Use of smlt0134 protein in inhibiting rice blast

Abstract

The application discloses application of smlt0134 protein in inhibiting rice Magnaporthe oryzae. Experiments prove that the smlt0134 protein can inhibit the rice Magnaporthe oryzae, mainly embodied in inhibiting growth of the rice Magnaporthe oryzae, inhibiting spore germination of the rice Magnaporthe oryzae and / or inhibiting spore growth of the rice Magnaporthe oryzae. Therefore, the smlt0134 protein is expected to develop into a new type of high-efficiency protein preparation. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of smlt0134 protein in inhibiting rice blast fungus. Background Technology

[0002] Rice blast, caused by Magnaphalthe oryzae, is one of the most destructive diseases of rice. Currently, the main control measures for rice blast are chemical control, with the most effective being 6% kasugamycin aqueous solution, which has a long duration of action; followed by 40% isoprothiolane emulsifiable concentrate; and lastly, 10% azoxystrobin microcapsule suspension (Jin Jian. Screening test of rice blast control agents [J]. Agricultural Technology and Equipment, 2023(6):193-194.). Although chemical control is highly effective, it causes great environmental pollution and easily leads to drug resistance, necessitating the exploration of greener and safer control methods. In recent years, research on the control of rice blast using biological control methods has become increasingly widespread, and many biocontrol strains have been discovered, such as Bacillus subtilis GB519 (Qi Shanyan, Zhu Feng, Wang Jichun, et al. Colonization of Bacillus subtilis GB519 in rice plants and its field control efficacy against rice blast [J]. Plant Protection, 2023, 49(2):48-56.) and Bacillus belyssus 5-8 (Li Shenyu, Jia Xiangzi, Guo Juntao, et al. Identification of biocontrol bacteria 5-8 against rice blast and its biocontrol mechanism). [J]. Journal of Northern Agriculture, 2021, 49(3):74-81.), Bacillus amyloliquefaciens HR-2 (Li Jin. Antagonistic effect of Bacillus amyloliquefaciens HR-2 on rice blast fungus and rapeseed sclerotinia rot fungus [D]: Master's thesis. Changsha: Hunan University, 2021.), Actinomycete Ahn109 (Hu Zhan, Cheng Wei, Li Yilu, et al. Isolation and identification of rice endophytic actinomycete Ahn109 and its inhibitory activity against rice blast [J / OL]. Jiangsu Agricultural Sciences: 1-8). These biocontrol strains show high biocontrol potential against rice blast. Biocontrol agents can be used to control the disease in a green and safe way. However, biocontrol agents are easily affected by environmental factors, which may cause them to lose their biocontrol effect.

[0003] In addition, exploring new rice varieties resistant to rice blast and selecting appropriate cultivation and management are also important measures for the prevention and control of rice blast. However, there are many problems such as the lack of resistant germplasm resources and the cumbersome and inefficient agricultural operations. There is an urgent need to develop plant-derived or microbial protein preparations to effectively prevent and control the occurrence of rice blast. Summary of the Invention

[0004] The purpose of this invention is to inhibit rice blast fungus.

[0005] This invention first protects the application of the smlt0134 protein, which can be S1) or S2):

[0006] S1) Inhibits rice blast fungus;

[0007] S2) Cultivate rice resistant to rice blast fungus.

[0008] The smlt0134 protein may be a1), a2), or a3):

[0009] a1) The amino acid sequence is that of the protein shown in SEQ ID No: 1;

[0010] a2) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1);

[0011] a3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No: 1.

[0012] The proteins in a2) above are labeled as shown in Table 1.

[0013] Table 1. Sequence of Labels

[0014] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tagII 8 WSHPQFEK c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0015] The protein in a3) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0016] The proteins mentioned in a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0017] The gene encoding the protein in a3) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID No: 2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0018] This invention also protects the application of nucleic acid molecules encoding any of the above-described smlt0134 proteins (i.e., the smlt0134 gene), which may be S1) or S2):

[0019] S1) Inhibits rice blast fungus;

[0020] S2) Cultivate rice resistant to rice blast fungus.

