Peanut disease-resistant gene achnhl24 and application thereof

Abstract

The application belongs to the technical field of molecular plant pathology, and particularly relates to a peanut disease-resistant gene AhNHL24 and application thereof. Through whole genome identification and bioinformatics analysis, 45 NHL family genes (AhNHL) are identified in peanuts, and gene expression analysis shows that most of the AhNHL genes can respond to the induction of biological stress and abiotic stress. Gene disease resistance identification finds that transient overexpression of AhNHL14, AhNHL24 and AhNHL31 can significantly improve the resistance of tobacco and peanuts to Pseudomonas solanacearum, and the disease resistance of AhNHL24 is the strongest. Through transgenic tobacco heterologous overexpression of AhNHL24 and inoculation experiments, it is shown that the peanut and tobacco plants overexpressing AhNHL24 not only have significantly enhanced resistance to Pseudomonas solanacearum, but also have significantly enhanced resistance to Sclerotium rofsii. The application provides an important basis for the cultivation of plant (peanut and tobacco, etc.) broad-spectrum disease-resistant varieties.

Description

Technical Field

[0001] This invention belongs to the field of molecular plant pathology technology, specifically relating to the peanut disease resistance gene AhNHL24 and its applications. Background Technology

[0002] Peanuts (Arachis hypogaea L.) are an important economic and oilseed crop worldwide, and a significant source of edible vegetable oil and protein, playing a crucial role in edible vegetable oil consumption and the snack food industry. However, peanut cultivation faces numerous challenges, including inefficient farming practices and insufficient variety. These factors collectively lead to the continuous outbreaks of diseases such as bacterial wilt and fungal white mold, with their severity increasing year by year, seriously affecting peanut quality and yield, and thus hindering the sustainable development of the peanut industry. Bacterial wilt and white mold are soil-borne diseases caused by Ralstonia solanacearum and Sclerotium rolfsii, respectively. Ralstonia solanacearum is a Gram-negative bacterium that can infect more than 400 plant species, including peanuts, soybeans, tomatoes, tobacco, potatoes, and peppers. *Sclerotium sclerotiorum*, belonging to the order Asporales, possesses both saprophytic and parasitic characteristics, capable of infecting over 200 plant species, including onions, peanuts, soybeans, alfalfa, clover, and peas. Peanut bacterial wilt and white mold occur in almost all peanut-producing areas, typically causing yield losses of 10-80%. Currently, disease resistance breeding is the most effective means of peanut disease control. Deeply exploring key functional genes for peanut disease resistance through molecular breeding technology will provide important germplasm resources for cultivating high-yielding, disease-resistant peanut varieties.

[0003] Studies have shown that the Nonrace-specific disease resistance gene 1 / Harpin-induced gene 1-like (NHL) family of genes are involved in pathogen-induced plant responses to biotic stress. These proteins are widely distributed in the plant kingdom and play a crucial role in plant resistance to biotic and abiotic stresses. NHL genes play an important role in plant-pathogen interactions and abiotic stress tolerance. Twenty-nine AtNHL genes have been identified in Arabidopsis thaliana, among which AtNHL1, AtNHL3, AtNHL5, AtNHL10, and AtNHL25 can mediate plant resistance to viral, bacterial, and fungal pathogens and play a key role in transducing pathogen infection signals in plant cells. The soybean GmNDR1 protein expressed on the plasma membrane can interact with pathogen effectors to transduce extracellular pathogen signals and regulate soybean's defense responses against *Phytophthora capsici* and *Pseudomonas syringae*. The soybean GmNHL1 and GmNHL8 genes can enhance Arabidopsis resistance to cyst nematodes by activating the jasmonic acid (JA) and ethylene (Eth) signaling pathways. Additionally, VvNHL1 mediates grape resistance to Botrytis cinerea by enhancing programmed cell death and systemic resistance. NbHIN1 confers tobacco resistance to Tobacco mosaic virus (TMV) by activating the JA signaling pathway; CaNHL4 enhances broad-spectrum resistance to TMV, Phytophthora capsici, and Pseudomonas aeruginosa by regulating reactive oxygen species production and the expression of genes related to the JA pathway. However, although NHL family genes have been found to participate in plant resistance to viral, bacterial, fungal, and nematode pathogens, peanut NHL family genes have not yet been cloned, and their role in peanut resistance to bacterial wilt and white mold remains unclear. Summary of the Invention

[0004] To overcome the shortcomings of the existing technology, this invention has found that plants overexpressing AhNHL24 not only show significantly enhanced resistance to bacterial wilt, but also significantly enhanced resistance to white mold. The results of this study are of great value for further research on the function of NHL family genes and for promoting the application of NHL genes in the breeding of broad-spectrum, highly resistant plants (especially tobacco and peanut).

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of this invention provides the application of the peanut disease resistance gene AhNHL24 in improving plant disease resistance and / or cultivating disease-resistant plant varieties, wherein the amino acid sequence encoded by the AhNHL24 gene is shown in SEQ ID NO: 5, and the disease resistance includes bacterial wilt and / or white mold.

[0007] Preferably, overexpression of the peanut disease resistance gene AhNHL24 in plants can improve the plant's resistance to bacterial wilt and white mold.

