid sequence having and having the glucose transfer activity of β-1 → 6 binding to the first glucose residue of SG2 (2). It consists of an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide consisting of a nucleotide sequence and under stringent conditions, and has a β-1 → 6 bond to the first glucose residue of SG2 (2). One or several amino acids have been deleted, substituted and / or added to the amino acid sequence of protein (P2-16) SEQ ID NO: 7 consisting of the amino acid sequence of protein (P2-15) SEQ ID NO: 7 having glucose transfer activity. 90 for the amino acid sequence of protein (P2-17) SEQ ID NO: 7, which consists of an amino acid sequence and has β-1 → 6 binding glucose transfer activity to the first glucose residue of SG2 (2). Of protein (P2-18) SEQ ID NO: 8, which consists of an amino acid sequence having% or more identity and has β-1 → 6 binding glucose transfer activity to the first glucose residue of SG2 (2). It consists of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence, and β to the first glucose residue of SG2 (2). Proteins with -1 → 6 binding glucose transfer activity Proteins (P2-11) and (P2-15) are the same as proteins (P2-1) and (P2-5). The proteins (P2-12) to (P2-14) and (P2-16) to (P2-18) are the proteins (P2-2) to (P2-4) and (P2-6) to (P2-8). This is an example of a preferred embodiment of the above, and preferred embodiments such as amino acid identity are the same as those of proteins (P2-2) to (P2-4) and (P2-6) to (P2-8).

In this first method, the step (i) of allowing the protein (P1) to act on SG1 and UDP-glucose is the step (i) in the method for producing SG2 (2) as the sesaminol disaccharide or trisaccharide glycoside. Since it is the same as i), the description here is omitted.

(Step (ii) of allowing protein (P2) to act on SG2 (2) and UDP-glucose)
In this step, by using a protein (P2), SG2 (2) is reacted with UDP-glucose, and the glucose transfer of β-1 → 6 bond to the first glucose residue of SG2 (2). SG3 (2,6) is synthesized by. The reaction is preferably carried out in an aqueous solution. The reaction conditions in this step are not particularly limited, but are preferably 10 to 50 ° C. and pH 5 to 8. The reaction temperature is more preferably 30 to 40 ° C. The ratio of the substrate and the protein (P2) may be appropriately set.

The second method for producing SG3 (2,6) comprises the step (i) of allowing the protein (P1) to act on SG2 (6) and UDP-glucose. It may also include a step of preparing SG2 (6). SG2 (6) can be produced, for example, by allowing protein (P2) to act on SG1 and UDP-glucose. Further, the step of preparing the SG1 may be included.

When the step of causing the protein (P2) to act on SG1 and UDP-glucose in this second method, the protein (P2) has a glucose transfer activity of β-1 → 6 binding to the glucose residue of SG1. Is preferable. As such a protein (P2), as the above (P2-1) to (P2-8), a protein having β-1 → 6 binding glucose transfer activity (IV) to the glucose residue of SG1 may be used. , A protein consisting of the amino acid sequence of SEQ ID NO: 5 and / or a protein consisting of the amino acid sequence of SEQ ID NO: 7 is preferably used. These may be used alone or in combination of two or more.
The reaction conditions are not particularly limited and may be the same as the reaction using the above protein (P2).

(Step of allowing protein (P2) to act on SG1 and UDP-glucose)
In this step, SG1 is reacted with UDP-glucose by using a protein (P2) to synthesize SG2 (6).
The preferable reaction conditions are the same as when the above protein (P2) is used.

In the first and second methods for producing SG3 (2,6), the protein (P2) is the glucose transfer activity of β-1 → 6 binding to the glucose residue of SG1 and SG2 (2). It is preferable to have a glucose transfer activity of β-1 → 6 bond to the first glucose residue of the above, and for example, a protein consisting of the amino acid sequence of SEQ ID NO: 5 or 7 is more preferable.

(Step (i) of allowing protein (P1) to act on SG2 (6) and UDP-glucose)
By using protein (P1), SG2 (6) is reacted with UDP-glucose, and SG3 (2, 2) is transferred by β-1 → 2 binding glucose transfer to the first glucose residue of SG2 (6). 6) is synthesized.
In the above, the protein (P1) functions as a protein having a glucose transfer activity (II) of β-1 → 2 binding to the first glucose residue of SG2 (6).

Next, a method for producing SG3 (2,2) as a sesaminol disaccharide or a trisaccharide glycoside will be described in more detail.
The method for producing SG3 (2,2) comprises the step (i) of allowing the protein (P1) to act on SG2 (2) and UDP-glucose. SG2 (2) may be produced by performing the step (i) of allowing the protein (P1) to act on SG1 and UDP-glucose described above. In this case, the production method is a step (i) of producing SG2 (2) by β-1 → 2 bond glucose transfer to the glucose residue of SG1, and the above protein in SG2 (2) and UDP-glucose. (P1) is allowed to act to generate SG3 (2,2). Further, the step of preparing the SG1 may be included.

(Step (i) of allowing protein (P1) to act on SG1 and UDP-glucose)
In this step, SG1 is reacted with UDP-glucose by using a protein (P1), and SG2 (2) is synthesized by a β-1 → 2 bond glucose transfer of SG1 to a glucose residue.

(Step (i) of causing protein (P1) to act on SG2 (2) and UDP-glucose)
By using protein (P1), SG2 (2) is reacted with UDP-glucose, and SG3 (2) is transferred by β-1 → 2 binding glucose transfer to the second glucose residue of SG2 (2). , 2) are synthesized.
In the above, the protein (P1) functions as a protein having a glucose transfer activity (III) of β-1 → 2 binding to the second glucose residue of SG2 (2).

The SG3 (2,2) thus obtained is also a novel compound of the present invention.

In the method for producing SG3 (2,2), if the above protein (P2) is present in the reaction system, SG3 (2,2) is synthesized together with SG3 (2,6) as the final product. NS.
Therefore, it is preferable that the protein (P2) is not present in the reaction system of the method for producing SG3 (2,2).

FIG. 1 is a reaction diagram schematically showing a route for synthesizing SG3 (2,6) or SG3 (2,2).
As shown in FIG. 1, a sesaminol disaccharide or trisaccharide glycoside can be produced from a sesaminol disaccharide or disaccharide glycoside using a protein (P1) and, if desired, a protein (P2). ..
Each of these reactions may be carried out in a separate reaction system or in the same reaction system.

In the above, as the sesaminol glycoside (1), when (S1) in the general formula (1) is a glucose residue, the sesaminol disaccharide is due to the glucose transfer of β-1 → 2 bond to the glucose residue. Alternatively, a method for producing a trisaccharide glycoside has been given as an example, but these are examples of the production method of the present invention and are not limited to the above.

The sesaminol disaccharide or trisaccharide glycoside produced by the production method of the present invention is usually more water-soluble than sesamin aglycone. That is, the sesaminol disaccharide or trisaccharide glycoside is easily dissolved in an aqueous solvent. Therefore, by using the sesaminol disaccharide or trisaccharide glycoside, for example, in the food and drink field and the medical field, an aqueous solution in which the sesaminol disaccharide or trisaccharide glycoside is dissolved can be easily obtained. ..

Next, the protein (P1), particularly the protein (P1-1), will be described in detail.

The protein (P1-1) is a protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2.
To date, the function of the protein consisting of the amino acid sequences of SEQ ID NOs: 1 and 2 has not been known.

