Spinach and method for producing the same

Abstract

To provide a technique capable of reducing the oxalic acid content in spinach.SOLUTION: Provided are spinach in which the enzymatic activity of ascorbic acid oxidase is reduced or inhibited, and / or in which the expression of a specific gene is suppressed or deleted; and a method for producing spinach that comprises a step of reducing or inhibiting the enzymatic activity of ascorbic acid oxidase and / or a step of suppressing or deleting the expression of a specific gene.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present disclosure relates to spinach. [Background technology]

[0002] Spinach is a nutritious vegetable that is rich in vitamins and minerals and is consumed worldwide. However, as disclosed in Non-Patent Document 1, spinach has a very high oxalic acid content, which may increase oxalic acid in urine and contribute to kidney stone formation. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Shi, A. et al., Euphytica, 2016 212(1), 17-28. Summary of the Invention [Problem to be solved by the invention]

[0004] While it is desirable to reduce the oxalic acid content of spinach, there are currently no reports on gene functional modification of spinach individuals, nor on the identification of genes that contribute to the reduction of oxalic acid content in spinach. Therefore, a technology that can reduce the oxalic acid content in spinach is desired. [Means for solving the problem]

[0005] The present disclosure can be realized in the following forms.

[0006] (1) According to one embodiment of the present disclosure, there is provided a spinach in which the enzyme activity of ascorbate oxidase is reduced or inhibited, and / or the expression of any one of the following genes (a) to (f) is suppressed or deleted: (a) a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, (b) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having the enzyme activity of ascorbic acid oxidase; (c) a gene consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and encoding a protein having the enzyme activity of ascorbic acid oxidase; (d) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; (e) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 and having the enzyme activity of ascorbic acid oxidase; (f) A gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in which one to several amino acid residues have been deleted, substituted or added and having the enzyme activity of ascorbic acid oxidase. This form of spinach allows for a lower oxalic acid content.

[0007] (2) In the spinach described in (1) above, any one of the following genes (g) to (l) may be suppressed in expression or deleted: (g) a gene consisting of the base sequence shown in SEQ ID NO: 1; (h) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO:1 and encoding a protein having the enzyme activity of ascorbic acid oxidase; (i) a gene consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1, and encoding a protein having the enzyme activity of ascorbic acid oxidase; (j) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3; (k) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 and having the enzyme activity of ascorbic acid oxidase; (l) A gene encoding a protein having the amino acid sequence shown in SEQ ID NO:3 in which one to several amino acid residues have been deleted, substituted or added and having the enzyme activity of ascorbic acid oxidase. This form of spinach allows the oxalic acid content to be further reduced.

[0008] (3) According to another aspect of the present disclosure, there is provided a method for producing spinach, comprising the steps of: reducing or inhibiting the enzyme activity of ascorbic acid oxidase; and / or suppressing or deleting the expression of any one of the following genes (a) to (f): (a) a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, (b) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having the enzyme activity of ascorbic acid oxidase; (c) a gene consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and encoding a protein having the enzyme activity of ascorbic acid oxidase; (d) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; (e) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 and having the enzyme activity of ascorbic acid oxidase; (f) A gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in which one to several amino acid residues have been deleted, substituted or added and having the enzyme activity of ascorbic acid oxidase. According to this form of the method for producing spinach, it is possible to produce spinach with a reduced oxalic acid content.

[0009] The present invention can be realized in various forms, for example, in the form of reduced oxalic acid spinach, a food product containing reduced oxalic acid spinach, a method for reducing the oxalic acid content of spinach, etc. [Brief description of the drawings]

