Application of apple pattern recognition receptor gene MdRE02 in regulation and control of plant disease resistance

Abstract

The invention belongs to the technical field of gene engineering, and relates to application of an apple pattern recognition receptor gene MdRE02 in regulation and control of plant disease resistance. The nucleotide sequence of the apple pattern recognition receptor gene MdRE02 is as shown in SEQ ID NO.1, the MdRE02 gene is silenced in apples, the expression of an immune gene induced by a pathogen-related molecular pattern VmE02 is remarkably reduced, and the disease resistance to valsa mali of apple trees is remarkably weakened. Through overexpression of the MdRE02 gene in the tobacco mutant re02, cell necrosis and active oxygen bursting induced by the VmE02 can be recovered, which indicates that the MdRE02 is a pattern recognition receptor for recognizing the VmE02. By overexpressing the MdRE02 gene in tobacco mutants re02 and oilseed rape, the resistance of the MdRE02 gene to sclerotinia sclerotiorum can be remarkably improved. The results show that the MdRE02 gene can be used for disease-resistant breeding and disease prevention and control of valsa ceratosperma and sclerotinia sclerotiorum of apple trees.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the apple pattern recognition receptor gene. MdRE02 Application in regulating plant disease resistance. Background Technology

[0002] Plants face various biotic stresses during their growth, and to resist infection by pathogenic microorganisms, they have evolved an innate immune system. Among these, pattern recognition receptors (PRRs) located on the cell membrane can sense pathogen-associated molecular patterns (PAMPs), thereby activating pattern-triggered immunity (PTI). PTI serves as the primary line of defense in plant immunity, enhancing the plant's basic resistance to various pathogens. Therefore, utilizing the PTI mechanism for crop disease control is one of the important approaches to achieving environmentally friendly plant protection strategies.

[0003] PRRs are widely present in plants and are mainly classified into receptor-like kinases (RLKs) and receptor-like proteins (RLPs) based on the presence or absence of intracellular kinase domains. Among them, extracellular leucine-rich repeat (LRR) PRRs are the most numerous. They recognize PAMPs through their extracellular LRR domains, thereby triggering a series of basic defense responses, such as reactive oxygen species (ROS) bursts, callose deposition, and defense gene expression. Studies have shown that overexpression or cross-species transfer of PRRs can confer broad-spectrum disease resistance in plants, making them a key target for genetic improvement of crop disease resistance. However, most known disease resistance-related PRRs are derived from model plants, and few similar genes have been identified in crops. Furthermore, research on genetic improvement based on endogenous crop PRR genes remains relatively limited.

[0004] As an important economic crop, apples lack highly disease-resistant varieties in their germplasm resources, making the discovery and utilization of their own disease-resistant genes an urgent need for genetic improvement. Since the first cloning of the disease-resistant gene encoding LRR-RLP from apples in 2004… HcrVf2 Since then, progress in identifying other disease resistance-related RLP genes in apples has been relatively slow. Therefore, identifying and functionally analyzing pattern recognition receptors involved in immune regulation in apples can not only provide key gene resources for creating disease-resistant apple germplasm, but also extend their disease resistance function to other crops through gene transfer, stacking, or synthetic biology strategies, providing important gene elements for cross-species disease resistance breeding. Summary of the Invention

[0005] To address the aforementioned technical problems and deficiencies, this invention provides an apple pattern recognition receptor gene. MdRE02 Application in regulating plant disease resistance.

[0006] In a first aspect, the present invention provides an apple pattern recognition receptor gene. MdRE02 Application in regulating plant disease resistance, the apple pattern recognition receptor gene MdRE02 The nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2;

[0007] The regulation of plant disease resistance includes: overexpressing the apple pattern recognition receptor gene. MdRE02 To improve plant disease resistance; to silence the apple pattern recognition receptor gene. MdRE02 This reduces the plant's disease resistance.

[0008] Furthermore, the apple pattern recognition receptor gene provided by this invention... MdRE02 In the application of regulating plant disease resistance, the improvement of plant disease resistance refers to enhancing the resistance of apple trees to apple tree rot pathogens. You are welcome. Resistance to Sclerotinia sclerotiorum, and / or improved resistance of tobacco and rapeseed to Sclerotinia sclerotiorum. Sclerotinia sclerotiorum Resistance.

[0009] Furthermore, the apple pattern recognition receptor gene provided by this invention... MdRE02 In the application of regulating plant disease resistance, the reduction of plant disease resistance is to downregulate the expression of apple defense-related genes induced by VmE02; The Apple defense-related genes include: MdPR1 , MdPR2 , MdPR5 , MdCYP81F2 .

[0010] Secondly, this invention provides an apple pattern recognition receptor gene. MdRE02 Application in breeding disease-resistant plant varieties, the apple pattern recognition receptor gene MdRE02 The nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2; The method for cultivating disease-resistant plant varieties involves overexpressing the apple pattern recognition receptor gene in plants. MdRE02 Thus, disease-resistant plant varieties were obtained.

[0011] Furthermore, the apple pattern recognition receptor gene provided by this invention... MdRE02 In the application of breeding disease-resistant plant varieties, the plants include: apple, tobacco and rapeseed.

