Application of smlt0134 protein in inhibition of pyricularia oryzae

Abstract

The invention discloses an application of smlt0134 protein in inhibition of pyricularia oryzae of rice. Experiments prove that the smlt0134 protein can inhibit the pyricularia oryzae, which is mainly embodied in inhibiting the growth of the pyricularia oryzae, inhibiting the spore germination of the pyricularia oryzae and / or inhibiting the spore growth of the pyricularia oryzae. Therefore, the smlt0134 protein is expected to be developed into a novel efficient protein preparation. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of smlt0134 protein in inhibiting rice blast fungus. Background Art

[0002] Rice blast, caused by Magnaporthe oryzae, is one of the most destructive diseases of rice. At present, the prevention and control measures for rice blast are mainly chemical agents. The best chemical agent is 6% kasugamycin aqueous solution, which has a longer duration of efficacy; followed by 40% pyraclostrobin emulsifiable concentrate; and finally 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 pollution to the environment and is very likely to cause drug resistance. It is necessary to explore more green and safe control methods. In recent years, research on the prevention and control of rice blast by biological control methods has become more and more extensive, 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 field control effect on rice blast [J]. Plant Protection, 2023, 49(2): 48-56.), Bacillus velez 5-8 (Li Shenyu, Jia Xiangzi, Guo Juntao, et al. Identification of rice blast biocontrol bacteria 5-8 and its biocontrol mechanism research Research [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 pathogen and rapeseed sclerotinia pathogen [D]: Master's degree 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 Science: 1-8). These biocontrol strains show high biocontrol potential against rice blast. The use of biocontrol agents can prevent and control the occurrence of diseases in a green and safe way, but biocontrol agents are easily affected by environmental factors and lose their biocontrol effect.

[0003] In addition, discovering new rice varieties resistant to rice blast and choosing appropriate cultivation management are also important measures to prevent and control rice blast. However, there are many problems such as the lack of resistant germplasm resources and cumbersome and inefficient farming operations. It is urgent to develop plant-based or microbial protein preparations to effectively prevent and control the occurrence of rice blast. Summary of the invention

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

[0005] The present invention firstly protects the application of smlt0134 protein, which can be S1) or S2):

[0006] S1) inhibiting rice blast fungus;

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

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

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

[0010] a2) a fusion protein obtained by connecting a tag to the N-terminus or / and the 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 specific labels of the proteins in a2) above are shown in Table 1.

[0013] Table 1. Sequences of tags

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

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

[0016] The protein in a3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

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

[0018] The present invention also protects the use of a nucleic acid molecule encoding any of the above-mentioned smlt0134 proteins (i.e., smlt0134 genes), which may be S1) or S2):

[0019] S1) inhibiting rice blast fungus;

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

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

[0022] (b1) a DNA molecule whose coding region is shown in SEQ ID NO: 2;

[0023] (b2) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2;

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

[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-mentioned smlt0134 proteins.

[0026] The stringent conditions are hybridization and membrane washing twice at 68°C in a 2×SSC, 0.1% SDS solution, each for 5 min, and hybridization and membrane washing twice at 68°C in a 0.5×SSC, 0.1% SDS solution, each for 15 min.

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

[0028] Among them, 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] A person skilled in the art can easily mutate the nucleotide sequence encoding any of the above-mentioned smlt0134 proteins by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 70% or higher identity with the nucleotide sequence of any of the above-mentioned smlt0134 proteins isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode any of the above-mentioned smlt0134 proteins.

[0030] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 70% or more, 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence encoding the amino acid sequence of the present invention, such as the smlt0134 protein shown in SEQ ID NO:1. Identity can be evaluated by the naked eye or by 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 applications, the inhibition of rice blast fungus may be embodied 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 may specifically be rice blast fungus (Magnaportheoryzae) Guy11.

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

[0034] The method for inhibiting rice blast fungus protected by the present invention may specifically be method 1, which may be to directly or indirectly treat rice blast fungus with any of the above-mentioned smlt0134 proteins.

