Application of NLP7 protein and coding gene thereof in improving botrytis cinerea resistance of plants

Abstract

The invention provides an application of NLP7 protein and a coding gene thereof in improving botrytis cinerea resistance of plants. Research finds that arabidopsis thaliana NLP7 up-regulates the JAZ1 gene expression level by targeting a promoter region of a jasmonic element inhibition factor JAZ1, further inhibits accumulation of jasmonic elements and expression of downstream resistance genes, and leads to enhancement of sensitivity of plants to botrytis cinerea. After the expression of the NLP7 in the plant is silenced or inhibited, the botrytis cinerea resistance of the plant can be effectively improved under a high-concentration nitrate culture condition, and the scab area is remarkably reduced. The invention provides a new target and method for improving the botrytis cinerea resistance of plants, and has important significance for preventing and treating botrytis cinerea infection of plants.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to the application of NLP7 protein and its encoding gene in improving plant resistance to gray mold. Background Art

[0002] Botrytis cinerea is the second most widely distributed fungal pathogen globally, infecting over 200 plant species, including fruits (such as grapes and strawberries), vegetables (such as tomatoes, cucumbers, and lettuce), flowers (such as roses and tulips), and field crops. A saprophytic fungus, Botrytis cinerea produces cell wall-degrading enzymes and many other toxic secondary metabolites. During hyphal invasion, it kills cells and obtains nutrients from the dead plant cells, allowing it to further grow and invade, causing devastating damage to plants. Botrytis cinerea damage occurs throughout the entire crop growth cycle, causing seedling damping-off, flower and fruit rot, and leaf drop in the field. The spores of Botrytis cinerea are extremely resilient, remaining dormant before harvest and causing disease when conditions are suitable after harvest, leading to fruit rot and significant losses. Given the severe damage Botrytis cinerea causes to plant growth, identifying effective targets and methods to enhance plant resistance to Botrytis cinerea is crucial for precise control of plant disease resistance and reducing the harm caused by Botrytis cinerea. Summary of the Invention

[0003] Based on this, the purpose of this invention is to provide the application of NLP7 protein and its encoding gene in improving plant resistance to gray mold.

[0004] To achieve the above objectives, the present invention adopts the following technical solution.

[0005] The first aspect of the present invention provides a gene encoding the Arabidopsis NLP7 protein or the application of the Arabidopsis NLP7 protein in improving plant resistance to Botrytis cinerea, wherein the nucleotide sequence of the gene encoding the Arabidopsis NLP7 protein is shown in SEQ ID NO:2; or is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:1;

[0006] The amino acid sequence of the Arabidopsis NLP7 protein is shown in SEQ ID NO:1.

[0007] The second aspect of the present invention provides a gene encoding the Arabidopsis NLP7 protein or the application of the Arabidopsis NLP7 protein in the breeding of plant varieties resistant to gray mold, wherein the nucleotide sequence of the gene encoding the Arabidopsis NLP7 protein is shown in SEQ ID NO:2; or is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:1;

[0008] A third aspect of the present invention provides a recombinant vector that can silence or inhibit the expression of a gene encoding the Arabidopsis NLP7 protein in plants; the nucleotide sequence of the gene encoding the Arabidopsis NLP7 protein is shown in SEQ ID NO:2; or is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:1.

[0009] In some embodiments, the recombinant vector silences or inhibits the expression of the gene encoding the Arabidopsis NLP7 protein in plants by gene knockout or T-DNA insertion.

[0010] In some embodiments, the recombinant vector is pROK2.

[0011] A fourth aspect of the present invention provides a host cell containing the recombinant vector as described above.

[0012] The fifth aspect of the invention provides any one of the following applications of the recombinant vector or the host cell as described above:

[0013] (1) In improving plant resistance to gray mold;

[0014] (2) Cultivate plant varieties resistant to gray mold.

[0015] In some embodiments, the plant is Arabidopsis thaliana.