[0012] Furthermore, the apple pattern recognition receptor gene provided by this invention... MdRE02 In the application of cultivating disease-resistant plant varieties, the disease-resistant plant varieties include those resistant to apple tree rot pathogens. You are welcome. Apple varieties, resistance to sclerotinia stem rot Sclerotinia sclerotiorum Tobacco and / or rapeseed varieties.

[0013] Thirdly, the present invention provides a method for improving plant disease resistance, comprising: constructing a gene containing an apple pattern recognition receptor. MdRE02 A transient expression vector was introduced into plant cells or tissues to activate the apple pattern recognition receptor gene. MdRE02 Transient overexpression to enhance plant disease resistance; The apple pattern recognition receptor gene MdRE02 The nucleotide sequence is shown in SEQ ID NO.1.

[0014] Furthermore, in the method for improving plant disease resistance provided by the present invention, the disease resistance includes: resistance of apples to apple tree rot pathogens. You are welcome. Resistance to Sclerotinia sclerotiorum, and / or resistance of tobacco and rapeseed to Sclerotinia sclerotiorum. Sclerotinia sclerotiorum Resistance.

[0015] Fourthly, the present invention provides a method for breeding disease-resistant plant varieties, comprising: constructing a structure containing an apple pattern recognition receptor gene. MdRE02 A transient expression vector was introduced into plant cells or tissues to activate the apple pattern recognition receptor gene. MdRE02 Transient overexpression yields disease-resistant plant varieties; The apple pattern recognition receptor gene MdRE02 The nucleotide sequence is shown in SEQ ID NO.1.

[0016] Furthermore, in the method for cultivating disease-resistant plant varieties provided by the present invention, the disease-resistant plant varieties include: those resistant to apple tree rot pathogens. You are welcome. Apple varieties, resistance to sclerotinia stem rot Sclerotinia sclerotiorum Tobacco and / or rapeseed varieties.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) The apple pattern recognition receptor gene provided by the present invention MdRE02 It is one of the few PRR genes identified in woody plants such as apples. Its key biological functions in immune regulation and disease resistance in apples have been clarified, providing a theoretical basis and important gene resources for creating new disease-resistant apple germplasm.

[0018] (2) The apple pattern recognition receptor gene provided by the present invention MdRE02It not only enhances resistance to rot pathogens in apples, but can also be transferred to tobacco and rapeseed through cross-species transfer, thereby enhancing their resistance to Sclerotinia sclerotiorum.

[0019] (3) VmE02 is a pathogen originating from apple tree rot ( V. mali The pathogen-associated molecular pattern of VmE02 belongs to the Eliciting plant response-like (ER) protein family in fungi. When the pattern recognition receptor on the plant cell membrane senses VmE02, it rapidly triggers the plant's basic immune response, including reactive oxygen species bursts, activation of defense genes, and hypersensitive cell necrosis. This invention reveals the apple pattern recognition receptor gene. MdRE02 This study mediated the recognition of the pathogen-associated molecular pattern VmE02. VmE02 exists across both fungi and oomycetes, thus this gene possesses broad-spectrum disease resistance potential and holds promising application prospects.

[0020] (4) This invention clarifies the gene MdRE02 Key biological functions in plant immune regulation and disease resistance. Transient silencing in apple leaves. MdRE02 It can inhibit the expression level of VmE02-induced immune genes and significantly reduce the resistance of apples to apple tree rot pathogen, while overexpression of this gene enhances the resistance. Heterologous overexpression in tobacco and rapeseed. MdRE02 This can enhance the resistance of both organisms to Sclerotinia sclerotiorum. Therefore, the gene provided by this invention... MdRE02 It can not only improve the resistance of apples to rot pathogens, but also be applied to tobacco and rapeseed through cross-species transfer to improve their resistance to Sclerotinia sclerotiorum. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 for MdRE02 Gel electrophoresis image of a gene clone fragment.

[0023] Figure 2 for MdRE02 The diagram illustrates the structure of the gene-encoded protein. A shows the predicted signal peptide and transmembrane domains; B is a schematic diagram of the domains; and C shows the predicted tertiary structure of the extracellular amino acid sequence.

[0024] Figure 3 for MdRE02Analysis of immune gene expression in apple tissue culture seedlings induced by VmE02 after transient gene silencing. Where A represents... MdRE02 Gene expression analysis; B represents immune-related genes. MdPR1 , MdPR2 , MdPR5 , MdCYP81F2 Analysis of the expression.

[0025] Figure 4 It is a tobacco mutant re02 Intermittent overexpression MdRE02 Immunophenotypic analysis induced by VmE02 after gene gene generation. In this diagram, A represents the cell necrosis phenotype; B represents the dynamic curve of real-time relative luminescence units of reactive oxygen species (ROS) emission.

[0026] Figure 5 for MdRE02 Apple's response after transient gene silencing V. mali Phenotypic analysis of the infection. A represents the phenotypic results of lesion area; B is a statistical graph of lesion area.

[0027] Figure 6 for MdRE02 Apple response after transient gene overexpression V. mali Phenotypic analysis of the infection. A represents expression level analysis; B represents the phenotypic results of lesion area; and C represents the statistical results of lesion area.

[0028] Figure 7 Tobacco mutant re02 overexpression MdRE02 Phenotypic analysis of gene resistance to Sclerotinia sclerotiorum. A represents the phenotypic results of lesion area; B represents the statistical results of lesion area; and C represents the expression of the MdRE02 protein.