[0021] The nucleic acid molecule encoding any of the smlt0134 proteins described above may be a DNA molecule as follows (b1) or (b2) or (b3) or (b4):

[0022] (b1) A DNA molecule with a coding region as shown in SEQ ID NO:2;

[0023] (b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO:2;

[0024] (b3) A DNA molecule that hybridizes under stringent conditions with a DNA molecule defined in (b1) or (b2) and encodes any of the smlt0134 proteins described above;

[0025] (b4) A DNA molecule derived from Stenotrophomonas maltophilia and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA molecule defined in (b1) or (b2) and encoding any of the above-described smlt0134 proteins.

[0026] The stringent conditions were: hybridization in a solution of 2×SSC and 0.1% SDS at 68°C, followed by two washes of 5 min each, and then hybridization in a solution of 0.5×SSC and 0.1% SDS at 68°C, followed by two washes of 15 min each.

[0027] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0028] Of these, SEQ ID NO:2 consists of 1017 nucleotides, and the nucleotides shown in SEQ ID NO:2 encode the amino acid sequence shown in SEQ ID NO:1.

[0029] Those skilled in the art can readily mutate the nucleotide sequence encoding any of the aforementioned smlt0134 proteins using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 70% or higher identity with the nucleotide sequence of any of the aforementioned smlt0134 proteins isolated by this invention, as long as they encode any of the aforementioned smlt0134 proteins, are derived from and are equivalent to the nucleotide sequence of this invention.

[0030] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 70% or higher, 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence of the smlt0134 protein shown in SEQ ID NO:1. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0031] In any of the above-described applications, the inhibition of rice blast fungus can be manifested as inhibiting the growth of rice blast fungus, inhibiting the spore germination of rice blast fungus, and / or inhibiting the spore growth of rice blast fungus.

[0032] In any of the above applications, the rice blast fungus can specifically be Magnaphalium praecoxii Guy11.

[0033] This invention also protects a method for inhibiting rice blast fungus.

[0034] The method for inhibiting rice blast fungus protected by this invention may specifically be Method 1, which may involve directly or indirectly treating rice blast fungus with any of the smlt0134 proteins described above.

[0035] The method for inhibiting rice blast fungus protected by this invention can specifically be Method 2, which involves directly or indirectly treating rice blast fungus with Stenotrophomonas maltophilia or a Stenotrophomonas maltophilia mutant. Compared with Stenotrophomonas maltophilia, the nucleic acid molecule encoding any of the above-mentioned smlt0134 protein in the genome of the Stenotrophomonas maltophilia mutant has been partially deleted, resulting in the inability to express the smlt0134 protein.

[0036] In the above method, the Stenotrophomonas maltophilia can specifically be Stenotrophomonas maltophilia CGMCC NO.1.1788.

[0037] In any of the methods described above, the inhibition of rice blast fungus can be manifested as inhibiting the growth of rice blast fungus, inhibiting the spore germination of rice blast fungus, and / or inhibiting the spore growth of rice blast fungus.

[0038] In any of the methods described above, the rice blast fungus may be Magnolia oryzae Guy11.

[0039] Experiments have shown that the smlt0134 protein can inhibit *Blastomyces oryzae*, with inhibition manifested in the suppression of *Blastomyces oryzae* growth, spore germination, and / or spore growth. Currently, antibiotic resistance caused by overuse poses a serious threat, and the smlt0134 protein shows promise as a novel, highly effective protein formulation. This invention has significant application value. Attached Figure Description

[0040] Figure 1 The results are obtained by agarose gel electrophoresis of some PCR amplification products from steps 7 and 8 of Example 1.

[0041] Figure 2 The results show the detection of the resistance of the smlt0134 mutant to rice blast fungus.

[0042] Figure 3 The SDS-PAGE results are of the post-column solution during the expression and purification of Smlt0134 protein.

[0043] Figure 4 The results are for the in vitro toxicity assay of the Smlt0134 protein. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0046] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0047] In the following examples, Stenotrophomonas maltophilia CGMCC NO. 1.1788 was purchased by the inventors of this application from the China General Microbiological Culture Collection Center (CGMCC). Hereinafter, Stenotrophomonas maltophilia CGMCC NO. 1.1788 is abbreviated as Stenotrophomonas maltophilia or Sma.

