Application of wheat Ms2 protein in broad-spectrum inhibition of division and / or growth of eukaryotic cells

Abstract

The invention discloses application of wheat Ms2 protein in broad-spectrum inhibition of eukaryotic cell division and / or growth, and belongs to the technical field of genetic engineering. The invention relates to a target protein Ms2, a wheat CDs sequence corresponding to the target protein Ms2 is shown as a sequence 1, and an amino acid sequence corresponding to the target protein Ms2 is shown as a sequence 5. A transient or stable genetic transformation technology is utilized for the first time to prove that the Ms2 protein has toxicity to plants, animals and fungal cells, cell division and / or growth of the plants, the animals and the fungal cells are inhibited, and a theoretical support is provided for application of the Ms2 protein to animal and plant disease resistance and gene therapy.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of a wheat Ms2 protein in inhibiting cell division and / or growth in animals, plants, and fungi. Background Technology

[0002] The basic building block of organisms in nature is the cell, and most active cells exist in a dynamic balance of the cell cycle, constantly dividing, growing, differentiating, maturing, and aging. During cell development, some cells begin to degenerate before they are fully formed, some begin to degenerate after reaching their maximum number of divisions, and some cells that cannot repair themselves after damage also degenerate. Cell degeneration during normal development is an active and orderly process regulated by genes under natural conditions, accompanying the growth and development of organisms. In animals, degenerated cells are located in normal tissues and are cleared by macrophages or neighboring cells without affecting the normal function of nearby cells. In plants, degenerated cells are not phagocytosed and become part of the plant organism.

[0003] Precise regulation of cell division and replication cycles is crucial for normal plant growth and development. In plants, the cell cycle is a major factor influencing meristematic tissue activity and growth rate. In animals, life activities such as wound healing and pathological tissue repair are related to cell cycle regulation (Ahuja et al., 2007; Zebrowski and Engel, 2013; Zhu et al., 2009). The eukaryotic cell cycle requires the synergistic regulation of proteins such as cyclin-dependent kinase (CDK), cyclin, cyclin-dependent kinase inhibitor (KRP), retinoblastoma gene 1 (RB1), and cyclin-dependent kinase inhibitor gene (CKI) (Nigg, 1995; Novakett et al., 1998). Cyclins can be classified into positive and negative regulatory proteins according to their function. Positive regulatory proteins include cyclin and CDK. Cyclin binds to CDK to form a complex that participates in events such as DNA replication and chromosome segregation, controlling cell cycle progression as well as cell proliferation and differentiation (Komaki and Sugimoto, 2012; Wood and Endicott, 2018). Negative regulatory proteins, such as CKI, RB, E2F transcription factor, and PTEN, inhibit cell cycle progression by blocking events such as DNA replication (Kumar et al., 2018; Wang et al., 2020).

[0004] Cytotoxic proteins can directly or indirectly cause cell cycle abnormalities, leading to inhibited cell division, slowed growth, and even unnatural cell degeneration. Based on their origin, they can be divided into endogenous and exogenous cytotoxic proteins. Endogenous cytotoxic proteins are mainly produced by the cell itself and participate in processes such as cell division, growth, and degeneration. For example, tau toxic protein oligomers can induce invagination of the nuclear lamina of neurons, thereby impairing nucleoplasmic transport and ultimately causing neuronal damage (Sun et al., 2024). Exogenous cytotoxic proteins originate from microorganisms such as bacteria and viruses and have bactericidal and antiviral effects. Cytotoxic proteins inhibit cell division, growth, or cause degeneration through various mechanisms: 1) They act directly on the cell membrane or organelle membranes, disrupting the integrity of membrane components; for example, BCL2-AssociatedX (BAX) protein can be converted from cytoplasmic monomers into a toxic oligomer that can penetrate the outer mitochondrial membrane, thereby disrupting body development, tissue homeostasis, and immune regulation, leading to diseases such as tumors, autoimmune diseases, neurodegenerative diseases, and heart failure (Hauseman et al., 2020); 2) They cause DNA damage, resulting in gene mutations and cellular abnormalities; for example, aflatoxin B1 (AFB1) combines with DNA after being metabolized in the body to form AFB1-DNA polymers, leading to severe DNA damage and exhibiting strong toxicity to the liver in humans and animals (Shirabe et al., 2011); 3) They interfere with intracellular signal transduction pathways, affecting normal cellular physiological functions, such as ricin and Shiga toxin. Some cytotoxic proteins can inhibit cell cycle growth, leading to tumor cell arrest or degeneration. For example, ectopic expression of the human cell cycle inhibitor p27 (a CKI inhibitor) and HIV-1 viral protein R (Vpr) can inhibit cell cycle growth. Other cytotoxic proteins can activate or inhibit degeneration-related proteins, resulting in cell degeneration. Some cytotoxic proteins can also inhibit the cell cycle, causing tumor cell growth arrest or degeneration. For instance, ectopic expression of plant RNA-dependent RNA polymerase (RDR1) can specifically block the cell cycle of cancer cells in solid tumors and leukemia (Qi et al., 2022). Some cytotoxic proteins can activate or inhibit immune responses, playing a role in the treatment of autoimmune diseases and transplant rejection in animals. Some cytotoxic proteins can inhibit the growth of plant host cells, suppressing the activity of invading pathogens and pests, thus enabling plant disease resistance applications, such as C12 family proteases (VPEs) and C14 family proteases (MCs). Therefore, the discovery of new cytotoxic proteins is of great significance for the prevention and control of plant, animal, and fungal diseases.

[0005] Fu Daolin et al. previously cloned the male nuclear sterility gene Ms2 from wheat (Ni et al., 2017), finding that the Ms2 gene is expressed only in wheat anther tissue, and its expression leads to the male sterility phenotype in wheat. This has significant application value in creating male-sterile wheat lines and developing hybrid wheat. As a high-performance male sterility gene, the Ms2 gene warrants further exploration to discover its novel functions. Summary of the Invention

[0006] This invention is the first to discover that the Ms2 protein is cytotoxic to wheat cells, directly or indirectly inhibiting wheat cell division and / or growth. More importantly, the wheat Ms2 protein exhibits broad-spectrum cytotoxicity in inhibiting the division and / or growth of animal, plant, and fungal cells, and has potential applications in areas such as tumor therapy, immune modulation, plant disease and pest resistance, and fungal control.

[0007] The purpose of this invention is to utilize the cytotoxic function of wheat Ms2 protein to inhibit the division and / or growth of eukaryotic cells.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the invention provides the use of wheat Ms2 protein as a toxic protein in either (1) or (2) below:

[0010] (1) Inhibits cell division and / or growth;

[0011] (2) Prepare products that inhibit cell division and / or growth;

[0012] The wheat Ms2 protein is the protein shown in (A1) or (A2) below:

[0013] (A1) A protein consisting of the amino acid sequence shown in sequence 5 of the sequence listing;

[0014] (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0015] In the above applications, to facilitate the purification of the protein in (A1), a molecular tag can be attached to the amino or carboxyl terminus of the protein in (A1). The tag can be Poly-Arg (typically 6 RRRRR), Poly-His (typically 6 HHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK), or c-myc (EQKLISEEDL).