[0010] [Figure 1] Graph showing the measurement results of the oxalic acid content in leafy vegetables. [Diagram 2] FIG. 1 is an explanatory diagram showing the albinism of plants associated with suppression of the expression of the PDS gene. [Diagram 3] Schematic diagram showing leaf phenotypes induced by VIGS. [Figure 4] Graph showing the expression level of PDS gene after VIGS treatment. [Diagram 5] FIG. 1 is an explanatory diagram showing genes used in searching for target genes. [Figure 6] FIG. 1 is an explanatory diagram showing the expression level of a target gene and the oxalic acid content after VIGS treatment. [Figure 7] FIG. 1 is an explanatory diagram showing the relationship between the expression level of the AAO1 gene and the oxalic acid content in improved Japan. [Figure 8] FIG. 1 is an explanatory diagram showing the relationship between AAO1 gene expression level and oxalic acid content in New Japan. [Figure 9] FIG. 2 is an explanatory diagram showing the relationship between the AAO1 gene expression level and the oxalic acid content in Justis. [Figure 10] FIG. 11 is an explanatory diagram showing the results of statistical processing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In general, the Agrobacterium method and CRISPR-Cas9 are sometimes used for plant transformation. However, the Agrobacterium method and CRISPR-Cas9 cannot be applied to plants of the Chenopodiaceae subfamily of the Amaranthaceae family, including spinach. For this reason, the present inventors attempted to establish a VIGS (virus-induced gene silencing) system in spinach as a method for analyzing gene functions in spinach, as shown in the examples described below. Furthermore, they found that the oxalic acid content can be reduced by knocking down the gene encoding ascorbate oxidase in spinach, and thus completed the present invention.

[0012] According to one embodiment of the present disclosure, there is provided spinach (Spinacia oleracea), in which the enzyme activity of ascorbic acid oxidase is reduced or inhibited, and / or the expression of any one of the following genes (a) to (f) is suppressed or deleted:

[0013] (a) a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, (b) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having the enzyme activity of ascorbic acid oxidase; (c) a gene consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and encoding a protein having the enzyme activity of ascorbic acid oxidase; (d) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; (e) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 and having the enzyme activity of ascorbic acid oxidase; (f) A gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in which one to several amino acid residues have been deleted, substituted or added and having the enzyme activity of ascorbic acid oxidase.

[0014] Ascorbic acid oxidase is an enzyme classified in EC1.10.3.3, and catalyzes a reaction in which ascorbic acid and O2 are used as substrates to produce monodehydroascorbic acid and H20. Monodehydroascorbic acid is metabolized to oxalic acid through hydrolysis and oxidation. The activity of ascorbic acid oxidase can be measured, for example, by adding an enzyme solution to a reaction solution containing L-ascorbic acid and a phosphate buffer, adding a hydrochloric acid solution after a predetermined period of time to stop the reaction, and then measuring the absorbance at 245 nm with a spectrophotometer.

[0015] The method for reducing or inhibiting the enzyme activity of ascorbic acid oxidase is not particularly limited, but for example, a method of treating a plant body with a compound that inhibits the enzyme activity of ascorbic acid oxidase can be mentioned. The above-mentioned compound is not particularly limited, but since ascorbic acid oxidase is a copper protein containing copper as a complex group, for example, a thiol compound such as cysteine, thiourea, NaCl, NaN3, and other compounds that form complexes with copper can be mentioned. In addition, the above-mentioned compound may be citric acid, phytin, EDTA (ethylenediaminetetraacetic acid), and the like, which are compounds that have the function of removing copper and iron ions, or trypsin, and the like. The plant body to be treated is not particularly limited, but for example, leaves, stems, flowers, roots, seeds, and combinations of two or more selected from these can be mentioned, and it is preferable that the plant body includes at least edible parts (leaves and stems) or is a seed.

[0016] Methods for suppressing the expression of a gene encoding ascorbic acid oxidase or for deleting a gene include, but are not limited to, a method for knocking down or knocking out the gene using VIGS, a method for transformation by genome editing targeting the gene, a method using mutation, and the like. Genes encoding ascorbic acid oxidase in spinach include AAO1 (Gene name: Spo22971) and AAO2 (Gene name: Spo10268). AAO1 is an ascorbate oxidase having a base sequence shown in SEQ ID NO: 1. AAO2 is an L-ascorbate oxidase having a base sequence shown in SEQ ID NO: 2. Sequence information for AAO1 and AAO2 can be confirmed in SpinachBase (http: / / www.spinachbase.org / ), a public database of the spinach genome. AAO1 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 3, and AAO2 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0017] In the present disclosure, the "identity" of a base sequence or an amino acid sequence means the maximum identity (%) of the sequences obtained by aligning the two sequences to be compared, with gaps introduced as necessary. The identity of a base sequence or an amino acid sequence can be calculated, for example, using blastn of NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / ) which implements the BLAST algorithm. In the present disclosure, the identity of a base sequence or an amino acid sequence is preferably 90% or more, more preferably 92% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, especially preferably 97% or more, particularly preferably 98% or more, and especially more preferably 99% or more.

