ARTIFICIAL Lsi1 PROTEIN

Abstract

The present invention provides a protein including an amino acid sequence that has at least 80% identity with the amino acid sequence represented by SEQ ID NO: 7 or 8. The protein has suppressed silicic acid transport activity.

Description

Artificial Lsi1 protein

[0001] The present invention relates to an artificial Lsi1 protein and the like.

[0002] In recent years, plant-derived biomass fuels have been attracting attention from the perspectives of preventing global warming and achieving carbon neutrality. While various plants have been used as raw materials for biomass fuels, large grasses such as sorghum are expected to be a new raw material because they have a higher biomass productivity per unit area than trees and can grow in harsh environments.

[0003] Among the components contained in grasses such as sorghum, lignin contained in the secondary cell walls of cells is considered to be a very useful component from the perspective of fuel use because it has a high carbon content and a high calorific value, and there is a demand for the development of plants with an increased lignin content as a raw material for biomass fuel. There is also a demand for the development of soft plants with a reduced silicon content that can be easily used as a fuel raw material.

[0004] It has been reported that mutation of a protein called Lsi1 in rice reduces the silicon content and increases the lignin content in rice plants that contain this protein (Non-Patent Document 1), and that suppressing the expression of the Lsi6 gene reduces the amount of silicon accumulated in rice (Patent Document 1).On the other hand, with regard to sorghum, which is gaining increasing attention as a raw material for biomass fuel, it has been reported that mutation of the Lsi1 protein reduces the silicon content in sorghum (Non-Patent Document 2).

[0005] Japanese Patent Application Laid-Open No. 2008-220306

[0006] S. Suzuki et al. , Silicon efficiency promotes lignin accumulation in rice, Plant Biotechnology, 29, 391-394 (2012). Oshry Markovich et al. , Silicification in Leaves of Sorghum Mutant with Low Silicon Accumulation, Silicon, 11, 2385-2391 (2019).

[0007] Therefore, the problem to be solved by the present invention is to provide a plant in which the amount of silicon accumulated is reduced and the amount of lignin accumulated is increased.

[0008] As a result of extensive research aimed at solving the above problems, the present inventors have discovered a novel artificial protein in plants that exhibits suppressed silica transport activity, and have also found that plants expressing the novel artificial protein with suppressed silica transport activity accumulate less silicon and more lignin.

[0009] That is, the present application encompasses the following inventions: [1] A protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 7 or 8, and having reduced silica transport activity. [2] The protein according to [1], comprising an amino acid sequence comprising an addition, substitution, and / or deletion of 1 to 20 amino acids at positions 60 to 160 of the amino acid sequence shown in SEQ ID NO: 6, and / or an amino acid sequence comprising an addition, substitution, and / or deletion of 1 to 20 amino acids at positions 170 to 260 of the amino acid sequence shown in SEQ ID NO: 6. [3] The protein according to [1] or [2], into which, at positions 60 to 160 and / or positions 170 to 260 of the amino acid sequence shown in SEQ ID NO: 6, (i) a mutation substituting a glycine with another polar amino acid, and / or (ii) a mutation substituting alanine with another hydrophobic amino acid has been introduced. [4] The protein according to [3], wherein the glycine is present at a position where it can interact with an extramolecular water molecule of the protein, and the alanine is present at a position where it can interact with an adjacent helix structure. [5] A protein consisting of the amino acid sequence set forth in SEQ ID NO: 7 or 8. [6] A nucleic acid encoding the protein according to any one of [1] to [5]. [7] A vector having the nucleic acid according to [6]. [8] A transformant having the nucleic acid according to [6] or the vector according to [7]. [9] A plant having the protein according to any one of [1] to [5].

[10] A plant having the nucleic acid according to [6] on its genome.

[0010] According to the present invention, it is possible to provide a plant in which the amount of silicon accumulated is reduced and the amount of lignin accumulated is increased.

[0011]

[0033] Figure 1 shows the measurement results of silicon and lignin content in Example 1. Values ​​are shown as the mean ± standard deviation (SD) of two individually combined strains (n ​​= 12), and asterisks indicate significant differences between the wild-type strain (WT) and the mutant strain (Student's t-test, * p < 0.05; ** p < 0.01; *** p < 0.001).

[0034] Figure 1 shows the alignment of the amino acid sequences of the wild-type strain silica transporter proteins, mutant 1 and mutant 2 (SEQ ID NOS: 6 to 8, respectively), with the amino acid sequence of the rice silica transporter protein (SEQ ID NOS: 9). A 3D model of the silica transporter protein, consisting of the amino acid sequence shown in SEQ ID NOS: 10, predicted by the AlphaFold Server model, is shown. A 3D model of the silica transporter protein, consisting of the amino acid sequence shown in SEQ ID NOS: 10, forming a tetramer is shown.

[0012] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0013] The protein of this embodiment is a protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7 or 8, and has suppressed silica transport activity, and is also referred to herein as an "artificial Lsi1 protein" or "the Lsi1 protein of this embodiment." As used herein, the term "artificial" in "artificial Lsi1 protein" means that it has been artificially created, in comparison with naturally occurring Lsi1 proteins. The "Lsi1 protein" herein is also referred to as a silicate transporter protein, a silicate channel protein, or simply Lsi1, etc.