[0029] Figure 8 Overexpression in rapeseed MdRE02 Phenotypic analysis of gene resistance to Sclerotinia sclerotiorum. A represents the phenotypic results of lesion area; B represents the statistical results of lesion area; and C represents the expression of the MdRE02 protein.

[0030] Figure 3 and Figure 5~8 All use Student- t Test calculation P value, express P <0.05, express P <0.01, express P <0.001. Detailed Implementation

[0031] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0032] The pK7GWIWG2D(Ⅱ) vector, pCAMBIA1300 vector, and apple tree rot pathogen involved in the following examples This is bad. ( V. mali ), Sclerotium Sclerotinia sclerotiorum ( S. sclerotiorum ) 、 The leaves of Gala apple tissue culture seedlings, tobacco Benzodiaceae, and rapeseed were all provided by the Fruit Tree Disease Research Team Laboratory of the College of Plant Protection, Northwest A&F University.

[0033] The LB solid culture medium involved in the following examples includes: adding 10 g tryptone, 5 g yeast extract, 10 g NaCl and 15 g agar powder to a 1 L measuring cup, adding water to make up to 1 L, and sterilizing at 121°C for 20 min.

[0034] LB liquid culture medium consists of: adding 10 g tryptone, 5 g yeast extract, and 10 g NaCl to a 1 L measuring cup, adding water to bring the volume to 1 L, and sterilizing at 121℃ for 20 min.

[0035] MS medium consists of: 30 g sucrose, 4.43 g MS medium (PhytoTechnology, catalog number: M519), 8 g agar powder, 200 µL 1 mg / mL IAA, 200 µL 1 mg / mL 6-BA, and autoclaved at 121°C for 20 min in a 1 L measuring cup.

[0036] PDA culture medium consists of: peeling and cutting 200 g of potatoes into pieces, boiling them in double-distilled water for 30 min, filtering the mixture through gauze into a 1 L measuring cup, adding 20 g of glucose and 15 g of agar powder, stirring well, and then bringing the volume to 1 L with double-distilled water. Autoclave at 121℃ for 20 min.

[0037] Example 1 This embodiment provides an apple pattern recognition receptor protein-coding gene. MdRE02 The method for obtaining it.

[0038] The apple pattern recognition receptor protein encoding gene described in this embodiment MdRE02 From the GDR database of Rosaceae plants Malus domesticaThe GDDH13 v1.1 genome (https: / / www.rosaceae.org / species / malus / malus_x_domestica / genome_GDDH13_v1.1) was obtained, with the gene number MD09G1131100. Its CDS sequence is shown in SEQ ID NO.1, with a full length of 2958 bp, encoding 985 amino acids. The amino acid sequence is shown in SEQ ID NO.2, and the protein molecular weight is 109.64 kDa.

[0039] Example 2 This embodiment provides an apple pattern recognition receptor protein-coding gene. MdRE02 Cloning methods.

[0040] RNA was extracted from apple leaves using an RNA kit (Beijing Huayueyang Biotechnology Co., Ltd., catalog number 0416-50); apple cDNA was obtained by reverse transcription using a reverse transcription kit (ThermoFisher Scientific, catalog number K1162). MdRE02 The CDS sequence of the gene was used to design a vector containing the homologous arm of the pCAMBIA1300 vector using SnapGene software. MdRE02 Primers for amplifying the gene CDS sequence (target fragment) (SEQ ID NO.5 and SEQ ID NO.6). Using cDNA as a template, PCR amplification of the target fragment was performed using a high-fidelity enzyme (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 10154ES03). The reaction system was: 25 μL 2×Hieff Canace® Plus PCR Master Mix (With Dye); 2 μL 1300-MdRE02-F and 2 μL 1300-MdRE02-R; 3 μL cDNA; 18 μL ddH2O. The PCR reaction program was: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 58℃ annealing for 20 s, 72℃ extension for 2 min, 35 cycles, 72℃ extension for 5 min. Agarose gel electrophoresis results are shown below. Figure 1 As shown, conforms to MdRE02 Size of the gene CDS sequence fragment.

[0041] 1300-MdRE02-F (SEQ ID NO.5): 5'-CGGGGGACGAGCTCGGTACCATGGATTCTTTGTTGTCC-3'; 1300-MdRE02-R (SEQ ID NO. 6): 5'-ACGTCGTATGGGTAGGTACCAGTTCGGCTCCCCTCGT-3'.

[0042] Example 3 This embodiment provides a method for analyzing the secondary and tertiary structures of the apple pattern recognition receptor protein MdRE02.

[0043] The structural features of the apple pattern recognition receptor protein MdRE02 were analyzed using bioinformatics methods. DeepTMHMM (https: / / dtu.biolib.com / DeepTMHMM) was used to predict the protein signal peptide and transmembrane domain. The results showed that amino acids 1-26 of the MdRE02 protein encode an exosensitive signal peptide, and amino acids 936-956 encode a transmembrane domain. Figure 2 A in Figure 2 (B in the original text). Analysis using LRRsearch (https: / / lrrsearch.com / ) showed that the MdRE02 protein encodes 28 leucine-rich repeat sequence motifs (LRR). Figure 2 (B in the original text). Tertiary structure prediction of the extracellular domain (MdRE02-ecto) of the MdRE02 protein using AlphaFold Server (https: / / alphafoldserver.com). Figure 2 Structural analysis of the MdRE02 protein indicates that it is an LRR-RLP type pattern recognition receptor.

