Cell death inhibitor and cell death inhibition method

Abstract

A cell death inhibitor that has as an effective component thereof ascorbic acid, a derivative thereof, a salt thereof, or a solvate thereof and inhibits cell death induced by transient protein expression in plants. The cell death inhibitor is useful due to being able to inhibit cell death that is induced by transient protein expression in plants.

Description

[Technical field]

[0001] The present invention relates to a cell death inhibitor and a method for inhibiting cell death, more specifically, to an inhibitor of cell death induced by transient protein expression in plants, and a method for inhibiting cell death induced by transient protein expression in plants. [Background technology]

[0002] There have been many studies on expressing a large amount of a target protein in a plant cell. For example, the inventors have previously developed an expression system as a technique for transiently expressing a large amount of a target protein, which comprises a first nucleic acid fragment including a geminivirus-derived long intergenic region (LIR), a geminivirus-derived small intergenic region (SIR), and an expression cassette for a target protein linked between the LIR and the SIR, and a second nucleic acid fragment including an expression cassette for a geminivirus-derived Rep / RepA protein, in which the expression cassette for the target protein includes, in this order, a promoter, a nucleic acid fragment encoding the target protein, and two or more linked terminators (see Patent Document 1).

[0003] However, it is known that transient expression of a large amount of a target protein in a plant cell induces cell death (necrosis) in the plant cell, and the expressed target protein is decomposed (see, for example, non-patent document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2018 / 220929 [Non-patent literature]

[0005] [Non-Patent Document 1] Pinkhasov J., et al., Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice, Plant Biotechnology Journal, 9, 991-1001, 2011. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a technique for suppressing cell death induced by transient protein expression in plants. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] An agent for suppressing cell death induced by transient protein expression in a plant, comprising ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof as an active ingredient. [2] The inhibitor according to [1], which is used by contacting 100 to 300 mM of ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof with a plant. [3] The inhibitor according to [1], wherein 25 to 80 mM of ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof is added to an Agrobacterium suspension for introducing an expression vector for a target protein into a plant, or is added to a culture solution for hydroponic cultivation of a plant. [4] The inhibitor according to any one of [1] to [3], wherein the plant is a Nicotiana plant. [5] A method for suppressing cell death induced by transient protein expression in a plant, comprising the step of administering to the plant ascorbic acid or a derivative thereof, or a salt or solvate thereof. [6] The method according to [5], wherein in the step, 100 to 300 mM of ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof is administered by contacting with the plant. [7] The method according to [5], wherein in the step, 25 to 80 mM ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof is added to an Agrobacterium suspension for introducing an expression vector for a target protein into the plant, or is added to a culture solution for hydroponic cultivation of the plant. [8] The method according to any one of [5] to [7], wherein the plant is a Nicotiana plant. Effect of the Invention

[0008] According to the present invention, a technique for suppressing cell death induced by transient protein expression in plants can be provided. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of the T-DNA region of the pBYR2HS-EGFP vector. [Diagram 2] Photographs (a) to (f) are representative photographs of leaves of each plant in Experimental Example 1. [Diagram 3] FIG. 1(a) is a photograph showing the results of Coomassie Brilliant Blue (CBB) staining in Experimental Example 1. FIG. 1(b) is a graph showing the results of quantifying the expression level of GFP protein based on FIG. [Figure 4] (a) is a photograph showing the results of Western blotting in which human Cullin 1 (hCul) protein was detected in Experimental Example 1. (b) is a graph showing the results of quantifying the expression level of hCul protein based on (a). [Diagram 5](a) is a photograph showing the results of Western blotting in which a fusion protein of Arabidopsis putative membrane lipoprotein (PLP) and GFP (PLP-GFP protein) was detected in Experimental Example 1. (b) is a graph showing the results of quantifying the expression level of PLP-GFP protein based on (a). [Figure 6] Photographs (a) and (b) are photographs of the leaves of each plant in Experimental Example 2. (a) shows the result when an aqueous solution of sodium ascorbate was sprayed, and (b) shows the result when the aqueous solution of sodium ascorbate was not sprayed. [Figure 7] FIG. 1(a) is a photograph showing the results of CBB staining in Experimental Example 2. FIG. 1(b) is a graph showing the results of quantifying the expression level of GFP protein based on FIG. [Figure 8] FIG. 1(a) is a photograph showing the results of Western blotting in which hCul protein was detected in Experimental Example 2. FIG. 1(b) is a graph showing the results of quantifying the expression level of hCul protein based on FIG. [Figure 9] 1(a) is a photograph showing the results of Western blotting in Experimental Example 2, in which Fbxw7 (hFbxw7) protein, a human F-box protein, was detected. [Figure 10] 6(a) to 6(c) are representative photographs of the leaves of each plant in Experimental Example 3. [Figure 11] Photographs (a) to (f) are representative photographs of the leaves of each plant in Experimental Example 4. [Figure 12] Photographs (a) to (f) are representative photographs of the entire plants in Experimental Example 4. [Figure 13] FIG. 1(a) is a photograph showing the results of Western blotting in which hCul protein was detected in Experimental Example 4. FIG. 1(b) is a graph showing the results of quantifying the expression level of hCul protein based on FIG. [Figure 14] Photographs (a) to (f) are representative photographs of the leaves of each plant in Experimental Example 5. [Figure 15] 13(a) is a photograph showing the results of CBB staining in Experimental Example 5. (b) is a graph showing the results of quantifying the expression level of GFP protein based on (a). [Figure 16] Photographs (a) to (d) are representative photographs of each plant taken in Experimental Example 6. [Figure 17] (a) to (d) are representative photographs of each plant taken in Experimental Example 7. (b) is a partially enlarged photograph of (a), and (d) is a partially enlarged photograph of (c). [Figure 18] 13(a) is a representative photograph of a plant that was not sprayed with an aqueous sodium ascorbate solution in Experimental Example 8. 13(b) is a photograph showing the results of Western blotting for detecting Cryj1 protein in Experimental Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] [Suppression of cell death induced by transient protein expression in plants] In one embodiment, the present invention provides an agent for suppressing cell death induced by transient protein expression in a plant, comprising ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof as an active ingredient.

[0011] As described later in the Examples, the inhibitor of this embodiment can suppress cell death (necrosis, gangrene) induced by transient protein expression in plants. Furthermore, as described later in the Examples, administration of the inhibitor of this embodiment suppresses cell death in plants, making it possible to mass-produce a target protein.

[0012] The inhibitor of the present embodiment may be ascorbic acid, a derivative of ascorbic acid, a salt of ascorbic acid, a salt of an ascorbic acid derivative, a solvate of ascorbic acid, a solvate of an ascorbic acid derivative, a solvate of a salt of ascorbic acid, or a solvate of a salt of an ascorbic acid derivative.

