Leguminous small peptide and receptor and application of leguminous small peptide and receptor in preparation of biopesticide

Abstract

The invention discloses a leguminous small peptide, a receptor and application of the leguminous small peptide and the receptor in preparation of biopesticides, and belongs to the technical field of biopesticides. The amino acid sequence of the leguminous small peptide is any one of SEQ ID NO.1-14, the amino acid sequence of the receptor of the leguminous small peptide is any one of SEQ ID NO.15-25, and the nucleotide sequence for coding the receptor of the leguminous small peptide is any one of SEQ ID NO.26-36. The leguminosae small peptide or / and the receptor of the leguminosae small peptide can be applied to preparation of biopesticide, and the leguminosae plant has a broad-spectrum disease-resistant effect.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, specifically relating to legume peptides and receptors and their application in the preparation of biopesticides. Background Technology

[0002] While chemically synthesized pesticides (such as organophosphates and pyrethroids) widely used in current agricultural production are effective in controlling pests and diseases, traditional pesticides inevitably cause non-biological problems such as environmental pollution: the half-life of chemical pesticides in the soil can be several months or even years (e.g., DDT takes 2-15 years to degrade), threatening ecosystems through bioaccumulation; about 70% of pesticides affect pollinating insects and aquatic organisms through drift, leading to a decline in biodiversity; pathogens develop new resistance variants on average every 5-8 years, forcing a continuous increase in pesticide use, creating a vicious cycle. Compared to chemical pesticides, biopesticides, represented by microbial preparations (such as Bacillus thuringiensis) and plant-derived pesticides (such as pyrethroids), have environmentally friendly characteristics, but still have some drawbacks, such as: narrow spectrum of action, most targeting only specific pathogens (e.g., Bt protein is only effective against lepidopteran larvae); and a single mechanism of action, relying on direct killing and lacking the ability to systematically induce resistance. Summary of the Invention

[0003] In view of the above-mentioned prior art, the present invention provides leguminous small peptides and receptors and their application in the preparation of biopesticides. Small peptide molecules with immune effects extracted from plants and similar immune small peptide molecules are used as novel pesticides to enable plants to have broad-spectrum disease resistance.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is to provide a legume peptide, wherein the amino acid sequence of the legume peptide is any one of SEQ ID NO.1~14.

[0005] Based on the above technical solution, the present invention can be further improved as follows.

[0006] Furthermore, the amino acid sequence of the receptor for the legume peptide is any one of SEQ ID NO.15~25.

[0007] Furthermore, the nucleotide sequence encoding the receptor for the legume peptide is any one of SEQ ID NO.26~36.

[0008] Furthermore, the application of the above-mentioned legume peptides and / or their receptors in the preparation of biopesticides.

[0009] Furthermore, biopesticides are being applied to legumes.

[0010] Furthermore, the biopesticide is a biopesticide that is resistant to damping-off or Phytophthora root rot.

[0011] Furthermore, the concentration of legume peptides in biopesticides is 0.5~1.5μM.

[0012] Furthermore, the concentration of legume peptides in the biopesticide was 1 μM.

[0013] The beneficial effects of this invention are: This invention identifies small peptide molecules with immune-boosting effects, which can be used as biopesticides, and screens for their receptors. Using small peptide molecules identified in plants, and extending this to the entire legume family, sequence alignment of these small peptides revealed small peptides with similar immune functions in soybeans and other legumes besides soybeans. Through screening, their receptors and potential receptors for other small peptides were identified, and finally, the generated reactive oxygen species (ROS) were used to verify these receptors in tobacco. Attached Figure Description

[0014] Figure 1 ROS burst levels in leaves (A) and roots (B) of soybean cultivar Wm82 treated with small peptide GmSubPEPs; Figure 2 To demonstrate the resistance of small peptides GmSubPEPs to damping-off (A) and Phytophthora root rot (B); Figure 3 A map of the p1300 plasmid containing the nucleotide sequence of the receptor GSPRs; Figure 4 The small peptide GmSubPEPs can induce ROS bursts in receptor proteins GSPRs; Figure 5 To enhance plant resistance to damping-off by using GSPR2 as a small peptide GmSubPEP3 receptor; Figure 6 GSPR1 was used as a small peptide GmSubPEP2 receptor to enhance the plant's resistance to Phytophthora root rot. Figure 7 The ROS burst level induced by SubPEPs of the legume family in tobacco, which transiently expresses its receptor; where A is LcuSubPEP1 and B is LerSubPEP1. Figure 8 The ROS burst level induced by SubPEPs of the legume family in tobacco, where the receptor is transiently expressed; where A is CaSubPEP1 and B is CaSubPEP2. Figure 9 The ROS burst level induced by leguminous SubPEPs in tobacco, where the receptor is transiently expressed; where A is MtSubPEP2, B is MtSubPEP3, C is MtSubPEP4, D is MtSubPEP5, and E is MtSubPEP6. Figure 10AhySubPEP induces ROS reactions in peanuts. Detailed Implementation

