Sitodiplosis mosellana anti-fusarium graminearum polypeptide SmAFP and application thereof

Abstract

The invention belongs to the technical field of crop disease prevention and treatment, and particularly relates to an anti-fusarium graminearum polypeptide SmAFP derived from Sitodiplosis mosellana, a nucleotide sequence coded by the same and application of the anti-fusarium graminearum polypeptide SmAFP. The invention discloses an anti-fusarium graminearum polypeptide SmAFP of Sitodiplosis mosellana. The amino acid sequence of the anti-fusarium graminearum polypeptide SmAFP is shown as SEQ ID NO: 2. The invention also provides an application of the Sitodiplosis mosellana anti-fusarium graminearum polypeptide SmAFP. The Sitodiplosis mosellana anti-fusarium graminearum polypeptide SmAFP is used for resisting wheat fungus fusarium graminearum, namely, the Sitodiplosis mosellana anti-fusarium graminearum polypeptide SmAFP can inhibit the growth of fusarium graminearum.

Description

Technical Field

[0001] The invention belongs to the technical field of crop disease prevention and treatment, and particularly relates to an anti-Fusarium graminearum polypeptide SmAFP derived from wheat red midge, a nucleic acid sequence encoding the same, and applications thereof. Background Art

[0002] Wheat is one of China's major grain crops. Head blight, primarily caused by the fungus Fusarium graminearum, is considered a "cancer" of wheat, severely impacting wheat yields and food safety. Currently, chemical control remains the primary preventive measure against the growing severity of head blight due to global warming. However, this is accompanied by a series of challenges, including drug residues, environmental pollution, and the rise of drug resistance. Therefore, the discovery of new, green active substances that effectively inhibit Fusarium graminearum while minimizing the development of drug resistance has become an urgent challenge.

[0003] Antimicrobial peptides are widely present naturally in the biological world. Based on their target, antimicrobial peptides can be categorized as antibacterial, antifungal, antiviral, and anticancer peptides. Due to their short length, broad-spectrum antimicrobial activity, low drug resistance, rapid bactericidal activity, and minimal toxic side effects, antimicrobial peptides have gradually become a potential alternative to traditional chemical pesticides, with enormous potential for application. For example, genetic engineering has been used to transfer the gene encoding bovine lactoferrin antimicrobial protein into wheat, significantly improving its resistance to ergot. The antimicrobial peptides MsrA2 and 10R exhibit fungicidal activity against Fusarium spp. Genetically modified versions of these peptides, when transferred into wheat, significantly improve resistance to ergot and powdery mildew. Furthermore, integrating them with emerging nanotechnology can enhance the stability of antimicrobial peptides and facilitate their application in crop disease prevention and control.

[0004] Antimicrobial peptides come from a wide variety of sources, including mammals, plants, amphibians, insects, and microorganisms. Insects, as the most numerous animal group on Earth, have become a valuable resource for the exploration of antimicrobial peptides. Since the isolation of the antimicrobial peptide cecropin from the pupae of the silkworm moth Hyalophora cecropia, research on insect antimicrobial peptides has continued unabated, involving hundreds of insect species. Based on their amino acid sequence and structural characteristics, insect antimicrobial peptides can be generally classified as cysteine-free α-helical peptides (e.g., cecropins), cysteine-rich β-sheet peptides (e.g., defensins), proline-rich peptides (e.g., apidaecins), and glycine-rich peptides (e.g., attacins). With the advancement of science and technology and the continuous accumulation of insect omics data, new insect antimicrobial peptides have been identified. However, compared to mammals and amphibians, research on insect antimicrobial peptides is currently limited to insects in the orders Lepidoptera, Diptera, Hemiptera, and Coleoptera. It can be seen that it is particularly important to utilize the existing large amount of insect resources, explore more insect antimicrobial peptides, and evaluate their antimicrobial activity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a wheat red midge anti-Fusarium graminearum polypeptide SmAFP gene and the protein encoded by it and its use.

[0006] In order to solve the above technical problems, the present invention provides a red wheat midge anti-Fusarium graminearum polypeptide SmAFP, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] As an improvement of the wheat midge anti-Fusarium graminearum polypeptide SmAFP of the present invention: the protein is its conservative variant protein, its active fragment or its active derivative.