[0013] Ascorbic acid is a type of organic compound with a lactone structure that functions as a nutrient vitamin C. Ascorbic acid is an optically active compound, and the L-form is known as vitamin C. In this embodiment, ascorbic acid may be either the L-form or the D-form, but is preferably the L-form.

[0014] Examples of the ascorbic acid derivative include ascorbic acid alkyl esters, ascorbic acid phosphate esters, ascorbic acid glucosides, and ascorbic acid alkyl ethers. More specific examples of the ascorbic acid derivative include ascorbic acid alkyl esters such as ascorbyl monostearate, ascorbyl monopalmitate, ascorbyl monoisopalmitate, ascorbyl monooleate, ascorbyl distearate, ascorbyl dipalmitate, ascorbyl monopalmitate, and ascorbyl tetrahexyldecanoate; ascorbic acid phosphate esters such as ascorbic acid monophosphate esters, ascorbic acid diphosphate esters, and ascorbic acid triphosphate esters; ascorbic acid monoglucosides such as ascorbic acid ethyl ether and ascorbic acid methyl ether; and ascorbic acid glucosides such as ascorbic acid diglucoside. In addition to these, examples of the ascorbic acid alkyl esters and ascorbic acid phosphate esters include palmitic acid ascorbyl phosphate. These ascorbic acid derivatives have a structure in which at least one of the hydroxyl groups at the 6-, 2-, 3- and 5-positions of ascorbic acid is substituted.

[0015] The salt of ascorbic acid or an ascorbic acid derivative is not particularly limited, and examples thereof include salts of ascorbic acid or an ascorbic acid derivative and an alkali metal (e.g., sodium, potassium, etc.), salts of ascorbic acid or an ascorbic acid derivative and an alkaline earth metal (e.g., calcium, magnesium, etc.), salts of ascorbic acid or an ascorbic acid derivative and a transition metal (e.g., zinc, iron, cobalt, copper, etc.), salts of ascorbic acid or an ascorbic acid derivative and a basic ammonium, salts of ascorbic acid or an ascorbic acid derivative and a triethanolamine, salts of ascorbic acid or an ascorbic acid derivative and an amino acid (e.g., L-histidine, L-arginine, L-lysine, etc.), etc. Among these, for example, sodium ascorbate, potassium ascorbate, calcium ascorbate, magnesium ascorbate, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, etc. can be suitably used.

[0016] Solvates of ascorbic acid or its derivatives or salts thereof include hydrates and organic solvent solvates, and more specific examples include sodium isoascorbate monohydrate and calcium ascorbate dihydrate.

[0017] The inhibitor of the present embodiment may be one of the above-mentioned ascorbic acid or its derivatives, salts thereof, or solvates thereof, or may be a mixture of two or more of them.

[0018] The inhibitor of the present embodiment may be administered by contacting the inhibitor with a plant at a concentration of 100 to 300 mM. Here, contacting the inhibitor with a plant may mean spraying a liquid in which ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof is dissolved or suspended on a part of the plant in which transient protein expression is induced, for example, on the leaves. Alternatively, the inhibitor may be immersed in the liquid in which the plant in which transient protein expression is induced is immersed.

[0019] In the liquid in which the inhibitor of this embodiment is dissolved or suspended, examples of the solvent include water, a buffer solution, and an isotonic solution.

[0020] In the liquid in which the inhibitor of the present embodiment is dissolved or suspended, the lower limit of the concentration of ascorbic acid or its derivatives or their salts or their solvates may be, for example, 80 mM, for example, 100 mM, for example, 150 mM, for example, 200 mM, or for example, 250 mM. In addition, in the liquid in which the inhibitor of the present embodiment is dissolved or suspended, the upper limit of the concentration of ascorbic acid or its derivatives or their salts or their solvates may be, for example, 300 mM, for example, 250 mM, for example, 200 mM, for example, 150 mM, or for example, 100 mM. These lower and upper limits can be combined arbitrarily.

[0021] When the inhibitor of this embodiment is sprayed, the spraying schedule can be, for example, once a day, once every two days, once every three days, or once every four days.

[0022] The inhibitor of the present embodiment may be added at a concentration of 25 to 80 mM to an Agrobacterium suspension for introducing an expression vector of a target protein into a plant, and the Agrobacterium suspension (agroinfiltration solution) may be used for agroinfiltration using a syringe or for vacuum infiltration, and administered to the plant. That is, the inhibitor of the present embodiment may be administered simultaneously with the introduction of an expression construct of a target protein into a plant. Alternatively, the inhibitor of the present embodiment may be administered by adding at a concentration of 25 to 80 mM to a culture solution for hydroponically cultivating a plant.

[0023] As described later in the Examples, when the inhibitor of this embodiment is added to an Agrobacterium suspension, the optimal concentration tends to be different from that when the inhibitor of this embodiment is brought into contact with a plant. Furthermore, when the inhibitor of this embodiment is added to a culture solution for hydroponic cultivation, it is preferable to add the inhibitor at the same concentration as when it is added to an Agrobacterium suspension.

[0024] More specifically, in the Agrobacterium suspension or the culture solution for hydroponic cultivation, the lower limit of the concentration of ascorbic acid or its derivatives or their salts or their solvates may be, for example, 25 mM, for example, 30 mM, for example, 40 mM, for example, 50 mM, or for example, 60 mM. In addition, in the Agrobacterium suspension or the culture solution for hydroponic cultivation, the upper limit of the concentration of ascorbic acid or its derivatives or their salts or their solvates may be, for example, 80 mM, for example, 60 mM, for example, 50 mM, for example, 40 mM, or for example, 30 mM. These lower and upper limits can be combined arbitrarily.

[0025] When the inhibitor of this embodiment is added to an Agrobacterium suspension, the inhibitor of this embodiment is administered only once during agroinfiltration.

[0026] The inhibitor of this embodiment may be administered by contact, by addition to an Agrobacterium suspension, or by addition to a culture solution for hydroponic cultivation of plants, either alone or in any combination of two or more thereof.

[0027] As used herein, cell death induced by transient protein expression in plants refers to cell death induced when a target protein is transiently (transiently) expressed in large amounts in plant cells.

[0028] The means for transient protein expression is not particularly limited, and examples thereof include a method using Agrobacterium and T-DNA. T-DNA is a specific region contained in Ti plasmid or Ri plasmid found in pathogenic strains of Agrobacterium, a pathogenic bacterium that causes crown gall tumors in dicotyledonous plants. T-DNA is a DNA region sandwiched between approximately 25 base sequences called Right Border (RB) and Left Border (LB). When Agrobacterium having T-DNA is allowed to coexist with plant cells, a nucleic acid fragment present between the RB and LB is transferred into the host plant cells.

