WO2023037620A1 - Plante tolérante au magnésium, procédé de production d'une plante tolérante au magnésium, procédé de culture d'une plante tolérante au magnésium et gène - Google Patents

Plante tolérante au magnésium, procédé de production d'une plante tolérante au magnésium, procédé de culture d'une plante tolérante au magnésium et gène Download PDF

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WO2023037620A1
WO2023037620A1 PCT/JP2022/012317 JP2022012317W WO2023037620A1 WO 2023037620 A1 WO2023037620 A1 WO 2023037620A1 JP 2022012317 W JP2022012317 W JP 2022012317W WO 2023037620 A1 WO2023037620 A1 WO 2023037620A1
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mtip1
protein
amino acid
acid sequence
magnesium
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PCT/JP2022/012317
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均 尾之内
憲哉 林
篤 海藤
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国立大学法人北海道大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

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  • the present invention relates to magnesium-tolerant plants, methods for producing magnesium-tolerant plants, methods for cultivating magnesium-tolerant plants, and genes.
  • This application claims priority based on Japanese Patent Application No. 2021-147788 filed in Japan on September 10, 2021, the content of which is incorporated herein.
  • Non-Patent Document 1 a method has already been developed for producing plants showing resistance to high concentrations of Na + by genetic engineering techniques.
  • Mg 2+ it has been reported that a quadruple mutant lacking four DELLA protein genes in Arabidopsis thaliana exhibits resistance to high concentrations of Mg 2+ (Non-Patent Document 2).
  • Non-Patent Document 1 since the DELLA protein shown in Non-Patent Document 1 is involved in the response to the plant hormone gibberellin, introduction of mutations into the DELLA protein gene has undesirable effects on crops, such as plant elongation and early flowering. Therefore, it is desired to develop a method for imparting resistance to high-concentration Mg 2+ by techniques other than the introduction of mutations into the DELLA protein gene.
  • the present invention was made to solve the above problems, and an object of the present invention is to provide a magnesium-tolerant plant. Another object of the present invention is to provide a method for producing a magnesium-tolerant plant. Another object of the present invention is to provide a method for cultivating the magnesium-tolerant plant. Another object of the present invention is to provide a gene capable of improving the magnesium tolerance of plants.
  • the present inventors found that the magnesium tolerance of plants can be improved by increasing the expression level of the MTIP1 (Mg transporter-interacting protein 1) protein. I have perfected my invention. That is, the present invention has the following aspects.
  • the MTIP1 protein is a protein selected from the group consisting of the following (A) to (C).
  • a protein that contributes to the improvement of magnesium tolerance in plants through enhanced expression or function C having an amino acid sequence with a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, and enhanced in expression or function
  • a method for producing a magnesium-tolerant plant which comprises enhancing the expression or function of MTIP1 protein in the plant.
  • MTIP1 protein is a protein selected from the group consisting of (A) to (C) below.
  • A a protein having the amino acid sequence represented by SEQ ID NO: 1;
  • B having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1;
  • a protein that contributes to the improvement of magnesium tolerance in plants through enhanced expression or function (C) having an amino acid sequence with a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, and enhanced in expression or function (9)
  • a method for cultivating a magnesium-tolerant plant which comprises cultivating the plant according to any one of (1) to (6) above in a high-concentration magnesium environment.
  • the uORF of the MTIP1 gene encodes a peptide selected from the group consisting of the following (D) to (F), and disruption of the uORF causes SEQ ID NO: All or part of the deletion, substitution, insertion, addition, or combination thereof in the region corresponding to the amino acid sequence represented by any one of 41 to 67, loss of the ability to suppress translation of the MTIP1 protein, or The gene according to (14) above, which is suppressed.
  • magnesium-tolerant plants can be provided. Moreover, according to the present invention, a method for producing a magnesium-tolerant plant can be provided. Moreover, according to this invention, the cultivation method of the said magnesium tolerance plant can be provided. Further, according to the present invention, genes capable of improving magnesium tolerance in plants can be provided.
  • FIG. 1 is a schematic diagram showing an example of the structure of MTIP1 mRNA containing uORF and mORF, and a diagram showing the results of comparison of amino acid sequences translated from uORF among plants.
  • FIG. 2 is a schematic diagram showing an example of a mechanism for maintaining homeostasis of Mg 2+ concentration in cells by uORF-mediated MTIP1 expression control.
  • FIG. 2 is a diagram explaining mutation sites introduced into the uORF of the MTIP1 gene in the mutants obtained in Examples.
  • FIG. 10 is a graph showing the effect of promoting mORF translation by mutations introduced into the uORF of the MTIP1 gene.
  • FIG. FIG. 10 is an image showing the results of improving plant tolerance to high concentrations of Mg 2+ by increasing the expression level of MTIP1 protein.
  • the plant of the embodiment is a magnesium-tolerant plant in which MTIP1 protein expression or function is enhanced.