As described above, the protein SiGGT21842 and the protein SiGGT21842R229Q, which are proteins consisting of the amino acid sequences of SEQ ID NOs: 1 and 2, have the following three activities (I) to (III).
That is, these proteins have a β-1 → 2 bond glucose transfer activity (I) to the glucose residue of SG1 and a β-1 → 2 bond glucose transfer activity (I) to the first glucose residue of SG2 (6). It has activity (II) and glucose transfer activity (III) of β-1 → 2 binding to the second glucose residue of SG2 (2). As mentioned above, the protein (P1) is a UDP-sugar specific glycosyltransferase.

The nucleotide sequence of SEQ ID NO: 3 encodes the amino acid sequence of SEQ ID NO: 1, and the nucleotide sequence of SEQ ID NO: 4 encodes the amino acid sequence of SEQ ID NO: 2.
The amino acid sequence of SEQ ID NO: 2 is a novel amino acid sequence. That is, the protein SiGGT21842R229Q is a novel protein. A protein consisting of the amino acid sequence of SEQ ID NO: 2 is also one of the present inventions.
A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4 is also one of the present inventions.

Next, an example of a method for producing a protein (P1) will be described.
Examples of the method for producing these proteins include the following methods.
For example, an expression vector is prepared by incorporating a polynucleotide containing the nucleotide sequence of SEQ ID NO: 3 or 4 into the vector. The above expression vector is introduced into a host, and the host is transformed into a transformant. The transformant and the method for producing the transformant will be described later.
Then, the protein (P1) can be obtained by expressing a protein in the transformant and purifying the protein.

The method for preparing a polynucleotide containing the nucleotide sequence of SEQ ID NO: 3 or 4 is not particularly limited. For example, it can be obtained by a known genetic engineering method or synthetic method. A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 or 4 can be obtained, for example, from a plant of the genus Sesamum (preferably sesame). The polynucleotide of the present invention includes naturally occurring polynucleotides isolated from plants and the like, recombinant polynucleotides produced by genetic engineering techniques, and chemically synthesized polynucleotides.

The vector is not particularly limited, and may be appropriately selected depending on, for example, the type of host to be introduced. When the above vector is transformed by using the Agrobacterium method, for example, a binary vector is preferable, and examples thereof include pBI121, pPZP202, pBINPLUS and pBIN19. When transforming a bacterium such as Escherichia coli, examples of the vector include a pET vector (Merck), a pCold vector (Takara Bio Co., Ltd.), and a PQE vector (QIAGEN). When transforming a eukaryote such as yeast, examples thereof include pYE22m and the like, and commercially available yeast expression vectors such as pYES (Invitrogen) and pESC (Stratagene) can also be used.

The expression vector preferably has, for example, a regulatory sequence that regulates the expression of the polynucleotide and the expression of the protein encoded by the polynucleotide. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, an origin of replication sequence (ori), and the like. For example, a conventional promoter (for example, trc promoter, tac promoter, lac promoter, etc.) can be used as the promoter of the expression vector for bacteria, and for example, the glyceraldehyde triphosphate dehydrogenase can be used as the promoter for yeast. Examples include promoters, PH05 promoters, and examples of promoters for filamentous fungi include amylases, trpC, and the like. As examples of promoters for expressing the target gene in plant cells, the enhancer sequences of the 35S RNA promoter of the Califlower mosaic virus, the rd29A gene promoter, the rbcS promoter, and the 35S RNA promoter of the Califlower mosaic virus are derived from agrobacterium. The mac-1 promoter added to the 5'side of the mannopine synthase promoter sequence of the above can be mentioned. Examples of promoters for animal cell hosts include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.).

In the expression vector, the arrangement of regulatory sequences is not particularly limited. The regulatory sequence may be arranged, for example, so as to be able to functionally regulate the expression of the polynucleotide and the expression of the protein encoded by the polynucleotide, and can be arranged based on a known method. In addition, the regulatory sequence may be appropriately selected according to the type of host to be introduced. As the regulatory sequence, for example, a sequence provided in advance by the vector may be used, a regulatory sequence may be further inserted into the vector, or the regulatory sequence contained in the vector may be replaced with another regulatory sequence. ..

The expression vector may further have, for example, one or more coding sequences for selectable markers or purified tags. Examples of the selectable marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, a cell surface receptor marker and the like. Examples of the purification tag include His tag, myc tag, FLAG tag, glutathione-S-transferase, maltose-binding protein and the like.

The method for introducing the expression vector into the host is not particularly limited, and a known method can be used. The introduction method can be appropriately determined depending on, for example, the type of host, the type of expression vector, and the like.

The host into which the expression vector is introduced is not particularly limited, but is preferably a non-human host, and examples thereof include plant bodies or parts thereof, microorganisms, animal cells, insect cells, and cultured cells thereof. Above all, it is preferable to use a plant, a part thereof, or a microorganism as a host.

When the host is a plant or a part thereof, the host plants include, for example, sesame plants (such as sesame), plants belonging to the genus Karahanasou (for example, Humulus lupulus), and rose family plants (for example, strawberries, sea urchins, etc.). Sakura, rose, blueberry, blackberry, bilberry, cassis, raspberry, etc.), Nadeshiko family plant (carnation, Kasumi sou, etc.), Kiku family plant (Kiku, Gerbera, sunflower, daisy, etc.), Orchid family plant (orchid, etc.), Sakura sou Family plants (cyclamen, etc.), Lindou family plants (Turkish gyo, lindo, etc.), Ayame family plants (Freesia, Ayame, Gradiolas, etc.), Gomanohagusa family plants (Kingyosou, Trenia, etc.) Tulip, etc.), Hirugao (Asagao, Momiji Hirugao, Yorgao, Sweet potato, Rukosou, Evolbles, etc.), Hydrangea (Ajisai, Utsugi, etc.), Urigao (Yugao, etc.) , Mokusei family plants (Lengyo, etc.), Vine family plants (eg, grapes, etc.), Swallow family plants (Cha, Tsubaki, Chanoki, etc.) , Hie, Kouryan, Satoukibi, Bamboo, Karasumugi, Shikokubie, Morokoshi, Makomo, Hatomugi, Grass, etc.) Family plants (nara, beech, kashiwa, etc.), citrus plants (eg, Daidai, Yuzu, Unshuumikan, Sansho), Abrana family plants (red cabbage, habutton, daikon, white nazuna, abrana, cabbage, broccoli, cauliflower, etc.) , Perilla family (salvia, perilla, lavender, tatsunamisou, etc.), eggplant family plants (eg, eggplant, tomato, capsicum, potato, tobacco, butterfly sagao, hozuki, petunia, calibracoa, nierenbergia, etc.), legumes (eg, soybean) , Azuki, Rakkasei, Ingenmame, Soramame, Miyakogusa, etc.).
Among these, a plant of the genus Sesamum is preferable, and a plant body of sesame or a part thereof is preferable.

In the present specification, the "plant body" means a plant individual indicating the whole plant, and the "plant body part" is a part of the above-mentioned plant individual, and examples thereof include organs, tissues, cells, and the like. It may be. Examples of the organs include petals, corollas, flowers, flowers, glandular hairs, leaves, seeds, fruits, stems, roots, propagules and the like. Examples of the stem include bulbs such as bulbs, tubers, corms, rhizomes, liners and the like. Examples of the root include a bulbous root, a horizontal root, and the like. The tissue is, for example, a part of the organ (for example, epidermis, phloem, parenchyma, xylem, vascular bundle, palisade tissue, sea surface tissue, etc.). The portion of the plant may be, for example, one type of organ, tissue and / or cell, or two or more types of organ, tissue and / or cell. The plant cell may be a cultured cell, and means any of various forms of cells (for example, suspended cultured cell), protoplast, callus, and the like.