[0018] In the present disclosure, "one to several bases" is preferably one to ten bases, more preferably one to eight bases, even more preferably one to six bases, and even more preferably one to three bases. It is preferable that the deletion, substitution, or addition of bases does not cause a frameshift. In addition, it is preferable that the substitution of bases is a substitution to a degenerate sequence that does not change the encoded amino acid. In addition, when the encoded amino acid is changed, it is preferable that it is a conservative substitution to an amino acid having a similar side chain.

[0019] In the present disclosure, "one to several amino acid residues" is preferably one to ten amino acid residues, more preferably one to eight amino acid residues, even more preferably one to six amino acid residues, and even more preferably one to three amino acid residues. The deletion, substitution or addition of amino acid residues is preferably in a region far from the active site of the encoded enzyme protein. Furthermore, the substitution of amino acid residues is preferably a conservative substitution with an amino acid having a similar side chain.

[0020] Conservative substitutions include, for example, substitutions of amino acid residues having basic side chains, such as lysine, arginine, and histidine; substitutions of amino acid residues having acidic side chains, such as aspartic acid and glutamic acid; substitutions of amino acid residues having non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions of amino acid residues having non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions of amino acid residues having β-branched side chains, such as threonine, valine, and isoleucine; and substitutions of amino acid residues having aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0021] In the spinach of the present disclosure, the expression of the AAO1 gene is preferably suppressed or deleted in order to further reduce the oxalic acid content. Therefore, in the spinach of the present disclosure, the expression of any one of the following genes (g) to (l) is preferably suppressed or deleted:

[0022] (g) a gene consisting of the base sequence shown in SEQ ID NO: 1; (h) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO:1 and encoding a protein having the enzyme activity of ascorbic acid oxidase; (i) a gene consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1, and encoding a protein having the enzyme activity of ascorbic acid oxidase; (j) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3; (k) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 and having the enzyme activity of ascorbic acid oxidase; (l) A gene encoding a protein having the amino acid sequence shown in SEQ ID NO:3 in which one to several amino acid residues have been deleted, substituted or added and having the enzyme activity of ascorbic acid oxidase.

[0023] As a method for suppressing the expression of any one of the above genes (a) to (f) or deleting a gene using VIGS, a method of infecting a spinach plant with a virus vector incorporating a target gene sequence is envisaged. Such a virus vector is not particularly limited, but examples thereof include Tobacco rattle virus (TRV), Tomato mosaic virus (ToMV), Apple latent spherical virus (ALSV), Tobacco ringspot virus (TRSV) and the like. As such a vector, it is preferable to use TRV from the viewpoint of infection efficiency into spinach. As a method for infecting a spinach plant with a virus vector, it is not particularly limited, but examples thereof include friction inoculation, particle gun method, and inoculation using Agrobacterium for spinach plant.