[0008] The present invention also provides a gene encoding the above-mentioned wheat red midge anti-Fusarium graminearum polypeptide SmAFP, whose nucleotide sequence is shown in SEQ ID NO: 1; or has at least 70% homology with the nucleotide sequence in SEQ ID NO: 1; or its nucleotide sequence can hybridize with the nucleotide sequence in SEQ ID NO: 1 under conditions of 40-55°C.

[0009] As an improvement of the gene of the present invention: the sequence comprises 8-66 consecutive nucleotides in SEQ ID NO: 1.

[0010] The present invention also provides a use of the above-mentioned wheat red midge anti-Fusarium graminearum polypeptide SmAFP: it is used to resist the wheat fungus Fusarium graminearum, that is, the wheat red midge anti-Fusarium graminearum polypeptide SmAFP can inhibit the growth of Fusarium graminearum.

[0011] Moreover, the antimicrobial peptide SmAFP has no cytotoxicity to the human embryonic kidney 293T cell line.

[0012] The wheat red midge anti-Fusarium graminearum polypeptide SmAFP and the nucleic acid sequence encoding it provided by the present invention enable the application of its amino acid sequence, coding sequence and its development into disease-resistant crops and biological pesticides with application value and application in multiple fields such as agricultural disease prevention and control.

[0013] The present invention is specifically implemented through the following technical solution: the present invention utilizes the genome of the wheat midge to obtain the gene sequence of the anti-Fusarium graminearum polypeptide SmAFP. The nucleic acid sequence is shown in SEQ ID NO. 1, and the corresponding amino acid sequence is shown in SEQ ID NO. 2. Based on the amino acid sequence, the polypeptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd. using solid-phase chemical synthesis. After purification by reverse-phase high-performance liquid chromatography and identification by electrospray ionization mass spectrometry, a pure polypeptide with a purity greater than 95% was obtained and desalted. The synthesized wheat midge anti-Fusarium graminearum polypeptide SmAFP can inhibit the growth of Fusarium graminearum and is non-cytotoxic.

[0014] The synthesized wheat midge anti-Fusarium graminearum polypeptide SmAFP of the present invention comprises: a protein having the amino acid sequence of SEQ ID NO: 2, or a conservative variant protein thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the protein is a protein having the sequence of SEQ ID NO: 2.

[0015] In the present invention, the nucleic acid sequence encoding the anti-Fusarium graminearum peptide SmAFP for wheat midge refers to a nucleotide sequence encoding the anti-Fusarium graminearum peptide SmAFP activity for wheat midge, such as the nucleotide sequence in SEQ ID NO:1 and its degenerate sequences. A degenerate sequence is one in which one or more codons in SEQ ID NO:1 are replaced with degenerate codons encoding the same amino acid. Due to codon degeneracy, degenerate sequences with as little as approximately 70% homology to the nucleotide sequence in SEQ ID NO:1 can also encode the sequence described in SEQ ID NO:1.

[0016] Also included are nucleotide sequences that hybridize to the nucleotide sequence in SEQ ID NO: 1 under moderately stringent conditions, more preferably under highly stringent conditions. Also included are nucleotide sequences that are at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identical to the nucleotide sequence in SEQ ID NO: 1. Also included are variants of the sequence in SEQ ID NO: 1 that encode proteins that have the same function as the native SmAFP anti-Fusarium graminearum polypeptide. These variants include (but are not limited to): deletions, insertions, and / or substitutions of several nucleotides (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10), and additions of several nucleotides (usually less than 60, preferably less than 30, more preferably less than 10, and most preferably less than 5) to the 5' and / or 3' ends.

[0017] In the present invention, the wheat midge anti-Fusarium graminearum polypeptide SmAFP refers to a protein having the sequence of SEQ ID NO: 2 that exhibits the activity of the wheat midge anti-Fusarium graminearum polypeptide SmAFP. This term also includes variants of SEQ ID NO: 2 that have the same function as the wheat midge anti-Fusarium graminearum polypeptide SmAFP. These variants include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10), as well as the addition of one or more amino acids (typically within 20, preferably within 10, and more preferably within 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids having similar or similar properties generally does not alter the function of the protein. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the anti-Fusarium graminearum polypeptide SmAFP of the wheat midge.

[0018] In the present invention, the conservative variant protein of the wheat midge anti-Fusarium graminearum polypeptide SmAFP refers to a protein formed by replacing at most 10, preferably at most 8, and more preferably at most 5 amino acids with similar or similar amino acids compared to the amino acid sequence of SEQ ID NO: 2.