The method for introducing the expression vector into the host is not particularly limited, and a known transformation method can be used. Examples of the introduction method include an Agrobacterium method, an introduction method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, and a DEAE-dextran method. , Direct injection method using a micro glass tube, hydrodynamic method, cationic liposome method, CRISPR-Cas method (for example, Woo et al., Nature Biotechnology 33, 1162-1164 (2015)), using an introduction aid. The method etc. can be mentioned. Examples of liposomes include lipofectamine and cationic liposomes. Examples of the introduction aid include atelocollagen, nanoparticles, polymers and the like. The introduction method may be appropriately selected according to the type of host and the like. For example, when the host is a plant or a part thereof, the Agrobacterium method is preferable.

When the host is a plant or a portion thereof, the expression vector may be introduced into, for example, the plant or a portion thereof, preferably a portion of the plant, more preferably a tissue or a cell. And more preferably cells. Examples of the above-mentioned tissue include sections cut out from a plant body, and specifically, sections such as leaves, stems, and roots. Examples of the cells include cells collected from a plant body or a tissue thereof, cultured cells of the cells, protoplasts, callus and the like.

Whether or not the expression vector has been introduced into the host can be confirmed by a PCR method, a Southern hybridization method, a Northern hybridization method, or the like. For example, DNA is prepared from transformants, DNA-specific primers are designed, and PCR is performed. The amplification product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplification product is detected as a single band. Can be confirmed to have been transformed. It is also possible to detect an amplification product by performing PCR using a primer labeled with a fluorescent dye or the like in advance. Further, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence, enzymatic reaction or the like can also be adopted.

The host into which the expression vector has been introduced is cultured in this way to express a protein (P1) such as a protein (P1-1).
As a method for culturing a host and a method for expressing a protein, a known method according to the type of host can be applied.

The method for purifying the protein (P1) is not particularly limited, and a known method can be used.
A protein (P1-1) can be produced by such a method.

The protein consisting of the amino acid sequence of SEQ ID NO: 2, the polynucleotide containing the nucleotide sequence of SEQ ID NO: 4, and the expression vector containing the polynucleotide are also the protein, polynucleotide, and expression vector of the present invention, respectively.

Further, in the method for producing the protein (P1), the following polynucleotides (N1-2) to (N1-4) are incorporated into a vector and expressed in place of the polynucleotide containing the nucleotide sequence of SEQ ID NO: 3 or 4. The protein (P1) can also be produced by producing a vector.
(N1-2) A nucleotide having a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added to the nucleotide sequence of SEQ ID NO: 3 or 4, and according to the above general formula (1). To the nucleotide sequence of nucleotide (N1-3) SEQ ID NO: 3 or 4, which encodes a protein having β-1 → 2 binding glycotide activity to the hexose residue of the sesaminol monosaccharide or disaccharide glycoside shown. On the other hand, β-1 to the hexsource residue of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1), which is a nucleotide having a nucleotide sequence having 90% or more identity. → Polynucleotide (N1-4) encoding a protein having 2-binding glycosyltransferase activity A polynucleotide that hybridizes with a nucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 4 under stringent conditions. A polynucleotide consisting of and encoding a protein having a β-1 → 2 binding glycotide transfer activity to a hexose residue of a sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1).

In the above (N1-2), "1 or several" means that, for example, the protein encoded by the polynucleotide of the above (N1-2) is β-to the hexose residue of the sesaminol glycoside (1). Any range may be used as long as it has a 1 → 2 bond glycosyl transfer activity. The "1 or several" in the above (N1-2) is preferably 1 to 50, 1 to 30, 1 to 20, more preferably 1 to 9, and even more preferably 1 to 1. The number is 5, particularly preferably 1 to 3, and most preferably 1 or 2. Addition also includes, for example, the meaning of insertion into a sequence. Deletion, substitution and / or addition of one or several nucleotides in the nucleotide sequence of a polynucleotide means that one or more nucleotides are located at any and one or more positions in the same sequence. It means that there are deletions, substitutions and / or additions, and two or more of deletions, substitutions and additions may occur at the same time.

In the above (N1-3), the identity (sequence identity) means that, for example, the protein encoded by the polynucleotide of the above (N1-3) is β to the hexose residue of the sesaminol glycoside (1). Any range may be used as long as it has a -1 → 2 bond glycosyl transfer activity. The identity is 90% or more, preferably 92% or more, more preferably 95% or more, 96% or more, 97% or more, still more preferably 98% or more, and particularly preferably 99% or more.

Next, proteins (P2), particularly proteins (P2-1) and (P2-5), will be described in detail.

The protein (P2-1) is a protein consisting of the amino acid sequence of SEQ ID NO: 5.
The protein (P2-5) is a protein consisting of the amino acid sequence of SEQ ID NO: 7.
To date, the function of the protein consisting of the amino acid sequence of SEQ ID NO: 5 has not been known.

The protein (P2-1) (protein SiGGT21646) and the protein (P2-5) (protein UGT94D1) have the following two activities.
That is, these proteins have β-1 → 6 binding glucose transfer activity (IV) to the glucose residue of SG1 and β-1 → 6 binding to the first glucose residue of SG2 (2). Has glucose transfer activity (V). As mentioned above, the protein (P2) is a UDP-sugar specific glycosyltransferase.

The nucleotide sequence of SEQ ID NO: 6 encodes the amino acid sequence of SEQ ID NO: 5, and the nucleotide sequence of SEQ ID NO: 8 encodes the amino acid sequence of SEQ ID NO: 7.

Next, a method for producing the protein (P2-1) (protein SiGGT21646) will be described.
In the method for producing the protein (P1), any or two or more of the following polynucleotides (N2-1) to (N2-8) are vectorized instead of the polynucleotide containing the nucleotide sequence of SEQ ID NO: 3 or 4. A protein (P2) such as a protein (P2-1) (protein SiGGT21646) can be produced by producing an expression vector by incorporating the expression vector into the protein (P2-1).
(N2-1) Nucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 Polynucleotide having a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added to the nucleotide sequence of (N2-2) SEQ ID NO: 6. It is a nucleotide and encodes a protein having a β-1 → 6 binding glycosyltransferase activity to the first hex source residue of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1). Nucleotide (N2-3) A sesaminol 1 sugar represented by the above general formula (1), which is a nucleotide having a nucleotide sequence having 90% or more identity with respect to the nucleotide sequence of SEQ ID NO: 6. Alternatively, a nucleotide complementary to the nucleotide sequence of SEQ ID NO: 6 of a polynucleotide (N2-4) encoding a protein having a β-1 → 6 binding glycotransferase activity to the first hex source residue of a disaccharide glycoside. To the first hexsource residue of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1), which consists of a polynucleotide consisting of a sequence and a polynucleotide that hybridizes under stringent conditions. One or a number in the nucleotide sequence of the nucleotide (N2-6) SEQ ID NO: 8 consisting of the nucleotide sequence of the nucleotide (N2-5) SEQ ID NO: 8 encoding the protein encoding the β-1 → 6 binding glycotide activity of The first hexadose of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1), wherein the nucleotide is a polynucleotide having a nucleotide sequence deleted, substituted and / or added. Polynucleotide (N2-7) Polynucleotide (N2-7) Polynucleotide having a nucleotide sequence having 90% or more identity with respect to the nucleotide sequence of SEQ ID NO: 8, which encodes a protein having β-1 → 6 binding glycotransferase activity to a residue. It is a nucleotide and encodes a protein having a β-1 → 6 binding glycosyltransferase activity to the first hex source residue of the sesaminol monosaccharide or disaccharide glycoside represented by the above general formula (1). Nucleotide (N2-8) Consists of a nucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 8 and a polynucleotide that hybridizes under stringent conditions, and is represented by the above general formula (1). A polynucleotide encoding a protein having a β-1 → 6-binding glycosyltransferase activity to the first hexose residue of a sesaminol monosaccharide or disaccharide glycoside

