Use of wheat ms2 protein in broad-spectrum inhibition of eukaryotic cell division and / or growth

Abstract

The present invention relates to the use of a wheat Ms2 protein in broad-spectrum inhibition of eukaryotic cell division and / or growth, and belongs to the technical field of genetic engineering. The present invention relates to a target protein Ms2, wherein a corresponding wheat CDs sequence thereof is as shown in sequence 1, and a corresponding amino acid sequence thereof is as shown in sequence 5. It is demonstrated for the first time using a transient or stable genetic transformation technique that the Ms2 protein is toxic to plant, animal and fungal cells, and inhibits cell division and / or growth thereof, thus providing theoretical support for the use of the Ms2 protein in disease resistance and gene therapy in animals and plants.

Description

Use of wheat Ms2 protein to inhibit eukaryotic cell division and / or growth

[0001] Cross-reference to Related Applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410598489.0, filed on May 15, 2024, and Chinese Patent Application No. 202510339029.0, filed on March 21, 2025, the contents of both of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of genetic engineering, and in particular to the use of wheat Ms2 protein to inhibit animal, plant and fungal cell division and / or growth. BACKGROUND

[0004] The basic unit of organisms in nature is cells, and most active cells are in a dynamic balance of cell cycle of continuous division, growth and differentiation, maturation and aging. During cell development, some cells begin to degenerate before they are fully formed, some cells reach the upper limit of the number of divisions and begin to degenerate, and some cells that are damaged and cannot be repaired also degenerate. Cell degeneration in normal development is a kind of active and orderly process regulated by genes under natural conditions, and is accompanied by the growth and development of organisms. In animals, degenerating cells are located in normal tissues and will be cleared by macrophages or adjacent cells, without affecting the normal function of adjacent cells. In plants, degenerating cells will not be phagocytosed and will become part of the plant body.

[0005] Accurate regulation of cell division and replication cycle is the key for plants to achieve normal growth and development. In plants, cell cycle is the main factor affecting the activity and growth rate of meristems. 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 cell cycle of eukaryotes requires the synergistic regulation of proteins such as cell cycle-dependent kinase (CDK), cyclin, cyclin-dependent kinase inhibitor (KRP), retinoblastoma gene 1 (RB1), and cyclin-dependent kinase inhibitor gene (CKI) (Nigg, 1995; Novak et al., 1998). Cyclins can be divided into positive and negative regulatory proteins according to their functions. The positive regulatory proteins are Cyclin and CDK. Cyclin combines with CDK to form a complex, participates in DNA replication, chromosome separation and other events, and controls cell cycle progression, 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 hindering DNA replication and other events (Kumar et al., 2018; Wang et al., 2020).

[0006] Cytotoxic proteins can directly or indirectly cause cell cycle abnormalities, leading to cell division obstruction, slow growth, and even non-natural cell degeneration. According to the source, they can be divided into endogenous and exogenous cytotoxic proteins. Endogenous cytotoxic proteins are mainly produced by cells themselves and are involved in cell division, growth, and degeneration. For example, tau toxic protein oligomers can induce the nuclear lamina of neurons to collapse, thereby damaging the nucleocytoplasmic transport, and ultimately leading to neuronal damage (Sun et al., 2024). Exogenous cytotoxic proteins are derived from bacteria, viruses, and other microorganisms, and have the functions of killing bacteria and resisting viruses. Cytotoxic proteins inhibit cell division, growth, or cause degeneration through various mechanisms: 1) directly acting on the cell membrane or organelle membrane, destroying the integrity of the membrane components; for example, BCL2-Associated X (BAX) protein can be converted from a cytosolic monomer to a toxic oligomer that can penetrate the outer membrane of mitochondria, thereby disrupting body development, tissue homeostasis, and immune regulation, causing diseases such as tumors, autoimmune diseases, neurodegenerative diseases, and heart failure (Hauseman et al., 2020); 2) causing DNA damage, leading to genetic mutations and cell abnormalities, such as aflatoxin B1 (AFB1) after metabolic conversion in the body, binds to DNA to form AFB1-DNA polymer, causing severe DNA damage, and has strong toxicity to the liver of humans and animals (Shirabe et al., 2011); 3) interfering with intracellular signal transduction pathways, affecting the normal physiological functions of cells, such as ricin and Shiga toxin, which can inhibit ribosome function and interfere with protein synthesis; 4) inhibiting the expression of cell proliferation-related genes, inhibiting cell division, such as human cell cycle CKI inhibitor protein p27 and HIV-1 viral protein R (Vpr); 5) directly activating degeneration-related proteins or inhibiting anti-degeneration-related proteins, leading to cell degeneration. Some cytotoxic proteins inhibit cell cycle, leading to tumor cell growth arrest or degeneration, such as 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 and play a role in the treatment of animal autoimmune diseases, transplant rejection, etc. Some cytotoxic proteins inhibit the growth of plant host cells to suppress the activity of invading pathogenic microorganisms, thereby achieving plant disease resistance applications, such as C12 family proteases (VPEs) and C14 family proteases (MCs). Therefore, it is of great significance to explore new cytotoxic proteins for the prevention and control of animals, plants, and fungal diseases.