[0029] Therefore, by introducing a vector carrying an expression construct for a target protein between the RB and LB into Agrobacterium and then introducing the Agrobacterium into a host plant, the expression construct for a target protein can be easily introduced into the host plant cells.

[0030] The vector in which an expression construct for a target protein exists between the RB and LB is preferably a vector that can be used in the binary vector method. The binary vector method is a method for introducing genes into plants using a vir helper Ti plasmid in which the original T-DNA of the Ti plasmid has been removed, and a small shuttle vector having an artificial T-DNA. Here, the shuttle vector is preferably one that can be maintained in both E. coli and Agrobacterium.

[0031] The vir helper Ti plasmid does not have the original T-DNA and is therefore unable to form crown galls in plants, but it does have the vir region necessary for transferring the T-DNA into the host plant cell.

[0032] Therefore, by introducing a T-DNA containing a desired nucleic acid fragment into an Agrobacterium having a vir helper Ti plasmid and then introducing the Agrobacterium into a host plant, the desired nucleic acid fragment can be easily introduced into a host plant cell.

[0033] In other words, a vector containing an expression construct for a target protein between the RB and LB has a replication origin for E. coli and a replication origin for Agrobacterium, and is conveniently a shuttle vector that can be maintained in both E. coli and Agrobacterium.

[0034] In the inhibitor of this embodiment, the means for transient protein expression is not particularly limited, and examples include a method of introducing into a plant an expression system previously developed by the inventors, which comprises a first nucleic acid fragment containing an LIR derived from a geminivirus, an SIR derived from a geminivirus, and an expression cassette for a target protein linked between the LIR and the SIR, and a second nucleic acid fragment containing an expression cassette for a Rep / RepA protein derived from a geminivirus, wherein the expression cassette for the target protein comprises, in this order, a promoter, a nucleic acid fragment encoding the target protein, and two or more linked terminators, for example, a method of introducing into a plant a commercial expression system based on the tobacco mosaic virus, known as the magnICON system.

[0035] In the inhibitor of the present embodiment, the plant is not particularly limited, and examples thereof include solanaceae plants such as tomato, eggplant, and capsicum, Asteraceae plants such as lettuce, Cucurbitaceae plants such as melon, and Orchidaceae plants such as Phalaenopsis. Among them, solanaceae plants are preferred, and Nicotiana plants are preferred. More specific examples of Nicotiana plants include Nicotiana benthamiana, Nicotiana tabacum, and Nicotiana canadensis.

[0036] [Method for suppressing cell death induced by transient protein expression in plants] In one embodiment, the present invention provides a method for suppressing cell death induced by transient protein expression in a plant, comprising the step of administering to the plant ascorbic acid or a derivative thereof, or a salt thereof, or a solvate thereof.

[0037] As described later in the Examples, the method of the present embodiment can suppress cell death (necrosis, necrosis) induced by transient protein expression in plants. In addition, suppressing cell death in plants makes it possible to mass-produce a target protein.

[0038] In the step of administering ascorbic acid, a derivative thereof, a salt thereof, or a solvate thereof, 100 to 300 mM of ascorbic acid, a derivative thereof, a salt thereof, or a solvate thereof may be administered by contacting the plant.

[0039] Here, contacting with a plant is the same as that described above, and may be spraying a liquid in which ascorbic acid or its derivatives, or their salts, or their solvates are dissolved or suspended on a part of a plant in which transient protein expression is induced, such as a leaf. Alternatively, the plant in which transient protein expression is induced may be immersed in the above-mentioned liquid. The liquid in which ascorbic acid or its derivatives, or their salts, or their solvates are dissolved or suspended, and the concentration of ascorbic acid or its derivatives, or their salts, or their solvates are the same as those described above.

[0040] In the step of administering ascorbic acid, a derivative thereof, a salt thereof, or a solvate thereof, 25 to 80 mM of ascorbic acid, a derivative thereof, a salt thereof, or a solvate thereof may be added to an Agrobacterium suspension for introducing an expression vector for a target protein into the plant, or may be added to a culture solution for hydroponically cultivating the plant.

[0041] Here, the concentrations of the Agrobacterium suspension, the culture solution for hydroponic cultivation, and the ascorbic acid or a derivative thereof or a salt or a solvate thereof are the same as those described above.

[0042] In the method of the present embodiment, the plants are the same as those described above, and examples thereof include solanaceae plants such as tomato, eggplant, and capsicum, Asteraceae plants such as lettuce, Cucurbitaceae plants such as melon, and Orchidaceae plants such as Phalaenopsis. Among them, solanaceae plants are preferred, and Nicotiana plants are preferred. More specific examples of Nicotiana plants include Nicotiana benthamiana, Nicotiana tabacum, and Nicotiana canadensis.

[0043] [Other embodiments] 2. Ascorbic acid or a derivative thereof, or a salt or solvate thereof for use in suppressing cell death induced by transient protein expression in a plant.

[0044] Use of ascorbic acid or a derivative thereof, or a salt or solvate thereof, for suppressing cell death induced by transient protein expression in a plant.

[0045] Use of ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof for the manufacture of an agent for suppressing cell death induced by transient protein expression in a plant. EXAMPLES

[0046] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0047] Materials and Methods (Vector Construction) 《Construction of pBYR2HS-EGFP vector》 The known vector pBYR2fp vector has a replication system derived from Bean yellow dwarf virus (BeYDV) and an expression cassette for the gene silencing inhibitor P19 derived from Tomato bushy stunt virus.

[0048] An EGFP gene fragment having the 5'-UTR of the alcohol dehydrogenase gene (hereinafter sometimes referred to as "AtADH5'") and the terminator of the Arabidopsis thaliana heat shock protein 18.2 gene (hereinafter sometimes referred to as "HSPter") was introduced into the pBYR2fp vector.

[0049] Specifically, first, an EGFP gene fragment was amplified by PCR using a primer (pRI201-EGFP-F, the nucleotide sequence of which is shown in SEQ ID NO: 1) and a primer (EGFP-pRI201-R, the nucleotide sequence of which is shown in SEQ ID NO: 2).

[0050] Subsequently, the obtained PCR product was cloned into pRI201-AN (Takara Bio) cleaved with restriction enzymes NdeI and SalI to prepare pRI201-EGFP vector.

[0051] Subsequently, using the pRI201-EGFP vector as a template and a primer (pBYR2fp-AtADH-F, the nucleotide sequence of which is shown in SEQ ID NO: 3) and a primer (pBYR2fp-HSPter-R, the nucleotide sequence of which is shown in SEQ ID NO: 4), an EGFP gene fragment having the 5'-UTR of the alcohol dehydrogenase gene and the terminator of the Arabidopsis thaliana heat shock protein 18.2 gene was amplified by PCR.