  • the enhanced expression of MTIP1 protein means that the expression level of MTIP1 protein is increased compared to the expression level of MTIP1 protein in the control plant.
  • the expression level of the MTIP1 protein can be confirmed, for example, by using an antibody against the MTIP1 protein or expression analysis using a reporter protein such as luciferase.
  • the enhancement of the function of the MTIP1 protein means that the function of the MTIP1 protein is improved as compared with the function of the MTIP1 protein of the control plant.
  • the MTIP1 protein causes Mg 2+ stored in the vacuoles to enter the cytoplasm through binding with the magnesium transporter MRS2-1 (AGI code: At1g16010), which is mainly present in the vacuoles, and promotes plant cell growth. It is presumed to increase the Mg 2+ concentration in the cytoplasm.
  • MTIP1 proteins with improved functions include, for example, MTIP1 proteins with improved binding ability to the above-described magnesium transporters compared to wild-type plant MTIP1 proteins.
  • Examples of the control plant that serves as a control for the enhancement of the plant of the embodiment include a wild-type plant of the plant.
  • Examples of the control plant for the enhancement in the event of artificial genetic modification described later include plants that have not undergone the above artificial genetic modification.
  • magnesium in magnesium tolerance is a concept including magnesium ion (Mg 2+ ) and magnesium salt.
  • magnesium salts include magnesium sulfate, magnesium chloride, magnesium oxide, magnesium hydroxide, and hydrates thereof.
  • Magnesium is a component of chlorophyll, so normally the right amount of magnesium is available for plant growth.
  • the magnesium concentration in the growth environment such as soil is excessive, dehydration or water absorption inhibition due to increased osmotic pressure in the extraroot region, growth inhibition due to excessive Mg 2+ uptake into plant cells, and excessive magnesium It is known that symptoms of disease appear.
  • Magnesium tolerant plants of embodiments have improved tolerance to magnesium due to the presence of excess magnesium in the growing environment compared to control plants of said plants. Therefore, the magnesium-tolerant plant of the embodiment can grow better than the control plant, even in a high-concentration magnesium environment.
  • improving the magnesium tolerance of plants includes strengthening and improving magnesium tolerance, and imparting magnesium tolerance to plants that do not have magnesium tolerance.
  • the fact that the magnesium tolerance of the plant is improved is, for example, by growing the plant A and the control plant of the plant A, and growing the plant A in an environment with a higher concentration of magnesium than the control plant. is also good, it can be evaluated that the magnesium tolerance of plant A is improved.
  • a magnesium-rich environment includes an amount of magnesium at a concentration that induces growth inhibition or hypermagnesium in control plants of the magnesium-tolerant plants of the embodiments.
  • the environment may be, for example, a root zone environment such as soil or a cultivation solution, or, in the case of cultured cells, a cell culture environment such as a medium.
  • a concentration varies depending on the target plant, considering the cultivation example of Arabidopsis thaliana on an agar medium (solid medium) shown in the examples below, for example, when the Mg 2+ molar concentration in the medium is 5 mM or more can be exemplified, and may be 5 to 100 mM, 10 to 80 mM, or 15 to 50 mM.
  • the concentration of magnesium corresponding to the Mg 2+ molar concentration in the medium can be exemplified.
  • a high concentration of magnesium in soil can be exemplified by, for example, a case where the content of exchangeable Mg 2+ in soil is 50 mg/100 g or more in terms of MgO, and may be 50 to 200 mg/100 g, or 80 to 150 mg/ It may be 100g.
  • the expression or function of the MTIP1 protein in the plant of the embodiment is preferably artificially enhanced, preferably enhanced by genetic modification, and preferably enhanced by genetic modification of the plant genome.
  • Genetic modification here refers to modifying a sequence that affects the expression or function of MTIP1 protein. It is preferable that the plant according to the embodiment has a modified genome sequence so that the expression or function of the MTIP1 protein is enhanced.
  • targets for genetic modification include the above-mentioned main ORF (also referred to as the main ORF or mORF) of the MTIP1 gene that encodes the MTIP1 protein, as well as the expression regulatory region of the MTIP1 gene.
  • main ORF also referred to as the main ORF or mORF
  • the modification may be a modification to the sequence of the endogenous MTIP1 gene and/or its expression regulatory region that the plant originally has, and the sequence-modified MTIP1 gene and/or its expression regulatory region is newly added. may have been introduced.
  • Sequence modifications include, for example, deletion, substitution, insertion, addition, and combinations thereof of part or all of the nucleotide sequence in the mORF of the MTIP1 gene and/or the expression regulatory region of the MTIP1 gene.
  • an MTIP1 protein with enhanced function can be obtained by modifying the mORF sequence of the MTIP1 gene.
  • sequence of the expression control region of the MTIP1 gene the transcription or translation of mRNA is enhanced, and the expression level of the MTIP1 protein can be enhanced.
  • the plant of the embodiment may have the modified region as a heterozygote or as a homozygote, as long as the effects of the present invention are exhibited.
