Use of rknr3 gene in improving plant resistance to root-knot nematode and a method for improving tomato resistance to root-knot nematode

Abstract

This invention discloses RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes and a method for improving tomato resistance to root-knot nematodes. The inventors of this invention previously discovered through extensive experiments and data analysis that tomato treatment with *Trichoderma* significantly increases its resistance to root-knot nematodes, and... RKNR3 Significant changes in gene expression levels suggest that this gene may play an important role in tomato root-knot nematode resistance. Furthermore, this invention utilizes virus-induced gene silencing technology to construct a silent gene... RKNR3 The VIGS vector of the gene was transformed into tomatoes and successfully inhibited. RKNR3 Silent plants exhibited significantly increased resistance to root-knot nematodes compared to the control group, indicating that this long non-coding RNA participates in the tomato's resistance to root-knot nematodes. This invention provides a theoretical basis and resistance resources for accelerating tomato disease resistance breeding and enriching the diversity of disease-resistant varieties.

Description

Technical Field

[0001] This invention relates to RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes and a method for improving tomato resistance to root-knot nematodes belong to the field of plant genetic engineering technology. Background Technology

[0002] Root-knot nematodes (RKN); Meloidogyne Root-knot nematodes (spp.) are a class of globally distributed, obligate plant parasitic nematodes, named for the characteristic root knots they cause infecting plant roots. Root-knot nematodes are among the most significant diseases causing damage to important agricultural crops worldwide, resulting in annual economic losses exceeding $100 billion in global agricultural production. More than 80 species of root-knot nematodes have been discovered and reported globally, exhibiting a wide host range and capable of harming over 3,000 plant species. Surveys indicate that the southern root-knot nematode (S. spp.) is a notable example. M. incognita ), Northern root-knot nematodes ( M. hapla ), peanut root-knot nematode ( M. arenaria Javan root-knot nematodes ( M. javanica These are four common nematodes that cause crop losses in my country.

[0003] tomato( Solanum lycopersicum Tomato (L.) is an annual or perennial herbaceous plant belonging to the genus *Solanum* of the family Solanaceae. Also known as tomato or foreign persimmon, it is not only the most widely cultivated and consumed vegetable crop worldwide, but also an important model organism in plant biology research. my country is the world's largest tomato producer, with an output exceeding 66 million tons in 2022, accounting for more than 35% of global production. Major producing areas include Shandong, Xinjiang, Henan, and Hebei provinces. In recent years, the area under protected cultivation of tomatoes has been increasing annually. Continuous cropping has led to the accumulation of soil pathogens in protected environments, resulting in a growing area affected by soil-borne diseases. Because tomatoes are highly susceptible to root-knot nematodes, their quality and yield are severely impacted, causing considerable economic losses.

[0004] The problem of root-knot nematodes damaging greenhouse vegetables is becoming increasingly serious. They can also cause significant yield reductions by spreading and inducing fungal and bacterial diseases. Southern root-knot nematodes, as the dominant species with the largest affected area and the most severe damage, seriously restrict tomato yield and quality. Although integrated pest management (agricultural operations, physical control, biological control, and chemical control) is widely used in greenhouse production, it has not fundamentally stopped the damage caused by root-knot nematodes and may even cause environmental damage to some extent. Therefore, accelerating research on root-knot nematode resistance mechanisms and the discovery of resistance genes is crucial for the molecular improvement of tomato nematode-resistant varieties and has significant scientific and practical implications.

[0005] Chinese invention patent CN11071423B, published on November 17, 2020, discloses a tomato CTI1 The application of genes in enhancing plant resistance to root-knot nematodes was disclosed, specifically demonstrating that infection with the southern root-knot nematode can rapidly induce nematode activity in tomato roots. CTI1 Gene expression: The cti1 mutant increased susceptibility to root-knot nematodes, while JA content and the expression of JA-related genes were also suppressed. CTI1 Overexpression has the opposite effect. These results indicate that... CTI1 It participates in the regulation of RKN-induced JA biosynthesis and signal transduction pathways.

