JP2000300279A - Low-temperature expression-type fructan-metabolizing enzyme gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene comprising a base sequence encoding a specific amino acid sequence, capable of improving the cold tolerance of plants and useful e.g. for producing an enzyme producing a fructo- oligosaccharide having the prophylactic effect of decayed tooth, the propagating action of intestinal bifidus bacillus or the like. SOLUTION: This enzyme gene is a new wheat invertase gene comprising a base sequence encoding an amino acid sequence expressed by the formula, can improve the cold tolerance and snow endurance of plants and is useful e.g. for producing an enzyme producing a fructo-oligosaccharide helpful for human health such as the prophylactic effect of decayed tooth, the propagating action of intestinal bifidus bacillus or the like. The enzyme gene is obtained by extracting mRNA from the crown part of winter habit wheat in a usual way, by producing a cDNA library using it and by screening the library through the hybridization assay utilizing a probe where a partial fragment of an invertase gene is labeled with a radioisotope.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the ability of plants to be cold-tolerant (freeze-tolerance <ability to survive for long periods at minus temperatures>) and snow-tolerance <to survive for long periods of time under snow cover at 0 ° C. in the dark. Capacity> etc.), and a gene having an enzyme involved in the metabolism of fructan that acts as a fructooligosaccharide that is extremely useful for human health.
A novel sucrose derived from autumn wheat (PI173438) having a high degree of cold tolerance: fructan 6-fructosyltransferase (sucrose: fructan 6)
-Fructosyltransferase) and a novel invertase (invertase) gene derived from wheat sown in the same autumn (PI173438).

[0002]

2. Description of the Related Art Among wintering crops, wheat and grasses express low-temperature-expressing fructan-metabolizing enzymes in a process of acclimating to a low temperature (hereinafter referred to as hardening) from fall to winter due to a decrease in temperature. As a result, fructan, a polysaccharide, is accumulated in vacuoles as an energy source in winter. Overwintering crops in the severe cold region are mainly cultivated in the long-term snowy region, so it is known that varieties with higher fructan accumulation have more wintering properties.

[0003] Fructan not only functions as an energy source during wintering, but also plays a major role in preventing freezing and controlling osmotic pressure in the freezing and drying resistance of plants.

[0004] In wheat, varieties having high frost resistance and snow resistance are being developed in the northern countries. However, the conventional breeding method based on crossing techniques obtains plants having a capability exceeding the existing varieties. It is difficult. So recently,
There is a need to improve varieties by biotechnological means.

[0005] Further, fructooligosaccharides are functional oligosaccharides useful for human health, such as anti-caries effects and intestinal bifidobacterial growth, and fructan-synthesizing genes are attracting attention in industrial use for fructo-oligosaccharide production. .

[0006] There are several types of enzymes related to fructan synthesis and their isozymes depending on the reaction stage and the binding mode. Specifically, sucrose: fructan 6-fructosyltransferase mainly acts on the synthesis of fructan of trisaccharide and tetrasaccharide from sucrose, and invertase acts as an enzyme for decomposing sucrose (disaccharide). Also, plant invertase having fructan synthesizing ability has a function of synthesizing trisaccharide under high concentration of sucrose.

[0007]

However, sucrose: fructan 6-fructosyltransferase has been reported from barley, and invertase has been reported from several other plants, all of which are expressed at room temperature. Since it is isolated under conditions, it is not a gene that is expressed at low temperatures and functions as an enzyme. Therefore, it is considered that it is difficult to improve the cold resistance of the plant even if the conventional sucrose: fructan 6-fructosyltransferase gene and invertase gene are used.

[0008] Accordingly, the present invention provides an autumn wheat seed line PI173438 (highly cold-tolerant line) which has been sufficiently hardened in order to isolate a fructan metabolizing enzyme gene which is expressed during hardening due to a temperature decrease from autumn to winter. )
And to provide the gene.

