Application of DMP3 gene and encoding protein thereof in improving plant disease resistance

Abstract

The invention discloses an application of a DMP3 gene and an encoding protein thereof in improving plant disease resistance, and belongs to the technical field of gene engineering. The DMP3 gene and the encoding protein thereof are used for improving the disease resistance of plants, the nucleotide sequence of the DMP3 gene is shown as SEQ ID NO: 1, and the amino acid sequence of the encoding protein of the DMP3 gene is shown as SEQ ID NO: 2, especially the disease resistance of the plants to pseudomonas syringae. The invention further provides a PAD3 gene promoter induced by pathogenic bacteria and a plant expression vector containing the PAD3 gene promoter induced by the pathogenic bacteria and a DMP3 gene, wherein the sequence of the PAD3 gene promoter is shown as SEQ ID NO: 3. Real-time quantitative PCR (polymerase chain reaction) shows that Pseudomonas syringae infection remarkably induces up-regulation of expression of a DMP3 gene of a plant, functional experiments show that DMP3 gene expression can activate immune response of the plant, and overexpression of DMP3 causes death of the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to the application of a DMP3 gene and its encoded protein in improving plant disease resistance. Background Art

[0002] Throughout their life cycle, plants continuously face complex biological stresses from multiple types of pathogenic microorganisms, including bacteria, fungi, viruses, and oomycetes. Among them, plant diseases caused by pathogenic bacteria are widely destructive. Studies have shown that there are currently more than 500 plant pathogens that pose a serious threat to the growth and development of crops and food security. As a typical representative of Gram-negative bacteria, Pseudomonas syringae pv. tomato DC3000 (PstDC3000) has attracted much attention due to its wide host adaptability. This pathogen can infect important economic crops and legumes such as corn, tobacco, and tomatoes, and has been listed as one of the top ten plant pathogens in the world by the international plant pathology community. In recent years, plant diseases caused by this pathogen have continued to break out in major grain-producing areas around the world. The development of new environmentally friendly and broad-spectrum resistant biological control technologies has become an urgent need for the sustainable development of global agriculture.

[0003] In the global plant protection system, chemical pesticides, as a core pest control measure, play a vital role in maintaining stable crop yields and increasing incomes, as well as ensuring food security. However, modern toxicological research indicates that current pesticide applications pose significant ecological risks. Their targeted utilization is low, and unused active ingredients can cause multiple environmental toxicological problems through non-target biological effects. According to WHO monitoring data, pesticide residues in farmland soils worldwide have generally exceeded international safety standards, with organophosphorus pesticides exhibiting bioaccumulation factors far exceeding baseline values ​​in the food chain. This long-term, low-dose exposure not only leads to declines in pollinator populations and disrupts the soil microbiome, but also exhibits a significant dose-response relationship with chronic diseases in humans, such as neurodevelopmental disorders and metabolic syndrome. Against this backdrop, developing precise disease resistance-inducing technologies based on activation of plant innate immunity, and shifting strategies from chemical control to biological defense, has become a key breakthrough in achieving the twin goals of "Zero Hunger" and "Land Ecosystem Protection" in the United Nations 2030 Agenda for Sustainable Development.

[0004] In recent years, numerous plant disease resistance (R) genes have been identified, most of which are NLR genes. These genes contain a nucleotide binding site and a leucine-rich region, and play a crucial role in plant disease resistance, particularly in ETI. Recent studies have reported another class of R genes, encoding proteins that can directly kill pathogen-infected plant cells, thereby achieving pathogen resistance. These R genes are known as Executor (E)R genes. While both proteins can activate plant immunity, their activation mechanisms differ significantly. NLR proteins are typically in an autoinhibitory state, becoming activated only upon recognition of pathogen effector proteins. Executor R genes, on the other hand, regulate their activity at the transcriptional level. Normally, these genes are silent. Only upon pathogen invasion do their effector proteins bind to the Executor R gene promoter, inducing expression and mediating cell death in infected plants, thereby resisting pathogen infection and enhancing plant disease resistance. Summary of the Invention

[0005] The main purpose of the present invention is to provide an application of the DMP3 gene and its encoded protein in improving plant disease resistance, in particular for improving plant disease resistance to Pseudomonas syringae.