[0007] Ni et al. (2017) cloned a male sterility gene Ms2 from wheat, and found that Ms2 gene only expressed in anther tissue of wheat, and its expression resulted in male sterility phenotype of wheat, which had great application value in the field of developing hybrid wheat. As a male sterility gene with excellent performance, Ms2 gene needs to be further developed for new functions. SUMMARY

[0008] The present application first found that Ms2 protein has cytotoxicity to wheat cells, which directly or indirectly inhibits the division and / or growth of wheat cells. More importantly, wheat Ms2 protein shows broad-spectrum cytotoxicity in inhibiting the division and / or growth of animal, plant and fungal cells, which can be widely used in the fields of tumor treatment, immune regulation, plant disease and pest resistance and fungal control in the future.

[0009] The purpose of the present application is to apply the cytotoxicity function of wheat Ms2 protein to inhibit the division and / or growth of eukaryotic cells.

[0010] To achieve the above purpose, the present application adopts the following technical solutions:

[0011] In a first aspect of the present application, wheat Ms2 protein is provided as a toxic protein for use in (1) or (2) as follows:

[0012] (1) inhibiting cell division and / or growth;

[0013] (2) preparing a product for inhibiting cell division and / or growth;

[0014] The wheat Ms2 protein is a protein as shown in (A1) or (A2) as follows:

[0015] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing;

[0016] (A2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0017] In the above use, in order to facilitate the purification of the protein in (A1), a molecular tag can be connected to the amino-terminal or carboxyl-terminal end of the protein in (A1). The tag can be Poly-Arg (usually 6 RRRRR), Poly-His (usually 6 HHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK) or c-myc (EQKLISEEDL).

[0018] In the above use, the cell is a plant cell, an animal cell or a fungal cell.

[0019] In a second aspect of the present application, the wheat Ms2 protein or the coding gene of the wheat Ms2 protein is used in the preparation of a plant product resistant to diseases.

[0020] Preferably, the coding gene of the wheat Ms2 protein is a nucleic acid molecule as shown in any one of (i)-(iv) below:

[0021] (i) the nucleic acid molecule as shown in SEQ ID NO: 1 in the sequence listing;

[0022] (ii) the nucleic acid molecule as shown in SEQ ID NO: 3 in the sequence listing;

[0023] (iii) the nucleic acid molecule as shown in SEQ ID NO: 4 in the sequence listing;

[0024] (iv) a nucleic acid molecule other than (i)-(iii) encoding the amino acid sequence as shown in SEQ ID NO: 5.

[0025] In the above use, the wheat Ms2 protein is a toxic protein that can inhibit the division and / or growth of cells. Therefore, the coding gene of the wheat Ms2 protein can be connected with an inducible promoter and introduced into a plant, and when the plant is attacked by a pathogenic fungus, the inducible promoter can receive a signal to cause the wheat Ms2 protein to be expressed in the cells of the plant attacked by the pathogenic fungus, thereby inhibiting the division and / or growth of the cells, so that the pathogenic fungus loses the "soil" for proliferation, and the plant achieves the effect of resisting diseases.

[0026] In a third aspect of the present application, the wheat Ms2 protein or the coding gene of the wheat Ms2 protein is used in the preparation of an animal cell therapy drug. The animal cell therapy drug is preferably a tumor therapy drug.

[0027] In the above use, the wheat Ms2 protein or the coding gene of the wheat Ms2 protein is expressed in animal cells, thereby inhibiting the division and / or growth of the animal cells; for example, it can be used to inhibit the division and / or growth of tumor cells, thereby achieving the effect of tumor therapy.

[0028] In a fourth aspect of the present application, the wheat Ms2 protein is used in the preparation of a product for killing harmful microorganisms.

[0029] In the above use, the wheat Ms2 protein can inhibit the division and / or growth of fungal cells, and therefore the 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 a fungal fungicide and the like.

[0030] In the above use, the fungal fungicide includes but is not limited to a Fusarium graminearum fungicide, a yeast fungicide, a Puccinia striiformis fungicide, and a powdery mildew fungicide.

[0031] In a fifth aspect of the present application, the wheat Ms2 protein is used in the preparation of a pesticide.

[0032] In the above use, the wheat Ms2 protein is administered orally or by injection, and the wheat Ms2 protein is used to inhibit the cell division and / or growth, thereby achieving the insecticidal effect.

[0033] In the above use, the insecticidal targets include, but are not limited to, aphids, cotton bollworms, rice planthoppers, and corn borers.

[0034] In a sixth aspect of the present application, the expression cassette, recombinant plasmid or recombinant bacteria containing the wheat Ms2 protein coding gene are used in any one of the following (1)-(5):

[0035] (1) inhibiting cell division and / or growth;

[0036] (2) preparing a product for inhibiting cell division and / or growth;

[0037] (3) preparing a tumor treatment drug;

[0038] (4) preparing a fungicide;

[0039] (5) preparing a pesticide.

[0040] In the above use, the nucleotide sequence of the wheat Ms2 protein coding gene is shown in any one of SEQ ID NOs. 1, 2, 3 and 4 in the sequence listing.