[0016] A sixth aspect of the present invention provides a method for improving plant resistance to gray mold, comprising the steps of: silencing or inhibiting the expression of a gene encoding the Arabidopsis NLP7 protein in the plant, and culturing the plant in a high concentration of nitrate supplementation;

[0017] The nucleotide sequence of the gene encoding the Arabidopsis NLP7 protein is shown in SEQ ID NO:2; or the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO:1.

[0018] In some embodiments, the method of silencing or inhibiting the expression of the gene encoding the Arabidopsis NLP7 protein in the plant includes at least one of gene knockout, mutagenesis, and T-DNA insertion.

[0019] Compared with the prior art, the present invention has the following beneficial effects.

[0020] This invention reveals that Arabidopsis NLP7 targets the promoter region of the jasmonic acid repressor JAZ1, upregulating JAZ1 gene expression and thereby inhibiting jasmonic acid accumulation and the expression of downstream resistance genes, leading to increased plant susceptibility to Botrytis cinerea. Silencing or inhibiting NLP7 expression in plants, under high-nitrate culture conditions, can effectively increase jasmonic acid content and promote the expression of resistance genes, thus effectively improving Botrytis cinerea resistance and significantly reducing lesion area.

[0021] This invention provides new targets and methods for improving plant resistance to gray mold, which is of great significance for the prevention and control of gray mold infection in plants. Attached Figure Description

[0022] Figure 1 The results of yeast monohybridization analysis for NLP7 and JAZ1 promoter binding are shown.

[0023] Figure 2 The results of ChIP-PCR analysis of NLP7 binding to the JAZ1 promoter are shown.

[0024] Figure 3 The results of dual-luciferase reporter gene detection analysis for NLP7-regulated JAZ1 expression.

[0025] Figure 4 Analysis of the identification results of NLP7 knockdown plants.

[0026] Figure 5 Analysis of the identification results of NLP7 overexpressing plants.

[0027] Figure 6 This is a real-time quantitative PCR analysis of JAZ1 expression levels in Col-0 (wild-type), NLP7 knockdown plants (nlp7-1), and NLP7 overexpression plants under high-nitrate culture conditions.

[0028] Figure 7 The results of jasmonic acid content analysis in Col-0 (wild type), NLP7 knockdown plants nlp7-1 and NLP7 overgrowth plants under high nitrate culture conditions.

[0029] Figure 8 The results show the expression of resistance genes in Col-0 (wild type), NLP7 knockdown plants (nlp7-1), and NLP7 overexpression plants under high nitrate culture conditions.

[0030] Figure 9 The results of botrytis cinerea resistance analysis in Col-0 (wild type), NLP7 knockdown plants (nlp7-1), and NLP7 overgrowth plants under high nitrate culture conditions. Detailed Implementation

[0031] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0032] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0033] In this invention, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent three cases: A existing alone, A and B existing simultaneously, and B existing alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0034] Nitrogen (N) is an essential nutrient element for plant growth and development. Nitrates (NO3) - Nitrate, as the main nitrogen source in soil, is not only a nutrient for plants but also a signaling molecule that regulates gene expression and metabolic processes. NLP7 (NIN-LIKEPROTEIN 7) is a key transcriptional regulator in the nitrate signaling pathway, participating in the regulation of primary nitrate response (PNR) and root-shoot growth and development.

[0035] The amino acid sequence of the NLP7 protein described in this invention is shown in SEQ ID NO:1.

[0036] SEQ ID NO:1:MCEPDDNSARNGVTTQPSRSRELLMDVDDLDLDGSWPLDQIPYLSSS 。

[0037] The nucleotide sequence of the gene encoding the NLP7 protein described in this invention is shown in SEQ ID NO:2.

[0038]

[0039] The nucleotide sequence of the promoter sequence 1164 bp upstream of the ATG of the JAZ1 gene described in this invention is shown in SEQ ID NO:3.

[0040]

[0041] The nucleotide sequence of the 2Kb upstream promoter sequence of JAZ1 described in this invention is shown in SEQ ID NO:4.