[0014] As used herein, "a protein with suppressed silica transport activity" means that the transport activity of a protein with silica transport activity is suppressed. Here, "suppressed" may refer to a protein with silica transport activity completely losing its function, or to a partial loss of its function. The Lsi1 protein of this embodiment has an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 7 or 8. That is, the Lsi1 protein is different from a protein in which truncation of the amino acid sequence in the amino acid sequence causes the protein to cease functioning, i.e., lacks silica transport activity. The suppression of silica transport activity may be confirmed by measuring the activity of taking up silica into plant cells containing the Lsi1 protein of this embodiment. Therefore, in this embodiment, "suppressed silica transport activity of a protein" may mean that the amount of silica uptake in plant cells containing the Lsi1 protein of this embodiment is suppressed compared to the activity of taking up silica in wild-type plant cells, specifically, plant cells containing the wild-type Lsi1 protein. As used herein, the term "wild-type" preferably refers to an Lsi1 protein consisting of the amino acid sequence shown in SEQ ID NO: 6. The amino acid sequence shown in SEQ ID NO: 6 is an example of an amino acid sequence constituting a wild-type sorghum-derived Lsi1 protein.

[0015] As used herein, "sorghum" refers to a plant belonging to the genus Sorghum of the family Poaceae, whose scientific name is Sorghum bicolor, and includes, for example, grain sorghum, sweet sorghum, etc. Sorghum is also known as corn, sorghum millet, sorghum, sorgo, etc.

[0016] In this embodiment, when a plant has the Lsi1 protein of this embodiment, silicon accumulation is suppressed in the plant body. Also, in this embodiment, when a plant has the Lsi1 protein of this embodiment, lignin accumulation is preferably promoted in the plant body. This embodiment also provides a protein comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7 or 8, which suppresses silicon accumulation and promotes lignin accumulation in a plant body.

[0017] As used herein, "suppressing silicon accumulation in a plant" refers to reducing the amount of silicon taken up into a plant, or reducing the amount of silicon accumulated in a plant, compared to a wild type. Alternatively, it may refer to completely eliminating silicon uptake into a plant. Here, silicon taken up and accumulated in a plant may be in any form, but is preferably in the form of silicic acid (Si(OH) ４ ) and silicon dioxide (SiO ２ ) and silicic acid (Si(OH) ４ Silicon may be accumulated in any part of the plant, but it is preferred that silicon be accumulated mainly on the surface of leaves, stems, husks, etc., other than the roots and endosperm.

[0018] As used herein, "promoting lignin accumulation in a plant" refers to increasing the amount of lignin synthesized in a plant, or increasing the amount of lignin accumulated in a plant, compared to a wild-type plant. Here, the lignin synthesized and accumulated in a plant may be in any form, and may be, for example, a mixture of guaiacyl lignin (G-lignin), syringyl lignin (S-lignin), and p-hydroxyphenyl lignin (H-lignin). In particular, the amounts of G-lignin and H-lignin accumulated may be increased. Furthermore, lignin may accumulate in any part of a plant, but it is preferable that it accumulates mainly in the secondary cell walls of cells.

[0019] In this embodiment, "comparison with the wild type" in "suppressing silicon accumulation in a plant" or "promoting lignin accumulation in a plant" may mean a comparison with a plant having a wild-type Lsi1 protein. "Comparison with the wild type" may also mean a comparison with a plant having an Lsi1 protein consisting of the amino acid sequence shown in SEQ ID NO:6.

[0020] As used herein, "identity" refers to the percentage (%) of amino acids that are the same at the same position or in the same row when two amino acid sequences are aligned. The percentage (%) of amino acids that are the same or similar at the same position or in the same row when two amino acid sequences are aligned is called homology, and is distinguished from this.

[0021] For example, an enzyme having 80% identity to a specific enzyme consisting of 100 amino acids may be an enzyme that, when aligned with the amino acid sequence of the specific enzyme consisting of 100 amino acids, has 80 positions where identical amino acids are lined up in the same position or row.

[0022] Furthermore, as used herein, "similar amino acids" refers to two or more amino acids that have similar chemical or physical properties. General classification of amino acids can be used as a reference for determining such similarity. For example, if two amino acids are classified into the same amino acid group, such as acidic amino acids, basic amino acids, aromatic amino acids, aliphatic amino acids, amino acids with hydroxyl groups, hydrophilic amino acids, or hydrophobic amino acids, the two amino acids are considered to have similar chemical or physical properties.

[0023] In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence shown in SEQ ID NO: 7 or 8.

[0024] Furthermore, in one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence comprising additions, substitutions, and / or deletions of 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids in the amino acid sequence set forth in SEQ ID NO: 6. Herein, when described as "X to Y (X and Y are 0 or a positive number)," X to Y are synonymous with at least X and at most Y, and indicate a range including the values ​​of X and Y.

[0025] In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence comprising an addition, substitution, and / or deletion of 1 to 20 amino acids at positions 60 to 160 of the amino acid sequence shown in SEQ ID NO: 6, and / or an amino acid sequence comprising an addition, substitution, and / or deletion of 1 to 20 amino acids at positions 170 to 260 of the amino acid sequence shown in SEQ ID NO: 6.

[0026] In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence comprising addition, substitution, and / or deletion of 1, 2, 3, 4, or 5 amino acids at positions 60 to 89, 90 to 110, 111 to 140, 141 to 160, 170 to 189, 190 to 210, and / or 211 to 260 of the amino acid sequence set forth in SEQ ID NO: 6.

