MtLOX24 gene and application thereof in improving resistance to diseases and pests of alfalfa

Abstract

The invention discloses an MtLOX24 gene and application of the MtLOX24 gene to improvement of alfalfa disease and pest resistance, a lipoxygenase gene MtLOX24 is cloned from medicago truncatula, an Agrobacterium tumefaciens-mediated method is adopted to obtain an MtLOX24 gene overexpressed medicago sativa strain, and wild type medicago sativa with overexpressed MtLOX24 is adopted as a material to carry out insect feeding and pathogenic bacterium infection experiments. By overexpressing the MtLOX24 gene, the resistance of medicago sativa to plant diseases and insect pests is remarkably improved, and compared with a wild plant, when the plant with the overexpressed MtLOX24 gene is fed by insects or infected by pathogenic bacteria, a defense mechanism can be more effectively activated, and the damage area and the weight loss are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a MtLOX24 gene and an application thereof in improving the disease and insect pest resistance of alfalfa. Background Art

[0002] Alfalfa (Medicago sativa) is one of the most important forages in my country. Its yield, quality and palatability are affected by insects and pathogens. The signal response pathway based on jasmonic acid (JA) plays an important role in the defense of plants against pests and diseases. Lipoxygenase (LOX) is a multifunctional enzyme that is mainly related to the growth and development of plant organs, the response to biotic and abiotic stresses, and the production of flavor substances. LOX can mediate the regulation of defense responses such as insects, pathogens, and trauma in plants by relying on JA signal transduction. Medicago truncatula and alfalfa are genetically similar and are often used as model plants to explore the molecular mechanisms of alfalfa genes. In this study, LOX family genes in Medicago truncatula and Medicago sativa were identified, and the expression patterns of their family members under methyl jasmonate (MeJA) treatment were analyzed. A MeJA-induced LOX gene (MtLOX24) was screened out from Medicago truncatula, and its function in the response process of pests and diseases was mainly analyzed. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a MtLOX24 gene and its application in improving alfalfa's resistance to diseases and insect pests.

[0004] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a Medicago truncatula MtLOX24 gene, the nucleotide sequence of the MtLOX24 gene is shown in SEQ ID NO.1.

[0005] Furthermore, the amino acid sequence of the protein encoded by the MtLOX24 gene is shown in SEQ ID NO.2.

[0006] In a second aspect, the present invention provides a transgenic vector containing the Medicago truncatula MtLOX24 gene.

[0007] In a third aspect, the present invention provides a recombinant strain or a recombinant cell containing the Medicago truncatula MtLOX24 gene.

[0008] In a fourth aspect, the present invention provides the use of overexpression of the Medicago truncatula MtLOX24 gene in improving plant disease and insect pest resistance.

[0009] Furthermore, the plants include Medicago truncatula and Medicago sativa.

[0010] Further, proceed as follows: S1. A lipoxygenase gene MtLOX24 was cloned from Medicago truncatula; S2, obtaining MtLOX24 gene overexpressing alfalfa strains by Agrobacterium-mediated method; S3. Insect feeding and pathogen infection experiments were conducted using wild-type and MtLOX24-overexpressing alfalfa as materials.

[0011] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects: The MtLOX24 gene provided by the present invention and its application in improving the resistance of alfalfa to diseases and insect pests significantly improves the resistance of alfalfa to diseases and insect pests by overexpressing the MtLOX24 gene. Compared with wild-type plants, plants overexpressing the MtLOX24 gene can more effectively activate defense mechanisms and reduce damage areas and weight losses when being fed by insects and infected by pathogens. The MtLOX24 gene can mediate the regulation of defense responses of insects, pathogens, etc. in plants by relying on the signal transduction of jasmonic acid (JA). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0013] Figure 1 It is the genetic transformation process of alfalfa; Figure 2 It is a schematic diagram of analyzing the expression level of alfalfa overexpressing MtLOX24 strains using qRT-PCR; Figure 3 This is a schematic diagram of the identification of resistance of MtLOX24-overexpressing alfalfa to Spodoptera litura; Figure 4 This is a schematic diagram of the resistance identification of MtLOX24-overexpressing alfalfa infected by Pst DC3000. DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0015] Example 1. Experimental Materials 1.1 Plant materials: The Medicago truncatula (R108, wild type) involved was preserved by our laboratory. The wild type alfalfa material was the variety “Zhongmu No. 1” bred in our laboratory, provided by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. The alfalfa overexpression strain was obtained by genetic transformation and grown in a plant incubator or artificial climate chamber (24 ℃ light / 20 ℃ dark, 16 h light / 8 h dark, relative humidity 70%). The plant materials are summarized in Table 1.