[0006] Another object of the present invention is to provide a PAD3 gene promoter induced by pathogens, which is inserted upstream of the DMP3 gene, around the enhancer or into a recombinant vector to regulate the expression of the DMP3 gene.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In a first aspect of the present invention, a DMP3 gene and its encoded protein are used for improving plant disease resistance. The nucleotide sequence of the DMP3 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2.

[0009] Preferably, the plant disease resistance is the plant's disease resistance to Pseudomonas syringae.

[0010] Preferably, the plant comprises crops selected from one or more of Arabidopsis thaliana, rapeseed, rice, tomato, potato, peanut, soybean, cotton, tobacco, cucumber and watermelon.

[0011] In a second aspect, the present invention further provides a PAD3 gene promoter induced by pathogens, the sequence of which is shown in SEQ ID NO: 3.

[0012] The third aspect of the present invention further provides a plant expression vector comprising the PAD3 gene promoter induced by pathogens and the DMP3 gene having a nucleotide sequence as shown in SEQ ID NO: 1.

[0013] Preferably, the pathogen is Pseudomonas syringae.

[0014] The fourth aspect of the present invention further provides an engineered Agrobacterium containing any of the aforementioned plant expression vectors.

[0015] The fifth aspect of the present invention further provides the use of the engineered Agrobacterium in improving plant disease resistance.

[0016] Preferably, the plant infected by the engineered Agrobacterium includes crops selected from one or more of Arabidopsis thaliana, rapeseed, rice, tomato, potato, peanut, soybean, cotton, tobacco, cucumber and watermelon.

[0017] Preferably, the plant disease resistance is the plant's disease resistance to Pseudomonas syringae.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This invention proposes the use of the DMP3 gene and its encoded protein to enhance plant disease resistance, particularly against Pseudomonas syringae. Real-time quantitative PCR revealed that infection with Pseudomonas syringae significantly upregulated DMP3 gene expression in Arabidopsis thaliana. Further functional experiments revealed that DMP3 gene expression can activate the immune response in Arabidopsis thaliana, and that overexpression of DMP3 can lead to plant death.

[0020] 2. The present invention provides a PAD3 gene promoter induced by pathogens, which can be used to regulate the appropriate expression of the DMP3 gene after pathogen induction, so as to avoid the failure to achieve the purpose of improving plant disease resistance due to low expression levels, and also avoid the death of plants due to excessive expression levels.

[0021] 3. The improvement of plant disease resistance in the present invention can reduce the use of chemical pesticides, reduce environmental pollution, and can be applied to the field of environmental protection engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the relative expression result of the DMP3 gene in Arabidopsis thaliana induced by Pseudomonas syringae infection in Example 1.

[0023] Figure 2 This is the activation of the DMP3 gene on the Arabidopsis immune response verified by ethylene production in Example 2.

[0024] Figure 3 This is to verify the activation of the DMP3 gene on the Arabidopsis immune response through phenotypic analysis in Example 2.

[0025] Figure 4 This is the activation of the DMP3 gene on the Arabidopsis immune response verified by MAPKs in Example 2.

[0026] Figure 5 This is the plant disease resistance test result of Example 3 in which the promoter of the PAD3 gene induced by pathogens regulates the expression of DMP3. DETAILED DESCRIPTION

[0027] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the following is a further detailed and complete description of the technical effects produced by the present invention in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present invention, and not all of them. It should be pointed out that for those skilled in the art, other embodiments obtained without departing from the concept of the present invention are all within the scope of protection of the present invention.

[0028] Example 1: Pseudomonas syringae infection induces relative expression of the DMP3 gene in Arabidopsis thaliana

[0029] (1) Analysis of relative gene expression by RT-qPCR

[0030] Wild-type Col-0 Arabidopsis seeds were evenly spread on 1 / 2MS solid culture medium plates and placed in a plant incubator at 22°C and 24h light. After culturing for 5 days, Arabidopsis seedlings with good growth were selected and transferred from the plates to glass vials containing 10mL SW liquid medium. They were then transferred to a plant incubator at 22°C and 24h light for 7 days. The seedlings were then treated with a suspension of Pseudomonas syringae. The final concentration of the suspension was OD 600 =0.02, samples were collected at 0 h, 6 h, and 12 h, and after quick freezing and grinding in liquid nitrogen, total RNA was extracted using a plant total RNA extraction kit. RT-qPCR was used to analyze the relative expression of DMPs gene family members at different time points after Arabidopsis thaliana was treated with Pseudomonas syringae. The primer sequences for RT-qPCR are as follows:

[0031] DMP3-qPCR-F:GTTTTCGACTACCCTGACCC

[0032] DMP3-qPCR-R: GCGCCGGATAAAAACAACTC

[0033] The results are as follows Figure 1As shown, infection with Pseudomonas syringae resulted in a significant upregulation of the expression level of the DMP3 gene in Arabidopsis thaliana.

[0034] Example 2: Activation of the DMP3 gene on the immune response in Arabidopsis

[0035] (I) Obtaining DMP3 overexpressing transgenic lines

[0036] Using the cDNA of wild-type Col-0 Arabidopsis thaliana treated with Pseudomonas syringae as a template, the DMP3 gene fragment was amplified by PCR. A clone was constructed using the bidirectional enzyme-dot primer introduction method. An XhoI-BglII restriction site was added to the 5' end of the target sequence, and an XbaI-SmaI restriction site was added to the 3' end of the target sequence. The primer sequences for amplifying the DMP3 gene are as follows:

[0037] DMP3-F: CCGctcgagagatctATGTCTTCACCATCTTCCCTAACGCAGAG

[0038] DMP3-R:GCtcttagacccgggACGACGACCCCCGTCTCCGG

[0039] The sequence of the DMP3 gene is as follows:

[0040] ATGTCTTCACCATCTTCCCTAACGCAGAGAAACCCAACTAGTTCGCAAGAGCAATCGGAGTCTGTTCCACAGCTAAGGAGGCAGACATCTCAACATGCAGTCATGTCACAGACGCTAACCTCAGCTGCAAACCTGGCAAATCTCCTTCCAACCGGAACGCTCTTAGCTTTCACGCTCCTCATACCCGTCTTTACATCCAATGGTTCATGCGATTACCCGACCCAGGTTTTAACCATAGTGCTCCTCACGCTTCTCTCCATCTCCTGCTTCCTTAGCTCCTTCACTGACAGCGTCAAGGCTGAAGACGGTAACGTTTATTACGGTTTTGCTACTCGTAAGGGCATGTGGGTTTTCGACTACCCTGACCCTGACGGTTTGGGTTTACCCAATCTTAGTAAATACCGGATAAGAATTATTGACTGGATCCACGCCGTTTTGTCGGTTCTTGTGTTTGGTGCGGTGGCTCTGAGGGACAAGAACGCCGTGAGTTGTTTTTATCCGGCGCCAGAGCAAGAAACCAAGAAGGTTTTGGACATTGTTCCAATGGGTGTTGGGGTTATCTGTGGCATGTTGTTCTTGGTTTTTCCGGCGAGAAGGCACGGTATTGGATATCCGGTCACCGGAGACGGGGGTCGTCGT(SEQ ID NO:1)。

[0041] The amino acid sequence of the protein encoded by the DMP3 gene is as follows:

[0042] MSSPSSLTQRNPTSSQEQSESVPQLRRQTSQHAVMSQTLTSAANLANLLP

[0043] TGTLLAFTLLIPVFTSNGSCDYPTQVLTIVLLTLLSISCFLSSFTDSVKAEDGNV

[0044] YYGFATRKGMWVFDYPDPDGLGLPNLSKYRIRIIDWIHAVLSVLVFGAVALRD

[0045] KNAVSCFYPAPEQETKKVLDIVPMGVGVICGMLFLVFPARRHGIGYPVTGDG

[0046] GRR (SEQ ID NO: 2).

[0047] The amplified DMP3 gene fragment was inserted into the estradiol (Est)-induced expression pER8 vector through the XhoI and XbaI restriction sites and fused with the 2HA tag to construct the pER8-Est:DMP3-Linker-2HA plant expression vector. After Agrobacterium-mediated transformation of wild-type Col-0 Arabidopsis thaliana, transformed T0 generation seeds were obtained for subsequent screening.

