Use of bacterial protein bGM to improve plant disease resistance

Abstract

The application provides application of a bacterial protein bGM to improve plant disease resistance, and relates to the technical field of biological gene engineering. The bGM protein with the amino acid sequence shown in SEQ ID No. 3 is used to improve plant disease resistance. The bGM protein in bacteria is used to improve plant disease resistance for the first time, and the available disease resistance gene resources can be greatly expanded. The bacterial bGM protein in the application can improve disease resistance in both dicotyledonous plants tobacco and monocotyledonous plants rice, while traditional disease resistance genes are often limited to specific species to play a function. The bGM protein in the application is a protein derived from bacteria, and the research on disease resistance in bacteria is relatively deep, and there are a large number of disease resistance gene resources, which are directly applied to plant disease resistance breeding, and time and labor are saved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to application of a bacterial protein bGM to improve plant disease resistance. BACKGROUND

[0002] Plants are threatened by various pathogens during growth and development. Rice is the staple food of more than half of the world's population and is often threatened by fungal diseases such as rice blast. Rice blast, caused by the filamentous fungus Magnaporthe oryzae, can cause a 10%-30% reduction in rice yield. Compared with spraying pesticides, exploring disease-resistant genes and cultivating disease-resistant varieties is an economical and effective method to prevent and control rice blast, greatly reducing damage to the environment. However, the traditional method of cloning disease-resistant genes requires relying on disease-resistant resource varieties, and cloning genes is time-consuming and laborious. Moreover, disease-resistant genes are usually limited to a single species, and will lose disease resistance after a few years of use, which leads to fewer and fewer available disease-resistant gene resources.

[0003] Bacteria have very rich disease-resistant gene resources. Gasdermin protein is an ancient disease-resistant protein that exists widely in bacteria, fungi and animals, but is absent in plants. There is no report on the use of this protein as an exogenous disease-resistant protein in plants for disease resistance. Therefore, we attempt to use this protein to provide a strategy for cultivating disease-resistant varieties and expand the available disease-resistant resources. SUMMARY

[0004] The purpose of the present application is to provide a bacterial protein bGM to improve plant disease resistance. The bacterial bGM protein is expressed in plants for the first time to improve plant disease resistance.

[0005] To achieve the above purpose, the present application provides a biological material containing a bacterial protein bGM for use in at least one of S1-S6:

[0006] The biological material is any of the following:

[0007] A1), a protein, which is any of the following:

[0008] B1) the amino acid sequence is a protein as shown in SEQ ID No. 3;

[0009] B2) a fusion protein obtained by connecting the N-terminal or / and C-terminal of the protein of B1) with a protein tag;

[0010] A2), a substance for regulating the expression of a gene encoding the protein of A1);

[0011] A3), a substance for regulating the activity and / or content of the protein of A1);

[0012] S1-S6 are:

[0013] S1) increasing disease resistance of plants;

[0014] S2) producing a product for increasing disease resistance of plants;

[0015] S3) breeding plants with increased disease resistance;

[0016] S4) producing a product for breeding plants with increased disease resistance;

[0017] S5) improving or producing a product for improving high disease resistance varieties;

[0018] S6) plant breeding.

[0019] In the present application, the disease resistance of the plants includes resistance to infection by at least one pathogen selected from the group consisting of Phytophthora capsici Leonian, Pyricularia oryzae Cavara; and the plants include tobacco and rice.

[0020] In the above uses, the substance for regulating expression of the gene encoding the protein includes the following biological materials:

[0021] C1) a nucleic acid molecule encoding a protein having the amino acid sequence shown in SEQ ID No. 3;

[0022] C2) an expression cassette containing the nucleic acid molecule of C1);

[0023] C3) a recombinant vector containing the nucleic acid molecule of C1) or a recombinant vector containing the expression cassette of C2);

[0024] C4) a recombinant microorganism containing the nucleic acid molecule of C1), or a recombinant microorganism containing the expression cassette of C2), or a recombinant microorganism containing the recombinant vector of C3).

[0025] The nucleic acid molecule of C1) is any one of the following:

[0026] a1) a nucleotide sequence shown in SEQ ID No. 1;

[0027] a2) a sequence obtained by optimizing the nucleotide sequence shown in SEQ ID No. 1 according to the rice codon.

[0028] In the present application, the sequence obtained by optimizing the rice codon is shown in SEQ ID No. 2.

[0029] The present application also provides a method for breeding a transgenic plant with increased disease resistance, comprising the following steps: introducing a protein shown in SEQ ID No. 3 into a plant of interest to obtain a transgenic plant with increased disease resistance.

[0030] In the method, the protein is introduced into the target plant by a protein expression vector, and the vector comprises pRHE.

[0031] In the method, the transgenic plant is constructed by using a pathogenic bacteria-induced gene promoter, and the method comprises the following steps: combining a regulatory element uORFs in the promoter of another pathogenic bacteria-induced gene TBF1 into a promoter PR10a-uORFs after the pathogenic bacteria-induced PR10a promoter; and using the PR10a-uORFs to construct a bGM protein plant transgenic strain.

[0032] In the method, the sequence of the pathogenic bacteria-induced PR10a promoter is shown in SEQ ID No. 18; and the nucleotide sequence of the regulatory element uORFs is shown in SEQ ID No. 19.

[0033] In the method, the target plant is rice.

[0034] The method improves the resistance of the bGM protein rice transgenic strain to Magnaporthe oryzae.

[0035] Compared with the prior art, the application has the following beneficial effects:

[0036] The application firstly expresses the bacterial bGM protein in a plant to enhance the disease resistance of the plant. The bGM protein is transiently expressed in Nicotiana benthamiana, which can cause the necrosis of tobacco cells, i.e., a hypersensitive necrosis reaction. After the bGM is transiently expressed in the Nicotiana benthamiana, there is accumulation of reactive oxygen species (ROS), which enhances the immunity of the tobacco cells. The transient expression of the bGM can also inhibit the infection of Phytophthora capsici and improve the disease resistance of the tobacco to the Phytophthora capsici.