[0024] In the spinach of the present disclosure, the enzyme activity of ascorbic acid oxidase may be reduced or inhibited, the expression of any one of the genes (a) to (f) above may be suppressed or deleted, or the enzyme activity of ascorbic acid oxidase may be reduced or inhibited and the expression of any one of the genes (a) to (f) above may be suppressed or deleted. In addition to the enzyme activity of ascorbic acid oxidase being reduced or inhibited and / or the expression of any one of the genes (a) to (f) above being suppressed or deleted, the expression of other genes may be suppressed or other genes may be deleted in the spinach of the present disclosure. Examples of such other genes include, but are not limited to, genes encoding glycolate oxidase and lactate dehydrogenase. Examples of genes encoding glycolate oxidase include, but are not limited to, GOX1 (Peroxisomal (S)-2-hydroxy-acid oxidase, gene name: Spo10076), GOX2 (Peroxisomal (S)-2-hydroxy-acid oxidase, gene name: Spo10074), GOX3 (Peroxisomal (S)-2-hydroxy-acid oxidase, gene name: Spo19861), and HAOX1 (Peroxisomal (S)-2-hydroxy-acid oxidase, gene name: Spo21282). Examples of genes encoding lactate dehydrogenase include, but are not limited to, LDH (L-lactate dehydrogenase, gene name: Spo26422), D-LDH (D-lactate dehydrogenase, putative, gene name: Spo22587), and the like. The sequence information of these genes is published in SpinachBase (http: / / www.spinachbase.org / ).

[0025] The origin and variety of the spinach disclosed herein are not particularly limited, and examples include Improved Japanese Spinach, New Japan, Justice, Nobel, Mirage, Chronos, Osiris, Reed, Solomon, Progress, Maromi, and the like.

[0026] The spinach of the present disclosure has a reduced oxalic acid content due to the above configuration. The reduced oxalic acid content means that the oxalic acid content is lower than that of the same variety of spinach. The oxalic acid content is preferably 10% or more lower than that of the same variety of spinach, more preferably 20% or more lower, even more preferably 30% or more lower, even more preferably 40% or more lower, and even more preferably 50% or more lower. In addition, the oxalic acid content in the edible part (the whole plant including the leaf blade and petiole) is preferably 5 mg / gFW or less, more preferably 4 mg / gFW or less, even more preferably 3 mg / gFW or less, even more preferably 2 mg / gFW or less, and even more preferably 1 mg / gFW or less. The oxalic acid content of spinach can be measured by high performance liquid chromatography (HPLC).

[0027] According to the spinach of the present disclosure, the content of oxalic acid is reduced, so that when spinach is ingested in a meal, the amount of oxalic acid that binds with calcium in the body can be reduced, and thus the formation of stones can be suppressed. In addition, according to the spinach of the present disclosure, the bitter taste caused by oxalic acid can be reduced, so that the taste of the spinach can be improved. In addition, since oxalic acid binds with calcium in the body, the spinach of the present disclosure, which has a reduced content of oxalic acid, can suppress the inhibition of calcium absorption. In addition, since the content of oxalic acid is reduced, it is expected that a cooking method that suppresses the loss of nutrition of spinach, such as eating spinach raw, will be possible. In addition, according to the spinach of the present disclosure, the metabolism of ascorbic acid (vitamin C), which is a substrate of ascorbic acid oxidase, may be suppressed, so that the content of ascorbic acid may increase, and therefore the nutritional value of spinach is expected to be improved.

[0028] According to another embodiment of the present disclosure, a method for producing spinach is provided. This method includes a step of reducing or inhibiting the enzyme activity of ascorbic acid oxidase, and / or a step of suppressing or deleting any one of the genes (a) to (f) above. The step of reducing or inhibiting the enzyme activity of ascorbic acid oxidase is not particularly limited, and for example, the step of treating a plant body with a compound having a function of inhibiting the enzyme activity of ascorbic acid oxidase as described above can be mentioned. The step of suppressing or deleting any one of the genes (a) to (f) above can be not particularly limited, and for example, the step of knocking down or knocking out the gene using VIGS as described above, transformation by genome editing targeting the gene, a method using mutation, and the like can be mentioned. EXAMPLES

[0029] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0030] <Experiment 1: Measurement of oxalic acid content in leafy vegetables> The oxalic acid content of 10 types of leafy vegetables designated as specific vegetables by the Ministry of Agriculture, Forestry and Fisheries Ordinance among vegetables commonly sold in Japan was measured.