[0048] After the harvested T0 generation seeds transformed with Agrobacterium are thoroughly dried, sterilized, purified, and evenly spread onto 1 / 2 MS solid culture medium plates containing the corresponding antibiotic. The plates are then incubated in a lighted plant incubator for approximately 10 days. Seedlings that show clear resistance, normal growth, and the development of true leaves are transplanted into soil to identify positive transgenic plants. After approximately two weeks of growth in soil, positive transgenic plants are tested for expression of the target gene by RT-qPCR or Western blot. Plants identified as expressing the target gene are cultured until maturity, after which seeds (T1 generation) are harvested.

[0049] Take an appropriate amount of fully dried T1 generation seeds, sterilize and purify them, evenly spread them on a 1 / 2MS solid culture medium plate containing the corresponding antibiotics, and place them in a plant light incubator for about 10 days. Statistical analysis is performed on the ratio of resistant seedlings to non-resistant seedlings, and strains with a resistant seedling: non-resistant seedling ratio of 3:1 are selected, that is, single-copy transgenic strains, and transplanted into the soil for continued cultivation. After maturity, seeds are collected from individual plants (T2 generation).

[0050] Take an appropriate amount of fully dried T2 generation seeds, sterilize and purify them, evenly spread them on 1 / 2MS solid culture medium plates containing corresponding antibiotics, and place them in a plant light incubator for about 10 days. Statistical analysis is performed on the ratio of resistant seedlings to non-resistant seedlings, and transgenic lines with all resistant seedlings are selected. RT-qPCR or Western Blot detection is used to confirm whether the target gene is stably expressed. Lines with all resistant seedlings and stably expressed target genes, i.e., homozygous and stably expressed transgenic plants, are transplanted into soil for further cultivation. After maturity, seeds (T3 generation) are collected for subsequent experiments.

[0051] (2) Ethylene determination

[0052] Take an appropriate amount of thoroughly dried T3 seeds, sterilize and purify them, and evenly spread them onto 1 / 2 MS solid culture medium plates containing the appropriate antibiotics. Incubate in a light-sensitive plant incubator. After 6 days of incubation, select well-growing Arabidopsis seedlings, transfer them from the plates to glass vials containing 10 mL of SW liquid culture medium, and transfer them to a plant incubator at 22°C with 24-hour light intensity for 7 days.

[0053] For the estradiol-treated group (-+Est group), estradiol was added to the glass vial that needed estradiol treatment, and DMSO was added to the glass vial of the control group. The cells were tightly sealed with a rubber cap and gently shaken to mix. Ethylene production was detected by gas chromatography at 3 h, 6 h, 12 h, and 24 h, respectively.

[0054] For the Pseudomonas syringae treatment group (-+Pst DC3000 group), 24 h before adding the Pseudomonas syringae suspension, the test samples were divided into two groups and treated with DMSO or Est respectively. After 24 h of treatment, the Pseudomonas syringae suspension was used again, and the final concentration of the Pseudomonas syringae suspension was OD 600 =0.02, tighten the sealing rubber cover, shake gently to mix, and detect the ethylene production by gas chromatography at 3h, 6h, 12h, and 24h.

[0055] The results are as follows Figure 2 As shown, for the estradiol treatment group (-+Est group), ethylene production increased significantly after DMP3 was induced by estradiol (Est) treatment alone, while DMSO treatment did not cause an increase in ethylene production. Ethylene is an important plant immune component, indicating that DMP3 expression activates plant immunity. For the Pseudomonas syringae treatment group (-+PstDC3000 group), even without DMP3 induction, Pseudomonas syringae can induce plant immune responses and produce ethylene. After inducing DMP3 expression, ethylene production increased significantly and was higher than that under sterile treatment, indicating that DMP3 can significantly increase ethylene production and activate plant immunity to resist Pseudomonas syringae infection, thereby achieving the purpose of disease resistance.

[0056] (III) Phenotypic analysis

[0057] An appropriate amount of thoroughly dried T3 seeds was sterilized and purified, then evenly spread onto 1 / 2 MS solid culture medium plates containing the appropriate antibiotics and cultured in a light-sensitive plant incubator. After 6 days of incubation, well-growing Arabidopsis seedlings were selected and transferred from the plates to glass vials containing 10 mL of SW liquid culture medium. The seeds were then transferred to a plant incubator at 22°C with 24-hour light intensity for 7 days. Estradiol was then added to the vials, and the treatment time was recorded. Changes in the plants after different treatment times were observed and photographed.