[0048] In the following examples, *Magnaporthe oryzae* Guy11 is described in the following literature: Andrew J. Foster, Magdalena Martin-Urdiroz, Xia Yan, Harriet Sabrina Wright, Darren M. Soanes & Nicholas J. Talbot. CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus. Scientific Reports volume 8, Article number: 14355 (2018), and its name in the literature is *M. oryzae* strain Guy11. *Magnaporthe oryzae* Guy11 is also known as GSE18361 in NCBI's GenBank database. In the following text, *Magnaportheoryzae* Guy11 will be simply referred to as *Magnaportheoryzae* Guy11 or Guy11.

[0049] In the following examples, the amino acid sequence of the smlt0134 protein is shown in SEQ ID NO:1. The nucleotide sequence of the gene encoding the smlt0134 protein (i.e., the smlt0134 gene) is shown in SEQ ID NO:2.

[0050] The culture media involved in the following examples are as follows:

[0051] The solutes and their concentrations in LB liquid culture medium were 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride, with water as the solvent and a pH of 5.8; it was sterilized by high-temperature steam sterilization at 121℃ for 20 min.

[0052] The solutes and their concentrations in LB solid medium were 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 15 g / L agar powder, with water as the solvent and a pH of 5.8; the medium was sterilized by steam at 121°C for 20 min.

[0053] The solutes and their concentrations in CM solid culture medium were: NaNO3 6 g / L, KCl 0.52 g / L, MgSO4·7H2O 0.52 g / L, KH2PO4 1.52 g / L, Peptone 2 g / L, Yeast extract 1 g / L, Casamino acid 1 g / L, D-Glucose 10 g / L, Trace elements 1 ml / L, Vitamin solution 1 ml / L, and agar powder 15 g / L. The solvent was water, and the pH was adjusted to 6.5 with 10M NaOH. The medium was then sterilized by steam at 121℃ for 20 min. Each 1L of Trace elements consists of 22g ZnSO4·7H2O, 11g H3BO3, 5g MnCl2·4H2O, 5g FeSO4·7H2O, 1.7g CoCl2·6H2O, 1.6g CuSO4·5H2O, 1.5g Na2MoO4·5H2O, 50g Na4EDTA, and water; each 100ml of Vitamin solution consists of 0.01g Biotin, 0.01g Thiamine, 0.01g Pyridoxin, 0.01g Riboflavin, 0.01g PABA (p-aminobenzonic acid), 0.01g Nicotinic acid, and water.

[0054] The vector pK18mobSacB is described in the following literature: Andreas Andreas Tauch, Wolfgang Kalinowski, Georg Thierbach and Alfred Pühler. Small mobilizable multi-purpose chromosome vectors derived from the Escherichia coiplasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene, 145(1994)69-73. The experimental materials are available to the public from the State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences (i.e., the applicant's location).

[0055] I. Obtaining the smlt0134 mutant

[0056] 1. Genomic DNA was extracted from *Stenotrophomonas maltophilia* and used as a template. Primer 0134-a: 5'-ACAGCTATGACATGATTAC GAATTC PCR amplification was performed using a primer pair consisting of AAGGCGCTGCATATCGTGCAGG-3' (underlined is the recognition site of restriction endonuclease EcoRI) and primer 0134-b: 5'-TCCGGGAGCATGCGGAATGCATCATCACGGCTGGGTGC-3', yielding the 907 bp left arm (0134LB) of the smlt0134 gene. Genomic DNA was extracted from *Stenotrophomonas maltophilia* and used as a template, and primer 0134-c: 5'-TCCGGGAGCATGCGGAATGCATCATCACGGCTGGGTGC-3' was used.

[0057] -ATTCCGCATGCTCCCGGATGACTTCCCCTCAAACGTGTCCG-3' and primer 0134-d:5'

[0058] -AAACGACGGCCAGTGCC AAGCTT PCR amplification was performed using a primer pair consisting of TACTTGCAGCGTCCCCCTCAATC-3' (the underlined part is the recognition site of the restriction endonuclease HindIII), and the right arm 0134RB of the 887bp smlt0134 gene was recovered.