[0052] Subsequently, the obtained PCR product was cloned into the pBYR2fp vector cleaved with the restriction enzymes XhoI and XbaI to obtain the pBYR2HS-EGFP vector. FIG. 1 is a schematic diagram of the T-DNA region of the pBYR2HS-EGFP vector. In FIG. 1, "35S-p×2" means the 35S promoter of the cauliflower mosaic virus (CaMV) having two enhancer elements, "AtADH5'" means the 5'-UTR of the Arabidopsis thaliana alcohol dehydrogenase gene, "EGFP" means enhanced green fluorescence protein, "Ext3'" means the terminator of the tobacco extensin gene, "HSPter" means the terminator of the Arabidopsis thaliana heat shock protein 18.2 gene, "LIR" means the Long Intergenic Region of the bean yellow dwarf virus (BeYDV) genome, and "SIR" means the Short Intergenic Region of the BeYDV genome. "Region," "C1" and "C2" refer to the open reading frames C1 and C2 encoding the Rep / RepA proteins, which are replication initiator proteins of BeYDV; "LB" and "RB" refer to the left and right border sequences of T-DNA, respectively; "Nos-p" refers to the NOS promoter; "p19" refers to the gene encoding the gene silencing inhibitor P19 derived from Tomato bushy stunt virus; and "Nos-t" refers to the NOS terminator.

[0053] <<Construction of pBYR2HS vector>> For convenience, the EGFP gene fragment was removed from the pBYR2HS-EGFP vector, and the restriction enzyme SalI site was introduced between AtADH5' and HSPter to create the pBYR2HS vector. By cleaving the pBYR2HS vector with the restriction enzyme SalI, the target gene can be introduced between AtADH5' and HSPter of the pBYR2HS vector.

[0054] 《Construction of pBYR2HS-HF-hCul vector》 A gene fragment (the base sequence of which is shown in SEQ ID NO: 5) encoding human Cullin 1 protein (hereinafter sometimes referred to as "hCul") optimized for the codons of Nicotiana benthamiana was chemically synthesized (Thermo Fisher Scientific). A 6x histidine tag and a FLAG tag were linked to the N-terminus of the synthesized gene fragment, and an endoplasmic reticulum retention signal, KDEL (SEQ ID NO: 6), was linked to the C-terminus.

[0055] Next, the hCul gene fragment was amplified by PCR using a primer (pBYR2HS-Hisx6-F, the nucleotide sequence of which is shown in SEQ ID NO: 7) and a primer (pBYR2HS-KDEL-R, the nucleotide sequence of which is shown in SEQ ID NO: 8). The hCul gene fragment was then introduced into the pBYR2HS vector cleaved with the restriction enzyme SalI to obtain the pBYR2HS-HF-hCul1 vector.

[0056] 《Construction of pBYR2HS-HF-hFbxw7 vector》 A gene fragment (the base sequence of which is shown in SEQ ID NO: 9) encoding the human F-box protein Fbxw7 protein (hereinafter sometimes referred to as "hFbxw7"), which was optimized for the codons of Nicotiana benthamiana, was chemically synthesized (Thermo Fisher Scientific). A 6x histidine tag and a FLAG tag were linked to the N-terminus of the synthesized gene fragment, and an endoplasmic reticulum retention signal, KDEL (SEQ ID NO: 6), was linked to the C-terminus.

[0057] Next, the hFbxw7 gene fragment was PCR amplified using primers (pBYR2HS-Hisx6-F, SEQ ID NO: 7) and (pBYR2HS-KDEL-R, SEQ ID NO: 8). Then, the hFbxw7 gene fragment was introduced into the pBYR2HS vector cleaved with the restriction enzyme SalI to obtain the pBYR2HS-HF-hFbxw7 vector.

[0058] 《Construction of pBYR2HS-CEGFP vector》 An EGFP gene fragment was amplified by PCR using primers (pBYR2HS-CEGFP-F, the nucleotide sequence of which is shown in SEQ ID NO: 10) and (pBYR2HS-CEGFP-R, the nucleotide sequence of which is shown in SEQ ID NO: 11). Subsequently, the EGFP gene fragment was introduced into the pBYR2HS vector cleaved with the restriction enzyme SalI to obtain the pBYR2HS-CEGFP vector.

[0059] 《Construction of pBYR2HS-PLP-GFP vector》 An Arabidopsis putative membrane lipoprotein (hereinafter sometimes referred to as "PLP") gene fragment was amplified by PCR using a primer (ADH-PLP-F, the base sequence of which is shown in SEQ ID NO: 12) and a primer (PLP-GFP-R, the base sequence of which is shown in SEQ ID NO: 13). Next, the PLP gene fragment was introduced into the pBYR2HS-CEGFP vector cleaved with the restriction enzyme SalI to obtain the pBYR2HS-PLP-GFP vector expressing a fusion protein of PLP and GFP.

[0060] 《Construction of pBYR2HS-Cryj1 vector》 A gene fragment (the base sequence is shown in SEQ ID NO: 14) encoding the Cryj1 protein (hereinafter sometimes referred to as "Cryj1"), a Japanese cedar allergen protein, was chemically synthesized (Thermo Fisher Scientific). Next, using the chemically synthesized gene fragment as a template, a primer (pBYR2HS-Cryj1Nt-F, the base sequence is shown in SEQ ID NO: 15) and a primer (pBYR2HS-Cryj1Nt-R, the base sequence is shown in SEQ ID NO: 16) was used to PCR amplify the gene fragment encoding the Cryj1 protein, a Japanese cedar pollen allergen. Next, the Cryj1 gene fragment was introduced into a pBYR2HS vector cleaved with the restriction enzyme SalI to obtain a pBYR2HS-Cryj1 vector.

[0061] (Plant growth conditions and agroinfiltration) N. benthamiana was grown for 5–6 weeks under conditions of 16 h light / 8 h dark at 25°C. Each of the above-mentioned vectors was introduced into Agrobacterium tumefaciens GV3101 strain carrying a binary vector, and the strain was cultured at 28°C until stationary phase in L-broth medium supplemented with 10 mM MES (pH 5.6), 20 μM acetosyringone, 50 mg / L kanamycin, 30 mg / L gentamicin, and 30 mg / L rifampicin.

[0062] The culture was then centrifuged to harvest the Agrobacterium tumefaciens, which was then cultured at OD in infiltration buffer (10 mM magnesium chloride, 10 mM MES (pH 5.6), 100 μM acetosyringone). 600 The cells were suspended in a solution with a dilution ratio of about 1. The Agrobacterium tumefaciens was then left in the solution for 2 to 3 hours.

