12-chain beta bucket protein SBR1 and application thereof in improving broad-spectrum disease resistance of plants

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a 12-chain beta bucket protein SBR1 and application thereof in improving broad-spectrum disease resistance of plants. According to the method, artificial intelligence deep learning is utilized, the beta tubby disease-resistant protein capable of being applied to plants is designed, the excavation time is greatly shortened, and manpower and material resources are saved. Meanwhile, the 12-chain beta buccal protein SBR1 designed from the beginning artificially is introduced into plant disease resistance for the first time, and the effect of the 12-chain beta buccal protein SBR1 in plant disease resistance is proved. A new plant disease-resistant strategy is introduced in the research, the number of applicable plant disease-resistant genes is greatly increased, and the cost of cloning the plant disease-resistant genes is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a 12-stranded beta-barrel protein SBR1 and an application thereof in improving the broad-spectrum disease resistance of plants. Background Art

[0002] The exploration of disease-resistant resources is an important part of plant pathology and breeding, but there are various challenges. First, there is great complexity in disease-resistant genes. Many plant diseases are caused by complex gene interactions, which makes the identification and cloning of disease-resistant genes difficult. Second, pathogens can mutate rapidly and produce new physiological subspecies, which quickly renders the disease resistance of crops ineffective. At the same time, many known disease-resistant genes are highly specific and can only fight against specific pathogens or physiological subspecies. They cannot be resistant to multiple pathogens or different physiological subspecies of the same pathogen. Therefore, such varieties with single disease resistance or easy loss of disease resistance are easily eliminated. While improving disease resistance, crop yield or quality may be sacrificed, and disease resistance needs to perform well under different environmental conditions. Therefore, it is necessary to develop genes that can remain effective for a long time and are not overcome by pathogens and are resistant to multiple pathogens. Summary of the invention

[0003] The purpose of the present invention is to provide a 12-stranded β-barrel protein SBR1 and its application in improving the broad-spectrum disease resistance of plants. The broad-spectrum disease resistance of plants can be improved by overexpressing the 12-stranded β-barrel protein SBR1.

[0004] The present invention provides a 12-stranded β-barrel protein SBR1, and the amino acid sequence of the 12-stranded β-barrel protein SBR1 is shown in SEQ ID NO.1.

[0005] The present invention also provides a nucleic acid molecule encoding the 12-stranded beta barrel protein SBR1.

[0006] The present invention also provides the use of the 12-stranded β-barrel protein SBR1 in improving the broad-spectrum disease resistance of plants, wherein the broad-spectrum disease resistance includes one or more of resistance to fungal diseases, resistance to bacterial diseases and resistance to viral diseases.

[0007] The present invention also provides a primer set for amplifying the nucleic acid molecule, wherein the primer set comprises a forward primer and a reverse primer;

[0008] The nucleotide sequence of the forward primer is shown in SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0009] The present invention also provides a biological material containing the nucleic acid molecule, including a recombinant expression vector containing the nucleic acid molecule and / or a recombinant microorganism containing the nucleic acid molecule.

[0010] As a preferred embodiment, the basic skeleton of the recombinant expression vector containing the nucleic acid molecule includes pCAMBIA1300 vector or pRHE vector.

[0011] The present invention also provides a pathogen-inducible plant expression vector, which comprises a PR10b promoter and the nucleic acid molecule.

[0012] As a preferred embodiment, the basic skeleton of the pathogen-inducible plant expression vector includes a pRHE vector; the gene accession number of the PR10b promoter is AF274851.1.

[0013] The present invention also provides the use of the primer set or the biological material or the pathogen-inducible plant expression vector in improving the broad-spectrum disease resistance of plants.

[0014] The present invention also provides a method for improving plant disease resistance, which involves overexpressing the nucleic acid molecule in the target plant genome.

[0015] Beneficial effects: The present invention provides a 12-stranded β-barrel protein SBR1. The present invention uses artificial intelligence deep learning to design a β-barrel disease-resistant protein that can be applied to plants, greatly reducing mining time and saving manpower and material resources. At the same time, the present invention introduces the artificially designed 12-stranded β-barrel protein SBR1 into plant disease resistance for the first time and proves its role in plant disease resistance. The research of the present invention introduces a new strategy for plant disease resistance, greatly increases the number of applicable plant disease resistance genes, and greatly reduces the cost of cloning plant disease resistance genes.

[0016] At present, disease-resistant genes are very complex, and many plant diseases are caused by complex gene interactions, which makes the identification and cloning of disease-resistant genes difficult. Secondly, pathogens can mutate rapidly, producing new physiological subspecies, which quickly render the disease resistance of crops ineffective. Therefore, the mining of disease-resistant genes is limited, and traditional disease-resistant gene mining schemes consume manpower and material resources. The present invention can use artificial intelligence deep learning to design β-barrel-shaped disease-resistant proteins that can be applied to plants, and the β-barrel-shaped disease-resistant proteins designed by artificial intelligence in the present invention are not limited in number, which can reduce mining time and save manpower and material resources.