[0019] The present invention also includes analogs of the wheat midge anti-Fusarium graminearum polypeptide SmAFP or protein. These analogs may differ from the wheat midge anti-Fusarium graminearum polypeptide SmAFP in terms of amino acid sequence, modifications that do not affect the sequence, or a combination of these. These proteins include natural or induced genetic variants. Induced variants can be obtained through various techniques, such as random mutagenesis through radiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biology techniques. Analogs also include those with residues other than naturally occurring L-amino acids (e.g., D-amino acids), as well as those with non-naturally occurring or synthetic amino acids (e.g., β- and γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins listed above.

[0020] Modifications (generally without altering the primary structure) include chemical derivatization of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation during protein synthesis and processing or during further processing steps. Such modifications can be accomplished by exposing the protein to glycosylation enzymes (e.g., mammalian glycosylases or deglycosylases). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Proteins modified to enhance proteolytic activity or optimize solubility are also included.

[0021] In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids, etc. When producing the wheat midge anti-Fusarium graminearum polypeptide SmAFP of the present invention, the wheat midge anti-Fusarium graminearum polypeptide SmAFP coding sequence can be operably linked to the expression control sequence to form a wheat midge anti-Fusarium graminearum polypeptide SmAFP expression vector.

[0022] As used herein, "operably linked to" refers to a situation where certain portions of a linear DNA sequence are able to influence the activity of other portions of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and participates in protein secretion, then the signal peptide (secretory leader) DNA is operably linked to the protein DNA; if a promoter controls transcription of the sequence, then it is operably linked to the coding sequence; if a ribosome binding site is positioned to enable translation, then it is operably linked to the coding sequence. Generally, "operably linked to" means adjacent, and for a secretory leader sequence, it means adjacent in reading frame.

[0023] Northern blotting or fluorescence quantitative PCR can also be used to analyze the expression of the SmAFP gene product of the wheat midge anti-Fusarium graminearum polypeptide, that is, to analyze the presence and quantity of the SmAFP RNA transcript in the cells.

[0024] Furthermore, the nucleic acid molecule used as a probe in the present invention typically comprises 8-66 consecutive nucleotides, preferably 15-50 consecutive nucleotides, of the nucleotide sequence encoding the SmAFP polypeptide, a peptide resistant to Fusarium graminearum, from the wheat midge. This probe can be used to detect the presence of a nucleic acid molecule encoding the SmAFP polypeptide, a peptide resistant to Fusarium graminearum, from the wheat midge in a sample.

[0025] The present invention relates to a method for detecting the presence of a nucleotide sequence for the anti-Fusarium graminearum polypeptide (SmAFP) from the wheat midge in a sample. The method comprises hybridizing the sample with the aforementioned probe and then detecting whether the probe binds. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence for the anti-Fusarium graminearum polypeptide (SmAFP) from the wheat midge and may be located on either side of or within the coding sequence. The primers are generally 15-50 nucleotides in length.

[0026] In addition, according to the nucleotide sequence and amino acid sequence of the wheat midge anti-Fusarium graminearum polypeptide SmAFP of the present invention, the wheat midge anti-Fusarium graminearum polypeptide SmAFP homologous genes or homologous proteins can be screened based on the nucleic acid homology or the homology of the expressed protein.

[0027] The full-length nucleotide sequence of the wheat midge anti-Fusarium graminearum polypeptide SmAFP of the present invention or a fragment thereof can generally be obtained by PCR amplification, recombination, or synthetic methods. For PCR amplification, the relevant sequence can be amplified based on the relevant nucleotide sequence disclosed herein and using a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template.

[0028] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0029] In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis. The wheat midge anti-Fusarium graminearum polypeptide SmAFP of the present invention can be used to screen out substances or receptors that interact with the wheat midge anti-Fusarium graminearum polypeptide SmAFP by various conventional screening methods.

[0030] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. The present invention exhibits a significant inhibitory effect against Fusarium graminearum in antibacterial tests and exhibits no cytotoxicity to the human embryonic kidney 293T cell line. Wheat head blight is a serious threat in my country, and the negative impact of chemical pesticides is significant. The wheat midge anti-Fusarium graminearum polypeptide SmAFP of the present invention has significant application value in preventing and treating crop diseases.

[0031] It should be emphasized that the currently known antimicrobial peptide AP00952 (Antimicrobial Peptide Database, https: / / aps.unmc.edu / AP / ), which has antibacterial, antifungal and anticancer uses, has a sequence similarity of only 46.43% with the polypeptide SmAFP of the present invention.