[0027] In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence comprising an addition, substitution, and / or deletion of 1, 2, 3, 4, or 5 amino acids at positions 90 to 110 and / or positions 190 to 210 of the amino acid sequence shown in SEQ ID NO: 6. In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence comprising an addition, substitution, and / or deletion of one amino acid at positions 95 to 105 and / or positions 195 to 205 of the amino acid sequence shown in SEQ ID NO: 6. In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence comprising an addition, substitution, and / or deletion of one amino acid at positions 103 and / or 201 of the amino acid sequence shown in SEQ ID NO: 6. In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence in which the glycine at position 103 is substituted with aspartic acid and / or the alanine at position 201 is substituted with valine in the amino acid sequence shown in SEQ ID NO: 6.

[0028] In one embodiment, the Lsi1 protein of this embodiment is a protein consisting of the amino acid sequence shown in SEQ ID NO: 7 or 8. SEQ ID NO: 7 represents the amino acid sequence in which the glycine at position 103 of the amino acid sequence shown in SEQ ID NO: 6 is substituted with aspartic acid. SEQ ID NO: 8 represents the amino acid sequence in which the alanine at position 201 of the amino acid sequence shown in SEQ ID NO: 6 is substituted with valine. The Lsi1 protein of this embodiment is a protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7 or 8, and preferably, the amino acid sequence that is 80% identical has aspartic acid at position 103 of the amino acid sequence shown in SEQ ID NO: 7 or valine at position 201 of the amino acid sequence shown in SEQ ID NO: 8. The Lsi1 protein of this embodiment may be a protein comprising an amino acid sequence shown in SEQ ID NO: 7, which has an aspartic acid at position 103 of the amino acid sequence shown in SEQ ID NO: 7 and has at least 80% identity to the amino acid sequence shown in SEQ ID NO: 7, or a protein comprising an amino acid sequence shown in SEQ ID NO: 8, which has a valine at position 201 of the amino acid sequence shown in SEQ ID NO: 8 and has at least 80% identity to the amino acid sequence shown in SEQ ID NO: 8. The Lsi1 protein of this embodiment may be a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence shown in SEQ ID NO: 7 or 8, which contains an addition, substitution, and / or deletion of 1 to 5 amino acids at positions 80 to 120 and / or 180 to 220 of the amino acid sequence shown in SEQ ID NO: 6, or a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence shown in SEQ ID NO: 7 or 8, which has an aspartic acid at position 103 of the amino acid sequence shown in SEQ ID NO: 7 and / or a valine at position 201 of the amino acid sequence shown in SEQ ID NO: 8. In the above description of the Lsi1 protein of this embodiment, the identity may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity.The Lsi1 protein of this embodiment may be a protein comprising the amino acid sequence shown in SEQ ID NO: 7, which has an aspartic acid at position 103 of the amino acid sequence shown in SEQ ID NO: 7 and includes additions, substitutions, and / or deletions of 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids; or a protein comprising the amino acid sequence shown in SEQ ID NO: 8, which has a valine at position 201 of the amino acid sequence shown in SEQ ID NO: 8 and includes additions, substitutions, and / or deletions of 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids. Furthermore, the Lsi1 protein of this embodiment may be a protein having an amino acid sequence shown in SEQ ID NO: 6, in which the 103rd amino acid, glycine, in the amino acid sequence shown in SEQ ID NO: 6, is aspartic acid, and / or the 201st amino acid, alanine, in the amino acid sequence shown in SEQ ID NO: 6, is valine, and which includes additions, substitutions, and / or deletions of 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids.

[0029] In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence comprising an addition, substitution, and / or deletion of one amino acid at positions 67, 68, 72, 86, 87, 97, 99, 103, 104, 109, 113, 115, 131, 136, 139, 154, 182, 189, 191, 194, 197, 201, 209, 210, 214, 220, 226, 228, and / or 250 of the amino acid sequence set forth in SEQ ID NO: 6.

[0030] In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence in which glycine at positions 72, 87, 99, 103, 154, 191, 210, and / or 214 of the amino acid sequence shown in SEQ ID NO: 6 is substituted with another polar amino acid. Examples of polar amino acids include aspartic acid, asparagine, glutamine, glutamic acid, histidine, lysine, and arginine. The glycines at positions 72, 87, 99, 103, 154, 191, 210, and 214 of the amino acid sequence shown in SEQ ID NO: 6 may be glycines located at positions capable of interacting with extramolecular water molecules of a protein comprising the amino acid sequence shown in SEQ ID NO: 6. Silica transport activity can be inhibited by substituting another polar amino acid for glycine at positions 72, 87, 99, 103, 154, 191, 210, and / or 214 of the amino acid sequence shown in SEQ ID NO: 6. Silica transport activity can also be inhibited by substituting another polar amino acid for the glycine present at the above-mentioned positions capable of interacting with water molecules, so that the polar amino acid interacts with water molecules or does not interact with water molecules.

[0031] In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence in which alanine at positions 67, 68, 86, 104, 109, 113, 115, 131, 136, 139, 182, 194, 197, 201, 209, 220, 226, 228, and / or 250 of the amino acid sequence set forth in SEQ ID NO: 6 is substituted with another hydrophobic amino acid. Examples of hydrophobic amino acids include valine, isoleucine, leucine, methionine, and phenylalanine. The alanines at positions 67, 68, 86, 104, 109, 113, 115, 131, 136, 139, 182, 194, 197, 201, 209, 220, 226, 228, and 250 of the amino acid sequence shown in SEQ ID NO: 6 may be alanines located at positions capable of interacting with an adjacent helix structure, preferably an adjacent helix structure within a protein molecule comprising the amino acid sequence shown in SEQ ID NO: 6. Silica transport activity can be inhibited by substituting another hydrophobic amino acid for the alanine at positions 67, 68, 86, 104, 109, 113, 115, 131, 136, 139, 182, 194, 197, 201, 209, 220, 226, 228, and / or 250 of the amino acid sequence shown in SEQ ID NO: 6. Silica transport activity can also be inhibited by substituting another hydrophobic amino acid for an alanine present in a position within the above protein molecule that is capable of interacting with an adjacent helix structure, thereby changing the three-dimensional structure of the protein.