[0031] (1) Method The leafy vegetables used were spinach, komatsuna, cabbage, lettuce, Chinese cabbage, bok choy, Chinese chives, green onions, onions, and broccoli that were sold at fresh food stores in January. 0.1 g of the edible part of the vegetables was sampled, frozen in liquid nitrogen, and then crushed in a mortar. Then, 1 ml of MilliQ water was added and boiled for 30 minutes. The sample was transferred to a 5 ml syringe and filtered with a membrane filter (0.22 μm, Merck Millipore). Then, spinach was diluted 10 times with MilliQ water, and the nine types of leafy vegetables other than spinach were diluted 6 times with MilliQ water, and subjected to high performance liquid chromatography (HPLC) under the following conditions. Oxalic acid dihydrate (Wako Pure Chemical Industries, Ltd.) was prepared to concentrations of 100 ppm, 50 ppm, 25 ppm, and 10 ppm and used to create a calibration curve. The oxalic acid peak in the calibration curve was detected around 15 minutes after the start of the measurement, so the HPLC measurement time was set to 20 minutes. The n number was set to 3, and the oxalic acid content relative to the fresh weight (FW) of leafy vegetables was calculated.

[0032] IC: ICS-900 (Thermo Scientific) Guard column: AG12A (Thermo Scientific) Column: AS12A (Thermo Scientific) Suppressor: ERS500 (recycle mode) (Thermo Scientific) Autosampler: AS-DV (Thermo Scientific) Eluent: Eluent for ion chromatography anion analysis AS12A (Kanto Chemical) Injection volume: 25μL Column temperature: 25℃ ·Flow rate: 1.5mL / min

[0033] (2) Results Figure 1 is a graph showing the results of measuring the oxalic acid content in leafy vegetables. Of the 10 types of leafy vegetables measured, 9 types of leafy vegetables other than spinach contained approximately 0.1 mg / g (FW) of oxalic acid. In contrast, the oxalic acid content of spinach was 7.6 mg / g (FW), which is more than 40 times the amount of oxalic acid compared to leafy vegetables other than spinach.

[0034] <Experiment 2: Establishment of a VIGS system in spinach> As a method to analyze gene functions in spinach, we attempted to establish a VIGS system in spinach.

[0035] (1) Viral vectors First, we investigated the viral vectors used for infection. Tomato mosaic virus (ToMV), Apple latent spherical virus (ALSV), Tobacco ringspot virus (TRSV), and Tobacco rattle virus (TRV) were used as viral vectors. The viral vectors were investigated as follows. A fragment of the spinach phytoen desaturase gene was incorporated into the genome of each virus, and the virus was inoculated into cotyledons of spinach (improved Japan) about two weeks after sowing. The presence or absence of whitening of newly developed leaves was used as an indicator to determine whether silencing of the PDS gene had occurred. As a result, the highest infection efficiency was observed when TRV was used.

[0036] (2) VIGS targeting the PDS gene Using TRV as a viral vector and improved Japanese spinach as a spinach variety, VIGS was performed targeting the Phytoene desaturase (PDS) gene, which is a carotenoid synthesis enzyme, and β-glucuronidase (GUS) gene as a control.

[0037] In order to prepare a TRV vector incorporating the sequence information of the target PDS gene, a gene fragment amplified using the following primers was inserted into pYL156 (Liu et al. Plant J. 2002 31(6), 777-786). A TRV vector incorporating the sequence information of the GUS gene was also prepared in the same manner. The sequences of the primers used to introduce the PDS gene and the primers used to introduce the GUS gene are shown in Table 1 below together with the sequence numbers. In Tables 1 to 4 below, primers with "-F" or "fw" in their names indicate forward primers, and those with "-R" or "rv" in their names indicate reverse primers, and the names include the target gene name. In Tables 1 and 3 below, the bold parts in the sequences indicate the restriction enzyme sites used for cloning, those with "Bam" in their names indicate that the restriction enzyme is BamHI, and those with "Sac" in their names indicate that the restriction enzyme is SacI.

[0038] [Table 1]

[0039] Seeds of improved Japanese spinach (Sakata Seed) were wrapped in water-soaked Kimwipes overnight and then sown in pods containing vermiculite. Two weeks later, cotyledons were inoculated with the TRV vector. TRV vector inoculation was performed according to the method described in Liu et al. Plant J. 2002 31(6), 777-786. The phenotype of the plant was confirmed 17 days after TRV vector inoculation. On the 20th day after TRV vector inoculation, newly formed true leaves (0.1 g) were sampled for gene expression analysis. The n number was 4 to 9.