[0035] The method for inhibiting rice blast fungus protected by the present invention can specifically be method 2, which can be to directly or indirectly treat rice blast fungus with Stenotrophomonas maltophilia or a mutant of Stenotrophomonas maltophilia; compared with Stenotrophomonas maltophilia, the nucleic acid molecule encoding any of the above-mentioned smlt0134 proteins in the genome of the mutant of Stenotrophomonas maltophilia is partially deleted, resulting in the inability to express the smlt0134 protein.

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

[0037] In any of the above methods, the inhibition of rice blast fungus may be embodied 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 above methods, the rice blast fungus may be rice blast fungus (Magnaporthe oryzae) Guy11.

[0039] Experiments have shown that the SMLT0134 protein can inhibit rice blast fungus, and the inhibition of rice blast fungus can be reflected in 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. At present, the drug resistance problem caused by the abuse of antibiotics has brought serious threats, and the SMLT0134 protein is expected to be developed into a new type of high-efficiency protein preparation. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the agarose gel electrophoresis detection result of some PCR amplification products in steps 7 and 8 of Example 1.

[0041] Figure 2 These are the results of testing the resistance of the smlt0134 mutant to rice blast fungus.

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

[0043] Figure 4 These are the results of in vitro toxicity testing of Smlt0134 protein. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0045] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0046] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

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

[0048] In the following examples, rice blast fungus (Magnaporthe oryzae) Guy11 is recorded in the following document: 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 document is M. oryzae strain Guy11. The Genbank of rice blast fungus (Magnaporthe oryzae) Guy11 in NCBI is GSE18361. Hereinafter, rice blast fungus (Magnaporthe oryzae) Guy11 is referred to as rice blast fungus 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 (ie, the smlt0134 gene) is shown in SEQ ID NO: 2.

[0050] The culture medium involved in the following examples is as follows:

[0051] The solutes and concentrations of the LB liquid culture medium were 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride, the solvent was water, and the pH value was 5.8; the medium was sterilized by high-temperature steam at 121°C for 20 min.

[0052] The solutes and their concentrations of the LB solid culture medium are 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 15 g / L agar powder, the solvent is water, and the pH value is 5.8; high-temperature steam sterilization at 121°C for 20 min.

[0053] The solutes and their concentrations of CM solid culture medium are NaNO36g / L, KCl 0.52g / L, MgSO4.7H2O 0.52g / L, KH2PO41.52g / L, Peptone 2g / L, Yeast extract 1g / L, Casamino acid 1g / L, D-Glucose10g / L, Trace elements 1ml / L, Vitamin solution 1ml / L and agar powder 15g / L. The solvent is water. The pH value is adjusted to 6.5 with 10MNaOH. High-temperature steam sterilization is carried out at 121℃ for 20min. Each 1L of Trace elements consists of 22gZnSO4.7H2O, 11g H3BO3, 5g MnCl2.4H2O, 5g FeSO4.7H2O, 1.7g CoCl2.6H2O, 1.6gCuSO4.5H2O, 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 cloning vectors derived from the Escherichia co / iplasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene, 145 (1994) 69-73. The public can obtain the experimental materials from the State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences (i.e., the applicant).

[0055] 1. Obtaining the smlt0134 mutant

[0056] 1. Extract genomic DNA of Stenotrophomonas maltophilia and use it as a template, using primer 0134-a: 5'-ACAGCTATGACATGATTAC GAATTC AAGGCGCTGCATATCGTGCAGG-3' (underlined is the recognition site of restriction endonuclease EcoRI) and primer 0134-b: 5'-TCCGGGAGCATGCGGAATGCATCATCACGGCTGGGTGC-3' were used for PCR amplification to recover the 907 bp left arm 0134LB of the smlt0134 gene. Genomic DNA of Stenotrophomonas maltophilia was extracted and used as a template, and primer 0134-c: 5'-TCCGGGAGCATGCGGAATGCATCATCACGGCTGGGTGC-3' was used for PCR amplification.

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

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

[0059] 2. Cut the vector PK18mobSacB with 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 an intermediate vector. Cut the intermediate vector with 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. The sequencing results showed that the recombinant vector PK18mobSacB-0134LB-0134RB was a recombinant plasmid obtained by inserting DNA sequence 1 (nucleotide sequence as shown in SEQ ID NO: 3) into the restriction endonuclease EcoRI recognition site of the vector PK18mobSacB and inserting DNA sequence 2 (nucleotide sequence as shown in SEQ ID NO: 4) into the restriction endonuclease HindIII recognition site.