[0032] In one embodiment, the Lsi1 protein of this embodiment is a protein comprising an amino acid sequence in which the alanine at positions 67, 86, 97, 182, 189, 197, and / or 201 of the amino acid sequence shown in SEQ ID NO: 6 is substituted with another hydrophobic amino acid. The alanines at positions 67, 86, 97, 182, 189, 197, and 201 of the amino acid sequence shown in SEQ ID NO: 6 may be alanines located at positions capable of interacting with an adjacent helix structure, preferably an adjacent helix structure outside the molecule of a protein comprising the amino acid sequence shown in SEQ ID NO: 6. Silica transport activity can be inhibited by substituting the alanine at positions 67, 86, 97, 182, 189, 197, and / or 201 of the amino acid sequence shown in SEQ ID NO: 6 with another hydrophobic amino acid. A protein comprising the amino acid sequence shown in SEQ ID NO: 6 may form a tetramer, in which case the extramolecularly adjacent helix structure of the protein comprising the amino acid sequence shown in SEQ ID NO: 6 may be the helix structure of another protein molecule that forms the tetramer. Alanine present at a position capable of interacting with the helix structure of another protein molecule that forms the tetramer may be substituted with another hydrophobic amino acid, thereby inhibiting tetramer formation and thereby suppressing silicate transport activity.

[0033] This embodiment also provides a nucleic acid encoding the Lsi1 protein of this embodiment. The nucleic acid may be DNA or RNA, or a chimeric nucleic acid comprising DNA and RNA. Furthermore, the nucleic acid is not particularly limited as long as it is a nucleic acid capable of expressing the Lsi1 protein of this embodiment. However, it is preferable that the nucleic acid be a nucleic acid in which codons encoding each amino acid in the amino acid sequence of the Lsi1 protein of this embodiment are consecutive, and it may also be a nucleic acid having a nucleotide sequence complementary to that of the nucleic acid encoding the Lsi1 protein of this embodiment. A nucleic acid having a nucleotide sequence complementary to that of the nucleic acid encoding the Lsi1 protein of this embodiment may produce a nucleic acid encoding the Lsi1 protein of this embodiment in a host cell.

[0034] The nucleic acid of this embodiment may also be a nucleic acid that can be contained in any appropriate vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. Accordingly, this embodiment also provides a vector containing the nucleic acid encoding the Lsi1 protein of this embodiment. In the vector, the nucleic acid may be incorporated together with factors necessary for transcription and translation, or peripheral sequences of the nucleic acid encoding the Lsi1 protein of this embodiment, and may exist contiguous or discontinuous therewith. The vector of this embodiment may also contain factors necessary for transcription and translation, or peripheral sequences of the nucleic acid encoding the Lsi1 protein of this embodiment. As used herein, the term "vector" refers to a concept that encompasses cloning vectors and expression vectors, and refers to a nucleic acid that carries a gene of interest to transform a host, preferably a cell, and promote the expression (e.g., transcription and translation) of the introduced sequence. In order for the vector to induce expression of the Lsi1 protein of this embodiment in a host cell, the vector may contain, in addition to the nucleic acid, transcription and translation regulatory factors such as a promoter, enhancer, or terminator. The type of vector is not particularly limited, and any vector known to those skilled in the art may be used.

[0035] This embodiment also provides a transformant into which the nucleic acid or vector has been introduced. As used herein, "transformation" refers to introducing a nucleic acid or vector into a cell or the like so as to produce a target peptide. The transformant in this embodiment may be in any form as long as it expresses the Lsi1 protein of this embodiment, but is preferably a cell (host cell), a cell cluster, a tissue, or a tissue cluster. Specifically, the cell may be a plant cell or the like, the cell cluster may be a callus or the like, the tissue may be a seed or the like, and the tissue cluster may be a plant or the like. This embodiment also provides a plant cell, callus, seed, or plant as a transformant. The plant is preferably sorghum. When the transformant in this embodiment is a host cell, the host cell may contain a vector expressing an endonuclease in addition to a vector containing a nucleic acid encoding the Lsi1 protein of this embodiment. The expression of the Lsi1 protein of this embodiment in the transformant may be derived from the introduced nucleic acid or vector, or may be derived from a genome modified by the vector. The seeds and plants as the transformants of this embodiment may be those into which the nucleic acid or vector of this embodiment has been directly introduced, or may be those differentiated from the callus as the transformant of this embodiment.Furthermore, the plants as the transformants of this embodiment may be those which have been grown from the seeds as the transformants of this embodiment.

[0036] The nucleic acids, vectors, and transformants of this embodiment may be produced by any method known to those skilled in the art, and the produced nucleic acids may be used to produce the Lsi1 protein of this embodiment or plants containing said protein. Furthermore, as the nucleic acid of this embodiment, a nucleic acid having a base sequence complementary to the nucleic acid encoding the Lsi1 protein of this embodiment may also be produced by any method known to those skilled in the art.

