JP2002281975A - Gene belonging to soy nitrate transporter 1 gene family - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new gene (fragment) belonging to NRT1 family in soy, because only the cDNA of NRT2 had been cloned with respect to soy although two families NRT1 and NRT2 that are induced by the exposure to a nitrogen source are known in plant. SOLUTION: The cDNA cloning and gene expression analysis of soy results is the isolation of a new soy gene (fragment) belonging to NRT1 family and the identification of a nucleotide sequence is achieved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a soybean gene and a gene and gene fragment belonging to the nitrate transporter 1 gene induced by exposure to a nitrogen source.

[0002]

2. Description of the Related Art Plants include a transporter, nitrate transporter 1, which is involved in the uptake of nitrate (NO ₃ ⁻ ) induced by exposure to a nitrogen source.
sporter 1) and nitrate transporter 2 (Nitra
te Transporter 2) is known to exist (hereinafter abbreviated as NRT1 and NRT2, respectively). In plants such as Arabidopsis thaliana and tomato, cDNAs of both families have already been cloned. However, in soybean, the NRT2 cDNA has only been cloned.

[0003]

An object of the present invention is to provide novel soybean genes and gene fragments belonging to the NRT1 family.

[0004]

Means for Solving the Problems In order to solve the above problems, the present inventors have conducted intensive studies, and as a result of soybean cDNA cloning and gene expression analysis, a novel soybean gene and gene fragment belonging to the NRT1 family have been identified. Isolation led to the identification of the nucleotide sequence.

[0005] Nitrogen sources are essential for many higher plants.
Nutrients, nitrate (NO _Three ^-) Of them
But it is the main nitrogen source. Night rate assimilation is the root
Of nitrate from soil
Is started by Following that,
Tolite (NO_Two ^-) Via ammonium (NH_Four ⁺)
Is reduced to Protoplasm of root epidermal and cortical cells
Transmembrane uptake is electrochemical nitrate
Proton-Ni
Treat (2H⁺/ NO_Three ^-) Done by symport
You.

[0006] Night rate transporter (NRT)
The identification of the gene that encodes began with studies on mutants lacking nitrate assimilation. Most of such eukaryotic mutants had impaired ability to reduce nitrate, while the remaining mutants were defective in their ability to incorporate nitrate. Characterization, isolation and homology analysis of these mutants defective in nitrate incorporation revealed the existence of two gene families in eukaryotes (Crawford et al., Trends Plant Sci. 199
8; 3: 389-395). These two gene families were named NRT1 and NRT2, respectively, but had a significant feature that there was no nucleotide sequence similarity between the families. The NRT1 and NRT2 families are conserved among higher plants, while the NRT2 family is also conserved in fungi and algae.

[0007] In Glycine max (soy), the high affinity nitrate transporter cDN
A has been cloned and named GmNRT2 as a gene belonging to the NRT2 family, and the expression of GmNRT2 in soybean roots is subject to different regulation depending on the type of nitrogen source supplied;
It has been reported that net nitrate uptake in roots increases with increased expression of the GmNRT2 gene (Amarasinghe et al., Planta 1998; 206: 44-5).
2). However, there is no report on cloning of the nitrate transporter gene of the soybean NRT1 family. Therefore, the present inventors have developed soybean c.
As a result of DNA cloning and gene expression analysis, NRT1
Novel soybean genes and gene fragments belonging to the family have been isolated and their nucleotide sequences identified.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described based on examples with reference to the drawings.

The cDNA cloning of the novel gene and gene fragment of the present invention was performed using a commercially available soybean cDNA library. The DNA manipulation method was in accordance with a conventional method (Sambrook et al., Molecular Cloning: a Laborato
^{ry Manual, 2 nd edn.,} 1989). The obtained cDNA clone was subjected to nucleotide sequence analysis to clarify the gene sequence, and then subjected to homology analysis with a known gene sequence. As a result, in comparison at the amino acid sequence level, there was no homology with the gene belonging to the NRT2 family, and the homology with the gene belonging to the NRT1 family was high. Therefore, it was confirmed that the gene belongs to the soybean NRT1 family. In addition, from the results of hydropathy analysis with known genes, it could be estimated that the gene was a nitrate transporter gene.

[0010] It is known that known nitrate transporter genes exhibit a complex gene expression pattern depending on the type of nitrogen source or the difference in exposure time to the nitrogen source. The case of the novel NRT1 gene of the present invention was similar in that it showed a complicated gene expression pattern.