[0058] The results are as follows Figure 3 As shown, at 0 h of estradiol treatment, the plants were normal green, with normal growth conditions. After being fished out, the plants stood upright and had no difference from wild-type plants. After 24 h of estradiol treatment, the leaves of the plants turned yellowish, and the growth conditions became abnormal. After being fished out, the plants were soft, showing cell death caused by immune activation. After 48 h of estradiol treatment, the plants turned completely yellow and white and died completely. After being fished out, they became soft and rotten and could not take shape. This shows that estradiol induces excessive expression of DMP3. Although it can activate plant immunity, its activation intensity is too high, leading to plant death. It is necessary to control its expression intensity so that it can not only play its immune and disease-resistant function but also maintain normal plant growth.

[0059] (IV) MAPKs detection

[0060] An appropriate amount of thoroughly dried T3 seeds was sterilized and purified, then evenly spread onto 1 / 2 MS solid culture medium plates containing the appropriate antibiotics and cultured in a light-sensitive plant incubator. After 6 days of culture, well-growing Arabidopsis seedlings were selected and transferred from the plates to glass vials containing 10 mL of SW liquid culture medium. The seeds were then transferred to a plant incubator at 22°C with 24-hour light intensity for 7 days. Estradiol was then added to the vials, and samples were collected at 0, 3, 6, 12, and 24 hours, snap-frozen in liquid nitrogen, and analyzed for activation of the MAPK cascade pathway using Western blotting.

[0061] The results are as follows Figure 4 As shown, with the increase of estradiol treatment time, the accumulation of DMP3 gradually increased, and the MAPKs cascade pathway was first significantly activated and then gradually weakened, indicating that DMP3 can significantly activate the immune response of Arabidopsis, but as the accumulation of DMP3 caused the plants to begin to die, the MAPKs cascade pathway also began to gradually weaken.

[0062] Example 3: The PAD3 gene promoter induced by pathogens regulates DMP3 expression and modulates plant disease resistance

[0063] (1) Obtaining transgenic lines

[0064] Using the wild-type Col-0 Arabidopsis genome as a template, the PAD3 promoter fragment was amplified by PCR. A clone was constructed using the bidirectional enzyme-dot primer introduction method. A Hind III restriction site was added to the 5' end of the target sequence, and an Xba I restriction site was added to the 3' end of the target sequence. The primer sequences for amplifying the PAD3 promoter are as follows:

[0065] PAD3pro-F:CCCaagcttGGTCACTGTTGTTGCGGTCT

[0066] PAD3pro-R: GCtctagaTTTCCTTGCCCTGTTCTTGTG

[0067] The sequence of the PAD3 promoter is as follows:

[0068] GGTCACTGTTGTTGCGGTCTTTACATCCAAAAAATGTATTTATTTGTGC

[0069] TCTACTCTTTTAAATACAACAGGAGGTGTGCAATATGGACTTTCTCAATTAT

[0070] GTTGTATCCTACCTATATAATGAAATAAAATCCAACGGAAGAAGGCCATTAA

[0071] TTCTCCTCCACGTGCGTTATGTCTTTTATTTGTTTTCTATGGGGCTCTATTAT [[ID=1​​​​​​​​​​​​​​​​​​​​​GTGTTTTTATTTATTATGAAATTATTACAAATAATCTAGTAAAGTTAAATTATAA