[0017] Traditional disease-resistant genes can only combat one or a few physiological races and cannot achieve combined disease resistance against major categories such as fungi, bacteria, and viruses. In the present invention, a protein designed by artificial intelligence is transiently expressed in tobacco by using the method of transient transformation, causing necrosis of tobacco cells, and broad-spectrum disease resistance across categories can be achieved by transferring a single gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0019] Figure 1 Figure for SBR1 in Example 1 causing cell death in tobacco. The left figure is the observation of Nicotiana benthamiana under white light for the hypersensitive response (HR) induced by transient expression of SBR1, the middle figure is the observation of Nicotiana benthamiana under ultraviolet light for the hypersensitive response (HR) induced by transient expression of SBR1, and the right figure is the DAB staining of the burst of reactive oxygen species (ROS) induced by SBR1 in Nicotiana benthamiana; the scale bar in the figure is 1 cm;

[0020] Figure 2 Figure for the burst of ROS induced by SBR1 in Nicotiana benthamiana - ROS measurement results in Example 1, where a is the relative light units at different times in different treatments, b is the total number of photons in different treatments, and c is the expression level of different disease-resistant genes in different treatments;

[0021] Figure 3 Figure for the resistance analysis of SBR1 transiently transformed tobacco to TuMV-GFP virus in Example 2, where a is the apparent figure of the resistance of SBR1 transiently transformed tobacco to TuMV-GFP virus, and b is the expression level of different genes of TuMV in different treatments; the scale bar in the figure is 1 cm;

[0022] Figure 4 Figure for the resistance analysis of SBR1 transiently transformed tobacco to Phytophthora capsici in Example 3, where a is the apparent figure of the resistance of SBR1 transiently transformed tobacco to Phytophthora capsici, b is the lesion area of Phytophthora capsici in different treatments, and c is the relative biomass of Phytophthora capsici in different treatments; the scale bar in the figure is 1 cm;

[0023] Figure 5 Figure for the resistance analysis of SBR1 stably transformed rice to Magnaporthe oryzae in Example 4, where a is the apparent figure of the resistance of SBR1 stably transformed rice to Magnaporthe oryzae, and b is the lesion area of rice blast in different treatments; the scale bar in the figure is 1 cm. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The present invention provides a 12-stranded β-barrel protein SBR1. As a specific embodiment, the amino acid sequence of the 12-stranded β-barrel protein SBR1 is shown in SEQ ID NO.1: MRPKPGSWKAGA GVTFYTDGTVTANVWYTYYLTENLALTAELTFKNGKLDSVSVGATYTFEPKLTVSVVGTYHTDGKWDVTVTVEGEVKINDKLTLYPGGKYTLSNTAPDTWAPYVKFEYKVTDKLSLLGKVEYNDDGTTTVSVGLKYKLTDNLSLTVTATFVNGKLDSVTVEVEYDLTK*.

[0025] As a specific embodiment, the 12-stranded β-barrel protein SBR1 can use an online codon optimization tool (https: / / www.novopro.cn / tools / codon-optimization.html) to optimize the SBR1 protein for plant codons, obtaining a nucleic acid molecule encoding the 12-stranded β-barrel protein SBR1. The nucleotide sequence of the nucleic acid molecule encoding the 12-stranded β-barrel protein SBR1 is shown as SEQ ID NO.4: 5’-ATGAGGCCAAAGCCAGGCTCTTGGAAGGCCGGAGCCGGAGTCACTTTCTACACCGACGGTACTGTGACCGCCAACGTGTGGTACACATACTATCTCACCGAAAACCTTGCACTCACAGCCGAGTTGACATTCAAGAACGGAAAGTTGGATAGCGTTTCAGTCGGCGCCACATATACCTTCGAGCCTAAGCTCACAGTGTCTGTCGTGGGTACATACCACACCGATGGCAAGTGGGATGTGACCGTGACAGTCGAAGGCGAAGTGAAGATCAACGACAAGCTCACATTGTATCCAGGCGGCAAGTACACTTTGAGCAATACTGCACCAGATACTTGGGCGCCTTACGTGAAGTTCGAGTACAAGGTGACTGATAAGCTCAGTCTGCTCGGCAAGGTCGAGTATAATGATGATGGCACCACAACTGTCTCAGTTGGCCTGAAGTACAAGCTCACTGATAATCTCTCTCTTACCGTCACTGCGACGTTCGTGAATGGCAAGCTCGATTCTGTGACTGTGGAGGTTGAGTATGACTTGACTAAGTGA-3’。

[0026] The present invention also provides the application of the above-mentioned 12-stranded β-barrel protein SBR1 in improving the broad-spectrum disease resistance of plants. The broad-spectrum disease resistance includes one or more of antifungal diseases, antibacterial diseases, and antiviral diseases. In the embodiments of the present invention, the broad-spectrum disease resistance includes that after transient transformation of tobacco with SBR1, the resistance of tobacco to TuMV-GFP virus can be improved; after transient transformation of tobacco with SBR1, the resistance of tobacco to Phytophthora capsici can be improved; after stable transformation of rice with SBR1, the resistance of rice to Magnaporthe oryzae can be improved.