  • MTIP1 protein The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of Arabidopsis thaliana MTIP1 (AGI code: At1g70780).
  • the MTIP1 protein in the present specification is not limited to the above-mentioned Arabidopsis thaliana MTIP1 protein, and broadly encompasses proteins having functions equivalent to those of the Arabidopsis thaliana MTIP1 protein.
  • the MTIP1 protein may be, for example, an Arabidopsis thaliana MTIP1 homolog or ortholog.
  • a person skilled in the art can obtain a protein having a function equivalent to that of Arabidopsis thaliana MTIP1 from the sequence information of Arabidopsis thaliana MTIP1. It has been confirmed that the amino acid sequence of the MTIP1 gene is widely conserved from angiosperms to pteridophytes.
  • MTIP1 proteins include proteins selected from the group consisting of (A) to (C) below.
  • A a protein having the amino acid sequence represented by SEQ ID NO: 1;
  • B having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1;
  • a protein that contributes to the improvement of magnesium tolerance in plants through expression or enhanced function (C) having an amino acid sequence with a sequence identity of 30% or more with the amino acid sequence represented by SEQ ID NO: 1, and enhanced in expression or function A protein that contributes to the improvement of magnesium tolerance in plants by
  • the Arabidopsis thaliana MTIP1 protein is known to have the ability to bind to magnesium transporters. Therefore, in the present specification, "a protein that contributes to the improvement of magnesium tolerance in plants by enhancing its expression or function" may be rephrased as "a protein that has the ability to bind to a magnesium transporter”.
  • examples of MTIP1 proteins include proteins selected from the group consisting of (A1) to (C1) below.
  • A1 a protein having the amino acid sequence represented by SEQ ID NO: 1;
  • B1 having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1;
  • a protein having the ability to bind to a magnesium transporter (C1)
  • magnesium transporter examples include MRS2-1 of Arabidopsis thaliana, which is a magnesium transporter that exists in vacuoles and is found to interact with the MTIP1 protein, and a magnesium transporter with an equivalent function.
  • the MTIP1 protein has a highly conserved region (14th to 100th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1) among the MTIP1 proteins of various plant species.
  • MTIP1 proteins include proteins selected from the group consisting of (A2) to (C2) below.
  • A2) a protein having the amino acid sequence represented by SEQ ID NO: 1;
  • a protein (C2 ) The sequence identity with the 14th to 100th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1 is 50% or more, and the sequence identity with the amino acid sequence represented by SEQ ID NO: 1 is 30%
  • the number of amino acids that may be deleted, substituted or added to SEQ ID NO: 1 is preferably 1 to 100, and 1 to 90. preferably 1 to 80, preferably 1 to 70, preferably 1 to 60, preferably 1 to 50, preferably 1 to 40, preferably 1 to 30, preferably 1 to 20 , preferably 1 to 15, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.
  • sequence identity of an amino acid sequence having a sequence identity of 50% or more with the 14th to 100th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1 preferably 60% to 100%, preferably 70% to 100%, preferably 80% to 100%, preferably 90% to 100%, preferably 95% to 100%, more preferably 98% % or more and 100% or less, more preferably 99% or more and 100% or less, can be exemplified.
  • the amino acid sequence having a sequence identity of 30% or more to the amino acid sequence represented by SEQ ID NO: 1 is , preferably 40% to 100%, preferably 50% to 100%, preferably 60% to 100%, preferably 70% to 100%, preferably 80% to 100%, preferably 90%
  • amino acid sequences having an identity of 95% or more and 100% or less, more preferably 98% or more and 100% or less, still more preferably 99% or more and 100% or less can be exemplified.
  • Amino acid sequence identity can be calculated using the BLASTP program provided by NCBI (National Center of Biotechnology Information).
  • the gene encoding the MTIP1 protein is a gene encoding any protein selected from the group consisting of (A) to (C), (A1) to (C1) and (A2) to (C2). and preferably a gene encoding any protein selected from the group consisting of (A) to (C).
  • Examples of the gene encoding the protein (A) include a polynucleotide having the nucleotide sequence represented by SEQ ID NO:2.
  • the nucleotide sequence represented by SEQ ID NO: 2 is the nucleotide sequence of the mORF coding region that encodes the Arabidopsis thaliana MTIP1 protein.
  • the expression regulatory region of the MTIP1 gene is not particularly limited as long as it has the function of regulating the expression level of the MTIP1 gene product. Examples include promoters, silencers, enhancers, and the like, and the upstream ORF of the MTIP1 gene described below is preferred.
  • the MTIP1 gene has an upstream ORF (also referred to as an upstream ORF or uORF) conserved among the MTIP1 genes of each plant on the 5' untranslated region side of the main ORF (major ORF) encoding the above MTIP1 protein. ing.
  • upstream ORF also referred to as an upstream ORF or uORF
  • Fig. 1 is a schematic diagram showing an example of the structure of MTIP1 mRNA containing uORF and mORF, and a diagram showing the results of comparing the amino acid sequences (SEQ ID NOS: 3 to 40) translated from uORF between plants.