[0037] The application constructs a vector by combining the recombinant PR10a-uORFs promoter with the bGM, transforms the rice, and obtains a transgenic rice plant. Through the identification of the reactive oxygen species burst of the transgenic rice plant, the expression of the resistance genes and the disease resistance to Magnaporthe oryzae, the expression amount of the disease-related genes PAL, OsPR1b, OsPR5 and OsPR10 in the transgenic rice plant is up-regulated. It is shown that the P PR10a-uORF : The disease resistance of the bGM strain is improved. The transgenic rice plant P PR10a-uORF : The lesion area of the bGM is obviously smaller than that of the wild type, which indicates that the strain improves the resistance to Magnaporthe oryzae. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is the cell hypersensitive necrosis reaction result of the tobacco leaf injected by the agrobacterium in Example 2.

[0039] Figure 2 It is the reactive oxygen accumulation and disease resistance gene expression determination result of the tobacco leaf injected in Example 2.

[0040] Figure 3 Results of testing tobacco for resistance to P. capsici for transient expression of bGM in Example 2.

[0041] Figure 4 Results of screening for Magnaporthe-induced expression genes in Example 3.

[0042] Figure 5 Plasmid map of recombinant vector pRHE-PR10a-uORFs-bGM in rice in Example 3.

[0043] Figure 6 Results of disease phenotype identification of rice and detection of disease-related genes in transgenic rice plants in Example 3.

[0044] Figure 7 Results of detection of Magnaporthe resistance of transgenic rice line PPR10a-uORF:bGM in Example 3. DETAILED DESCRIPTION

[0045] The present application provides use of a biological material comprising bacterial protein bGM in at least one of S1-S6:

[0046] The biological material is any one of the following:

[0047] A1), a protein, which is any one of the following:

[0048] B1) a protein with an amino acid sequence as shown in SEQ ID No. 3;

[0049] B2) a fusion protein obtained by connecting the N-terminal or / and C-terminal of the protein of B1) with a protein tag;

[0050] A2), a substance that regulates expression of a gene encoding the protein of A1);

[0051] A3), a substance that regulates activity and / or content of the protein of A1);

[0052] The S1-S6 are:

[0053] S1) increasing plant disease resistance;

[0054] S2) preparing a product for improving plant disease resistance;

[0055] S3) breeding plants with improved disease resistance;

[0056] S4) preparing a product for breeding plants with improved disease resistance;

[0057] S5) improving high disease resistance varieties or preparing a product for high disease resistance varieties;

[0058] S6) Plant breeding.

[0059] In order to facilitate purification or detection of the protein in A1), a tag protein can be attached to the amino-terminal or carboxy-terminal end of the protein consisting of the amino acid sequence shown in SEQ ID No. 3 in the sequence listing.

[0060] The tag protein includes a GST (glutathione S-transferase) tag protein, a 6xHis tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a 5Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0061] In the present application, the substance that regulates the activity and / or content of the protein can be a substance that regulates the expression of a gene encoding the protein bGM.

[0062] In the present application, the purpose of the plant breeding can include improving the disease resistance of the plant. The improvement of the disease resistance of the plant can be achieved by expressing a gene encoding the protein bGM.

[0063] In the above use, the disease resistance of the plant includes resistance to infection by at least one pathogen of Phytophthora capsici Leonian, Pyricularia oryzae Cavara; and the plant includes tobacco, rice.

[0064] In the above use, the substance that regulates the expression of a gene encoding the protein includes the following biological material:

[0065] C1) a nucleic acid molecule encoding the protein shown in the nucleotide sequence of SEQ ID No. 3;

[0066] C2) an expression cassette containing the nucleic acid molecule of C1);

[0067] C3) a recombinant vector containing the nucleic acid molecule of C1), or a recombinant vector containing the expression cassette of C2);

[0068] C4) a recombinant microorganism containing the nucleic acid molecule of C1), or a recombinant microorganism containing the expression cassette of C2), or a recombinant microorganism containing the recombinant vector of C3).

[0069] The nucleic acid molecule of C1) above is any one of:

[0070] a1) the nucleotide sequence shown in SEQ ID No. 1;

[0071] a2) a sequence obtained based on the nucleotide sequence shown in SEQ ID No. 1 according to plant codon optimization.

[0072] The protein bGM gene described in the present application can be any nucleotide sequence capable of encoding the protein bGM. Considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0073] As an implementable manner, the sequence obtained according to rice codon optimization in the present application is shown in SEQ ID No. 2.

[0074] The bGM protein described in the present application can improve the disease resistance in both dicotyledonous tobacco and monocotyledonous rice.

[0075] In the present application, the N-terminal (bGM) sequence of bacterial bGSDM is synthesized according to rice codon optimization as shown in SEQ ID No. 2, and then transiently expressed in plants to improve the immunity of the plants. The bGM sequence in the present application is preferably derived from the bacterium Runella zeae DSM 19591. In the present application, the bGM sequence is connected to the tobacco expression vector pCAMBIAsuper1300-GFP by homologous recombination method, and then transiently expressed in plants. In the present application, the host bacteria used for transient expression in plants is preferably Agrobacterium GV3101. The bGM of the present application can induce hypersensitive necrosis reaction in tobacco. The accumulation of reactive oxygen species (ROS) in the leaf position expressing bGM enhances the immunity of tobacco cells.

[0076] The present application also provides a method for cultivating a transgenic plant with improved disease resistance, comprising the following steps: introducing the protein as shown in SEQ ID No. 3 into the target plant to obtain a transgenic target plant with improved disease resistance.

[0077] In the above method, the protein is introduced into the target plant by a protein expression vector to obtain the transgenic target plant, and the vector comprises pRHE.

[0078] In the above method, the transgenic target plant is constructed using a pathogen-induced gene promoter, and the method comprises the following steps: combining the regulatory elements uORFs in the promoter of another pathogen-induced gene TBF1 with the pathogen-induced PR10a promoter to form a new promoter PR10a-uORFs, and using PR10a-uORFs to construct a bGM protein plant transgenic strain.

[0079] In the above method, the promoter sequence of the pathogen-induced PR10a is shown in SEQ ID No. 18; and the nucleotide sequence of the regulatory element uORFs is shown in SEQ ID No. 19.