[0059] 2. Digest the vector PK18mobSacB with the restriction endonuclease EcoRI and recover the linearized vector PK18mobSacB. Perform homologous recombination between the linearized vector PK18mobSacB and the left arm (0134LB) of the smlt0134 gene to obtain the intermediate vector. Digest the intermediate vector with the restriction endonuclease HindIII and recover the linearized intermediate vector. Perform homologous recombination between the linearized intermediate vector and the right arm (0134RB) of the smlt0134 gene to obtain the recombinant vector PK18mobSacB-0134LB-0134RB.

[0060] The recombinant vector PK18mobSacB-0134LB-0134RB was sequenced. Sequencing results showed that the recombinant vector PK18mobSacB-0134LB-0134RB was obtained by inserting DNA sequence 1 (nucleotide sequence as shown in SEQ ID NO:3) into the EcoRI restriction enzyme recognition site of the vector PK18mobSacB and DNA sequence 2 (nucleotide sequence as shown in SEQ ID NO:4) into the HindIII restriction enzyme recognition site.

[0061] 3. Transform the recombinant vector PK18mobSacB-0134LB-0134RB into competent cells of Stenotrophomonas maltophilia, then spread them on LB solid medium containing 50 mg / L kanamycin and incubate at 28°C for 2-3 days to obtain several single colonies.

[0062] 4. Using genomic DNA of Stenotrophomonas maltophilia as a template, the O134-F:5' sequence was used.

[0063] PCR amplification was performed using primers consisting of 0134-F: 5'-ATTCGGCCGTACTGAAGAGC-3' and 0134-R: 5'-CCCAAGAGTGTTTCCGACAGA-3', yielding PCR amplification products. The results showed that the PCR amplification products obtained using *Stenotrophomonas maltophilia* genomic DNA as a template contained a 2891 bp DNA fragment. Using the single colonies obtained in step 3 as templates, PCR amplification was performed using primers consisting of 0134-F: 5'-ATTCGGCCGTACTGAAGAGC-3' and 0134-R: 5'-ATTCGGCCGTACTGAAGAGC-3' and 0134-R: 5'-ATTCGGCCGTACTGAAGAGC-3', respectively.

[0064] The primer pair consisting of -CCCAAGAGTGTTTCCGACAGA-3' was used for PCR amplification to obtain the PCR amplification product. Then, the following judgment was made: if the PCR amplification product of a single colony does not contain a DNA fragment of size 2891bp, then the single colony is a single colony that is positive for a single exchange.

[0065] 5. Inoculate the single colonies obtained in step 4 that have undergone a first exchange positive reaction into LB liquid medium containing 50 mg / L kanamycin, incubate overnight at 28°C with shaking, centrifuge, and collect the bacterial cells. Then wash the bacterial cells twice with LB liquid medium, and inoculate again into LB liquid medium, incubate at 28°C with shaking for 2-4 hours (to allow for a second exchange reaction), and obtain the culture solution. Spread the culture solution onto LB solid medium containing 20% ​​sucrose, and incubate at 28°C for 2-3 days to obtain several single colonies.

[0066] 6. Inoculate the single colonies obtained in step 5 onto LB solid medium (i.e., resistant plate) and LB solid medium (i.e., non-resistant plate) containing 50 mg / L kanamycin, and incubate overnight at 28°C. If a single colony can grow on the non-resistant plate but not on the resistant plate, then the single colony is preliminarily identified as a positive colony.

[0067] 7. Using the positive colonies or *Stenotrophomonas maltophilia* colonies initially identified in step 6 as templates, perform PCR amplification using primer pairs 0134-F: 5'-ATTCGGCCGTACTGAAGAGC-3' and 0134-R: 5'-CCCAAGAGTGTTTCCGACAGA-3' to obtain PCR amplification products. Then, perform the following judgment: if the PCR amplification product of a preliminarily identified positive colony contains a 2120 bp DNA fragment (with a 771 bp sequence deleted from the smlt0134 gene), the colony is determined to be a positive colony; otherwise, the colony is determined to be a non-positive colony. The PCR amplification product of *Stenotrophomonas maltophilia* colonies contains a 2891 bp DNA fragment.

[0068] The results of partial PCR amplification product detection by agarose gel electrophoresis are shown below. Figure 1 The left-middle image shows (WT represents *Stenotrophomonas maltophilia*, and the others are positive colonies initially identified in step 6). The results indicate that positive colonies 1, 17, and 30 initially identified in step 6 (in...) Figure 1 The left and right images (labeled 1, 17 and 30 respectively) were all identified as positive colonies.