[0016] Table 1 Summary of plant materials 1.2 Culture medium formulation LM medium (LB modified medium): tryptone 10 g / L, yeast extract 6 g / L, KH2PO4 1.5 g / L, NaCl 0.6 g / L, MgSO4 0.35 g / L, agar powder 15 g / L, pH adjusted to 7.0.

[0017] Potato dextrose agar medium (PDA medium): potato extract powder 6 g / L, glucose 20 g / L, agar powder 20 g / L.

[0018] Potato dextrose broth (PDB medium): potato extract 6 g / L, glucose 20 g / L.

[0019] Murashige & Skoog culture medium (Phytotech) was purchased from Beijing Ximeijie Company.

[0020] 1.3 Test strains Fusarium was provided by the College of Grassland Science, Gansu Agricultural University, and Pst DC3000 was provided by the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0021] 1.4 Test insects Spodoptera litura breeding: The Spodoptera litura species were purchased from Keyun Biological Company and cultured for multiple generations using the indoor artificial culture method at a temperature of (25±2)°C, a relative humidity of 30%-40%, a light period of 14 h, and a dark period of 10 h. The larvae of Spodoptera litura acclimated indoors were used as test insects.

[0022] 2. MtLOX24 gene cloning and genetic transformation process 2.1 Construction of MtLOX24 overexpression vector (1) First, primers were designed based on the CDS sequence of the MtLOX24 (MTR_8g018690) gene identified in the NCBI database. Medicago truncatula cDNA was used as a template and then amplified using a high-fidelity enzyme. The PCR reaction system was: 95 ºC for 5 min, 95 ºC for 30 s, 53 ºC for 30 s, 72 ºC for 3 min, 72 ºC for 5 min, and 4 ºC∞.

[0023] (2) Afterwards, the amplified product is excised from the gel and recovered. This process uses a purification kit. The concentration of the recovered product is measured using a UV spectrophotometer.

[0024] (3) Then, the cloning vector pEASY-T5 and the purified MtLOX24 gene product were ligated for transformation. Specifically, the cloning vector pEASY-T5 and the purified target gene product were mixed and ligated at 25°C for 30 min.

[0025] (4) When transforming E. coli, use a pipette to draw up the above mixture and add it to the freshly thawed competent E. coli, react on ice for 30 min, heat shock at 42 ℃ for 25-30 s, and place on ice for 2 min. Then add 500 μL of the antibody-free LB solution to the centrifuge tube, incubate at 220 rpm and 37 ℃ for 1 h, take 200 μL of the bacterial solution and spread it on LB medium (kanamycin, 50 mg / L), and incubate it in a 37 ℃ incubator overnight. Pick a single clone of the bacterial solution into a 1.5 mL centrifuge tube, incubate it in LB liquid medium at 220 rpm and 37 ℃, and perform PCR positive identification using M13 F / R primers. The reaction system is as follows: 1 μL of bacterial solution, 0.5 μL of M13 F, 0.5 μL of M13 R, 5 μL of 2×PCR Mix, and 3 μL of ddH2O. The PCR reaction system was: 95 ºC for 5 min, 95 ºC for 30 s, 55 ºC for 30 s, 72 ºC for 2 min, 72 ºC for 5 min, and 4 ºC∞. After the bacterial solution PCR identification was completed, the positive clone bacterial solution was selected for sequencing in the company, and then the sequenced bacterial solution was mixed with 50% glycerol in a 1:1 ratio and stored in a -80 ºC refrigerator.

