Synthetic disease-resistant gene nlp27 and its application

Abstract

This invention discloses the artificial synthesis of the disease-resistant gene NLP27 and its application, belonging to the field of plant genetic engineering technology. This invention selects a conserved 27-amino acid fragment from Verticillium dahliae NLP1, modifies its nucleotide sequence, and synthesizes the NLP27 gene, which is then overexpressed in potato plants through genetic transformation. Inoculation analysis shows that transgenic plants with the NLP27 gene exhibit high resistance to potato late blight and scab. Quantitative PCR analysis shows that excessively high NLP27 transcript levels significantly inhibit potato plant growth. After screening for expression levels and disease resistance, three transgenic lines with moderate NLP27 expression levels (such as PT1, PT6, and PT7) were obtained. These lines significantly enhanced resistance to potato late blight and scab without sacrificing yield.

Description

Technical Field

[0001] This invention relates to the artificial synthesis of the disease resistance gene NLP27 and its application, belonging to the field of plant genetic engineering technology. Background Technology

[0002] Necrosis and ethylene-inducible peptide-1 (NLP)-like proteins are one of the largest families of microbial effectors. These effectors have been found in bacteria, fungi, and oomycetes, and can be present in both pathogenic and non-pathogenic microorganisms. NLPs contain a typical NPP1 domain, which is involved in various physiological processes in microorganisms, such as pathogenicity, sporulation, attachment, and tolerance to abiotic stress. NLPs can induce leaf necrosis and ethylene production in plants, hence their name. NLPs are generally divided into two categories: those that are cytotoxic to the host plant and those that are non-cytotoxic. For example, cytotoxic NLPs can help pathogens infect plants, including potatoes, soybeans, cotton, tomatoes, tobacco, and grapes, causing diseases that can result in significant economic losses. Simultaneously, both cytotoxic and non-cytotoxic NLPs can stimulate plant immune responses, such as the expression of immune response genes, activation of the MAPK signaling cascade, ROS bursting, SA induction, and necrosis and ethylene induction, thereby enhancing plant immunity to pathogens.

[0003] NLP has been reported as a microbial-associated molecular pattern, similar to chitin, flagellin, and elongation factor Tu, which can be sensed by plant pattern recognition receptors to trigger immune responses, thereby inducing defense gene expression, generating reactive oxygen species, and other protective mechanisms. Typically, MAMPs are reported to contain a short motif that can be recognized by PRRs; for example, in Arabidopsis, FLS2 and EFR recognize flg22 and elf18, respectively. Small peptide fragments of 20 / 24 amino acids present in NLP are sufficient to trigger innate immune responses in plants. This conserved motif, when synthesized into a small peptide, can stimulate plant immunity but appears to inhibit plant growth. Oome et al. identified growth-inhibiting regions by overexpressing different truncated mutants of Arabidopsis thaliana NLP, regions that coincide with the conserved NLP24-triggered immune response region. However, the correlation between the expression levels of these different mutants and plant resistance and growth inhibition requires further investigation.

[0004] Potato is an annual herbaceous tuber crop belonging to the Solanaceae family. It is the world's fourth largest food crop after rice, wheat, and corn, with diverse uses as a food, vegetable, and feed crop. Currently, diseases such as potato late blight, black embryo disease, and common scab are increasing year by year, causing huge losses to potato yields. Therefore, identifying new disease-resistant genes and technologies, and breeding new disease-resistant varieties are important measures for current potato disease control. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides the artificially synthesized disease-resistant gene NLP27 and its applications, aiming to solve the technical problem of how to prevent and control diseases such as potato late blight, black embryo disease, and common scab by cultivating new disease-resistant varieties.

[0006] The first technical solution provided by the present invention is an NLP27 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] SEQ ID NO. 1: MGIMYAWYWPKDQPADGNLASGHRHDWE.

[0008] The second technical solution provided by the present invention is a gene encoding the NLP27 protein described in the first technical solution.

[0009] In some embodiments, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.

[0011] In some embodiments, the recombinant plasmid is expressed using plasmid pBI121 as an expression vector.

[0012] The fourth technology provided by this invention is to express the NLP27 protein described in the first technical solution, contain the gene described in the second technical solution, or transform recombinant cells with the recombinant plasmid described in the third technical solution.

[0013] In some embodiments, the recombinant cells are plant cells or microbial cells.