[0063] The Agrobacterium tumefaciens suspension was then infiltrated onto the underside of N. benthamiana leaves using a 1 mL syringe without a needle, or, in some cases, N. benthamiana leaves were vacuum infiltrated by immersing them in the Agrobacterium suspension (agroinfiltration solution) and leaving them under a pressure of 736 mmHg for 5 min, after which the pressure was returned to atmospheric pressure.

[0064] In some experiments, the leaves of the plants were sprayed with various concentrations of sodium ascorbate or sodium citrate solutions once every two days. In some experiments, sodium ascorbate was added to the infiltration buffer. The expression of heat shock proteins was induced by incubating the plants at 37°C for 1 hour 1 and 3 days after infiltration. In the following experimental examples, sodium ascorbate was used as sodium L-ascorbate.

[0065] [Experimental Example 1] (Ascorbic acid spray 1) GFP, hCul, hFbxw7, PLP-GFP, and Cryj1 were expressed in N. benthamiana leaves by agroinfiltration using a syringe. For the expression of PLP-GFP, 1 / 100 amount of pBYR2HS-EGFP was also agroinfiltrated. After agroinfiltration, each plant was cultured at 18°C ​​for 7 days. In addition, 0, 30, 50, 100, 200, and 300 mM sodium ascorbate solution was sprayed once every two days.

[0066] Figures 2(a)-(f) are representative photographs of the leaves of each plant taken 7 days after agroinfiltration. The scale bar is 1 cm. Figure 2(a) shows the results without spraying sodium ascorbate solution, Figure 2(b) shows the results with 30 mM sodium ascorbate solution, Figure 2(c) shows the results with 50 mM sodium ascorbate solution, Figure 2(d) shows the results with 100 mM sodium ascorbate solution, Figure 2(e) shows the results with 200 mM sodium ascorbate solution, and Figure 2(f) shows the results with 300 mM sodium ascorbate solution. In Figures 2(a)-(f), the areas surrounded by dotted circles are the areas where GFP, hCul, hFbxw7, PLP-GFP, and Cryj1 were expressed, respectively.

[0067] As a result, the leaves were observed to turn black, especially in the areas expressing hCul, hFbxw7, PLP-GFP, and Cryj1, and it was revealed that the transient protein expression induced cell death (necrosis).In addition, it was revealed that necrosis was suppressed when 100, 200, and 300 mM sodium ascorbate solution was sprayed.

[0068] <<Measurement of GFP protein expression level>> Next, soluble proteins were extracted from the GFP-expressing region of the leaves of each plant. The prepared soluble proteins were then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Coomassie Brilliant Blue (CBB) staining. For comparison, 100ng, 200ng, and 400ng of purified GFP protein were also subjected to SDS-PAGE and stained with CBB.

[0069] Figure 3(a) is a photograph showing the results of CBB staining. In Figure 3(a), "NT" indicates the results for N. benthamiana leaves that were not subjected to gene introduction, and "FW" indicates fresh weight. In Figure 3(a), the band at approximately 30 kDa indicated by the arrowhead is the GFP protein band, and the band at approximately 50 kDa is the Rubisco large subunit band. Figure 3(b) is a graph showing the results of quantifying the expression level of GFP protein based on Figure 3(a). In Figure 3(b), "*" indicates that there is a significant difference at p<0.05 as a result of Student's T-test.

[0070] As a result, an increase in the expression of GFP protein was observed in samples sprayed with a sodium ascorbate solution at a concentration of 100 mM or more. In addition, an increase in the expression of Rubisco large subunit was also observed in samples sprayed with a sodium ascorbate solution at a concentration of 100 mM or more. These results demonstrated that spraying with a sodium ascorbate solution reduced plant necrosis and increased the production of the target protein.

[0071] 《Measurement of hCul protein expression level》 In addition, soluble proteins were extracted from the area of ​​the leaves of each plant where the hCul protein was expressed. The soluble proteins were then subjected to SDS-PAGE and transferred to a PVDF membrane. As mentioned above, the N-terminus of the hCul protein was tagged with a FLAG tag. Therefore, the hCul protein was detected by Western blotting using an anti-FLAG antibody.

[0072] FIG. 4(a) is a photograph showing the results of Western blotting. In FIG. 4(a), "NT" indicates the results for N. benthamiana leaves that were not subjected to gene introduction, and "FW" indicates fresh weight. In FIG. 4(a), the band indicated by the arrowhead indicates the hCul protein band. FIG. 4(b) is a graph showing the results of quantifying the expression level of hCul protein based on FIG. 4(a). In FIG. 4(b), "*" indicates that there is a significant difference as a result of Student's T-test with p<0.05, and "**" indicates that there is a significant difference as a result of Student's T-test with p<0.01.

[0073] As a result, an increase in the expression level of hCul protein was observed in the samples sprayed with a sodium ascorbate solution at a concentration of 100 mM or more. In addition, a peak in the expression level of hCul protein was observed in the samples sprayed with a sodium ascorbate solution at a concentration of 200 mM or more. This result further supports the idea that spraying with a sodium ascorbate solution reduces plant necrosis and increases the production of the target protein.

[0074] 《Measurement of PLP-GFP protein expression level》 Soluble proteins were extracted from the area of ​​the leaves of each plant where the PLP-GFP protein was expressed. The soluble proteins were then subjected to SDS-PAGE and transferred to a PVDF membrane. For comparison, 100ng, 200ng, and 300ng of purified GFP protein were also subjected to SDS-PAGE and transferred to a PVDF membrane. The PLP-GFP protein was then detected by Western blotting using an anti-GFP antibody.

[0075] Figure 5(a) is a photograph showing the results of Western blotting. In Figure 5(a), "NT" indicates the results for N. benthamiana leaves that were not subjected to gene introduction, and "FW" indicates the fresh weight. In Figure 5(a), the band indicated by the arrowhead is the band of PLP-GFP protein. In addition, the band at approximately 30 kDa is the band of GFP protein. As described above, in the expression of PLP-GFP, GFP protein was detected because 1 / 100 amount of pBYR2HS-EGFP was simultaneously agroinfiltrated.

[0076] Figure 5(b) is a graph showing the results of quantifying the expression level of PLP-GFP protein based on Figure 5(a). In Figure 5(b), "**" indicates that there is a significant difference at p<0.01 as a result of Student's T-test.

[0077] As a result, the expression level of PLP-GFP protein was increased in the samples sprayed with sodium ascorbate solution at a concentration of 100 mM or more, which further supports the idea that spraying with sodium ascorbate solution reduces plant necrosis and increases the production of the target protein.

[0078] [Experimental Example 2] (Ascorbic acid spray 2) Agroinfiltration was performed using a syringe to express GFP, hCul, and hFbxw7 in the leaves of Nicotiana benthamiana. After agroinfiltration, each plant was cultured at 25°C for 7 days. In addition, 0 or 200 mM sodium ascorbate solution was sprayed once every two days. This experimental example differed from experimental example 1 mainly in the growth temperature of the plants.