[0006] Chinese invention patent CN112322651B, published on April 1, 2022, discloses a tomato autophagy gene. ATGs In its application to improving plant resistance to root-knot nematodes, the study specifically disclosed that infection with the southern root-knot nematode can rapidly induce the expression of autophagy genes in tomato roots, and that mutants increase susceptibility to root-knot nematodes. ATG10 Conversely, overexpression, i.e., autophagy, plays a role in tomato root-knot nematode resistance.

[0007] Chinese invention patent application document with publication date of October 20, 2023 and publication number CN116903718A has been published. SlWRKY80 The application of this technology in improving resistance to root-knot nematode disease in tomatoes specifically addresses the current situation of a lack of available resistance sources for the Mi family of major resistance genes against root-knot nematodes in tomatoes. SlWRKY80 It can participate in the tomato's resistance to root-knot nematodes at the transcriptional regulatory level, and overexpression SlWRKY80 This can significantly improve the resistance of tomato plants to pathogens.

[0008] Chinese invention patent CN115976099B, published on August 8, 2025, discloses the knockout process. JAMs Genes were used to enhance tomato resistance to southern root-knot nematodes. Jams mutant plants were constructed using CRISPR / CAS9 technology. Four weeks after inoculation of wild-type tomato (WT) and Jams mutant plants with nematodes, the number of root knots in the Jams mutant plants was found to be less than in wild-type tomatoes, and the root knots in wild-type tomatoes were also more prominent than those in the Jams mutant plants. This indicates that the Jams mutant enhances tomato resistance to root-knot nematodes. JAMs Negative regulation of resistance to southern root-knot nematode in tomatoes.

[0009] In summary, existing technologies have identified genes related to autophagy and ubiquitination that can enhance the resistance of tomatoes to root-knot nematodes, but there are no reports on whether long non-coding RNAs can enhance the resistance of tomatoes to root-knot nematodes. Summary of the Invention

[0010] The first objective of this invention is to provide RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes aims to address the limited mining of long non-coding RNAs involved in regulating plant resistance to root-knot nematodes in existing technologies.

[0011] The second objective of this invention is to provide a method for improving the resistance of tomatoes to root-knot nematodes, thereby addressing the problem in the prior art that the resistance of tomatoes to root-knot nematodes needs to be improved.

[0012] To achieve the above objectives, in this invention RKNR3 The technical approach used in applying genes to improve plant resistance to root-knot nematodes is as follows: RKNR3 The application of genes in improving plant resistance to root-knot nematodes, the aforementioned RKNR3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; In plants, it inhibits... RKNR3 Gene expression enhances a plant's resistance to root-knot nematodes.

[0013] The beneficial effects of the above technical solution are as follows: This invention RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes is a pioneering invention. Through extensive experiments and data analysis, the inventors of this invention discovered that tomato plants treated with Trichoderma exhibited significantly increased resistance to root-knot nematodes, and... RKNR3 Significant changes in gene expression levels suggest that this gene may play an important role in tomato root-knot nematode resistance. Furthermore, this invention utilizes virus-induced gene silencing (VIGS) technology to construct a silent gene... RKNR3 The VIGS vector of the gene was transformed into tomatoes and successfully inhibited. RKNR3 Silent plants exhibited significantly increased resistance to root-knot nematodes compared to the control group, indicating that this long non-coding RNA participates in the process of tomato resistance to root-knot nematodes. This provides a theoretical basis and resistance resources for accelerating the breeding process of tomato disease resistance and enriching the diversity of disease-resistant varieties.

[0014] As a further improvement, the suppression is for constructing RKNR3 Gene silencing vectors were used to transform plants, and plants with increased resistance to root-knot nematodes were screened and identified.

[0015] As a further improvement, the aforementioned RKNR3 Gene silencing vectors RKNR3 The specific sequence in a gene is the guide sequence.

[0016] As a further improvement, the nucleotide sequence of the guide sequence is shown in SEQ ID NO.2.

[0017] As a further improvement, the plant is a tomato.

[0018] As a further improvement, the root-knot nematode is the southern root-knot nematode.