[0014] The fifth technical solution provided by the present invention is a method for enhancing the disease resistance of potatoes. The method involves linking the gene containing the gene described in the second technical solution to a vector or the recombinant plasmid described in the third technical solution, and transforming it into potatoes through Agrobacterium-mediated infection; or transfecting potatoes with the recombinant plasmid described in the fourth technical solution.

[0015] In some embodiments, the disease resistance includes resistance to late blight, scab, Alternaria alternata, Verticillium dahliae, and / or Fusarium oxysporum.

[0016] In some implementations, the copy number of the gene is 1.

[0017] The sixth technical solution provided by this invention is the application of the NLP27 protein described in the first technical solution, the gene described in the second technical solution, the recombinant plasmid described in the third technical solution, or the recombinant cell described in the fourth technical solution in improving the disease resistance of potatoes.

[0018] The seventh technical solution provided by the present invention is a method for cultivating disease-resistant potatoes. The method involves linking the gene contained in the second technical solution to a vector or the recombinant plasmid contained in the third technical solution, and transforming it into potatoes through Agrobacterium-mediated infection; or transfecting potatoes with the recombinant plasmid contained in the fourth technical solution to obtain disease-resistant potato lines.

[0019] In some embodiments, the resistance includes resistance to late blight, scab, Alternaria solanacea, Verticillium dahliae, and / or Fusarium oxysporum.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention selects a conserved 27-amino acid fragment from Verticillium dahliae NLP1, modifies its nucleotide sequence, and synthesizes the NLP27 gene, which is then overexpressed in potato plants through genetic transformation. Inoculation analysis showed that the NLP27 transgenic plants exhibited high resistance to potato late blight and scab. Quantitative PCR analysis showed that excessively high NLP27 transcript levels significantly inhibited potato plant growth. After screening for expression levels and disease resistance, three transgenic lines with moderate NLP27 expression levels (such as PT1, PT6, and PT7) were obtained. These lines significantly enhanced resistance to potato late blight and scab without sacrificing yield. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the pBI121-NLP27 vector.

[0023] Figure 2 To estimate the developmental process of transgenic NLP27 potato plants: (A) Induction of sterile potato tubers, 'DXR' potato plantlets were cultured in sterile potato tuber induction medium for about 60 days; (B) Inoculated potato tuber sections grown in co-medium; (C) Kanamycin-resistant callus induced on treated tuber sections in selection medium; (D) Shoot differentiation from callus in selection medium.

[0024] Figure 3 For the detection of transgenic NLP27 plants; (A) PCR amplification detection of transgenic NLP27 plants; (B) Detection of NLP27 gene transcriptional expression by RT-PCR; (C) Relative expression level of NLP27 gene analyzed by qRT-PCR; aj, different letters indicate statistically significant differences; M, DL2000 DNA molecular marker; PT1-PT12, twelve positive transgenic potato plants; CK, transgenic potato plants transformed with empty vector; P, PBI121-NLP27 plasmid.

[0025] Figure 4 To analyze the resistance of transgenic NLP27 plants to Phytophthora blight infection; (A) Ex vivo leaves inoculated with Phytophthora zoospores show the development of late blight symptoms; PT1, PT6 and PT7 are three transgenic plants with moderate NLP27 expression levels; CK, transgenic potato plants transformed with empty vector, scale bar: 1 cm; (B) The size of necrotic spots on treated leaves was quantified using ImageJ software, ac represents statistically significant differences; (C) The relative biomass of Phytophthora blight was analyzed by qPCR.

[0026] Figure 5 To analyze the resistance of transgenic NLP27 plants to *Streptomyces scabica* infection; (A) Slices of small potato tubers inoculated with *Streptomyces scabica* showed scab symptoms over time. PT1, PT6, and PT7 represent different transgenic lines. CK and control transgenic lines (including empty vector) are shown. Scale bar: 2 cm (left); 3 mm (right); (B) Relative biomass of *Streptomyces scabica* pathogen analyzed by qPCR. ac represents statistically significant differences; (C) Whole potato tubers inoculated with *Streptomyces scabica* showed scab symptoms on day 5. The area within the blue circle represents scab symptoms. Scale bar: 5 mm; (D) The area of ​​necrotic spots on treated tubers was quantified using ImageJ software.