[0079] Figures 6(a) and (b) are photographs of the leaves of each plant taken 7 days after agroinfiltration. The scale bar is 1 cm. Figure 6(a) shows the result of spraying with a 200 mM aqueous solution of sodium ascorbate, and Figure 6(b) shows the result of not spraying with the aqueous solution of sodium ascorbate (control, Mock).

[0080] As a result, it was found that when an aqueous solution of sodium ascorbate was sprayed, gangrene tended to be suppressed.

[0081] <<Measurement of GFP protein expression level>> Three, five, and seven days after agroinfiltration, soluble proteins were extracted from the GFP-expressing area of ​​the leaves of each plant, and then subjected to SDS-PAGE and detected by CBB staining.

[0082] Figure 7(a) is a photograph showing the results of CBB staining. In Figure 7(a), "NT" indicates the results of N. benthamiana leaves that were not subjected to gene introduction, and "FW" indicates the fresh weight. "Mock" indicates the results when no sodium ascorbate aqueous solution was sprayed, and "AsA" indicates the results when 200 mM sodium ascorbate aqueous solution was sprayed.

[0083] In Figure 7(a), the band at approximately 30 kDa indicated by the arrowhead is the GFP protein band, and the band at approximately 50 kDa is the Rubisco large subunit band. Figure 7(b) is a graph showing the results of quantifying the expression level of GFP protein based on Figure 7(a). In Figure 7(b), "M" indicates the result when no sodium ascorbate aqueous solution was sprayed, and "A" indicates the result when a 200 mM sodium ascorbate aqueous solution was sprayed. Additionally, "*" indicates that there is a significant difference at p<0.05 as a result of the Student's T-test.

[0084] Although the GFP protein was stable to a certain extent, necrosis was observed when the culture was continued for 7 days after agroinfiltration. In contrast, necrosis was reduced by spraying the plants with a 200 mM aqueous solution of sodium ascorbate, and the expression level of the GFP protein tended to be maintained. This result further supports the idea that spraying the plants with an aqueous solution of sodium ascorbate reduced necrosis and increased the production of the target protein.

[0085] 《Measurement of hCul protein expression level》 Three, five, and seven days after agroinfiltration, soluble proteins were extracted from the area of ​​the leaves of each plant where hCul protein was expressed. The soluble proteins were then subjected to SDS-PAGE and transferred to a PVDF membrane. As mentioned above, the N-terminus of the hCul protein was tagged with a FLAG tag. Therefore, the hCul protein was detected by Western blotting using an anti-FLAG antibody.

[0086] Figure 8(a) is a photograph showing the results of Western blotting. Figure 8(a) also shows the results of CBB staining. In Figure 8(a), "NT" indicates the results for N. benthamiana leaves that were not subjected to gene introduction, and "FW" indicates the fresh weight. "Mock" indicates the results when no sodium ascorbate aqueous solution was sprayed, and "AsA" indicates the results when a 200 mM sodium ascorbate aqueous solution was sprayed. The band indicated by the arrowhead is the hCul protein band. "RbcL" indicates the Rubisco large subunit.

[0087] Figure 8(b) is a graph showing the results of quantifying the expression level of hCul protein based on Figure 8(a). In Figure 8(b), "M" indicates the result when no sodium ascorbate aqueous solution was sprayed, and "A" indicates the result when a 200 mM sodium ascorbate aqueous solution was sprayed. Additionally, "*" indicates that there is a significant difference at p<0.05 as a result of the Student's T-test.

[0088] As a result, almost no expression of hCul protein was observed in the sample that was not sprayed with sodium ascorbate solution, whereas in the sample sprayed with 200 mM sodium ascorbate solution, the expression level of hCul protein peaked 3 days after agroinfiltration and was maintained even 7 days after agroinfiltration.

[0089] This result further supports that spraying with an aqueous solution of sodium ascorbate reduced plant necrosis and increased the production of the target protein.

[0090] 《Measurement of hFbxw7 protein expression level》 Three days after agroinfiltration, soluble proteins were extracted from the area of ​​the leaves of each plant where the hFbxw7 protein was expressed. The soluble proteins were then subjected to SDS-PAGE and transferred to a PVDF membrane. As mentioned above, the N-terminus of the hFbxw7 protein was tagged with a FLAG tag. Therefore, the hFbxw7 protein was detected by Western blotting using an anti-FLAG antibody.

[0091] FIG. 9 is a photograph showing the results of Western blotting. FIG. 9 also shows the results of CBB staining. In FIG. 9, "NT" indicates the results for N. benthamiana leaves that were not subjected to gene introduction, and "FW" indicates the fresh weight. "Mock" indicates the results when no sodium ascorbate aqueous solution was sprayed, and "AsA" indicates the results when a 200 mM sodium ascorbate aqueous solution was sprayed. The band indicated by the arrowhead is the band of hFbxw7 protein. "RbcL" indicates Rubisco large subunit.

[0092] As a result, little expression of hFbxw7 protein was observed in the sample that was not sprayed with sodium ascorbate solution. In contrast, expression of hFbxw7 protein was observed 3 days after agroinfiltration in the sample sprayed with 200 mM sodium ascorbate solution. This result further supports the idea that spraying with sodium ascorbate solution reduces plant necrosis and increases production of the target protein.

[0093] [Experimental Example 3] (Addition of ascorbic acid to agroinfiltration solution 1) We investigated whether sodium ascorbate could be added to the agroinfiltration solution to suppress necrosis. Specifically, N. benthamiana plants were immersed in the agroinfiltration solution, left to stand for 5 minutes under a pressure of 736 mmHg, and then the pressure was returned to atmospheric pressure for vacuum infiltration, resulting in the expression of hCul protein. Here, sodium ascorbate was added to the agroinfiltration solution at final concentrations of 80 mM, 100 mM, and 200 mM. After agroinfiltration, each plant was cultured at 25°C for 7 days.

[0094] Figures 10(a)-(c) are representative photographs of the leaves of each plant taken 7 days after agroinfiltration. Figure 10(a) shows the results of adding 80 mM sodium ascorbate to the agroinfiltration solution, Figure 10(b) shows the results of adding 100 mM sodium ascorbate to the agroinfiltration solution, and Figure 10(c) shows the results of adding 200 mM sodium ascorbate to the agroinfiltration solution. In Figures 10(a)-(c), "vacuum" indicates the results of vacuum infiltration.

[0095] As a result, it was found that when sodium ascorbate at a concentration of 80 mM or more was added to the agroinfiltration solution, necrosis was induced, which was thought to be due to osmotic stress.