[0044] Example 4 This embodiment provides apple tissue culture seedlings. MdRE02 Methods for transient gene silencing.

[0045] (1) MdRE02 Construction of gene silencing vectors Obtained in Example 2 MdRE02 Using the gene as a template, its specific silencing fragment (SEQ ID NO.3) was cloned into the pK7GWIWG2D(Ⅱ) vector to construct the vector. MdRE02 Gene silencing vectors. A gene silencing vector was constructed using the Gateway recombination cloning system. MdRE02 The gene expression vector was constructed stepwise using Gateway BP Clonase and Gateway LR Clonase (Invitrogen, USA). The specific construction process included: first, under the catalysis of Gateway BP Clonase, expressing the gene containing the attB site... MdRE02The gene was subjected to in vitro recombination with the donor vector pDONR222 containing the attP site, using primers shown in SEQ ID NO. 7-8, to obtain the entry clone. The entry clone was then sequenced and verified using primers SEQ ID NO. 8 and M13-F (SEQ ID NO. 9). Subsequently, the correctly sequenced entry clone (containing the attL site) was subjected to in vitro recombination with the pK7GWIWG2D(Ⅱ) vector (target vector) containing the (attR) site under the catalysis of Gateway LR Clonase to obtain the expression clone. The recombination reaction volume was 1 µL. MdRE02 Gene fragment or introductory clone, 0.5 µL donor or target vector, 1 µL TE buffer (pH 8.0), and 0.5 µL Gateway BP cloning enzyme or GatewayLR cloning enzyme. The recombination reaction was performed overnight at 25°C. The expression clone was transformed into *E. coli* DH5α competent cells (the resulting recombinant plasmid was designated pK7-MdRE02), and single colonies were screened for colony PCR identification (primers used are shown in SEQ ID NO. 10-11). The empty pK7GWIWG2D(Ⅱ) vector was transformed into *E. coli* DH5α competent cells in the same manner (the resulting recombinant plasmid was designated empty pK7 vector). Positive clones of the expected size (including the pK7-MdRE02 clone and the empty pK7 vector clone) were amplified, and the recombinant plasmid was extracted and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing comparison.

[0046] SEQ ID NO.3: GTTTCCGCAGGCCAATTCTTAGTTCCTGATTCCTTGTCTTGTTTTGGAAAATTAACCAAGCTCAACCACTTGCGTCTTTCGCATATTAACTTACAAGGGAATTTCCCACGTTTTGTGGCTAACCTCACCCAACTTGTGTTTCTAGACTTGTTTGTCAATTCAATAACTGGTGAAATCCCATCTTGGCTCACATTAGAG p222- MdRE02 -RNAi-F (SEQ ID NO.7): 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGTTTCCGCAGGCCAATTCTTAGTT-3'; p222- MdRE02-RNAi-R (SEQ ID NO. 8): 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCTCTAATGTGAGCCAAGATGGGA-3'.

[0047] M13-F (SEQ ID NO. 9): 5'-TGTAAAACGAGCGGCCAGT-3'.

[0048] pK7-knan-F (SEQ ID NO. 10): 5'-GTGACTCCCTTAATTCTCATGTATAATTCGC-3'; pK7-intron-R (SEQ ID NO. 11); 5'-CCGTAAGAAGAGGCAAGAGTATGA-3'.

[0049] (2) Agrobacterium-mediated transient transformation The correctly sequenced pK7-MdRE02 and empty pK7 vectors were transformed into Agrobacterium competent cells EHA105 (Shanghai Weidi Biotechnology Co., Ltd.). The transformed EHA105 strains were plated on LB solid medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL) and cultured at 28°C for 48–72 h until single colonies appeared. Single colonies of pK7-MdRE02 and empty pK7 vectors were selected and inoculated into LB liquid medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL), and cultured with shaking at 220 rpm for 48–72 h. The bacterial culture was collected and centrifuged at 6000 rpm for 3 min to obtain bacterial cells. The bacterial cells were washed three times with infection solution (containing 10 mM MgCl2·6H2O, 10 mM MES, and 100 μM acetylsyleugenone). The bacterial cells were then resuspended and the OD was adjusted. 600 The pH was set at 0.6–0.8, and the culture was allowed to stand at room temperature in the dark for 2–3 hours to obtain Agrobacterium tumefaciens cultures carrying pK7-MdRE02 and empty pK7 vectors. Four to five-week-old Gala apple tissue culture seedlings were immersed in Agrobacterium tumefaciens cultures carrying pK7-MdRE02 and empty pK7 vectors, respectively, and placed in a sealed vacuum chamber for vacuum permeation treatment. Each permeation lasted 5 minutes, and two permeation operations were performed. After permeation, the apple tissue culture seedlings were reinserted into MS medium and cultured for another 3 days to achieve… MdRE02 Gene silencing.