[0080] In the method, the target plant is rice.

[0081] The disease resistance of the bGM protein plant transgenic line obtained by the method is improved, and in particular, the disease resistance of the bGM protein rice transgenic line to Magnaporthe oryzae is improved.

[0082] The present application directly uses the bGM protein in bacteria to improve the disease resistance of tobacco and rice, which can greatly expand the available disease resistance gene resources. In the present application, the bacterial bGM protein can improve the disease resistance in both dicotyledonous tobacco and monocotyledonous rice. Traditional disease resistance genes are often limited to specific species for function. The bGM protein in the present application is a protein derived from bacteria, and the research on disease resistance in bacteria is relatively deep, and there are a large number of disease resistance gene resources. Direct application in plant disease resistance breeding can save time and effort.

[0083] In order to further illustrate the present application, the application of the bacterial protein bGM to improve the disease resistance of plants provided by the present application will be described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0084] The test materials used in the present application and their sources include the following:

[0085] Strain material:

[0086] Bacteria Runella zeae, purchased from BioVector NTCC typical culture preservation center, item number DSM 19591.

[0087] Phytophthora capsici LT263, given by Professor Luo Zhaoxi of Huazhong Agricultural University.

[0088] Magnaporthe oryzae EA18 (Magnaporthe oryzae EA18), reference: Wang Y, Yang L, Ma C, Zhou Y, Zhao M, Bi R, Liang X, Peng YL, Yang J, Kang Z, Li G. Genome Sequence of Magnaporthe oryzae EA18 Virulent to Multiple Widely Used Rice Varieties. Mol Plant Microbe Interact. 2022 Aug; 35(8): 727-730.

[0089] The experimental methods used in the following examples are conventional molecular biology methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified. Example 1 Sequence optimization of the N-terminal domain of bacterial bGSDM protein (bGM) for plants

[0090] The bacterial bGM (Gene accession in the IMG database: 2525253496) was optimized for rice codon using an online codon optimization tool (https: / / www.novopro.cn / tools / codon-optimization.html).

[0091] The N-terminal sequence of the bacterial bGSDM before optimization is (bGM, 741 bp):

[0092] ATGGAATGTAATGACCCGTTTGTAGTAGCATTGAAGGATAAAGGATATAGCCTTGTCGCCTATCCTAAGACGTCTATCAGACCTTTGCATATTTATGAGCATACTATTAAAAATGCTTTTAAAAGAATATGGATTCAATCGGAAGCACAACCAACAAGTGGTTTTATAAAGTCGCTATTTTCTGATAAGATTCATGGCGCAATTGGCTTAAGTGATGGGCAAGGCATAGATATAGATTTACGAAAAACGAATAGTTTGTCTTCAGCCGTTGCCGCTAAGATACTGGAAAGCTATTTTCAAGATTCAGCACCAAGTTTTGACTTAGCTTTTGAAAACAGTAGTTCCGTTATTTTTCATATAGAAGAAATTATAACTACGGATGCGGATGAAATAAGCTTAAGAAATTGGCTTAATGACAATCAAAATGAGTTGAGGGAAATTTATAAAGAAGAAATAAAAAAAGGGAATTTCTTTGTGGCTACGTCTTTACTAAGAGCTAAGAAAATGCGAATGCAATTTGAGAGAAAGAACAAAGGCGAGTTAGGAGTTGATGTGAGTAAAATCAAGAATTTACCTGTTGATGCAAAATTAGAATCAAAGATAGAGGGTTCAACCTATGATAGGTTGGTATTTGAAACTCCTGATGAAGGGATTGTCTTTGGGGTCAAATTAGTTCGACTTTTTTTTAGCGATAATGGTATTCTTACAATTGATAAAAAACAAGACTTTAACCGAGTTTTA (SEQ ID NO. 1).

[0093] The bacterial bGM nucleotide sequence optimized according to the rice codon preference is as follows (741 bp):

[0094] ATGGAGTGCAACGACCCGTTCGTGGTGGCCCTCAAGGACAAGGGCTACAGCCTCGTGGCGTACCCAAAGACGAGCATCCGCCCACTCCACATCTACGAGCACACCATCAAGAACGCCTTCAAGCGCATCTGGATTCAATCCGAGGCCCAACCGACCAGCGGCTTCATTAAGTCCCTCTTCTCCGACAAGATCCACGGCGCCATCGGCCTCTCTGACGGCCAAGGCATTGACATTGACCTCCGCAAGACTAACTCCCTCAGCTCCGCCGTGGCCGCTAAGATTCTCGAGTCCTACTTCCAGGACAGCGCCCCATCTTTCGACCTCGCTTTCGAGAACTCATCTTCCGTGATCTTCCACATCGAGGAGATCATCACCACCGACGCCGATGAGATCAGCCTCAGGAACTGGCTCAACGACAACCAAAACGAGCTCCGCGAGATCTATAAGGAGGAGATCAAGAAGGGCAACTTCTTCGTGGCCACCTCCCTCCTCAGAGCCAAGAAGATGCGCATGCAATTCGAGCGCAAGAACAAGGGCGAGCTCGGCGTTGACGTGTCCAAGATCAAGAACCTCCCGGTGGACGCCAAGCTCGAGTCTAAGATCGAGGGCAGCACCTACGACCGCCTCGTTTTCGAGACACCGGACGAGGGCATTGTGTTCGGCGTGAAGCTCGTCAGACTCTTCTTCTCCGACAACGGCATCCTCACAATTGACAAGAAGCAGGACTTCAACAGGGTGCTC (SEQ ID NO. 2).

[0095] Amino acid sequence (247 aa) optimized according to rice codon preference:

[0096] MECNDPFVVALKDKGYSLVAYPKTSIRPLHIYEHTIKNAFKRIWIQSEAQPTSGFIKSLFSDKIHGAIGLSDGQGIDIDLRKTNSLSSAVAAKILESYFQDSAPSFDLAFENSSSVIFHIEEIITTDADEISLRNWLNDNQNELREIYKEEIKKGNFFVATSLLRAKKMRMQFERKNKGELGVDVSKIKNLPVDAKLESKIEGSTYDRLVFETPDEGIVFGVKLVRLFFSDNGILTIDKKQDFNRVL (SEQ ID NO. 3).