[0026] (5) Take the above positive bacterial solution for amplification and then use a plasmid extraction kit (Omega, D6943-01) to extract the plasmid pEASYT5-MtLOX24.

[0027] (6) Then, the pCAMBIA3301 expression vector plasmid was double-digested with NcoI and BglⅡ. The reaction system was as follows: plasmid template amount ≤ 2500 ng, NcoI 1 μL, BglⅡ 1 μL, 10×M buffer 5 μL, ddH2O supplemented to 50 μL. The digested product was subjected to agarose gel electrophoresis, and the target band was purified and recovered. The concentration of the recovered product was measured by UV spectrophotometer.

[0028] (7) Design seamless cloning primers 3301-MtLOX24-F / R (Table 2), using the pEASYT5-MtLOX24 plasmid extracted in step (5) as the template. The PCR reaction system is: 95 ºC for 5 min, 95 ºC for 30 s, 57 ºC for 30 s, 72 ºC for 3 min, 72 ºC for 5 min, 4 ºC∞. Cut the target band and recover it in the same way as step (2).

[0029] (8) Seamless cloning was performed using a seamless cloning kit. The product recovered from the gel in step (7) was mixed with the linearized vector in step (6) and the 2× Seamless Master Mix in the kit. The mixture was ligated according to the instructions. Escherichia coli was then transformed according to the method in step (4). A single clone was picked (kanamycin, 50 mg / L) and identified by colony PCR using 35S F and linker-free MtLOX24 R primers.

[0030] (9) After sequencing of colonies that were positive for colony PCR, the plasmid extracted from the bacterial solution with correct sequencing was finally extracted and named pCAMBIA3301-MtLOX24.

[0031] (10) Use the freeze-thaw method to transform the overexpression vector pCAMBIA3301-MtLOX24 into Agrobacterium EHA105 competent cells. Mix the Agrobacterium culture solution with 50% glycerol in a 1:1 ratio and store in a -80 ºC refrigerator.

[0032] Table 2 Primer sequences 2.2 Transformation of alfalfa by overexpression of MtLOX24 The leaf infection method was used for the transformation of alfalfa, and the alfalfa material was the cutting material of the mother plant of Zhongmu No. 1 preserved in this laboratory. The specific transformation method is as follows: (1) Cut the alfalfa strain Zhonglu No. 1 stored in our laboratory, cut the alfalfa stems for cuttings, culture them in vermiculite for 15-20 days until they take root, and then transfer them to soil (nutrient soil: vermiculite = 1:1). After 3-4 weeks of culture, cut the healthy and stretched alfalfa leaves and put them in a conical flask containing sterile ultrapure water as the infection material.

[0033] (2) Place the prepared alfalfa leaves in a clean bench and sterilize with 10% NaClO for 7-8 minutes, then wash them 4-5 times with sterile ultrapure water for later use.

[0034] (3) One day before transformation, take 20 μL of Agrobacterium culture medium carrying the pCAMBIA3301-MtLOX24 overexpression vector stored at -80 ºC and add it to 20 mL YEB liquid culture medium containing 20 μL Kan and 20 μL Rif. Shake and activate the culture medium at 28 ºC at 200 rpm in a shaker in the dark.

[0035] (4) Take 50 μL of the activated bacterial solution in the previous step and add it to 200 mL YEB medium (200 μL Kan, 200 μL Rif) for further activation. Incubate in a shaker at 28°C and 200 rpm in the dark until the OD reaches 600 Between 0.6-0.8.

[0036] (5) The activated bacterial solution was divided into 50 mL centrifuge tubes and centrifuged at 4000 g for 10 min. After discarding the supernatant, add one volume of SH3a liquid culture medium to resuspend the culture to obtain the resuspended Agrobacterium infection solution.

[0037] (6) Under sterile conditions, place the disinfected leaves in a new sterile tissue culture bottle and add the Agrobacterium infection solution resuspended in the previous step. After sealing the tissue culture bottle with a sealing film, place the tissue culture bottle in an ultrasonic cleaner and ultrasonicate at 20 Hz for 5 seconds. Adjust the number of ultrasonications according to the condition of the leaves until the infection solution turns green.