[0041] Advantages of the present application:

[0042] The present application has found that the wheat Ms2 protein has cytotoxicity and can inhibit the cell division and / or growth of plant cells, animal cells and fungal cells. The toxicity of the wheat Ms2 protein can be used in the fields of plant and animal disease resistance, gene therapy and fungal control. BRIEF DESCRIPTION OF DRAWINGS

[0043] Fig. 1 is a schematic diagram of the partial plasmid structure.

[0044] Fig. 2 is the mitochondrial localization of the wheat Ms2 protein.

[0045] Fig. 3 is the heat-induced expression of the transgenic wheat and Ms2 gene. In the figure, A is a transgenic wheat plant; and B is the PCR detection of the heat-induced transgenic expression.

[0046] Fig. 4 is the heat-induced expression of Ms2 in the transgenic wheat roots, which causes the inhibition of the cell division and / or growth of the root cells. In the figure, A is the number of lateral roots after heat induction; and B is the color of the root tips after heat induction.

[0047] Fig. 5 is the heat-induced expression of Ms2 in the transgenic wheat roots, which causes the inhibition of the cell division and / or growth of the lateral root cells.

[0048] Figure 6: Heat-induced expression of Ms2 in transgenic wheat 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.

[0049] 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 after heat induction; B represents the proportion of embryogenic callus after heat induction; and C represents the size of callus after heat induction.

[0050] Figure 8: Heat-induced expression of Ms2 in transgenic tobacco 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 after infection with Agrobacterium carrying the target plasmid.

[0051] Figure 9: Ms2 gene transfection in animal cells inhibits cell division and / or growth after 72 hours; in the figure, A represents transfection with 293; B represents transfection with huh7.

[0052] Figure 10: Ms2 gene transfection of 293 cell line inhibits cell division and / or growth 96 h; 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.

[0053] 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.

[0054] 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 plaques; B represents the statistical analysis of Gibberella fuciformis plaque diameters.

[0055] Figure 13: Effects 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. 600 Values; 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

[0056] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the application. Unless otherwise defined, 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 application belongs.

[0057] To reveal the function of wheat Ms2 protein, the present application uses heat shock protein gene promoters Lehsp23.8, 35S promoter, Rp27 promoter and CMV promoter to construct related plasmids of wheat Ms2 gene, and uses transient and stable genetic transformation technology to clarify the cytotoxicity of Ms2 protein to plants, animals and fungi.

[0058] The present application constructs 25 kinds of plasmids for 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 * is the sequence optimized for fungi (Gibberella); cMs2 #The sequence in animals is codon-optimized; the Ms2 gene in SVpc530 and SVpc681 is implanted with a sense point mutation, and the mutation site of the cDNA is C158T. The plasmids SVpc337, SVpc348, SVpc350 and SVpc351 are driven by the heat shock promoter Lehsp23.8, and are used for genetic transformation of wheat and tobacco. The plasmids SVpc369, SVpc375, SVpc391 and SVpc424 are driven by the 35S promoter, and are used for stable or transient transformation of tobacco. The plasmids SVpc398, SVpc399 and SVpc432 are used for prokaryotic expression, and are induced by IPTG for expression, and are used for prokaryotic expression and inhibition experiment of Gibberella. The plasmids SVpc437, SVpc438, SVpc441, SVpc443, SVpc529 and SVpc530 are driven by the GAL1 promoter, and are used for transformation of yeast; the plasmids SVpc677, SVpc678, SVpc679, SVpc680 and SVpc681 are driven by the TDH3 promoter, and are used for transformation of yeast. The plasmids SVpc419, SVpc420 and SVpc421 are driven by the CMV promoter, and are used for transient transformation of animal cells.

[0059] The present application proves that the wheat Ms2 protein has cytotoxicity to animals, plants and fungal organisms, and can inhibit the division and / or growth of animal, plant and fungal cells.

[0060] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0061] The test materials used in the examples of the present application are all conventional test materials in the art, and can be purchased through commercial channels. The experimental methods not specified in detail are carried out according to the conventional test methods or according to the operation instructions recommended by the suppliers.

[0062] Example 1: Construction of wheat Ms2 gene related plasmids

[0063] The genetic transformation plasmids involved in the present application include: SVpc337, SVpc348, SVpc350, SVpc351 and 25 plasmids in total (Table 1, Figure 1). The plasmids SVpc337, SVpc348, SVpc350 and SVpc351 all use tomato heat shock promoter Lehsp23.8 (1611bp) to drive the expression of target genes (Yi et al., 2006, Plant Science, 171:398-407), which is a heat shock inducible promoter that starts the expression of downstream genes at high temperature of 38-39°C, and is used for genetic transformation of wheat and tobacco. The plasmids SVpc369, SVpc375, SVpc391 and SVpc424 use 35S promoter to drive the expression of downstream target genes, and are used for stable or transient transformation of tobacco. The plasmids SVpc419, SVpc420 and SVpc421 use CMV promoter to drive the expression of downstream target genes, and are used for transient transformation of animal cells. The plasmids SVpc398, SVpc399 and SVpc432 are prokaryotic expression plasmids, and are induced to express by IPTG, and are used for prokaryotic expression and antibacterial experiment of Gibberella. The plasmids SVpc437, SVpc438, SVpc441, SVpc443, SVpc529 and SVpc530 use GAL1 promoter to drive the expression of downstream target genes, and are used for transformation of yeast. The plasmids SVpc677, SVpc678, SVpc679, SVpc680 and SVpc681 use TDH3 promoter to drive the expression of downstream target genes, and are used for transformation of yeast. The Ms2 gene in SVpc530S and Vpc681 carries a point mutation, and the cDNA mutation site is C158T.