[0097] Example 2 The N-terminal domain of bacterial bGSDM protein (bGM) induces cell death in tobacco and increases resistance to Phytophthora capsici.

[0098] 1. Construction of bacterial bGM expression vector

[0099] The bGM fragment with rice codon optimization was amplified and connected to tobacco expression vector pCAMBIAsuper1300-GFP by homologous recombination.

[0100] Specific steps:

[0101] (1) Amplification of target fragment

[0102] The plasmid containing the target gene (optimized bGM fragment) was used as the template, and the PCR reaction system was as follows:

[0103] 2x PCR Buffer, 25 μL

[0104] dNTPs, 1 μL

[0105] bGM-P1300 / F (10 mM), 2 μL

[0106] bGM-P1300 / R (10 mM), 2 μL

[0107] Template, 0.5 μL

[0108] Taq enzyme, 1 μL

[0109] ddH2O, 18.5 μL

[0110] Primer sequence:

[0111] bGM-P1300 / F: ccaaatcgactctagaaagctt atggagtgcaacgacccgt (SEQ ID NO. 4);

[0112] bGM-P1300 / R: ttgctcaccatggtctc tcagagcaccctgttgaagtcc (SEQ ID NO. 5).

[0113] Amplification procedure: pre-denaturation, 95℃, 3min; denaturation, 95℃, 30sec; annealing, 55℃, 30sec; extension temperature, 72℃, determined according to the size of the amplified fragment, generally calculated as 60sec / kb; extension to denaturation process for 35 cycles; then 72℃, 5min; finally 16℃, 10min.

[0114] (2) Gel recovery of target fragment product

[0115] Agarose gel loading, 130V electrophoresis for 30min, and the target fragment was recovered by using a gel recovery kit (Vazyme #DC301-01);

[0116] (3) The target vector pCAMBIAsuper1300-GFP binary vector was single-enzyme cut using HindIII endonuclease (NEB #R3104S) according to the enzyme cutting conditions in the instruction manual, and the enzyme cut vector was purified using a gel recovery kit;

[0117] (4) The target fragment was connected using 2xSeamless Cloning Mix (BioRun), and the nucleotide concentration of the target fragment was measured using a micro UV-visible spectrophotometer (NanoDrop One / Onec) before connection, and the connection system was calculated, and the connection conditions were referred to the instruction manual;

[0118] (5) After the connection was completed, 1μL of the connection product was mixed into 50μL of E. coli DH5α competent cells, then added to an electric shock cup, placed on ice, and subjected to electric shock under the conditions of 1.8KV, 5ms;

[0119] (6) The cells after electric shock were washed out with 0.5mL of LB liquid medium, and recovered and cultured at 37℃, 120r / min for 45min to obtain the transformed bacteria solution;

[0120] (7) The transformed bacteria solution was evenly coated on an LB solid culture plate containing Kana antibiotic, blown dry in a clean bench, and then cultured overnight at 37℃ in an incubator;

[0121] (8) In a 2 mL centrifuge tube, add 400 μL of LB liquid medium containing Kana antibiotic, pick a single colony into the liquid medium, and incubate at 37°C for 4 h at 200 r / min. Take 0.6 μL of the bacterial solution and use specific primers P1300 / SeqF and P1300GFP / SeqR at both ends of the enzyme digestion site on the carrier for PCR detection. Correctly clone and expand to 5-6 mL culture at 37°C for overnight incubation at 200 r / min.

[0122] The above PCR detection primers are:

[0123] P1300 / SeqF: CGCCATTTCGCCTTTTCAG (SEQ ID NO. 6);

[0124] P1300GFP / SeqR: AGAAGTCGTGCTGCTTCATG (SEQ ID NO. 7).

[0125] Extract the plasmid according to the instructions of the plasmid extraction kit (Vazyme) and send the sample for sequencing. After sequencing, obtain the bacterial bGM expression vector plasmid (pCAMBIAsuper1300-GFP-bGM) containing the target fragment; use the plasmid for the following experiments.

[0126] After sequencing, transfer the plasmid containing the bacterial bGM target fragment from E. coli DH5α to Agrobacterium GV3101. The specific transformation steps are as follows:

[0127] ① Thaw the GV3101 Agrobacterium competent cells stored at -80°C on ice or at room temperature;

[0128] ② Under sterile conditions, add 1 μg of pCAMBIAsuper1300-GFP-bGM plasmid DNA to be transformed to the just-thawed GV3101 Agrobacterium competent cell suspension, mix gently, and incubate in ice water bath for 10 min;

[0129] ③ Place the centrifuge tube in liquid nitrogen and freeze quickly for 5 min;

[0130] ④ Quickly place the centrifuge tube in a 37°C water bath for 5 min, without shaking the water surface;

[0131] ⑤ Place the centrifuge tube back in the ice water bath and keep for another 5 min;

[0132] ⑥ Under sterile conditions, add 800 μL of LB liquid medium without antibiotic and incubate at 28°C for 2-3 h with shaking;

[0133] ⑦ 5000r / min centrifugal 1 min to collect bacteria, take about 100 μL supernatant to resuspend the bacteria by blowing gently, add to the LB solid medium containing Rif and Kana antibiotics, and use a sterile bacterial spreader or glass beads to evenly spread the cells. After the liquid in the plate is completely absorbed, invert the plate and incubate at 28°C for 2-3 days.

[0134] And use P1300 / SeqF and P1300GFP / SeqR primer pairs for verification sequencing, and preserve for use after sequencing correctly.

[0135] 2, transient expression of bGM causes tobacco cell death:

[0136] Use the Agrobacterium GV3101 constructed in Example 1 to inject tobacco, transiently express the bGM of the bacteria, the full-length bGSDM gene (bGMF) and the empty pCAMBIAsuper1300-GFP (EV) as a negative control, and use the previously reported Executor R gene Xa23 gene as a positive control. The full-length bGSDM gene (bGMF) cannot induce cell death in the previous report, while the Xa23 gene can cause tobacco cell necrosis when transiently expressed in tobacco.