  • the inventors have clarified that a peptide encoded by the uORF of the MTIP1 gene is involved in the translational regulation of the MTIP1 protein depending on the intracellular Mg 2+ concentration. They also found that disruption of the uORF of the MTIP1 gene facilitates the enhancement of the expression of the MTIP1 protein even in a high-concentration magnesium environment, and that the magnesium tolerance of plants can be improved.
  • Examples of magnesium-tolerant plants of the embodiments include plants in which the MTIP1 protein expression is enhanced to improve magnesium tolerance and the MTIP1 protein expression is enhanced by disruption of the uORF of the MTIP1 gene.
  • a magnesium-tolerant plant in which the uORF of the MTIP1 gene is disrupted can be exemplified as the magnesium-tolerant plant of the embodiment.
  • the plant species whose uORF amino acid sequences are shown in Fig. 1 are representative species of each taxonomic group (Order) in seed plants.
  • the inventors have revealed that the conserved region and initiation codon of the uORF of the MTIP1 gene are involved in the ability to suppress translation of the mORF of the MTIP1 gene (previous report: Hayashi et al. 2017 Nucleic Acids Res. 45:8844 -8858). As shown in Fig.
  • the peptide encoded by the uORF of the MTIP1 gene according to the embodiment has the ability to suppress the translation of MTIP1 protein in response to Mg 2+ .
  • Examples of the uORF of the MTIP1 gene include polynucleotides encoding peptides selected from the group consisting of (D) to (F) below.
  • E in the amino acid sequence represented by any one of SEQ ID NOs: 41 to 67
  • one or more amino acids A peptide having a deleted, substituted, or added amino acid sequence and having the ability to inhibit translation of the MTIP1 protein
  • F having a sequence identity of 70 with the amino acid sequence represented by any one of SEQ ID NOS: 41 to 67 % or more and having the ability to suppress the translation of the MTIP1 protein
  • the uORF of the MTIP1 gene is a polynucleotide encoding a peptide selected from the group consisting of (D1) to (F1) below.
  • D1 a peptide having the amino acid sequence represented by SEQ ID NO: 41
  • E1 having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 41, Peptide having the ability to suppress the translation of the MTIP1 protein
  • F1 A peptide having an amino acid sequence having a sequence identity of 70% or more with the amino acid sequence represented by SEQ ID NO: 41 and having the ability to suppress the translation of the MTIP1 protein
  • the number of amino acids that may be deleted, substituted, or added is preferably 1 to 6, preferably 1 to 5, 1 to 3 is more preferred, and 1 or 2 is even more preferred.
  • the amino acid sequence having a sequence identity of 70% or more is preferably 75% or more to the amino acid sequence represented by any one of SEQ ID NOs: 41 to 67. % or less, preferably 80% or more and 100% or less, preferably 85% or more and 100% or less, more preferably 90% or more and 100% or less, and still more preferably 93% or more and 100% or less .
  • the conserved region is preferably on the C-terminal side.
  • the terminal amino acid of the amino acid sequence (conserved region) represented by any one of SEQ ID NOs: 41 to 67 is 1 from the C-terminal side of the peptide encoded by the uORF of the MTIP1 gene. It is preferably in the 1st to 20th positions, more preferably in the 1st to 15th positions, and further preferably in the 1st to 10th positions.
  • it preferably has terminal amino acids of the amino acid sequence (conserved region) represented by any one of SEQ ID NOs: 41 to 67 at positions 1 to 20 from the C-terminal side, and A polynucleotide encoding a peptide having a total length of preferably 20 to 100 amino acids, more preferably 25 to 90 amino acids, and even more preferably 30 to 80 amino acids can be exemplified.
  • the MTIP1 gene is a gene encoding any protein selected from the group consisting of (A) to (C), and the uORF is any one of SEQ ID NOs: 41 to 67.
  • the terminal amino acid of the amino acid sequence (conserved region) represented by is 1 to 20 from the C-terminal side, and preferably has a total length of 20 to 100 amino acids, more preferably a total length of 25 to 90 amino acids, More preferably, a polynucleotide encoding a peptide with a total length of 30 to 80 amino acids can be exemplified.
  • An example of the uORF of the MTIP1 gene is a polynucleotide encoding a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 3 to 40 shown in FIG.
  • the uORF encoding the peptide having the amino acid sequence represented by SEQ ID NO: 3, which has the amino acid sequence represented by SEQ ID NO: 41 in (D) above, is a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 68. mentioned.
  • the nucleotide sequence represented by SEQ ID NO: 68 is the uORF nucleotide sequence of MTIP1 of Arabidopsis thaliana.
  • Disruption of the uORF includes deletion, substitution, insertion, addition, or a combination of all or part of the base sequence of the uORF of the MTIP1 gene, resulting in loss or suppression of the ability to suppress translation of the MTIP1 protein. can be exemplified. Disruption of the uORF is sufficient as long as the ability to suppress translation of the MTIP1 protein is lost or suppressed.