[0019] To achieve the above objectives, the technical solution adopted in this invention for improving the resistance of tomatoes to root-knot nematodes is as follows: A method for improving the resistance of tomatoes to root-knot nematodes includes the following steps: (1) Inhibits the growth of tomato RKNR3 Gene expression levels, obtained RKNR3 Tomato plants with reduced gene expression; RKNR3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; (2) Inoculate the Trichoderma as described in step (1) RKNR3 On tomato plants with reduced gene expression.

[0020] The beneficial effects of the above technical solution are as follows: This invention provides a method for achieving silent... RKNR3 A method for genetically enhancing the resistance of Trichoderma acicularis to tomato root-knot nematodes. This invention utilizes VIGS technology to construct TRV- RKNR3 Silent plants, and in TRV-empty (control) and TRV- RKNR3 After inoculating silent plants with root-knot nematodes for 45 days, TRV- was found to be present. RKNR3 The number of root knots in silent plants was significantly reduced compared to tomato plants in the control group; after treatment with Trichoderma acicularis, TRV- RKNR3 The number of root knots in the silent plants was also significantly reduced compared to the control group of tomato plants.

[0021] As a further improvement, in step (2) the following steps are to be implemented. RKNR3 The inoculation was performed on tomato plants with reduced gene expression when they grew to the stage of four leaves and one bud; the Trichoderma was Trichoderma echinosporum.

[0022] As a further improvement, the inoculation involves irrigating the tomato plants with a suspension of Trichoderma spores, thereby establishing Trichoderma on the tomato plants.

[0023] As a further improvement, the concentration of the Trichoderma spore suspension is (1~3)×10⁻⁶. 8 10-15 mL of the Trichoderma spore suspension per tomato plant. Attached Figure Description

[0024] Figure 1 Silence in Embodiment 1 of the present invention RKNR3 Genetic sequencing results of E. coli culture; Figure 2 In Embodiment 1 of the present inventionRKNR3 Gene silencing efficiency test results; Figure 3 The TRV blank control group and TRV- in Example 2 of this invention RKNR3 Root knot phenotype of gene-silenced group plants 45 days after inoculation with root-knot nematodes (where the red-stained part is the root knot); Figure 4 The TRV blank control group and TRV- in Example 2 of this invention RKNR3 Statistical results of root knot number, root knot index and control effect 45 days after inoculation of root-knot nematodes in gene-silenced group plants; Figure 5 The following are the times after root-knot nematode infection of Cooperative 903 tomato plants in Example 3 of this invention: 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h. RKNR3 Gene expression. Detailed Implementation

[0025] Root-knot nematodes are a devastating soil-borne disease in tomato production. They are highly parasitic and reproduce rapidly. After infection, they cause root knots to form in the roots, hindering nutrient absorption. Affected plants exhibit slow growth, stunted growth, and reduced fruit production, resulting in yield reductions of 30% to 80%. In severe cases, they can even lead to premature plant death. Moreover, nematodes are prone to developing resistance to pesticides, and the damage is exacerbated in fields where crops are continuously cropped.

[0026] In terms of prevention and control, chemical control methods result in pesticide residues that pollute the environment, harm human health, and severely damage soil microbial communities; disease-resistant varieties and highly effective, low-toxicity nematode insecticides are relatively scarce. With the development of molecular biology techniques, researchers have begun to analyze the regulatory mechanisms of plant resistance to root-knot nematodes at the gene level, and have discovered several genes related to root-knot nematode resistance, such as the cyclic E3 ubiquitin ligase gene. CTI1 Genes such as autophagy-related genes provide targets for molecular marker-assisted selection, accelerating the breeding process of root-knot nematode-resistant varieties.

[0027] Given their importance in the growth and development of eukaryotes, lncRNAs have become a research hotspot in recent years. Although the exploration of plant lncRNAs is still in its early stages compared to animals, their basic functional forms and mechanisms have been understood in various model plants. Besides model plants such as Arabidopsis thaliana and rice, lncRNAs have also been identified in a variety of horticultural plants, such as apples, grapes, tomatoes, and cabbages. Horticultural plants play an important role in people's lives, and horticultural plant research is an important component of agricultural development. Research on horticultural plant lncRNAs can not only improve the regulatory mechanisms of plant lncRNAs but also promote molecular breeding of horticultural plants. Studies have shown that long non-coding RNAs (lncRNAs) play a key role in plant disease resistance through pathways such as regulating gene expression, participating in reactive oxygen species metabolism, and interacting with miRNAs.