[0137] The transient expression process of N. benthamiana is as follows:

[0138] S1. Preparation of tobacco: N. benthamiana is cultured in potting soil (peat soil / vermiculite = 1:3, v / v), and the growth environment is temperature 21-22°C, humidity 60%, light / dark alternating time 16 / 8h, and light intensity LED 80 μM m -2 s -1 . Select 4-6 week old N. benthamiana leaves with vigorous growth on the upper part for transient expression experiment.

[0139] S2. Preparation of Agrobacterium GV3101 injection solution: add 2-3 mL of LB liquid medium containing Kana antibiotic in a test tube, use a toothpick to pick the Agrobacterium clone constructed correctly in the previous Example 1, and culture at 28°C, 200 r / min until OD 600 = 1-2;

[0140] S3. Centrifuge the above Agrobacterium cells at room temperature at 3500g for 5 min, discard the supernatant, and resuspend the Agrobacterium cells with MES resuspension solution containing acetosyringone, and the final concentration of acetosyringone is 100 uM;

[0141] S4. Measure the OD value of the cells with a UV spectrophotometer, the wavelength of the UV spectrophotometer is 600, and adjust before injection as needed, generally OD 600 = 0.8-1;

[0142] S5. After the above-mentioned Agrobacterium cell resuspension solution was incubated at room temperature for 1 h, the tobacco was injected using a 1 mL disposable syringe with a removed needle and was labeled.

[0143] S6. The necrosis of the tobacco cells was observed under a UV lamp.

[0144] The Agrobacterium injection was sampled for gene expression detection 15 h after expression, and the detection results are shown in B of FIG. 6. Figure 1 Figure 1 B of FIG. 6 is the qRT-PCR detection of the expression of the target gene (n = 3). n represents the number of technical repeats used in the analysis. Figure 1 The results of B of FIG. 6 show that the genes in the tobacco leaves transfected by the Agrobacterium bGM and bGMF are normally expressed.

[0145] The necrosis of the tobacco leaves was observed under a UV lamp 24 h after the Agrobacterium injection, and the leaves were photographed under white light and a UV lamp. Figure 1 In A of FIG. 7, Xa23 is a positive control, and empty vector (EV) and full-length bGSDM gene (bGMF) are negative controls. Scale, 1 centimeter. Figure 1 The results of A of FIG. 7 show that the bGM gene induces cell death 33 h after transient expression; and that the bGM of the transiently expressed bacteria causes the HR response of tobacco, and the full-length bGSDM gene (bGMF) does not cause the HR response.

[0146] In summary, the N-terminal domain of bGSDM (bGM) can induce the hypersensitive necrosis response of tobacco.

[0147] 3. Transient expression of bGM promotes accumulation of reactive oxygen species:

[0148] As described above, the bGM, bGMF, empty vector negative control (EV) and positive control Xa23 gene were injected into N. benthamiana leaves, respectively. The injected tobacco leaves were subjected to DAB staining 28 h after injection.

[0149] The specific DAB staining method is as follows:

[0150] a. Use the enhanced DAB color developing kit (Solarbio) to prepare DAB staining solution, which is used immediately after preparation;

[0151] b. Soak the infected leaves in DAB staining solution, and stain at 28°C in the dark for 36 h;

[0152] c. Soak the leaves in 95% alcohol, and replace the alcohol every 24 h until the discoloration is complete or 95°C heating is added to accelerate the discoloration;

[0153] d. Stretch the leaves in water and take pictures to record the staining effect. ​

[0154] Figure 2 A is the result of DAB staining of active oxygen in leaves after 28h of transient transformation. Figure 2 The results in A show that there is accumulation of active oxygen (ROS) in the leaf position expressing bGM, and there is no accumulation of active oxygen in the leaf position expressing full-length bGSD M (bGMF) and empty vector (EV). The expression of bGM enhances the immunity of tobacco cells.

[0155] 4. Up-regulation of disease resistance-related genes:

[0156] After 15h of Agrobacterium injection into tobacco leaves, the expression of disease resistance genes was detected, and the specific method was as follows:

[0157] (1) The leaves expressing bGM, bGMF and empty vector obtained were quickly ground into powder with liquid nitrogen, and an appropriate amount was taken into a sterile 2mL centrifuge tube, 1mL Trizol was added, and after shaking evenly, it was placed on ice for 5min.

[0158] (2) Centrifuge at 12000r / min for 10min at 4℃, and take the supernatant into a sterile 2mL centrifuge tube, add 200μL chloroform and 200μL water-saturated phenol, and shake evenly for 10-15s.

[0159] (3) Centrifuge at 12000r / min for 10min at 4℃, and take 500μL supernatant into a sterile 2mL centrifuge tube, add DEPC water to 800μL, and add the same volume of chloroform, and shake evenly.

[0160] (4) Centrifuge at 12000r / min for 10min at 4℃, and take 500μL supernatant into a sterile 2mL centrifuge tube, add 2 times the volume of pre-cooled anhydrous ethanol, mix gently, and precipitate at -20℃ for 2h.

[0161] (5) Centrifuge at 12000r / min for 10min at 4℃, remove the supernatant, add 1mL of 75% ethanol, and suspend the precipitate by tapping the bottom of the centrifuge tube.

[0162] (6) Centrifuge at 12000r / min for 10min at 4℃, remove the supernatant, and blow dry on a clean bench.

[0163] (7) Add 54μL of DEPC water to dissolve the precipitate, add 6μL of 10×Reaction Buffer and 1.5μL of RNAase-free DNAase to each 54μL sample, and incubate at 37℃ for 40-60min.

[0164] (8) Add DEPC water to 800 μL, add equal volume of chloroform, mix gently, centrifuge at 12000 r / min for 10 min at 4°C, take 500 μL supernatant to a new sterile 2 mL centrifuge tube, add 2 times volume of pre-cooled anhydrous ethanol, mix gently, precipitate at -20°C for 2 h.

[0165] (9) Centrifuge at 12000 r / min for 10 min at 4°C, discard supernatant, add 75% ethanol 1 mL, suspend the precipitate.