[0011] The NRT1 gene of the present invention is a known NRT1 gene.
The difference from one gene lies in the difference in the tissues that express it. Known NRT1 genes, such as Arabidopsis (Ar
abidopsis thaliana), rape (Brassica napus),
In the case of tomato (Lycopersicon esculentum), induction of gene expression by a nitrogen source is mainly limited to the root. The NRT1 gene of the soybean of the present invention is clearly different from the known NRT1 gene in that expression was confirmed in both roots and leaves, or only leaves, depending on the conditions of induction by the nitrogen source. It can be said that it is a gene.

The novel gene of the present invention can be used for plant breeding by genetic recombination. For example, it can be used for the creation of a new recombinant plant that can grow with a small amount of nitrogen fertilizer or on a soil with low nitrogen content. Nitrogen fertilizers have been used in large quantities as synthetic fertilizers, and have greatly contributed to increasing food production.
On the other hand, nitrate derived from nitrogen fertilizer exudes from farmland, causing environmental pollution due to eutrophication of lakes and bays, and acidification of soil by nitrogen compounds remaining in farmland, It has been noticeable in recent years. Genetically modified plants that can grow with small amounts of nitrogen fertilizers can help reduce such harm.

When the soybean NRT1 gene of the present invention is used for genetic engineering for improving plant varieties, it is necessary to introduce the gene into plant tissues. For gene transfer, electroporation, polyethylene glycol,
Known methods that do not use a vector, such as a microinjection method and a microprojectile method, can be used. Alternatively, a transformation method using a vector may be used.

The soybean NRT1 gene fragment of the present invention can be used as a probe for obtaining a full-length cDNA clone.

[0015]

EXAMPLES Specific embodiments of the present invention will be shown below as examples, but the present invention is not limited to only these examples.

[0016]

Example 1 As a soybean cDNA library, Stratagene was used. Epicoty of soybean (Glycine max cv. Williams 83) on day 12
l) A cDNA library prepared using the mRNA derived from it.

As a host bacterium, Escherichia coli XL1-Blue was used. For PCR (Polymerase Chain Reaction)
Two primers AAT-N and AAT-C (Table 1)
When GmNRT1-1 cDNA is amplified,
It was cloned from a soybean cDNA library.

PCR for cloning full-length cDNA was performed using 0.1 μg of a soybean cDNA library as a template, 2.5 units of Taq DNA polymerase, and 100 pmol of primers. GmN
The 5'-region of the RT1-1 cDNA is labeled with the primer 5'V
Each cDNA was amplified using a set of EC and 3 ′ VEC (Table 1) and a primer of 5 ′ SBON1 and 3 ′ SBON1 (Table 1) for the 3′-region.
The 5′-ends of the primers 5′SBON1 and 3′SBON1 have cleavage sites for restriction enzymes BamHI and HindIII, respectively. After cutting the amplified PCR fragment with restriction enzymes BamHI or HindIII,
It was inserted into the multiple cloning site of the vector pUC118. Next, each PCR fragment was recombined with an overlapping region having a HindIII cleavage site, and then inserted into the BamHI-HindIII cleavage site of the pBluescript vector. The final recombinant was confirmed by nucleotide sequence analysis.

[0019]

[Table 1]

CDN having homology to GmNRT1-1
In order to search for A, about 1 × 10 ⁵ phage clones of the aforementioned soybean cDNA library were screened.
The screening probe was GmNRT1-1cD.
Using a 452 bp DNA fragment corresponding to nucleotides 816 to 1,267 of NA, detection was carried out using an ECL kit (Amersham-Pharmacia Biotech). As a result, two full-length cDNAs of GmNRT1-1 and GmNRT1-2 could be detected. The respective cDNA base sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

GmNRT1-1 cDNA has a stop codon at the 17th-19th nucleotide site in the same frame upstream of the open reading frame (ORF), and the first methionine residue (Met) in that frame. The presence of a poly (A) tail in the untranslated region (3′-UTR) at the 3 ′ end indicated that the cDNA was a full-length cDNA.
The ORF consisting of 1,794 bp encodes a peptide of 597 amino acids and has a molecular weight of 65,82.
ODa was calculated.