[0028] Based on this, the present invention first provides RKNR3 The study explores the application of genes in enhancing plant resistance to root-knot nematodes, and then uses this as a basis to provide a method for improving tomato resistance to root-knot nematodes.

[0029] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0030] Unless otherwise specified, the following examples were conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or as recommended by the manufacturer's instructions.

[0031] Experimental materials: The plant materials used in this invention include: the tomato variety is Hezuo 903, provided by Shanghai Hongqiao Tianlong Seed Industry Co., Ltd.; Trichoderma echinosporum T141 was donated by Professor Xu Tong of Zhejiang University and purified and preserved in our laboratory.

[0032] The specific procedures for preparing the Trichoderma spore suspension in the following examples are as follows: (1) The Trichoderma spp. used was Trichoderma spp. T141. The Trichoderma spp. was made into a cake using a sterile punch in a clean bench, then inoculated onto a PDA plate for activation and cultured at 28°C for 7 days. (2) After 7 days, Trichoderma was scraped and filtered through 8 layers of sterile gauze to obtain a suspension of Trichoderma spores; (3) Calculate the spore concentration under a microscope using a hemocytometer, dilute to 1×10⁸ spores / mL, and inoculate and use within 24 hours if possible.

[0033] The specific procedures for culturing root-knot nematodes in the following examples are as follows: (1) The root-knot nematode used was the southern root-knot nematode. The nematode was bred in the substrate soil of ordinary cultivated tomatoes in the greenhouse, and the room temperature was maintained at 22-26℃. (2) After rinsing the harvested root knots with tap water, pick out the egg sacs; (3) Transfer the egg suspension into a cell culture plate and place it in a 28°C constant temperature incubator, keeping it moist during the process; (4) After 2 to 3 days, J2 stage nematodes are obtained. To avoid the death of the nematodes, they should be inoculated and used within 24 hours.

[0034] The following examples illustrate the method for observing the number of root knots (using acid fuchsin staining for observing root phenotypes in root-knot nematode infection): (1) After rinsing the tomato roots infected by root-knot nematodes with tap water, use a sodium hypochlorite solution with an effective chlorine content of more than 1.5% to bleach the roots for 5 minutes. (2) Remove the root system, rinse it repeatedly with tap water for about 2 minutes to remove the residual sodium hypochlorite solution, and then soak it in distilled water for 15 minutes. (3) Remove the root system, dry the root system with absorbent paper, and stain the tomato root system with 3.5% acid fuchsin solution (0.35 g acid fuchsin dissolved in 25 mL acetic acid, and distilled water added to make up to 100 mL) at room temperature for about 1 hour. (4) Remove the root system, rinse the root system with tap water to remove excess magenta liquid on the surface, and decolorize it overnight in distilled water; (5) Examine the nematode invasion under a stereomicroscope, take photos and count the number of root knots.

[0035] This invention RKNR3 Application of genes in enhancing plant resistance to root-knot nematodes; a specific embodiment of a method for improving tomato resistance to root-knot nematodes: Example 1 RKNR3 Construction and detection of gene-silenced tomato plants This embodiment constructs a tomato VIGS silent TRV- RKNR3 The vector was then transferred into tomatoes to construct a... RKNR3 The specific steps for creating gene-silenced tomato plants are as follows: 1. Total RNA extraction and cDNA synthesis from tomatoes Total RNA was extracted from young tomato root tips using the FreeZol Reagent R711 method by Novizan, and the first strand of cDNA was synthesized by reverse transcription using the M5 Hiper lncRNA cDNA Synthesis Kit gDNA remover from Polymer.