[0069] 8. Using genomic DNA from Stenotrophomonas maltophilia, positive colony 1, positive colony 17, or positive colony 30 identified in step 7 as templates, PCR amplification was performed using primer pairs consisting of 0134-F: 5'-ATTCGGCCGTACTGAAGAGC-3' and 0134-R: 5'-CCCAAGAGTGTTTCCGACAGA-3' to obtain PCR amplification products; the PCR amplification products were then sequenced.

[0070] Agarose gel electrophoresis results of PCR amplification products of positive colonies 1, 17, and 30 are shown in the figure. Figure 1 The image in the middle right (WT represents Stenotrophomonas maltophilia, and 1, 17, and 30 represent positive colony 1, positive colony 17, and positive colony 30, respectively).

[0071] Sequencing results showed that the nucleotide sequences of the PCR amplification products of positive colonies 1, 17, and 30 were all as shown in SEQ ID NO:5. A 771 bp sequence from the smlt0134 gene was deleted from the genomic DNA of all three positive colonies.

[0072] Positive colony 1, positive colony 17 and positive colony 30 are all smlt0134 mutants.

[0073] II. Detection of the relative expression levels of the smlt0134 gene in the smlt0134 mutant obtained in Step 1 and in Stenotrophomonas maltophilia.

[0074] 1. Extract total RNA from the test bacteria (smlt0134 mutant or Stenotrophomonas maltophilia).

[0075] 2. Reverse transcribe the total RNA of the bacteria to be tested to obtain the cDNA of the bacteria.

[0076] 3. Detect the relative expression level of the smlt0134 gene in the cDNA of the test bacteria by real-time PCR (using the tmRNA gene as an internal reference gene).

[0077] The results showed that, compared with Stenotrophomonas maltophilia, the relative expression level of the smlt0134 gene was significantly reduced in the smlt0134 mutant obtained in step one.

[0078] III. Detection of the resistance of the smlt0134 mutant to rice blast fungus

[0079] The smlt0134 mutant in this experiment was the positive colony 1 identified in step one.

[0080] 1. Activate rice blast fungus

[0081] The preserved rice blast fungus Guy11 was inoculated onto CM solid medium and cultured at 28℃ for 5 days. Then, tender mycelia at the edge of the colony were picked and inoculated onto CM solid medium and cultured under light at 28℃. When the mycelia turned from white to black, the spores, i.e., the spores of rice blast fungus Guy11, were collected.

[0082] 2. Preparation of Guy11 spore solution

[0083] After completing step 1, add 2-3 ml of sterile water to the culture dish containing spores of *Guy11* blast fungus. Gently scrape the *Guy11* colonies with a glass spreader, filter the hyphae through a 40 μm filter, and finally collect the filtrate in a centrifuge tube. Count the number of spores in the filtrate using a hemocytometer, then dilute with water to obtain a *Guy11* spore solution with a concentration of 9-12 spores / μl.

[0084] Guy11 spore solution is best used immediately after washing to avoid germination due to prolonged storage.

[0085] 3. Preparation of bacterial culture of Stenotrophomonas maltophilia

[0086] (1) Add 1 ml of LB liquid medium and 10 μl of the test bacterial solution (Stenotrophomonas maltophiliae or smlt0134 mutant bacterial solution) to a 1.5 ml centrifuge tube, and incubate overnight at 28°C with shaking at 220 rpm to obtain the bacterial culture. Use a toothpick to streak the bacterial culture onto LB solid medium, and incubate at 28°C for 3 days to obtain a single colony.

[0087] (2) Inoculate a single colony into 5 ml of LB liquid medium and incubate overnight at 28°C with shaking at 220 rpm to obtain culture solution 1. Then, add LB liquid medium to culture solution 1 to obtain OD. 600nm 2. Culture medium with a value of 1.0.

[0088] 4. Co-cultivation

[0089] Mix 50 μl of Guy11 spore suspension with 10 μl of culture medium 2 or LB liquid medium (as a blank control) to obtain a mixture. Then spread the mixture on LB solid medium and incubate at 28°C for 5-6 days to observe the spore germination of Guy11.