[0008] This invention identified 45 AhNHL genes in the peanut genome through whole-genome identification, mainly distributed at the ends of 15 chromosomes. Bioinformatics analysis showed that this family is relatively conserved during evolution, and its functions include regulating broad-spectrum resistance to various plant diseases such as viruses, bacteria, and fungi, as well as participating in plant stress responses to abiotic stresses such as drought, salinity, chilling injury, and heavy metal pollution. It also plays a role in plant growth and development processes such as seed germination, flowering, fruiting, and sugar translocation. Further promoter cis-regulatory element prediction and gene expression analysis showed that most AhNHL genes can respond to the induction of both biotic stress (R. solanacearum infection) and abiotic stress (cadmium stress). Based on the above information, four genes—AhNHL14, AhNHL24, AhNHL31, and AhNHL33—were selected for cloning, recombinant expression vector construction, subcellular localization analysis, transient overexpression in tobacco and peanut leaves, disease resistance identification, and trypan blue staining for quantitative cell necrosis assays. The results showed that transient overexpression of AhNHL14, AhNHL24, and AhNHL31 significantly improved the resistance of tobacco and peanut to *Ralstonia solanacearum*, with AhNHL24 exhibiting the strongest resistance. Heterologous overexpression of AhNHL24 in transgenic tobacco and inoculation experiments demonstrated that tobacco plants overexpressing AhNHL24 showed significantly enhanced resistance not only to *Ralstonia solanacearum* but also to *Sclerotium oxysporum*. These results not only deepen our understanding of the function of the plant NHL gene family but also provide important evidence for further exploring the disease resistance mechanisms of peanuts to *Ralstonia solanacearum* and *Sclerotium oxysporum*, as well as for breeding broad-spectrum disease-resistant varieties of plants (such as peanuts and tobacco).

[0009] Preferably, the nucleotide sequence of the AhNHL24 gene is shown in SEQ ID NO: 2.

[0010] Preferably, the pathogen of bacterial wilt is Ralstonia solanacearum (of the Solanaceae family), and the pathogen of white rot is Sclerotium rolfsii.

[0011] Preferably, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0012] More preferably, the plants include peanuts, soybeans, tomatoes, tobacco, cucumbers, potatoes, peppers, alfalfa, clover, peas, or Arabidopsis thaliana. Among them, peanuts and tobacco may also be different varieties of cultivated species.

[0013] Furthermore, the plants include peanuts and tobacco.

[0014] The second aspect of this invention provides a method for improving broad-spectrum disease resistance in plants, specifically by overexpressing the peanut disease resistance gene AhNHL24, thereby improving the plant's resistance to bacterial wilt and white mold; the amino acid sequence encoded by the AhNHL24 gene is shown in SEQ ID NO: 5.

[0015] Preferably, the above-mentioned method for improving broad-spectrum disease resistance in plants includes the following steps:

[0016] S1. The AhNHL24 gene was cloned into the pCambia1300-YFP plant expression vector, transformed into Escherichia coli competent cells, and after screening for positive clones, the recombinant plasmid pCambia1300-AhNHL24-YFP was extracted.

[0017] S2. The recombinant plasmid was transformed into Agrobacterium competent cells. After resistance selection, the recombinant bacteria were introduced into plants for overexpression, thus obtaining transgenic plants overexpressing AhNHL24.

[0018] More preferably, the primers used to amplify the AhNHL24 gene from peanut are shown in SEQ ID NO: 26 and SEQ ID NO: 27.

[0019] More preferably, recombinant bacteria are introduced into plants via leaf disc transformation.

[0020] More preferably, the competent Escherichia coli cells are DH5α competent Escherichia coli cells, and the competent Agrobacterium cells are Agrobacterium tumefaciens EHA105 competent cells.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention, through whole-genome identification and bioinformatics analysis, identified 45 NHL family genes (AhNHL) in peanut. Gene expression analysis showed that most AhNHL genes can respond to biotic and abiotic stress induction. Disease resistance identification revealed that transient overexpression of AhNHL14, AhNHL24, and AhNHL31 significantly improved the resistance of tobacco and peanut to *Ralstonia solanacearum*, with AhNHL24 exhibiting the strongest resistance. Heterologous overexpression of AhNHL24 in transgenic tobacco and inoculation experiments showed that peanut and tobacco plants overexpressing AhNHL24 not only showed significantly enhanced resistance to *Ralstonia solanacearum* but also significantly enhanced resistance to *Sclerotium affine*. This invention provides important evidence for the breeding of broad-spectrum disease-resistant varieties of plants (such as peanuts and tobacco).

[0023] Specifically, the present invention has the following advantages:

[0024] (1) Through whole-genome gene family identification, gene and encoded protein characteristic analysis, promoter cis-regulatory element analysis, gene expression analysis and functional verification, this invention found that the gene AhNHL24 has a significant effect on improving plant disease resistance.

[0025] (2) The gene AhNHL24 can not only improve the plant’s resistance to R. solanacearum, but also improve the plant’s broad-spectrum resistance to S. rolfsii. Based on the sequence conservation and phylogenetic analysis of the AhNHL gene, it can be inferred that AhNHL24 may also have certain resistance to other pathogens, and may even participate in regulating the plant’s response to abiotic stress, as well as the plant’s growth and development.

[0026] (3) The gene AhNHL24 can not only improve the resistance of peanuts to R. solanacearum, but also improve the resistance of tobacco to R. solanacearum and S. rolfsii.