[0042]

[0043] The following description is based on specific implementation methods.

[0044] Reagent preparation

[0045] MS medium: 6.2mg / L Boirc Acid, 332.2mg / L Calcium Chloride, 0.025mg / LCobalt Chloride·6H2O, 0.025mg / L Cupric Sulfate·5H2O, 37.26mg / L Na2EDTA·2H2O, 27.8mg / L Ferrous Sulfate·7H2O, 180.7mg / L Magnesium Sulfate,16.9mg / L ManganeseSulfate·H2O,0.25mg / L Molybdic Acid·2H2O,0.83mg / L Potassium Iodide,1900mg / L Potassium Nitrate,170mg / L Potassium Phosphate,8.6mg / L Zinc Sulfate·7H2O,2mg / L Glycine,100mg / L myo-Inositol,0.5mg / L Nicotinic Acid,0.5mg / L Pyridoxine·HCI,0.1mg / L Thiamine·HCI,1650mg / L Ammonium Nitrate.

[0046] Nitrogen-free liquid culture medium: 332.2 mg / L CaCl2, 180.7 mg / L MgSO4, 170 mg / L KH2PO4, 6.2 mg / L H3BO3, 0.025 mg / L CoCl2·6H2O, 0.025 mg / L CuSO4·5H2O, 27.8 mg / L FeSO4·7H2O, 37.3 mg / L Na2EDTA·2H2O, 16.9 mg / L MnSO4·H2O, 0.25 mg / L Na2MoO4·2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 5 mM KCl.

[0047] High-concentration nitrate liquid culture medium: 332.2 mg / L CaCl2, 180.7 mg / L MgSO4, 170 mg / L KH2PO4, 6.2 mg / L H3BO3, 0.025 mg / L CoCl2·6H2O, 0.025 mg / L CuSO4·5H2O, 27.8 mg / L FeSO4·7H2O, 37.3 mg / L Na2EDTA·2H2O, 16.9 mg / L MnSO4·H2O, 0.25 mg / L Na2MoO4·2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 5 mM KNO3.

[0048] Example 1: Yeast monohybridization analysis of NLP7 and JAZ1 promoter binding

[0049] The coding sequence of NLP7 (CDS, as shown in SEQ ID NO:2) was cloned into the pGADT7 vector, and the promoter sequence 1164 bp upstream of the ATG of the JAZ1 gene (as shown in SEQ ID NO:3) was inserted into the pLacZi2μ vector. Both vectors were co-transformed into yeast strain HA105 and cultured for 72 hours on SD / -Ura / -Leu medium (without uracil (Ura) and leucine (Leu)). Subsequently, 1 mL of yeast suspension (one clone from each sample resuspended in 1 mL of sterile water) was incubated at 30°C for 12 hours, diluted with sterile water at 1:10, 1:100, and 1:1000, and then inoculated onto SD / -Ura / -Leu medium containing BU salt and X-Gal and cultured for 12 hours.

[0050] The results are as follows Figure 1 As shown, in the yeast single-hybrid experiment, the experimental group (AD-NLP7+pJAZ1-LacZ) grew well on SD / -Ura / -Leu+BU salts medium, and X-gal staining was blue; the negative controls (AD+pJAZ1-LacZ, AD-NLP7+LacZ, and AD+LacZ) could grow on SD / -Ura / -Leu+BU salts medium, but no staining was observed. In conclusion, NLP7 can bind to the JAZ1 promoter.