[0040] (3) Confirmation of phenotype FIG. 2 is an explanatory diagram showing the bleaching of a plant body due to the suppression of the expression of the PDS gene. In FIG. 2, "TRV_GUS" shows a control plant body, and "TRV-PDS" shows a plant body that underwent VIGS targeting the PDS gene. Since the PDS gene is a carotenoid synthesis enzyme, when the expression of the PDS gene is suppressed, chlorophyll is photooxidized by excess light, resulting in bleaching of the plant tissue. As shown in the area surrounded by the dashed line in FIG. 2, bleaching was observed in the plant body that underwent VIGS targeting the PDS gene, indicating that the expression of the PDS gene was suppressed by VIGS. This result indicates that the expression of a target gene can be suppressed in spinach by VIGS, and indicates that the establishment of a VIGS system in a spinach individual was successful.

[0041] Figure 3 is an explanatory diagram showing the leaf phenotype caused by VIGS. In individuals in which the PDS gene was targeted, in addition to white leaves ("PDS-white" in Figure 3), mosaic-colored leaves ("PDS" in Figure 3) were observed.

[0042] (4) Analysis of the expression level of the PDS gene To investigate the effect of VIGS in spinach, the phenotype of VIGS-treated individuals and the expression level of the PDS gene were evaluated. As samples, we used bleached leaves (PDS-white), mosaic-colored leaves (PDS), and control leaves (TRV_GUS) as shown in Figure 3. Leaves in which more than 90% of the leaf area was bleached were designated "PDS-white." Analysis of the expression level of the PDS gene was performed as follows.

[0043] Total RNA was extracted using RNAiso plus (Takara Bio) and ISOSPIN Plant RNA Kit (Nippon Gene), and reverse transcription was performed at 37°C for 30 minutes using ReverTra ACE kit (Toyobo), followed by inactivating reverse transcriptase by incubating at 98°C for 5 minutes to prepare single-stranded complementary DNA (cDNA). The expression level was quantified by qRT-PCR using the prepared cDNA as a template and corrected for the expression level of actin-11. qRT-PCR was performed using Thunderbird SYBR qPCR Mix (Toyobo) with a LightCycler 96 system (Roche Diagnostics) (LightCycler is a registered trademark). The PCR reaction conditions were 95°C / 300 seconds heat treatment, followed by 45 cycles of 95°C / 10 seconds, 60°C / 30 seconds, and 72°C / 25 seconds. The sequences of the primers used in qRT-PCR are shown below in Table 2 together with their SEQ ID NOs. Student's t-test was performed for the expression level of each sample.

[0044] [Table 2]

[0045] FIG. 4 is a graph showing the expression level of the PDS gene after VIGS treatment. The vertical axis in FIG. 4 shows the relative expression level of the PDS gene to the expression level of actin-11. In FIG. 4, the average ± standard deviation is shown. From the results shown in FIG. 4, the following was found. That is, in the bleached leaves, the expression level of the PDS gene was suppressed to 1 / 10 or less compared to the control, and a significant difference was found at the 1% significance level. In contrast, the mosaic-colored leaves showed a large variation among individuals, and it was found that there was a difference in the degree of suppression of the PDS gene. These findings suggest that VIGS for spinach may not have a uniform effect on the entire plant body.

[0046] <Experiment 3: Searching for target genes using VIGS> Genes involved in oxalic acid reduction in spinach were screened using VIGS.

[0047] FIG. 5 is an explanatory diagram showing the genes used in the search for target genes. The search for target genes was carried out with a view to attempting gene knockout by genome editing, targeting spinach homologs of oxalic acid biosynthesis-related genes reported in model plants such as Arabidopsis thaliana. As shown in FIG. 5, in Arabidopsis thaliana, there are metabolic pathways from the TCA cycle, synthesis pathways from glycolic acid, and synthesis pathways from ascorbic acid, and it is believed that oxalic acid is produced as a metabolic product. Among such oxalic acid biosynthesis-related genes, we attempted to suppress gene expression and reduce oxalic acid content by VIGS for genes with homologs in the spinach genome.