[0027] The present invention also provides a primer set for amplifying the nucleic acid molecule, and the primer set includes a forward primer and a reverse primer; the nucleotide sequence of the forward primer is as shown in SEQ ID NO.2; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.3.

[0028] The sequences of the primer set of the present invention are shown as follows:

[0029] Forward primer (SEQ ID NO.2): 5’-CCAAATCGACTCTAGAAAGCTTATGAGG CCAAAGCCAGG-3’;

[0030] Reverse primer (SEQ ID NO.3): 5’-TTGCTCACCATGGTCTCTCACTTAGTCA AGTCATACTCAAC-3’.

[0031] The present invention also provides a biological material containing the nucleic acid molecule, including a recombinant expression vector containing the nucleic acid molecule and / or a recombinant microorganism containing the nucleic acid molecule. The basic backbone of the recombinant expression vector containing the nucleic acid molecule of the present invention includes the pCAMBIA1300 vector or the pRHE vector. As a specific embodiment, the recombinant expression vector containing the nucleic acid molecule includes inserting the nucleic acid molecule into the pCAMBIA1300 vector to obtain the p1300-SBR1 overexpression vector or inserting the nucleic acid molecule into the pRHE vector to obtain the pRHE-SBR1 overexpression vector. As an embodiment, the recombinant microorganism containing the nucleic acid molecule includes Agrobacterium tumefaciens GV3101 containing the nucleic acid molecule.

[0032] The present invention also provides a pathogen-inducible plant expression vector, and the pathogen-inducible plant expression vector includes the PR10b promoter and the nucleic acid molecule described above. As a specific embodiment, the basic backbone of the pathogen-inducible plant expression vector includes the pRHE vector; the gene accession number of the PR10b promoter is AF274851.1. As a specific embodiment, Magnaporthe oryzae is the pathogen that induces rice to be infected with rice blast. After Magnaporthe oryzae invades rice, it will induce the up-regulation of many defense-related genes in rice. Among them, PR10b, as a rice defense-related gene, has an increased expression level during the infection of Magnaporthe oryzae to defend against the infection of Magnaporthe oryzae. Therefore, PR10b will be induced to express during the infection of Magnaporthe oryzae, and the expression level is very low under non-infection induction conditions. In order to achieve the induced expression of SBR1 in rice during the infection of Magnaporthe oryzae, the PR10b promoter and the nucleic acid molecule encoding the 12-stranded β-barrel protein SBR1 are constructed into the pRHE vector, thereby obtaining the pathogen-inducible plant expression vector, enabling the recombinant expression of the PR10b promoter and SBR1 in rice.

[0033] The present invention also provides the use of the primer set, the biological material, or the pathogen-inducible plant expression vector in enhancing the broad-spectrum disease resistance of plants.

[0034] The present invention also provides a method for enhancing the disease resistance of plants, which is to overexpress the nucleic acid molecule in the genome of the target plant.

[0035] In a specific embodiment, the present invention transferred the overexpression vector p1300-SBR1 containing the nucleic acid molecule into Agrobacterium tumefaciens GV3101, and after injecting the Agrobacterium tumefaciens into tobacco, tobacco with enhanced disease resistance was obtained; the overexpression vector pRHE-SBR1 containing the nucleic acid molecule was transformed into the target rice plants by Aidianjing Biotechnology Co., Ltd., and rice with enhanced disease resistance was obtained.

[0036] To further illustrate the present invention, the following examples are used to describe in detail a 12-stranded β-barrel protein SBR1 provided by the present invention and its application in enhancing the broad-spectrum disease resistance of plants, but they should not be construed as limiting the protection scope of the present invention.

[0037] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products well-known to those skilled in the art can be used.

[0038] Example 1 SBR1 induces tobacco cell death and has the immune characteristics of plant disease resistance genes

[0039] S1. Vector construction:

[0040] Through artificial intelligence deep learning, a β-barrel protein was designed from scratch, and an artificially de novo designed 12-stranded β-barrel protein (shown in SEQ ID NO.1) was obtained. The SBR1 protein was optimized for plant codons using an online codon optimization tool (https: / / www.novo pro.cn / tools / codon-optimization.html) to obtain the SBR1 sequence optimized for plants (shown in SEQ ID NO.4), and the SBR1 fragment was synthesized by Beijing Tsingke Biotechnology Co., Ltd. The full-length SBR1 fragment was amplified and constructed into the pCAMBIA1300 vector (doi: 10.3389 / fmicb.2024.1507998) by homologous recombination.

[0041] The primer sequences were designed as follows:

[0042] P1300-SBR1-F: 5’-CCAAATCGACTCTAGAAAGCTTATGAGGCCAAAGCC AGG-3’ (shown in SEQ ID NO.2);

[0043] P1300 - SBR1 - R: 5'-TTGCTCACCATGGTCTCTCACTTAGTCAAGTCATACTCAAC3' (as shown in SEQ ID NO.3);

[0044] P1300 - SBR1 - R2: 5'-TTGCTCACCATGGTCTCCTTAGTCAAGTCATACTCAACCTCC - 3' (as shown in SEQ ID NO.5).