[0050] (3) In apple leaves MdRE02 Gene expression analysis after gene silencing Apple leaf samples were collected 3 days after pK7-MdRE02 and empty pK7 vectors were infecting apple tissue culture seedlings. RNA was extracted from the apple leaf samples using a polysaccharide-polyphenol plant RNA extraction kit (Beijing Huayueyang Biotechnology Co., Ltd.). RNA concentration was measured using an ultra-micro spectrophotometer. cDNA was obtained from different samples after reverse transcription using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific). The PrimerQues Tool was used to design cDNA. MdRE02 Primers for quantitative PCR of genes (SEQ ID NO.12~13). MdActin The gene was used as an internal reference gene (primers used are shown in SEQ ID NO. 14-15). The cDNA was diluted 10-fold and used as a template. RT-qPCR was used to detect the gene in different apple leaf samples. MdRE02 Gene expression levels. The RT-qPCR system (20 μL) consists of: adding 10 μL of 2×ChamQ SYBR qPCRMaster Mix (Nanjing Novizan Biotechnology Co., Ltd.) and 0.5 μL of qRT-qPCR to ice. MdRE02 -F and qRT- MdRE02 -R, 1 μL cDNA, 8 μL ddH2O. The RT-qPCR reaction program included: pre-deformation at 95℃ for 30 s, cycling at 95℃ for 10 s, and 60℃ for 30 s for 40 cycles. Each RT-qPCR assay was performed with three biological replicates and three experimental replicates, using 2... –ΔΔCT The qPCR results were analyzed. RT-qPCR quantification results showed that, compared with apple leaf samples infected with empty pK7 vector (CK), pK7-MdRE02 (RNAi::MdRE02) was significantly more effective than pK7-MdRE02 in apple leaf samples 3 days after infection. MdRE02 Gene expression was downregulated by 88% ( Figure 3 The percentage in A represents the relative change in expression level, calculated by infecting apple leaves with the empty pK7 vector. MdRE02 The expression level was calculated as a control (to meet the silencing requirement).

[0051] qRT- MdRE02 -F (SEQ ID NO. 12): 5'-TGTGGCTAACCTCACCCAAC-3'; qRT- MdRE02 -R (SEQ ID NO. 13): 5'-AGGTGAGCCGCAGAAAAGTT-3'.

[0052] qRT- MdActin-F (SEQ ID NO.14): 5'-AGGCGCGAAATTACCAATCC-3'; qRT- MdActin -R (SEQ ID NO. 15): 5'-GCCCTCCAATTGTTCCTCGTTAAG-3'.

[0053] Example 5 This embodiment provides MdRE02 Analysis of VmE02-induced expression of apple defense-related genes after transient gene silencing.

[0054] The pathogen-associated molecular pattern (PAMP) used in this embodiment is the VmE02 protein, derived from the apple tree rot pathogen (…). This is bad. The VmE02 protein used was obtained from recombinant expression and purification, and its amino acid sequence is shown in SEQ ID NO.4. Apple tissue culture seedlings infected with empty pK7 vector and pK7-MdRE02 for 3 days were immersed in 1 μM VmE02 protein solution and placed in a sealed vacuum chamber for vacuum permeation treatment. Subsequently, the apple tissue culture seedlings were reinserted into MS medium for culture, and total RNA was extracted from apple leaves after 12 h. After reverse transcription into cDNA, RT-qPCR was performed according to the method in Example 4 to detect apple defense-related genes. MdPR1 (The primers used are shown in SEQ ID NO.16~17) MdPR2 (The primers used are shown in SEQ ID NO.18~19) MdPR5 (The primers used are shown in SEQ ID NO.20~21) MdCYP81F2 The expression of (primers used are shown in SEQ ID NO.22~23). Figure 3 As shown in B, compared with apple tissue culture seedlings infected with the empty pK7 vector (CK), the silence... MdRE02 Following the gene (pK7-MdRE02, i.e., RNAi::MdRE02), VmE02 induces apple defense-related genes. MdPR1 , MdPR2 , MdPR5 , MdCYP81F2 Both showed significant downregulation of expression.

[0055] SEQ ID NO.4:MHITSLFAILAAPVMVLATLDPATSNTKGSCPSTYSCDASKVSNSIQAAECAYNTRTSETETFAVFVTEHKYDDVEGAPYGTCSAYTCTAPTSSEMETNSDCWTFFWSDDGESSGVGTDCIKDPSSGECGCEDSDGTFIVGSSSCT q MdPR1 -F (SEQ ID NO. 16): 5'-CGTGGGATGACAATGTAGCAGG-3'; q MdPR1 -R (SEQ ID NO. 17): 5'-GCAAGGTTTTCACCGTATGGC-3'.

[0056] qRT- MdPR2 -F (SEQ ID NO. 18): 5'-CCTGCCATCAAAACATCCAC-3'; qRT- MdPR2 -R (SEQ ID NO. 19): 5'-GCTGAAGTAAGGGTACACATTCACAA-3'.

[0057] qRT- MdPR5 -F (SEQ ID NO.20): 5'-TGCGGCAAACGGGGGTC-3'; qRT- MdPR5 -R (SEQ ID NO. 21): 5'-GCAGGGCAAGACGAGGGCT-3'.

[0058] q MdCYP81F2 -F (SEQ ID NO. 22): 5'-AACCAGCCCATTTCAAAGCC-3'; q MdCYP81F2 -R (SEQ ID NO. 23): 5'-TCACGCTGCTCCTGTCATATC-3'.

[0059] Example 6 This embodiment provides a tobacco mutant. re02 overexpression MdRE02 Genes restore VmE02-induced cell necrosis and reactive oxygen species bursts.