GmNRT1-2 cDNA also has a stop codon at a site 105 nucleotides upstream from the start codon in the same frame upstream of the open reading frame (ORF), and the first methionine residue (Met ), And the presence of a poly (A) tail in the untranslated region (3′-UTR) at the 3 ′ end, indicated that the cDNA was a full-length cDNA. An ORF consisting of 1,818 bp contains 60
A peptide of 5 amino acids was encoded and its molecular weight was calculated to be 66,720 Da.

[0023]

Example 2 It was considered that a gene belonging to the soybean NRT1 family may exist in addition to GmNRT1-1 and GmNRT1-2, and further cDNA cloning was attempted. The experimental method is substantially the same as in Example 1. As a result, three new types of cDNAs could be detected.

The full-length GmNRT1-3 (SEQ ID NO: 3 in the sequence listing) was prepared by RACE (Rapid Amplification of cDNA).
Ends) method, and the SMART RA
CEcDNA Amplification kit
(Clontech) and primer 5'-TTGGATTC
This was performed using ACAACGTTGGACACGG-3 '. In the same frame upstream of the open reading frame (ORF), a stop codon is present at nucleotides 138-140, indicating that the first methionine residue (Met) is present in the clone; Further, since the poly (A) tail exists in the untranslated region (3′-UTR) at the 3 ′ end, GmNRT1-1 and GmNRT1
As in -2, it was confirmed that the cDNA was full-length. The ORF consisting of 1,728 bp encodes a peptide of 575 amino acids and has a molecular weight of 64,
It was calculated to be 120 Da.

On the other hand, GmNRT1-4 (SEQ ID NO: 4 in the sequence listing) and GmNRT1-5 (SEQ ID NO: 5 in the sequence listing) were not full-length cDNAs, but gene fragments. GmN
RT1-4 lacked the 5 'untranslated region (5'-UTR), the 3' untranslated region (3'-UTR) and the poly (A) tail. GmNRT1-5 lacks the 5′-end untranslated region (5′-UTR), but the 3′-end untranslated region (3′-UTR).
-UTR) and poly (A) tail. For the coding region, GmNRT1-4 is approximately 30 N-terminal.
0 amino acids and about 50 amino acids on the C-terminal side were deleted. The coding region of GmNRT1-5 is N
About 20 terminal amino acids were deleted.

[0026]

Example 3 The obtained novel cDNA, GmNRT1-
In order to prove that 1 to GmNRT1-5 belong to the NRT1 gene family, homology analysis with NRT1 proteins of other known plants was performed. G
Based on the sequence of mNRT1-2, the software BL
Analysis was performed using AST (FIG. 1). Homology is AtNR compared to Arabidopsis thaliana
T1 is 31% and AtNRT3 is 31% (Fig. 1). In the case of tomato (Lycopersicon esculentum), LeNRT1-
1 was 31% and LeNRT1-2 was 32% (Table 2), all showing high homology. The homology between the soybean NRT1 families was as high as 97 to 29% (Table 2).

[0027]

[Table 2]

As a result of the hydropathy analysis, similar to the AtNRT1 protein belonging to the NRT1 family of Arabidopsis thaliana, it has two characteristics that it has 12 transmembrane segments and a low hydrophobic region in the center. Was. FIG. 2 shows an example of the result of the hydropathy analysis.

On the other hand, the homology with the NRT2 family of soybeans and other plants based on GmNRT1-2 is as low as 4 to 3% (Table 2), and it can be said that there is substantially no homology. Therefore, the NRT1 family and NR
It was proved that there was a clear difference in the amino acid sequence from the T2 family.

When the above results are combined, the comparison at the amino acid sequence level shows that the novel gene and gene fragment of the present invention belong to the soybean NRT1 family.

[0031]

Example 4 In order to confirm the expression pattern of a gene belonging to the soybean NRT1 family of the present invention, the following test was conducted.

In a photo chamber, soybeans (Glyc
ine max cv. Williams) was grown under a light / dark cycle condition of 22 ° C. and 16 hours / 8 hours. NRT1 with nitrogen source
The result of the gene induction was determined by RT-PCR (Reve
rse Transcriptase-Polymerase Chain Reaction).