[0027] In summary, this invention can provide valuable genetic resources for broad-spectrum disease resistance molecular breeding of plants such as peanuts, and therefore has important theoretical and practical significance. Attached Figure Description

[0028] Figure 1 Chromosomal distribution and collinearity analysis of AhNHL family genes;

[0029] Figure 2 Phylogenetic analysis of NHL family genes from different plants;

[0030] Figure 3Prediction and expression analysis of cis-regulatory elements for the AhNHL gene;

[0031] Figure 4 To analyze the expression of the AhNHL gene under Ralstonia solanacearum infection and plant hormone treatment using real-time quantitative fluorescence technology;

[0032] Figure 5 To analyze the subcellular localization of the AhNHL gene and validate its transient overexpression function;

[0033] Figure 6 The creation of transgenic tobacco with heterologous overexpression of AhNHL24 and the identification of its resistance to bacterial wilt and white mold. Detailed Implementation

[0034] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0036] To further investigate the function of NHL family genes and promote their application in breeding broad-spectrum, highly resistant plants (especially tobacco and peanut), this invention conducts whole-genome identification of the NHL family gene (AhNHL) in cultivated peanut (A. hypogaea), and performs bioinformatics analysis on its gene structure, physicochemical properties of encoded proteins, chromosome distribution, multi-species collinear evolutionary relationships, promoter cis-regulatory elements, and other characteristics. Using public databases and transcriptome gene expression data, combined with real-time quantitative expression analysis, tissue-specific and... The expression patterns induced by non-biotic stress and hormone responses were analyzed. Genes with significantly different expression levels were cloned and recombinant expression vectors were constructed. Transient overexpression and subcellular localization analysis were performed in tobacco leaves using the Agrobacterium tumefaciens infiltration method. After inoculating tobacco leaves transiently overexpressing AhNHL with Ralstonia solanacearum, the resistance of the AhNHL gene to Ralstonia solanacearum was assessed by observing leaf cell morphology and measuring necrotic cells using trypan blue staining. The same method was used to verify the resistance of AhNHL to Ralstonia solanacearum in peanut leaves. Finally, the AhNHL gene with the best resistance was selected to construct a homozygous transgenic tobacco family material with its heterologous overexpression. The resistance of the AhNHL gene to Ralstonia solanacearum and Sclerotium rolfsii was further evaluated by inoculation with Ralstonia solanacearum and Sclerotium rolfsii.

[0037] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Identification and phylogenetic analysis of the peanut AhNHL gene family

[0039] 1.1 Identification of the peanut AhNHL gene family

[0040] Peanut protein datasets (JBrowse2arahy.Tifrunner.gnm2.x.aradu.K30065.gnm1.DWM1.paf.gz) were obtained from the Peanutbase database (https: / / dev.peanutbase.org / genomics / #hypogaea), and hidden Markov model files (PF01436) related to conserved domains of peanut NHL proteins were obtained from the PFAM database. HMMER software was used to screen for peanut NHL candidate genes. Coding sequences (CDS) and predicted amino acid (AA) sequences of peanut NHL genes were retrieved from peanut databases. The chromosomal locations of peanut NHL family genes obtained from PeanutBase were mapped to the corresponding chromosomes using the MG2C software platform.

[0041] The results showed that 45 peanut NHL family genes (named AhNHL) were identified in the peanut genome, and they were unevenly distributed across the 15 chromosomes of peanut. Figure 1 A). Most AhNHL genes are located at the ends of chromosomes and are named AhNHL1-AhNHL45 according to their position on the chromosome. There are 6 AhNHL genes on chromosomes A02 (AhNHL3-8) and B02 (AhNHL26-31), respectively, while only 1 AhNHL gene is present on chromosomes A05 (AhNHL15) and A08 (AhNHL19). Five AhNHL genes (AhNHL32-36) were found on chromosome B03, followed by chromosomes A10 (AhNHL20-23), A03 (AhNHL9-11), A04 (AhNHL12-14), and B04 (AhNHL37-39), each containing 3-4 AhNHL genes. In addition, there are two AhNHL genes on chromosomes A01, A05, A07, B01, B05, B08, and B10 respectively.

[0042] 1.2 Physicochemical Properties Analysis of Proteins Encoded by Peanut AhNHL Family Genes

[0043] To confirm the members of the NHL gene family, conserved domains in each candidate amino acid sequence were identified using the SMART and CDD online software. The physicochemical properties of peanut NHL family genes, such as molecular weight, amino acid number, and isoelectric point, were predicted using the online software ProtParam. The results showed that the genomic sequence length of these AhNHL genes ranged from a maximum of 1679 base pairs (bp) (AhNHL20) to a minimum of 3430 bp (AhNHL35). Most AhNHL genes contained 2-3 exons, approximately 40% contained only 1 exon, and only AhNHL35 contained 5 exons.

[0044] The lengths of AhNHL-encoded proteins range from 96 amino acids (AhNHL39) to 344 amino acids (AhNHL18), with molecular weights ranging from 10385.17 to 38088.31 kilodaltons (kDa) and isoelectric points from 4.75 to 10.38. Conserved motif analysis revealed that AhNHL proteins possess three conserved domains: the late embryo abundant protein 2 (LEA_2) domain, a low-complexity domain, and a transmembrane region. These genes exhibit largely similar protein structural features. The LEA-2 domain is present in all these AhNHL proteins, while AhNHL12, AhNHL37, AhNHL11, and AhNHL33 contain two LEA_2 domains. The low-complexity region was found only in a subset of these genes. Apart from seven AhNHL proteins (AhNHL3, AhNHL26, AhNHL35, AhNHL12, AhNHL37, AhNHL11, and AhNHL33), the rest contain transmembrane regions, indicating that they primarily function as membrane proteins.

[0045] To verify this hypothesis, the subcellular localization and domain motifs of AhNHL proteins were predicted using WoLF POSRT and MEME, respectively (Table 1). The results showed that most AhNHL proteins were located in chloroplasts (44%) and cytoplasm (31%), with a few located in the plasma membrane (AhNHL3, AhNHL24), nucleus (AhNHL4, AhNHL6, and AhNHL20), extracellular space (AhNHL9, AhNHL13, AhNHL31, and AhNHL37), vacuole (AhNHL38), and cytoskeleton (AhNHL44). These results suggest that these peanut NHL genes may have different biological functions.