[0166] (10) Centrifuge at 12000 r / min for 12 min at 4°C, remove supernatant, dry in a clean bench.

[0167] (11) Add about 50 μL DEPC water, mix gently, measure concentration and electrophoresis, and use the extracted RNA for reverse transcription or subsequent storage at -80°C.

[0168] Synthesis of cDNA:

[0169] (1) Reverse system:

[0170] 5x PrimeScript Buffer, 4 μL

[0171] PrimeScript RT Enzyme Mix I, 1 μL

[0172] Oligo dT primer (50 mM), 1 μL

[0173] Random 6Mers (100 mM), 1 μL

[0174] RNA, 4 μL (1 μg)

[0175] Add RNase-free water to 20 μL

[0176] (2) Reaction conditions:

[0177] 50°C, 15 min

[0178] 85°C, 5 sec

[0179] Fluorescence real-time quantitative PCR:

[0180] (1) qRT-PCR system:

[0181] SYBR II, 10 μL

[0182] RNase-Free ddH2O, 8 μL

[0183] Primer F, 0.5 μL

[0184] Primer R, 0.5 μL

[0185] cDNA (diluted 10 times), 1 μL

[0186] Add RNase-free water to 20 μL

[0187] Primer used in qRT-PCR system of Table 1

[0188] qRT-EFla / F agaggccctcagacaaac (SEQ ID NO. 8) qRT-EFla / R taggtccaaaggtcacaa (SEQ ID NO. 9) qRT-ERFl / F gctcttaacgtcggatggtc (SEQ ID NO. 10) qRT-ERFl / R agccaaaccctagctccatt (SEQ ID NO. 11) qRT-LOXl / F aaaacctatgcctcaagaac (SEQ ID NO. 12) qRT-LOXl / R actgctgcataggctttgg (SEQ ID NO. 13) qRT-NPRl / F acatcagcggaagcagtag (SEQ ID NO. 14) qRT-NPRl / R gtcggcgaagtagtcaaac (SEQ ID NO. 15) qRT-PALl / F gttatgctcttagaacgtcgccc (SEQ ID NO. 16) qRT-PALl / R ccgtgtaatgccttgtttcttga (SEQ ID NO. 17)

[0189] (2) qRT-PCR reaction condition: (two-step method)

[0190] 95°C, 30 sec cycle 1 cycle; 95°C 5 sec, 60°C 32 sec cycle 40 cycles;

[0191] EF-1α as internal reference, 3 technical repeats for each sample, 2 -ΔΔCt method was used to calculate the expression difference of genes. The experiment was conducted for 3 biological repeats.

[0192] The expression results of defense-related genes after transient expression of bGM gene for 15 h (n=3) are shown in Fig. 1B. Figure 1 Figure 1 In Fig. 1B, the scale is 1 cm. n represents the number of technical repeats used in the analysis. The asterisk represents the significance statistical method Student's t-test (*P<0.05, ***P<0.001). Figure 2 The results of Fig. 1B show that the expression of disease resistance-related genes ERF1, LOX1, NPR1, PAL1 in tobacco leaves after expression of bGM is significantly increased compared with injection of empty load.

[0193] 5. Transient expression of bGM can inhibit the infection of P. capsici:

[0194] As described in 1, P. capsici was inoculated at 15 h after injection. The specific method is as follows:

[0195] (1) Prepare 4-6 weeks old N. benthamiana with good growth and P. capsici flat that is not full-grown. The N. benthamiana is cultured at 25°C under 12h / 12h light condition, and the P. capsici is cultured on PDA flat in dark;

[0196] (2) The N. benthamiana leaves injected with Agrobacterium are taken off, and the leaves are placed in a culture dish with filter paper soaked with ultrapure water. A 3 mm diameter P. capsici plug is inoculated at the center of the inoculation site, and then the culture dish is sealed with plastic wrap;

[0197] (3) The culture dish is placed in a 28°C constant temperature biochemical incubator and cultured in dark for 36 h;

[0198] ​(4) Observe and photograph under ultraviolet light and record the data. The results are as follows: Figure 3 As shown.

[0199] Figure 3 In the figure, A represents transient expression of bGM enhancing tobacco resistance to *Phytophthora capsici* (oomycetes). This result was obtained by photographing the image 36 hours after inoculation. Scale bar: 1 cm. Figure 3 In the figure, B represents the statistical results of lesion area after transient expression of bGM followed by inoculation with Phytophthora capsici (n=13). Figure 3 In the figure, C represents the relative biomass statistic of *Phytophthora capsici* after transient expression of bGM (n=3). n represents the number of replicates of the technique used in the analysis. An asterisk indicates the significance statistical method used: Student's t-test (**P<0.01, ****P<0.0001). (Summary) Figure 3 As a result, injecting bGM expression relative to the empty vector reduced the spread of Phytophthora capsici, and significantly reduced the lesion area and the relative biomass of the pathogen.

[0200] Example 3: Enhancing Rice Resistance to Rice Blast by the N-Terminal Domain (bGM) of Bacterial bGSDM Protein 1. Screening of Pathogen-Inducing Promoters

[0201] This embodiment uses transcriptional data of rice leaf sheath infection by *Magnaporum oryzae* from the NCBI database to screen for genes induced by *Magnaporum oryzae*. Based on different induction levels, five genes were selected: OsCYP450, OsPR1a, OsPR10b, OsGABA, and OsPR10a. Figure 4 ). Figure 4 The transcriptional data for the rice genes upregulated when rice blast fungus infects the leaf sheath were obtained from the rice leaf sheath transcriptome infected with rice blast fungus.

[0202] When rice is infected by *Strombus haematobium* and *Bacterium oryzae*, OsPR1a is upregulated to defend against pathogen infection. The rice protein OsWRKY6 directly activates the expression of OsWRKY45 and OsWRKY47, and through these two OsWRKY proteins, activates OsPR1a and OsPR1b. This signaling pathway is involved in Xa1-mediated defense against Xoo. OsPR1b is upregulated in rice varieties resistant to *Bacterium oryzae*, thereby enhancing rice resistance to *Strombus haematobium*. *Strombus haematobium* infection, mechanical damage, and ultraviolet radiation induce the expression of OsGABA-T in rice leaves, while this expression is undetectable in normal rice organs.