[0016] In the above applications, the cells are plant cells, animal cells, or fungal cells.

[0017] In a second aspect, the present invention provides the application of the above-mentioned wheat Ms2 protein or the encoding gene of wheat Ms2 protein in the preparation of disease-resistant plant products.

[0018] Preferably, the gene encoding the wheat Ms2 protein is a nucleic acid molecule as shown in any one of the following (i)-(iv):

[0019] (i) The nucleic acid molecule shown in sequence 1 of the sequence listing;

[0020] (ii) The nucleic acid molecule shown in sequence 3 of the sequence listing;

[0021] (iii) The nucleic acid molecule shown in sequence 4 of the sequence listing;

[0022] (iv) Nucleic acid molecules with the amino acid sequences shown in Figure 5, excluding (i)-(iii).

[0023] In the above applications, wheat Ms2 protein is a toxic protein that can inhibit cell division and / or growth. Therefore, the gene encoding wheat Ms2 protein can be linked to an inducible promoter and introduced into plants. When plants are attacked by pathogens, the inducible promoter can receive the signal, causing wheat Ms2 protein to be expressed in cells attacked by pathogens, inhibiting cell division and / or growth, thereby depriving pathogens of the "soil" for proliferation and achieving the effect of plant resistance to disease.

[0024] A third aspect of the present invention provides the use of the aforementioned wheat Ms2 protein or the gene encoding the wheat Ms2 protein in the preparation of an animal cell therapeutic drug. The animal cell therapeutic drug is preferably a tumor therapeutic drug.

[0025] In the above applications, wheat Ms2 protein or the gene encoding wheat Ms2 protein is expressed in animal cells, thereby inhibiting the division and / or growth of animal cells; for example, it can be used to inhibit the division and / or growth of tumor cells to achieve the effect of tumor treatment.

[0026] In a fourth aspect, the present invention provides the use of the above-mentioned wheat Ms2 protein in the preparation of products for killing harmful microorganisms.

[0027] In the above applications, wheat Ms2 protein can inhibit the division and / or growth of fungal cells. Therefore, wheat Ms2 protein can be developed into a product for killing harmful microorganisms. The product for killing harmful microorganisms can be in the form of fungicides or other products.

[0028] In the above applications, the fungicides include, but are not limited to: Fusarium graminearum fungicide, yeast fungicide, stripe rust fungicide, and powdery mildew fungicide.

[0029] A fifth aspect of the invention provides the use of wheat Ms2 protein in the preparation of insecticides.

[0030] In the above applications, wheat Ms2 protein can be administered orally or by injection, utilizing its ability to inhibit cell division and / or growth to achieve insecticidal effects.

[0031] In the above applications, the targets of insect control include, but are not limited to: aphids, cotton bollworms, rice planthoppers, and corn borers.

[0032] In a sixth aspect, the invention provides the use of an expression cassette containing the wheat Ms2 protein-coding gene, a recombinant plasmid, or a recombinant bacterium in any of the following (1)-(5):

[0033] (1) Inhibits cell division and / or growth;

[0034] (2) Prepare products that inhibit cell division and / or growth;

[0035] (3) Preparation of tumor treatment drugs;

[0036] (4) Preparation of fungicides;

[0037] (5) Preparation of insecticides.

[0038] In the above applications, the nucleotide sequence of the wheat Ms2 protein encoding gene is shown in any one of sequences 1, 2, 3 and 4 in the sequence listing.

[0039] The beneficial effects of this invention are:

[0040] This invention has discovered that wheat Ms2 protein has cytotoxic properties, exhibiting broad-spectrum inhibition of plant cell, animal cell, and fungal cell division and / or growth. Utilizing the toxic effects of wheat Ms2 protein, it can be widely applied in fields such as plant and animal disease resistance, gene therapy, and fungal control. Attached Figure Description

[0041] Figure 1 Schematic diagram of partial plasmid structure.

[0042] Figure 2 Mitochondrial localization of wheat Ms2 protein.

[0043] Figure 3 Transgenic wheat and heat-shock-induced expression of the Ms2 gene. In the figure, A represents the transgenic wheat plant; B represents the PCR detection of heat-shock-induced transgenic expression.

[0044] Figure 4 In transgenic wheat roots, heat-induced expression of Ms2 leads to inhibited root cell division and / or growth. In the figure, A represents the number of lateral roots after heat induction; B represents the color of the root tip after heat induction.

[0045] Figure 5 Thermo-induced expression of Ms2 in transgenic wheat roots leads to inhibition of lateral root cell division and / or growth.

[0046] Figure 6 In transgenic wheat, heat-induced expression of Ms2 inhibits coleoptile cell division and / or growth. In the figure, A represents seedling growth after heat induction; B represents coleoptile length and plant height of seedlings after heat induction.

[0047] Figure 7 Transient heat-induced expression of the Ms2 gene inhibits the division and / or growth of wheat callus cells. In the figure, A represents the growth state of callus tissue after heat induction; B represents the proportion of embryogenic callus tissue after heat induction; and C represents the size of callus tissue after heat induction.

[0048] Figure 8 In transgenic tobacco, heat-induced expression of Ms2 inhibits root and leaf division and / or growth. In the figure, A shows the growth of genetically transformed tobacco in rooting medium; B shows the detection of Ms2-GFP protein in transgenic tobacco; and C shows the state of tobacco leaves infected with Agrobacterium carrying the target plasmid.

[0049] Figure 9 Ms2 gene transfection of animal cells for 72 hours inhibited cell division and / or growth; in the figure, A represents transfection of 293; B represents transfection of huh7.

[0050] Figure 10 Ms2 gene transfection of 293 cell line for 96 h inhibited cell division and / or growth; in the figure, A is a confocal microscope image of 293 cell line 96 h after transfection; B is the proportion of fluorescent positive cells.

[0051] Figure 11 : Detection of Ms2 prokaryotic protein expression; In the figure, A is the detection of Coomassie brilliant blue staining; B is the detection of Western blot based on Ms2 antibody.

[0052] Figure 12 Ms2 prokaryotic expression inhibits the division and / or growth of Gibberella fuciformis cells. In the figure, A represents the growth status of Gibberella fuciformis patches; B represents the statistical analysis of Gibberella fuciformis patch diameters.