[0046] Table 1. Genetic information related to the AhNHL family in peanuts.

[0047]

[0048]

[0049]

[0050] 1.3 Collinearity and phylogenetic analysis of peanut AhNHL family genes

[0051] Collinearity analysis of NHL family genes in peanut and other plant species (such as rice, maize, Arabidopsis thaliana, and soybean) was performed using MCScanx software. The results showed that 40, 45, 11, and 21 genes with collinearity with AhNHLs were identified in Arabidopsis thaliana, soybean (Glycinemax), maize (Zea mays), and rice (Oryza sativa), respectively. Figure 1 (B) Although Arabidopsis and soybean have more collinear gene pairs with AhNHLs, significant collinearity was also found between maize, rice, and AhNHLs. This indicates that the NHL gene family has been conserved between dicotyledonous and monocotyledonous plants over a long evolutionary period.

[0052] Furthermore, a phylogenetic tree containing 45 AhNHL proteins and 34 reported NHL proteins from other plant species was constructed using MEGA 7.0 software (Table 2). Figure 2 These species include soybean, pepper (Capsicum annuum), rice, Arabidopsis thaliana, and grape (Vitis vinifera). Phylogenetic analysis of the amino acid sequences was performed using MEGA 5.10 software, and a phylogenetic tree was constructed using the neighbor-joining method with a bootstrap value of 1000, hiding values ​​with confidence levels less than 70%. The results showed that these 79 NHL proteins were divided into two groups (A and B). Group A consisted of 17 AhNHL proteins and 11 homologous NHLs. Among them, AhNHL12, AhNHL17, AhNHL33, AhNHL35, and AhNHL37 clustered closely with disease resistance genes including GmNDR1, AtNDR1, and CsNDR1. AhNHL33 was closely related to LjNHL13a, indicating that they have similar roles in nodule formation and nitrogen fixation. The remaining 28 AhNHL proteins and another 23 NHL proteins were assigned to Group B. AhNHL8 and AhNHL31 are closest to AtNHL3, AeNHL3, AtNHL10, and NbHIN1, conferring resistance to bacterial and fungal diseases in plants. AhNHL2 and AhNHL24 are closest to CaNHL4, which positively modulates plant defense responses to viral, fungal, and bacterial pathogens (such as TMV, P. capsici, and P. syringae).

[0053] Table 2. NHL gene information used for phylogenetic analysis

[0054]

[0055]

[0056]

[0057] Example 2: Cis-regulatory elements and expression analysis of peanut AhNHL family gene promoters

[0058] 2.1 Prediction of cis-regulatory elements in peanut AhNHL family gene promoters

[0059] The 2000bp upstream sequence of the start codon of 45 peanut NHL family genes shown in Table 1 was extracted, and the cis-regulatory elements were analyzed using the online PlantCARE software. Figure 3 A). The results showed that the various elements involved in plant responses to plant hormones and stress were not evenly distributed in the promoter, with the majority being abscisic acid (ABA) responsive elements. AhNHL16 and AhNHL24 had the most ABA and JA responsive elements, respectively. AhNHL45 had 9 elements, while AhNHL44 contained only 4 ABA responsive elements. Defense and stress response elements were present in the promoters of the AHNHL genes, including AhNHL24 and AhNHL45.

[0060] 2.2 Analysis of tissue-specific and stress-response expression of peanut AhNHL family genes

[0061] The tissue-specific expression pattern of the peanut AhNHL gene was studied using data from the Peanutbase database. Figure 3 B). The results showed that one-quarter of the AhNHL genes were highly expressed in various tissues, while one-third were lowly expressed. Specifically, AhNHL2, AhNHL19, AhNHL21, AhNHL24, and AhNHL31 were highly expressed in the roots. In particular, AhNHL10, AhNHL16, AhNHL34, and AhNHL41 were highly expressed in the shoot apex. Furthermore, using our published transcriptome data (see "Zhao, Kai, et al." Dynamic N...),... 6β-methyladenosine RNA modification regulates peanut resistance to bacterial wilting. (New Phytologist 242.1(2024):231-246). This study analyzed the expression patterns of peanut NHL genes under biotic stress (R. solanacearum infection) and abiotic stress (Cd stress). The results showed that AhNHL2, AhNHL9, AhNHL18, AhNHL21, AhNHL24, and AhNHL32 were significantly upregulated in H108 under R. solanacearum infection, while AhNHL3, AhNHL7, AhNHL10, AhNHL18, and AhNHL30 were upregulated under cadmium stress.

[0062] 2.3 Inoculation of peanut plants with Ralstonia solanacearum and treatment with exogenous hormones

[0063] Seeds of the highly resistant bacterial wilt peanut variety "NDH108" and the highly susceptible variety "NDH107," bred by the peanut breeding team of the College of Agriculture at Henan Agricultural University, were placed in petri dishes containing sterile water and germinated overnight at room temperature in the dark. Germinated peanut seeds were then transplanted into plastic pots (10×10×10cm, 4 seedlings per pot) and grown in a suitable greenhouse. Peanut seedlings were grown in the greenhouse in a culture medium containing vermiculite and Danish Pindl nutrient substrate (volume ratio 2:1); 16 hours of light at 28℃ and 8 hours of darkness at 26℃.