Another object of the present invention is to provide a wheat-derived fructan synthesis gene which is useful for the production of a functional polysaccharide useful for human health, such as a caries prevention effect and an intestinal bifidobacteria growth effect.

It is a further object of the present invention to provide a method for isolating the above gene that functions as an enzyme in a low-temperature environment.

[0011]

In order to achieve the above object, the present invention provides a wheat sucrose: fructan 6-character sequence comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 shown in FIG. Provide a fructosyltransferase gene.

The wheat sucrose: fructan 6-fructosyltransferase gene consisting of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 shown in FIG. 1 has a protein consisting of 1648 bases and 616 amino acids, and is derived from barley. It has 93% (amino acid sequence level) homology with the gene.

The wheat sucrose: fructan 6-fructosyltransferase gene having a base sequence encoding the amino acid sequence of SEQ ID NO: 1 shown in FIG. 1 has an enzymatic function under a low temperature environment and is introduced into a plant. This gives the plant cold resistance.

Further, the above-mentioned wheat sucrose: fructan 6-fructosyltransferase gene comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 shown in FIG. 1 has a function of synthesizing fructooligosaccharides useful for human health. Things.

Further, in order to achieve the above-mentioned object, the present invention provides a wheat invertase gene comprising a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 shown in FIG.

The wheat invertase gene consisting of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 shown in FIG. 2 is 55% (amino acid sequence level) of the same gene derived from 1986 bases, 662 amino acids and sugarcane. They have homology.

The wheat invertase gene consisting of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 shown in FIG. 2 has an enzymatic function under a low-temperature environment, and is introduced into plants to produce It gives cold resistance.

Further, the wheat invertase gene comprising the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 shown in FIG. 2 has a function of synthesizing fructooligosaccharides useful for human health.

Accordingly, the present invention relates to a single gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 shown in FIG. 1 and a gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 shown in FIG. The present invention also provides a method for isolating a low-temperature-expressed fructan metabolizing enzyme gene that is involved in improving cold resistance by hardening (low-temperature acclimation). Extracting mRNA from the autumn wheat line PI173438 (having high snow resistance);
CDNA and cDNA library based on EMBL / Genebank / DDBJDN
A. Specific primers for the two types of sucrose derived from several plants, which are published by Databank, with reference to the nucleotide sequences highly conserved in the fructan 6-fructosyltransferase gene and the invertase gene. And using the designed specific primers of the two genes,
A PCR (polymerase chain reaction) reaction is performed using the above cDNA as a template to amplify the fragments of the two genes to obtain an amplified fragment, and the above cDNA library is probed with the above amplified fragment as a probe. By a hybridization assay to isolate a clone containing the full length of the gene.

One of the specific primers of the above two genes has one of the following nucleotide sequences (Forward): 5'ATGGATAT / C
-GAT / CCCNATC / CG-
G (SEQ ID NO: 3), and the other has the following base sequence (Reverse): 5′CCNGGTTNG
-CA / GTTA / GTTA / GA-
A (SEQ ID NO: 4).

That is, the fructan metabolizing enzyme gene of the present invention is a low-temperature-expressing fructan metabolizing enzyme gene.
As a method for isolating these low-temperature-expressed fructan metabolizing enzyme genes, the following process is specifically used.

For more information, please refer to the
Autumn wheat wheat PI hardened (cold acclimated) until 2 days
MRNA is extracted from 173438 (having high snow resistance). Then, based on the mRNA, cDNA and c
Create a DNA library.

Next, EMBL / Genebank / D
Detailed analysis of the nucleotide sequences of the sucrose: fructan 6-fructosyltransferase gene and the invertase gene derived from several plants disclosed in the DBJ DNA Data Bank, and the highly conserved nucleotide sequences in all genes For reference, one set (the two types of genes) of specific primers is designed to enable simultaneous amplification of the sucrose: fructan 6-fructosyltransferase gene and the invertase gene.