[0090] Observation results are shown in Figure 2 (Guy11 served as the blank control; Sma+Guy11 was a co-culture of *Stenotrophomonas maltophilia* bacterial suspension and Guy11 spore suspension; Δsmlt0134+Guy11 was a co-culture of smlt0134 mutant bacterial suspension and Guy11 spore suspension). The results showed that both *Stenotrophomonas maltophilia* and the smlt0134 mutant significantly inhibited *Blastomyces oryzae* Guy11, and compared with *Stenotrophomonas maltophilia*, the smlt0134 mutant showed a significantly reduced (more than 10,000-fold) resistance to *Blastomyces oryzae* Guy11. Therefore, the smlt0134 protein can inhibit the growth of *Blastomyces oryzae* Guy11.

[0091] Example 2: Expression, purification, and in vitro toxicity assay of Smlt0134 protein.

[0092] I. In vitro expression and purification of Smlt0134 protein using a prokaryotic system

[0093] 1. Construction of recombinant plasmid pET30a-smlt0134

[0094] The small DNA fragment between the restriction endonucleases NdeⅠ and HindⅢ in plasmid pET30a(GenStar) was replaced with the smlt0134 gene, whose nucleotide sequence is shown in SEQ ID NO:2, to obtain the recombinant plasmid pET30a-smlt0134.

[0095] 2. Obtaining recombinant Escherichia coli

[0096] The recombinant plasmid pET30a-smlt0134 was transformed into E. coli BL21(DE3) to obtain recombinant E. coli.

[0097] 3. Expression and purification of Smlt0134 protein

[0098] (1) The recombinant Escherichia coli monoclonal obtained in step 2 was inoculated into 5 ml of LB liquid medium and cultured overnight at 28°C and 220 rpm to obtain culture solution 1.

[0099] (2) After completing step (1), inoculate the culture solution 1 into 500ml LB liquid medium and culture overnight at 28℃ and 220rpm to obtain culture solution 2.

[0100] (3) After completing step (2), take the culture liquid 2, centrifuge, and collect the bacterial cells.

[0101] (4) After completing step (3), resuspend the bacterial cells in protein lysis buffer (the solute and its concentration are 100mM NaCl and 1mM EDTA, and the solvent is pH 8.0, 50mM Tris-HCl buffer) (the ratio of protein lysis buffer to bacterial cells is 3mL:1g) to obtain a resuspension.

[0102] (5) After completing step (4), place the resuspended solution on ice and sonicate for 30-50 minutes (to disrupt cells), then centrifuge at 4°C and 14000g for 30 minutes and collect the supernatant. Detect the protein content in the supernatant.

[0103] (6) Equilibrate the Ni-NTA beads (Novagen) resin with binding buffer (solute and concentration of 300mM NaCl and 10-20mM imidazole, solvent of pH 8.0 and 50mM phosphate buffer), then add the supernatant collected in step (5) (the ratio of resin to supernatant is 1mL resin: 5-10mg protein in supernatant), mix at 4℃ for 1-4h.

[0104] (7) After completing step (6), pass the supernatant containing the resin through a manual chromatography column. Proteins containing the His×6 tag and other proteins are bound to the resin. Then wash the resin with wash buffer (solute and concentration of 300mM NaCl and imidazole, solvent of pH 8.0, 50mM phosphate buffer) with increasing concentrations of imidazole (20-50mM) for 4-8 column volumes until no other proteins flow out.

[0105] (8) After completing step (7), first wash the resin with 5 column volumes of elution buffer 1 (solute and concentration of 300mM NaCl and 250mM imidazole, solvent of pH 8.0 and 50mM phosphate buffer), and then wash the resin with 2 column volumes of elution buffer 2 (solute and concentration of 300mM NaCl and 350mM imidazole, solvent of pH 8.0 and 50mM phosphate buffer) to obtain the protein solution.

[0106] The solutions obtained after column chromatography were collected and subjected to SDS-PAGE sequentially. The results are as follows: Figure 3As shown in the figure. The results indicate that the Smlt0134 protein was obtained after the above steps.