[0203] Infection by pathogens, and deficiencies in salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), NaCl, and phosphorus all induce the upregulation of OsPR10a expression in rice. In the transcriptome data of rice blast fungus infection, OsPR10a showed the highest induced expression level; therefore, the promoter of this gene was selected and combined with bGM.

[0204] 2. Construction of transgenic rice plants

[0205] The promoter of OsPR10a gene was selected to be added in combination with bGM, and homologous recombination was performed on the vector pRHE (He F, Zhang F, Sun W, Ning Y, Wang GL. A Versatile Vector Toolkit for Functional Analysis of Rice Genes. Rice (N Y). 2018 Apr 20; 11(1): 27.) The specific steps are as follows:

[0206] (1) Amplification of bGM fragments and OsPR10a promoters using rice codon optimization, i.e. pathogen-induced PR10a promoter and uORFs elements of TBF1 gene;

[0207] The promoter sequence of pathogen-induced PR10a (2186 bp) is as follows:

[0208]

[0209] uORFs element sequence of TBF1 (477bp):

[0210] AACAGCATCCGTTTTTATAATTTAATTTTCTTACAAAGGTAGGACCAACATTTGTGATCTATAAATCTTCCTACTACGTTATATAGAGACCCTTCGACATAACACTTAACTCGTTTATATATTTGTTTTACTTGTTTTGCACATACACACAAAAATAAAAAAGACTTTATATTTATTTACTTTTTAATCACACGGATTAGCTCCGGCGAAGTATGGTCGTCGTCTTCATCTTCTTCCTCCATCATCAGATTTTTCCTTAAATGGAAGAAACCAAACGAAACTCCGATCTTCTCCGTTCTCGTGTTTTCCTCTCTGGCTTTTATTGCTGGGATTGGGAATTTCTCACCGCTCTCTTGCTTTTTAGTTGCTGATTCTTTTTCCTTCGACTTTCTATTTCCAATCTTTCTTCTTCTCTTTGTGTATTAGATTATTTTTAGTTTTATTTTTCTGTGGTAAAATAAAAAAAGTTCGCCGGAG (SEQ ID NO. 19).

[0211] The plant codon-optimized bGM fragment was amplified using a plasmid containing the target gene as a template, the OsPR10a promoter was amplified using DNA of wild-type rice variety Kitaake as a template, and the uORFs element of the TBF1 gene was amplified using DNA of wild-type Arabidopsis thaliana Col-0 as a template. The PCR reaction system was as follows:

[0212] 2x PCR Buffer, 25 μL

[0213] dNTPs, 1 μL

[0214] bGM-P1300 / F (10 mM), 2 μL

[0215] bGM-P1300 / R (10 mM), 2 μL

[0216] Template, 0.5 μL

[0217] Taq enzyme, 1 μL

[0218] ddH2O, 18.5 μL

[0219] Primer sequences:

[0220] Primers for amplifying bGM fragment:

[0221] OsPR10a-P / F: CAGATCCAGTGGGATCCCGCCATTTCTGATCCGTTCTTG (SEQ ID NO. 20);

[0222] OsPR10a-P / R: TTATAAAAACGGATGCTGTTCACTGAAGATATAATCTAA CTAGCTAG (SEQ ID NO. 21);

[0223] uORF / F: AACAGCATCCGTTTTTATAA (SEQ ID NO. 22);

[0224] uORF / R: ACGGGTCGTTGCACTCATGGTGATGGTGATGATGCATCTCCG GCGAACTTTTTTTATTTT (SEQ ID NO. 23);

[0225] bGM-Nhis / F: ATGCATCATCACCATCACCATGAGTGCAACGACCCGT (SEQ ID NO. 24);

[0226] bGM-PRHE / R: GCACTAGTAAGCTTGGTACCTCAGAGCACCCTGTTGAA GTCC (SEQ ID NO. 25);

[0227] Amplification procedure: pre-denaturation, 95 °C, 3 min; denaturation, 95 °C, 30 sec in the second step; annealing is the third step, 55 °C, 30 sec; extension temperature is the fourth step, 72 °C, and the extension time is calculated according to 60 sec / kb in general; the process from the second step to denaturation is performed in the fourth step, 35 cycles; then 72 °C, 5 min in the fifth step; finally, 16 °C, 10 min in the sixth step.

[0228] (2) Gel recovery of target fragment product

[0229] Point sample in agarose gel, 130 V electrophoresis for 30 min, and the target fragment is recovered by cutting the gel using a gel recovery kit (Vazyme #DC301-01);

[0230] (3) According to the enzyme digestion conditions of NEB endonuclease (NEB#R3104S) instructions, the empty pRHE binary vector was double-digested by BamH I and Kpn I, and the digested vector was purified by a gel recovery kit;

[0231] (4) The target fragment nucleotide concentration was measured using a micro UV-visible spectrophotometer (NanoDrop One / Onec) before connection, and the connection system was calculated. The connection conditions were referred to the instructions;

[0232] (5) After the connection was completed, 1 μL of the connection product was mixed into 50 μL of E. coli DH5α competent cells, and then an electric shock cup was added. The electric shock was performed at 1.8 KV and 5 ms on ice;

[0233] (6) The cells after electric shock were washed out with 0.5 mL of LB liquid medium, and recovered and cultured at 37°C and 120 r / min for 45 min;

[0234] (7) The transformed bacterial liquid was evenly coated on an LB solid culture plate containing Kana antibiotic. After being blown dry in a clean bench, it was cultured overnight in a 37°C incubator;

[0235] (8) 400 μL of LB liquid medium containing Kana antibiotic was added to a 2 mL centrifuge tube, and a single colony was picked into the liquid medium. It was cultured at 37°C and 200 r / min for 4 h. 0.6 μL of bacterial liquid was used to perform PCR detection using specific primers of the target fragment at both ends of the enzyme digestion site of the vector. The correct clone was expanded to 5-6 mL and cultured overnight at 37°C and 200 r / min;

[0236] PCR detection primers:

[0237] PR10aP-JC / F: TGCCCAGGTCTCAAATGTC (SEQ ID NO. 26);

[0238] bGM-JC / R: TGATGATCTCCTCGATGTGG (SEQ ID NO. 27).