In the above (N2-2) and (N2-6), "1 or several" means that, for example, the protein encoded by the polynucleotides of the above (N2-2) and (N2-6) is a sesaminol glycoside. Any range may be used as long as it has a β-1 → 6-binding glycosyltransferase activity to the first hexose residue of the body (1). The "1 or several" in the above (N2-2) and (N2-6) is preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10, and more preferably 1 to 9. , More preferably 1 to 5, particularly preferably 1 to 3, and most preferably 1 or 2.

In the above (N2-3) and (N2-7), the identity (sequence identity) is that, for example, the protein encoded by the polynucleotides of the above (N2-3) and (N2-7) is a sesaminol component. Any range may be used as long as it has the glycosyltransferase activity of β-1 → 6 binding to the first hexose residue of the sugar body (1). The identity is 90% or more, preferably 92% or more, more preferably 95% or more, 96% or more, 97% or more, still more preferably 98% or more, and particularly preferably 99% or more.

In addition, an expression vector containing at least one polynucleotide selected from the group consisting of the above-mentioned polynucleotides (N1-1) to (N1-4) and polynucleotides (N2-1) to (N2-4) was introduced. A transformant that is a non-human host is also a transformant of the present invention. The method for producing such an expression vector and transformant is the same as the method for producing the expression vector and transformant in the above-mentioned production of protein (P-1).
Preferably, it is a transformant which is a non-human host into which an expression vector containing at least one polynucleotide selected from the group consisting of polynucleotides (N1-1) to (N1-4) has been introduced, and this transformation The body may be further introduced with an expression vector containing at least one polynucleotide selected from the group consisting of polynucleotides (N2-1) to (N2-4).

As a method for cultivating or culturing the transformant, a known method according to the type of the transformant can be applied. By cultivating or culturing the transformant, the above-mentioned polynucleotide can be expressed in the transformant to synthesize a protein (P1) and / or a protein (P2). At this time, the protein (P1) and / or the protein (P2) synthesized in the transformant can be isolated and used for the production of the above-mentioned sesaminol disaccharide or trisaccharide glycoside. The proteins (P1) and (P2) may be purified proteins or unpurified proteins. Further, in the transformant, the synthesis of the protein and the synthesis of the sesaminol disaccharide or trisaccharide glycoside may be carried out. By culturing the transformant in this way, the protein (P1) and / or (P2) synthesis step and the sesaminol disaccharide or trisaccharide glycoside synthesis step can be performed, and the sesaminol disaccharide can be performed. Alternatively, a trisaccharide glycoside can be synthesized.

So far, proteins (P1) and (P2) have been prepared using transformants prepared using polynucleotides (N1-1) to (N1-4) and polynucleotides (N2-1) to (N2-8). Has been explained such as a method of manufacturing.
In addition, for example, proteins (P1) and (P2) may be obtained by extracting and purifying proteins (P1) and (P2) from sesame seeds.

Next, according to the present invention, a polynucleotide containing at least a part of the nucleotide sequence of SEQ ID NO: 3, 4 or 6 is used to select plants having different sesaminol disaccharide or trisaccharide glycoside synthesizing ability. The method of selecting plants will be described.
Examples of plants having different sesaminol disaccharide or trisaccharide glycoside synthesizing ability include plants having high or low sesaminol disaccharide or trisaccharide dividend synthesizing ability as compared with existing varieties.
Examples of the sesaminol disaccharide or trisaccharide glycoside include sesaminol disaccharide or trisaccharide glycoside produced by the above-mentioned production method, preferably SG2 (2), SG3 (2,6), and SG3 (2,6). One or more selected from the group consisting of SG3 (2,2) and SG2 (6).

The method for selecting a plant of the present invention may be any method as long as it is a method for selecting a plant using a polynucleotide containing at least a part of the nucleotide sequence of SEQ ID NO: 3, 4 or 6. good. The method for selecting plants of the present invention will be described below with reference to two examples (first example and second example).

The first example of the method for selecting a plant of the present invention includes (1) a polynucleotide extraction step, (2) a DNA amplification step, (3) a genetic variation selection step, and (4) a comparison step. The method can be given.
Each step will be described in detail below.

(1) Polynucleotide Extraction Step First, a polynucleotide which is DNA or RNA is extracted from a plant or a part thereof.
When the extracted polynucleotide is RNA, RNA is reverse transcribed to synthesize cDNA.
Known methods can be used for the extraction of polynucleotides and the synthesis of cDNA.
The site of the test plant from which DNA or RNA is extracted is not particularly limited, but for example, when selecting a sesame plant, sesame seeds or the like are preferable.

(2) DNA Amplification Step Next, a polynucleotide containing at least a part of the nucleotide sequence of SEQ ID NO: 3, 4 or 6 is amplified from DNA or cDNA to prepare an amplified DNA fragment.
The method for amplifying the polynucleotide is not particularly limited, but PCR can be used, for example.
DNA amplification methods and the design and synthesis of primers used in PCR are well known in the art and can be easily carried out by those skilled in the art based on sequences.

(3) Genetic variation selection step Next, by comparing the nucleotide sequence of the amplified DNA fragment with the nucleotide sequence of SEQ ID NO: 3, 4 or 6, the genetic of the gene encoding the protein having the activity of sesaminol GGT The mutation is identified and the mutant plant having the gene containing the genetic variation is selected.
In addition, in this specification, "genetic variation" means a mutation and / or a gene polymorphism.

(4) Comparison step Next, the expression level of the gene containing the genetic mutation in the mutant plant, or the activity of sesaminol GGT of the gene encoded by the gene containing the genetic mutation, and SEQ ID NOs: 3 and 4 of the existing varieties. Or, the expression level of the gene containing the nucleotide sequence of 6 or the activity of sesaminol GGT of the protein encoded by the gene containing the nucleotide sequence of SEQ ID NO: 3, 4 or 6 is compared.

When the expression level of the gene containing the genetic variation in the mutant plant is higher than the expression level of the gene containing the nucleotide sequence of SEQ ID NO: 3, 4 or 6 of the existing variety, or the protein encoded by the gene containing the genetic variation. When the activity of sesaminol GGT in Sesaminol GGT is higher than the activity of sesaminol GGT in the gene encoded by the gene containing the nucleotide sequence of SEQ ID NO: 3, 4 or 6, such mutant plants are compared to existing varieties. It can be selected as a plant having a high ability to synthesize sesaminol disaccharide or trisaccharide glycoside.
In addition, when the expression level of the gene containing the genetic variation in the mutant plant is lower than the expression level of the gene containing the nucleotide sequence of SEQ ID NO: 3, 4 or 6 of the existing variety, or when the gene containing the genetic variation is encoded. If the activity of the sesaminol GGT of the resulting protein is lower than the activity of the sesaminol GGT of the gene encoded by the gene containing the nucleotide sequence of SEQ ID NO: 3, 4 or 6, such mutant plants are compared with existing varieties. Therefore, it can be selected as a plant having a low ability to synthesize sesaminol disaccharide or trisaccharide glycoside.