[0032] In summary, the SmAFP peptide against Fusarium graminearum discovered in this patent has broad application prospects and is expected to become a biological pesticide for the efficient prevention and control of important crop diseases, laying the foundation for the future cultivation of transgenic crops with this disease-resistant gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0034] Figure 1The absorbance value of the fungus after incubation of the synthesized gradient concentrations of the wheat midge anti-Fusarium graminearum polypeptide SmAFP with Fusarium graminearum for 24 hours is shown in FIG. 2 , and the negative control is double distilled water (0 μg / mL) in which the wheat midge anti-Fusarium graminearum polypeptide SmAFP is dissolved. The measurement wavelength is 600 nm.

[0035] Note: Figure 1 The experimental principle is that the absorbance value measured at a wavelength of 600nm is often used to measure the concentration of bacterial solution, and the absorbance value is proportional to the concentration of bacterial solution in the solution.

[0036] Figure 2 The relative fluorescence value of the cells after incubation of the synthesized gradient concentrations of the wheat midge anti-Fusarium graminearum polypeptide SmAFP with human embryonic kidney 293T cells for 24 hours, the negative control is double distilled water (0 μg / mL) in which the wheat midge anti-Fusarium graminearum polypeptide SmAFP is dissolved, the excitation wavelength Ex is 485 nm, and the emission wavelength Em is 525 nm;

[0037] Note: Figure 2 The experimental principle is CellTox TM CellTox Green Cytotoxicity Assay (Promega, Madison, USA) TM Green Dye cannot enter living cells, but can firmly bind to DNA in dead cells, and the fluorescent signal generated is proportional to the cytotoxicity. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with laboratory specific test data and specific examples. These examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions or the conditions recommended by the manufacturer.

[0039] The amino acid sequence of the anti-Fusarium graminearum polypeptide SmAFP of the wheat red midge is set as shown in SEQ ID NO: 2.

[0040] Example 1: Antibacterial test of the wheat midge anti-Fusarium graminearum polypeptide SmAFP against Fusarium graminearum

[0041] Different concentrations of the anti-Fusarium graminearum peptide SmAFP of wheat red midge were incubated with Fusarium graminearum, and the fungal absorbance was detected using a microplate reader Thermo Scientific Varioskan Flash (Thermo Scientific, Vantaa, Finland) (the absorbance measured at a wavelength of 600 nm is often used to measure the concentration of the bacterial solution, and the absorbance is proportional to the concentration of the bacterial solution in the solution) to evaluate the antibacterial activity of the anti-Fusarium graminearum peptide SmAFP of wheat red midge; the details are as follows:

[0042] 1) Weigh 2 g of glucose (Macklin, Shanghai, China), 34.53 g of 3-morpholinepropanesulfonic acid (Aladdin, Shanghai, China), and 10.45 g of RPMI 1640 medium powder (Procell, Wuhan, China), dissolve them in 900 mL of double-distilled water, and stir thoroughly.

[0043] 2) The solution obtained in step 1) was adjusted to pH 7.0 ± 0.1 with sodium hydroxide powder or hydrochloric acid solution (SCR, Shanghai, China), and then the volume was adjusted to 1 L with double distilled water.

[0044] 3) The solution obtained in step 2) was sterilized by positive pressure filtration using a 0.22 μm pore size filter membrane (Millipore, Darmstadt, Germany) in a clean bench. The filtered liquid culture medium was subjected to a bacterial test and used only after passing the test.

[0045] 4) The conidia of Fusarium graminearum were diluted into the liquid culture medium obtained in step 3) and inoculated into 96-well plates (LABSELECT, Anhui, China) at the same density (about 2500-5000 conidia per well), with 50 μL per well.

[0046] Note: Fusarium graminearum is PH-1, Jian, Y., Liu, Z., Wang, H., Chen, Y., Yin, Y., Zhao, Y., & Ma, Z. (2021). Interplay of two transcription factors for recruitment of the chromatin remodeling complex modulates fungal nitrosative stressresponse. Nature communications, 12(1), 2576-2592. https: / / doi.org / 10.1038 / s41467-021-22831-8.

[0047] 5) Dissolve the synthesized SmAFP peptide against Fusarium graminearum in double-distilled water to an initial concentration of 1024 μg / mL. Then, perform a two-fold gradient dilution to a maximum concentration of 512 μg / mL and a minimum concentration of 0.5 μg / mL.