[0027] Figure 6 The analysis included the expression levels of defense-related genes in transgenic plants; (A) the relative expression levels of defense-related genes PR1, PR5, LOX1, PAL1, WRKY8, and NAC2 in transgenic plants infected with Phytophthora infestans and CK plants, where PT1, PT6, PT7, and CK represent three different transgenic lines and control lines, respectively, and af represents the statistically significant differences between treatment samples in each gene expression group; (B) the relative expression levels of defense-related genes in plants infected with Streptomyces scabica; and (C) the relative expression levels of defense marker genes exogenously sprayed on wild-type plants.

[0028] Figure 7 Analysis of reactive oxygen species accumulation and callose deposition; (A) Nitroblue tetrazolium staining analysis of detached leaves of transgenic and CK plants; (B) Determination of superoxide anion content in leaves of transgenic and CK plants; (C) 3,3'-diaminobenzidine staining analysis of detached leaves of transgenic and CK plants; (D) Determination of H2O2 content in leaves of transgenic plants; (E) Callose deposition analysis of detached leaves of transgenic and CK plants; (F) Quantification of the number of callose deposition points per square millimeter in detached leaves of transgenic and CK plants using ImageJ, scale bar: 50 micrometers.

[0029] Figure 8 (A) Expression levels of ROS-related genes and callosine synthesis-related genes in transgenic lines; (B) Expression levels of four ROS scavenging genes (StSOD1, StCAT1, StAPX3, and StAOX1) and one ROS-producing gene (StRboh-B) in transgenic lines; ad: different letters indicate statistically significant differences; ag: different letters indicate statistically significant differences.

[0030] Figure 9 The correlation between transformant growth phenotype and NLP27 gene expression level is shown in the figure above; the numbering order of the transformants in the figure above corresponds to the growth phenotype of the transformants shown in the figure below. The blue triangles represent changes in NLP27 expression level.

[0031] Figure 10 The phenotype of transgenic NLP27 plants grown in net bags.

[0032] Figure 11 Phenotypic responses of transgenic potatoes to different pathogens; (A) Ex vivo leaves inoculated with Alternaria alternata showed early blight symptoms, PT1, PT6, and PT7 were three transgenic plants with moderate NLP27 expression levels, CK was a transgenic potato plant transformed with an empty vector, scale bar: 1 cm; (B) Transgenic plants infected with Verticillium dahliae showed Verticillium wilt symptoms; (C) Transgenic plants infected with Fusarium oxysporum showed Fusarium wilt symptoms; (D) Relative biomass of Alternaria alternata in transgenic plants analyzed by qPCR; (E) Relative biomass of Verticillium dahliae in transgenic plants analyzed by qPCR; (F) Relative biomass of Fusarium oxysporum in transgenic plants analyzed by qPCR; ad: different letters indicate statistically significant differences. Detailed Implementation

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0034] Test method:

[0035] 1. DNA extraction and PCR reaction

[0036] To analyze transgenic plants, genomic DNA was extracted from potato leaves using the CTAB method. PCR was performed using NLPF and NLPR primers to detect the components of the transgenic plant genomic DNA. The NLPF primers were designed to target the CaMV35S promoter, and the NLPR primers were located within the NLP27 gene. Relevant primers are listed in Supplementary Table 1. The PCR reaction program was: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 30 seconds; 58°C annealing / extension for 30 seconds, for a total of 35 cycles.

[0037] 2. RNA extraction, reverse transcription, and real-time quantitative PCR

[0038] Total RNA was extracted from potato leaf tissue using a plant total RNA extraction kit. RNA quality was assessed by agarose gel electrophoresis and spectrophotometry. Two μg of total RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit. The diluted cDNA template was used for quantitative real-time PCR to detect resistance-related marker genes, ROS-related genes, and callosine synthesis-related genes. RT-PCR was performed using Easy Taq polymerase to detect NLP27 gene expression. The corresponding DNA from transgenic plants transformed with the empty vector was used as a negative control. Stactin was used as an internal control gene to normalize gene expression. All primers are listed in Table 1.

[0039] 3. Analyze the copy number of transgenic plants using qPCR.

[0040] The copy number of T-DNA in transgenic NLP27 potatoes was analyzed using qPCR. The potato Patatin gene was used as an internal control, a plasmid containing the NLP27 gene served as a positive control, and non-transgenic potatoes served as a negative control. The CT values ​​of the Patatin and NLP27 genes in each sample were obtained by qRT-PCR, and the copy number of NLP27 transgenic potatoes was calculated using the Pfaffl method.