[0053] Figure 13 The effect of Ms2 protein on yeast cell division and / or growth; in the figure, A represents the OD between different GAL1-driven expression vectors after different induction times. 600Values; B represents the expression of different vectors driven by the constitutive promoter TDH3 after different induction times; C represents the green fluorescence of GFP (SVpc678), Ms2-GFP (SVpc679), and GFP-Ms2 (SVpc680); D represents the protein signals of GFP (SVpc678), Ms2-GFP (SVpc679), and GFP-Ms2 (SVpc680). Detailed Implementation

[0054] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0055] To elucidate the function of the wheat Ms2 protein, this invention employs the heat shock protein gene promoters Lehsp23.8, 35S, Rp27, and CMV to construct plasmids related to the wheat Ms2 gene. Transient and stable genetic transformation techniques are then used to elucidate the cytotoxicity of the Ms2 protein to plant, animal, and fungal organisms.

[0056] This invention constructed 25 plasmids targeting the Ms2 gene: SVpc337(Lehsp23.8::cMs2:3*Flag), SVpc348(Lehsp23.8::GFP), SVpc350(Lehsp23.8::cMs2(-C1 / 2AA):GFP), SVpc351(Lehsp23.8::cMs2:GFP), SVpc369(35S::GFP), SVpc375(35S::cMs2:GFP), SVpc391(35S::cMs2), SVpc398(Tac::GST), SVpc399(Tac::GST:cMs2), SVpc419(CMV::EGFP), SVpc420(CMV::cMs2). # ),SVpc421(CMV::cMs2 # :EGFP),SVpc424(35S::AtAOX:mCherry),SVpc432(Tac::cMs2),SVpc437(GAL1::terminator),SVpc438(GAL1 ::cMs2),SVpc441(GAL1::GFP), SVpc443(GAL1::cMs2:GFP), SVpc529(GAL1::GFP:cMs2), SVpc530(GAL1::cMs2 C158T:GFP), SVpc677(TDH3::cMs2), SVpc678(TDH3::GFP), SVpc679(TDH3::cMs2:GFP), SVpc680(TDH3::GFP:cMs2), SVpc681(TDH3::cMs2 C158T :GFP)(Table 1). cMs2 * The codon-optimized sequence for fungi (Fusarium graminearum); cMs2 # The sequences were optimized for codons in animals; a sense point mutation was inserted into the Ms2 gene in SVpc530 and SVpc681, with the cDNA mutation site being C158T. Plasmids SVpc337, SVpc348, SVpc350, and SVpc351, driven by the heat shock promoter Lehsp23.8, were used for genetic transformation in wheat and tobacco. Plasmids SVpc369, SVpc375, SVpc391, and SVpc424, driven by the 35S promoter, were used for stable or transient transformation in tobacco. SVpc398, SVpc399, and SVpc432 were used for prokaryotic expression, induced by IPTG, for prokaryotic expression and antibacterial experiments against *Fusarium graminearum*. Plasmids SVpc437, SVpc438, SVpc441, SVpc443, SVpc529, and SVpc530 are driven by the GAL1 promoter and are used for yeast transformation; plasmids SVpc677, SVpc678, SVpc679, SVpc680, and SVpc681 are driven by the TDH3 promoter and are used for yeast transformation. Plasmids SVpc419, SVpc420, and SVpc421 are driven by the CMV promoter and are used for transient transformation of animal cells.

[0057] This invention confirms that wheat Ms2 protein is cytotoxic to animal, plant and fungal organisms, and can inhibit the division and / or growth of animal, plant and fungal cells.

[0058] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0059] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0060] Example 1: Construction of wheat Ms2 gene-related plasmid

[0061] The genetic transformation plasmids involved in this invention include 25 plasmids such as SVpc337, SVpc348, SVpc350, and SVpc351 (Table 1). Figure 1 Plasmids SVpc337, SVpc348, SVpc350, and SVpc351 all use the tomato heat shock promoter Lehsp23.8 (1611 bp) to drive the expression of target genes (Yi et al., 2006, Plant Science, 171:398-407). This promoter is a heat shock-inducible promoter, initiating downstream gene expression at 38℃-39℃, and is used for genetic transformation in wheat and tobacco. Plasmids SVpc369, SVpc375, SVpc391, and SVpc424 use the 35S promoter to drive the expression of downstream target genes and are used for stable or transient transformation in tobacco. Plasmids SVpc419, SVpc420, and SVpc421 use the CMV promoter to drive the expression of downstream target genes and are used for transient transformation in animal cells. SVpc398, SVpc399, and SVpc432 are prokaryotic expression plasmids, induced by IPTG, used for prokaryotic expression and antibacterial experiments against *Fusarium graminearum*. SVpc437, SVpc438, SVpc441, SVpc443, SVpc529, and SVpc530 plasmids, driven by the GAL1 promoter, express downstream target genes and are used for yeast transformation. SVpc677, SVpc678, SVpc679, SVpc680, and SVpc681 plasmids, driven by the TDH3 promoter, express downstream target genes and are used for yeast transformation. Among them, SVpc530S and SVpc681 contain a point mutation in the Ms2 gene, with the cDNA mutation site being C158T.

[0062] Wild-type cMs2 gene (sequence 1) was obtained by PCR amplification from a cDNA template using primers MS2-F: 5'-ATG GCA GGG CAC CAC AG-3' and MS2-R: 5'-TCA ACT TGA GAA TGC TGC GGA TAA G-3'. The cDNA template was derived from LM15. RMs2 Young spikelets at the S2 stage. The cMs2 gene in plasmid SVpc350 has a 432bp deletion at the 3' end, resulting in a halved cMs2 gene sequence (Sequence 2); the cMs2 gene in plasmids SVpc417 and SVpc418... * (Sequence 3) is an optimized codon sequence suitable for fungal expression, commercially synthesized (Sangon Biotech); cMs2 in plasmids SVpc420 and SVpc421 # (Sequence 4) is an optimized codon sequence suitable for animal expression. It was commercially synthesized (Vezhen Biotechnology) and still encodes the same Ms2 protein after optimization (Sequence 5).

[0063] Construction of genetic transformation plasmids: A linearized vector backbone was obtained through enzyme digestion or PCR; homologous arms were introduced at both ends of the insert fragment via PCR; and in-fusion cloning was used to ligate the gene fragment to the linearized vector backbone. Vector backbone information, restriction sites, and homologous arms are detailed in Table 2.

[0064] Specifically, plasmid SVpc337 expresses cMs2 fused with 3*Flag; plasmids SVpc351 and SVpc375 express cMs2 fused with GFP; and plasmids SVpc417 and SVpc421 express cMs2 fused with EGFP. Plasmid SVpc348 serves as a strict control for SVpc351; plasmid SVpc369 serves as a strict control for SVpc375; plasmid SVpc419 serves as a strict control for SVpc421; plasmid SVpc441 serves as a strict control for SVpc443; and plasmid SVpc678 serves as a strict control for SVpc679.

[0065] Table 1. Plasmid construction involved in this invention

[0066]

[0067]

[0068] cMs2 * It is an optimized codon sequence adapted for fungal expression; cMs2 # It is an optimized codon sequence adapted to animal expression.