[0036] 2. Gene cloning and construction of engineered Agrobacterium tumefaciens bacteria Gene-specific primers were designed, and the silencing target fragment was obtained from the Tomato Database VIGS Tool website (SGN-VIGS). Specific primers were designed using Primer 5, and the silencing gene fragment with a length of 180-250 bp was amplified by PCR (avoiding conserved regions to prevent off-target silencing). Specifically, the nucleotide sequence of the silencing target fragment used is shown in SEQ ID NO.2.

[0037] The specific primer sequences are as follows: TRV-lnc-F: 5'-gtgagtaaggttaccgaattcCCACATCTACAGTGAGCGGGAAC-3' (shown in SEQ IDNO.3); TRV-lnc-R: 5'-gggacatgcccgggcctcgagTGCTCTGTACGTTGTTGAACGGTAT-3' (shown in SEQ ID NO.4).

[0038] Recovery using high-fidelity enzyme cloning RKNR3 Gene silencing fragment, PCR reaction system (20 μL): 1 μL high-fidelity enzyme, 1 μL each of forward and reverse primers, 1 μL cDNA template, 10 μL 2×PCR Mix, 7 μL ddH2O; reaction program: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, Tm for 30 s, 72℃ for 30 s, 28-35 cycles; maintenance at 4℃.

[0039] Using restriction endonucleases EcoR I and Xho The pTRV2 vector was double-digested with enzymes at 37°C for 5-15 min, and the linearized pTRV2 vector was recovered. The gene cloning products were recovered via gel electrophoresis and mixed at a fragment:vector molar ratio of 3:1 / 3:2. The mixture was then purified using Novizan's ClonExpress. ® II. One-Step Cloning Kit ligase: Ligate at 37°C for 30 min to construct recombinant vector TRV- RKNR3 .

[0040] The ligation product was transformed into *E. coli* DH5α competent cells, cultured overnight at 37°C in LB solid medium containing kanamycin, and single colonies were picked and used... RKNR3 During annealing, the upstream primer (i.e., the TRV-lnc-F primer mentioned above) and the universal TRV2 downstream primer (i.e., the TRV-R primer shown in SEQ ID NO.6 below) were used for bacterial culture PCR. Positive single clones were screened and identified by sequencing. Sequencing results are shown below. Figure 1 Sequencing results showed that the sequence alignment was successful.

[0041] The successfully sequenced TRV- RKNR3 Escherichia coli plasmid was transformed into Agrobacterium tumefaciens GV3101 competent cells, which were then plated on LB agar plates containing kanamycin and rifampin (Rif) and cultured at 28°C for 48 h. The positive strain was verified by PCR.

[0042] The primer sequences used are as follows: TRV-F:5'-GGTCAAGGTACGTAGTAGAG-3' (shown in SEQ ID NO.5); TRV-R:5'-CGAGAATGTCAATCTCGTAGG-3' (shown in SEQ ID NO.6).

[0043] Plasmids containing ptrv1 and TRV- RKNR3 The bacterial culture was inoculated into 20 mL of LB resistant liquid medium and cultured at 28℃ and 250 r / min for 2 days with shaking. 20 μL of the turbid bacterial culture was then transferred to 50 mL of LB resistant liquid medium and cultured for another day.

[0044] Collect bacterial cells by centrifugation at 4000 r / min for 10 min, resuspend the cells in infection buffer (10 mmol / L MgCl2, 10 mmol / L LMES, 20 μmol / L AS, pH 5.6), and adjust OD. 600 =0.8-1.2, incubate at room temperature in the dark for 3 h; mix the bacterial culture containing pTRV1 plasmid with TRV- RKNR3 The bacterial suspensions were mixed at a 1:1 volume ratio to obtain the VIGS silencing infection solution; simultaneously, TRV-empty (empty vector) was set as a negative control, TRV- PDS As a silent contrast.

[0045] 3. Construction of VIGS Silent Plants in Tomato Seedlings Select tomato seedlings with two fully expanded cotyledons that are growing in the same direction. Use a disposable sterile syringe to draw up the infection solution and slowly inject it from the back of the cotyledons until the infection solution seeps out from the edge of the leaf.