[0041] 4. Analysis of reactive oxygen species accumulation

[0042] To visualize the accumulation of reactive oxygen species (ROS) in transgenic potato plants, 3,3'-diaminobenzidine staining and nitroblue tetrazolium staining were performed. DAB staining was used to analyze H2O2 levels, and NBT staining was used to analyze O2 levels. - Horizontal incubation. Immerse fresh plant leaves in NBT or DAB staining solution and incubate at room temperature in the dark until blue or dark brown substances appear on the leaves. Then, decolorize the leaves in 95% ethanol to remove the leaf background. Determine H2O2 and O2 according to the described method. - content.

[0043] 5. Callose determination

[0044] Mature leaves from 10-week-old plants were collected for callose deposition analysis. The leaves were completely immersed in a 3:1 ethanol:lactophenol solution, the clarified lactophenol solution being a mixture of equal volumes of phenol, glycerol, lactic acid, and water. The mixture was then vacuum filtered for 15 minutes and incubated at 65°C for 30 minutes to ensure complete chlorophyll release. The bleached leaves were washed with 50% ethanol and stained with aniline blue solution. The leaves were then mounted in 70% glycerol and observed using a Zeiss inverted fluorescence microscope.

[0045] Materials used in the examples:

[0046] 1. Selective culture medium formulation: 1 x MS + 30 g sucrose + 1 mg / L auxin + 0.2 mg / L gibberellin + 0.5 mg / L 6-benzylaminopurine + 2 mg / L zeatin + 50 mg / L kanamycin + 300 mg / L cefotaxime, pH 5.8.

[0047] 2. Rooting medium formula: 1 / 2 x MS + 30 g sucrose + 300 mg / L cefotaxime, pH 5.8.

[0048] 3. YDA medium formulation: yeast extract (1%), peptone (2%), glucose (2%) and adenine (0.004%).

[0049] 4. Pathogenic fungus 88069 Reference: "An RLP23–SOBIR1–BAK1 complex mediates NLP-triggered immunity."

[0050] 5. Streptomyces scabica CGMCC 4.1765 strain was purchased from the China General Microbiological Culture Collection Center.

[0051] 6. Current progress on pathogenicity-related genes in Fusarium oxysporum f. sp. tropical cubense race 4.》(Chen, D., Ju,M., Xie, J. et al. Current progress on pathogenicity-related genes inFusarium oxysporum f. sp. cubense tropical race 4. Phytopathol Res 6, 53(2024). https: / / doi.org / 10.1186 / s42483-024-00274-5).

[0052] 7. Introduction to JY Enzyme Vayg1 is required for microsclerotium formation and melanin production in Verticillium dahliae. Fungal Genet Biol.》(Fan R, Klosterman SJ, Wang C, Subbarao KV, Xu X, Shang W, Hu X. Vayg1 is required for microsclerotium formation and melanin production in Verticilliumdahliae. Fungal Genet Biol. 2017 Jan;98:1-11. doi: 10.1016 / j.fgb.2016.11.003.Epub 2016 Nov 17. PMID: 27866941.).

[0053] 8. Alternaria solani HWC-168 Reference "Transcriptome sequencing leads to an improved understanding of the infection mechanism of Alternaria solani in potato. BMC Plant Biol." (Jiang J, Guo X, Tan H, Ding M, Liu F, Yang Z, Zhu J. Transcriptome sequencing leads to an improved understanding of the infection mechanism of Alternaria solani in potato. BMC Plant Biol. 2023 Mar 1;23(1):120. doi: 10.1186 / s12870-023-04103-3. PMID: 36859112; PMCID: PMC9976505.).

[0054] The primers used in the following examples are shown in Table 1.

[0055] Table 1 Primer Information

[0056] Example 1: Construction of NLP27 short peptide gene expression vector

[0057] The coding sequence of the NLP27 gene is derived from the *Verticillium dahliae* NLP1 gene (nucleotide sequence shown in SEQ ID NO.3, amino acid sequence of the encoded protein shown in SEQ ID NO.4). A conserved 27-amino acid fragment was selected, and its coding sequence was optimized to suit the codon usage preferences of dicotyledonous plants. An ATG start codon was added to the 5' end of this fragment, and a TGA stop codon was added to the 3' end. This fragment was artificially synthesized by Beijing Qingke Biotechnology Co., Ltd. (nucleotide sequence shown in SEQ ID NO.2). Subsequently, this synthesized fragment was cloned into the pBI121 vector to obtain a recombinant plasmid named pBI121-NLP27 (e.g., pBI121-NLP27). Figure 1 (As shown). The upstream of the NLP27 fragment is the cauliflower mosaic virus 35S promoter, and the downstream is the carmine synthase terminator. All primers are listed in Table 1.