[0064] The *Ralstonia solanacearum* strain isolated and preserved in our laboratory (specific isolation method referenced in "Dai Xiaoqiu et al., 2022. Isolation and genetic diversity identification of the pathogen of peanut bacterial wilt in Guangdong Province, Journal of Zhongkai University of Agriculture and Engineering, 2022, 35(03): 26-33") was streaked on triphenyltetrazole chloride (TTC) agar containing 10.0 g / L peptone, 1 g / L casein hydrate, 5.0 g / L D-glucose and 15.0 g / L agar. Single colonies were picked with sterile toothpicks and inoculated into TTC liquid medium, and cultured for 2 days in a shaker at 28℃ and 200 rpm / min. The resuspended bacteria were then adjusted to OD using a Nanodrop 2000c spectrophotometer. 600 It is around 0.5, and its concentration is approximately 10. 8CFU / mL. Subsequently, peanut seedlings were inoculated with Ralstonia solanacearum using the root-damage method previously studied (Zhao K, Ren R, Ma X, et al. Genome-wide investigation of defensin genesin peanut (Arachis hypogaea L.) reveals AhDef2.2 conferring resistance to bacterial wilt[J]. The Crop Journal, 2022, 10(3):809-819.). When the peanuts had three true leaves, the tip of the main root was cut off, and a concentration of 10 CFU / mL was used. 8 CFU / mL Ralstonia solanacearum inoculum was used for inoculation, followed by placement in an incubator at 28°C, 60% humidity, and a 16-hour light / 8-hour light cycle. Inoculation was performed using the osmosis method. For each leaf, 100 μL of different bacterial concentrations (10⁻⁶ CFU / mL) was inoculated using a needleless syringe. 8 10 7 and 10 6 Inoculum of CFU / mL was used, with TTC liquid medium inoculation serving as a control. Roots of H108 and H107 lines were collected at 0, 0.5, 1.0, and 7.0 days post-inoculation (dpi). Seedlings of H107 and H108 lines were sprayed with 3 mmol / L salicylic acid (SA), 100 mmol / L methyl jasmonic acid (Me-JA), and 10 μmol / L abscisic acid (ABA), respectively. Root tissue was collected at 0, 0.5, 1.0, and 7.0 days post-treatment (dpt). Each sample was collected independently, with three biological replicates. Collected samples were cryopreserved in liquid nitrogen at -80°C for subsequent quantitative fluorescence expression analysis.

[0065] 2.4 Quantitative Fluorescence Expression Analysis of Peanut AhNHL Family Genes

[0066] Based on phylogenetic and expression pattern analysis, eight AhNHL genes were selected, including AhNHL3, AhNHL14, AhNHL23, AhNHL24, AhNHL28, AhNHL31, AhNHL33, and AhNHL42, and their expression was further analyzed using quantitative real-time analysis. Figure 4A). Gene-specific primers were designed using Primer-BLAST online software, with AhActin7 (XM_025826875) as the internal reference gene (Table 3). Only primers amplifying a single product of approximately 150-250 bp were selected for quantitative real-time expression analysis. Total RNA was extracted using the DP441 RNA extraction kit, and the concentration and integrity of RNA were further detected using an Agilent 2100 bioanalyzer. RNA reverse transcription and first-strand cDNA synthesis were performed according to the instructions of the Prime Script RT kit and gDNA Eraser kit. The quantitative real-time reaction system consisted of 1.0 μL of first-strand cDNA (10-fold dilution), 1.0 μL of forward and reverse primers (10.0 μM), 10.0 μL of LTakara in 2×SYBR Green I Master Mix, and 7.0 μL of ddH2O, with a final reaction volume of 20 μL. All reactions were performed in 96-well plates using a Bio-Rad CFX-96 real-time PCR system, with three replicates for each reaction. Polymerase chain reaction (PCR) amplification conditions included: an initial denaturation step at 95°C for 3 minutes, followed by 40 cycles, each cycle consisting of denaturation at 95°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 65°C for 5 seconds. Finally, a final extension step was performed at 68°C for 5 minutes. Quantitative analysis was then performed by comparing the results with other methods (2...). -ΔΔCT The expression levels of target genes were assessed. Results showed that among the genes induced by *Ralstonia solanacearum*, AhNHL24, AhNHL28, and AhNHL28 were significantly upregulated from 0.5–7.0 dpi in H108 cells, while significantly downregulated in H107 cells. The expression levels of other genes showed some differences between H107 and H108 cells. Further investigation was conducted using qRT-PCR to study the expression patterns of these AhNHL genes in response to exogenous plant hormones. Figure 4 (B) Among them, AhNHL3 and AhNHL14 showed higher expression levels after treatment. AhNHL3, AhNHL14, AhNHL23, AhNHL24, and AhNHL33 were significantly upregulated by SA, JA, and ABA in H107 and H108 cells, respectively, while AhNHL33 was upregulated only by SA. This indicates that these AhNHL genes are involved in the plant response to Ralstonia solanacearum and plant hormone signaling.

[0067] Table 3 Gene-specific primer sequences used for quantitative real-time expression analysis

[0068]

[0069] Example 3: Cloning, subcellular localization analysis, and verification of disease resistance function of peanut AhNHL family genes

[0070] 3.1 Cloning of the AhNHL gene

[0071] Based on expression analysis, AhNHL14, AhNHL24, AhNHL31, and AhNHL33 were selected for subcellular localization analysis. Homologous recombination primers were designed using Primer Premier 5.0 software based on the complete coding sequences of the four genes (Table 4).

[0072] The nucleotide sequences of the AhNHL14, AhNHL24, AhNHL31, and AhNHL33 genes are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[0073] AhNHL14 (SEQ ID NO: 1):

[0074] .