[0064] Obtaining of wild type cMs2 gene (sequence 1): PCR amplification from 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', and the cDNA template is from LM15 RMs2 Young ear at S2 stage. The 3' end of cMs2 gene in plasmid SVpc350 is removed by 432bp, which is a half sequence of cMs2 gene (sequence 2); the cMs2 * (sequence 3) is an optimized codon sequence suitable for fungal expression, which is synthesized by commercialization (Shenguo Bioengineering); the cMs2 # (sequence 4) is an optimized codon sequence suitable for animal expression, which is synthesized by commercialization (Weizhen Bioengineering), and after optimization, it still encodes the same Ms2 protein (sequence 5).

[0065] Construction of genetic transformation plasmid: linearized vector skeleton was obtained by enzyme digestion or PCR, homologous arms were introduced at both ends of the insert by PCR, and the gene fragment was connected with the linearized vector skeleton by In-Fusion cloning. The vector skeleton information, enzyme digestion sites and homologous arms are shown in Table 2.

[0066] Among them, cMs2 is fused and expressed with 3*Flag in plasmid SVpc337; cMs2 is fused and expressed with GFP in plasmids SVpc351 and SVpc375; cMs2 is fused and expressed with EGFP in plasmids SVpc417 and SVpc421. Plasmid SVpc348 is a strict control of SVpc351; plasmid SVpc369 is a strict control of SVpc375; plasmid SVpc419 is a strict control of SVpc421; plasmid SVpc441 is a strict control of SVpc443; plasmid SVpc678 is a strict control of SVpc679.

[0067] Table 1. Plasmid construction involved in the present application

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

[0069] Table 2. Vector skeleton information, enzyme digestion sites and homologous arms involved in plasmid construction

[0070] Sequence 1: Full-length sequence of cMs2 gene Note: Bold bases are the positions of mutant bases in plasmids SVpc530 and SVpc681

[0071] Sequence 2: Half-length sequence of cMs2 gene

[0072] Sequence 3: Sequence of cMs2 gene after codon optimization in fungi (Gibberella)

[0073] Sequence 4: Sequence of cMs2 gene after codon optimization in animals (cMs2 # )

[0074] Sequence 5: Amino acid sequence of Ms2 protein

[0075] Example 2: Subcellular localization based on Ms2 heat-induced expression

[0076] Subcellular localization of Ms2 protein involved the use of plasmid SVpc351 and mitochondria co-localization plasmid SVpc424 (35S::AtAOX:mCherry). In Arabidopsis, AOX protein is localized in mitochondria (Chris, et al., FEBS Letters, 2008: 582, 3073-3079). After the two plasmids were each introduced into Agrobacterium AGL1 strain, co-infection was performed by injecting tobacco leaves, and after 48 hours of culture, 1-2 leaves were taken for in vitro high temperature treatment (38°C, 2h, dark). After the treatment, localization was observed using a laser confocal microscope. The results showed that Ms2 and AOX were co-localized in mitochondria (Figure 2).

[0077] Example 3: Obtaining of Ms2 transgenic wheat and heat-induced expression of Ms2 gene

[0078] Genetic transformation of wheat used plasmids SVpc337, SVpc348 and SVpc351. The recipient material was wheat Fielder, and the Bar gene was used as a selection gene. The specific operation process included:

[0079] (a) Planting and pretreatment of wheat: the grains of wheat 14 days after flowering (embryos about 1.5-2mm) were selected, surface sterilized with sterilization solution, and then the embryos were peeled in a clean bench and placed in liquid induction medium (1 / 10 Linsmaier and Skoog (LS) salts, 1 / 10 Murashige and Skoog (MS) vitamins, 10g / L glucose, 0.5g / L MES 2-(N-morpholino)ethanesulfonic acid, 100mM acetosyringone) for 5min for standby, and then the embryos were collected by centrifugation;

[0080] (b) Agrobacterium infection: the constructed plasmid was transformed into Agrobacterium AGL1, and fresh Agrobacterium (OD=0.6) infection solution was used to infect the wheat embryos;

[0081] (c) Callus induction, selection and regeneration: The infected proembryos were 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 ascorbic acid, 250 mg / L carbenicillin, 100 mg / L cefotaxime, 1.95 g / L MES and 5 g / L agar) for 5 days in the dark at 25°C, callus selection medium 1 (LS salts, MS vitamins, 40 g / L maltose, 0.5 mg / L 2,4-D, 2.2 mg / L picloram, 0.85 mg / L AgNO3, 100 mg / L L-ascorbic acid, 250 mg / L carbenicillin, 100 mg / L cefotaxime, 5 mg / L phosphinothricin, 1.95 g / L MES and 5 g / L agar) for 2 weeks in the dark at 25°C, callus selection medium 2 (LS salts, MS vitamins, 40 g / L maltose, 0.5 mg / L 2,4-D, 2.2 mg / L picloram, 0.85 mg / L AgNO3, 100 mg / L L-ascorbic acid, 250 mg / L carbenicillin, 100 mg / L cefotaxime, 10 mg / L phosphinothricin, 1.95 g / L MES and 5 g / L agar) for 3 weeks in the dark at 25°C, and regeneration medium (LS salts, LS vitamins, 20 g / L sucrose, 0.5 g / L MES, 2.5 mg / L CuSO4 5H2O, 250 mg / L carbenicillin, 100 mg / L cefotaxime, 5 mg / L phosphinothricin and 8 g / L agar) under 16 hours light / 8 hours dark at 25°C until leaf and shoot elongation, and then 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 phytagel) under 16 hours light / 8 hours dark at 25°C until root elongation, and then transplanted to a greenhouse for growth;