[0051] Example 2: ChIP-PCR analysis of NLP7 binding to JAZ1 promoter

[0052] Two grams of 14-day-old 35S::NLP7-myc transgenic overexpression seedlings carrying the myc tag were cross-linked in 1% formaldehyde solution for 15 minutes under vacuum on ice. The cross-linking reaction was terminated by adding 2M glycine solution. Cell nuclei were collected using M1, M2, and M3 buffers, and then chromatin fragmented into DNA fragments with an average length of 250-500 bp by sonication on ice (15 cycles, 30 s on / 30 s off). Immunoprecipitation was performed using anti-myc antibody and Protein G magnetic beads. ChIP samples were digested with proteinase K overnight at 65°C. The immunoprecipitated DNA was purified by phenol extraction and then detected by qPCR. The reaction system included the purified product, specific primers, and ChamQ SYBR Color qPCR premix. Specific primers included: upstream primer: TAAATTAGGCTCGTGAAGGAGGG (SEQ ID NO:5); downstream primer: ATCGGGAATGTGTCTCGGTT (SEQ ID NO:6). Reaction procedure: 95 degrees for 2 minutes; 95 degrees for 15 seconds; 60 degrees for 30 seconds; 95 degrees for 15 seconds; 60 degrees for 1 minute; 95 degrees for 15 seconds. 40 cycles.

[0053] The results are as follows Figure 2 As shown, in the ChIP-PCR experiment, the experimental group (35S::NLP7-myc) showed a significantly higher enrichment fold in the JAZ1 promoter region after immunoprecipitation with myc antibody compared to the IgG control group, while the negative control (wild-type plant Col-0) showed no significant enrichment. In conclusion, NLP7 is significantly enriched in the JAZ1 promoter region, indicating an interaction between NLP7 and the JAZ1 promoter.

[0054] Example 3: Detection and analysis of dual-luciferase reporter genes regulating JAZ1 expression by NLP7

[0055] The coding sequence of NLP7 (as shown in SEQ ID NO:2) was cloned into the PGREENII62-SK expression vector. The 2Kb upstream promoter sequence of JAZ1 (as shown in SEQ ID NO:4) was cloned between the XmaI and HindIII restriction sites of the pGreenII 0800-LUC vector. The recombinant plasmids were co-transformed into Arabidopsis mesophyll protoplasts. After transfected cells were cultured for at least 15 hours, the relative luciferase (LUC) activity was analyzed using a dual-luciferase reporter gene assay system (Promega).

[0056] The results are as follows Figure 3As shown, the luciferase ratio of the experimental group (NLP7+JAZ1) was significantly higher than that of the control group (empty vector+JAZ1), indicating that NLP7 can significantly upregulate (upregulate by about 4.6 times, P value <0.0001) LUC expression driven by the JAZ1 promoter.

[0057] Example 4: Preparation and Identification of NLP7 Knockdown Plants

[0058] T-DNA insertion mutation is one of the most classic, widely used, and authoritative methods for studying gene function in Arabidopsis thaliana. It utilizes the natural gene transfer mechanism of Agrobacterium tumefaciens to randomly insert a specific DNA fragment (T-DNA) into the Arabidopsis genome, thereby disrupting or altering the function of the target gene and inhibiting its expression.

[0059] In this embodiment, the NLP7 knockdown plant (nlp7-1) was obtained by infiltration transformation of Columbia ecotype (Columbia, Col) plants using Agrobacterium tumefaciens carrying the pROK2 vector. Kanamycin was used to screen for plants carrying T-DNA; each T3 generation plant was a seedling grown from seeds of the same origin. Each line contained one or more T-DNA insertion elements. The T-DNA insertion mutant primers LP: GTTTTCTTTAGACCGCCACC (SEQ ID NO:7) were used.

[0060] RP: AAGAATCAACCGAACAACACG (SEQ ID NO:8); primer T-DNA-LBb1.3: ATTTTGCCGATTTCGGAAC (SEQ ID NO:9) was used to identify the knockdown lines. The PCR amplification program was: 94°C for 5 seconds; 55°C for 15 seconds; 72°C for 20 seconds; 36 cycles.

[0061] The results are as follows Figure 4 a and Figure 4 As shown in b, primer LP+RP amplified bands in both wild-type Col-0 and nlp7-1 plants; while primer T-DND-LBb1.3+RP amplified bands only in nlp7-1 plants, indicating that the T-DNA sequence in nlp7-1 plants is inserted into the NLP7 gene sequence.