[0058] The recombinant plasmid pBI121-NLP27 was transformed into Agrobacterium tumefaciens GV3101 competent cells by heat shock transformation to obtain Agrobacterium tumefaciens GV3101 strain carrying the pBI121-NLP27 plasmid.

[0059] Example 2: Agrobacterium tumefaciens-mediated genetic transformation of potato

[0060] Potato transformation was performed using *Agrobacterium tumefaciens* strain GV3101 carrying the pBI121-NLP27 plasmid, as described in Example 1. First, 'DXR' potato plantlets were cultured in a small tuber induction medium. Small tuber sections were used as explants and co-cultured with *Agrobacterium tumefaciens* carrying the pBI121-NLP27 vector for 48 hours. After infection, the treated tuber sections were cultured in a kanamycin-containing selective medium until callus differentiation and shoot emergence. When the regenerated shoots reached 1-2 cm in length, they were transferred to a rooting medium for plant regeneration, yielding healthy plants. Healthy regenerated plants were transplanted into pots for growth until the transgenic potato tubers were harvested, completing one life cycle of the transgenic NLP27 potato (e.g., ...). Figure 2 (As shown). A total of 45 independent putative transgenic regenerated plants were obtained.

[0061] Total DNA was extracted from leaves of 45 presumed transgenic plants as templates for PCR detection. Due to the short NLP27 gene fragment, the forward primer for PCR was designed on the CaMV35S promoter, and the reverse primer was located on the NLP27 gene (as shown in Table 1). The results showed that 43 out of the 45 independently transgenic plants contained the NLP27 gene, while the plants transformed with the empty vector (control, CK) did not. Figure 3 A) indicates that the transformation efficiency of the NLP27 gene in potatoes is very high.

[0062] To detect NLP27 gene expression in these transgenic potatoes, total RNA was extracted from leaves of PCR-positive plants for RT-PCR and qRT-PCR analysis. RT-PCR results showed that 35 out of 43 transgenic plants exhibited NLP27 mRNA bands, while the control group did not. Figure 3 B). Based on qRT-PCR analysis, the expression levels of different transformed plants differed significantly ( Figure 3 (C) indicates that different insertion sites of the NLP27 gene may affect its expression level.

[0063] Compared to the control, transgenic plants with high NLP27 expression exhibited stunted growth. Three transgenic plants with moderate NLP27 expression levels, PT1, PT6, and PT7, showed similar growth to the control and were selected for gene insertion copy number analysis via qPCR. As shown in Table 2, compared to the potato patatin gene, an internal reference gene in previously reported potato transgenic gene copy number studies, the relative copy number of NLP27 in the three samples (PT1, PT6, and PT7) was approximately "1", indicating that they were single-copy transgenic plants.

[0064] Table 2. Real-time quantitative PCR estimation of copy number in transgenic NLP27 plants.

[0065] Example 3: Overexpression of NLP27 enhanced plant resistance to late blight.

[0066] To clarify whether NLP27 is involved in plant disease resistance responses, three transgenic plants (PT1, PT6, and PT7) with moderate NLP27 expression levels were selected for late blight resistance analysis. Extruded leaves from transgenic plants and controls were inoculated with *Phytophthora 88069*. Specifically, *Phytophthora 88069* mycelium was collected and suspended in pre-cooled sterile water, and zoospore release was induced at 4°C for approximately 1 hour. Several transgenic potato leaves (PT1, PT6, and PT7) were placed face down in petri dishes, and 10 µL of zoospore suspension was added, diluting the spore concentration to 10,000–20,000 spores / mL. The dishes were then incubated at 18°C ​​for 2 days. The resistance of the transgenic plants to *Phytophthora 88069*, which causes late blight, was then observed and photographed. Genomic DNA was extracted from infected leaves using the CTAB method. Following Albert's report, the DNA biomass of *Phytophthora 88069* in infected leaves was quantified by qPCR using the PiO8 gene. Statin was used as an internal control gene. Primers are listed in Supplementary Table 1. The experiment was performed in three biological replicates.