[0015] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0016] Biomaterials used in the examples: 1. Phytophthora strains causing soybean root rot Phytophthora sojae (P. sojae) P7076 is a commonly used research strain obtained from public sources; *Rhizoctonia solani*, the pathogen causing soybean damping-off. Rhizoctonia solani Kuhn These are commonly used research strains, obtained from publicly available sources.

[0017] 2. Soybean Cultivation Varieties Glycine Max Williams 82 (Wm82) was preserved in the laboratory; mutant. gspr1 From Weimi Bio, gspr2 From Song Qingxin's laboratory at Nanjing Agricultural University.

[0018] 3. Primer synthesis and sequencing were completed by Beijing Qingke Biotechnology Co., Ltd.

[0019] 4. Cultivation conditions for soybean seedlings: Soybean seeds are cultivated in an incubator at 25℃ (16h light / 8h darkness, humidity about 60%) for about two weeks. The cultivation soil used is 100% vermiculite, and water is applied every three days.

[0020] 5. Cultivation conditions for soybean etiolated seedlings: Soybean seeds are cultivated in a 25℃ incubator (humidity of about 60%) in complete darkness for about a week. The cultivation soil used is 100% vermiculite, and water is applied every three days.

[0021] 6. The small peptides GmSubPEPs (GmSubPEP1, GmSubPEP2, GmSubPEP3 and GmSubPEP4), LcuSubPEP, LerSubPEP, CaSubPEPs (CaSubPEP1 and CaSubPEP2), MtSubPEPs (MtSubPEP2, MtSubPEP3, MtSubPEP4, MtSubPEP5, MtSubPEP6), AhySubPEP, and control small peptides (flg22, GmSubPEP5-12, GmSubPEP5-13 and AhySubPEP-2) were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. The purity of the synthesized peptides was above 90%. They were in powder form, aliquoted into 1mg tubes, and stored at -80℃ for long-term use. Once the small peptide powder is obtained, take out one tube and dissolve it with ddH2O to a concentration of 1mM according to the molecular weight and purity of the small peptide. Store it in a -20℃ freezer for short-term use (six months). Each time it is used, prepare a working solution with a lower concentration of the small peptide.

[0022] The amino acid sequences of GmSubPEP1, GmSubPEP2, GmSubPEP3, GmSubPEP4, CaSubPEP1, MtSubPEP2, MtSubPEP3, MtSubPEP4, and AhySubPEP are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, LerSubPEP, LcuSubPEP, and AhySubPEP are shown in SEQ ID NO.14.

[0023] Experimental reagents: Quantitative PCR kit was purchased from Novizan Biotechnology Co., Ltd.; V8 vegetable juice was purchased from Beijing Xile Trading Co., Ltd.; calcium carbonate (analytical grade) was purchased from Xilong Scientific Co., Ltd.; potato dextrose agar (PDA) medium was purchased from Oxoid; agar powder was purchased from Beijing Solarbio Technology Co., Ltd.; Tween-20 was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Silwet L-77 was purchased from GE; peroxidase (V900503-100MG) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; luminol (A8511) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; tap water and sterile double-distilled water (ddH2O) were obtained from our unit.

[0024] Experimental consumables: Vermiculite was purchased from Beijing Jixiang Feiyun Landscape Engineering Co., Ltd.; 150mm disposable round dishes were purchased from Beijing Yiyan Shengke Biotechnology Co., Ltd.; 96-well white microplates (3922) were purchased from CORNING; Eppendorf pipettes were purchased from Beijing Shengang Technology Co., Ltd.; culture boxes and culture caps were obtained from our unit's greenhouse; Erlenmeyer flasks, markers, scissors, blades, tweezers, centrifuge tubes, pipette tips, and hole punches were all purchased from CASMA Mall.

[0025] Experimental equipment: PCR instrument purchased from Bio-Rad; quantitative PCR instrument purchased from Bio-Rad; MLS-3750 high-temperature autoclave purchased from Sanyo Corporation, Japan; light incubator purchased from Fujian Jiupu Biotechnology Co., Ltd.; ELISA reader (Envision) purchased from PerkinElmer; small low-temperature incubator (Asone) purchased from Beijing Jianqiang Weiye Technology Co., Ltd.; Eppendorf centrifuge purchased from Eppendorf (Shanghai) International Trading Co., Ltd.; spectrophotometer purchased from Beckman Coulter; shaker purchased from Shanghai Zhichu Instrument Co., Ltd.; -20℃ freezer and ultra-low temperature freezer were both purchased from Haier Corporation.