  • disruption of the uORF includes: A state in which the ability to suppress translation of the MTIP1 protein is lost or suppressed by deletion, substitution, insertion, addition, or a combination thereof of all or part of the nucleotide sequence can be exemplified.
  • the ability to suppress the translation of the MTIP1 protein can be lost or suppressed.
  • insertion or deletion of another sequence into or from the uORF sequence of the MTIP1 gene causes a frameshift, which can result in loss or suppression of the ability to suppress translation of the MTIP1 protein.
  • the addition or substitution of another sequence to the uORF sequence of the MTIP1 gene causes a structural change in the gene product, which can result in the loss or suppression of the ability to suppress translation of the MTIP1 protein.
  • conserveed regions corresponding to the amino acid sequences represented by SEQ ID NOs: 41 to 67 can be easily identified by a person skilled in the art by aligning the amino acid sequences of the uORFs of MTIP1 mRNA of each plant, as illustrated in FIG. Identifiable.
  • the magnesium-tolerant plant of the embodiment may have improved magnesium tolerance by disruption of the uORF, the uORF in the magnesium-tolerant plant can be confirmed, for example, in a control plant of the plant of the embodiment.
  • a polynucleotide having a uORF encoding any peptide selected from the group consisting of (D) to (F) and (D1) to (F1) as the MTIP1 gene encoding the MTIP1 protein more preferably Polynucleotides having a uORF encoding any peptide selected from the group consisting of (D1) to (F1) are exemplified.
  • the MTIP1 gene encoding the MTIP1 protein encodes any protein selected from the group consisting of (A) to (C), (A1) to (C1) and (A2) to (C2); Examples of polynucleotides having a uORF encoding any peptide selected from the group consisting of (D) to (F) and (D1) to (F1) are shown below.
  • the MTIP1 protein is one in which the MTIP1 gene encoding the MTIP1 protein has a uORF encoding any peptide selected from the group consisting of (D) to (F) and (D1) to (F1). Illustrate.
  • the MTIP1 protein is any protein selected from the group consisting of (A) to (C), (A1) to (C1), and (A2) to (C2), and encodes the MTIP1 protein.
  • the MTIP1 gene has a uORF encoding any peptide selected from the group consisting of (D) to (F) and (D1) to (F1).
  • wild-type MTIP1 mRNA has a uORF upstream of the MTIP1 mORF.
  • short peptides of 33-70 amino acids are synthesized when the uORF is translated by the ribosome.
  • Mg 2+ concentration in the cytoplasm is high, it is thought that the ribosomes that synthesized themselves are stagnated in the uORF in response to Mg 2+ .
  • FIG. 2 is a schematic diagram showing an example of a mechanism for maintaining intracellular Mg 2+ concentration homeostasis by uORF-mediated MTIP1 expression control.
  • Mg 2+ concentration in the cytoplasm is low (Fig. 2, bottom)
  • ribosomes start scanning from the upstream of uORF, and ribosomes skip the uORF and resume translation at the mORF, resulting in translation of the mORF. is considered to be
  • Interaction of the translated MTIP1 protein with the Mg 2+ transporter is thought to result in the influx of Mg 2+ from the vacuole to the cytoplasm through the Mg 2+ transporter.
  • the Mg 2+ concentration in the cytoplasm is sufficient (Fig.
  • ribosomes are stagnant in the uORF, which suppresses the above-mentioned skipping and translation restart, and the mORF-encoded MTIP1 protein is suppressed. It is believed that translation is suppressed. In this way, it is thought that the uORF suppresses the translation of the MTIP1 protein and suppresses the influx of Mg 2+ from the vacuole to the cytoplasm via the Mg 2+ transporter.
  • the Mg 2+ concentration in the cytoplasm is likely to be further increased.
  • enhancement of homeostatic maintenance that keeps the Mg 2+ concentration in the cytoplasm constant contributes to the improvement of magnesium tolerance in plants. Being able to improve performance is an unexpected result that goes beyond normal assumptions.
  • the expression level of the wild-type MTIP1 gene is controlled by the uORF in a high-concentration magnesium environment. It is very useful as a technique for enhancing the expression of MTIP1 protein in . For example, it is considered that the expression or function of MTIP1 protein can be more effectively enhanced in a high-concentration magnesium environment, compared to the case of enhancing the transcription level of MTIP1 mRNA using an overexpression promoter.
  • the type of plant used in this embodiment is not particularly limited as long as the effects of the present invention can be obtained. Since the MTIP1 gene and its uORF peptide sequence are widely conserved among plant species, they are widely applicable to plants in general.
  • the plant of the present embodiment is preferably a seed plant, more preferably an angiosperm.