[0075] AhNHL24 (SEQ ID NO: 2):

[0076] 。

[0077] AhNHL31(SEQ ID NO:3):

[0078] ATGTCGCAACTGAACGGCGCATATTACGGCCCTTCCATACCTCCGCCGCAACACAAACGTCACTACCGTGGCGATGGCGGCGGCAGCAACTGCTGCTGCTGCTGCGGAATCTTCCGCTGCTGCTGCGGCTGCATTTTTGACTGCATCTTCGGCCTCATCTGCAAGATCTTAACCACACTTCTCATCCTTCTCATCATCGTAGCCGTTCTTCTCTACTTCATCGTACGGCCAAATCTCGTCAAGTTCCACGTCAGCAACGCCGTCCTCACTCAGTTCAATTTCTCCTCCGACGCCGCCGCCACCAACAACAACACGCTCCACTACAACCTCATGCTAAACTTCACGATCCGAAACCCTAACAAGCGCGTGGGAATCTACTACGACTACATCGAGGCGCGTGGATTCTACCACGACGTTGGCTTCGGTAACGTCACTATGCAACCGTTTTTTCAGGGAACGAAGAACACGACGGTGGTTGCGACGACTCTCAAGGGTGAGAATGAGGTGGTTTTGGGATCGGATAAAGGATCCAAGGTGGAGGAAGAACGAGGATCTGGTGTTTATGGCATTGATTTGAAGGTGTTCATGAAAGTGAGGTTTAAGTTTTGGTTTATGAAAACTGGGAAGGTGAAACCCAAGGCTATTTGTGTGTTGAAGGTTCCGTTGATGACGAGATCGAAGAATGGAACTTTTACGGCGGAGCACGGCGGCGCGTTTCAAGATACCGCGTGCGACTGGGGTTACCGGTGGTTGTGGTTTCATCATTAG。

[0079] AhNHL33(SEQ ID NO:4):

[0080] ATGTCGACGTCTGACAAGCCGGAAGTAGTGGAAAGGGGTACTAAGGATGAGAAGCACAAAGATGATGATAAAGAGGAGGGTGAGAAGGGTGGATTTATTGAGAAGGTGAAGGATTTCATTCATGACATTGGTGAGAAGATTGAGGGGGCTATAGGGTTTGGGAAGCCAAGTGCAGATGTTAAAGCAATCCACATTCCCAAGATCAATCTTCACCAGGCAGACCTTGTTGTTGATGTGCTTGTAAAGAATCCTAATCCGGTGCCGATCCCTCTGATTGACATAAACTACTTGGTTGAGAGTGATGGAAGGAAGCTAGTTTCTGGATTGATACCGGATGCTGGCACAATTCATGCGCATGGAGAGGAGACTGTCAAGGTTCCGGTTACTTTGATTTATGATGACATTAAGCAAACATATGATGATATTCAACCGGGGAGCATCATTCCTTATAGGTTGAAGGTTGATCTCATTGTTGATGTTCCGATCTTGGGGAGGCTGACTCTACCTCTGGAGAAGACTGGAGAAATCCCCATACCTTACAAGCCAGATATTGATCTTGAGAAGATCCATTTTGATAAGTTCTCTTTTGAAGAGACAATTGCAACTCTTCATTTGAAATTGGAAAACAAGAATGATTTCGACCTTGGCCTCAATACGCTTGATTATGAGGTTTGGCTTGGTGATGTCAGCATTGGAGCTGCTGAACTCTCCAAGTCTGCGAAAATCGAGAAAAGTGGTATTAGTTACATTGATATTCCAGTTACCTTTAGGCCCAAGGATTTCGGCTCTGCATTGTGGGATATGATCAGAGGAAAAGGAGTGAAGTAG。

[0081] Table 4 Specific primer sequences for AhNHL gene cloning

[0082]

[0083]

[0084] PCR amplification was performed using Primer STAR Max DNA polymerase with H108 cDNA as a template. The PCR reaction system and conditions were as follows: 1 μL each of forward and reverse primers, 25 μL of polymerase, 2 μL of template cDNA, 21 μL of ddH2O, and a total reaction volume of 50 μL. The PCR reaction was performed according to the following program: 94℃ for 2 min; 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 2 min, for 32 cycles; 72℃ for 7 min, and then isothermal at 4℃. The PCR product was detected by 1% agarose gel electrophoresis. After the detection result was correct, the PCR product was recovered from the gel using a DNA recovery kit to obtain the target gene fragment.

[0085] 3.2 Construction of recombinant vector for the AhNHL gene

[0086] The pCambia1300-YFP plant expression vector was double-digested with Kpn I and Sca I restriction endonucleases to produce sticky ends, resulting in a linearized pCambia1300-YFP vector. Subsequently, the target gene fragment was cloned into the pCambia1300-YFP plant expression vector using the CloneSmarter Seamless Assembly Cloning Kit. Further, the recombinant vector was transformed into DH5α *E. coli* competent cells using a heat shock method. Positive clones were picked and cultured, and after colony PCR detection, 3–5 correct single clones were selected, cultured, and plasmids were extracted using a plasmid mini-extraction kit and sent for sequencing. Sequencing was performed by BGI Genomics (Shenzhen) Co., Ltd. Single *E. coli* colonies that were correctly sequenced were inoculated into a solution containing 50 ng / mL. -1 Incubate in 50 mL LB broth with kanamycin. After culturing at 37°C for 12–16 hours, collect bacterial cells by centrifugation at 3000 × g for 10 minutes. Extract plasmid DNA using an Omega Bio-tek kit to obtain recombinant plasmids pCambia1300-AhNHL14-YFP, pCambia1300-AhNHL24-YFP, pCambia1300-AhNHL31-YFP, and pCambia1300-AhNHL33-YFP.