[0067] The results showed that two days after inoculation, the wild-type showed large areas of necrosis around the inoculation site, while PT1, PT6, and PT7 only showed minor necrotic spots. Figure 4 A). The numerical values ​​for the size of necrotic spots showed that the PT1 plant had the lowest damage diameter of 2.4±0.6 mm, and the area of ​​necrotic spots on the leaves of the three transgenic plants was significantly smaller than that of the control (14.2±2.1 mm). Figure 4 A and B). DNA analysis using qPCR and counting the relative biomass of *Phytophthora infestans* in the treated leaves revealed that the relative biomass of *Phytophthora infestans* in transgenic plants PT1, PT6, and PT7 compared to the control group were 0.18±0.05, 0.38±0.08, and 0.16±0.04, respectively. Figure 4C). These results indicate that NLP27 can stimulate an immune response and inhibit the pathogenicity of Phytophthora infestans.

[0068] Example 4: Overexpression of NLP27 enhanced plant resistance to scab.

[0069] To further elucidate the function of NLP27 in plant disease resistance, the response of transgenic plants to *Streptomyces scabica* infection was measured. Potato tubers were sliced ​​and inoculated with *Streptomyces scabica* CGMCC 4.1765, as follows: potato tubers were surface-sterilized with 75% ethanol solution for 1 minute and rinsed with sterile water. Micro-wounds were then created on the tuber surface and placed in petri dishes containing 25 μL of *Streptomyces scabica* suspension. The slices were incubated for 5 days at 28°C and above 85% relative humidity using moistened sterile filter paper. A similar method was used to assess disease resistance by inoculating potato tuber slices with *Streptomyces scabica*. Potato tuber slices, 1.3 cm in diameter and 4 mm thick, prepared using a sterile punch, were placed in petri dishes containing 25 μL of *Streptomyces scabica* suspension and incubated at 28°C for 48 hours. Transgenic potatoes transformed with an empty vector were used as a control; according to Wong's report, DNA biomass was measured by qPCR using the COX1 gene of *Streptomyces scabica*, with the Stactin gene used as an internal control. Each treatment was repeated three times.

[0070] As the inoculation time increased, the tuber slices of the control group gradually showed infection symptoms, with the tubers turning black and necrosis. However, the tubers of the three transgenic potatoes, PT1, PT6, and PT7, only showed mild disease symptoms, with the tuber slices becoming slightly darker in color. Figure 5 A). Pathogen counting was performed on inoculated tuber slices by qPCR analysis 48 hours after treatment. The results showed that the relative pathogen content in the three potato slices was significantly lower than the control, at 0.19±0.04, 0.22±0.05, and 0.17±0.03, respectively. Figure 5 B). As a parallel experiment, whole tubers were inoculated with *Streptomyces scabica* to assess the function of NLP27 in plant resistance. On day 5, the control showed obvious pitting and browning symptoms at the inoculation site, while the three NLP27-overexpressing potato tubers showed only mild pitting and browning symptoms. Figure 5 C). Lesion diameter measurements showed that the lesion diameters of the three transgenic plants were 4.1±1.6 mm, 4.9±0.8 mm, and 4.0±0.9 mm, respectively. Compared with the control (14.1±1.8 mm), the three transgenic plants showed significantly higher resistance to scab. Figure 5 D). These results indicate that NLP27 transgenic potatoes exhibit high resistance to scab disease.

[0071] Example 5: Overexpression of NLP27 in transgenic plants upregulated the expression of resistance-related marker genes.