[0038] (7) After the ultrasound is completed, transfer the tissue culture bottle to a vacuum pump and evacuate for 10 minutes. After taking it out, shake it in a shaker at 50 rpm for 30 minutes.

[0039] (8) Under sterile conditions, remove the leaves from the tissue culture bottle and place them on filter paper that has been sterilized and dried in advance. After absorbing the moisture on the surface of the leaves, spread the leaves flat on the non-resistant SH3a co-culture medium.

[0040] (9) Incubate the cells at 24°C in the dark for 1-2 days.

[0041] (10) If there are many colonies around the leaves, wash the leaves in sterile ultrapure water containing Cef. After washing, place the leaves on sterile filter paper.

[0042] (11) The leaves from which the surface water has been absorbed are transferred to SH3a selection medium (containing 400 mg / L Cef and 4 mg / L PPT) for further cultivation. After 1-2 weeks of cultivation, the leaves swell and callus tissue gradually appears. After 1-2 months of cultivation, larger fluffy granular callus tissue is obtained. These callus tissues are transferred to MSBK medium and green buds grow after about 14 days of cultivation. The callus tissues are then transferred to SH9a medium (containing 400 mg / L Cef and 4 mg / L PPT). After the seedlings grow, they are transferred to 1 / 2 SH9a medium (containing 400 mg / L Cef and 2 mg / L PPT) for rooting. During this process, the culture medium needs to be changed every 1-2 weeks.

[0043] (12) The seedlings with strong root systems in the tissue culture bottles were removed from the culture medium, placed in soil (nutrient soil: vermiculite = 1:1), and positive identification was performed on each individual plant.

[0044] 2.3 Positive identification of MtLOX24 overexpressing alfalfa Extract T0 generation alfalfa leaf genomic DNA, and use two pairs of primers, vector primer 35S F and MtLOX24-R without linker, for PCR amplification (Table 2). Use pCAMBIA3301-MtLOX24 plasmid as positive control, and wild-type alfalfa leaves as negative control. If the PCR product shows the same target band as the positive control, the corresponding alfalfa plant is identified as a positive strain. Take the leaves of the positive seedlings for RNA extraction, cDNA inversion, and use qRTPCR to detect the expression level of the MtLOX24 gene in the overexpression strain. The sequence of the quantitative primer qRTPCR-MtLOX24-F / R is shown in Table 2. The internal reference of alfalfa in quantitative detection is MsActin-F / R, and the sequence is shown in Table 2.

[0045] The positive plants of the T0 generation of transgenic alfalfa overexpressing MtLOX24 were cut and rooted in vermiculite, and then transferred to soil for further cultivation as subsequent experimental materials.

[0046] The nucleotide sequence of the MtLOX24 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0047] SEQ ID NO.1:

[0048] SEQ ID NO.2: MFSGITGIINRGEKLKGTVVLMQKNVLDVNELTSIKSNPVGGIIGGAIGGAFGAVGTVLDTATAFLGRNVALKLISATSADGSGKGKVGKQTFLEGVITSIPTLGDKQNAYSVHFEWGSDMGIPGAFYIENFLQHEFFLVSLTLEDVPNHGTINFVCNSWVYNDKKYKSDRIFFANKTYLPSETPAPLVYYRQEELKTLRGDGKGERKEWERIYDYDVYNDLGEPDSKPTLGRPVLGGSSTLPYPRRGRTGRKPAKKDPKSESRSGTVYLPRDEAFGHTKSSDFLAFILKSASQNIIPSLKSVVSKEFNNFEDVRSLYDGGIKLPTNFLSNVSPIPLFTELFRSDGASTLKFSPPKVVQVDHSAWMTDEEFAREMIAGVNPHIIKKLTEFPHKSKLDSQKYGDNTSTITKEQLEPNLGGITVEQAIQNNKLYILDHYDIVYPYLRKINATETKAYAARTILFLQNDGTLKPLAIELSKPHPDDDSYGPVSEVYFPASEGVEASIWLLAKAYVVVNDSCHHQLVSHWLNTHAVVEPFIIATNRHLSTVHPVHKLLLPHYRDTMNINSLARNVLVNAEGIIESTFLWGGYALEMSAVAYRDWVFTEQGLPNDLLKRGVAVEDPASPHGIRLLIEDYPYASDGLEIWAAIKTWVGEYVSFYYKSDAAIAQDAELQAFWKELVEVGHGDLKSATWWFKMQTRAELIEASTILIWIASALHAAVNFGQYPYGGYILNRPTKSRRFMPEKGSAEYAELSKDYQKAYLKTITPKSDTLTDLTIIEVLSRHASDEQYLGERIEGDLWTSDAQPKEAYKKFARKLAEIEEKLVKRNADESLRNRYGPVKMPYTLLYPSSEEGLTFRGIPNSVSI*。