[0082] (d) Identification of transgenic plants: DNA and RNA were extracted from leaves, respectively, and PCR amplification was performed on the target gene to screen positive transgenic plants. DNA identification results showed that 13, 21 and 24 independent transgenic positive plants were obtained for SVpc337, SVpc348 and SVpc351 plasmids, respectively. RT-PCR detection results showed that among the 13 SVpc337 positive plants, 7 plants detected Ms2 gene expression after heat treatment (38°C, 3h) of the in vitro leaves; among the 24 SVpc351 positive plants, 16 plants detected Ms2 gene expression after heat treatment (38°C, 3h) of the in vitro leaves.

[0083] The growth status of 8 T0 generation SVpc351 positive plants (5583, 5584, 5585, 5592, 5593, 5594, 5612 and 5613) and 2 T0 generation SVpc348 control positive plants (5470 and 5471) is shown here (Figure 3A). To detect the heat-induced expression of the Ms2 gene, the leaves of 8 T0 generation SVpc351 positive plants and 1 T0 generation SVpc348 positive plant were heat treated at 38°C for 3h, and RNA was extracted and subjected to RT-PCR for the Ms2 gene. Among them, 6 T0 generation transgenic plants could detect the expression of the Ms2 gene, while 5583, 5585, 5470 (SVpc348 positive plant) and wild type Fielder plants did not detect the expression of the Ms2 gene (Figure 3B).

[0084] Example 4: Ms2 gene expression inhibits the growth of wheat seedlings

[0085] The T1 generation transgenic plants 5593 (SVpc351), 5594 (SVpc351), 5651 (SVpc337), 5677 (SVpc337), 5471 (SVpc348), 5565 (SVpc348) and Fielder constructed in Example 3 were used to test the effect of the Ms2 gene on the growth and development of wheat seedlings.

[0086] The above transgenic seeds were germinated for 5 days to the first leaf unfolded, and control group (25°C, Control) and treatment group (36°C 2h, Treatment) were set up respectively (Figure 4). The treatment group was placed at 25°C after heat shock was completed. After 3 days, the growth of wheat seedlings of different strains and different treatment groups was observed and counted. The results showed that there were obvious differences in the number of lateral roots, root tip color and root hair growth between different strains and different treatment groups. For SVpc351 and SVpc337 strains containing Ms2 transgene, the number of lateral roots of the treatment group was significantly less than that of the control group. For SVpc348 and Fielder strains not containing Ms2 transgene, there was no obvious difference in the number of lateral roots between the treatment group and the control group (Figure 4A, Figure 5). In the treatment group, the number of lateral roots of SVpc351 and SVpc337 was significantly reduced compared with SVpc348 and Fielder, while there was no obvious difference in the number of lateral roots among different strains in the control group (Figure 4A, Figure 5). In the treatment group, the root tips of SVpc351 and SVpc337 strains showed less root hair, growth inhibition (local contraction), and accompanied by browning compared with SVpc348 and Fielder (Figure 4B). The above results showed that Ms2 gene had an inhibitory effect on the normal division and / or growth of wheat seedling root cells.

[0087] In order to further test the effect of Ms2 gene on the elongation of wheat coleoptile and the growth of seedlings, T1 generation transgenic strains of SVpc351 and SVpc348 were selected respectively (Figure 6), and strain 5583 (SVpc351) was a transgenic negative control material. After the seeds were germinated for 2 days (the length of coleoptile was about 5mm), control group (25°C, Control) and treatment group (38°C 2h, Treatment) were set up respectively. The treatment group was placed at 25°C after heat shock was completed. After 2 days of treatment, the growth of 5594 (SVpc351) and 5626 (SVpc351) treatment groups was significantly weaker than that of the control group (Figure 6A), while there was no obvious difference between the control group and the treatment group of 5470 (SVpc348), 5471 (SVpc348) and Fielder. After 3 days of heat shock treatment, the plant height and coleoptile length of different strains were counted. The plant height and coleoptile length of 5594 (SVpc351) and 5626 (SVpc351) treatment groups (Figure 6B) were significantly lower than those of their own control groups and the control groups and treatment groups of other strains. The above results showed that Ms2 gene expression had an inhibitory effect on the normal division and / or growth of wheat coleoptile cells.