[0087] 3.3 Subcellular localization analysis

[0088] The recombinant and control vectors were transformed into *Agrobacterium tumefaciens* EHA105 competent cells using a freeze-thaw method, followed by *Agrobacterium* culture on LB solid medium containing the corresponding antibiotics. Single colonies were picked and inoculated into 1 mL of LB liquid medium containing 50 ng / mL kanamycin and cultured at 28°C for 18 hours. After incubation at 37°C with shaking at 200 rpm for 12-16 hours, the cells were collected by centrifugation at 3000×g for 10 minutes. 50 mL of the bacterial culture was inoculated into 50 mL of freshly prepared infection medium (containing 10 mM MgCl2, 5 mM MES, 5 μM acetylsalicylic acid, and 50 ng / mL kanamycin) and incubated at 28°C for 2-3 hours before infection. Simultaneously, *Agrobacterium* medium containing the AtPIP2a-RFP membrane-labeled vector was mixed with an equal volume of the recombinant and control vectors for subcellular localization analysis. Healthy *Nicotiana benthamiana* plants (purchased from Beijing Huayueyang Biotechnology Co., Ltd.) were placed under an incandescent lamp for 2 hours to open the stomata of the leaves. The lower epidermis of the leaves was punctured with a needle, and a 0.5 mL syringe was used to draw up the bacterial suspension and gently inject it into the leaves from the back. The injection area was marked, and the plants were then placed in a culture chamber for 48-72 hours. Finally, the yellow-green and red fluorescence of the fusion protein and the control were observed using an LSM710 confocal laser scanning microscope, and 3-5 images were randomly collected. The results showed that the YFP control was uniformly expressed in cells, while the AhNHL-YFP fusion was expressed not only in the nucleus but also on the plasma membrane. Figure 5 A).

[0089] Example 4: Verification of disease resistance function of peanut AhNHL family genes

[0090] 4.1 Identification of bacterial wilt resistance in AhNHL14, AhNHL24, AhNHL31 and AhNHL33

[0091] To determine potential resistance to bacterial wilt (BW), a BW inoculation experiment was conducted on leaves of *Ralstonia benthamiana* and peanut variety NDH107, which overexpress AhNHL14, AhNHL24, AhNHL31, and AhNHL33. Specifically, *Agrobacterium* cells carrying the recombinant vector and the control vector were injected into fully expanded tobacco and peanut leaves, respectively. *Ralstonia benthamiana* was cultured in TTC liquid medium until OD... 600nm =0.8, using a needleless syringe to inject healthy tobacco leaves into tobacco and peanut leaves that transiently express the AhNHL gene, and observe symptoms 3 days after inoculation.

[0092] Since Ralstonia solanacearum infection causes leaf cell death and necrotic lesions, the resistance level resulting from the overexpression of candidate genes was assessed by measuring the induced necrotic lesions. Trypan blue (TB) was used for visualization of cell death and assessment of resistance levels. The procedure was as follows: Tobacco and peanut leaves were immersed in trypan blue staining solution, then immersed in a boiling water bath for 2 minutes and cooled to room temperature. Afterwards, the stained leaves were immersed in a chloral hydrate solution (1.25 g / mL) at 25°C and rinsed at 50 rpm. To ensure complete decolorization, the solution was changed every 3 hours until the leaves became transparent. The leaves were then immersed in 75% ethanol for 20 minutes, followed by observation and photography. Three inoculation sites were set on each leaf, and the expansion and area of ​​lesions were observed, measured, photographed, and recorded daily. Furthermore, the area of ​​lesions on peanut and tobacco leaves was measured using the ImageJ (153-win-java8) digital image analysis program. The results showed that tobacco leaves overexpressing AhNHL14, AhNHL24, and AhNHL31 exhibited milder necrotic lesions compared to AhNHL33 and the control group. Figure 5 B). Necrotic lesions caused by cell death could be observed by TB staining, and the lesion area was measured using ImageJ software. Results showed that in the blank control group, peanut leaves exhibited curling and yellowing, and this phenomenon became more pronounced with increasing duration of Ralstonia solanacearum infection. Transient overexpression of AhNHL24 resulted in the least cell death and the smallest lesion area, followed by AhNHL14 and AhNHL31. Figure 5 D). Furthermore, these four AhNHL genes were transiently overexpressed in leaves of the susceptible peanut variety NDH107 for resistance identification. Minimal cell death and necrotic lesions were observed in leaves overexpressing AhNHL24. Figure 5 (CE). In conclusion, overexpression of AhNHLs, especially AhNHL24, enhances the resistance of tobacco and peanut plants to Ralstonia solanacearum.

[0093] The amino acid sequence encoded by the AhNHL24 gene is shown in SEQ ID NO: 5:

[0094] AhNHL24 (SEQ ID NO: 5):

[0095] MADNQRIHPDIEASPRPSAPLVPGNIAKSENGDPNNSPLPPPLPQRTLPVMHSKPPRRRRSCCCRFLCCTFTTLLILIIAITAGILFLAFRPKIPKYSVDKLRITEFNFSSGTNILSVTSNVRIT ARNPNKKIGIYYEGGSHISAWYSGSQLCEGSMIKFYQGHKNTTVLDLPLRGQIQDASGLVSKIQQQIQDTNNIPLDIKVKQPVRVKFGKLKLFKVNFRVRCKLVVDSLSANNDIKISSSSCKFRFRL.