[0062] NLP7 expression was detected by real-time quantitative PCR using SYBR Select premixed buffer on an ABi7500 real-time PCR system (Life Technologies). ACTIN8 was used as an internal reference gene, and normalization was performed using the ΔΔCt method. Primers for NLP7 were: upstream primer: GGCGTCACTACTCAACCTTC (SEQ ID NO:10); downstream primer: GCCCATTACCATTAGGATTA (SEQ ID NO:11). Primers for ACTIN8 were: upstream primer: TCAGCACTTTCCAGCAGATG (SEQ ID NO:12); downstream primer: CGTGGACAATGCCTGGAC (SEQ ID NO:13). The reaction program was: 95°C for 2 min; 95°C for 15 s; 60°C for 30 s; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s. 40 cycles were performed.

[0063] The results are as follows Figure 4 As shown in c, the expression level of the NLP7 gene in wild-type Col-0 plants was significantly higher than that in nlp7-1 plants, indicating that the insertion of T-DNA significantly inhibited the transcription of the NLP7 gene.

[0064] Example 5: Preparation and Identification of NLP7 Overexpressing Plants

[0065] NLP7 overexpressing plants were obtained by transforming Columbia (Col) ecotype plants with Agrobacterium tumefaciens carrying the pJG186-myc vector using a vacuum infiltration method. NLP7 expression was detected by real-time quantitative PCR using SYBR Select premixed buffer on an ABi7500 real-time PCR system (Life Technologies). ACTIN8 was used as an internal reference gene, and normalization was performed using the ΔΔCt method. Primers for NLP7 were: upstream primer: GGCGTCACTACTCAACCTTC (SEQ ID NO:10); downstream primer: GCCCATTACCATTAGGATTA (SEQ ID NO:11). Primers for ACTIN8 were: upstream primer: TCAGCACTTTCCAGCAGATG (SEQ ID NO:12); downstream primer: CGTGGACAATGCCTGGAC (SEQ ID NO:13). Reaction procedure: 95 degrees for 2 minutes; 95 degrees for 15 seconds; 60 degrees for 30 seconds; 95 degrees for 15 seconds; 60 degrees for 1 minute; 95 degrees for 15 seconds. 40 cycles.

[0066] The results are as follows Figure 5As shown, the NLP7 gene expression level in the NLP7 overexpressing plant (35S::NLP7-myc) was significantly higher than that in the Col-0 (wild type) plant.

[0067] Example 6: Real-time quantitative PCR analysis of JAZ1 gene expression level

[0068] Col-0 (wild-type), NLP7 knockdown plants (nlp7-1), and NLP7 overexpression plants (35S::NLP7-myc) were cultured on MS medium for 11 days, then transferred to nitrogen-free liquid medium and cultured with gentle shaking for 2 days. Subsequently, they were treated with high-nitrate liquid medium and nitrogen-free liquid medium for 30 minutes each. Total RNA was extracted from seedlings using the TRIzol method. Using 2 μg of total RNA as a template, first-strand cDNA was synthesized using a reverse transcription system (TransScript one-step genomic DNA removal and cDNA synthesis super-mix). JAZ1 expression was detected by real-time quantitative PCR using ChamQ SYBR Color qPCR premix (low ROX) on an ABi7500 real-time PCR system (Life Technologies) with SYBR Select premix. ACTIN8 was used as an internal control gene and normalized using the ΔΔCt method. The primers for JAZ1 are: upstream primer: TCAGCGACAACAACCATGA (SEQ ID NO:14); downstream primer: GCTATTAAGCGGTGCCTTTGC (SEQ ID NO:15). The primers for ACTIN8 are: upstream primer: TCAGCACTTTCCAGCAGATG (SEQ ID NO:12); downstream primer: CGTGGACAATGCCTGGAC (SEQ ID NO:13). Reaction program: 95°C for 2 min; 95°C for 15 s; 60°C for 30 s; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s. 40 cycles.