[0048] (1) Method Using TRV as a viral vector and improved Japanese spinach as a spinach variety, VIGS was performed targeting each gene. The target genes were isocitrate lyase (ICL), glycolate oxidase (GOX1, GOX2, GOX3, HAOX1, LDH, D-LDH), and ascorbate oxidase (AAO1, AAO2). The GUS gene was used as a control.

[0049] As in the above method, a TRV vector incorporating the sequence information of the target gene was created. The sequences of the primers used for the introduction of each gene are shown in Table 3 below along with the sequence numbers. Note that since GOX3 and HAOX1 have high sequence homology, we attempted to knock them down simultaneously.

[0050] [Table 3]

[0051] Cotyledons of improved Japanese spinach were inoculated with the TRV vector in the same manner as described above. Twenty days after TRV vector inoculation, newly formed true leaves (0.1 g) were sampled for analysis of gene expression levels and measurement of oxalic acid content. Expression levels of target genes were analyzed in the same manner as described above. The sequences of the primers used in qRT-PCR are shown in Table 4 below along with their sequence numbers. The n number was set to 5. The relative expression levels of target genes were calculated when the expression level of each gene in the control plant inoculated with TRV-GUS was set to 1.

[0052] [Table 4]

[0053] The oxalic acid content was determined in the same manner as described above using the sampled leaves of 4-week-old spinach plants, with the n number being 5. The relative content was calculated when the oxalic acid content in the control was set at 1.

[0054] (2) Results FIG. 6 is an explanatory diagram showing the expression level of a target gene and the content of oxalic acid after VIGS treatment. In FIG. 6, the relative expression level of the target gene, the relative content of oxalic acid, and the average value of the relative content of oxalic acid are arranged in correspondence with each target gene. The relative expression level of the target gene means the relative expression level of the target gene when the expression level of each gene in the control plant inoculated with TRV-GUS is set to 1. The relative content of oxalic acid means the relative content when the content of oxalic acid in the control is set to 1. Note that in FIG. 6, among the target genes tested, those in which the growth of the plant body was hindered by VIGS treatment and those in which the effect of suppressing gene expression was not observed are not disclosed.

[0055] As shown in Figure 6, the gene expression suppression effect was observed in multiple lines for the target genes. In particular, gene expression was significantly suppressed in the AAO1 gene and GOX1 gene. In addition, the oxalic acid content was significantly reduced in the AAO1 gene expression suppression strain compared to the control. In addition, the AAO2 gene, which is an ascorbate oxidase like the AAO1 gene, the GOX1 gene, and the LDH gene also tended to reduce the oxalic acid content compared to the control, while the oxalic acid content did not decrease due to the suppression of the ICL gene expression. In general, the isocitrate pathway is thought to play an important role in the synthesis of oxalic acid, but the results shown in Figure 6 suggest that the ICL gene, which does not have a homolog and acts alone, does not affect the oxalic acid content.

[0056] Here, according to the above-mentioned Experiment 2, in the VIGS for spinach, it is possible that the effect is not uniform throughout the plant body. And since the suppression of ascorbic acid oxidase expression is not expressed phenotypically, it is not always possible to collect leaves in which the expression of ascorbic acid oxidase is suppressed during sampling. Furthermore, from the viewpoint of the detection limit, it is not possible to use the same leaves to analyze the gene expression level and to measure the oxalic acid content. For these reasons, it may be difficult to find a clear correlation between the suppression of gene expression level and the reduction in oxalic acid content.

[0057] <Experiment 4: Examination of the effect of suppressing AAO1 gene expression in other varieties> We investigated whether similar results could be obtained for the suppression of AAO1 gene expression in other spinach varieties. As mentioned above, due to the nature of the experiment, it is difficult to find a clear correlation between the suppression of gene expression and the reduction of oxalic acid content. Therefore, we investigated the suppression of AAO1 gene expression by ensuring the n number.