  • Plants of the present embodiment include, for example, grasses such as rice, wheat and corn; solanaceous plants such as eggplants, tomatoes, capsicums, potatoes and tobacco; cucurbitaceous plants such as cucumbers, melons and pumpkins; , soybeans, beans and other leguminous plants, Asteraceous plants such as chrysanthemum and lettuce, Chenopodiaceous plants such as spinach, Rosaceous plants such as strawberries and apples, Grape plants such as grapes, Rutaceous plants such as mandarin oranges, Arabidopsis thaliana , rape, cabbage, broccoli, cauliflower, Chinese cabbage, Japanese radish, and the like, and among the above examples, cruciferous plants belonging to the cruciferous family are preferred.
  • the term "plant” may be the entire plant body or a part thereof.
  • plants according to embodiments include seeds, plant cells, plant cultured cells, cell masses, callus, plant tissues, and organs.
  • next-generation plants (progeny) or clones of the magnesium-tolerant plants of the embodiments are also included in the magnesium-tolerant plants of the embodiments, as long as they have magnesium tolerance.
  • the plant of the present embodiment may have sodium tolerance to high concentrations of sodium (sodium salt or Na + ion) in addition to magnesium tolerance.
  • a high sodium concentration is a concentration of sodium that induces stunting or hypernatremia in control plants.
  • the sodium tolerance may be possessed by conventionally known sodium-tolerant plants such as those shown in Non-Patent Document 1, for example.
  • the main cause of salt damage caused by sea water is the high concentrations of Na + and Mg 2+ ions contained in sea water. can be suitably used for the cultivation of
  • a method for producing a magnesium-tolerant plant of the embodiment is a method including enhancing the expression or function of MTIP1 protein in the plant. Magnesium tolerance can be improved by enhancing the expression or function of the MTIP1 protein in the plant of interest. According to this production method, the magnesium-tolerant plant of the above embodiment can be produced.
  • a method for improving magnesium tolerance in plants which includes enhancing the expression or function of MTIP1 protein in plants.
  • the enhancement of MTIP1 protein expression means that the expression level of MTIP1 protein is increased as compared with the expression level of MTIP1 protein in the control plant.
  • the enhanced function of MTIP1 protein means that the function of MTIP1 protein is improved as compared with the function of MTIP1 protein in the control plant.
  • MTIP1 proteins examples include those exemplified in the magnesium-tolerant plants of the above embodiments.
  • the expression or function enhancement of MTIP1 protein in plants can be artificially enhanced by genetic engineering techniques. For example, it is preferable to enhance the expression or function of the MTIP1 protein by genetically modifying the target plant to improve magnesium tolerance.
  • the enhancement improves the magnesium tolerance of the plant by enhancing the expression of the MTIP1 protein, and disrupting the uORF of the MTIP1 gene of the plant to reduce the MTIP1 protein. It is preferable to increase the expression level of
  • Examples of uORFs and their destruction include those exemplified in the magnesium-tolerant plants of the above embodiments.
  • the method for producing a magnesium-tolerant plant of the embodiment can be exemplified by a method for producing a magnesium-tolerant plant comprising disrupting the uORF of the MTIP1 gene.
  • the method for producing a magnesium-tolerant plant of the embodiment includes: enhancing the expression or function of the MTIP1 protein in a plant; and evaluating the magnesium tolerance of the plant and selecting a magnesium-tolerant plant.
  • Magnesium tolerance can be evaluated by growing plants in a high-concentration magnesium environment and observing the growth state. If the growth is good, it can be evaluated as having magnesium tolerance.
  • the plant whose magnesium tolerance is improved is not particularly limited, but is preferably a seed plant, more preferably an angiosperm. Plants include those exemplified in the magnesium-tolerant plants of the above embodiments.
  • the plant species produced may be magnesium-tolerant as well as sodium-tolerant to high concentrations of sodium (sodium salts or Na + ions).
  • the sodium tolerance may be possessed by conventionally known sodium-tolerant plants such as those shown in Non-Patent Document 1, for example.
  • a plant with improved sodium tolerance and magnesium tolerance can be produced by further enhancing the expression or function of MTIP1 protein in an existing plant with improved sodium tolerance by genetic engineering techniques.
  • a plant with improved sodium tolerance and magnesium tolerance can be produced by crossing an existing plant with improved sodium tolerance and the magnesium-tolerant plant of the present embodiment.
  • a method for cultivating a magnesium-tolerant plant of the embodiment includes cultivating the magnesium-tolerant plant of the embodiment in a high-concentration magnesium environment.
  • a high concentration of magnesium is an amount of magnesium at a concentration that induces growth inhibition or hypermagnesium in control plants of magnesium-tolerant plants of interest.
  • Examples of high-concentration magnesium environments include media with a Mg 2+ molar concentration of 5 mM or more, which may be 5 to 100 mM, 10 to 80 mM, or 15 to 50 mM. There may be.
  • a high-concentration magnesium environment in soil can be exemplified by, for example, a case where the content of exchangeable Mg 2+ in soil is 50 mg / 100 g or more in terms of MgO, and may be 50 to 200 mg / 100 g. It may be 150mg/100g.