The existing varieties in the present invention are usually existing varieties included in the species of the test plant, and are preferably taxonomically the same genus. Existing varieties refer to all varieties that exist when the test plant is obtained, and include varieties created by artificial manipulation such as wild type, crossing, and genetic manipulation. Plants having different sesaminol disaccharide or trisaccharide glycoside synthesizing ability in the present invention have high or low expression level of the above gene or sesaminol GGT activity of the protein encoded by the above gene with respect to all existing varieties. No need. If the expression level of the gene or the activity of sesaminol GGT of the protein is high with respect to a specific existing variety, it can be selected as a plant having a high ability to synthesize sesaminol disaccharide or trisaccharide glycoside.

Next, another example (second example) of the method for selecting plants of the present invention will be described.
The second example of the method for selecting a plant of the present invention includes a step of measuring the expression level of a polynucleotide containing at least a part of the nucleotide sequence of SEQ ID NO: 3, 4 or 6, and uses the expression level as an index. , Sesaminol disaccharide or trisaccharide Glycoside Plants with different ability to synthesize are selected.
The polynucleotide is a polynucleotide containing the nucleotide sequence of SEQ ID NO: 3, 4 or 6, and more preferably a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, 4 or 6. In the present invention, it is preferable to use the expression level of the polynucleotide of the nucleotide sequence of SEQ ID NO: 3, 4 or 6 as an index. The expression of these two or more nucleotide sequences may be used as an index. The method for measuring the expression level of the polynucleotide of the present invention is not particularly limited, and RNA may be extracted from the plant body or a portion thereof by the method described above, and the mRNA of the polynucleotide may be quantified.
A plant having a high expression level of the gene having the nucleotide sequence of SEQ ID NO: 3, 4 or 6 has a high expression level of the protein SiGGT21842, the protein SiGGT21842R229Q, or the protein SiGGT21646.
Therefore, a plant having a high expression level of a gene having the nucleotide sequence of SEQ ID NO: 3, 4 or 6 can be selected as a plant having a high ability to synthesize sesaminol disaccharide or trisaccharide glycoside.
In addition, a plant having a low expression level of the gene having the nucleotide sequence of SEQ ID NO: 3, 4 or 6 has a low expression level of the protein SiGGT21842, the protein SiGGT21842R229Q, or the protein SiGGT21646.
Therefore, a plant having a low expression level of the gene having the nucleotide sequence of SEQ ID NO: 3, 4 or 6 can be selected as a plant having a low ability to synthesize sesaminol disaccharide or trisaccharide glycoside.

In the selection method of the present invention, the expression level of the polynucleotide is compared among a plurality of plants, and the plant having a high expression level is regarded as a plant having a high ability to synthesize sesaminol disaccharide or trisaccharide glycoside. Can be sorted. It is preferable that the plurality of plants to be compared are taxonomically the same species or genus.
A plant having a high ability to synthesize sesaminol disaccharide or trisaccharide glycoside is preferably a plant having a high ability to synthesize sesaminol disaccharide or trisaccharide glycoside with respect to the existing varieties contained in the seed of the plant. be. For example, by comparing the expression level of the above-mentioned polynucleotide between a certain plant body (test plant body) and a plant body of an existing cultivar and selecting a plant body having a higher expression level than the existing cultivar, the existing cultivar On the other hand, a plant having a high ability to synthesize sesaminol disaccharide or trisaccharide glycoside can be selected.
Further, for example, by comparing the expression level of the above polynucleotide between the test plant body and the plant body of the existing cultivar and selecting the plant body having the expression level lower than that of the existing cultivar, the existing cultivar can be compared. Plants having a low ability to synthesize sesaminol disaccharide or trisaccharide glycosides can be selected.

The method for selecting plants of the present invention is preferably used for selecting plants of the genus Sesamum, and more preferably used for selecting sesame seeds. The plant may be a wild-type plant, a mutated plant, a plant obtained by mating selection, or the like.

Hereinafter, examples will be shown in which the present invention will be described in more detail. The present invention is not limited to these examples.

The abbreviations of the compounds used in Examples and the like will be described below.
SG1: Sesaminol 2'-O-β-D-glucoside Epi-SG1: Sesaminol 2'-O-β-D-glucoside epimer SG2 (2): Sesaminol 2'-O-β-D-glucosyl ( 1 → 2) -β
-D-Glucoside SG2 (6): Sesaminol 2'-O-β-D-Glucoside (1 → 6) -β
-D-Glucoside Epi-SG2 (6): Sesaminol 2'-O-β-D-Glucoside (1 → 6) -β
-D-Glucoside Epimer SG3 (2,2): Sesaminol 2'-O-β-D-Glucoside (1 → 2) -O
-[Β-D-Glucoside (1 → 2)]-β-D-Glucoside SG3 (2,6): Sesaminol 2'-O-β-D-Glucoside (1 → 2) -O
-[Β-D-Glucoside (1 → 6)]-β-D-Glucoside Epi-SG3 (2,6): Sesaminol 2'-O-β-D-Glucoside (1 → 2) -O
-[Β-D-Glucoside (1 → 6)]-β-D-Glucoside
Epimer

<Isolation of candidate genes for specific glycosyltransferases of sesaminol glycosides>
The molecular biology techniques used herein followed the methods described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 2001), unless otherwise detailed.
A UGT-like sequence different from the glycosylated enzyme gene (UGT) of the prior art document (Non-Patent Document 1) was searched from the Expressed Tag Seq (EST) of sesame seeds by homology search. As a result, UGT94D1 consisting of the amino acid sequence of SEQ ID NO: 7 and the protein SiGGT21842 consisting of the amino acid sequence of SEQ ID NO: 1 showing sequence identity of 54% at the DNA level and 42% at the amino acid level, and 56% at the DNA level, amino acid level. The protein SiGGT21646 consisting of the amino acid sequence of SEQ ID NO: 5 showing 45% sequence identity was found in. In addition, these proteins are also described as "SiGGT protein", and the genes encoding these proteins are also described below as "SiGGT gene".
Since both of these SiGGT genes have a nucleotide length of about 1400 bp and are considered to contain a full-length open reading frame (ORF), the following primer sets (SEQ ID NOs: 9 and 10 and SEQ ID NOs: 11 and 12) The cDNA prepared from sesame seeds was used as a template for amplification by PCR.
The sesame seed cDNA was obtained by extracting total RNA from sesame seeds using the RNeasy Plant Mini kit (QIAGEN) and reverse transcribing (RT) 0.5 μg of it with a Random Oligo-dT primer.