[0060] (1) Construction of the carrier Using Kpn I and Sal I double restriction sites, the apple tree rot pathogen ( V. mali The coding sequence of the secretory protein VmE02 was constructed into a flag-tagged pCAMBIA1300 vector (primers used are shown in SEQ ID NO. 24-25, and the resulting recombinant plasmid is denoted as 1300-VmE02). The plasmid obtained in Example 2 was then used... MdRE02The gene clone was constructed into the pCAMBIA1300 vector with an HA tag (the resulting recombinant plasmid is designated 1300-MdRE02), using only the Kpn I single enzyme restriction site. GFP was used as a negative control (primers used are shown in SEQ ID NO. 26-27, and the resulting recombinant vector is designated 1300-GFP). The gene was then cloned into NbRE02, a synonymous mutant of the receptor protein NbRE02 in Nicotiana benthamiana. syn As a positive control (i.e., the NbRE02 receptor protein obtained by synonymous mutation targeting the CRISPR-Cas9 knockout site, the recombinant plasmid is designated 1300-NbRE02), syn The specific operating procedure is as follows: First, the pCAMBIA1300 vector was linearized. The reaction system included 1 μg pCAMBIA1300 plasmid, 5 μL 10×QuickCut Buffer, 1 μL of the corresponding rapid restriction enzyme, and ddH2O was added to bring the volume to 50 μL. The PCR reaction program was set to 37℃ for 10 min, then 80℃ for 10 min. After the reaction, the linearized vector was placed on ice for later use. Subsequently, the amplified target gene fragment was ligated to the linearized vector using a homologous recombinase (Nanjing Novizan Biotechnology Co., Ltd., catalog number C112). The ligation product was transformed into E. coli DH5α competent cells (Sangon Biotech (Shanghai) Co., Ltd.) and plated on LB solid medium containing 50 μg / mL kanamycin, and incubated upside down at 37℃ for 12 h. Single colonies were selected for colony PCR identification (using gene-specific F primers and SEQ ID NO. 28). Positive clones with amplified bands consistent with the target gene were selected and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking for 16 h. Recombinant plasmids were extracted using a plasmid extraction kit and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification.

[0061] 1300-VmE02-F (SEQ ID NO. 24): 5'-CGGGGGACGAGCTCGGTACCATGCATATCACCAGCCTCT-3'; 1300-VmE02-R (SEQ ID NO. 25): 5'-TGGTCTTTGTAGTCGTCGACAGTGCAGCTGGAGCTGCCA-3'.

[0062] 1300-GFP-F (SEQ ID NO. 26): 5'-CGGGGGACGAGCTCGGTACCATGGTGAGCAAGGGCGAGG-3'; 1300-GFP-R (SEQ ID NO. 27): 5'-ACGTCGTATGGGTAGGTACCCTTGTACAGCTCGTCCAT-3'; 1300-R (SEQ ID NO. 28): 5'-TTCTGGTGTGTGCGCAATGA-3'.

[0063] (2) Agrobacterium-mediated transient transformation The correctly sequenced recombinant plasmids (1300-VmE02, 1300-MdRE02, and 1300-NbRE02, respectively) were used. syn GV3101 Agrobacterium was transformed and treated according to the method described in Example 4, and 1300-GFP was used. The OD of Agrobacterium was adjusted. 600 The pH was adjusted to 0.6–0.8, and the mixture was allowed to stand at room temperature in the dark for 2–3 hours. The experimental setup is shown in Table 1. Following the combinations in Table 1, the Agrobacterium tumefaciens bacterial suspensions were mixed 1:1 (v / v) to obtain Agrobacterium tumefaciens mixed bacterial suspensions containing recombinant plasmid combinations. Four to five-week-old tobacco mutant plants were used. re02 Take 1 mL of Agrobacterium mixed bacterial solution and inject it into the tobacco mutant plant using a sterile syringe. re02 The underside of the leaves was then further cultured in a greenhouse.

[0064] Table 1 Experimental Setup

[0065] (3) Phenotypic results of cell necrosis Five days after injection of Agrobacterium mixed bacterial suspension, cell death phenotypes were observed visually. Compared with the positive control group... NbRE02 syn Consistency, shared expression MdRE02 It can restore the VmE02-induced cell necrosis phenotype (MdRE02+VmE02), and is co-expressed. GFP It cannot restore the VmE02-induced cell necrosis phenotype (GFP+VmE02), but only when expressed... GFP , NbRE02 syn or MdRE02 Neither of them could induce cell necrosis. Figure 4 A in the figure indicates heterogeneous replenishment. MdRE02 It can restore the cell necrosis response induced by VmE02.