[0045] Amplify the SBR1 fragment: The primer pairing is as follows: P1300 - SBR1 - F and P1300 - SBR1 - R (SBR1 fragment), P1300 - SBR1 - F and P1300 - SBR1 - R2 (SBR1 - eGFP fragment). Use the synthesized SBR1 fragment as a template for PCR amplification. The reaction conditions are: pre - denaturation at 95°C for 5 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, for 35 cycles; extension at 72°C for 5 min.

[0046] Homologous recombination ligation: Digest the pCAMBIA1300 vector with HindIII restriction endonuclease (NEB#R3104). Ligate the digested product with the corresponding fusion fragment to construct a tobacco expression vector, and then transform Escherichia coli DH5α (TOLOBIO; CC96102) to obtain the P1300 - SBR1 vector expressing SBR1 and the P1300 - SBR1 - eGFP vector expressing SBR1 - eGFP protein.

[0047] Construct Xa23 (GenBank accession number: KP123634.1) into the pCAMBIA1300 vector. Construction method: Use primers P1300 - Xa23 - F: 5'-ATCGACTCTAGAAAGCTTATGTTGCATCATCTCAAGGAGCT - 3' (as shown in SEQ ID NO.6), P1300 - Xa23 - R: 5'-CACCA TGGTCTCAAGCTTTTAAACAGGGAGAATAACCATCTTGTCG - 3' (as shown in SEQ ID NO.7), and use the Nipponbare rice genome as a template for PCR amplification. The reaction conditions are: pre - denaturation at 95°C for 5 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, for 35 cycles; extension at 72°C for 5 min.

[0048] Homologous recombination ligation: The pCAMBIA1300 vector was digested with HindIII, and the digested product was ligated with the corresponding fusion fragment to construct a tobacco expression vector. Subsequently, Escherichia coli DH5α was transformed to obtain the P1300-Xa23 vector containing Xa23.

[0049] S2. Immune characteristic analysis:

[0050] 1. Transient expression of SBR1 in Nicotiana benthamiana induces the hypersensitive response (HR) in plants: This experiment was divided into 3 groups: SBR1, SBR1-eGFP, the pCAMBIA1300 empty vector (EV), and the positive control Xa23 that induces the HR response. The P1300-SBR1 vector, P1300-SBR1-eGFP vector, P1300-Xa23 vector, and the pCAMBIA1300 empty vector (EV) were transferred into Agrobacterium tumefaciens GV3101. After culturing with shaking for 16 h, the bacterial cells were resuspended with the infection solution (10 mM MgCl2, 10 mM MES, and 100 μM acetosyringone) to obtain Agrobacterium tumefaciens containing the P1300-SBR1 vector, P1300-SBR1-eGFP vector, P1300-Xa23 vector, and the pCAMBIA1300 empty vector (EV). The OD of the Agrobacterium tumefaciens expressing the P1300-SBR1 vector, P1300-SBR1-eGFP vector, P1300-Xa23 vector, and the pCAMBIA1300 empty vector (EV) was adjusted 600 to 1.0. The Agrobacterium tumefaciens containing the P1300-SBR1 vector, P1300-SBR1-eGFP vector, P1300-Xa23 vector, and the pCAMBIA1300 empty vector (EV) was respectively injected into the leaves of 4-week-old Nicotiana benthamiana. 48 h after injection, the tobacco leaves were observed and photographed under white light ( Figure 1 left figure) and ultraviolet ( Figure 1 middle figure) (UV) light. Figure 1 The results showed that SBR1 and SBR1-eGFP could induce tobacco cell necrosis and cause the HR response in tobacco cells, just like the positive control Xa23, while the empty vector (EV) did not trigger the HR response in tobacco cells.

[0051] 2. SBR1 induces the burst of reactive oxygen species (ROS) in Nicotiana benthamiana - DAB staining: Agrobacterium containing the P1300 - SBR1 vector, P1300 - SBR1 - eGFP vector, P1300 - Xa23 vector, and the pCAMBIA1300 empty vector (EV) was injected into the leaves of four - week - old Nicotiana benthamiana plants according to the method of step 1 in S2. 15 h after injection, the injected tobacco leaves were immersed in 20 mL of DAB staining solution (Solarbio, catalog number: G2370), horizontally shaken in the dark for 8 h, then placed in 95% ethanol for decolorization, horizontally shaken for 48 h, and photographed under white light. Figure 1 The results of the figure on the right show that SBR1, SBR1 - eGFP, and the positive control Xa23 can all stain the phenomenon of ROS burst, while the empty vector (EV) does not have this phenomenon. This indicates that the expression of SBR1 and SBR1 - eGFP in tobacco cells can induce ROS burst like the positive control Xa23.