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

[0062] 4. Using the genomic DNA of Stenotrophomonas maltophilia as a template, 0134-F: 5'

[0063] PCR amplification was performed using a primer pair consisting of 0134-F: 5'-ATTCGGCCGTACTGAAGAGC-3' and 0134-R: 5'-CCCAAGAGTGTTTCCGACAGA-3' to obtain a PCR amplification product; the results showed that the PCR amplification product obtained using the genomic DNA of Stenotrophomonas maltophilia as a template contained a DNA fragment of 2891 bp in size. The single colony obtained in step 3 was used as a template, using 0134-F: 5'-ATTCGGCCGTACTGAAGAGC-3' and 0134-R: 5'

[0064] -CCCAAGAGTGTTTCCGACAGA-3' was used for PCR amplification to obtain a PCR amplification product; then the following judgment was made: if the PCR amplification product of a single colony did not contain a DNA fragment of 2891 bp in size, the single colony was a single colony with a positive exchange.

[0065] 5. Inoculate the single colony obtained in step 4 with the first exchange positive into LB liquid medium containing 50 mg / L kanamycin, culture at 28°C overnight with shaking, centrifuge and collect the cells. Then wash the cells twice with LB liquid medium, inoculate into LB liquid medium again, culture at 28°C with shaking for 2-4h (for the purpose of secondary exchange), and obtain the culture solution. Then spread the culture solution on LB solid medium containing 20% ​​sucrose, culture at 28°C for 2-3 days, and obtain several single colonies.

[0066] 6. The single colonies obtained in step 5 were inoculated into LB solid medium (i.e., resistant plate) and LB solid medium (i.e., non-resistant plate) containing 50 mg / L kanamycin, and cultured overnight at 28°C. If a single colony can grow on a non-resistant plate but cannot grow on a resistant plate, the single colony is preliminarily judged as a positive colony.

[0067] 7. Using the positive colonies or the Stenotrophomonas maltophilia colonies preliminarily determined in step 6 as templates, PCR amplification was performed using the primer pair consisting of 0134-F: 5'-ATTCGGCCGTACTGAAGAGC-3' and 0134-R: 5'-CCCAAGAGTGTTTCCGACAGA-3' to obtain PCR amplification products; then the following judgment was made: if the PCR amplification product of a preliminarily determined positive colony contained a DNA fragment of 2120 bp (771 bp sequence in the smlt0134 gene was deleted), the colony was determined to be a positive colony; otherwise, the colony was determined to be a non-positive colony. The PCR amplification product of the Stenotrophomonas maltophilia colony contained a DNA fragment of 2891 bp.

[0068] The results of agarose gel electrophoresis of some PCR amplification products are shown in Figure 1 Middle left picture (WT is Stenotrophomonas maltophilia, the others are positive colonies preliminarily determined in step 6). The results show that positive colonies 1, 17 and 30 (in the Figure 1 The colonies marked in the middle left figure are 1, 17 and 30, respectively, and are all identified as positive colonies.

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

[0070] The agarose gel electrophoresis results of the PCR amplification products of positive colony 1, positive colony 17 and positive colony 30 are shown in Figure 1 Middle right picture (WT is Stenotrophomonas maltophilia, 1, 17 and 30 are positive colony 1, positive colony 17 and positive colony 30, respectively).

[0071] The sequencing results showed that the nucleotide sequences of the PCR amplification products of positive colony 1, positive colony 17 and positive colony 30 were all shown in SEQ ID NO: 5. In the genomic DNA of positive colony 1, positive colony 17 and positive colony 30, a 771 bp sequence in the smlt0134 gene was deleted.

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

[0073] 2. Detection of the relative expression of the smlt0134 gene in the smlt0134 mutant obtained in step 1 and in Stenotrophomonas maltophilia

[0074] 1. Extract the total RNA of 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 to be tested.