Forward primer for protein SiGGT21842 (SiGGT-21842-pET-FW):
5'-TGCCGCGCGCGCAGCCATATGGAGAAAAAAAGAAGCTAAATTCAG-3'(SEQ ID NO: 9)
Reverse primer for protein SiGGT21842 (SiGGT-21842-pET-RV):
5'-GTTAGCAGCCGGGATCCTCACTTTTCACTGCCTAATTG-3'(SEQ ID NO: 10)

Forward primer for protein SiGGT21646 (SiGGT-21646-pET-FW):
5'-TGCCGCGCGCGCAGCCATATTGGGTTCAGACAAAGAGTGC-3'(SEQ ID NO: 11)
Reverse primer for protein SiGGT21646 (BamHI-SiGGT-21646-pET-RV):
5'-GTTAGCAGCCGGGATCCTTAATTCCCACAAATTTGTGCTAACTTG-3'(SEQ ID NO: 12)

The PCR reaction solution (50 μL) had a composition of 1 μL of sesame seed-derived cDNA, 1 × ExTaq buffer (TaKaRaBio), 0.2 mM dNTPs, 0.4 pmol / μL of each primer, and 2.5 U of ExTaq polymerase. The PCR reaction was carried out at 94 ° C. for 3 minutes, followed by a reaction at 94 ° C. for 1 minute, 50 ° C. for 1 minute, and 72 ° C. for 2 minutes for a total of 30 cycles to amplify the DNA. The obtained PCR product was electrophoresed on a 0.8% agarose gel and stained with ethidium bromide. As a result, a band of amplified DNA was confirmed in the vicinity of the 1400 bp size estimated from each template DNA. The results are shown in FIGS. 2 (a) and 2 (b).
FIG. 2A is a photograph of agarose gel electrophoresis of a PCR product obtained using a sesame seed cDNA as a template and a forward primer for protein SiGGT21842 and a reverse primer for protein SiGGT21842, and FIG. 2B is a sesame gel electrophoresis. It is a photograph of agarose gel electrophoresis of a PCR product obtained by using a forward primer for protein SiGGT21646 and a reverse primer for protein SiGGT21646 using seed cDNA as a template.
In FIG. 2A, lane A is a PCR product using a primer for protein SiGGT21842, and lane B in FIG. 2B is a PCR product using a primer for protein SiGGT21646. M in FIGS. 2 (a) and 2 (b) is a marker.
In addition, the bands near 1400 bp in FIGS. 2 (a) and 2 (b) are indicated by arrows.
In FIG. 2, M is a size marker.

<Construction of expression vector>
The PCR product was then subcloned into the E. coli expression vector pET15b (Novagene) using the GeneArt Seamless Cloning and Enzyme Mix (Thermo Fisher Scientific) by the method recommended by the manufacturer. The site encoding the His tag located upstream of the NdeI site of this vector matches the ORF of the SiGGT gene, and it was designed to be expressed as a chimeric protein in which the His tag is fused to the N-terminal of the SiGGT protein.

Next, the nucleotide sequence of the subcloned expression vector was analyzed with a capillary sequencer (Genetic Analyzer 3130xl, Applied Biosystems).

As a result, it was confirmed that a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 was incorporated into the expression vector in which the PCR product using the primer for protein SiGGT21842 was subcloned. The nucleotide sequence of SEQ ID NO: 4 is a nucleotide sequence in which the 686th guanine residue of the nucleotide sequence of SEQ ID NO: 3 is an adenine residue, and the nucleotide sequence of SEQ ID NO: 4 is an allele of the nucleotide sequence of SEQ ID NO: 3. It was considered. The protein encoded by SEQ ID NO: 4 consists of the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 is an amino acid sequence in which the 229th arginine residue of the amino acid sequence of SEQ ID NO: 1 is a glutamine residue.

In addition, it was confirmed that a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 was incorporated into the expression vector in which the PCR product using the primer for protein SiGGT21646 was subcloned.

The expression vector in which the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is incorporated is designated as the SiGGT21842 expression vector, and the expression vector in which the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 is incorporated is designated as the SiGGT21842R229Q expression vector, and the expression vector of SEQ ID NO: 6 is used. An expression vector in which a polynucleotide consisting of a nucleotide sequence was incorporated was designated as a SiGGT21646 expression vector.
It was also confirmed that there were no errors in the connection part of each expression vector.

<Expression and purification of recombinant protein>
Escherichia coli BL21 (DE3) strain was transformed according to a conventional method using the SiGGT21842 expression vector, the SiGGT21646 expression vector, and the expression vector pET15b (empty pET15b vector) as a negative control.
Each of the obtained transformants was cultured with shaking at 37 ° C. overnight in 4 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 1 g / L NaCl) containing 50 μg / mL ampicillin. 2 mL of the culture solution reached the quiescent period was inoculated into 98 mL of a medium having the same composition, and cultured with shaking at 25 ° C. for 20 hours using Overnight Expression Expression System 1 (Novagen).

All the following operations were performed at 4 ° C.
The cultured transformant was collected by centrifugation (5,000 × g, 10 min), and Buffer S [20 mM HEPES buffer (pH 7.5), 20 mM imidazole, 14 mM β-mercaptoethanol] was added to 1 mL / g cell. It was added and suspended so as to become.
Subsequently, ultrasonic crushing (15 sec × 8 times) was performed, and centrifugation (15,000 × g, 15 min) was performed. The obtained supernatant was recovered as a crude enzyme solution.
The crude enzyme solution was loaded on His SpinTrap (GE Healthcare) equilibrated with Buffer S and centrifuged (70 × g, 30 sec).
After washing with Buffer S, the protein bound to the column was eluted stepwise with 5 mL each of Buffer S containing 100 mM and 500 mM imidazole.
Each eluted fraction was buffer-substituted with 20 mM HEPES buffer (pH 7.5) and 14 mM β-mercaptoethanol using Microcon YM-30 (Amicon). This buffer-substituted product enzyme solution was used for the following reaction.

Each eluted fraction was separated by SDS-PAGE, and the separated polyacrylamide gel was stained with CBB.
In the 500 mM imidazole elution fraction obtained from the crude enzyme solution obtained using the SiGGT21842 expression vector and the SiGGT21646 expression vector, a protein band was observed at a position near the molecular weight of 50 kDa, which is the estimated molecular weight of the HisTag-fused SiGGT protein. rice field.

Furthermore, Western blotting analysis using an anti-His tag-antibody was performed.
In the 500 mM imidazole-eluting fraction obtained from each crude enzyme solution obtained by using the SiGGT21842 expression vector and the SiGGT21646 expression vector, a protein was detected at a molecular weight of about 50 kDa. On the other hand, the protein having a molecular weight of about 50 kDa was not detected in the eluted fraction (hereinafter, also referred to as “negative control fraction”) obtained from the crude enzyme solution obtained by using the expression vector pET15b.

Therefore, from the results of SDS-PAGE and the results of Western blotting analysis, it was confirmed that each transformant expressed the protein SiGGT21842 or the protein SiGGT21646.

Further, a transformant was prepared in the same manner as described above except that the SiGGT21842R229Q expression vector was used instead of the SiGGT21842 expression vector and the SiGGT21646 expression vector, and the protein SiGGT21842R229Q was purified.

(Example 1: Functional analysis of protein SiGGT21842 1)
The reaction between the protein SiGGT21842 and SG1 was analyzed by the method shown below.

(LC condition)
Device name: LCMS-IT-TOF (Manufacturer: Shimadzu: Prominence LC20 series)
Column: Waters Sunfire C18 3.5um 2.0mm I. D. × 20 mm
Mobile phase: A: 10 mM ammonium acetate aqueous solution B: Acetonitrile flow rate: 0.4 mL / min
Column oven: 40 ° C
Gradient: B (v / v%): 20% (0 min) -80% (6 min) -80% (8 min) -20% (8.1 min) -20% (12 min)
Under this condition, the peak of SG2 (2) of the sesaminol disaccharide glycoside has a retention time of 3.91 minutes, and the peak of SG2 (6) has a retention time of 3.77 minutes, and these are separated.