[0052] 3. SBR1 Induces ROS Burst in Nicotiana benthamiana - ROS Assay: This experiment was divided into four groups, namely the SBR1 / Chitin group (treatment group 1), the EV / Chitin group (treatment group 2), the SBR1 / mock group (control group 1), and the EV / mock group (control group 2). In the SBR1 / Chitin group (treatment group 1), Agrobacterium containing the P1300-SBR1 vector was first used to inject Nicotiana benthamiana leaves. After 10 h of injection, small round discs of Nicotiana benthamiana leaves with a diameter of 3 mm were collected and immersed in distilled water overnight in a 96-well plate. The distilled water was discarded, and 100 μL of Chitin mixed solution (50 μM luminol (Merck; 123072), 10 μg / mL horseradish peroxidase (Merck; SRE0082), and 6 mM Chitin (GLPBIO; GB57663)) was added to each well. In the EV / Chitin group (treatment group 2), Agrobacterium containing the empty vector (EV) was first used to inject Nicotiana benthamiana leaves. After 10 h of injection, small round discs of Nicotiana benthamiana leaves with a diameter of 3 mm were collected and immersed in distilled water overnight in a 96-well plate. The distilled water was discarded, and 100 μL of Chitin mixed solution was added to each well. In the SBR1 / mock group (control group 1), Agrobacterium containing the P1300-SBR1 vector was first used to inject Nicotiana benthamiana leaves. After 10 h of injection, small round discs of Nicotiana benthamiana leaves with a diameter of 3 mm were collected and immersed in distilled water overnight in a 96-well plate. The distilled water was discarded, and 100 μL of distilled water was added to each well as a control. In the EV / mock group (control group 2), Agrobacterium containing the empty vector (EV) was first used to inject Nicotiana benthamiana leaves. After 10 h of injection, small round discs of Nicotiana benthamiana leaves with a diameter of 3 mm were collected and immersed in distilled water overnight in a 96-well plate. The distilled water was discarded, and 100 μL of distilled water was added to each well as a control. Each sample group had 11 biological replicates. Chemiluminescence was measured at intervals of 500 ms for 60 min in a SPARK-10M microplate reader (TECAN).

[0053] From Figure 2 As shown by a, b in and the results of Table 1 and Table 2, after induction with Chitin, it was found that the reactive oxygen species in tobacco after injection of Agrobacterium containing the SBR1 expression plasmid increased sharply, significantly higher than other groups, indicating that SBR1 expression can induce the production of reactive oxygen species, and the total amount of reactive oxygen species in this group was much higher than that of control groups 1 and 2.

[0054] Table 1 Relative light units at different times in different treatments

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Table 2 Total number of photons in different treatments

[0062]

[0063]

[0064] 4. Detection of disease resistance-related genes: Experimental method: This experiment was divided into two groups, that is, injecting Agrobacterium containing the P1300-SBR1 vector into Nicotiana benthamiana as the experimental group and injecting Agrobacterium with an empty vector (EV) expression plasmid into Nicotiana benthamiana as the control group. At least three biological replicates were performed for each sample.

[0065] RNA extraction and reverse transcription quantitative PCR analysis: Collect tobacco leaves containing the P1300-SBR1 vector and the control EV 15 h after Agrobacterium injection, quickly place them in liquid nitrogen for freezing, and use them for RNA extraction after grinding in liquid nitrogen. Total RNA was extracted from the samples using Vazyme's VeZol Reagent (R411-01 / 02). RNA was purified from total RNA using Vazyme's 4×gDNA wiper Mix, and complementary DNA (cDNA) was synthesized using Vazyme's 5×HiScriptⅡ qRT SuperMix cDNA synthesis kit (R123-01). qRT-PCR was performed on a Bio-Rad CFX96 real-time system and a C1000 thermal cycler (Bio-Rad) using Tolobio's 2×Q3 SYBR qPCR Master Mix (Universal) (Tolobio, 22204). 2 -ΔΔCT This method was used to calculate the gene expression level.

[0066] Detection primer sequences:

[0067] PR1b: qRT-NbPR1b-F: 5’-GTGGACACTATACTCAGGTG-3’ (as shown in SEQ ID NO.8);

[0068] qRT-NbPR1b-R: 5’-TCCAACTTGGAATCAAAGGG-3’ (as shown in SEQ ID NO.9);

[0069] PR2b: qRT-NbPR2b-F: 5’-AGGTGTTTGCTATGGAATGC-3’ (as shown in SEQ ID NO.10);

[0070] qRT-NbPR2b-R: 5'-CTGTACCCACCATCTTGC-3' (as shown in SEQ ID NO.11);

[0071] PR4: qRT-NbPR4-F: 5'-GGCCAAGATTCCTGTGGTAGAT-3' (as shown in SEQ ID NO.12);

[0072] qRT-NbPR4-R: 5'-CACTGTTGTTTGAGTTCCTGTTCCT-3' (as shown in SEQ ID NO.13);

[0073] LOX: qRT-NbLOX-F: 5'-AAAACCTATGCCTCAAGAAC-3' (as shown in SEQ ID NO.14);

[0074] qRT-NbLOX-R: 5'-ACTGCTGCATAGGCTTTGG-3' (as shown in SEQ ID NO.15);

[0075] Reference gene: NbEF1α

[0076] Detection primers: qRT-NbEF1α-F: 5'-AGAGGCCCTCAGACAAAC-3' (as shown in SEQ ID NO.16);

[0077] qRT-NbEF1α-R: 5'-TAGGTCCAAAGGTCACAA-3' (as shown in SEQ ID NO.17).