  • the plant can be cultivated satisfactorily with improved resistance to magnesium stress even in a high-concentration magnesium environment.
  • Wild-type MTIP1 mRNA has a uORF upstream of the mORF of MTIP1, but the gene of one embodiment of the present invention is an MTIP1 gene in which the uORF is disrupted, and the MTIP1 protein encoded by the MTIP1 gene is Provided is the MTIP1 gene, which is a protein selected from the group consisting of the following (A) to (C).
  • A a protein having the amino acid sequence represented by SEQ ID NO: 1;
  • B having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1;
  • a protein that contributes to the improvement of magnesium tolerance in plants through enhanced expression or function (C) having an amino acid sequence with a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, and enhanced in expression or function A protein that contributes to the improvement of magnesium tolerance in plants by
  • the uORF of the MTIP1 gene encodes a peptide selected from the group consisting of (D) to (F) below, and the disruption of the uORF is any one of SEQ ID NOS: 41 to 67
  • the ability to suppress translation of the MTIP1 protein is lost or suppressed by deletion, substitution, insertion, addition, or a combination thereof of all or part of the nucleotide sequence in the region corresponding to the amino acid sequence represented by Preferably.
  • a gene in the present embodiment may be a nucleic acid such as DNA or RNA, and includes polynucleotides on the genome, as well as polynucleotides, mRNA, cDNA, and isolated gene clones of gene products thereof.
  • the expression of MTIP1 protein is easily enhanced in plants, and the magnesium tolerance of plants can be improved.
  • a plant having or introduced with the gene of the embodiment has improved magnesium tolerance.
  • the oligonucleotide pair of sequences gR2for (SEQ ID NO: 69: 5′-ATTGCGTTCACGGGTCGAGGCCA-3′) and gR2rev (SEQ ID NO: 70: 5′-AAACTGGCCTCGACCCGTGAACG-3′) and gR3for (SEQ ID NO: 71: 5'-ATTGGATCCGAAAGCGAAAACAG-3') and gR3rev (SEQ ID NO: 72: 5'-AAACCTGTTTTCGCTTTCGGATC-3') were annealed to generate a genome-editing binary vector plasmid pKI1.1R (previous report: Tsutsui, H.
  • the T-DNA region of pKI1.1R contains an expression cassette that expresses gRNA, an expression cassette that expresses Cas9 protein in meristems, and an expression cassette that expresses red fluorescent protein RFP in seeds.
  • the prepared plasmid was introduced into the Agrobacterium C58C1RifR (pGV2260) strain, and Arabidopsis thaliana (ecotype Col-0) was infected with the Agrobacterium by the floral dip method.
  • Arabidopsis thaliana ecotype Col-0
  • T1 seeds collected from Agrobacterium-infected plants seeds emitting red fluorescence were selected as transgenic seeds, sown in Jiffy Seven (manufactured by Sakata Seed Co., Ltd.) and cultivated under constant light conditions at 22°C. bottom.
  • DNA was extracted from rosette leaves of transgenic plants of the T1 generation and subjected to heteroduplex mobility assay (HMA) and sequence analysis to detect mutations introduced into the uORF of the MTIP1 gene.
  • HMA heteroduplex mobility assay
  • Transient expression analysis A transient expression system was used to investigate how the mutations in the MTIP1 uORF caused by genome editing affect the expression of the MTIP1 protein.
  • the gene encoding the luciferase NanoLuc (Nluc) derived from Oplophorus gracilirostris (Nluc) with a PEST sequence added to the protein (NlucP) was transferred to the cauliflower mosaic virus 35S RNA promoter. Plasmid pNH007 was constructed which was ligated downstream of (35S promoter).
  • plasmid pNL1.2 manufactured by Promega
  • primers NlucPfor SEQ ID NO: 79: 5'-GAAAGATGGCGTCGACGGTCTTCACACTCGAAGA-3'
  • NlucPrev SEQ ID NO: 80: 5'-TCATCTTCATCTTCGAGCTCTTAGACGTTGATGCGAGCTG-3
  • Plasmid pNH007 was prepared by inserting the resulting PCR fragment between the SalI and SacI sites of plasmid pKM56 (previous report: Hayashi et al. 2017) using the SLiCE method.
  • primers AT1G70780 5′UTRfor (SEQ ID NO: 81: 5′-ATTTGGAGAGAACCAAACAATCACATTCTTCTC-3′) and AT1G70780 5′UTRrev (SEQ ID NO: 82: 5′) were obtained from wild-type Arabidopsis thaliana (ecotype Col-0) and mutant genomic DNA.
  • -AGAGTCGACAACATCTTCGAAATCGAGA-3' was used to amplify the 5' untranslated region of the MTIP1 gene by PCR.
  • the 35S promoter region was amplified using plasmid pBI221 as a template and primers pUC19rev3 (SEQ ID NO: 83: 5'-GACCATGATTACGCCAAGCT-3') and AT1G70780 35Srev (SEQ ID NO: 84: 5'-TGTGATTGTTGGTTCTCTCCAAATGAAATGAACT-3').