[0049] III. Experimental methods 3.1 Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection (1) Incubate the Pst DC3000 strain in modified LM medium at 30°C for 2 days.

[0050] (2) Pick a single colony and place it in 5 mL of liquid LM medium and shake it at 30°C overnight.

[0051] (3) Pipette 100 μL of the microplate broth into 10 mL of liquid LM medium and shake at 30°C and 200 rpm until the OD600 reaches about 0.8.

[0052] (4) Centrifuge the shaken bacterial solution at 4000 rpm for 10 min at room temperature, and then resuspend the bacterial solution in MgCl2 solution (containing 0.02% Silwet L-77) to a concentration of 0.5 × 10 5 cfu / mL. The remaining Pst DC3000 bacterial solution was added with 25% glycerol and stored at -80°C.

[0053] (5) Six-week-old wild-type (WT) and MtLOX24 transgenic alfalfa lines (L3 and L4) were inoculated with Pseudomonas syringae pv. tomato. Three plants were replicated in each group, and four leaves were selected from each plant. The bacterial suspension was infiltrated into the leaves with a sterile syringe. After the residual bacterial liquid on the surface of the leaves was dried, the leaves were placed in an artificial climate chamber under weak light (24 ℃ light / 20 ℃ dark, 16 h light / 8 h dark, relative humidity 70%) for incubation. After 72 hours of treatment, the leaves infected with the bacterial liquid were removed and the size of the bacterial plaques was counted.

[0054] 3.2 Feeding treatments for Spodoptera litura By observing the feeding of Spodoptera litura larvae on plants, the resistance of different materials to Spodoptera litura was determined. Specific methods: Take 15 detached leaves of 1-month-old alfalfa with consistent growth, place them on wet filter paper in an insect box and culture them at a constant temperature of 25°C, 16h / darkness 8h light, and 70% relative humidity. Repeat five boxes for each material. Select 4 5th-instar larvae with consistent growth and weight, starve them for 2 hours, put them in alfalfa to feed for 36 hours, and weigh them with a balance with an accuracy of one ten-thousandth. Compare with the fresh weight weighed before treatment to calculate the fresh weight of the leaves lost, and use Image J software to count the bitten area of ​​the leaves.

[0055] At the same time, the in vitro branches of 1-month-old wild-type (WT) and transgenic alfalfa plants were taken and placed in square culture dishes (13×13 cm) containing wet filter paper, and the 5th instar larvae of Spodoptera litura were used for feeding experiments. Three branches of WT, L3 and L4 were mixed in each culture dish, and 5 larvae were placed in each culture dish. Five replicates were set up for the experiment. The leaves of alfalfa plants were collected 12 hours after feeding, and weighed with a balance with an accuracy of one ten-thousandth. The fresh weight of the leaves was calculated by comparing with the fresh weight weighed before treatment, and the bitten area of ​​the leaves was counted using Image J software.

[0056] 3.4 Data Analysis GraphPad Prism 7 software was used to draw bar graphs. SPSS19.0 was used for one-way analysis of variance, and the differences among treatments were evaluated by Duncan analysis at the 0.05 level. Different lowercase letters indicate significant differences (P<0.05). All experiments were repeated three times, and the data used are the average of the three repetitions.