[0048] 6) Add 50 μL of the anti-Fusarium graminearum peptide SmAFP of the wheat red midge to each well of the 96-well plate obtained in step 4) to make the final concentration of the peptide be 256-128-64-32-16-8-4-2-1-0.5-0.25 μg / mL.

[0049] 7) After incubation at 30°C for 24 h, the 96-well plate was placed in a microplate reader Thermo Scientific Varioskan Flash (Thermo Scientific, Vantaa, Finland) to measure the absorbance of the fungus in each well at a wavelength of 600 nm.

[0050] The negative control group consisted of double-distilled water (0 μg / mL) containing the anti-Fusarium graminearum peptide SmAFP from the wheat midge; the experimental treatment groups were treated with different concentrations of SmAFP (256-128-64-32-16-8-4-2-1-0.5-0.25 μg / mL). Each treatment (experimental and control groups) had three biological replicates. One-way analysis of variance (ANOVA) was performed using the DPS data processing system (Tang and Feng, 2007), and multiple comparisons were performed using the Tukey test. Figure 1 The a and b in the figure are marked with letters. When the significance level is 0.05, the statistical significance of the data is judged: if there is one same letter between the treatments, the difference is considered to be insignificant; if there is no same letter, the difference is considered to be significant. At the same time, the plot was drawn using GraphPad Prism 8.3.0 (GraphPad Prism, CA, USA). The specific results are as follows: Figure 1 As shown. Figure 1The data show that after incubating Fusarium graminearum with different concentrations of wheat red midge anti-Fusarium graminearum peptide SmAFP, the absorbance values between treatments were different from those in the negative control group (double distilled water as the solvent for dissolving wheat red midge anti-Fusarium graminearum peptide SmAFP), and there were statistically significant differences: after incubating Fusarium graminearum with 0.5 / 1 / 2 / 4 / 8 / 16 / 32 / 64 / 128 / 256 μg / mL of wheat red midge anti-Fusarium graminearum peptide SmAFP, the absorbance values between treatments were significantly decreased compared with the negative control group, and the decrease in absorbance caused by 4 / 8 / 16 / 32 / 64 / 128 / 256 μg / mL of wheat red midge anti-Fusarium graminearum peptide SmAFP was more significant than that caused by 0.5 / 1 / 2 μg / mL of wheat red midge anti-Fusarium graminearum peptide SmAFP. Combined with the experimental principle that the absorbance value at a wavelength of 600nm is proportional to the concentration of the bacterial solution, this shows that compared with the negative control group, the growth of Fusarium graminearum incubated with the anti-Fusarium graminearum peptide SmAFP of the wheat red midge was inhibited, and the minimum concentration of the inhibitory effect caused by the anti-Fusarium graminearum peptide SmAFP of the wheat red midge was 0.5μg / mL.

[0051] Therefore, the above test results prove that the anti-Fusarium graminearum polypeptide SmAFP of the wheat red midge of the present invention can inhibit the growth of Fusarium graminearum.

[0052] Example 2: Toxicity determination of the anti-Fusarium graminearum peptide SmAFP of the wheat midge on human embryonic kidney 293T cell line

[0053] The anti-Fusarium graminearum peptide SmAFP from the wheat midge was incubated with human embryonic kidney 293T cells and the expression of the peptide was detected by CellTox. TM Cell death rate was detected by Green Cytotoxicity Assay (Promega, Madison, USA) (asymmetric cyanine fluorescent dye CellTox TM Green Dye cannot enter living cells, but can firmly bind to DNA in dead cells. The generated fluorescent signal is proportional to the cytotoxicity) to evaluate the cytotoxicity of the anti-Fusarium graminearum peptide SmAFP of the wheat red midge, as follows:

[0054] 1) Human embryonic kidney 293T cells were cultured in DMEM high-glucose medium (Life Technologies Corporation, NY, USA) in a constant-temperature CO2 incubator at 37°C and 5% CO2 for 24 hours. The culture medium was supplemented with 10% fetal bovine serum (WISENT, Nanjing, China) and 1% double-streptomycin (Solarbio, Beijing, China), using a 100× penicillin and streptomycin mixture specifically for cell culture.

[0055] 2) Take out CellTox from -20℃ freezer TM Green Dye (Promega, Madison, USA), equilibrated to 37°C.

[0056] 3) In a clean bench, add 1 μL CellTox TM Green Dye was added to 499 μL DMEM high glucose medium. TM The concentration of Green Dye was 2×.