[0069] Table 2. Vector backbone information, restriction sites, and homologous arms involved in plasmid construction

[0070]

[0071]

[0072] Sequence 1: Full-length sequence of the cMs2 gene

[0073] ATGGCAGGGCACCACAGCCCCTCGGCGGCCTCGGCACTGCGTGAAAAAGACACGCTGGTGAGGT

[0074] GTCTCGTGGGATCAGGTCCCGGCGGCGGCGCTCATGCCGGGACCTTCGGCGCTGTGCGGGACTT

[0075] CCTCATCCAGTTCCGCGACCAAGGAATCCCCTGGGTCCGCATCTACGAGTCAACCCCGGCTTGG

[0076] CAGCAGCAATCCGGCGGGCTGCTGATCCAGGATTGGGACGGAGACGCCGCGGCGGAGGGAGCCA

[0077] AGGTGTTCTTCACGCTCATCACCACAAGGAGGGGCGGCGCCATTAACAGGAGGGCACTGGGAGG

[0078] CGGGACGTGGACAAGCAAGGCCGCGCCCAGGGTAGGGGACGAGGTCGCCGTCAGCACACTGTAC

[0079] TTCAAACGGGGCGGGTCCAGCGGCAGATTATTCACCGCCTTGGAGATCCATCTCAGAAACGAGC

[0080] CCCAAGTTGCTATCTGCCTGCTGCATCCGACTAACTATCTGTATAGCATTCGGGATTTGAGGCT

[0081] CTACATCGACCAGGGATGGTTCCCGGGAGGAACTCAAGCAAACCTGGGCGCGGAGCAATATCAA

[0082] GATCCTGATGTTCCTGGATTCGTGAGTGGTTCACGTGCTGATTACACCACTATTCTGTTTTCTA

[0083] GCAGTGAGACTATTTACGACCAGCAATCGATTCATTCCTCCGGGGCTGCTCTGCCACCTCATGA

[0084] TGCATCTCTGGATGCTATTTCTCACCACCTGTTTTCAGAAAACAACTCAACGCCAGAGTTTGGT

[0085] GGACAGTATTCTCATGCTGATGAAATATCAATCCTTAATGAATACTACAATACCTTGATGGGGA

[0086] CCAACTCCAACTCAGGATTGCATGCCTTATCCGCAGCATTCTCAAGTTGA

[0087] Note: The bolded bases represent the positions of mutant bases in plasmids SVpc530 and SVpc681.

[0088] Sequence 2: Half-length sequence of cMs2 gene

[0089] ATGGCAGGGCACCACAGCCCCTCGGCGGCCTCGGCACTGCGTGAAAAAGACACGCTGGTGAGGT

[0090] GTCTCGTGGGATCAGGTCCCGGCGGCGGCGCTCATGCCGGGACCTTCGGCGCTGTGCGGGACTT

[0091] CCTCATCCAGTTCCGCGACCAAGGAATCCCCTGGGTCCGCATCTACGAGTCAACCCCGGCTTGG

[0092] CAGCAGCAATCCGGCGGGCTGCTGATCCAGGATTGGGACGGAGACGCCGCGGCGGAGGGAGCCA

[0093] AGGTGTTCTTCACGCTCATCACCACAAGGAGGGGCGGCGCCATTAACAGGAGGGCACTGGGAGG

[0094] CGGGACGTGGACAAGCAAGGCCGCGCCCAGGGTAGGGGACGAGGTCGCCGTCAGCACACTGTAC

[0095] TTCAAACGGGGCGGGTCCAGCGGCAGATTATTCACCGCCTTGGAGATCCATCTCAGAAACGAGC

[0096] CCTGA

[0097] Sequence 3: The codon-optimized sequence of the cMs2 gene in the fungus (Fusarium graminearum) (cMs2*)

[0098] ATGGCCGGCCATCATTCTCCTTCTGCCGCCTCTGCCCTTCGCGAGAAGGACACTCTTGTGCGCT

[0099] GTCTTGTGGGCTCTGGCCCTGGCGCGGCCGCCCATGCCGGCACTTTCGGCGCCGTGCGCGATTT

[0100] CCTTATTCAGTTCCGCGATCAGGGCATTCCTTGGGTGCGCATCTATGAGTCAACTCCTGCCTGG

[0101] CAGCAGCAGTCTGGCGGCCTTCTGATTCAGGATTGGGATGGCGACGCCGCCGCCGAGGGCGCCA

[0102] AGGTTTTCTTCACTCTTATTACTACCAGGCGCGGCGGCGCCATTAATCGCCGCGCCTTGGGCGG

[0103] CGGCACTTGGACCTCTAAGGCCGCCCCTAGGGTGGGTGACGAGGTGGCCGTTTCTACTCTTTAT

[0104] TTTAAGAGGGGCGGCTCTTCTGGCCGCCTTTTTACTGCCCTTGAGATTCATCTTAGGAATGAGC

[0105] CTCAGGTGGCCATTTGTCTTTTGCATCCTACTAATTATCTTTATTCTATTCGCGATCTTAGGTT

[0106] GTATATTGATCAGGGCTGGTTCCCTGGCGGTACTCAGGCCAATCTTGGCGCCGAGCAGTATCAG

[0107] GATCCTGATGTTCCTGGCTTTGTTTCTGGCTCTCGCGCCGATTATACTACCATTCTTTTTAGCT

[0108] CTTCTGAGACTATCTATGACCAGCAGTCTATTCATTCTTCTGGCGCCGCATTGCCTCCTCATGA

[0109] TGCCTCTCTTGATGCCATTTCTCATCATTTGTTTTCTGAGAATAATTCTACTCCTGAGTTTGGC

[0110] GGCCAGTATTCTCATGCCGATGAGATTTCTATTCTTAATGAGTATTATAATACTCTGATGGGCA

[0111] CTAATTCTAATTCTGGCCTTCATGCCCTTTCTGCCGCCTTCTCTTCTTGA

[0112] Sequence 4: The sequence of cMs2 gene after codon optimization in animals (cMs2 # )