For short-term exposure to a nitrogen source, the soybean seeds are sterilized wet vermiculite.
Germinated in the tray of e). The seedlings after 6 days were transferred to a hydroponics pot containing a nutrient solution and treated for 3 to 6 hours.
A nutrient solution substantially free of nitrogen source is 2 mM CaSO
₄ , 1 mM MgSO ₄ , 0.94 mM CaCl ₂ ,
_{0.5mM K 2 SO 4, 0.16mM K} 2 HPO 4,
0.082mM KH ₂ PO _4, 0.024mM Fe-
EDTA, 1.08 mM MES buffer, 0.8
mM KOH, 5 μM H ₃ BO ₃ , 2 μM MnS
O ₄ , 0.8 μM ZnSO ₄ , 0.033 μM CoC
1, 0.65 nM (NH ₄ ) ₆ MO ₇ O ₂₄ . The nutrient solution supplemented with nitrogen contains 10 mM KNO ₃ or 10 mM NH ₄ Cl or 10 mM KNO ₃ .
Both 10 mM NH ₄ Cl were added to the nutrient solution substantially free of the nitrogen source. The pH of the culture was adjusted to 6.0.

For prolonged exposure to a nitrogen source, soybean seeds are sterilized by wet vermiculit
The seeds were germinated in the tray of e) and, after adding one of the three nutrient solutions, they were grown for 7-14 days. The composition of nitrate and ammonium (NH ₄ ⁺ ) replenisher is 25 mM
NH ₄ NO ₃ , 2 mM CaCl ₂ , 3 mM KCl,
1.6 mM MgCl ₂ , 0.375 mM MgSO ₄ ,
0.375mM NaH ₂ PO _4, 0.05mM Fe-
EDTA, 0.01 mM boric acid, 0.045 mM M
nSO ₄ , 0.7 μM ZnSO ₄ , 0.2 μM CuS
O ₄ , 0.2 μM KI. The composition of the ammonium replenisher is the same as the nitrate and ammonium replenisher, except that it contains 25 mM ammonium succinate instead of NH ₄ NO ₃ . The pH of the culture was 6.0
Was adjusted.

RT-PCR was performed using Takara Shuzo's mRNA Se.
The active RT-PCR kit was used. Total RNA was isolated using TRIzol (manufactured by GIBCO BRL). For brief exposure to a nitrogen source, total RNA was isolated from soybean roots. In the case of long-term exposure to nitrogen sources, total RNA from soybean roots and leaves
Was isolated.

GmNRT1-1 and GmNRT1-2
To analyze the expression of both, a primer set of 5 ′ SBON1 and 3 ′ SBON1 (Table 1) was used.
The reverse transcription reaction was performed using 0.1 μg of total RNA and 3′SB
The test was performed at 50 ° C. for 15 minutes using the ON1 primer.
In the subsequent PCR reaction, 25 to 40 cycles of the reaction were performed using a primer set of 5′SBON1 and 3′SBON1 (FIGS. 3 and 4 described below show 25, 30, 3
The results are shown for samples from each cycle of 5, 40, and the time course of the RT-PCR product can be seen). Reactions per cycle were performed at 85 ° C. for 1 minute heat denaturation, 45 ° C. for 1 minute annealing.
ng) at 72 ° C. for 1 minute. Reaction products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. The concentration of the band on the gel was determined using a densitometer (Bio-Rad Mole
Quantification using a cular imager).

In order to distinguish the expression of GmNRT1-1 and GmNRT1-2, 5′Ndif and 3′Ndif
Another primer set (Table 1) was used. These are each one of the nucleotide sequences of GmNRT1-2.
27th to 143rd and 313th to 329th. The products of this reaction were separated by 12% polyacrylamide gel electrophoresis.

The product of RT-PCR is GmNRT1-
1 and GmNRT1-2, the same total RNA and the same primer set 5′Ndif and 3′Ndif were used to determine the RT-PCR using the same Takara Shuzo RNA PCR kit (AMV). Was done. The product of this reaction is a T-vector (Promega
), And nucleotide sequence analysis was performed.

When a transcript of GmNRT2 is to be detected, 5'-
GNT and 3'-GNT (Table 1) were used. mRNA S
Under the experimental conditions of the present invention using the selective RT-PCR kit, no band was detected under the conditions excluding reverse transcriptase. Therefore, it was shown that the band amplified in this example was derived from RNA. FIG. 3 shows the results. The position of the target band is indicated by a wedge-shaped arrow mark.