Using the designed one set (two kinds of genes) of specific primers, PCR was performed using the above cDNA as a template.
(Polymerase chain reaction) reaction to amplify the expected fragments of the sucrose: fructan 6-fructosyltransferase gene and the invertase gene (both around 1500 bases for both enzyme genes) to obtain a fragment of the amplified portion .

A clone containing the full length gene is isolated by screening the above cDNA library by hybridization assay using each amplified fragment as a probe. The nucleotide sequence of the isolated clone was analyzed, and a novel sucrose: fructan was used in wheat.
The 6-fructosyltransferase gene and the invertase gene are determined.

[0026]

[Examples] 1) Snow-resistant autumn-sown wheat line PI173438
Preparation of cDNA and cDNA library from cultivated wheat in late September seeded in a bat and hardened under natural conditions until November 22 (Triticum)
astivum L. ) MRNA was extracted from the crown portion of PI173438 (having high snow resistance) by a conventional method. Using 5 μg of the obtained mRNA, cDNA synthesis kit (manufactured by STRATAGENE) was used for cD
NA was synthesized. After connecting adapters to both ends of the cDNA, λZAP Expression vector
(Stratagene), about 6x10
A 6 pfu cDNA library was prepared.

2) Using primers of the above two genes
And a sucrose of known PCR reaction (published in the EMBL / Genebank / DDBJ DNA data bank) using the above cDNA as a template : a region highly conserved in both the nucleotide sequences of the fructan 6-fructosyltransferase gene and the invertase gene (Forward): 5'ATGAAT / C
-GAT / CCCNATC / CG-
G (SEQ ID NO: 3), a specific primer having a base sequence, and (Reverse): 5′CCNGTGTNG.
-CA / GTTA / GTTA / GA-
Using a specific primer having a base sequence of A (SEQ ID NO: 4) and one set (specifically, the two types of genes), a PCR reaction was performed using the cDNA synthesized as described above as a template.

The above PCR reaction was carried out in a 50 μl reaction system. 1 μl of Taq DNA polymerase (5 units / μl) manufactured by Nippon Gene, 5 μl of 10 × polymerase buffer (including MgCl 2), dNTP
5 μl of the solution (10 mM) and the above primers (12 μl)
M) and the cDNA synthesized above was
After adding ng, the total volume of the reaction solution was adjusted to 50 μl with distilled water. P
The reaction conditions and the number of times of CR are shown in Table 1 below.

[0029]

[Table 1] (In Table 1, "denaturation" means a reaction that changes the double strand of DNA into each single strand, and "extension reaction"
(Amplification reaction of NA fragment means "30 times" means that a cycle including denaturation, annealing and extension reaction is repeated 30 times.)

As a result of the PCR reaction, one set having the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4 (2
Known sucrose from the primers of
Fragments of the expected length from the fructan 6-fructosyltransferase gene and the invertase gene (both genes around 1500 bases) were amplified. After isolation and purification of each, a novel sucrose: fructan 6-fructosyl is determined by determining the base sequence of each amplified fragment using a commercially available automatic DNA sequencer (ABI, model 373S) based on a conventional method. It was confirmed that partial fragments of the transferase gene and the invertase gene were isolated.

3) Sucrose of the present invention: fructan 6
Fructosyltransferase gene and inbell
Isolation and nucleotide sequence of a cDNA clone containing the enzyme
Using a filter in which about 100,000 clones of the cDNA library obtained above were plotted, partial fragments of the novel sucrose: fructan 6-fructosyltransferase gene and invertase gene obtained above were radioisotoped. A hybridization assay was performed using the labeled probe.

The hybridization reaction was performed using 50% formamide, 5 × SSPE solution, 5 × Denhardt solution, 0 ×
A 5% SDS solution and 0.2 mg / ml salmon sperm DNA were added, and the reaction was performed at 42 ° C. for 16 hours.