[0113] ATGGCCGGCCACCACAGCCCCAGCGCCGCCTCTGCCCTGCGGGAAAAGGACACCCTCGTGCGGT

[0114] GCCTGGTGGGCAGCGGTCCTGGCGGCGGCGCCCACGCTGGCACATTCGGCGCTGTGCGGGACTT

[0115] CCTGATTCAGTTCAGAGATCAGGGCATCCCCTGGGTTAGAATCTACGAGAGCACACCTGCCTGG

[0116] CAGCAGCAATCTGGCGGCCTGCTGATCCAAGATTGGGACGGCGATGCCGCTGCCGAGGGCGCAA

[0117] AGGTGTTCTTCACCCTGATCACAACAAGAAGGGGCGGAGCCATCAACAGACGGGCCCTGGGCGG

[0118] AGGAACCTGGACCTCAAAGGCCGCCCCTAGAGTCGGCGACGAGGTGGCCGTGTCCACCCTGTAC

[0119] TTCAAAAGAGGCGGCAGCTCTGGGAGACTGTTTACCGCCCTGGAAATCCACCTGAGAAACGAGC

[0120] CTCAGGTGGCCATTTGTCTGCTGCACCCTACAAACTACCTGTATTCTATCAGAGATCTGCGGCT

[0121] GTACATCGACCAGGGATGGTTTCCAGGCGGCACCCAGGCCAATCTGGGAGCTGAACAGTACCAG

[0122] GACCCCGACGTGCCCGGCTTCGTGTCTGGATCTAGAGCCGACTACACCACCATCCTGTTCAGCA

[0123] GTAGCGAGACAATCTACGACCAGCAGAGCATCCACTCCAGCGGAGCCGCTCTGCCTCCACACGA

[0124] CGCCAGCCTGGACGCCATCAGCCATCACCTGTTTAGCGAAAACAACTCCACCCCTGAGTTCGGC

[0125] GGCCAGTACTCCCACGCCGATGAGATCAGCATCCTGAACGAATACTACAACACCCTGATGGGCA

[0126] CCAACAGCAATAGCGGCCTCCATGCCCTGAGCGCCGCTTTCAGCAGCTGA

[0127] Sequence 5: Ms2 protein amino acid sequence

[0128] MAGHHSPSAASALREKDTLVRCLVGSGPGGGAHAGTFGAVRDFLIQFRDQGIPWVRIYESTPAW

[0129] QQQSGGLLIQDWDGDAAAEGAKVFFTLITTRRGGAINRRALGGGTWTSKAAPRVGDEVAVSTLY

[0130] FKRGGSSGRLFTALEHLRNEPQVAICLLHPTNYLYSIRDLRLYIDQGWFPGGTQANLGAEQYQ

[0131] DPDVPGFVSGSRADYTTILFSSSETIYDQQSIHSSGAALPPHDASLDAISHHLFSENNSTPEFG

[0132] GQYSHADEISILNEYYNTLMGTNSNSGLHALSAAFSS

[0133] Example 2: Subcellular localization based on Ms2 heat-induced expression

[0134] Subcellular localization of the Ms2 protein involved the use of plasmid SVpc351 and the mitochondrial co-localization plasmid SVpc424 (35S::AtAOX:mCherry). In Arabidopsis thaliana, the AOX protein is localized in the mitochondria (Chris, et al., FEBS Letters, 2008:582, 3073–3079). Two plasmids were separately introduced into Agrobacterium AGL1 strain, and tobacco leaves were co-infected by injection and cultured for 48 hours. One to two leaves were then subjected to in vitro high-temperature treatment (38°C, 2 h, in darkness). After treatment, localization was observed using a laser confocal microscope. The results showed that Ms2 and AOX were co-localized in the mitochondria. Figure 2 ).

[0135] Example 3: Obtaining Ms2 transgenic wheat and heat-induced expression of the Ms2 gene

[0136] Genetic transformation of wheat was performed using plasmids SVpc337, SVpc348, and SVpc351. The recipient material was wheat Fielder, and the Bar gene was used as the selection gene. The specific operational procedures included:

[0137] (a) Wheat planting and pretreatment: Wheat grains (embryos about 1.5-2 mm) were selected 14 days after flowering. After surface sterilization with disinfectant, the embryos were removed in a clean bench and placed in liquid induction medium (1 / 10 Linsmaier and Skoog (LS) salts, 1 / 10 Murashige and Skoog (MS) vitamins, 10 g / L glucose, 0.5 g / L MES 2-(N-morpholino)ethanesulfonic acid, 100 mM acetylsyl syringone) for 5 min for later use. The embryos were then collected by centrifugation.

[0138] (b) Agrobacterium infection: The constructed plasmid was transformed into Agrobacterium AGL1, and fresh Agrobacterium (OD=0.6) infection solution was used to infect wheat embryos;

[0139] (c) Callus induction, selection, and regeneration: Infected embryos were successively transferred to callus induction medium (LS salts, MS vitamins, 0.5 mg / L 2,4-D, 2.2 mg / L picloram, 0.85 mg / L AgNO3, 100 mg / L ascorbicacid, 250 mg / L carbenicillin, 100 mg / L cefotaxime, 1.95 g / L MES and 5 g agarose) and cultured in the dark at 25°C for 5 days. Callus selection medium 1 (LS salts, MS vitamins, 40 g / L maltose, 0.5 mg / L 2,4-D, 2.2 mg / L thiamethoxam, 0.85 mg / L AgNO3, 100 mg / L L-ascorbic acid, 250 mg / L carbenicillin, 100 mg / L cefotaxime, 5 mg / L glufosinate, 1.95 g / L acetic acid) was then used. MES and 5 g / L agarose) were cultured in the dark at 25°C for 2 weeks; induction and selection medium 2 (LS salts, MS vitamins, 40 g / L maltose, 0.5 mg / L 2,4-D, 2.2 mg / L chlorhexidine, 0.85 mg / L AgNO3, 100 mg / L L-ascorbic acid, 250 mg / L carbenicillin, 100 mg / L cefotaxime, 10 mg / L glufosinate, 1.95 g / L MES and 5 g / L agarose) were cultured in the dark at 25°C for 3 weeks; regeneration medium (LS salts, LS vitamins, 20 g / L sucrose, 0.5 g / L MES, 2.5 mg / L CuSO4·5H2O) was cultured in the dark. O, 250 mg / L carbenicillin, 100 mg / L cefotaxime, 5 mg / L glufosinate and 8 g / L agarose) were incubated at 25°C under 16 hours of light / 8 hours of darkness until the leaves and stems elongated. When the regenerated seedlings were vigorous, they were transferred to rooting medium (LS salts, LS vitamins, 15 g / L sucrose, 0.5 g / L MES, 250 mg / L carbenicillin and 3 g / L plant gel) under 25°C under 16 hours of light / 8 hours of darkness until the roots elongated. After the regenerated seedlings had well-developed roots, they were transplanted to a greenhouse for planting.

[0140] (d) Identification of transgenic plants: DNA and RNA were extracted from leaves, and PCR amplification of the target gene was performed to screen for positive transgenic plants. DNA identification results showed that 13, 21, and 24 independent positive transgenic plants were obtained for the three plasmids SVpc337, SVpc348, and SVpc351, respectively. RT-PCR detection results showed that in 7 of the 13 SVpc337 positive plants, the expression of the Ms2 gene was detected in detached leaves after heat shock treatment (38℃, 3h); in 16 of the 24 SVpc351 positive plants, the expression of the Ms2 gene was detected in detached leaves after heat shock treatment (38℃, 3h).