[0096] [Experimental Example 4] (Addition of ascorbic acid to agroinfiltration solution 2) We investigated whether sodium ascorbate could be added to the agroinfiltration solution to suppress necrosis. Specifically, N. benthamiana plants were immersed in the agroinfiltration solution, left to stand for 5 minutes under a pressure of 736 mmHg, and then vacuum infiltrated by returning the pressure to atmospheric pressure to express the hCul protein. Here, sodium ascorbate was added to the agroinfiltration solution at final concentrations of 0, 10, 20, 30, and 40 mM. Sodium ascorbate was administered only once during agroinfiltration. For comparison, a sample was also prepared in which sodium ascorbate was not added to the agroinfiltration solution and a 200 mM aqueous solution of sodium ascorbate was sprayed once every two days. Each plant was cultured at 25°C for 7 days after agroinfiltration.

[0097] Figures 11(a) to (f) are representative photographs of the leaves of each plant taken 7 days after agroinfiltration. Figure 11(a) shows the results when sodium ascorbate was not added to the agroinfiltration solution, Figure 11(b) shows the results when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 10 mM, Figure 11(c) shows the results when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 20 mM, Figure 11(d) shows the results when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 30 mM, Figure 11(e) shows the results when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 40 mM, and Figure 11(f) shows the results when sodium ascorbate was not added to the agroinfiltration solution and a 200 mM aqueous solution of sodium ascorbate was sprayed once every two days.

[0098] In addition, Figures 12(a) to (f) are representative photographs of the entire plants taken 7 days after agroinfiltration. Figure 12(a) shows the results when sodium ascorbate was not added to the agroinfiltration solution, Figure 12(b) shows the results when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 10 mM, Figure 12(c) shows the results when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 20 mM, Figure 12(d) shows the results when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 30 mM, Figure 12(e) shows the results when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 40 mM, and Figure 12(f) shows the results when sodium ascorbate was not added to the agroinfiltration solution and a 200 mM aqueous solution of sodium ascorbate was sprayed once every two days.

[0099] Next, 7 days after agroinfiltration, soluble proteins were extracted from the leaves of each plant. The prepared soluble proteins were then subjected to SDS-PAGE and transferred to a PVDF membrane. As mentioned above, the N-terminus of the hCul protein was tagged with a FLAG tag. Therefore, the hCul protein was detected by Western blotting using an anti-FLAG antibody.

[0100] Figure 13(a) is a photograph showing the results of Western blotting. Figure 13 also shows the results of CBB staining. In Figure 13(a), "NT" indicates the results for N. benthamiana leaves that were not subjected to gene introduction, and "FW" indicates the fresh weight. "Vacuum" indicates the results of vacuum infiltration, and "spray" indicates the results of spraying an aqueous solution of sodium ascorbate without adding sodium ascorbate to the agroinfiltration solution. The band indicated by the arrowhead is the band of hCul protein. "RbcL" indicates the Rubisco large subunit.

[0101] Figure 13(b) is a graph showing the results of quantifying the expression level of hCul protein based on Figure 13(a). In Figure 13(b), "vacuum" and "spray" have the same meanings as in Figure 13(a).

[0102] As a result, when sodium ascorbate was added to the agroinfiltration solution at a final concentration of 40 mM and then vacuum infiltration was performed, the expression of hCul protein was observed to the same extent as when no sodium ascorbate was added to the agroinfiltration solution and an aqueous solution of 200 mM sodium ascorbate was sprayed once every two days.

[0103] This result indicates that the addition of sodium ascorbate to the agroinfiltration solution can also reduce plant necrosis and increase the production of the target protein.

[0104] [Experimental Example 5] (Sodium citrate spray) We investigated whether spraying sodium citrate solution instead of sodium ascorbate solution could reduce plant necrosis. Specifically, we used a syringe to perform agroinfiltration to express GFP, hCul, hFbxw7, and PLP-GFP proteins in Nicotiana benthamiana leaves. After agroinfiltration, each plant was cultured at 18°C ​​for 7 days. In addition, 0, 30, 50, 100, 200, and 300 mM sodium citrate solution was sprayed once every two days.

[0105] Figures 14(a)-(f) are photographs of the leaves of each plant taken 7 days after agroinfiltration. The scale bar is 1 cm. Figure 14(a) shows the result of not spraying sodium citrate solution, Figure 14(b) shows the result of spraying 30 mM sodium citrate solution, Figure 14(c) shows the result of spraying 50 mM sodium citrate solution, Figure 14(d) shows the result of spraying 100 mM sodium citrate solution, Figure 14(e) shows the result of spraying 200 mM sodium citrate solution, and Figure 14(f) shows the result of spraying 300 mM sodium citrate solution.

[0106] As a result, it was found that when the leaves were sprayed with a sodium citrate solution having a concentration of 100 mM or more, the leaves became wilted.

[0107] Next, soluble proteins were extracted from the GFP-expressing regions of the leaves of each plant, and then subjected to SDS-PAGE and detected by CBB staining.

[0108] FIG. 15(a) is a photograph showing the results of CBB staining. In FIG. 15(a), "NT" indicates the results for Nicotiana benthamiana leaves that were not subjected to gene introduction, and "FW" indicates fresh weight. In FIG. 15(a), the lower band is the approximately 30 kDa GFP protein band, and the upper band is the approximately 50 kDa Rubisco large subunit band. FIG. 15(b) is a graph showing the results of quantifying the expression level of GFP protein based on FIG. 15(a).

[0109] As a result, it was found that the amount of GFP protein expression increased when the plant was sprayed with a sodium ascorbate solution, whereas the amount of GFP protein expression decreased when the plant was sprayed with a sodium citrate solution. This result indicates that administering sodium citrate to plants cannot suppress cell death induced by transient protein expression.

[0110] [Experimental Example 6] (Relief of endoplasmic reticulum stress 1) We investigated whether spraying endoplasmic reticulum stress inhibitors, 4-phenylbutyrate (4-PBA), taurine-conjugated ursodeoxycholic acid (TUDCA), and trimethylamine-N-oxide (TMAO), instead of sodium ascorbate solution could reduce plant necrosis.

[0111] Specifically, Nicotiana benthamiana was immersed in the agroinfiltration solution, left to stand for 5 minutes under a pressure of 736 mmHg, and then vacuum infiltrated by returning the pressure to atmospheric pressure to express the hFbxw7 protein. After agroinfiltration, each plant was cultured at 20°C for 7 days. In addition, 1 mM of each endoplasmic reticulum stress inhibitor was sprayed 1 day and 3 days after agroinfiltration.

[0112] Figures 16(a)-(d) are photographs of each plant taken 7 days after agroinfiltration. Figure 16(a) shows the results when no endoplasmic reticulum stress inhibitor was sprayed, Figure 16(b) shows the results when 1 mM 4-PBA was sprayed, Figure 16(c) shows the results when 1 mM TUDCA was sprayed, and Figure 16(d) shows the results when 1 mM TMAO was sprayed.