[0057] 4. Results and Analysis 4.1 Obtaining transgenic alfalfa MtLOX24 material After the MtLOX24 gene was overexpressed in alfalfa Zhongmu 1, a total of 25 overexpression transgenic lines (MtLOX24-OE) were obtained. The genetic transformation process of alfalfa was as follows: Figure 1 As shown, Figure 1 A is 3 days after induction with SH3a medium, B is 7 days after induction with SH3a medium, C is callus induced by SH3a, D is cultured with MSBK medium, and E is after transplanting. The expression levels of MtLOX24 gene in 6 transgenic lines (L3, L4, L8, L14, L17, and L20) were identified by qRT-PCR. Figure 2 Two overexpression lines, L3 and L4, were selected for subsequent experiments.

[0058] 4.2 Overexpression of the MtLOX24 gene enhances plant defense against Spodoptera litura In order to understand the role of alfalfa LOX genes when chewing insects bite plants, we used Spodoptera litura to conduct a feeding experiment on alfalfa. We conducted an insect feeding experiment on detached leaves of alfalfa WT and two MtLOX24-OE strains L3 and L4. After alfalfa leaves were fed for 36 hours, the area of ​​alfalfa WT eaten was larger than that of the MtLOX24-OE overexpressing strain, and there was a significant difference, such as Figure 3 At the same time, the fresh weight loss of alfalfa WT leaves after larvae feeding was significantly higher than that of the MtLOX24-OE overexpression strain (P<0.05). Figure 3C. Furthermore, in the feeding experiment of Spodoptera litura larvae on the branches of WT and MtLOX24-OE overexpressing strains of alfalfa, it was found that compared with the branches of the overexpressing strains, more leaves in the WT branches were fed by the larvae, such as Figure 3 D. By counting the weight loss of each branch after insect feeding, it was found that the fresh weight loss of alfalfa WT was significantly higher than that of the overexpression strain (P<0.05). Figure 3 E. The above results showed that Spodoptera litura had a higher preference for wild-type alfalfa than for the overexpression strain, that is, overexpression of the MtLOX24 gene increased the resistance of the plant to Spodoptera litura.

[0059] 4.3 MtLOX24 gene is involved in plant resistance to pathogens We studied the role of Pst DC3000 in the L3 and L4 leaves of wild-type alfalfa WT and MtLOX24-OE overexpressing strains. After 72 h of infiltration with Pst DC3000, WT leaves showed yellowing, such as Figure 4 As shown in A, the area of ​​leaf damage was significantly higher than that of MtLOX24-OE strains L3 and L4. Figure 4 As shown in B, it shows that the MtLOX24 gene can positively regulate the plant's resistance to the model bacterium Pst DC3000, promote plant immunity, and inhibit the infection of pathogens.

[0060] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements fall within the scope of the present invention to be protected. The scope of the present invention to be protected is defined by the attached claims and their equivalents.

Claims

1. A Medicago truncatula MtLOX24 gene, characterized in that: The nucleotide sequence of the MtLOX24 gene is shown in SEQ ID NO.

1.

2. The Medicago truncatula MtLOX24 gene, characterized in that: The amino acid sequence encoded by the MtLOX24 gene is shown in SEQ ID NO.

2.

3. A transgenic vector containing the Medicago truncatula MtLOX24 gene according to claim 1.

4. A recombinant strain or recombinant cell containing the Medicago truncatula MtLOX24 gene according to claim 1.

5. Use of the Medicago truncatula MtLOX24 gene overexpression according to claim 1 in improving plant disease and insect pest resistance.

6. The use according to claim 5, characterized in that: The plants include Medicago truncatula and Medicago sativa.

7. The use according to claim 5, characterized in that: Follow these steps: S1. A lipoxygenase gene MtLOX24 was cloned from Medicago truncatula; S2, obtaining MtLOX24 gene overexpressing alfalfa strains by Agrobacterium-mediated method; S3. Insect feeding and pathogen infection experiments were conducted using wild-type and MtLOX24-overexpressing alfalfa as materials.

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