[0037] When a plant, preferably sorghum, contains the Lsi1 protein of this embodiment, the amount of silicon accumulated in the plant decreases, while the amount of lignin increases. When a plant contains the Lsi1 protein of this embodiment, preferably when part or all of the Lsi1 protein contained in the roots, etc., is the Lsi1 protein of this embodiment, the amount of silicon accumulated in the plant decreases and the amount of lignin increases. The amount and proportion of the Lsi1 protein of this embodiment contained in the plant, and the location where the protein is contained, may be arbitrary, but it is preferably contained in a manner that reduces the amount of silicon accumulated in the plant and increases the amount of lignin. If the amount of silicon in a plant containing the Lsi1 protein of this embodiment is lower than that of the wild-type, the protein can be evaluated as a protein that can suppress silicon accumulation in the plant. Similarly, if the amount of lignin in a plant containing the Lsi1 protein of this embodiment is higher than that of the wild-type, the protein can be evaluated as a protein that can promote lignin accumulation in the plant. The amount of silicon accumulated in a plant having the Lsi1 protein of this embodiment is preferably suppressed to 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less compared to the wild type. Furthermore, the amount of silicon accumulated in the wild type may be suppressed to 0% or more, 10% or more, 20% or more, 30% or more, or 40% or more, for example, 0-50%, 0-40%, 0-30%, 0-20%, 10-50%, 10-40%, 10-30%, 20-50%, 20-40%, or 30-50%. The amount of silicon accumulated in the wild type is preferably suppressed to 40-50%, 30-40%, 20-30%, 10-20%, or 0-10%, and more preferably to 10-20%. Furthermore, it is preferable that the amount of lignin accumulated in a plant having the Lsi1 protein of this embodiment increases by 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more compared to the wild type.Furthermore, compared to the amount of accumulated lignin in the wild type, the increase is preferably 10 to 60%, more preferably 10 to 50%, and may be, for example, an increase of 10 to 20%, 20 to 30%, 30 to 40%, or 40 to 50%.

[0038] The decrease in the amount of silicon and the increase in the amount of lignin accumulated in a plant having the Lsi1 protein of this embodiment may be evaluated by any method known to those skilled in the art. The silicon content can be evaluated, for example, by absorptiometry, inductively coupled plasma atomic emission spectroscopy, etc. Specific examples of absorptiometry include the colorimetric molybdenum method, and the method described in Reference 1 can be used as a reference. The lignin content can also be evaluated, for example, by a method of quantification using an acid or a method of quantification by spectroscopy. Specific examples of methods of quantification using an acid include the Klason method. Specific examples of methods of quantification by spectroscopy include the thioglycolic acid lignin method and the acetyl bromide method. When evaluating by the thioglycolic acid lignin method, for example, the method described in Reference 3 can be used as a reference. The plant used to measure the silicon and lignin content may be a plant having the Lsi1 protein of this embodiment, or may be a next-generation plant obtained by cultivating seeds from the plant.

[0039] Furthermore, the amino acid sequence of the Lsi1 protein of this embodiment, as well as the amino acid sequence and nucleotide sequence of a plant containing the protein, may be evaluated by any method known to those skilled in the art. To identify the amino acid sequence of the Lsi1 protein, for example, DNA extracted from a plant containing the amino acid sequence of the Lsi1 protein is amplified using any primers that amplify a region containing part or all of the region encoding the Lsi1 protein, and the resulting amplicon sequence is subjected to sequence analysis to identify the DNA sequence, thereby identifying the amino acid sequence from the DNA sequence.

[0040] The present embodiment also provides a plant having the Lsi1 protein of the present embodiment. The plant is preferably sorghum, but may be any other plant. Furthermore, the plant having the Lsi1 protein of the present embodiment can be used for any purpose depending on the intended purpose. For example, a plant having the Lsi1 protein of the present embodiment can be cultivated and used for the industrial production of lignin. Furthermore, for example, since a plant having the Lsi1 protein of the present embodiment has a reduced amount of non-combustible silicon and an increased amount of lignin containing a large amount of combustible carbon, the Lsi1 protein of the present embodiment can also be used for biofuel production. In particular, it is preferably used for the production of biofuel pellets. Fuels made from plants having the Lsi1 protein of the present embodiment can have a higher calorific value than fuels made from wild-type plants. The present embodiment also provides a plant having the Lsi1 protein of the present embodiment for use as a biofuel feedstock.

[0041] The Lsi1 protein of this embodiment may be produced by any method known to those skilled in the art. For example, it may be produced by introducing a vector containing a nucleic acid encoding the Lsi1 protein of this embodiment into a host, transforming the host, and allowing the host to express the protein, or it may be produced by extracting the protein from a plant that has been modified to contain the Lsi1 protein of this embodiment. This embodiment also provides a method for producing the Lsi1 protein of this embodiment.

[0042] Plants having the Lsi1 protein of this embodiment may be produced by any method known to those skilled in the art. Here, the plant having the Lsi1 protein of this embodiment may be a plant having a nucleic acid encoding the Lsi1 protein of this embodiment, and the plant having a nucleic acid encoding the Lsi1 protein of this embodiment may be a plant having the Lsi1 protein of this embodiment. This embodiment also provides a plant having a nucleic acid encoding the Lsi1 protein of this embodiment. The plant is preferably sorghum, but may be any other plant.