[0078] Figure 2 The results in Figure c and Table 3 showed that the expression levels of disease-resistant genes such as PR1b, PR2b, PR4, and LOX in tobacco samples injected with Agrobacterium expressing SBR1 were significantly increased, significantly higher than those in the control group (after t-test, P value < 0.0001), indicating that the expression of SBR1 activated the expression of tobacco disease-resistant genes, demonstrating that the SBR1 gene has the immune characteristics of tobacco R genes.

[0079] Table 3 Expression levels of different disease-resistant genes in different treatments

[0080]

[0081] From the above results, it can be seen that the SRB1 protein has the basic characteristics of plant disease-resistant proteins.

[0082] Example 2 Analysis of the resistance of SBR1 transiently transformed tobacco to TuMV-GFP virus

[0083] The TuMV infectious clone vector carrying GFP (doi: https: / / doi.org / 10.1111 / mpp.13146), P1300-SBR1 vector, and pCAMBIA1300 empty vector (EV) plasmids were transformed into Agrobacterium tumefaciens strain GV3101. After shaking culture at 28 °C for 16 h, the bacterial cells were resuspended with an infection solution (10 mM MgCl2, 10 mM MES, and 100 μM acetosyringone) to obtain Agrobacterium containing the P1300-SBR1 vector, pCAMBIA 1300 empty vector (EV), and TuMV expression plasmid. The OD of Agrobacterium expressing the P1300-SBR1 vector, pCAMBIA1300 empty vector (EV), and TuMV was adjusted 600To 2.0, the Agrobacterium containing the P1300-SBR1 vector / TuMV expression plasmid and the Agrobacterium containing the EV expression plasmid / TuMV expression plasmid were mixed at a volume ratio of 1:1 respectively, and then the young two leaves under the systemic leaves of wild-type tobacco (WT) were injected. The tobacco injected with the 1:1 mixture of the Agrobacterium containing the P1300-SBR1 vector / TuMV expression plasmid was used as the experimental group, and the tobacco injected with the 1:1 mixture of the Agrobacterium containing the EV expression plasmid / TuMV expression plasmid was used as the control group. Nine days post inoculation (dpi), photographs were taken and counted under ultraviolet light (UV). At the same time, the RNA of the systemic leaves above the injected tobacco leaves was extracted, and the tobacco EF1α gene was used as an internal reference for qRT-PCR to measure the expression levels of the TuMV virus GFP (detection primers: TuMV-GFP-F: 5'-GAAGCGGCACGACTTCTTCAAGAG-3' (as shown in SEQ ID NO.18); TuMV-GFP-R: 5'-GCCGAGGATGTTTCCGTCCTCC-3' (as shown in SEQ ID NO.19)) and the coat protein CP gene (detection primers: TuMV-CP-F: 5'-CACGCC GGAGCAGACGGATC-3' (as shown in SEQ ID NO.20); TuMV-CP-R: 5'-CTGATCGTCGCCGTCCATCATC-3' (as shown in SEQ ID NO.21)). The systemic leaves were quickly placed in liquid nitrogen for freezing and ground in liquid nitrogen for RNA extraction. Total RNA was extracted from the samples using Vazyme VeZol Reagent. RNA was purified from the total RNA using Vazyme 4×gDNA wiper Mix, and complementary DNA (cDNA) was synthesized using Vazyme 5×HiScriptⅡqRT SuperMix cDNA synthesis kit. qRT-PCR was performed using TOLOBIO 2×Q3 SYBR qPCR Master Mix (Universal) on a Bio-Rad CFX96 real-time system and a C1000 thermal cycler (Bio-Rad). 2 -ΔΔCT The method was used to calculate the gene expression levels. Five biological replicates were performed for each group.

[0084] Figure 3 The results in a showed that the content of TuMV-GFP in the SBR1 transiently transformed tobacco systemic leaves was significantly lower than that in the control group. Figure 3Figure b and Table 4 show that the expression levels of the GFP and coat protein CP genes of the TuMV virus are also significantly lower than those of the control group (after t-test, P value < 0.0001). In summary, transient transformation of tobacco with SBR1 confers significant resistance to the TuMV virus.