When exposed to a nitrogen source for a short time (3 hours), GmNRT1-1 and / or GmNRT1-2
No amplified band was detected without a nitrogen source (Nitrogen
-free), growth in the presence of nitrate (Nitrate), growth in the presence of nitrate and ammonium (Nitrate / Am
monium), and when grown in the presence of ammonium (Ammonium), no amplified band was detected (FIG. 3A). The relative intensity of the amplified band was higher in the presence of nitrate, no nitrogen source, and coexistence of nitrate and ammonium.

As a control, the expression of GmNRT2 of the NRT2 family was examined using the same total RNA. As a result, the amplified band of GmNRT2 is
The highest in the presence of nitrate and the next highest in the absence of a nitrogen source, but no amplified band was detected in the presence of ammonium (FIG. 3B)). The GmNRT2 results were in agreement with the literature values (Amarasinghe et al.,
Planta 1998; 206: 44-52). When using total RNA after long-term (6 hours) exposure to a nitrogen source, Gm
NRT1-1 and / or GmNRT1-2 and G
Almost the same results were obtained for mNRT2.

In order to distinguish the expression of GmNRT1-1 and GmNRT1-2, RT-PCR was performed using the primer sets 5′Ndif and 3′Ndif (Table 1).
In this case, a 179 bp band is detected from GmNRT1-1, and a 203 bp band is detected from GmNRT1-2. For total RNA from roots grown in the presence of nitrate, primer set 5'Ndif
RT-PCR using 3'Ndif and
Only the p band was detected (FIG. 3C). further,
Nucleotide sequence analysis confirms that GmNRT1-2
Was confirmed as a transcript. Therefore, in soybean roots grown in the presence of nitrate, GmNRT1-2
Was found to have been expressed. Similarly, in the case without a nitrogen source and in the presence of nitrate and ammonium, only the amplified band of GmNRT1-2 was observed.

For 14 days of prolonged exposure to a nitrogen source, only roots grown in the presence of ammonium contained GmN
Only the RT1-2 band was observed, and nucleotide sequence analysis confirmed that it was derived from GmNRT1-2, but no band was observed in the presence of nitrate and ammonium (FIG. 3D), lane 1 And 2). On the other hand, GmNRT1-1 and GmNRT
Two bands of 1-2 were observed and confirmed by nucleotide sequence analysis (FIG. 3D), lanes 4 and 5). From the above results, GmNRT1-1 and GmNRT
1-2 have different expression patterns in tissues,
GmNRT1-1 is expressed in leaves and GmNRT1
-2 was found to be expressed in both roots and leaves.

The data shown in FIG. 3E was used to check the quality of the total RNA used in the test and to assure the quantitativeness of the results.
As a result of determination using the states of the 28S and 18S bands in A) as an index, it was shown that there was no problem.

[0045]

Example 5 GmNRT1-3 and GmNRT1-4
Was also tested for confirming the gene expression pattern. Unless otherwise stated, the test conditions are the same as in Example 4.

Total R obtained from root after 3 hours treatment
In the case of NA, GmNRT1-
Three amplified bands were detected (FIG. 4A)). This result indicates that the GmNRT1-3 gene is constitutive
e). GmNRT2
As for the result, a result consistent with Example 4 was obtained (FIG. 4).
B) and FIG. 3 B)). In addition, in the case of total RNA obtained from the root after the treatment for 6 hours, almost the same results were obtained for both GmNRT1-3 and GmNRT2. The data shown in FIG. 4C) was performed for the purpose of checking the quality of the total RNA used in the test and ensuring the quantification of the results.
As a result of determining using the state of the S band as an index, it was shown that there was no problem.

In the case of long-term exposure to a nitrogen source, the exposure was carried out under two conditions: in the presence of ammonium and in the presence of nitrate and ammonium. Total RNA was prepared on days 5, 7, 11, and 14 from soybean roots, and 11 and 11 from leaves.
After preparation on day 14, they were subjected to RT-PCR. As a primer set, 5'S for GmNRT1-3
BON2 and 3'SBON2 (Table 1) were converted to GmNRT1-
For 4, 5′SBON3 and 3′SBON3 (Table 1) were used.

In the roots, amplified bands of GmNRT1-3 were equally detected in all samples (FIG. 5A). This result also indicated that the GmNRT1-3 gene was constitutively expressed. GmNRT1-
The band No. 2 was observed in all the soybean samples grown in the presence of ammonium, but no band was observed in the presence of nitrate and ammonium (FIG. 5B). Band intensity gradually increased with growth, clearly indicating that the increase in band intensity was associated with root growth.