[0113] As a result, it was revealed that spraying of endoplasmic reticulum stress inhibitors could not suppress cell death induced by transient protein expression.

[0114] [Experimental Example 7] (Alleviation of endoplasmic reticulum stress 2) We investigated whether endoplasmic reticulum stress could be alleviated by inducing the expression of heat shock proteins, and thus alleviate plant necrosis. Specifically, N. benthamiana plants were immersed in an agroinfiltration solution, left to stand for 5 minutes under a pressure of 736 mmHg, and then vacuum infiltrated by returning the pressure to atmospheric pressure to express the hFbxw7 protein. After agroinfiltration, each plant was cultured at 20°C for 7 days. In addition, one day after agroinfiltration, the plants were incubated at 37°C for 1 hour to induce the expression of heat shock proteins.

[0115] Figures 17(a) to (d) are representative photographs of each plant taken 7 days after agroinfiltration. Figure 17(a) shows the results when heat shock protein expression induction was not performed, and Figure 17(b) is a partially enlarged photograph of Figure 17(a). Figure 17(c) shows the results when heat shock protein expression induction was performed, and Figure 17(d) is a partially enlarged photograph of Figure 17(c). In Figures 17(c) and (d), "HS" indicates that heat shock protein expression induction was performed.

[0116] As a result, it was revealed that spraying of endoplasmic reticulum stress inhibitors could not suppress cell death induced by transient protein expression.

[0117] [Experimental Example 8] (Ascorbic acid spray 3) N. benthamiana was immersed in the agroinfiltration solution, left to stand for 5 minutes under a pressure of 736 mmHg, and then vacuum infiltrated by returning the pressure to atmospheric pressure to express the Cryj1 protein. After agroinfiltration, each plant was cultured at 24°C or 20°C for 7 days. In addition, 0 mM or 200 mM sodium ascorbate solution was sprayed once every two days.

[0118] Figure 18(a) is a representative photograph of plants 4 days after agroinfiltration that were not sprayed with an aqueous solution of sodium ascorbate. As shown in Figure 18(a), necrosis was observed when the plant was not sprayed with an aqueous solution of sodium ascorbate.

[0119] Then, 7 days after agroinfiltration, soluble proteins were extracted from the leaves of each plant. The prepared soluble proteins were then subjected to SDS-PAGE and transferred to a PVDF membrane. Cryj1 protein was then detected by Western blotting using anti-Cryj1 antibody.

[0120] Figure 18(b) is a photograph showing the results of Western blotting. In Figure 18(b), "P" indicates the results of SDS-PAGE and Western blotting of Cryj1 protein derived from a natural product as a positive control, "M" indicates a molecular weight marker, "WT" indicates the results of N. benthamiana leaves that were not subjected to gene introduction, "24°C" indicates the results of culturing at 24°C, "20°C" indicates the results of culturing at 20°C, and "+AsA" indicates the results of spraying 200 mM sodium ascorbate aqueous solution once every two days.

[0121] As a result, in both cases of culturing at 24°C and 20°C, when the aqueous solution of sodium ascorbate was not sprayed, no expression of Cryj1 protein was observed. On the other hand, when the aqueous solution of sodium ascorbate was sprayed, expression of Cryj1 protein was observed.

[0122] As with the natural Cryj1 protein, two bands were observed. Of the two bands, the band with the higher molecular weight is the band of the Cryj1 protein bound to a glycan. In other words, it was confirmed that the Cryj1 protein expressed in Nicotiana benthamiana also has a glycan attached thereto. [Industrial Applicability]

[0123] According to the present invention, a technique for suppressing cell death induced by transient protein expression in plants can be provided.

Claims

1. 1. An inhibitor of cell death induced by transient protein expression in Nicotiana plants, comprising ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof as an active ingredient, Ascorbic acid or a derivative thereof or a salt or solvate thereof is used in a concentration of 100 to 300 mM for contacting the Nicotiana plant; or ascorbic acid or a derivative thereof, or a salt or solvate thereof, at a concentration of 25 to 80 mM, is added to an Agrobacterium suspension for introducing an expression vector for a target protein into a Nicotiana plant, or is added to a culture solution for hydroponically cultivating a Nicotiana plant; the derivative is an ascorbic acid alkyl ester, an ascorbic acid phosphate, an ascorbic acid glucoside, or an ascorbic acid alkyl ether; The inhibitor, wherein the solvate is a hydrate or an organic solvate.

2. 2. The inhibitor of claim 1, wherein the derivative is ascorbyl monostearate, ascorbyl monopalmitate, ascorbyl monoisopalmitate, ascorbyl monooleate, ascorbyl distearate, ascorbyl dipalmitate, ascorbyl monopalmitate, ascorbyl tetrahexyldecanoate, ascorbic acid monophosphate, ascorbic acid diphosphate, ascorbic acid triphosphate, ascorbic acid ethyl ether, ascorbic acid methyl ether, ascorbic acid diglucoside, or ascorbyl palmitate phosphate.

3. 3. The inhibitor of claim 1 or 2, wherein the solvate is sodium isoascorbate monohydrate or calcium ascorbate dihydrate.

4. A method for suppressing cell death induced by transient protein expression in a Nicotiana plant, the method comprising the step of administering to the Nicotiana plant ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof, In the step, ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof is administered to the Nicotiana plant by contacting the plant with the ascorbic acid or a derivative thereof at a concentration of 100 to 300 mM; or In the step, ascorbic acid or a derivative thereof, a salt thereof, or a solvate thereof is added at a concentration of 25 to 80 mM to an Agrobacterium suspension for introducing an expression vector for a target protein into the Nicotiana plant, or is added to a culture solution for hydroponically cultivating the Nicotiana plant, the derivative is an ascorbic acid alkyl ester, an ascorbic acid phosphate, an ascorbic acid glucoside, or an ascorbic acid alkyl ether; The method, wherein the solvate is a hydrate or an organic solvate.

5. 5. The method of claim 4, wherein the derivative is ascorbyl monostearate, ascorbyl monopalmitate, ascorbyl monoisopalmitate, ascorbyl monooleate, ascorbyl distearate, ascorbyl dipalmitate, ascorbyl monopalmitate, ascorbyl tetrahexyldecanoate, ascorbic acid monophosphate, ascorbic acid diphosphate, ascorbic acid triphosphate, ascorbic acid ethyl ether, ascorbic acid methyl ether, ascorbic acid diglucoside or ascorbyl palmitate phosphate.

6. 6. The method of claim 4 or 5, wherein the solvate is sodium isoascorbate monohydrate or calcium ascorbate dihydrate.

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