(MS condition)
ESI (negative mode)
Selected ion monitoring: m / z 317,479,641,687,803 and 849

Reaction solution according to Example 1 (2 mM UDP-glucose, 0.1 mM SG 1,100 mM potassium phosphate buffer (pH 7.5) as a glycosyl acceptor substrate, and 25 μL of a purified protein SiGGT21842 solution as an enzyme solution were prepared to 50 μL with distilled water). Was prepared and reacted at 30 ° C. for 1 hour. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions.

Further, as a positive control, a standard product of SG2 (2) was used, and reverse phase LC-MS analysis was performed under the above conditions.

As a negative control, a reaction solution (2 mM UDP-glucose, 0.1 mM SG1,100 mM potassium phosphate buffer (pH 7.5) as a glycosyl acceptor substrate, and 25 μL of the negative control fraction was prepared to 50 μL with distilled water) was prepared. Then, the reaction was carried out at 30 ° C. for 1 hour. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions.

The results of these reversed-phase LC-MS analyzes are shown in FIG.
FIG. 3A is a chart of reverse phase LC-MS analysis of the reaction solution using SG1 and the purified protein SiGGT21842 solution, and FIG. 3B is a standard reverse phase LC of SG2 (2). -MS analysis chart, FIG. 3 (c) is a chart of reverse phase LC-MS analysis of the reaction solution using SG1 and the negative control fraction.
That is, FIG. 3A is a chart of reverse phase LC-MS analysis of the reaction solution of Example 1, and FIG. 3B is a standard SG2 (2) as a positive control of Example 1. It is a chart of the reverse phase LC-MS analysis using the above, and FIG. 3 (c) is a chart of the reverse phase LC-MS analysis of the reaction solution of the negative control of Example 1.

As shown in FIG. 3A, when the protein SiGGT21842 was reacted with SG1, a new peak having a molecular weight that seems to have added one glucose to SG1 was detected. This coincided with the peak of SG2 (2) of the standard product with a positive control holding time of 3.91 minutes shown in FIG. 3 (b). On the other hand, as shown in FIG. 3C, in the negative control of Example 1, SG1 of the glycosyl acceptor substrate and its epimer remained, and in Example 1, the specific reaction of the protein SiGGT21842. It was confirmed that SG2 (2) was generated.
The peak with a retention time of 4.39 minutes shown in FIG. 3C is considered to be SG1, and the peak with a retention time of 4.46 minutes is considered to be the peak of Epi-SG1.

(Example 2: Functional analysis of protein SiGGT21842 2)
The reaction between the protein SiGGT21842 and SG2 (2) was analyzed by the method shown below.

50 μL of the reaction solution (2 mM UDP-glucose, 0.1 mM SG2 (2) as the glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5)), and 25 μL of the purified protein SiGGT21842 solution as the enzyme solution according to Example 2 with distilled water. Was prepared and reacted at 30 ° C. for 1 hour. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions.

As a negative control, a reaction solution was prepared using 25 μL of a negative control fraction instead of the enzyme solution in the above reaction solution, and reacted at 30 ° C. for 1 hour. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions.

The results of these reversed-phase LC-MS analyzes are shown in FIG.
FIG. 4A is a chart of reverse phase LC-MS analysis of the reaction solution using SG2 (2) and the purified protein SiGGT21842 solution, and FIG. 4B is a chart of SG2 (2) and a negative control image. It is a chart of the reverse phase LC-MS analysis of the reaction solution using a minute.
That is, FIG. 4 (a) is a chart of the reverse phase LC-MS analysis of the reaction solution of Example 2, and FIG. 4 (b) is the reverse phase LC- of the reaction solution of the negative control of Example 2. It is a chart of MS analysis.

As shown in FIG. 4A, when the protein SiGGT21842 and SG2 (2) were reacted, a new peak thought to be a trisaccharide glycoside in which one glucose was added to SG2 (2) was retained for a retention time of 3. Detected at .32 minutes. From the position specificity of the protein SiGGT21842, it was inferred that this product was SG3 (2,2) in which the 2-position of the second glucose residue of SG2 (2) was further glucosylated. There are no reports of such sugar chain-bound sesaminol glycosides in sesame seeds.
The peak with a holding time of 3.90 minutes in FIG. 4A is the peak of SG2 (2).
The peak with a holding time of 3.89 minutes shown in FIG. 4 (b) is the peak of SG2 (2).

(Example 3: Functional analysis of protein SiGGT21842 3)
The reaction between the protein SiGGT21842 and SG2 (6) was analyzed by the method shown below.

50 μL of the reaction solution according to Example 3 (2 mM UDP-glucose, 0.1 mM SG2 (6) as a glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5)), and 25 μL of purified protein SiGGT21842 solution as an enzyme solution with distilled water. Was prepared and reacted at 30 ° C. for 1 hour. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions.

As a positive control, a reaction solution (2 mM UDP-glucose, 0.1 mM SG3 (2,6) as a glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5)), and a negative control fraction of 25 μL were added to 50 μL of distilled water. Was prepared and reacted at 30 ° C. for 1 hour. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions.

As a negative control, a reaction solution (2 mM UDP-glucose, 0.1 mM SG2 (6) as a glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5)), and a negative control fraction of 25 μL were prepared to 50 μL with distilled water. ) Was prepared and reacted at 30 ° C. for 1 hour. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions.

The results of these reversed-phase LC-MS analyzes are shown in FIG.
FIG. 5 (a) is a chart of reverse phase LC-MS analysis of the reaction solution using SG2 (6) and the purified protein SiGGT21842 solution, and FIG. 5 (b) is SG3 (2, 6) and negative. It is a chart of the reverse phase LC-MS analysis of the reaction solution using the control fraction, and FIG. 5 (c) shows the reverse phase LC-MS analysis of the reaction solution using SG2 (6) and the negative control fraction. It is a chart.
That is, FIG. 5 (a) is a chart of the reverse phase LC-MS analysis of the reaction solution of Example 3, and FIG. 5 (b) is the reverse phase LC-MS of the reaction solution of the positive control of Example 3. It is a chart of MS analysis, and FIG. 5 (c) is a chart of reverse phase LC-MS analysis of the reaction solution of the negative control of Example 3.
The peak with a holding time of 3.30 minutes in FIG. 5 (b) is the peak of SG3 (2, 6).

As shown in FIG. 5 (a), when the protein SiGGT21842 and SG2 (6) were reacted, a trisaccharide glycoside in which one glucose was added to the 2-position of the first glucose residue of SG2 (6). A new peak with a molecular weight that appears to be a glycoside was detected with a retention time of 3.31 minutes. The new peak generated from the product retention time was the SG3 (2,6) peak.
The peak with a holding time of 3.80 minutes in FIG. 5 (c) is the peak of SG2 (6).

(Example 4: Functional analysis of protein SiGGT21842 4)
A method for producing SG3 (2,6) using sesaminol as a starting material was examined by the method shown below.

(Negative control)
A reaction solution (2 mM UDP-glucose, 0.1 mM sesaminol as a glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5), 25 μL of negative control fraction prepared to 50 μL with distilled water) was prepared at 30 ° C. for 1 hour. It was reacted. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions. The results are shown in FIG. 6 (a).
FIG. 6A is a chart of reverse phase LC-MS analysis of the reaction solution of the negative control of Example 4.