[0107] (9) After completing step (8), concentrate the protein solution using an ultrafiltration tube (Millipore) of appropriate size and replace the buffer solution with protein storage solution (solute and its concentration is 0.5mM EDTA, 50mM NaCl and 5% glycerol, solvent is pH 8.0, 50mM Tris-HCl buffer) to obtain Smlt0134 protein solution that can be stored for a long time.

[0108] The concentration of Smlt0134 protein in the Smlt0134 protein solution is 180 μM.

[0109] II. In vitro toxicity assay of Smlt0134 protein

[0110] 1. Add 2-3 ml of sterile water to a culture dish containing spores of *Guy11* blast fungus. Gently scrape the *Guy11* colonies with a glass spreader, filter the hyphae through a 40 μm filter, and finally collect the filtrate in a centrifuge tube. Count the number of spores in the filtrate using a hemocytometer, then dilute with water to obtain a *Guy11* spore solution with a concentration of 9-12 spores / μl.

[0111] Guy11 spore solution is best used immediately after washing to avoid germination due to prolonged storage.

[0112] 2. Preparation of the mixture

[0113] Add 30 μl Guy11 spore solution, 30 μl water and 60 μl Smlt0134 protein solution to a PCR tube, and mix well with a pipette to obtain a 90 μM mixture 1.

[0114] Add 30 μl Guy11 spore solution and 90 μl Smlt0134 protein solution to a PCR tube, and mix well with a pipette to obtain a 120 μM mixture 2.

[0115] Add 30 μl of Guy11 spore solution and 90 μl of water to a PCR tube, and mix well with a pipette to obtain a 90 μM mixture 3 (as a negative control).

[0116] 3. Take a food storage container, first lay three layers of filter paper, then add sterile water until the filter paper is completely wetted but does not form streams of water. Then place a hydrophobic glass slide on the filter paper, and drop the mixture (mixture 1, mixture 2, or mixture 3) onto the hydrophobic glass slide (5 μl per drop), with 3 drops on each slide, which constitutes 3 replicates. Finally, cover the food storage container and incubate at 28°C in the dark for 12 hours.

[0117] 4. After completing step 3, invert the hydrophobic slide onto the slide and observe the germination and morphology of the spores under a microscope.

[0118] Observation results are shown in Figure 4 (Guy11 was the negative control; 90 μM was mixture 1, and 120 μM was mixture 2). The results showed that when spores of *Blastoma rice* Guy11 were treated with Smlt0134 protein in vitro, the treated spores could not germinate normally, were elongated, and showed signs of damage. Therefore, smlt0134 protein can inhibit the germination and normal growth of *Blastoma rice* Guy11 spores.

[0119] Based on the above experimental results, smlt0134 protein can resist rice blast fungus and is expected to become a new type of high-efficiency protein preparation.

[0120] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of smlt0134 protein in inhibiting rice blast fungus; The smlt0134 protein is either a1 or a2). a1) The amino acid sequence is that of the protein shown in SEQ ID No: 1; a2) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1).

2. The application of the nucleic acid molecule encoding the smlt0134 protein of claim 1 in inhibiting rice blast fungus.

3. The application according to claim 2, characterized in that: The nucleic acid molecule encoding the smlt0134 protein of claim 1 is a DNA molecule with the coding region as shown in SEQ ID NO:

2.

4. The application according to claim 2, characterized in that: The nucleic acid molecule encoding the smlt0134 protein of claim 1 is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:

2.

5. The application according to claim 2, characterized in that: The nucleic acid molecule encoding the smlt0134 protein of claim 1 is a DNA molecule derived from Stenotrophomonas maltophilia and having at least 70% homology with the DNA molecule shown in SEQ ID NO:2 and encoding any of the aforementioned smlt0134 proteins.

6. The application according to claim 1 or 2, characterized in that: The inhibition of rice blast fungus is manifested in inhibiting the growth of rice blast fungus, inhibiting the germination of rice blast fungus spores and / or inhibiting the spore growth of rice blast fungus.

7. The application according to claim 1 or 2, characterized in that: The rice blast fungus is *Pyroblastus oryzae* (rice blast fungus). Magnaporthe oryzae Guy11.

8. A method for inhibiting rice blast fungus, comprising directly treating rice blast fungus with the smlt0134 protein described in claim 1.

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