  • pUC19rev3 SEQ ID NO: 83: 5'-GACCATGATTACGCCAAGCT-3'
  • AT1G70780 35Srev SEQ ID NO: 84: 5'-TGTGATTGTTGGTTCTCTCCAAATGAAATGAACT-3'
  • the PCR fragment of the 35S promoter region and the PCR fragment of the MTIP1 5' untranslated region were fused using overlap PCR.
  • the fused PCR fragment was cut with EcoRV and SalI and inserted between the EcoRV site in the 35S promoter of plasmid pNH00
  • the reporter plasmid thus prepared and the internal standard plasmid pKM56 (Hayashi et al. 2017) (a plasmid with a construct in which the luciferase [Eluc-PEST] gene derived from the Brazilian click beetle (Pyrearinus termitilluminans) is linked downstream of the 35S promoter ) was introduced into protoplasts of Arabidopsis thaliana cultured cells MM2d using the polyethylene glycol (PEG) method.
  • PEG polyethylene glycol
  • washing buffer 0.4 M mannitol, 5 mM CaCl 2 , and 0.5 M 2-(N-morpholino)ethanesulfonic acid, pH 5.8 was added and mixed.
  • Protoplasts were harvested by centrifugation and resuspended in 1000 ⁇ l modified liquid LS medium containing 0.4 M mannitol without MgSO 4 .
  • 450 ⁇ l of the protoplast suspension was dispensed into two microtubes, 3 ⁇ l of 300 mM MgSO 4 (final concentration: 2.0 mM) was added to one, and the same amount of water was added to the other.
  • Wild-type MTIP1 5′ was observed in both media containing 2 mM MgSO 4 (Mg + ), which is sufficient for the growth of wild-type Arabidopsis thaliana, and in media without Mg salts (Mg ⁇ ).
  • Reporter plasmids with mutant MTIP1 5' untranslated regions showed higher Nluc activity than reporter plasmids with untranslated regions.
  • the difference between the wild-type and mutants was remarkable when they were cultured in a medium (Mg + ) containing 2 mM MgSO 4 . This result suggests that genome-edited mutations in the MTIP1 uORF had a stimulatory effect on the translation of the major ORF and increased the expression of the MTIP1 protein.
  • Mg resistance test of MTIP1 uORF disruption strain Selected by PCR, HMA, and sequence analysis. In addition, lines with no red fluorescence of seeds were selected to establish mutant homozygous lines in which the T-DNA was deleted. Seeds of the homozygous mutant lines thus established were grown together with seeds of wild-type Arabidopsis thaliana (ecotype Col-0) on MGRL agar medium containing 20 mM MgSO 4 (previous report: Fujiwara, T., Hirai, M. Y., Chino, M., Komeda, Y. & Naito, S. Effects of Sulfur Nutrition on Expression of the Soybean Seed Storage Protein Genes in Transgenic Petunia. Plant Physiol. 99, (1992). Cultivated for 2 weeks. The composition of the medium used is shown below.

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Abstract

La présente invention concerne une plante tolérante au magnésium et dont la fonction ou l'expression d'une protéine MTIP1 est améliorée. La présente invention concerne un procédé pour produire une plante tolérante au magnésium incluant l'amélioration de l'expression ou de la fonction d'une protéine MTIP1 chez ladite plante.
PCT/JP2022/012317 2021-09-10 2022-03-17 Plante tolérante au magnésium, procédé de production d'une plante tolérante au magnésium, procédé de culture d'une plante tolérante au magnésium et gène WO2023037620A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Journal of the American College of Cardiology", vol. 69, 25 March 2021, ELSEVIER, AMSTERDAM, NL, ISSN: 0735-1097, article HAYASHI NORIYA: "Studies on upstream open reading frame-eoncoded peptides that cause ribosome stalling in plants [an abstract of entire text]", pages: 1 - 4, XP093046000 *
DATABASE Uniprot Uniprot; . . : "At1g70780", XP093046028 *
HAYASHI NORIYA, SASAKI SHUN, TAKAHASHI HIRO, YAMASHITA YUI, NAITO SATOSHI, ONOUCHI HITOSHI: "Identification of Arabidopsis thaliana upstream open reading frames encoding peptide sequences that cause ribosomal arrest", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 45, no. 15, 6 September 2017 (2017-09-06), GB , pages 8844 - 8858, XP093046008, ISSN: 0305-1048, DOI: 10.1093/nar/gkx528 *
UM TAEYOUNG, PARK TAEHYEON, SHIM JAE SUNG, KIM YOUN SHIC, LEE GANG-SEOB, CHOI IK-YOUNG, KIM JU-KON, SEO JUN SUNG, PARK SOO CHUL: "Application of Upstream Open Reading Frames (uORFs) Editing for the Development of Stress-Tolerant Crops", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 22, no. 7, pages 3743, XP093046007, DOI: 10.3390/ijms22073743 *

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