[0088] Example 5: Transient expression of Ms2 gene inhibits the normal division and / or growth of wheat callus cells

[0089] To investigate the effect of Ms2 gene on the growth of wheat callus, the experiments of Agrobacterium infection and callus induction were performed on the immature embryos of wheat Fielder using SVpc348 and SVpc351 (the tissue culture method was referred to steps a-c in Example 3). After the immature embryos infected by SVpc348 and SVpc351 were transferred to "induction and selection medium 1" (same as Example 3), they were subjected to heat shock treatment (38°C, 3h) and then continued to be cultured in dark at 25°C. The growth of callus was recorded by taking pictures at 0dah, 2dah, 4dah, 7dah and 10dah (dah=day after heat-shock) and the proportion of embryogenic callus was calculated (Fig. 7A-B). The size of callus after infection by different plasmids was quantitatively analyzed at 10dah (Fig. 7C). The results showed that at 10dah, the proportion of embryogenic callus corresponding to SVpc351 was significantly lower than that of SVpc348, which was reduced by about 33% (Fig. 7B). Compared with the callus corresponding to SVpc348, the callus infected by SVpc351 was weak in vitality, had no obvious differentiation, and the cell division and / or growth was inhibited (Fig. 7A). The above results indicated that the expression of Ms2 gene had an inhibitory effect on the normal division and / or growth of wheat callus cells.

[0090] Example 6: Ms2 protein inhibits the normal division and / or growth of tobacco cells

[0091] The stable genetic transformation of tobacco involved five plasmids in Example 1: SVpc337, SVpc348, SVpc351, SVpc369 and SVpc375, among which SVpc337, SVpc348 and SVpc351 were driven by the heat shock promoter Lehsp23.8, and SVpc369 and SVpc375 were driven by the 35S promoter.

[0092] Genetic transformation of tobacco was performed using Agrobacterium-mediated leaf disc transformation. During tobacco transformation, compared to the control plasmid SVpc348 (Lehsp23.8::GFP), genetically transformed plants associated with SVpc351 (Lehsp23.8::cMs2:GFP) showed inhibited rooting in rooting media (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 shows slight leakage 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 inhibited rooting in SVpc351 transformed plants. Therefore, the Ms2 gene exhibits cytotoxicity in tobacco root cells, inhibiting normal root cell division and / or growth.

[0093] To detect whether Ms2 protein was successfully expressed in tobacco, SVpc348 and SVpc351 transgenic plants and wild-type non-transgenic controls (WT) were subjected to heat shock 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 group (SVpc351-H, SVpc348-H, and WT-H) and the room temperature group (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 KD) was successfully expressed in SVpc351 transgenic plants (Figure 8B), and GFP was highly expressed in SVpc348 transgenic plants; however, in the room temperature group, some leakage was observed in both SVpc351 and SVpc348 transgenic plants (Figure 8B). During the genetic transformation of tobacco using two plasmids driven by 35S, SVpc369 (35S::GFP) and SVpc375 (35S::cMs2:GFP), tobacco leaves infected with Agrobacterium SVpc375 exhibited a browning phenotype compared to the control SVpc369, and no new bud differentiation was observed (Fig. 8C). These results indicate that the expression of the Ms2 gene inhibits normal cell division and / or growth in tobacco leaves.

[0094] Example 7: Ms2 gene expression inhibits normal cell division and / or growth in animal cells

[0095] 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 optimization sequence) gene C-terminal fusion eGFP fluorescent protein; SVpc419 does not contain the Ms2# gene, which is a strict control of SVpc421. The above plasmids are respectively transfected into 293 and huh7 cell lines, and the transfection steps are as follows:

[0096] 1. Preheat the water bath to 37°C; take the recipient cells from the liquid nitrogen tank or the refrigerator and quickly put them into the preheated water bath; use tweezers to hold the frozen tube and shake it to ensure uniform heating;

[0097] 2. When the frozen tube is completely melted, centrifuge at 4°C for 5 min to remove the supernatant and leave the precipitate;

[0098] 3. Suspend the precipitate with 1 ml of 90% DMEM + 10% FBS complete medium, and inoculate into a culture dish containing 9 ml of complete medium. After cross mixing, place it in a CO2 incubator for culture. Pass 3-4 times to ensure that the cell state is good and there is no contamination;

[0099] 4. On the day of transfection, plate the cells, remove the culture medium from a 10 cm dish, wash off excess serum with pbs, add 1-2 ml of trypsin and digest for 30 s, add 10 ml of medium to wash off the adherent cells and mix well;

[0100] 5. Take 10 ul of cell suspension and count it with a cell counting plate. Each well of a 6-well plate contains 10,000 cells, and 1 ml of cell suspension is distributed per well. Place it in a CO2 incubator for culture;

[0101] 6. Prepare the transfection preparation solution with a sterile EP tube: A solution = dilute 0.5 ug plasmid with 200 ul DMEM, B solution = dilute 1.5 ul PEI with 200 ul DMEM. Mix A and B solutions gently and separately, and let stand for 5 min. Add B solution to A solution, mix gently, and let stand at room temperature for 20 min. Add the transfection reagent to the medium in each well, mix crossly, and replace the complete medium after overnight culture.

[0102] Take pictures at 24h, 48h, 72h and 96h after transfection with an ordinary fluorescence microscope. At 96h, use a confocal microscope to take pictures, and use a cell flow cytometer to detect the fluorescence rate of SVpc419 and SVpc421 transfected 293 cells. Each plasmid sets three repeated groups, and each group of repeated cells is counted to 10,000 for detection. Untransfected 293 cells are used as a blank control.