[0096] 4.2 Creation and Disease Resistance Identification of Transgenic Tobacco Overexpressing AhNHL24

[0097] Based on the verification results of disease resistance function of the AhNHL family genes in peanuts, AhNHL24 was selected for heterologous overexpression in tobacco transgenic plants. Transgenic tobacco plants overexpressing AhNHL24 were created using the leaf disc transformation method, and the expression level of AhNHL24 in homozygous T3 generation tobacco lines was semi-quantitatively detected. Using NbActin as an internal reference gene, the expression level of the AhNHL24 gene in wild-type (WT) and three overexpressing AhNHL24 transgenic families (OE-AhNHL24#11, OE-AhNHL24#15, and OE-AhNHL24#17) was detected by qRT-PCR. The results showed that, compared with WT, all three overexpressing lines showed obvious specific bands, indicating that the AhNHL24 gene was successfully overexpressed in transgenic tobacco. Figure 6 A).

[0098] To determine the role of AhNHL in plant resistance to BW and SR (white rot), wild-type and transgenic tobacco plants were inoculated with *Ralstonia solanacearum* and *Sclerotium rolfsii* isolated and preserved in our laboratory (the isolation method for *Ralstonia solanacearum* can be found in the literature "Li Liangliang et al., 2021. Identification, biological characteristics and indoor screening of *Ralstonia solanacearum* causal agent for peanuts in Runan County." *Henan Science*. 2021, 39(04): 551-558."). Approximately 100 ml of *Ralstonia solanacearum* culture solution was added to each pot of tobacco plant, and the disease index (DI) was checked after 2, 4, 8, 12, and 16 days. Simultaneously, *S. rolfsii* was inoculated into tobacco plants using an oat-mediated inoculation method. Briefly, *S. rolfsii* mycelia were inoculated onto autoclaved oat seeds and cultured at 28°C for 3 days. Subsequently, equal amounts of oat seeds covered with mycelium were placed near the stems of tobacco plants, and disease incidence was checked after 1, 2, 3, 4, and 5 days. The level of resistance was determined based on the disease incidence and survival rate of the tobacco plants.

[0099] Twelve days after inoculation with Ralstonia solanacearum, phenotypic changes in WT and overexpression lines showed that WT plants exhibited severe wilting symptoms, while the symptoms of the overexpression lines were significantly reduced, indicating that AhNHL24 gene overexpression enhanced plant resistance to Ralstonia solanacearum. Figure 6 B). Further comparison of the local phenotypes of the OE-AhNHL24#17 line and WT plants revealed that the overexpressing line exhibited better overall growth and significantly lower leaf wilting compared to WT, further validating the disease resistance function of this gene. Figure 6 C). The dynamic change curve of incidence rate showed that, compared with WT, the incidence rate of the OE-AhNHL24#17 strain was significantly reduced at all time points, and the upward trend over time was slower, quantitatively demonstrating that AhNHL24 overexpression can significantly reduce the incidence rate of bacterial wilt. Figure 6 D).

[0100] After inoculation with *Sclerotium rolfsii*, the control group (uninoculated plants) grew normally, while the experimental group (WT plants) showed obvious disease symptoms. The symptoms of the overexpressing strain OE-AhNHL24#17 were significantly reduced, indicating that AhNHL24 overexpression also conferred resistance to this pathogen. Figure 6 E). Incidence curve analysis showed that the incidence rate of the OE-AhNHL24#17 strain was lower than that of WT at all time points, and the increase over time was smaller, indicating that AhNHL24 overexpression enhanced the plant's resistance to *Sclerotinia sclerotiorum*. Figure 6 F). These results indicate that the AhNHL24 gene is involved in enhancing plant resistance to Sclerotium truncatum.

[0101] In summary, the AhNHL24 gene exhibits some resistance to both bacterial wilt and white mold pathogens in peanuts. Overexpression of this gene significantly enhances peanut resistance to both bacterial wilt and Sclerotium truncatum, making it an ideal gene for enhancing plant disease resistance. Furthermore, overexpression of this gene in tobacco through genetic transformation can confer resistance to multiple pathogens, thereby improving the crop's field disease resistance.

[0102] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Use of the peanut disease resistance gene AhNHL24 for increasing disease resistance of plants and / or for breeding disease resistant plant varieties, characterized in that, Overexpression of the peanut disease resistance gene AhNHL24 in plants can improve the plant's resistance to bacterial wilt and white mold. The amino acid sequence encoded by the AhNHL24 gene is shown in SEQ ID NO: 5, the disease resistance is resistance to bacterial wilt and / or white mold, and the plant is peanut or tobacco.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the AhNHL24 gene is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, The pathogen of bacterial wilt is Ralstonia solanacearum (Solanaceae family), and the pathogen of white rot is Sclerotium rolfsii (Solanaceae family).

4. A method for improving plant disease resistance, characterized in that, Overexpression of the peanut disease resistance gene AhNHL24 enhances the plant's resistance to bacterial wilt and white mold; the amino acid sequence encoded by the AhNHL24 gene is shown in SEQ ID NO: 5, and the plant is peanut or tobacco.

5. The method for improving plant disease resistance according to claim 4, characterized in that, Includes the following steps: S1. The AhNHL24 gene was cloned into the pCambia1300-YFP plant expression vector, transformed into Escherichia coli competent cells, and after screening for positive clones, the recombinant plasmid pCambia1300-AhNHL24-YFP was extracted. S2. The recombinant plasmid was transformed into Agrobacterium competent cells. After resistance selection, the recombinant bacteria were introduced into plants for overexpression, thus obtaining transgenic plants overexpressing AhNHL24.

6. The method for improving plant disease resistance according to claim 5, characterized in that, The primers used to amplify the AhNHL24 gene from peanut are shown in SEQ ID NO: 26 and SEQ ID NO:

27.

7. The method for improving plant disease resistance according to claim 5, characterized in that, Recombinant bacteria were introduced into plants using the leaf disc transformation method.

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