[0141] This section shows the growth status of eight T0 generation SVpc351 positive plants (5583, 5584, 5585, 5592, 5593, 5594, 5612, and 5613) and two T0 generation SVpc348 control positive plants (5470 and 5471). Figure 3 A). To detect heat-induced expression of the Ms2 gene, leaves from 8 T0 generation SVpc351 positive plants and 1 T0 generation SVpc348 positive plant were taken, heat-shocked at 38℃ for 3 h, and RNA was extracted. RT-PCR was performed targeting the Ms2 gene. Ms2 gene expression was detected in 6 T0 generation transgenic plants, while no Ms2 gene expression was detected in 5583, 5585, 5470 (SVpc348 positive plants) and wild-type Fielder plants. Figure 3 B).

[0142] Example 4: Ms2 gene expression inhibits wheat seedling growth

[0143] The effects of the Ms2 gene on wheat seedling growth and development were tested using the T1 generation transgenic plants 5593(SVpc351), 5594(SVpc351), 5651(SVpc337), 5677(SVpc337), 5471(SVpc348), 5565(SVpc348) constructed in Example 3 and Fielder.

[0144] Five days after germination of the above-mentioned transgenic seeds until the first leaf unfolded, a control group (25℃, Control) and a treatment group (36℃, 2h, Treatment) were set up. Figure 4After heat shock, the treatment groups were placed at 25℃ for 3 days, and the growth of wheat seedlings from different lines and treatment groups was observed and statistically analyzed. The results showed significant differences in the number of lateral roots, root tip color, and root hair growth among different lines and treatment groups. For the two Ms2 transgenic lines SVpc351 and SVpc337, the number of lateral roots in the treatment groups was significantly less than that in the control group; for the two Ms2 transgenic lines SVpc348 and Fielder, there was no significant difference in the number of lateral roots between the treatment and control groups. Figure 4 A, Figure 5 In the treatment groups, compared with SVpc348 and Fielder, the number of lateral roots in SVpc351 and SVpc337 was significantly reduced, while there was no significant difference in the number of lateral roots among the lines in the control group. Figure 4 A, Figure 5 In the treatment groups, the root tips of lines SVpc351 and SVpc337 showed fewer root hairs and inhibited growth (local shrinkage) compared to SVpc348 and Fielder, accompanied by browning. Figure 4 B). The above results indicate that the Ms2 gene has an inhibitory effect on the normal division and / or growth of wheat seedling root cells.

[0145] To further test the effects of the Ms2 gene on wheat coleoptile elongation and seedling growth, T1 generation transgenic lines of SVpc351 and SVpc348 were selected. Figure 6 Among them, strain 5583 (SVpc351) served as the transgenic negative control. Two days after seed germination (coleoptile length approximately 5 mm), control (25℃, Control) and treatment (38℃, 2h, Treatment) groups were established. The treatment groups were placed at 25℃ after heat shock. Two days after treatment, the seedlings of the 5594 (SVpc351) and 5626 (SVpc351) treatment groups showed significantly weaker growth than the control group. Figure 6 A), while the control and treatment groups of 5470(SVpc348), 5471(SVpc348) and Fielder showed no significant differences. Three days after heat shock treatment, plant height and coleoptile length were statistically analyzed for different lines. The seedling height and coleoptile length of the 5594(SVpc351) and 5626(SVpc351) treatment groups were significantly higher. Figure 6 B) Both were significantly lower than their own control group and the control and treatment groups of other strains. These results indicate that Ms2 gene expression inhibits normal cell division and / or growth in wheat coleoptiles.

[0146] Example 5: Transient expression of the Ms2 gene inhibits normal division and / or growth of wheat callus cells

[0147] To investigate the effect of the Ms2 gene on wheat callus growth, Agrobacterium infection and callus induction experiments were conducted on wheat Fielder embryos using SVpc348 and SVpc351 (tissue culture method as described in Example 3, step ac). Embryos infected with SVpc348 and SVpc351 were transferred to "Callus Induction Selection Medium 1" (same as in Example 3) and subjected to heat shock treatment (38℃, 3h), followed by dark culture at 25℃. Callus growth was photographed and recorded at 0dah, 2dah, 4dah, 7dah, and 10dah (dah = day after heat-shock), and the proportion of embryogenic callus tissue was calculated. Figure 7 AB); the size of callus tissue infected with different plasmids was quantitatively analyzed at 10 dah. Figure 7 C). The results showed that at 10 dah, the proportion of embryogenic callus corresponding to SVpc351 was significantly lower than that of SVpc348, decreasing by approximately 33%. Figure 7 B); Compared with the callus corresponding to SVpc348, SVpc351 leads to weak viability, lack of obvious differentiation, and inhibited cell division and / or growth in infected callus. Figure 7 A). The above results indicate that Ms2 gene expression inhibits the normal division and / or growth of wheat callus cells.

[0148] Example 6: Ms2 protein inhibits normal division and / or growth of tobacco cells

[0149] Stable genetic transformation of tobacco involved five plasmids from Example 1: SVpc337, SVpc348, SVpc351, SVpc369, and SVpc375, of which SVpc337, SVpc348, and SVpc351 were driven by the heat shock promoter Lehsp23.8, and SVpc369 and SVpc375 were driven by the 35S promoter.

[0150] Genetic transformation of tobacco was performed using Agrobacterium-mediated leaf disc transformation. During the transformation process, compared to the control plasmid SVpc348 (Lehsp23.8::GFP), genetically transformed plants associated with SVpc351 (Lehsp23.8::cMs2:GFP) showed inhibited rooting in rooting medium (30 g / L sucrose, 4.405 g / L MS, 6 g / L agarose, 300 mg / L cefotaxime, 8 mg / L PPT). Figure 8A). Literature reports indicate that Lehsp23.8 is slightly leaked in tomato root tips under normal temperature conditions (Yi et al., 2006, Plant Science, 171:398-407). Tobacco and tomato belong to the Solanaceae family, and the Ms2 gene may be slightly expressed in tobacco roots, leading to rooting inhibition in SVpc351 transformed plants. Therefore, the Ms2 gene exhibits cytotoxicity in tobacco root cells, inhibiting normal root cell division and / or growth.

[0151] To detect whether Ms2 protein was successfully expressed in tobacco, transgenic plants of SVpc348 and SVpc351, as well as wild-type non-transgenic controls (WT), were subjected to heat shock treatment at 38℃ for 3 h, with a room temperature group serving as a control. After treatment, leaf samples were collected from each plant in the heat shock groups (SVpc351-H, SVpc348-H, and WT-H) and the room temperature groups (SVpc351, SVpc348, and WT) to prepare protein samples, which were then detected by Western blot using a GFP antibody. The results showed that after heat shock treatment, Ms2-GFP protein (60 kDa) was successfully expressed in the SVpc351 transgenic plants. Figure 8 B), GFP was highly expressed in SVpc348 transgenic plants; while in the normal temperature group, SVpc351 and SVpc348 transgenic plants showed partial leakage ( Figure 8 B). During the genetic transformation of tobacco using two 35S-driven plasmids, SVpc369 (35S::GFP) and SVpc375 (35S::cMs2:GFP), tobacco leaves infected with Agrobacterium SVpc375 showed a browning phenotype compared to the control SVpc369, with no new bud differentiation observed. Figure 8 C). The above results indicate that the expression of the Ms2 gene has an inhibitory effect on the normal division and / or growth of tobacco leaf cells.