[0076] 3. Fluorescence quantitative PCR was used to detect the relative expression level of the smlt0134 gene in the cDNA of the test bacteria (with the tmRNA gene as the internal reference gene).

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

[0078] 3. Detection of the resistance of smlt0134 mutant to rice blast fungus

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

[0080] 1. Activate rice blast fungus

[0081] The preserved rice blast fungus Guy11 was inoculated into CM solid culture medium, cultured at 28°C for 5 days, young mycelium at the edge of the colony was picked and inoculated into CM solid culture medium, cultured in the light at 28°C, and spores, i.e., spores of rice blast fungus Guy11, were collected when the mycelium turned from white to black.

[0082] 2. Preparation of Guy11 spore solution

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

[0084] It is best to wash the Guy11 spore liquid before use to avoid germination due to long-term storage.

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

[0086] (1) Add 1 ml of LB liquid medium and 10 μl of the test bacterial solution (S. maltophilia bacterial solution or smlt0134 mutant bacterial solution) to a 1.5 ml centrifuge tube, and culture overnight at 28°C and 220 rpm to obtain a cultured bacterial solution. Use a toothpick to streak the cultured bacterial solution on LB solid medium, and culture at 28°C for 3 days to obtain a single clone.

[0087] (2) Inoculate a single clone into 5 ml LB liquid medium and culture overnight at 28°C and 220 rpm to obtain culture solution 1. Then add LB liquid medium to culture solution 1 to obtain OD 600nm The value is 1.0 for culture broth 2.

[0088] 4. Co-cultivation

[0089] 50 μl of Guy11 spore solution and 10 μl of culture solution 2 or LB liquid medium (as a blank control) were mixed to obtain a mixed solution, which was then spread on LB solid medium and cultured at 28° C. for 5-6 days to observe the germination of Guy11 spores.

[0090] Observation results are shown in Figure 2 (Guy11 is a blank control, Sma+Guy11 is a co-culture of the bacterial solution of S. maltophilia and the spore solution of Guy11, and Δsmlt0134+Guy11 is a co-culture of the bacterial solution of the smlt0134 mutant and the spore solution of Guy11). The results showed that both S. maltophilia and the smlt0134 mutant could significantly inhibit the rice blast fungus Guy11, and compared with S. maltophilia, the ability of the smlt0134 mutant to resist the rice blast fungus Guy11 was significantly reduced (reduced by more than 10,000 times). This shows that the smlt0134 protein can inhibit the growth of the rice blast fungus Guy11.

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

[0092] 1. Prokaryotic expression and purification of Smlt0134 protein in vitro

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

[0094] The small DNA fragment between the restriction endonucleases NdeⅠ and HindⅢ of the plasmid pET30a (GenStar) was replaced with the smlt0134 gene with a nucleotide sequence as 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 E. coli monoclone obtained in step 2 was inoculated into 5 ml of LB liquid culture medium and cultured overnight at 28° C. and 220 rpm with shaking to obtain culture solution 1.

[0099] (2) After completing step (1), culture solution 1 was inoculated into 500 ml of LB liquid medium and cultured overnight at 28° C. and 220 rpm with shaking to obtain culture solution 2.

[0100] (3) After completing step (2), take the culture solution 2, centrifuge it, and collect the bacteria.

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

[0102] (5) After completing step (4), the resuspension was placed on ice and sonicated for 30-50 min (to disrupt cells), and then centrifuged at 4°C and 14,000 g for 30 min to collect the supernatant. The protein content in the supernatant was detected.

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

[0104] (7) After completing step (6), the supernatant containing the resin is passed through a manual chromatography column, and the protein containing the His×6 tag and the impurity protein are bound to the resin. The resin is then washed with a wash buffer (solute and its concentration are 300 mM NaCl and imidazole, and the solvent is pH 8.0, 50 mM phosphate buffer) with increasing imidazole concentrations (20-50 mM) for 4-8 column volumes until no impurity protein flows out.

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

[0106] The above column solutions were collected and subjected to SDS-PAGE in sequence. The results were as follows: Figure 3The results showed that after the above steps, Smlt0134 protein was obtained.