[0103] The results show that the GFP fluorescence rate of SVpc421 transfected cells is significantly lower than that of SVpc419 at 72h and 96h after transfection of 293 (Figure 9A) and huh7 (Figure 9B) animal cell lines (Figures 9-10). The results of flow cytometry detection of 293 cell line at 96h show that 12.96% of the cells corresponding to SVpc421 present fluorescence among more than 30,000 cells detected, which is significantly lower than the average fluorescence rate of cells corresponding to SVpc419 (31.6%) (Figure 10B). The successfully transfected cells of SVpc421 have reduced green fluorescence area and are in a condensed state of dots, which cannot normally divide to form new cells; while the successfully transfected cells of SVpc419 have normal morphology and green fluorescence fills the entire cell (Figures 9-10); both 293 and huh7 cell lines show similar results. Therefore, Ms2 gene expression is toxic to animal cells and can inhibit the normal division and / or growth of animal cells.

[0104] Example 8: Ms2 protein inhibits the division and / or growth of Gibberella cell

[0105] In order to test the bacteriostatic effect of Ms2 protein, the prokaryotic expression plasmids SVpc398, SVpc399 and SVpc432 were transformed into E. coli DE3 strain suitable for expressing toxic proteins, and protein identification and bacteriostatic test were performed after induction of expression, and the specific operation steps are as follows:

[0106] 1. Take the positive monoclonal in 20 ml LB liquid with ampicillin, 37°C, 200 rpm overnight culture;

[0107] 2. Take 1 ml of bacterial solution in 100 ml of LB liquid with ampicillin, continue to culture to OD value of 0.6-1;

[0108] 3. Add 110 ul of 1M IPTG, 37°C, 200 rpm induction culture for 5h;

[0109] 4. Adjust the OD value of the bacterial solution to a uniform value, take 200 ul and evenly spread on PDA medium, 10 repeats for each plasmid, 37°C overnight culture;

[0110] 5. Collect the conidia of F. graminearum PH-1, adjust the concentration to 1 x 10 6 6. Take 50 ul of conidia and add to the center of the PDA medium, 25°C culture for 3 days, and observe the growth of F. graminearum PH-1;

[0111] 6. Collect the bacterial cell pellets after induction in step 3, and wash with pre-cooled 1 x PBS three times;

[0112] 7. Add 20 mL pre-cooled 1 x PBS solution to resuspend the bacterial pellet, then add 200 μl PMSF, 40 μl DTT, and sonicate on ice for 30 minutes. Centrifuge at 12000 rpm for 5 minutes at 4°C, and discard the supernatant. Wash the bacterial pellet with pre-cooled 1 x PBS three times;

[0113] 8. Repeat step 7, add 20 mL pre-cooled 1 x PBS solution to resuspend the bacterial solution, then add 200 μl PMSF, 40 μl DTT, and 20 mL 2 x SDS protein loading buffer. Mix and boil for 10 minutes, and store at -20°C.

[0114] 9. Take 15 μL of the sample for Coomassie blue staining detection, and use Ms2 antibody for Western-blot verification.

[0115] The results show that after 5 hours of IPTG induction, Coomassie blue staining detection shows that GST (26 KD) corresponding to SVpc398 and GST-Ms2 (55 KD) corresponding to SVpc399 are both expressed in large quantities (Figure 11A), but Ms2 (29 KD) protein corresponding to SVpc432 is not expressed in large quantities (only an unknown protein <26 KD is induced; Figure 11A). However, Western-blot detection confirms that GST-Ms2 protein corresponding to SVpc399 is normally expressed, and Ms2 protein corresponding to SVpc432 is expressed in small quantities (Figure 11B).

[0116] The results of the inhibition experiment on F. graminearum PH-1 show that the F. graminearum PH-1 plaque (average diameter 2.1 cm) on the culture medium containing bacteria corresponding to SVpc432 is significantly smaller than the plaque (average diameter 2.9 cm) on the culture medium containing bacteria corresponding to SVpc398 and the plaque (average diameter 3.1 cm) on the culture medium containing bacteria corresponding to SVpc399 (Figure 12). Moreover, the F. graminearum plaque on the culture medium containing bacteria corresponding to SVpc432 is mostly white or yellow, while the F. graminearum plaque on the culture medium containing bacteria corresponding to SVpc398 and SVpc399 is mostly red (Figure 12A), suggesting that the former (SVpc432) causes the growth, physiology, and maturation state of F. graminearum to slow down. It can be seen that the prokaryotically expressed Ms2 protein has an inhibitory effect on the normal division and / or growth of F. graminearum cells.