[0043] For example, (i) a plant mutated to have the Lsi1 protein of this embodiment may be selected from nature, (ii) a plant having the Lsi1 protein of this embodiment may be selected from artificially mutated plants, (iii) a plant having the Lsi1 protein of this embodiment may be obtained by crossbreeding, (iv) a plant having the desired Lsi1 protein of this embodiment may be obtained by genetic recombination or genome editing, or (v) a plant having the Lsi1 protein of this embodiment may be obtained by a combination of these methods. This embodiment also provides a method for producing a plant having the Lsi1 protein of this embodiment, and a method for producing a plant with reduced silicon accumulation and increased lignin accumulation. In the above (i) and (ii), etc., a method for selecting a plant having the Lsi1 protein of this embodiment is, for example, a method for selecting by confirming the germanium tolerance of the plant. By utilizing the fact that germanium is a homologous element of silicon, a plant having the Lsi1 protein of this embodiment can be selected by selecting a strain that can grow even in the presence of germanium, which is toxic to plants (Oshry Markovich et al., Silicification in Leaves of Sorghum Mutant with Low Silicon Accumulation, Silicon, 11, 2385-2391 (2019)). In (ii) above, methods for inducing artificial mutations include, for example, methods using chemical substances such as radiation or ethyl methanesulfonate (EMS). Specifically, artificial mutations can be induced by treating seeds with radiation, EMS, or the like, and in this case, it is preferable to use sterilized seeds. As described above in (iii), one method for obtaining a plant having the Lsi1 protein of the present embodiment by crossbreeding is, for example, a method called DNA marker-assisted selection, which uses a DNA marker linked to the gene encoding the Lsi1 protein of the present embodiment. By performing selection using such a genetic marker, the crossbreeding process can be shortened, and plants having the gene of interest can be obtained quickly.As described in (iv) above, a method for obtaining a plant having the Lsi1 protein of this embodiment by genetic recombination includes, for example, a method using Agrobacterium. When using Agrobacterium, for example, Agrobacterium carrying a nucleic acid encoding the Lsi1 protein of this embodiment or a vector containing the nucleic acid is infected into a plant cell, preferably a callus, and the nucleic acid is integrated into the plant genome within the plant cell, allowing the plant cell to redifferentiate, thereby obtaining a plant having the Lsi1 protein of this embodiment. Alternatively, a plant having the Lsi1 protein of this embodiment can be obtained by directly introducing a nucleic acid encoding the Lsi1 protein of this embodiment into a plant using a particle gun or the like. Furthermore, as described in (iv) above, a method for obtaining a plant having the Lsi1 protein of this embodiment by genome editing includes, for example, a method using endonucleases such as ZFN, TALEN, and CRISPR / Cas9. When such an endonuclease is used, for example, a plant having the Lsi1 protein of this embodiment can be obtained by introducing the endonuclease and a gRNA that recognizes the target sequence into a plant and then introducing a nucleic acid encoding the Lsi1 protein of this embodiment into the target site in the plant genome, or by introducing a mutation into the target site in the plant genome so as to encode the Lsi1 protein of this embodiment. A vector may be used to introduce the endonuclease and gRNA into the plant.

[0044] By the above-described method, a plant having the Lsi1 protein of this embodiment and a plant having a nucleic acid encoding the Lsi1 protein of this embodiment can be obtained. When a nucleic acid encoding the Lsi1 protein of this embodiment is introduced into a plant genome to obtain a plant having the Lsi1 protein of this embodiment, a plant having a nucleic acid encoding the Lsi1 protein of this embodiment on its genome can also be obtained. This embodiment also provides a plant having a nucleic acid encoding the Lsi1 protein of this embodiment on its genome. The plant is preferably sorghum, but may be any other plant.

[0045] Whether a certain plant has the Lsi1 protein of this embodiment or a nucleic acid encoding the Lsi1 protein of this embodiment can be assessed by any method known to those skilled in the art. For example, whether a certain plant has the Lsi1 protein of this embodiment can be assessed by, for example, amplifying cDNA synthesized from RNA extracted from the plant using any primers that amplify a region containing part or all of the region encoding the Lsi1 protein and analyzing the resulting amplicon sequence. Furthermore, whether a certain plant has a nucleic acid encoding the Lsi1 protein of this embodiment can be assessed by, for example, amplifying DNA extracted from the plant using any primers that amplify a region containing part or all of the region encoding the Lsi1 protein and analyzing the resulting amplicon sequence. As a non-limiting example, if the Lsi1 protein of this embodiment contains aspartic acid at position 103 of the amino acid sequence shown in SEQ ID NO: 7 and / or valine at position 201 of the amino acid sequence shown in SEQ ID NO: 8, these may be used as indicators to confirm whether the plant being tested contains the Lsi1 protein of this embodiment or a nucleic acid encoding the Lsi1 protein of this embodiment. That is, the Lsi1 protein of this embodiment may be evaluated using a mutation that results in suppression of silica transport activity as an indicator.

[0046] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0047] Example 1: Mutation Induction (Seed Sterilization) 360 g of sorghum (USDA ID: NSL 87411) seeds were sterilized for 5 minutes with a 1% (effective chlorine concentration) aqueous sodium hypochlorite solution (Junsei Chemical Co., Ltd., sodium hypochlorite). (Mutagen Treatment) Three 1000 mL tubes were prepared, each containing 500 mL of sterilized water and 120 g of sterilized seeds. To each tube, 1.25 mL (final concentration: 0.25%), 1.5 mL (final concentration: 0.3%), and 1.75 mL (final concentration: 0.35%) of EMS (Junsei Chemical Co., Ltd., ethyl methanesulfonate) were added and mixed thoroughly. After further incubation at room temperature for 16 hours with shaking, the seeds were washed six times with sterilized water. The sixth wash involved 30 minutes of mixing with sterilized water. The seeds were then washed for another hour with running water. (M1 cultivation and M2 seed acquisition) 4,000 seeds of each EMS treatment concentration were sown in plug trays (TO plug trays, Tokai Kasei Co., Ltd.) (total of 12,000 seeds), and a total of approximately 10,000 germinated individuals (M1) were planted in a field, and M2 seeds were harvested.