[0239] (9) The plasmid was extracted according to the instructions of the plasmid extraction kit (Vazyme), and was sent for sequencing. After the sequencing was correct, the recombinant vector pRHE-PR10a-uORFs-bGM was obtained. The plasmid map is shown in Figure 5 . The correct vector was sent to the company to transform rice variety Kitaake, and transgenic rice plant P PR10a-uORF :bGM was obtained.

[0240] 3. Phenotype identification of rice disease resistance

[0241] Firstly, the expression of disease-related genes in transgenic rice plants was detected by qRT-PCR. Phenotype observation found that there were disease-like spots on the leaves of transgenic plants. The leaves with disease-like spots were selected to extract RNA for detection of disease-related genes. According to the results of qRT-PCR, the expression of PAL, OsPR1b, OsPR5 and OsPR10 in transgenic plants was up-regulated. It was shown that P Figure 6 :bGM strain improved disease resistance. PR10a-uORF Figure 6 In the table, A is the growth phenotype of T0 generation plants; WT represents wild type rice Kittake, and 1, 9, 14 and 15 are transgenic rice with positive sequencing. Figure 6 In the table, B is the expression detection result of bGM gene. Figure 6 In the table, C is the expression detection of defense-related genes of positive plants. Actin is the internal reference gene. Asterisk indicates the significance statistical method Student's t-test (**p<0.01, ***p<0.01, ****p<0.0001).

[0242] Then, the transgenic rice plants were inoculated with the laboratory isolated Magnaporthe oryzae race EA18 to detect the resistance to Magnaporthe oryzae.

[0243] The specific detection method is as follows: the rice leaf pieces containing Magnaporthe oryzae race EA18 spores were inoculated on oat medium and cultured at 25°C in the dark for 2-3 days; after the hyphae grew out, they were inoculated on new oat medium and cultured for one week. The upper extended leaves of transgenic rice plants P PR10a-uORF :bGM and wild type Kitaake were taped with transparent tape on the back, and the front of the leaves was pressed with a special tweezer. A 3mm diameter fungus cake was placed on the wound, and then the fungus cake was fixed with transparent tape. After 14 days of inoculation, the disease incidence was observed and photographed to count the lesion length. Figure 7 The results showed that the lesion area of transgenic rice plants P PR10a-uORF :bGM was significantly smaller than that of the wild type, which indicated that the transgenic strain improved the resistance to Magnaporthe oryzae. Figure 7 In the table, the transgenic rice strain P PR10a-uORF :bGM has resistance to Magnaporthe oryzae. WT is wild type rice Kittake.

[0244] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.​

Claims

1. Application of biomaterials in at least one of S1-S4: The biomaterial is a protein, and the protein is any one of the following: B1) The amino acid sequence is that of the protein shown in SEQ ID No. 3; B2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in B1); S1-S4 are: S1) Increases plant disease resistance; S2) Prepare products that enhance plant disease resistance; S3) Cultivate plants with enhanced disease resistance; S4) Prepare products from plants with enhanced disease resistance; The plant is tobacco or rice; the disease resistance is resistance to Phytophthora capsici or Phytophthora oryzae; when the plant is tobacco, the disease resistance is resistance to Phytophthora capsici, and when the plant is rice, the disease resistance is resistance to Phytophthora oryzae.

2. The application of biological materials that regulate the expression of the gene encoding the protein as described in claim 1 in at least one of S1-S6. The biological material used to regulate the expression of the gene encoding the protein as described in claim 1 is any one or more of the following: C1) Nucleic acid molecules encoding proteins with amino acid sequences as shown in SEQ ID No. 3; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette described in C2; C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); S1-S6 are: S1) Increases plant disease resistance; S2) Prepare products that enhance plant disease resistance; S3) Cultivate plants with enhanced disease resistance; S4) Prepare products from plants with enhanced disease resistance; S5) Improve disease-resistant varieties or prepare products of disease-resistant varieties; S6) Breeding related to tobacco resistance to Phytophthora capsici or rice resistance to rice blast; The plant is tobacco or rice; the disease resistance is resistance to Phytophthora capsici or Phytophthora oryzae; when the plant is tobacco, the disease resistance is resistance to Phytophthora capsici, and when the plant is rice, the disease resistance is resistance to Phytophthora oryzae.

3. The application according to claim 2, characterized in that, C1) The nucleic acid molecule is any of the following: a1) The nucleotide sequence shown in SEQ ID No. 1; a2) The sequence obtained by optimizing the nucleotide sequence shown in SEQ ID No. 1 based on plant codons.

4. The application according to claim 3, characterized in that, The sequence obtained by optimizing the rice codons is shown in SEQ ID No.

2.

5. A method for cultivating transgenic plants with enhanced disease resistance, characterized in that, The process includes the following steps: introducing a nucleic acid molecule encoding a protein with an amino acid sequence as shown in SEQ ID No. 3 into the target plant to obtain a transgenic plant with enhanced disease resistance; When the plant is tobacco, the disease resistance is against Phytophthora capsici; when the plant is rice, the disease resistance is against Pythium oryzae.

6. The method according to claim 5, characterized in that, The transgenic plant is obtained by transferring the protein expression vector into the target plant, wherein the protein expression vector includes pRHE.

7. The method according to claim 6, characterized in that, The method for constructing the transgenic plant using a pathogen-induced gene promoter includes: adding a regulatory element uORFs from the promoter of another pathogen-induced gene TBF1 after the pathogen-induced PR10a promoter to form the promoter PR10a-uORFs, and using PR10a-uORFs to construct a bGM protein transgenic plant line.

8. The method according to claim 7, characterized in that, The promoter sequence for PR10a induced by the pathogen is shown in SEQ ID No. 18; the nucleotide sequence of the regulatory element uORFs is shown in SEQ ID No. 19.

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