[0117] Example 9: Effect of Ms2 protein on the division and / or growth of yeast cells

[0118] Yeast is a single-celled eukaryotic microorganism. In order to study the effect of Ms2 protein on the growth of yeast, GAL1 was used to drive GFP (SVpc441), cMs2 (SVpc438), cMs2-GFP (SVpc443), GFP-cMs2 (SVpc529), and cMs2C158T -GFP(SVpc530) expression, and glycerolaldehyde-3-phosphate dehydrogenase constitutive promoter (TDH3) driven GFP(SVpc678), cMs2(SVpc677), cMs2-GFP(SVpc679), GFP-cMs2(SVpc680) and cMs2 C158T -GFP(SVpc681) expression, the expression vector is pYES2, the plasmid is transformed into Saccharomyces cerevisiae strain INVSc1 respectively. The yeast transformation refers to the Clontech yeast transformation method, after the transformation is completed, the bacteria liquid is respectively coated on the solid culture medium plate containing 2% glucose SC-U (for GAL1 promoter related vector) and SC-U (for TDH3 promoter related vector), and is placed in a 30°C incubator for culture for 2-3 days. After the positive clones grow, protein expression is induced respectively, and whether the fluorescence and the protein are successfully expressed is detected, and the influence of different Ms2 protein types on the growth of the yeast bacteria is detected. The specific method is as follows:

[0119] 1. Take the yeast positive single clone in 50 mL containing 2% glucose SC-U (induction medium) or SC-U liquid medium (non-induction medium), and culture at 30°C and 200 rpm overnight;

[0120] 2. Take the bacteria liquid after overnight culture, centrifuge at 3000 rpm for 5 min at 4°C, collect the bacteria, and wash with sterile water for three times to completely remove the culture medium in the previous step;

[0121] 3. Discard the supernatant, resuspend the bacteria with 10-20 mL induction / non-induction medium, and then transfer to 150 mL medium, adjust the initial OD600 to 0.4, and induce protein expression at 30°C and 200 rpm;

[0122] 4. Measure the OD600 value at different times (0h, 4h, 8h, 12h, 24h, 36h, 48h) after induction, and draw the yeast growth curve;

[0123] 5. After inducing protein expression, centrifuge at 3000 rpm for 5 min at 4°C, discard the supernatant, and use the bacteria for protein extraction;

[0124] 6. Resuspend the yeast bacteria with 500 μL lysis buffer (50 mmol / L sodium phosphate, pH 7.4; 1 mmol / L EDTA; 5% glycerol; 1 mmol / L PMSF), centrifuge at 3000 rpm for 5 min at 4°C, and discard the supernatant;

[0125] 7. Add an equal volume of glass beads (0.4-0.6 mm, Sigma), and vortex vigorously for 30 s, then place on ice for 30 s; repeat 4 times until the cells are completely lysed;

[0126] 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.

[0127] 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 13A). 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 trend among the vectors driven by TDH3 (Figure 13B). After 12 h of yeast expression, we detected green fluorescence and protein signal in yeast strains carrying GFP (SVpc678), Ms2-GFP (SVpc679), or GFP-Ms2 (SVpc680). The results showed that strong green fluorescence was detected in the TDH3::GFP strain, weak green fluorescence was detected in the TDH3::GFP:cMs2 strain, and no green fluorescence was detected in the TDH3::cMs2:GFP strain (Figure 13C). Western blotting using GFP antibody showed that the control plasmid GFP strain had a strong signal; the plasmid GFP:Ms2 strain had a weak signal, and the plasmid Ms2:GFP strain had only a weak signal (Figure 13D). 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.

[0128] 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. Use of wheat Ms2 protein as a toxic protein in (1) or (2) below: (1) inhibiting cell division and / or growth; (2) producing a product for inhibiting cell division and / or growth; said wheat Ms2 protein is a protein as shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing; (A2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein defined in (A1).

2. Use according to claim 1, characterized in that, said cell is a plant cell, an animal cell and a eukaryotic microbial cell.

3. Use of wheat Ms2 protein or a gene encoding the wheat Ms2 protein in producing a disease-resistant plant product; said wheat Ms2 protein is a protein as shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing; (A2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein defined in (A1).

4. Use according to claim 3, characterized in that, said gene encoding the wheat Ms2 protein is a nucleic acid molecule as shown in any one of (i) to (iv) below: (i) a nucleic acid molecule shown in SEQ ID NO: 1 in the sequence listing; (ii) a nucleic acid molecule shown in SEQ ID NO: 3 in the sequence listing; (iii) a nucleic acid molecule shown in SEQ ID NO: 4 in the sequence listing; (iv) a nucleic acid molecule other than (i) to (iii) which encodes the amino acid sequence shown in SEQ ID NO:

5.

5. Use of wheat Ms2 protein or a gene encoding the wheat Ms2 protein in producing an animal cell therapeutic drug; said wheat Ms2 protein is a protein as shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing; (A2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein defined in (A1).

6. Use according to claim 5, characterized in that, said animal cell therapeutic drug is a tumor therapeutic drug.

7. Use of wheat Ms2 protein in producing a product for killing harmful microorganisms; said wheat Ms2 protein is a protein as shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing; (A2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein defined in (A1).

8. Use according to claim 7, characterized in that, said product for killing harmful microorganisms includes a fungicide.

9. Use of wheat Ms2 protein in producing an insecticide; said wheat Ms2 protein is a protein as shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing; (A2) a fusion protein obtained by linking a tag to the N terminus and / or C terminus of the protein defined in (A1).

10. Use of an expression cassette, a recombinant plasmid or a recombinant bacterium containing a gene encoding wheat Ms2 protein in any one of (1) to (5) below: (1) inhibiting cell division and / or growth; (2) producing a product for inhibiting cell division and / or growth; (3) producing a tumor therapeutic drug; (4) producing a fungicide; (5) producing an insecticide.

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