[0048] Example 2: Selection of germanium-resistant mutants The M2 seeds obtained in Example 1 were sown, and after 5 days, the seeds were grown in hydroponic solution 1 (Ca(NO ３ ) ２ 236.2mg / L, KCl 74.6mg / L, MgSO ４ ・7H ２ O 123.2mg / L, (NH ４ ) ２ HPO ４ 33mg / L, Fe(III)-EDTA 37.7mg / L, H ３ BO ３ 5.72mg / L, MnCl ２ ・4H ２ O 3.62mg / L, CuSO ４ ・5H ２ O 0.16mg / L, ZnSO ４ ・7H ２ O 0.44mg / L, (NH ４ ) 2Mo ７ O ２４ ・4H ２Four days after adding the hydroponic solution 1, only healthy individuals were selected and continued to be cultivated. After three days, germanic acid (GeO 0.2 mg / L) was added at a concentration of 50 μM. ２ Hydroponic solution 1 (hydroponic solution 2) containing 100% Lsi1 protein was added. After 7 days, hydroponic solution 2 was added and the plants were cultivated. After 7 days, the phenotype was observed and germanium-resistant mutants were selected. The selected germanium-resistant mutants were used in the next experiment as individuals with a mutated Lsi1 protein.

[0049] Example 3: Identification of Mutation Sites DNA extracted from the germanium-resistant mutants selected in Example 2 was amplified by PCR using primer set 1 (SEQ ID NOs: 1 and 2) and primer set 2 (SEQ ID NOs: 3 and 4), respectively, to obtain 2 kB and 1.6 kB DNA fragments, respectively. The DNA sequences of the resulting 2 kB and 1.6 kB DNA fragments were determined using primer 3 (SEQ ID NO: 5), and the mutation sites were identified by comparing the determined sequences with the amino acid sequence of the wild-type strain (SEQ ID NO: 6). As a result, the presence of two types of mutants (mutant 1 and 2) was revealed. Table 1 shows the amino acid sequences of the Lsi1 proteins of mutant 1 and 2 (SEQ ID NOs: 7 and 8, respectively, with the mutation sites underlined in bold). The sequences of the silica transporter proteins of mutant 1 and 2 are shown in Table 1.

[0050]

[0051] Example 4: Evaluation of silicon and lignin content Two mutants (mutant 1 and 2) selected as germanium-resistant mutants were transplanted into pots (Wagner pots, Fujiwara Manufacturing Co., Ltd.) containing culture soil (a mixture of Honen Agri Co., Ltd.'s Honen's Culture Soil No. 1 and Takii Seed Co., Ltd.'s Takii Seed Culture Soil in a ratio of 2:3), and cultivated in a greenhouse to collect seeds. The silicon and lignin content of the obtained seeds was measured using cultivated individuals cultivated under the same conditions.

[0052] The shoots and roots of the cultivated individuals and wild-type sorghum (USDA ID: NSL 87411) obtained above were dried and crushed into fine powder. Approximately 10 mg of the crushed powder was incinerated in a nickel crucible and added to 100 mg of Na ２ CO ３ The solution was dissolved by heating with HCl and diluted to 10 mL with distilled water. The silicon concentration in the resulting solution was evaluated using the colorimetric molybdenum blue method (Reference 1). Furthermore, following the method described in Reference 2, approximately 200 mg of the ground powder was extracted sequentially with 1 mL of methanol at 60°C 20 times, with hexane at room temperature 5 times, and with distilled water at 60°C 5 times, followed by freeze-drying to obtain cell wall residue samples. The lignin content in the samples was evaluated using the thioglycolic acid lignin method (Reference 3). Measurement of silicon and lignin content using the above method revealed that the lignin content in mutants 1 and 2 was increased by 22-28% compared to the wild type (WT), and the silicon content in the shoot was significantly reduced (Figure 1).

[0053] Reference 1: Rivai, R. R. et al. (2022) Limiting silicon supply alters lignin content and structures of sorghum seedling cell walls, Plant Science, 321 (111325). Reference 2: Yamamura, M. et al. (2012) Microscale thioacidolysis method for the rapid analysis of β-O-4 structures in lignin, Plant Biotechnology, 29(4), pp. 419-423. Reference 3: Suzuki, S. et al. (2009) High-throughput determination of thioglycolic acid lignin from rice, Plant Biotechnology, 26(3), pp. 337-340.

[0054] Example 5: Analysis using AlphaFold Server model The three-dimensional protein structures of mutants 1 and 2 were analyzed using the AlphaFold Server model. As a result, the 103rd residue in the amino acid sequence shown in SEQ ID NO: 7, which was mutated from glycine to aspartic acid in mutant 1, was predicted to be a residue located at a position capable of interacting with water molecules in the silica channel. It was thought that the mutation of the residue located at a position capable of interacting with water molecules from glycine to polar aspartic acid suppressed silica transport activity and reduced the silicon content in plants. Furthermore, the 201st residue in the amino acid sequence shown in SEQ ID NO: 8 in mutant 2, which was mutated from alanine to valine, was predicted to be a residue located in a position where it can interact with an adjacent helix structure within the mutant 2 protein molecule, or a residue located in a position where it can interact with a helix structure in another adjacent protein molecule that forms the tetramer.It was thought that the mutation of the residue located in a position where it can interact with a helix structure to the more hydrophobic valine suppressed silica transport activity and reduced the silicon content in the plant.