[0072] To elucidate new insights into the types of defense mechanisms, the transcription levels of resistance-related marker genes (including PR1, PR5, LOX, PAL, WRKY8, and NAC2) were monitored in transgenic NLP27 potato plants and controls inoculated with Phytophthora or Streptomyces scabica. In the case of Phytophthora inoculation, the expression levels of six defense-related genes in the infected tissues of transgenic plants were significantly higher than those in the control. Figure 6 A). Similar results were obtained in these transgenic plants infected with *Streptomyces scabica* as with those treated with *Phytophthora*. Figure 6 B). Therefore, overexpression of NLP27 enhances plant resistance to two pathogens, potentially involving the pathogenesis-related protein (PR1 and PR5), salicylic acid (SA; PR1, PR5, and PAL1) and jasmonic acid (JA; PR4, PDF1.2, and LOX1) signaling pathways, as well as basal defense responses (WRKY8 and NAC2). Furthermore, exogenous spraying of NLP27 peptide, synthesized by Tsingke (Beijing, China), onto wild-type plants was conducted to verify its function in plant immune responses. Results showed that compared to the control without NLP27 peptide spraying, the expression of six resistance-related marker genes—PR1, PR5, LOX1, PAL1, WRKY8, and NAC2—was significantly upregulated, comparable to the effect of NLP27 overexpression in transgenic plants. Figure 6 C).

[0073] Example 6: Overexpression of NLP27 increased the accumulation of reactive oxygen species and callose in leaves.

[0074] To further explore the disease resistance mechanism of transgenic NLP27 plants, the accumulation of reactive oxygen species was analyzed. Based on nitroblue tetrazolium staining analysis, large blue areas appeared on the leaves of the three transgenic plants, while the control only showed a few blue spots. Figure 7 A). Quantitative analysis also showed that superoxide anions (O2) were present in transgenic NLP27 plants. - The accumulation levels of these substances were 10.3±0.8 μM / g FW, 8.6±0.7 μM / g FW, and 7.8±0.6 μM / g FW, respectively, significantly higher than the control (2.2±0.4 μM / g FW). Figure 7 B). DAB staining analysis showed that the leaf staining area and color depth of the three transgenic plants were significantly higher than those of the control (B). Figure 7 C). Quantitative analysis showed that the H2O2 content in the leaves of transgenic plants was significantly higher than that in the control ( Figure 7D). To investigate the expression levels of ROS-related genes in these transgenic lines, four ROS scavenging genes (StSOD1, StCAT1, StAPX1, and StAOX1) and one ROS-producing gene (StRboh-B) were selected to detect their expression levels. The results showed that the expression levels of the four ROS scavenging genes were similar to the control, while the expression level of StRboh-B was significantly higher. Figure 8 A).

[0075] To further investigate whether NLP27 induces callose deposition to enhance plant defense against pathogens, callose staining analysis was performed. The results showed that callose deposition in the leaves of the three transgenic plants was significantly higher than that in the control. Figure 7 E). Quantitative analysis showed that the density of callose spots accumulated in the leaves of transgenic plants was 51±4 / mm. 2 72±8 pieces / mm 2 49±5 pieces / mm 2 The number of samples was significantly higher than that of the control (12±5 samples / mm). 2 () Figure 7 F). Associated with callose accumulation in transgenic plants, the expression of four callose synthesis-related genes, StGSL1, StUDP1, StPLDα, and StMYB103, was significantly upregulated compared to the control. Figure 8 B).

[0076] High expression levels of NLP27 in the transformant of Example 7 reduced plant growth and tuber yield.

[0077] Existing technology reports indicate that the NLP24 peptide can trigger plant immunity and appears to inhibit plant growth (Oome et al., 2014; Seidl et al., 2019). For example... Figure 9 As shown, there were significant differences in the growth phenotypes of various T1 generation potato transformants. Therefore, 23 transformants with relative NLP27 transcription levels ranging from 0.1 to 32.5 (Table 3) and controls were cultured in pots to assess the correlation between NLP27 expression levels and growth. Figure 9 and Figure 10 As shown, the growth of the transformants gradually increased with decreasing NLP27 transcription levels. Furthermore, the potato tuber yield per pot gradually increased with decreasing NLP27 expression levels (Table 3). Correlation analysis showed a negative correlation between NLP27 transcription levels and tuber yield (correlation coefficient R0). 2= -0.8781). However, transformants with relative expression levels below 8.1, including PT1, PT6, and PT7, exhibited similar tuber yields to the control. Therefore, these three transformants with moderate expression levels, PT1, PT6, and PT7, are ideal transgenic NLP27 lines for potato disease resistance breeding.

[0078] Table 3. Relative expression levels of NLP27 and potato tuber yield in different T1 generation transformants.