[0152] Example 7: Ms2 gene expression inhibits normal cell division and / or growth in animal cells

[0153] To test the toxicity of the Ms2 gene to animal cells, transient transformations were performed on the human kidney epithelial cell line (293) and the human liver cancer cell line (huh7), using plasmids SVpc419 and SVpc421, with the animal constitutive promoter CMV. The Ms2 gene was incorporated into the SVpc421 plasmid. # (Ms2 animal optimized sequence) gene C-terminus fused with eGFP fluorescent protein; SVpc419 does not contain Ms2. # The gene serves as a strict control for SVpc421. The above plasmid was transfected into the 293 and huh7 cell lines, respectively. The transfection steps are as follows:

[0154] 1. Preheat the water bath to 37°C; remove the recipient cells from the liquid nitrogen tank or refrigerator and quickly place them into the preheated water bath. Use tweezers to hold the cryovial and shake it to ensure even heating.

[0155] 2. When the cryovial is completely thawed, centrifuge at 4°C for 5 minutes, remove the supernatant, and keep the precipitate;

[0156] 3. Suspend the precipitate in 1 ml of 90% DMEM + 10% FBS complete medium and inoculate it into a culture dish containing 9 ml of complete medium. Mix thoroughly in a cross-hatching manner and incubate in a CO2 incubator. Subculture 3-4 times to ensure that the cells are in good condition and free from contamination.

[0157] 4. On the day of transfection, plate the cells, remove the culture medium from the 10cm plate, wash away the excess serum with PBS, add 1-2ml of trypsin to digest for 30s, add 10ml of culture medium to wash off the adherent cells and mix well.

[0158] 5. Take 10 μL of cell suspension and count the cells using a cell counting chamber. For a 6-well plate, distribute 10,000 cells per well and dispense 1 ml of cell suspension per well. Incubate in a CO2 incubator.

[0159] 6. After approximately 4-6 hours, when cells have fully adhered to the culture dish, prepare for transfection. Aspirate the culture medium from the dish and replace it with serum-free medium. Prepare the transfection solution using sterile EP tubes: Solution A = 0.5 μg plasmid diluted in 200 μL DMEM; Solution B = 1.5 μL PEI diluted in 200 μL DMEM. Gently mix Solutions A and B separately, let stand for 5 minutes, then add Solution B to Solution A, gently mix, and let stand at room temperature for 20 minutes. Add the transfection reagent to the culture medium in each well, mix in a crosswise direction, and incubate overnight. Replace with complete culture medium for further culture.

[0160] Images were taken using a conventional fluorescence microscope at 24h, 48h, 72h, and 96h post-transfection. At 96h, images were taken using a confocal microscope. The fluorescence rate of SVpc419 and SVpc421 transfected 293 cells was detected using flow cytometry. Three replicates were set up for each plasmid, with 10,000 cells counted in each replicate. Untransfected 293 cells were used as a blank control.

[0161] The results showed that in 293 transfections ( Figure 9 A) and huh7 Figure 9 B) At 72h and 96h, the GFP fluorescence rate of SVpc421 transfected cells was significantly lower than that of SVpc419. Figure 9-10Cell flow cytometry results after 96 hours of transfection into the 293 cell line showed that 12.96% of the cells corresponding to SVpc421 exhibited fluorescence in more than 30,000 cells tested, significantly lower than the average fluorescence rate of cells corresponding to SVpc419 (31.6%). Figure 10 B). Cells successfully transfected with SVpc421 showed reduced green fluorescence area, appearing as punctate condensations, and were unable to divide normally to form new cells; while cells successfully transfected with SVpc419 had normal morphology, with green fluorescence filling the entire cell. Figure 9-10 The 293 and huh7 cell lines showed similar results. Therefore, Ms2 gene expression is toxic to animal cells, inhibiting normal cell division and / or growth.

[0162] Example 8: Ms2 protein inhibits Fusarium graminearum cell division and / or growth

[0163] To test the antibacterial activity of the Ms2 protein, prokaryotic expression plasmids SVpc398, SVpc399, and SVpc432 were transformed into Escherichia coli DE3 strain suitable for expressing the virulence protein. After induction of expression, protein identification and antibacterial tests were performed. The specific steps are as follows:

[0164] 1. Take a positive single clone and place it in 20 ml of LB liquid containing ampicillin. Incubate overnight at 37°C with gentle shaking at 200 rpm.

[0165] 2. Take 1 ml of bacterial culture and add it to 100 ml of LB liquid containing ampicillin. Continue culturing until the OD value is 0.6-1.

[0166] 3. Add 110 μL of 1M IPTG and incubate at 37°C and 200 rpm for 5 hours.

[0167] 4. Adjust the OD value of the bacterial culture to a uniform value, take 200 μL and spread it evenly on PDA medium, with 10 replicates for each plasmid, and incubate overnight at 37°C.

[0168] 5. Collect conidia of Fusarium graminearum PH-1 and adjust the concentration to 1×10⁻⁶. 6 50 μL of conidia were added to the center of PDA medium and cultured at 25°C for 3 days. The growth of Fusarium graminearum in PH-1 was then observed.

[0169] 6. Collect the bacterial cell pellets induced in step 3 and wash them three times with pre-cooled 1×PBS;

[0170] 7. Resuspend the bacterial pellet in 20 mL of pre-chilled 1×PBS solution, then add 200 μL PMSF and 40 μL LTT, sonicate on ice for 30 minutes, centrifuge at 12000 rpm for 5 minutes at 4°C, and discard the supernatant; wash the bacterial pellet three times with pre-chilled 1×PBS.

[0171] 8. Repeat step 7, add 20 mL of pre-cooled 1×PBS solution to resuspend the bacterial culture, then add 200 μl PMSF, 40 μl DTT, and 20 mL 2×SDS protein loading buffer, mix well, boil for 10 minutes, and store at -20℃.

[0172] 9. Take 15 μL of sample for Coomassie brilliant blue staining and verify with Ms2 antibody using Western blot.

[0173] The results showed that, 5 hours after IPTG induction, Coomassie brilliant blue staining revealed high levels of expression of GST (26 KD) corresponding to SVpc398 and GST-Ms2 (55 KD) corresponding to SVpc399. Figure 11 A), but the Ms2 (29KD) protein corresponding to SVpc432 was not expressed in large quantities (only an unknown protein <26KD was induced); Figure 11 A). However, Western blot analysis confirmed that the GST-Ms2 protein corresponding to SVpc399 was normally expressed, while the Ms2 protein corresponding to SVpc432 was expressed in small amounts. Figure 11 B).