[0058] (1) Method The spinach varieties used were Improved Japanese Spinach, New Japan, and Justice (all manufactured by Sakata Seed Co., Ltd.). Inoculation with the TRV vector and sampling were performed in the same manner as described above, and the mRNA expression level of the AAO1 gene and the oxalic acid content were analyzed. The n number was 4 to 10.

[0059] (2) Results FIG. 7 is an explanatory diagram showing the relationship between the expression level of the AAO1 gene in Improved Japan and the oxalic acid content. FIG. 8 is an explanatory diagram showing the relationship between the expression level of the AAO1 gene in New Japan and the oxalic acid content. FIG. 9 is an explanatory diagram showing the relationship between the expression level of the AAO1 gene in Justice and the oxalic acid content. In FIGS. 7 to 9, the vertical axis in the diagram showing the expression level of the AAO1 gene indicates the relative expression level (relative expression) of the AAO1 gene with respect to the expression level of the Actin gene, and the vertical axis in the diagram showing the oxalic acid content indicates the oxalic acid content (mg / gFW). As shown in FIGS. 7 to 9, suppression of the expression of the AAO1 gene and a decrease in the oxalic acid content were observed in all varieties.

[0060] FIG. 10 is an explanatory diagram showing the results of statistical processing. FIG. 10 shows the results of a Student's t-test on the oxalic acid contents shown in FIGS. 7 to 9. FIG. 10 shows the average±standard deviation. The results shown in FIG. 10 reveal the following. That is, in all varieties, the oxalic acid content was reduced by about 30% compared to the control, and a significant difference was observed at the 1% significance level.

[0061] These results suggest that ascorbate oxidase may be a target gene for reducing oxalic acid oxidation in spinach. In addition, as described above, VIGS of spinach showed that the oxalic acid content was reduced by about 30% despite the variation in expression suppression in leaves, so it is expected that ascorbate oxidase may be involved in further reduction of oxalic acid content.

[0062] The present invention is not limited to the above-mentioned embodiment, and can be realized in various configurations without departing from the spirit of the present invention. For example, the technical features in the embodiments and examples corresponding to the technical features in each form described in the Summary of the Invention column can be replaced or combined as appropriate to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. Spinach, The enzyme activity of ascorbate oxidase is reduced or inhibited, and / or Any one of the following genes (a) to (f) is suppressed or deleted: Spinach: (a) a gene consisting of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, (b) a gene consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having the enzyme activity of ascorbic acid oxidase; (c) a gene consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and encoding a protein having the enzyme activity of ascorbic acid oxidase; (d) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; (e) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 and having the enzyme activity of ascorbic acid oxidase; (f) A gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in which one to several amino acid residues have been deleted, substituted or added and having the enzyme activity of ascorbic acid oxidase.

2. The spinach according to claim 1, Any one of the following genes (g) to (l) is suppressed or deleted: Spinach: (g) a gene consisting of the base sequence shown in SEQ ID NO: 1; (h) a gene having a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 1 and encoding a protein having the enzyme activity of ascorbic acid oxidase; (i) a gene consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1, and encoding a protein having the enzyme activity of ascorbic acid oxidase; (j) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3; (k) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 and having the enzyme activity of ascorbic acid oxidase; (l) A gene encoding a protein having the amino acid sequence of SEQ ID NO:3 in which one to several amino acid residues have been deleted, substituted or added and having the enzyme activity of ascorbic acid oxidase.

3. A method for producing spinach, comprising the steps of: A step of reducing or inhibiting the enzyme activity of ascorbic acid oxidase; and / or The method includes a step of suppressing or deleting any one of the following genes (a) to (f): How to make spinach: (a) a gene consisting of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, (b) a gene consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having the enzyme activity of ascorbic acid oxidase; (c) a gene consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and encoding a protein having the enzyme activity of ascorbic acid oxidase; (d) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; (e) a gene encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 and having the enzyme activity of ascorbic acid oxidase; (f) A gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in which one to several amino acid residues have been deleted, substituted or added and having the enzyme activity of ascorbic acid oxidase.

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