[0046] Infected tomato seedlings were cultured in an incubator with 16 h of light / 8 h of darkness and a temperature of 22℃ / 19℃. They were then transferred to an artificial climate chamber for normal culture (25℃, 14 h of light / 10 h of darkness) for subsequent experiments.

[0047] 4. RKNR3 Determination of gene silencing efficiency qRT-PCR was performed using the PolymerM5 Hiper lncRNA Quantitative Detection Kit MF881-01.

[0048] To contain TRV- PDS When tomato seedlings infected with plasmid Agrobacterium exhibit an albino phenotype, the effect of TRV- RKNR3 Plant leaves were sampled, total RNA was extracted and reverse transcribed into cDNA for further processing. RKNR3 Detection of silent efficiency.

[0049] Detection RKNR3The primer sequences used for the gene are as follows: F:5'-GAAGTTCAAGATGACTTTCAC-3' (shown in SEQ ID NO.7); R: 5'-AAGTCTCCTGCTTAATACA-3' (shown in SEQ ID NO. 8).

[0050] The primer sequences used for the internal reference gene are as follows: actin-F:5'-GTCCTCTTCCAGCCATCCAT-3' (shown in SEQ ID NO.9); actin-R:5'-ACCACTGAGCACAATGTTACCG-3' (shown in SEQ ID NO. 10).

[0051] Real-time quantitative PCR (qRT-PCR) was performed using the ABI Prism 7500 real-time quantitative PCR system, and gene expression analysis was performed using the PolymerMidea M5 Hiper lncRNA fluorescence quantitative detection kit MF881-01.

[0052] A 20 μL reaction mixture contained 10 μL of 2×LncR SYBR QPCR Mixture, 0.4 μL of forward and reverse primers (10 μM), 2.5 μL of cDNA template, and 6.7 μL of ddH2O. PCR conditions were: 95℃ for 30 s; 95℃ denaturation for 10 s, 60℃ annealing for 34 s, for 35-45 cycles. Fluorescence data were collected at the end of the extension phase of each cycle. Tomato actin gene was used as an internal control. Relative gene expression levels were calculated using a 22... -∆∆CT The calculations were performed using methods that included three replicates of each experiment.

[0053] RKNR3 See gene silencing efficiency results Figure 2 Using VIGS silent technology, a successful construction was achieved. RKNR3 Silencing vector, obtaining silent strains. RKNR3 Silent plants showed a 65.37% decrease in gene expression compared to the empty vector, indicating that... RKNR3 The gene was successfully silenced.

[0054] Example 2 RKNR3 Identification of gene function This embodiment uses the tomato TRV- constructed in Example 1. RKNR3 Both silent plants and TRV-empty empty vector plants were divided into four treatment groups: control, Trichoderma (T), root-knot nematode (R), and Trichoderma plus root-knot nematode (T+R) to validate the results. RKNR3 The function of genes, specifically implemented as follows: 1. For tomato TRV- RKNR3 Silent plants and TRV-empty empty plants were inoculated with Trichoderma echinococcus T141, and the specific procedures were as follows: When the tomato plants reach the stage of four leaves and one bud, inoculate them with Trichoderma through root irrigation using T and T+R methods, applying approximately 10 mL per plant at a concentration of 1×10⁻⁶. 8 Trichoderma spore suspension per mL, with normal watering during the period.

[0055] The plants were cultivated in nutrient pots filled with high-temperature sterilized substrate soil. The plant growth conditions were 14 h light / 10 h dark, and cultured at 25℃ for 7 days to allow Trichoderma acicularis to colonize the tomato roots.

[0056] 2. Regarding TRV- RKNR3 Silent plants and TRV-empty plants were inoculated with root-knot nematodes, and the specific procedures were as follows: Seven days after inoculation with *Trichoderma hygroscopicum*, R and T+R plants were treated with nematode inoculation, with approximately 800 J2-stage nematodes inoculated per plant. Normal watering was maintained during this period. Nematode treatment was carried out over 4-6 weeks. Finally, the tomato roots were rinsed, and root scans were performed using an Epson Perfection V800 Photo root scanner to calculate the number of root knots, root knot index, and control efficacy. See the root knot phenotype diagram below. Figure 3 The number of root knots, root knot index, and control effect are shown in [reference needed]. Figure 4 .