[0057] 4) Gently pipette to mix, add 500 μL of DMEM high glucose medium containing human embryonic kidney 293T cells to the mixed system obtained in step 3) to make CellTox TM The final concentration of Green Dye is 1×.

[0058] 5) Gently pipette to mix, and seed human embryonic kidney 293T cells at the same density (about 500-1000 cells per well) in a 384-well plate (PerkinElmer, MA, USA), with 40 μL per well.

[0059] 6) Place the cell culture plate in a cell culture incubator and culture overnight at 37°C for 12 hours.

[0060] 7) The next day, the synthesized wheat midge anti-Fusarium graminearum peptide SmAFP was dissolved in double-distilled water to an initial concentration of 1024 μg / mL. Then, it was diluted in a two-fold gradient to a maximum concentration of 512 μg / mL and a minimum concentration of 0.5 μg / mL.

[0061] 8) Then, the culture plate was removed from the cell culture incubator and 40 μL of the anti-Fusarium graminearum peptide SmAFP from the wheat midge was added to each well, so that the final peptide concentration was 256-128-64-32-16-8-4-2-1-0.5-0.25 μg / mL.

[0062] 9) After incubation at 37°C for 24 hours, the supernatant from each well was aspirated and the cells were immediately placed in a Thermo Scientific Varioskan Flash (Thermo Scientific, Vantaa, Finland) microplate reader to measure the fluorescence of the adherent cells at the bottom of each well. The excitation wavelength (Ex) was 485 nm, and the emission wavelength (Em) was 525 nm.

[0063] The negative control group consisted of double-distilled water (0 μg / mL) containing the anti-Fusarium graminearum peptide SmAFP from the wheat midge; the experimental treatment groups were treated with different concentrations of SmAFP (256-128-64-32-16-8-4-2-1-0.5-0.25 μg / mL). Each treatment (experimental and control groups) had three biological replicates. One-way analysis of variance (ANOVA) was performed using the DPS data processing system (Tang and Feng, 2007), and multiple comparisons were performed using the Tukey test. Figure 2 The a and b in the figure are marked with letters. When the significance level is 0.05, the statistical significance of the data is judged: if there is one same letter between the treatments, the difference is considered to be insignificant; if there is no same letter, the difference is considered to be significant. At the same time, the plot was drawn using GraphPad Prism 8.3.0 (GraphPad Prism, CA, USA). The specific results are as follows: Figure 2 As shown. Figure 2 The data showed that after incubating human embryonic kidney 293T cells with different concentrations of wheat midge anti-Fusarium graminearum peptide SmAFP, the relative fluorescence values of each treatment were no different from those of the negative control group (double distilled water, the solvent for dissolving wheat midge anti-Fusarium graminearum peptide SmAFP), and there was no statistically significant difference. TM The experimental principle of Green Dye dye (the dye cannot enter living cells, but can firmly bind to the DNA in dead cells, and the fluorescent signal generated is proportional to the cytotoxicity) shows that compared with the negative control group, human embryonic kidney 293T cells incubated with the anti-Fusarium graminearum peptide SmAFP of the wheat red midge did not produce cytotoxicity.

[0064] Therefore, the above test results prove that the anti-Fusarium graminearum polypeptide SmAFP of the red wheat midge of the present invention has no cytotoxicity to the human embryonic kidney 293T cell line.

[0065] Finally, it should be noted that the above examples are merely specific implementation examples of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The red wheat midge anti-Fusarium graminearum peptide SmAFP is characterized by: The amino acid sequence is shown in SEQ ID NO:

2.

2. The wheat midge anti-Fusarium graminearum polypeptide SmAFP according to claim 1, characterized in that: The protein is a protein, a conservative variant protein thereof, an active fragment thereof or an active derivative thereof.

3. The gene encoding the wheat red midge anti-Fusarium graminearum polypeptide SmAFP according to claim 1 or 2, characterized in that: The nucleotide sequence is as shown in SEQ ID NO: 1; or it has at least 70% homology with the nucleotide sequence in SEQ ID NO: 1; or its nucleotide sequence can hybridize with the nucleotide sequence in SEQ ID NO: 1 at 40-55°C.

4. The gene according to claim 3, characterized in that: The nucleotide sequence contains 8 to 66 consecutive nucleotides.

5. The use of the wheat red midge anti-Fusarium graminearum polypeptide SmAFP according to claim 1 or 2, characterized in that: Used to inhibit the growth of Fusarium graminearum.

Images