[0055] Furthermore, in addition to the residue at position 103, the AlphaFold Server model predicted that the following residues in the amino acid sequence shown in SEQ ID NO: 6, namely, glycine at positions 72, 87, 99, 154, 191, 210, and 214, could interact with water molecules within the silica channel. This suggests that, in addition to or instead of glycine at position 103, mutation of at least one of glycine at positions 72, 87, 99, 154, 191, 210, and 214 to a polar amino acid such as aspartic acid could suppress silicate transport activity, as in mutant 1. Similarly, in addition to the residue at position 201, the following residues are present in the amino acid sequence shown in SEQ ID NO: 6: alanine at positions 67, 68, 86, 104, 109, 113, 115, 131, 136, 139, 182, 194, 197, 209, 220, 226, 228, and 250, which are capable of interacting with adjacent helix structures within the protein molecule: alanine at positions 67 (chain D), 86 (chain D), 97 (chain D), 182 (chain B), 189 (chain B), and 197 (chain D). B) was predicted by the AlphaFold Server model as a residue present in a position that can interact with a helix structure in another adjacent protein molecule that constitutes the tetramer.Thus, in addition to or instead of alanine at position 201, at least one of alanine at position 67, alanine at position 68, alanine at position 86, alanine at position 104, alanine at position 109, alanine at position 113, alanine at position 115, alanine at position 131, alanine at position 136, alanine at position 139, alanine at position 182, alanine at position 194, alanine at position 197, alanine at position 209, alanine at position 220, alanine at position 226, alanine at position 228, and alanine at position 250 may be used, and / or at least one of alanine at position 67 (chain D), alanine at position 86 (chain D), alanine at position 97 (chain D), alanine at position 182 (chain B), alanine at position 189 (chain B), and alanine at position 197 (chain B). It was suggested that by mutating at least one of B) to a more hydrophobic amino acid, the silica transport activity would be suppressed, as in mutant 2.

[0056] FIG. 2 shows the alignment of the amino acid sequences of the wild-type silicate transporter proteins, mutant 1 and mutant 2 (SEQ ID NOS: 6 to 8, respectively), with the amino acid sequence of the rice silicate transporter protein (SEQ ID NOS: 9 (Table 2)). In FIG. 2, "TM1" to "TM6" indicate transmembrane helical structures, and "HB" and "HE" indicate other helical structures (the same applies to FIG. 3 described below). FIG. 3 shows a 3D model of the silicate transporter protein consisting of the amino acid sequence shown in SEQ ID NOS: 10 (Table 2), predicted by the AlphaFold Server model. The amino acid sequence shown in SEQ ID NOS: 10 is the amino acid sequence of the silicate transporter protein in which all glycines predicted above to be present at positions capable of interacting with water molecules in the silicate channel have been mutated to aspartic acid, and further, all alanines predicted above to be present at positions capable of interacting with the helix structure have been mutated to valine. The 3D model in Figure 3 shows the positions of each mutated glycine and each alanine, with "EXT." indicating the outside of the cell, "Cyt." indicating the inside of the cell, and spheres representing water molecules in the silica channel. Figure 4 shows a 3D model of a silica transporter protein consisting of the amino acid sequence shown in SEQ ID NO: 10, which forms a tetramer. Figure 4 also shows the positions of each mutated alanine, which is present at a position where it can interact with the helix structure of another adjacent protein molecule that forms the tetramer. The 3D model in Figure 4 was created by superimposing the monomer structure of the silica transporter protein consisting of the amino acid sequence shown in SEQ ID NO: 10, obtained using the AlphaFold Server model, onto the tetramer of the rice-derived silica transporter protein (PDB: 7CJS).

[0057]

Claims

1. A protein comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7 or 8, and having suppressed silica transport activity.

2. The protein according to claim 1, comprising an amino acid sequence comprising an addition, substitution, and / or deletion of 1 to 20 amino acids at positions 60 to 160 of the amino acid sequence shown in SEQ ID NO: 6, and / or an amino acid sequence comprising an addition, substitution, and / or deletion of 1 to 20 amino acids at positions 170 to 260 of the amino acid sequence shown in SEQ ID NO:

6.

3. The protein according to claim 1, wherein the following mutations have been introduced at positions 60 to 160 and / or 170 to 260 of the amino acid sequence shown in SEQ ID NO: 6: (i) a mutation that substitutes glycine with another polar amino acid, and / or (ii) a mutation that substitutes alanine with another hydrophobic amino acid.

4. The protein according to claim 3, wherein the glycine is a glycine present in a position capable of interacting with an extramolecular water molecule of the protein, and the alanine is an alanine present in a position capable of interacting with an adjacent helix structure.

5. A protein consisting of the amino acid sequence shown in SEQ ID NO: 7 or 8.

6. A nucleic acid encoding a protein according to any one of claims 1 to 5.

7. A vector having the nucleic acid of claim 6.

8. A transformant having the nucleic acid according to claim 6.

9. A transformant having the vector according to claim 7.

10. A plant having the protein according to any one of claims 1 to 5.

11. A plant having the nucleic acid according to claim 6 on its genome.

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