[0069] The results are as follows Figure 6 As shown, compared with wild-type Col-0, the expression level of JAZ1 in NLP7-1 was significantly reduced, while the expression level of JAZ1 in NLP7 overexpressing plants was significantly increased. These results indicate that under high nitrate conditions, NLP7 positively regulates the expression of the JAZ1 gene.

[0070] Example 7: Analysis of Jasmonic Acid and JA-Ile Content

[0071] Col-0 (wild-type), NLP7 knockdown plant nlp7-1, and NLP7 overgrowth plant (35S::NLP7-myc) were cultured on MS medium for 11 days, then transferred to nitrogen-free liquid medium and cultured with gentle shaking for 2 days. Subsequently, they were treated with high-nitrate liquid medium and nitrogen-free liquid medium for 30 minutes each. The stored samples were ground into powder in liquid nitrogen, and extraction buffer (isopropanol:formic acid = 99.5:0.5) was added. The samples were then resuspended in 1 mL of methanol (85:15). Using a Waters Sep-Pak C18 solid-phase extraction column (SPE), 2 mL of eluent was collected from each sample. The eluent was concentrated under vacuum using a Labconco CentriVap vacuum centrifuge and then redissolved in 100 μL of methanol (60:40) for subsequent liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. LC-MS / MS analysis was performed using a Waters-equipped... UPLC TM AB of C18 column (50×2.1mm, 1.7μm) 4500QTRAP mass spectrometer The mobile phase solvents were 0.1% formic acid acetonitrile solution (A) and 0.1% formic acid acetonitrile solution (B), with gradient elution conditions of 0–10 min and a linear gradient of 50%–100%. Quantification was performed using multiple reaction monitoring (MRM) mode, with the MRM ion pair for jasmonic acid being 209.1180 > 59.0134 and the MRM ion pair for JA-Ile being 322.2020 > 130.0087. Mass spectrometry data were acquired and processed using AB SCIEX Analyst 1.6.3 software (Applied Biosystems). Quantification of JA and JA-Ile levels was performed using the internal standard method and compared with standards.

[0072] The results are as follows Figure 7 As shown, compared with wild-type Col-0, the contents of jasmonic acid and jasmonic acid-isoleucine (JA-Ile) in NLP7-1 were significantly increased, while the contents of jasmonic acid and JA-Ile in NLP7 overgrowth plants were significantly decreased. These results indicate that under high nitrate conditions, NLP7 negatively regulates the jasmonic acid content in plants.

[0073] Example 8: Real-time quantitative PCR analysis of resistance gene expression levels

[0074] Col-0 (wild-type), NLP7 knockdown plants (nlp7-1), and NLP7 overexpression plants (35S::NLP7-myc) were cultured on MS medium for 11 days, then transferred to nitrogen-free liquid medium and cultured with gentle shaking for 2 days. Subsequently, they were treated with high-nitrate liquid medium and nitrogen-free liquid medium for 30 minutes each. Total RNA was extracted from seedlings using the TRIzol method. Using 2 μg of total RNA as a template, first-strand cDNA was synthesized using a reverse transcription system (TransScript one-step genomic DNA removal and cDNA synthesis super-mix). Real-time quantitative PCR experiments were performed on an ABi7500 real-time PCR system (Life Technologies) using ChamQ SYBR Color qPCR premix (low ROX) with SYBR Select premix to detect the expression of JAZ1, PDF1.2, and VSP2. ACTIN8 was used as an internal reference gene and normalized using the ΔΔCt method. The primers for PDF1.2 are: upstream primer: GTTTGCGAGAAGCCAAGTG (SEQ ID NO:16); downstream primer: GCATGATCCATGTTTGGCTCC (SEQ ID NO:17). The primers for VSP2 are: upstream primer: TCAGTGACCGTTGGAAGTTGTG (SEQ ID NO:18); downstream primer: GTTCGAACCATTAGGCTTCAATATG (SEQ ID NO:19). The primers for ACTIN8 are: upstream primer: TCAGCACTTTCCAGCAGATG (SEQ ID NO:12); downstream primer: CGTGGACAATGCCTGGAC (SEQ ID NO:13).