The expression of GmNRT1-4 was weak and was observed only in the samples on the 5th and 7th days in the presence of ammonium (FIG. 5C). Therefore, this result indicates that in soybean grown in the presence of ammonium, the GmNRT1-4 gene is expressed at an early stage of growth, and the expression of the GmNRT1-2 gene increases with root growth. Was done. No amplified band of GmNRT1-5 could be observed in all test samples. Probably, expression is too low under the current test conditions. GmN
The RT2 band was detected on days 7 and 11 in the presence of ammonium.
As seen in the day sample, a very weak band was observed only in the sample on day 11 in the presence of nitrate and ammonium (FIG. 5D). The data in FIG. 5E) was used to check the quality of the total RNA used in the test and to assure the quantitativeness of the results.
As a result, it was shown that there was no problem.

In leaves, amplified bands of the GmNRT1-3 gene were similarly detected in samples both in the presence of ammonium and in the presence of nitrate and ammonium,
The band intensities of the samples on the 11th and 14th days were almost the same (A in FIG. 6). However, GmNRT
A band of 1-1 and / or GmNRT1-2 was also detected under both conditions, and the intensity of the band at day 14 was 1
It was much stronger than the day 1 sample (FIG. 6B)). The data shown in FIG. 6C) was performed for the purpose of checking the quality of the total RNA used in the test and assuring the quantitativeness of the results. , Showed no problem.

[0051]

According to the present invention, as a result of soybean cDNA cloning and gene expression analysis, novel genes and gene fragments of soybeans belonging to the NRT1 family have been isolated and their nucleotide sequences have been identified. From the comparison with the known NRT1 family genes, particularly the difference in expression tissues, etc., the genes and gene fragments of the present invention could certainly be identified as novel genes.

[0052]

[Table 3]

[0053]

[Table 4]

[0054]

[Table 5]

[0055]

[Table 6]

[0056]

[Table 7]

[Brief description of the drawings]

FIG. 1. Soybean GmNRT1-1 and GmNRT1-
2 and Arabidopsis, Brassica
sica) and the tomato (Lycopersicon) NRT1 family. Amino acids corresponding to GmNRT1-1 are indicated by dots (.), Portions where no corresponding amino acid is present are indicated by-marks, and amino acids conserved in all are indicated by * marks.

FIG. 2 shows a hydropathy plot of the protein. A) is a hydropathy plot of soybean GmNRT1-2, and B) is an Arabidopsis thaliana NRT1. The bar (-) shown at the top of the figure indicates 12 transmembrane regions.

FIG. 3. GmNRT1 and / or GmNRT1-2
9 is an electrophoretic photograph showing the results of expression analysis of GmNRT2 and GmNRT2. The location of the main band is indicated by a wedge-shaped arrow.

FIG. 4. Under short-term (3 hours) exposure to a nitrogen source,
It is a comparison of the expression of GmNRT1-3 and GmNRT2 in soybean roots. A) is GmNRT1-3, B) is G
This is the result of mNRT2, and the position of the target band is indicated by a wedge-shaped arrow.

FIG. 5 is a comparison of expression in soybean roots under conditions of long-term exposure to a nitrogen source. A) GmNRT1-3,
B) is GmNRT1-2, C) is GmNRT1-4,
D) is the result of GmNRT2. The duration of exposure to the nitrogen source is shown on days 5, 7, 11, and 14 respectively. The position of the band of interest is indicated by a wedge-shaped arrow. In addition, since there is a part where the band is thin and difficult to confirm, in the data of FIGS. 5A to 5D, the presence of the band is indicated when there is a wedge-shaped arrow. Indicates that it does not exist.

FIG. 6 is a comparison of expression in soybean leaves under conditions of long-term exposure to a nitrogen source. A) GmNRT1-3,
B) is GmNRT1-1 and / or GmNRT1-
2 is the result. Each exposure period was 11
Results from days 14 and 14, with the position of the band of interest indicated by a wedge-shaped arrow.

Claims (3)

[Claims] 1. A soybean gene belonging to the nitrate transporter 1 gene family induced by exposure to a nitrogen source. 2. The soybean gene according to claim 1, wherein the nucleotide sequence is represented by any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the sequence listing. 3. The nucleotide sequence is SEQ ID NO: 4 in the sequence listing.
Alternatively, the soybean gene fragment according to claim 1 represented by SEQ ID NO: 5.