[0066] (4) Detection of reactive oxygen species bursts Containing 1300-GFP and 1300-NbRE02 syn Alternatively, Agrobacterium tumefaciens solution of 1300-MdRE02 can be injected into tobacco mutant plants. re02Transient expression was carried out in leaves (the corresponding treatment groups were designated as OE-GFP and OE-NbRE02, respectively). syn The treated tobacco was cultured in a greenhouse for 48 h using OE-MdRE02. Leaf discs were punched out using a 4 mm diameter punch and gently placed into 96-well microplates containing 100 μL of ultrapure water. Eight leaf discs were used for each treatment. The discs were wrapped in aluminum foil and left to stand in the dark overnight. Luminol derivative L-012 (Wako, catalog number 120-04891), horseradish peroxidase HRP (Solepro), and VmE02 purified protein were prepared and the reaction solution was prepared as follows: 5 μL 20 mM L-012, 5 μL 2 mg / mL HRP, 100 μL 10 μM VmE02 protein, and 890 μL ultrapure water. The ultrapure water in the microplate was gently removed with a pipette, taking care to avoid damaging the leaf discs. Then, 100 μL of reaction solution was added to each well, and the plate was immediately placed in a molecular device reader. The kinetic cycle was set (total duration 1 h, kinetic interval 1 min), and the chemiluminescence fluorescence value was read. Figure 4 As shown in B, with OE-GFP ( re02 Compared to +OE-GFP, in overexpression MdRE02 Gene (OE-MdRE02, i.e.) re02 +OE-MdRE02) or NbRE02 syn (OE-NbRE02) syn ,Right now re02 +OE-NbRE02 syn Tobacco mutants re02 VmEO2 on plants can induce the release of reactive oxygen species, indicating that... MdRE02 The gene can respond to VmE02 and generate an immune response; it is a pattern recognition receptor for VmE02.

[0067] Example 7 This embodiment provides MdRE02 Genes in response V. mali Analysis of disease resistance function during infection.

[0068] (1) MdRE02 overexpression of genes MdRE02Gene overexpression was performed using *Agrobacterium* containing the recombinant plasmid 1300-MdRE02 constructed in Example 6, with *Agrobacterium* containing the recombinant plasmid 1300-GFP used as a negative control. Apple tissue culture seedlings aged 4-5 weeks were immersed in *Agrobacterium* culture containing the recombinant plasmid and placed in a sealed vacuum chamber for vacuum infiltration. Each infiltration lasted 5 minutes, and two infiltration operations were performed. After infiltration, the apple tissue culture seedlings were reinserted into MS medium and cultured for another 3 days to achieve overexpression of the target gene. Apple leaves were processed according to the method in Example 4. MdRE02 Gene expression analysis after overexpression (the corresponding treatment groups were denoted as OE-GFP and OE-MdRE02, respectively). Quantitative results showed that, compared with overexpression... GFP Compared to (OE-GFP), overexpression MdRE02 The expression level of the gene (OE-MdRE02) was upregulated approximately 19-fold after 3 days of infection into apple leaves. Figure 6 (A) in the text meets the overexpression requirement.

[0069] (2) MdRE02 Gene response V. mali Functional analysis of infection Apple tissue culture seedlings infected with the empty pK7 vector in Example 4 and silent... MdRE02 Post-genetic inoculation of apple tissue culture seedlings (pK7-MdRE02) V. mali Simultaneously, the overexpressing GFP apple tissue culture seedlings (OE-GFP) pretreated with VmE02 and the overexpressing GFP were compared with those of the apple tissue culture seedlings. MdRE02 Inoculation of genetically modified apple tissue culture seedlings (OE-MdRE02) V. mali ,analyze MdRE02 The function of genes in regulating apple rot resistance. First, activation was performed on PDA agar plates. V. mali Incubate at 25℃ in the dark for 2 days for later use. Insert apple leaves into water agar to maintain humidity. Using a sterile syringe needle, make one incision on each leaf. Introduce the activated [plants / leaves]. V. mali Using a 2 mm diameter punch, collect mycelial cakes from the edge of the colony and place them on the wound. Spray with water and keep moist. Take photos of the leaves 24–36 h after inoculation based on disease symptoms, and use ImageJ software to count the lesion area. Each treatment had at least six biological replicates, and the experiment was repeated three times. Results showed that silencing... MdRE02 The disease incidence in apple tissue culture seedlings infected with the pK7-MdRE02 gene (i.e., RNAi::MdRE02) was significantly more severe than that in apple tissue culture seedlings infected with the empty pK7 vector (CK). Figure 5 In the A section, the lesion area increased by approximately 36% compared to apple tissue culture seedlings infected with the empty pK7 vector. Figure 5B in the figure; this percentage is the relative increase rate of area, calculated using the lesion area of ​​apple tissue culture leaves infected with the empty pK7 vector as a control; while overexpression MdRE02 Apple tissue culture seedlings pretreated with VmE02 after gene expression (OE-MdRE02, i.e., OE-MdRE02+VmE02) and expression GFP Compared to apple tissue culture seedlings (OE-GFP, i.e., OE-GFP+VmE02), apple tissue culture seedlings showed a significant reduction in leaf disease. Figure 6 In B), the lesion area was significantly smaller compared to the control ( Figure 6 (C in the middle).

[0070] Example 8 This embodiment provides a tobacco mutant. re02 overexpression MdRE02 Analysis of disease resistance function after gene expression.

[0071] Take tobacco mutant plants that are 4-5 weeks old re02 Agrobacterium bacterial suspension containing recombinant plasmids 1300-MdRE02 or 1300-GFP was injected into the abaxial surface of tobacco leaves using a 1 mL sterile syringe (without needle). The mixture was then cultured in a greenhouse for 48 h (the corresponding treatment groups were designated OE-GFP and OE-MdRE02, respectively). *Sclerotinia sclerotiorum* was activated in PDA medium for later use. Fresh *Sclerotinia sclerotiorum* mycelial cakes were inoculated onto the abaxial surface of detached tobacco leaves using a 2 mm punch and cultured at 25°C. The size of lesions was observed and photographed after 24–36 h. The lesion area was statistically analyzed using ImageJ software. Simultaneously, leaf samples after 48 h of transient expression were placed in liquid nitrogen for Western blotting experiments to analyze expression levels. The results showed that compared with overexpression… GFP Compared to (OE-GFP), in tobacco mutants re02 Intermittent overexpression MdRE02 The gene (OE-MdRE02) can enhance its resistance to Sclerotinia sclerotiorum. Figure 7 ).