[0080] TTCACCAAAAGTTTGCGGTTGCCAAATATTCTTCATTATTATTATTTTTCATA

[0081] TCTCAAACAAAGTAGTTATGCAACCTTGCTTAAAATATATGATCCCCATAAAT

[0082] TTTCAAAACCGGTTTTATATGCAAAGTACATGTCAAAACGTTATCTTATTTA

[0083] CATCCATTTACTAATCAAATATCTATTTTACCACAAAAGGTGTGTTTTATACC

[0084] CTATCATGTTTTACTCTTGAGATATGTTCTTTGACACCACCCACAAAATATCT

[0085] CTACGAAATACGAAGCCACTATATGTCTCTTTAATTTCACTTTTTATAGTTTT

[0086] TTTCACCGCTAAAATTGTTGACTAAAAAAATATATTGCATAAAAATAATTGAT

[0087] AATATATTTATAAGAAAAACTATGATAAGAAAAAATATAATCGGTTGAATG

[0088] AGTCATGACTAACAATAATTAAAGGTTAGGAAATTAAAGAAAATAAATTC

[0089] TGAATAACTAAAAAAAAAAAAATAGAAGATGATGATATATGGATCCCTAT

[0090] ACTAATATTTTGGAAGTACATTGAAAAACTAACTTTCAAAGACCCAATTAAT

[0091] TAAGCTCATTAAGGATAAACATGTTAAATACTAACTTATGGACATTAATTAA

[0092] ATTAAAATTAAAACGAAAATAAATTGATGACAAAAAAAAACTATGAA

[0093] TTTTCTTATTGATTTGTAATCTACTTTCTTGAAAAAATTTGAAGTTTACTG

[0094] ACGGCTTCCTTTTTTTGGAAACTCCAAAATAACAAAACATATGAAGAAGTT

[0095] TTGGAATAGCCTTTGACTCAACAACTTTAACAATAGAAAGAAAACATGTTT

[0096] AATTAATGTTCATGCACTTCGTCTCGGCTGCCCCTTGTGGCCTGTGGGGTT

[0097] GCCGGGTTGGCTTAGCTTGAGACGACCCAATACTGAATTTGTTAGCTCGGT

[0098] CAGTGAAGTCTACATGCATGATACAAAAAGATTGACTAGTGTTTAAGTTTT

[0099] TTTTTTTTTTTTTTTTTTTTCATAAATGGTAGTGTCTCATATTGAAAATGGT

[0100] AGTTTGAAAAGTATTCAGTTTGTTTGTTCACTTTGGATTATTTGATTTTGGTT

[0101] TTGTTAATTGAATCAGTTTTGTTTGAAAGTATTCACTTTTGAAAGTGTTC

[0102] ACTTTAAAAAGTTTTGTTTGAAAGTGTTCACTTTGAAAAGTATTCTTTGA

[0103] GAAGTGCTCAGTTTTGGTTTGTCCACTTACGATTATTTCACAAGCTACAG

[0104] CGGATAGTAGTGACTAGTGACTTATGACTTTGAATAAAGAATTTCCCTCTAA

[0105] AGGAATGAATACATTATAAATAGATTATTAACCTAAGCTTGATAGAGAAGAC

[0106] AGAACAAAAAAAAACACAAGAACAGGGCAAGGAAA (SEQ ID NO: 3).

[0107] Using the cDNA of wild-type Col-0 Arabidopsis thaliana treated with Pseudomonas syringae as a template, the DMP3 gene fragment was amplified by PCR. A clone was constructed using the bidirectional enzyme-dot primer introduction method. A SmaI restriction site was added to the 5' end of the target sequence, an XhoI restriction site was added to the 3' end of the target sequence, and an HA tag sequence and a stop codon sequence were added before the XhoI restriction site. The primer sequences for amplifying the DMP3 gene are as follows:

[0108] DMP3-121-F:TCCCcccgggATGTCTTCACCATCTTCCCTAAC

[0109] DMP3-121-R:

[0110] CCGctcgagTTAAGCGTAGTCTGGAACGTCGTATGGGTAACGACGACCCC CGTCTCCGG

[0111] The sequence of DMP3 is shown in SEQ ID NO: 1.

[0112] The amplified PAD3 promoter fragment was inserted into the resistance-modified pBI121 (hyg) vector through the Hind III and Xba I restriction sites. The amplified DMP3-HA-Stop fragment was inserted into the above promoter fragment through the Sma I and Xho I restriction sites to construct the pBI121-PAD3pro:DMP3-HA-Stop plant expression vector. After Agrobacterium-mediated transformation of wild-type Col-0 Arabidopsis thaliana, transformed T0 generation seeds were obtained for subsequent screening.