(First reaction)
The reaction solution (2 mM UDP-glucose, 0.1 mM sesaminol as a glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5), UGT71A9, UGT94D1 adjusted to 50 μL with distilled water) was reacted at 30 ° C. for 1 hour. rice field. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions. The results are shown in FIG. 6 (b).
FIG. 6B is a chart of reverse phase LC-MS analysis of the reaction solution of the first reaction of Example 4.
UGT71A9 and UGT94D1 were prepared by the method described in Non-Patent Document 1.

(Second reaction)
Prepare 25 μL of a reaction solution (2 mM UDP-glucose, 0.1 mM sesaminol as a glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5), UGT71A9, UGT94D1, purified protein SiGGT21842 solution) in distilled water to 50 μL, and prepare at 30 ° C. The reaction was carried out for 1 hour. Reverse-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions. The results are shown in FIG. 6 (c).
FIG. 6C is a chart of reverse phase LC-MS analysis of the reaction solution of the second reaction of Example 4.

In FIG. 6A, peaks were observed at the positions where the holding time was 6.06 minutes and the holding time was 6.28 minutes.
In FIG. 6B, peaks were observed at the positions where the holding time was 3.77 minutes and the holding time was 3.85 minutes.
In FIG. 6 (c), peaks were observed at the positions where the holding time was 3.30 minutes and the holding time was 3.40 minutes.

The peak with a retention time of 6.06 minutes in FIG. 6 (a) is the peak of sesaminol, and the peak with a retention time of 6.28 minutes is the peak of episesaminol, which is an epimer of sesaminol.

The peak with a retention time of 3.77 minutes in FIG. 6 (b) is the peak of SG2 (6), and the peak with a retention time of 3.85 minutes is the peak of Epi-SG2 (6).
From this result, it was confirmed that SG2 (6) was produced by UGT71A9 and UGT94D1. It was also suggested that these enzymes did not strictly distinguish epimers.

The peak with a retention time of 3.30 minutes in FIG. 6 (c) is the peak of SG3 (2,6), and the peak with a retention time of 3.40 minutes is the peak of Epi-SG3 (2,6).

From these results, it was confirmed that when UGT71A9, UGT94D1 and the protein SiGGT21842 were reacted with sesaminol as enzymes, glycosylation at three sites occurred consecutively and SG3 (2,6) was produced.
That is, as the first reaction, UGT71A9 and UGT94D1 react with sesaminol, and the reaction occurs.
It was confirmed that SG2 (6) was produced, and as a second reaction, the proteins SiGGT21842 and SG2 (6) reacted to produce SG3 (2,6).
It was also suggested that the protein SiGGT21842 did not strictly distinguish epimers.

The combinations of the glycosyl acceptor, the enzyme solution, and the like used in the reaction solution and the like for the functional analysis of the protein SiGGT21842 in Examples 1 to 4 are summarized in Table 1 below.

(Example 5: Functional analysis of protein SiGGT21842R229Q)
When the same tests as above (functional analysis 1 of protein SiGGT21842 1) to (functional analysis 4 of protein SiGGT21842) were performed using protein SiGGT21842R229Q instead of protein SiGGT21842, the protein SiGGT21842R229Q has the same function as protein SiGGT21842. It has been found.

Based on the above, the protein SiGGT21842 and the protein SiGGT21842R229Q are UDP-sugar-specific glycosyltransferases that have not been identified so far, and their functions are the second glucose of SG2 (2) (the second glucose residue of SG1). It was found that it has a function of glucosylating the 2-position of the residue and the 2-position of the first glucose residue of SG2 (6).

(Functional analysis of protein SiGGT21646)
The function of the protein SiGGT21646 was analyzed by the methods shown in Test Examples 1 to 7 below.

(Test Example 1) to (Test Example 7)
The reaction solutions of Test Examples 1 to 7 were prepared as shown in Table 2 for the combination of the glycosyl acceptor substrate and the enzyme.
Each reaction solution contained 25 μL of 2 mM UDP-glucose, 0.1 mM glycosyl acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5), enzyme solution or negative control fraction, and was prepared to 50 μL with distilled water.
That is, the reaction solution of Test Example 1 contains sesaminol as a glycosyl acceptor substrate and the protein SiGGT21646 as an enzyme.
The reaction solution of Test Example 2 contains SG2 (2) as a glycosyl acceptor substrate and the protein SiGGT21646 as an enzyme.
The reaction solution of Test Example 3 contains sesaminol as a glycosyl acceptor substrate, and UGT71A9 and protein SiGGT21646 as enzymes.
The reaction solution of Test Example 4 contains SG2 (2) as a glycosyl acceptor substrate and UGT94D1 as an enzyme.
The reaction solution of Test Example 5 contains sesaminol as a glycosyl acceptor substrate and contains UGT71A9 and UGT94D1 as enzymes.
The reaction solution of Test Example 6 contains SG2 (6) as a glycosyl acceptor substrate and the protein SiGGT21646 as an enzyme.
The reaction solution of Test Example 7 contains SG2 (6) as a glycosyl acceptor substrate and UGT94D1 as an enzyme.

Next, each reaction solution was reacted at 30 ° C. for 1 hour. Reversed-phase LC-MS analysis was performed on 5 μL of the reaction solution after the reaction under the above conditions. The results are shown in FIGS. 7 to 13.
FIG. 7 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 1.
FIG. 8 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 2.
FIG. 9 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 3.
FIG. 10 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 4.
FIG. 11 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 5.
FIG. 12 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 6.
FIG. 13 is a chart of reverse phase LC-MS analysis of the reaction solution of Test Example 7.

As shown in FIG. 7, when the protein SiGGT21646 was reacted with sesaminol, no product was observed.
On the other hand, as shown in FIG. 8, when the protein SiGGT21646 was reacted with SG2 (2), a peak to which one molecule of glucose was added was observed, which was identified as Sesamino SG3 (2,6) from the retention time.
Further, as shown in FIG. 9, when UGT71A9 and the protein SiGGT21646 were reacted with sesaminol, a peak to which two molecules of glucose were added was observed, which was identified as sesaminol SG2 (6) from the retention time.
As shown in FIGS. 10 and 11, these reactions are similarly observed in UGT94D1, and as shown in FIGS. 12 and 13, these proteins have almost no activity to glucosylate SG2 (6). , Protein SiGGT21646, like UGT94D1, is a sugar-specific glycosyltransferase that glucosylates the 6-position of the glucose residue of SG1 and the 6-position of the first glucose residue of SG2 (2). Shown.

According to the present invention, the protein (P1) can be used to glucosylate the 2-position of the glucose residue of SG1 and the 2-position of the first glucose residue of SG2 (6). SG2 (2) and SG3 (2,6) can be produced.
Further, the protein (P1) can be used to glucosylate the 2-position of the second glucose residue of SG2 (2), and SG3 (2,2) can be produced.
This time, by analyzing the functions of the protein SiGGT21842 and the protein SiGGT21842R229Q contained in the protein (P1), the entire biosynthetic pathway from sesaminol to SG3 (2,6) was clarified. Therefore, it is possible to provide breeding markers and molecular tools for producing sesaminol triglycosides, which are water-soluble lignan glycosides, and other related compounds not only in plants but also in microorganisms.

Claims (1)

Sesaminol 2'-O-β-D-glucosyl (1 → 2) -O- [β-D-glucosyl (1 → 2)] -β-D-glucoside represented by the following formula (5).

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