[0072] Example 9 This embodiment provides heterologous expression in rapeseed. MdRE02 Analysis of disease resistance function after gene expression.

[0073] Healthy, appropriately sized rapeseed leaves were selected. Agrobacterium tumefaciens suspension carrying the 1300-MdRE02 or 1300-GFP plasmids was injected transiently onto the underside of the leaves using a 1 mL sterile syringe (the corresponding treatment groups were designated OE-GFP and OE-MdRE02, respectively). The treated rapeseed leaves were then cultured in a greenhouse for 72 h. The infected rapeseed leaves were then placed in water agar for humidification. A wound was made on each leaf using a sterile syringe needle. Activated Sclerotinia sclerotiorum was used to collect mycelial cakes from the colony edge using a 2 mm diameter punch, and the mycelial cakes were placed on the wounds. The area was then sprayed with water and kept moist. The size of the lesions was observed and photographed after 24–36 h. The lesion area was statistically analyzed using ImageJ software. Simultaneously, leaf samples from 72 h after transient expression were placed in liquid nitrogen for Western blotting experiments to analyze the expression status. The results showed that compared with overexpression… GFP Compared to (OE-GFP), transient overexpression in rapeseed MdRE02 The gene (OE-MdRE02) can enhance its resistance to Sclerotinia sclerotiorum. Figure 8 ),show MdRE02 Genes can be transferred across species to rapeseed to improve its resistance to Sclerotinia sclerotiorum.

[0074] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Apple pattern recognition receptor gene MdRE02 Its application in regulating plant disease resistance is characterized by, The apple pattern recognition receptor gene MdRE02 The nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2; The regulation of plant disease resistance includes: overexpressing the apple pattern recognition receptor gene. MdRE02 To improve plant disease resistance; to silence the apple pattern recognition receptor gene. MdRE02 This reduces the plant's disease resistance.

2. The apple pattern recognition receptor gene according to claim 1 MdRE02 Its application in regulating plant disease resistance is characterized by, The improvement of plant disease resistance refers to enhancing the resistance of apples to apple tree rot pathogens. Valsa mali Resistance to Sclerotinia sclerotiorum, and / or improved resistance of tobacco and rapeseed to Sclerotinia sclerotiorum. Sclerotinia sclerotiorum Resistance.

3. The apple pattern recognition receptor gene according to claim 1 MdRE02 Its application in regulating plant disease resistance is characterized by, The reduction in plant disease resistance is achieved by downregulating the expression of VmE02-induced apple defense-related genes. The Apple defense-related genes include: MdPR1 , MdPR2 , MdPR5 , MdCYP81F2 .

4. Apple pattern recognition receptor gene MdRE02 Its application in breeding disease-resistant plant varieties is characterized by... The apple pattern recognition receptor gene MdRE02 The nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2; The method for cultivating disease-resistant plant varieties involves overexpressing the apple pattern recognition receptor gene in plants. MdRE02 Thus, disease-resistant plant varieties were obtained.

5. The apple pattern recognition receptor gene according to claim 4 MdRE02 Its application in breeding disease-resistant plant varieties is characterized by... The plants mentioned include: apple, tobacco and rapeseed.

6. The apple pattern recognition receptor gene according to claim 4 MdRE02 Its application in breeding disease-resistant plant varieties is characterized by... The plant disease-resistant varieties include those resistant to apple tree rot pathogen. Valsa mali Apple varieties, resistance to sclerotinia stem rot Sclerotinia sclerotiorum Tobacco and / or rapeseed varieties.

7. A method for improving plant disease resistance, characterized in that, include: Constructing a gene containing apple pattern recognition receptor MdRE02 A transient expression vector was introduced into plant cells or tissues to activate the apple pattern recognition receptor gene. MdRE02 Transient overexpression to enhance plant disease resistance; The apple pattern recognition receptor gene MdRE02 The nucleotide sequence is shown in SEQ ID NO.

1.

8. The method for improving plant disease resistance according to claim 7, characterized in that, The disease resistance includes: resistance of apples to apple tree rot pathogens. Valsa mali Resistance to Sclerotinia sclerotiorum, and / or resistance of tobacco and rapeseed to Sclerotinia sclerotiorum. Sclerotinia sclerotiorum Resistance.

9. A method for cultivating disease-resistant plant varieties, characterized in that, include: Constructing a gene containing apple pattern recognition receptor MdRE02 A transient expression vector was introduced into plant cells or tissues to activate the apple pattern recognition receptor gene. MdRE02 Transient overexpression yields disease-resistant plant varieties; The apple pattern recognition receptor gene MdRE02 The nucleotide sequence is shown in SEQ ID NO.

1.

10. The method for cultivating disease-resistant plant varieties according to claim 9, characterized in that, The plant disease-resistant varieties include those resistant to apple tree rot pathogen. Valsa mali Apple varieties, resistance to sclerotinia stem rot Sclerotinia sclerotiorum Tobacco and / or rapeseed varieties.

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