Thereafter, 2 × S
Washing was performed twice using the SC solution and the 0.1% SDS solution at 65 ° C. for 10 minutes.
Next, washing was performed twice using a 0.1 × SSC solution and a 0.1% SDS solution at 65 ° C. for 15 minutes. Using the washed filter and the X-ray film, a positive clone bound to the probe labeled with a radioisotope was screened.

The nucleotide sequence of each of the about 35 positive clones thus obtained was determined using a DNA sequencer manufactured by ABI. From the analysis of the nucleotide sequence of the obtained candidate clone, a novel sucrose consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 shown in FIG.
It was found that the fructan 6-fructosyltransferase gene and the novel invertase gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 shown in FIG. 2 were isolated.

Specifically, the isolated protein has a protein consisting of 1648 bases and 616 amino acids and has 93% (amino acid sequence level) homology with the barley sucrose: fructan 6-fructosyltransferase gene. A novel sucrose of wheat having: a fructan 6-fructosyltransferase gene (consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 shown in FIG. 1), and a protein consisting of 1986 bases and 662 amino acids, and Invertase gene and 55
% (Amino acid sequence level) of a novel invertase gene of wheat (consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 shown in FIG. 2).

In order to confirm the enzymatic activity of the thus obtained genes, an enzymatic reaction was carried out under the following conditions using a yeast culture into which these genes were incorporated. <Enzyme reaction> 20 mM citrate / phosphate buffer pH 5.5 Final substrate concentration 50 mM sucrose Reaction conditions 4 ° C, 17 hours

As a result, in the culture medium of yeast into which the novel sucrose: fructan 6-fructosyltransferase gene has been incorporated, trisaccharide kesttriose and tetrasaccharide kesttetraose using sucrose, which is a disaccharide, as a substrate. It was found that it had the ability to synthesize. Also,
It has been found that the culture medium of yeast incorporating the novel invertase gene has the ability to synthesize trisaccharide kesttriose using the disaccharide sucrose as a substrate and the ability to decompose sucrose into glucose and fructose. Was.

The nucleotide sequence of the gene obtained as described above is as follows.

[0039]

According to the present invention, a novel sucrose: fructan 6-fructosyltransferase gene derived from wheat having an amino acid sequence different from that of a known sucrose: fructan 6-fructosyltransferase gene and a known invertase gene and amino acid sequence Novel invertase genes from different wheats are provided.

Since the novel sucrose: fructan 6-fructosyltransferase gene and the novel invertase gene of the present invention have a fructan metabolizing ability in a low-temperature environment, the sucrose: fructan 6-fructosyltransferase gene or the invertase gene of the present invention can be planted. By being introduced into the body, the cold tolerance of the plant is improved, and a plant variety having high cold tolerance can be provided.

Further, the novel sucrose: fructan 6-fructosyltransferase gene and the novel invertase gene of the present invention have a capability of synthesizing fructooligosaccharides, and thus are useful for human health, such as a caries prevention effect and an intestinal bifidobacteria growth effect. It is also effectively applied as a useful fructan synthase gene to the production of various functional oligosaccharides.

[Brief description of the drawings]

FIG. 1 is a view showing an amino acid sequence described in SEQ ID NO: 1.

FIG. 2 is a view showing an amino acid sequence of SEQ ID NO: 2.

Claims (4)

[Claims] 1. A wheat invertase gene comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 2 shown in FIG. 2. The wheat invertase gene according to claim 1,
The invertase gene according to claim 5, which has a protein consisting of 86 bases and 662 amino acids and has 55% (amino acid sequence level) homology with the same gene derived from sugarcane. 3. The plant according to claim 5, wherein the wheat invertase gene has an enzyme function in a low-temperature environment and imparts cold resistance to the plant by being introduced into the plant.
The described wheat invertase gene. 4. The wheat invertase gene according to claim 5, wherein the wheat invertase gene has a function of synthesizing a fructooligosaccharide useful for human health.