[0079] Example 8: NLP27 enhances broad-spectrum plant resistance

[0080] Given that the function of NLP27 overexpression in potato resistance to late blight and scab involves the accumulation of ROS and callosity, the study further evaluated whether NLP27 overexpression conferred broad-spectrum resistance to other pathogens. The NLP27 resistance function was also investigated for three other major potato diseases—early blight, Verticillium wilt, and Fusarium wilt. Furthermore, the resistance of transgenic plants to *Alternaria alternata*, *Verticillium dahliae*, and *Fusarium oxysporum*, the pathogens corresponding to these three diseases, was tested, specifically as follows: *Fusarium oxysporum* TR4 strain was cultured and homogenized on PDA solid medium plates, then suspended in sterile water, and the spore density was adjusted to 1.5 × 10⁻⁶. 7 mL -1 Three-week-old potato seedlings were treated with root dipping, immersing them in spore solution for 30 minutes, and then cultured in sterile sand moistened with sterile Hoogland's solution. The biomass of *Fusarium oxysporum* in transgenic potatoes was analyzed by qPCR. StActin was used as an internal control gene. The elongation factor 1-α gene was used to quantify fungal colonization. *Verticillium dahliae* strain JY was homogenized in PDA liquid medium for 7 days. Conidia were then collected by suspending the medium in sterile water and adjusted to a concentration of 10⁻⁶. 7 Conidia / mL. Roots of 3-week-old transgenic potato seedlings were dipped for 30 minutes. The biomass of *Verticillium dahliae* in transgenic potatoes was analyzed according to previously described methods. *Verticillium dahliae* biomass was quantified using the VdEF-1α gene. Detached leaves of transgenic potatoes were infected with *Alternaria solanacea* strain HWC-168, suspended in sterile water, and spore density was adjusted to 10-1. 6 Spores / mL: An equal volume of spore suspension was evenly applied to detached transgenic leaves, kept moist, and disease progress was monitored under the same growth conditions. The AsCytb gene of *Alternaria solanacearum* was used as a reference, and DNA biomass was analyzed by qPCR. All primers used in the qPCR analysis are listed in Table 1.

[0081] Regarding plant resistance to early blight, two days after inoculation with Alternaria solanacea, large-scale necrosis occurred in the control detached leaves around the inoculation site, while only slight necrosis was observed in PT1, PT6, and PT7 transgenic plants. Figure 11 A). Inoculating transgenic plants with two fungi, *Verticillium dahliae* and *Fusarium oxysporum*, showed that the disease severity in the control group was significantly higher than that in the three transgenic plants (PT1, PT6, and PT7), with leaves turning yellow and even dying. Figure 11 B and C). The transgenic plants showed supported resistance to all three pathogens, and the pathogen biomass in the transgenic plants was significantly lower than that in the control (B and C). Figure 11 (D, E, and F). These results indicate that the NLP27 gene plays an important role in enhancing broad-spectrum plant resistance.

[0082] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An NLP27 protein, characterized in that, The amino acid sequence of the NLP27 protein is shown in SEQ ID NO.

1.

2. The gene encoding the NLP27 protein of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmid was expressed using plasmid pBI121 as the expression vector.

5. A recombinant cell expressing the NLP27 protein of claim 1, containing the gene of claim 2, or transformed with the recombinant plasmid of claim 3 or 4.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells are plant cells or microbial cells.

7. A method for enhancing the disease resistance of potatoes, characterized in that, The method involves linking the gene described in claim 2 to a vector or the recombinant plasmid described in claim 3 or 4, and then transforming it into potatoes via Agrobacterium-mediated infection. Alternatively, the recombinant plasmid described in claim 5 or 6 can be transfected into potatoes; The copy number of the gene described in claim 2 is 1.

8. The method according to claim 7, characterized in that, The disease resistance includes resistance to late blight, scab, Alternaria alternata, Verticillium dahliae, and / or Fusarium oxysporum.

9. The application of the NLP27 protein of claim 1, the gene of claim 2, the recombinant plasmid of any one of claims 3-4, or the recombinant cell of any one of claims 5-6 in improving the disease resistance of potatoes.

10. A method for cultivating disease-resistant potatoes, characterized in that, The method involves linking the gene described in claim 2 to a vector or the recombinant plasmid described in claim 3 or 4, and transforming it into potatoes through Agrobacterium-mediated infection to obtain disease-resistant potato lines. Alternatively, the recombinant plasmid described in claim 5 or 6 can be transfected into potatoes to obtain disease-resistant potato lines; The copy number of the gene described in claim 2 is 1.

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