[0075] The results are as follows Figure 8 As shown, compared with wild-type Col-0, the expression levels of PDF1.2 and VSP2 were significantly increased in NLP7-1, while the expression levels of PDF1.2 and VSP2 were significantly decreased in NLP7 overexpressing plants. These results indicate that under high nitrate concentrations, NLP7 negatively regulates the expression levels of resistance genes.

[0076] Example 9: Analysis of Botrytis cinerea resistance

[0077] Leaves of Col-0 (wild type), nlp7-1 (knockdown plants), and NLP7 overgrowth plants (35S::NLP7-myc) were infected using a drip inoculation method. Each leaf was inoculated with 3 μL of gray mold conidial suspension (1×10⁻⁶). 6 (1 conidia / mL). Leaves were photographed after drip inoculation and kept under high humidity for 48 hours. Lesion area was assessed using ImageJ software.

[0078] The results are as follows Figure 9 As shown, compared with the wild-type Col-0, the lesion area of ​​nlp7-1 was significantly reduced (P < 0.05), while the lesion area of ​​NLP7 over-exposed plants was significantly increased (P < 0.05); the lesion areas of Col-0, nlp7-1, and NLP7 over-exposed plants were 11.2 mm. 2 7.1mm 2 and 15.2mm 2 The results indicate that under high nitrate conditions, NLP7 negatively regulates plant resistance to Botrytis cinerea.

[0079] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0080] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. The gene encoding the Arabidopsis NLP7 protein or the application of the Arabidopsis NLP7 protein in improving plant resistance to Botrytis cinerea, characterized in that, The nucleotide sequence of the gene encoding the Arabidopsis NLP7 protein is shown in SEQ ID NO:2; or the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO:

1. The amino acid sequence of the Arabidopsis NLP7 protein is shown in SEQ ID NO:

1.

2. The application of the Arabidopsis NLP7 protein encoding gene or the Arabidopsis NLP7 protein in the breeding of gray mold-resistant plant varieties, characterized in that, The nucleotide sequence of the gene encoding the Arabidopsis NLP7 protein is shown in SEQ ID NO:2; or the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO:

1. The amino acid sequence of the Arabidopsis NLP7 protein is shown in SEQ ID NO:

1.

3. A recombinant vector, characterized in that, The recombinant vector can silence or inhibit the expression of the gene encoding the Arabidopsis NLP7 protein in plants; the nucleotide sequence of the gene encoding the Arabidopsis NLP7 protein is shown in SEQ ID NO:2; or the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO:

1.

4. The recombinant vector as described in claim 3, characterized in that, The recombinant vector silences or inhibits the expression of the gene encoding the Arabidopsis NLP7 protein in plants by gene knockout or T-DNA insertion.

5. The recombinant vector as described in claim 4, characterized in that, The recombinant vector is pROK2.

6. A host cell, characterized in that, The host cell contains the recombinant vector as described in any one of claims 3 to 5.

7. Any of the following applications of the recombinant vector as described in any one of claims 3 to 5 or the host cell as described in claim 6: (1) In improving plant resistance to gray mold; (2) Cultivate plant varieties resistant to gray mold.

8. The application as described in claims 1 to 2 or 7, characterized in that, The plant in question is Arabidopsis thaliana.

9. A method for improving plant resistance to gray mold, characterized in that, Includes the following steps: Silencing or inhibiting the expression of the gene encoding the Arabidopsis NLP7 protein in plants and culturing the plants in a high concentration of nitrate; The nucleotide sequence of the gene encoding the Arabidopsis NLP7 protein is shown in SEQ ID NO:2; or the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO:

1.

10. The method as described in claim 9, characterized in that, The method for silencing or inhibiting the expression of the gene encoding the Arabidopsis NLP7 protein in plants includes at least one of gene knockout, mutagenesis, and T-DNA insertion.

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