[0057] As shown in the figure, compared with TRV (control group), TRV- RKNR3 The number of root knots and the root knot index were significantly reduced in silent plants. TRV -RKNR3 In silent plants, the number of root knots and the root knot index in the T+R group were reduced by 59.73% and 60.48%, respectively, compared to the R group. In TRV-free plants, the control efficacy against Trichoderma was 48%, while in TRV... -RKNR3 A significant increase in control efficacy was observed in silent plants, at approximately 60%.

[0058] The above results fully demonstrate that inhibiting RKNR3 Gene expression significantly enhances tomato plants' resistance to southern root-knot nematodes. Meanwhile... RKNR3 Inoculating gene-silenced tomato plants with Trichoderma can further enhance the tomato's resistance to southern root-knot nematodes.

[0059] Example 3 RKNR3 Function of genes in Trichoderma-induced resistance response In this embodiment, the nematode infection of plants was detected at 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h. RKNR3The gene expression levels in Tomato Cooperation 903 were determined through the following specific procedures: Four treatment groups were set up for tomatoes: control, Trichoderma (T), root-knot nematode (R), and Trichoderma plus root-knot nematode (T+R). When the tomatoes reached the four-leaf stage, the plants were inoculated with Trichoderma, with each plant inoculated with approximately 10 ml of a 1×10⁻⁶ ml solution. 8 After 7 days, approximately 800 J2-stage nematodes were inoculated into each plant using a Trichoderma spore suspension at a concentration of 1 / mL. Root samples were then collected for qRT-PCR detection. RKNR3 The expression level was determined. The qRT-PCR detection was as described in Example 1 and will not be repeated here.

[0060] Results of expression levels at different times are shown in Figure 5 After inoculation with root-knot nematodes, RKNR3 Gene expression was significantly upregulated at 24 h and 36 h, and the highest expression level was observed in the treatment with Trichoderma pretreatment followed by nematode inoculation, indicating that... RKNR3 Genes play an important role in the resistance response induced by Trichoderma.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes is characterized by: The RKNR3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; In plants, it inhibits... RKNR3 Gene expression enhances a plant's resistance to root-knot nematodes.

2. As described in claim 1 RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes is characterized by: The suppression is for construction RKNR3 Gene silencing vectors were used to transform plants, and plants with increased resistance to root-knot nematodes were screened and identified.

3. As described in claim 2 RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes is characterized by: The RKNR3 Gene silencing vectors RKNR3 The specific sequence in a gene is the guide sequence.

4. The method according to claim 3 RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes is characterized by: The nucleotide sequence of the guide sequence is shown in SEQ ID NO.

2.

5. The method according to any one of claims 1 to 4 RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes is characterized by: The plant in question is a tomato.

6. The method according to claim 5 RKNR3 The application of genes in enhancing plant resistance to root-knot nematodes is characterized by: The root-knot nematode is the southern root-knot nematode.

7. A method for improving the resistance of tomatoes to root-knot nematodes, characterized in that: Includes the following steps: (1) Inhibits the growth of tomato RKNR3 Gene expression levels, obtained RKNR3 Tomato plants with reduced gene expression; RKNR3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; (2) Inoculate the Trichoderma as described in step (1) RKNR3 On tomato plants with reduced gene expression.

8. The method for improving the resistance of tomatoes to root-knot nematodes according to claim 7, characterized in that: In step (2) RKNR3 The inoculation was performed on tomato plants with reduced gene expression when they grew to the stage of four leaves and one bud; the Trichoderma was Trichoderma echinosporum.

9. The method for improving the resistance of tomatoes to root-knot nematodes according to claim 8, characterized in that: The inoculation involves irrigating the tomato plants with a suspension of Trichoderma spores, allowing the Trichoderma to colonize the tomato plants.

10. The method for improving the resistance of tomatoes to root-knot nematodes according to claim 9, characterized in that: The concentration of the Trichoderma spore suspension was (1~3)×10 8 10-15 mL of the Trichoderma spore suspension per tomato plant.

Images