[0174] The antibacterial experiment on Fusarium graminearum PH-1 showed that, on a culture medium containing the bacteria corresponding to SVpc432, the bacterial plaques of Fusarium graminearum PH-1 (average diameter 2.1 cm) were significantly smaller than those of the corresponding bacteria SVpc398 (average diameter 2.9 cm) and SVpc399 (average diameter 3.1 cm). Figure 12 Furthermore, on the culture medium corresponding to SVpc432, Fusarium graminearum plaques were mostly white or yellow, while the Fusarium graminearum plaques corresponding to SVpc398 and SVpc399 were mostly red. Figure 12 A) It is speculated that the former (SVpc432) slows down the growth, physiology, and maturation of Fusarium graminearum. Therefore, the Ms2 protein expressed in prokaryotes has an inhibitory effect on the normal division and / or growth of Fusarium graminearum cells.

[0175] Example 9: Effects of Ms2 protein on yeast cell division and / or growth

[0176] Yeast is a single-celled eukaryotic microorganism. To study the effect of the Ms2 protein on yeast growth, the galactose-inducible promoter (GAL1) was used to drive GFP (SVpc441), cMs2 (SVpc438), cMs2-GFP (SVpc443), GFP-cMs2 (SVpc529), and cMs2... C158T -GFP(SVpc530) expression, and the constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase (TDH3) driving GFP(SVpc678), cMs2(SVpc677), cMs2-GFP(SVpc679), GFP-cMs2(SVpc680) and cMs2 C158T -GFP (SVpc681) expression was performed using the pYES2 expression vector, and the plasmid was transformed into the Saccharomyces cerevisiae strain INVSC1. Yeast transformation followed the Clontech yeast transformation method. After transformation, the bacterial culture was plated onto SC-U (for the GAL1 promoter-related vector) and SC-U (for the TDH3 promoter-related vector) solid medium plates containing 2% glucose, and incubated at 30°C for 2-3 days. After positive clones appeared, protein expression was induced, and fluorescence and successful protein expression were detected. The effects of different Ms2 protein types on yeast growth were also examined. The specific methods are as follows:

[0177] 1. Take a positive yeast clone and place it in 50 mL of SC-U (induction medium) or SC-U liquid medium (non-induction medium) containing 2% glucose, and incubate overnight at 30℃ and 200 rpm.

[0178] 2. Take the overnight culture medium, centrifuge at 3000 rpm for 5 min at 4℃, collect the bacterial cells, sterilize and wash three times with water to completely remove the culture medium from the previous step;

[0179] 3. Discard the supernatant, resuspend the bacterial cells in 10-20 mL of induction / non-induction medium, transfer them to 150 mL of medium, adjust the initial OD600 to 0.4, and induce protein expression at 30℃ and 200 rpm.

[0180] 4. Measure the OD600 values ​​at different times after induction (0h, 4h, 8h, 12h, 24h, 36h, 48h) and plot the yeast growth curve.

[0181] 5. After inducing protein expression, centrifuge at 3000 rpm for 5 min at 4℃, discard the supernatant, and use the bacterial cells for protein extraction;

[0182] 6. Resuspend the yeast cells in 500 μL of lysis buffer (50 mmol / L sodium phosphate, pH 7.4; 1 mmol / L EDTA; 5% glycerol; 1 mmol / L LMSF), centrifuge at 3000 rpm for 5 min at 4 °C, and discard the supernatant;

[0183] 7. Add an equal volume of glass beads (0.4-0.6 mm, Sigma), vigorously vortex for 30 seconds, then place on ice for 30 seconds; repeat 4 times until all cells are lysed;

[0184] 8. Collect the supernatant, add an equal volume of SDS sample buffer, and boil for 5 minutes; it is then ready for SDS-PAGE electrophoresis and Western blot detection.

[0185] The results showed that, after different induction times, the OD values ​​of different GAL1-driven expression vectors varied. 600 The values ​​showed no significant difference and all conformed to the normal growth pattern of yeast. Figure 13 A). We also examined the expression of different vectors driven by the constitutive promoter TDH3. Similar to GAL1, there was no significant difference in the growth trends among the vectors driven by TDH3. Figure 13 B). Twelve hours after yeast expression, we detected green fluorescence and protein signals in yeast lines carrying GFP (SVpc678), Ms2-GFP (SVpc679), or GFP-Ms2 (SVpc680). The results showed that strong green fluorescence was detected in the TDH3::GFP strain, weaker green fluorescence in the TDH3::GFP:cMs2 strain, and no green fluorescence was detected in the TDH3::cMs2:GFP strain. Figure 13 C). Western blotting using GFP antibody showed a strong signal in the control plasmid GFP strain; a weak signal in the plasmid GFP:Ms2 strain; and only a very weak signal in the plasmid Ms2:GFP strain. Figure 13 D). In conclusion, the Ms2 protein may not be cytotoxic in yeast, and the reason for the difference between green fluorescence and protein signal is still unclear.

[0186] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of wheat Ms2 protein as a toxic protein in the following (1) or (2): (1) Inhibits cell division and / or growth; (2) Prepare products that inhibit cell division and / or growth; The wheat Ms2 protein is the protein shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in sequence 5 of the sequence listing; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. The application according to claim 1, characterized in that, The cells are plant cells, animal cells, and eukaryotic microbial cells.

3. The application of wheat Ms2 protein or the gene encoding wheat Ms2 protein in the preparation of disease-resistant plant products; wherein the wheat Ms2 protein is the protein shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in sequence 5 of the sequence listing; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

4. The application according to claim 3, characterized in that, The gene encoding the wheat Ms2 protein is any one of the following nucleic acid molecules (i)-(iv): (i) The nucleic acid molecule shown in sequence 1 of the sequence listing; (ii) The nucleic acid molecule shown in sequence 3 of the sequence listing; (iii) The nucleic acid molecule shown in sequence 4 of the sequence listing; (iv) Nucleic acid molecules with the amino acid sequences shown in Figure 5, excluding (i)-(iii).

5. The application of wheat Ms2 protein or the gene encoding wheat Ms2 protein in the preparation of animal cell therapeutic drugs; wherein the wheat Ms2 protein is a protein as shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in sequence 5 of the sequence listing; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

6. The application according to claim 5, characterized in that, The animal cell therapy drug is a tumor therapy drug.

7. Application of wheat Ms2 protein in the preparation of products for killing harmful microorganisms; wherein the wheat Ms2 protein is a protein as shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in sequence 5 of the sequence listing; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

8. The application according to claim 7, characterized in that, The products for killing harmful microorganisms include fungicides.

9. Application of wheat Ms2 protein in the preparation of insecticides, wherein the wheat Ms2 protein is the protein shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in sequence 5 of the sequence listing; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

10. The application of expression cassettes, recombinant plasmids, or recombinant bacteria containing the wheat Ms2 protein-coding gene in any of the following (1)-(5): (1) Inhibits cell division and / or growth; (2) Prepare products that inhibit cell division and / or growth; (3) Preparation of tumor treatment drugs; (4) Preparation of bactericides; (5) Preparation of insecticides.

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