[0107] (9) After completing step (8), the protein solution was concentrated using an ultrafiltration tube of appropriate size (Millipore) and the buffer in which it was located was replaced with a protein storage solution (solutes and their concentrations were 0.5 mM EDTA, 50 mM NaCl and 5% glycerol, and the solvent was pH 8.0, 50 mM Tris-HCl buffer) to obtain a Smlt0134 protein solution that can be stored for a long time.

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

[0109] 2. In vitro toxicity detection of Smlt0134 protein

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

[0111] It is best to wash the Guy11 spore liquid before use to avoid germination due to long-term storage.

[0112] 2. Preparation of mixed solution

[0113] Add 30 μl of Guy11 spore solution, 30 μl of water and 60 μl of Smlt0134 protein solution to the PCR tube, and pipette to mix them evenly to obtain a mixed solution 1 with a concentration of 90 μM.

[0114] Add 30 μl of Guy11 spore solution and 90 μl of Smlt0134 protein solution to the PCR tube, and pipette to mix them evenly to obtain a mixed solution 2 with a concentration of 120 μM.

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

[0116] 3. Take a fresh-keeping box, first lay 3 layers of filter paper, then add sterile water until the filter paper is completely wet but not gathered into a stream, then place a hydrophobic glass slide on the filter paper, drop the mixed solution (mixed solution 1, mixed solution 1 or mixed solution 3) on the hydrophobic glass slide (5 μl per drop), drop 3 drops on each glass slide, i.e. 3 replicates; finally, cover the fresh-keeping box with a lid and incubate at 28°C in the dark for 12 h.

[0117] 4. After completing step 3, place the hydrophobic glass slide upside down on a glass slide and observe the germination and morphology of the spores under a microscope.

[0118] Observation results are shown in Figure 4 (Guy11 is the negative control, 90μM is the mixture 1, and 120μM is the mixture 2). The results showed that when the spores of the rice blast fungus Guy11 were treated with Smlt0134 protein in vitro, the spores of the rice blast fungus Guy11 could not germinate normally, but were elongated and damaged. This shows that the smlt0134 protein can inhibit the spore germination and normal growth of the rice blast fungus Guy11.

[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. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of smlt0134 protein, S1) or S2): S1) inhibiting rice blast fungus; S2) cultivating rice resistant to rice blast fungus; The smlt0134 protein is a1) or a2) or a3): a1) the amino acid sequence is the protein shown in SEQ ID No: 1; a2) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of a1); 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.

2. The use of a nucleic acid molecule encoding the smlt0134 protein of claim 1, which is S1) or S2): S1) inhibiting rice blast fungus; S2) Cultivating rice resistant to rice blast fungus.

3. The use according to claim 2, characterized in that: The nucleic acid molecule encoding the smlt0134 protein in claim 1 is a DNA molecule of (b1) or (b2) or (b3) or (b4): (b1) a DNA molecule whose coding region is shown in SEQ ID NO: 2; (b2) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b3) a DNA molecule that hybridizes with the DNA molecule defined in (b1) or (b2) under stringent conditions and encodes any of the above-mentioned smlt0134 proteins; (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-mentioned smlt0134 proteins.

4. The use according to claim 1 or 2, characterized in that: The inhibition of rice blast fungus is embodied in 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.

5. The use according to any one of claims 1 to 4, characterized in that: The rice blast fungus is Magnaporthe oryzae Guy11.

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

7. A method for inhibiting rice blast fungus, comprising directly or indirectly treating rice blast fungus with Stenotrophomonas maltophilia or a mutant of Stenotrophomonas maltophilia; Compared with Stenotrophomonas maltophilia, the nucleic acid molecule encoding the smlt0134 protein in claim 1 is partially deleted in the genome of the Stenotrophomonas maltophilia mutant, resulting in the inability to express the smlt0134 protein.

8. The method according to claim 7, characterized in that: The Stenotrophomonas maltophilia is Stenotrophomonas maltophilia CGMCC NO.1.1788.

9. The method according to any one of claims 6 to 8, characterized in that: The inhibition of rice blast fungus is embodied in 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.

10. The method according to any one of claims 6 to 9, characterized in that: The rice blast fungus is Magnaporthe oryzae Guy11.

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