[0085] Table 4 Expression levels of different TuMV genes in different treatments

[0086]

[0087] Example 3 Analysis of the resistance of SBR1 transiently transformed tobacco to Phytophthora capsici

[0088] The P1300-SBR1 vector and the pCAMBIA1300 empty vector (EV) plasmid were transformed into Agrobacterium tumefaciens strain GV3101. After shaking culture at 28 °C for 16 h, the cells were resuspended with an infection solution (10 mM MgCl2, 10 mM MES, and 100 μM acetosyringone) to obtain Agrobacterium tumefaciens containing the P1300-SBR1 vector and the pCAMBIA1300 empty vector (EV) expression plasmid. Adjust the OD of Agrobacterium tumefaciens expressing the P1300-SBR1 vector and the pCAMBIA1300 empty vector (EV) 600 to 1.0. Tobacco injected with Agrobacterium tumefaciens containing the P1300-SBR1 expression plasmid was used as the experimental group, and tobacco injected with Agrobacterium tumefaciens containing the EV expression plasmid was used as the control group. 12 biological replicates were performed for each sample.

[0089] At 15 h after injection, the injected leaves in different treatments were cut off, and the leaves were placed in an inoculation tray lined with wet absorbent paper towels. A 3-mm diameter Phytophthora capsici strain LT263 fungal disc was inoculated at the injection site, 10 μL of sterilized distilled water was added dropwise at the inoculation site, and the inoculation site was covered with plastic wrap and kept moist and dark for 48 h. The tobacco leaves were observed and photographed under ultraviolet (UV) light. The results ( Figure 4 ) showed that the lesion area of the treatment group injected with Agrobacterium tumefaciens expressing the SBR1 plasmid was significantly smaller than that of the control group injected with Agrobacterium tumefaciens expressing the EV plasmid. Moreover, by detecting the relative biomass of the pathogen, it was found that the biomass of the pathogen detected in the treatment group injected with Agrobacterium tumefaciens expressing the SBR1 plasmid was significantly lower than that of the control group.

[0090] Using ImageJ to statistically analyze the lesion area, it was found that the infection lesions formed by the treatment group injected with SBR1 were significantly smaller than those of the control group. Samples of inoculated leaves were taken with a diameter of 2 cm centered on the point where the inoculation mycelial disc was placed. DNA was extracted by the CTAB method, and the DNA concentration was adjusted. Using tobacco gene EF1α as an internal reference, the expression level of Phytophthora capsici tubulin gene was detected. The primer information was as follows: qRT-NbEF1α-F: 5’-AGAGGCCCTCAGACAAAC-3’ (as shown in SEQ ID NO.22) and qRT-NbEF1α-R: 5’-TAGGTCCAAAGGTCACAA-3’ (as shown in SEQ ID NO.23); Pc-tubulin-F: 5’-CAGAGGGTGCTGAGCTTATTGAC-3’ (as shown in SEQ ID NO.24) and Pc-tubulin-R: 5’-GAGAGTGGGTGATCTGGAAACCC-3’ (as shown in SEQ ID NO.25); qRT-PCR was performed on a Bio-Rad CFX96 real-time system and a C1000 thermal cycler (Bio-Rad) using the Tolobio 2×Q3SYBR qPCR Master Mix (Universal). The relative biomass of Phytophthora capsici in tobacco leaf tissues was calculated using the 2 -ΔΔCT method.

[0091] As can be seen from Figure 4 , Table 5 and Table 6, after the same time of inoculation with Phytophthora capsici and detection, it was found that the area of infection lesions of Phytophthora capsici on the leaves of Agrobacterium injected with SBR1 expression plasmid was significantly smaller than that on the tobacco leaves of Agrobacterium injected with EV expression plasmid. In tobacco leaves of the same weight, the relative pathogen biomass of the sample of Agrobacterium injected with SBR1 expression plasmid was significantly lower than that of the tobacco leaf sample of Agrobacterium injected with EV expression plasmid (after t-test, P value < 0.0001). Therefore, the SBR1 protein can function in tobacco and enhance the immune level of tobacco and the ability to resist Phytophthora capsici.

[0092] Table 5 Lesion area of Phytophthora capsici in different treatments (unit: cm 2 )

[0093]

[0094]

[0095] Table 6 Relative biomass of Phytophthora capsici in different treatments

[0096]

[0097] Analysis of the Resistance of SBR1-Stably Transformed Rice to Magnaporthe oryzae in Example 4

[0098] S1. Screening and Application Strategy of Pathogen-Inducible Promoters

[0099] Magnaporthe oryzae is the main fungal disease affecting rice yield and quality and is considered the most serious threat to global rice production. Magnaporthe oryzae is the pathogen that induces rice to be infected with rice blast. When Magnaporthe oryzae invades rice, it induces the upregulation of many defense-related genes in rice. Among them, PR10b (AF274851.1), as a rice defense-related gene, increases its expression level during the infection of Magnaporthe oryzae to defend against the infection of Magnaporthe oryzae. Therefore, PR10b is induced to express during the infection of Magnaporthe oryzae and has a very low expression level under non-infection-induced conditions. In order to achieve the inducible expression of SBR1 in rice during the infection of Magnaporthe oryzae, the PR10b promoter and SBR1 are recombinantly expressed in rice and homologously recombined onto the vector pRHE (doi: https: / / doi.org / 10.1038 / s41586-023-06205-2). The vector with correct sequencing is sent to AidyGen Biotech Co., Ltd. to transform the wild-type rice variety Kitaake (Kit) (doi: https: / / doi.org / 10.1038 / s41586-023-06205-2).