[0113] After the collected Agrobacterium-transformed T0 seeds are fully dried, sterilized and purified, and evenly coated on a 1 / 2MS solid culture medium plate containing the corresponding antibiotics. They are placed in a plant light incubator and cultured for about 10 days. The seedlings with obvious resistance, normal growth and true leaves are transplanted into the soil to be positive transgenic plants. After growing in the soil for about 2 weeks, use a hole puncher to punch holes on the leaves and take samples. The final concentration is OD 600= 0.02 suspension of Pseudomonas syringae, soaked for 24 hours, then removed and dried, total protein was extracted, and Western Blot was used to detect whether the target gene was expressed in the positive transgenic plants. Plants that were identified to express the target gene were cultured until maturity and seeds were collected (T1 generation).

[0114] Take an appropriate amount of fully dried T1 generation seeds, sterilize and purify them, evenly spread them on a 1 / 2MS solid culture medium plate containing the corresponding antibiotics, and place them in a plant light incubator for about 10 days. Statistical analysis is performed on the ratio of resistant seedlings to non-resistant seedlings, and strains with a resistant seedling: non-resistant seedling ratio of 3:1 are selected, that is, single-copy transgenic strains, and transplanted into the soil for continued cultivation. After maturity, seeds are collected from individual plants (T2 generation).

[0115] Take an appropriate amount of fully dried T2 generation seeds, sterilize and purify them, evenly spread them on a 1 / 2MS solid culture medium plate containing the corresponding antibiotics, and culture them in a plant light incubator for about 10 days. Statistic the ratio of resistant seedlings to non-resistant seedlings, select transgenic lines with all resistant seedlings, pick 3 seedlings, and use a final concentration of OD 600 = 0.02 Pseudomonas syringae suspension, soaked for 24 hours, then removed and dried, total protein extracted, and Western Blot analysis was used to confirm whether the target gene was stably expressed. The lines that were all resistant seedlings and had stable expression of the target gene, i.e., homozygous and stably expressed transgenic plants, were transplanted into soil for further cultivation. After maturity, seeds (T3 generation) were collected for subsequent experiments.

[0116] (2) Pseudomonas syringae resistance test

[0117] The stable transgenic Arabidopsis thaliana strain expressing PAD3pro:DMP3-HA was sown in soil and grown in a culture room at 21°C with 14h light / 10h dark for 3-4 weeks until the seedling stage. The final concentration was OD 600 = 0.0005 of the Pseudomonas syringae suspension was injected into leaves with good growth conditions. After 3 days, samples were taken using a punch and the number of Pseudomonas syringae colonies was statistically analyzed.

[0118] The results are as follows Figure 5 As shown, compared with the wild-type Col-0 Arabidopsis thaliana, the number of Pseudomonas syringae per unit area in the transgenic plants was significantly reduced, and the leaves were green, the growth condition was good, and there was no obvious abnormality in appearance, indicating that the PAD3 promoter regulating the expression of the DMP3 gene can significantly improve the disease resistance of Arabidopsis thaliana to Pseudomonas syringae without affecting its growth.

[0119] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Application of the DMP3 gene and its encoded protein in improving plant disease resistance. The nucleotide sequence of the DMP3 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The plant disease resistance is the plant's disease resistance to Pseudomonas syringae.

3. The use according to claim 1 or 2, characterized in that The plants include crops, selected from one or more of Arabidopsis, rapeseed, rice, tomato, potato, peanut, soybean, cotton, tobacco, cucumber and watermelon.

4. A PAD3 gene promoter induced by pathogens, the sequence of which is shown in SEQ ID NO:

3.

5. A plant expression vector, characterized in that It comprises the PAD3 gene promoter induced by pathogens as claimed in claim 4 and the DMP3 gene with a nucleotide sequence as shown in SEQ ID NO:

1.

6. The use according to claim 5, characterized in that The pathogen is Pseudomonas syringae.

7. An engineered Agrobacterium, characterized in that: Contains the plant expression vector according to claim 5 or 6.

8. Use of the engineered Agrobacterium according to claim 7 in improving plant disease resistance.

9. The use according to claim 8, characterized in that The engineered Agrobacterium infects the plant, which includes crops selected from one or more of Arabidopsis thaliana, rapeseed, rice, tomato, potato, peanut, soybean, cotton, tobacco, cucumber and watermelon.

10. The use according to claim 8, characterized in that The plant disease resistance is the plant's disease resistance to Pseudomonas syringae.

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