[0100] S2. Identification of the Disease Resistance of the Transgenic Lines in the T0 Generation

[0101] Finally, a total of 13 independent T0-generation rice lines were obtained from the company. The obtained transgenic rice T0 lines (genetic background is Kitaake) were numbered 1-13 respectively, and the wild-type rice line was used as a control. The DNA samples of the above 13 lines were extracted by the CTAB method. Using the extracted DNA as a template and the primers of the target gene as the detection primers (the specific primer information is: PR10b-JC-F: 5’-CCACATCAG ATAAGCTCAGC-3’ (shown as SEQ ID NO.26), and PR10b-JC-R: 5’-TGTGAGC TTAGGCTCGAAG-3’ (shown as SEQ ID NO.27)). After the above PCR detection process, 4 positive lines were finally detected.

[0102] Disease resistance identification: At the 3-5 leaf stage of transgenic rice, use a hole punch to damage the leaf at the vein of a healthy leaf without punching through. Then, use a sterile hole punch to cut a small 3-mm circular mycelium from Magnaporthe oryzae (EA18) grown on OTA medium (150 mL of tomato juice, 40 g of oats, 0.6 g of calcium carbonate, and 18 g of agar powder added to each liter of distilled water) for 7 days. Place the mycelial block on the damaged area of the leaf and seal the punched area with transparent tape. The inoculation conditions in the greenhouse are 12 / 12 h of day / night light, a temperature of 28 °C, and a relative humidity of 80%. After 14 days of inoculation, cut the leaves for the statistical analysis of the disease area and the determination of fungal biomass. Among them, pPR10b::SBR1 is SBR1 transgenic rice, serving as the treatment group, and Kit serves as the control group.

[0103] The use of ImageJ to statistically analyze the lesion area found that the infection lesions formed by the treatment group were significantly smaller than those of the control group. As can be seen from Figure 5 Table 7, after inoculating Magnaporthe oryzae for the same time and detecting, it was found that the lesion area of Magnaporthe oryzae infection on the leaves of SBR1 transgenic rice was significantly smaller than that of the leaves of the control Kit (after t-test, P value < 0.0001). Therefore, the SBR1 protein can perform its function in rice and enhance the plant's immune level and the ability to resist Magnaporthe oryzae.

[0104] Table 7 Lesion area of rice blast in different treatments (unit: cm 2 )

[0105]

[0106] Thus, it can be seen that the present invention can use artificial intelligence deep learning to design a β-barrel disease-resistant protein that can be applied to plants, greatly reducing the mining time and saving manpower and material resources. At the same time, the present invention first introduced the artificially de novo designed 12-stranded β-barrel protein into plant disease resistance and proved its role in plant disease resistance. The research of the present invention introduced a new strategy for plant disease resistance, greatly increasing the number of plant disease-resistant genes that can be applied and greatly reducing the cost of cloning plant disease-resistant genes.

[0107] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A 12-stranded β-barrel protein SBR1, characterized in that The amino acid sequence of the 12-stranded β-barrel protein SBR1 is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the 12-stranded β-barrel protein SBR1 according to claim 1.

3. Use of the 12-stranded β-barrel protein SBR1 according to claim 1 in improving the broad-spectrum disease resistance of plants, characterized in that: The broad-spectrum disease resistance includes one or more of resistance to fungal diseases, resistance to bacterial diseases and resistance to viral diseases.

4. A primer set for amplifying the nucleic acid molecule according to claim 2, characterized in that: The primer set includes a forward primer and a reverse primer; The nucleotide sequence of the forward primer is shown in SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

3.

5. A biological material comprising the nucleic acid molecule according to claim 2, characterized in that: It includes a recombinant expression vector containing the nucleic acid molecule and / or a recombinant microorganism containing the nucleic acid molecule.

6. The biomaterial according to claim 5, characterized in that The basic framework of the recombinant expression vector containing the nucleic acid molecule includes pCAMBIA1300 vector or pRHE vector.

7. A pathogen-inducible plant expression vector, characterized in that: The pathogen-inducible plant expression vector comprises a PR10b promoter and the nucleic acid molecule according to claim 2.

8. The pathogen-inducible plant expression vector according to claim 7, characterized in that: The basic framework of the pathogen-inducible plant expression vector includes a pRHE vector; The gene accession number of the PR10b promoter is AF274851.

1.

9. Use of the primer set according to claim 4, the biological material according to claim 5 or 6, or the pathogen-inducible plant expression vector according to claim 7 or 8 in improving the broad-spectrum disease resistance of plants.

10. A method for improving plant disease resistance, characterized in that: Overexpressing the nucleic acid molecule of claim 2 in the genome of a target plant.

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