[0026] Example 1 Treatment of leaves and roots of soybean cultivar Wm82 with small peptide GmSubPEPs resulted in ROS burst levels. GmSubPEPs can activate the immune response in soybeans, and the surge of reactive oxygen species (ROS) is one of the earliest observable responses in plants defending against pathogen infection. The specific experimental steps are as follows: (1) Soybean cultivar Wm82 was planted in 100% vermiculite and placed in a 25℃ light incubator (60% humidity) for about 14 days under normal light conditions. (2) Add 200 μL ddH2O to each well of a 96-well white microplate, and then place the sample into the corresponding well of the microplate; leaf samples: use a punch to take leaves, and take 8 replicates for each treatment (i.e., take 8 small round leaves of 4 mm); root samples: cut 3 roots of 4 mm, and take 8 replicates for each treatment. (3) Cover the microplate with a piece of white paper and place it under a fluorescent light for overnight incubation; (4) Add 200 μL of detection solution to each well of the microplate. 1600 μL of detection solution needs to be prepared for each of the 8 replicates. The detection solution is divided into control solution and small peptide solution. The ROS reaction detection solution is prepared by adding 1600 μL ddH2O, 1.6 μL peroxidase and 1.6 μL substrate luminol to a 2 mL centrifuge tube and mixing well to obtain the control solution. Add small peptide (final concentration of 1 μM, small peptide is small peptide GmSubPEPs or small peptide flg22) to the control solution and mix well to obtain the small peptide solution. (5) After adding the detection solution, the ROS burst level was detected by an enzyme-linked immunosorbent assay (ELISA) reader after different treatment times (0~160 min). (6) Use Excel and Prism 9 to draw a curve graph.

[0027] like Figure 1As shown in the figure, the amino acid sequence of GmSubPEP5-12 is NVDVSQDKDIDE (SEQ ID NO.37); the amino acid sequence of GmSubPEP5-13 is NVDVSQDKDIDEH (SEQ ID NO.38). These two serve as negative controls, indicating that GmSubPEP1, GmSubPEP3, and GmSubPEP4 can induce ROS in soybean leaves, while GmSubPEP1 and GmSubPEP2 can induce ROS in soybean roots.

[0028] Example 2 GmSubPEPs, a small peptide, can be used as a biopesticide to enhance plant resistance to damping-off disease. Detection of small peptide pretreatment to improve soybean resistance to Rhizoctonia solani, the soybean damping-off disease. Rhizoctonia solani Kuhn The specific experimental steps for determining resistance are as follows: (1) Soybean cultivar Wm82 was planted in 100% vermiculite and placed in a 25℃ light incubator (60% humidity) for about 14 days under normal light conditions. (2) Preparation of potato dextrose agar medium: Prepare potato dextrose agar medium (take 39g PDA and dilute to 1L with ddH2O solution), dispense into small triangular flasks, sterilize at 121℃ for 15min, and store in a 4℃ cold room for later use. (3) Rhizoctonia solani Rhizoctonia solani Kuhn Activation: While waiting for the soybean seedlings to grow, the bacterial strains need to be activated simultaneously. Rhizoctonia solani Kuhn The specific method is as follows: Take a piece of Rhizoctonia solani stored in a 10℃ incubator. Rhizoctonia solani Kuhn For the bacterial culture plate, gently poke a small amount of mycelium with a pipette tip and place it on a new PDA medium that does not contain antibiotics. Incubate at room temperature for about 3 days. (4) Pretreatment with surfactant Silwet L-77: After soybeans have grown for 14 days, soybean leaves are removed from the plant and placed in 15 mL of 0.002% Silwet L-77 solution (prepared with ddH2O) for 12 h. (5) Add GmSubPEPs to the pretreated Silwet L-77 solution to make the final concentration 1 μM, which is the small peptide solution treatment. The control is not treated. Then soak soybean leaves for 24 h, with about 20 leaves in each treatment. (6) Rhizoctonia solani Rhizoctonia solani Kuhn Infecting soybean leaves with the strain: Soybean leaves were laid flat in a round dish and placed in contact with sterile filter paper moistened with ddH2O; Rhizoctonia solani, which had been cultured for about 3 days, was then introduced into the dish. Rhizoctonia solani Kuhn The strain was made into small discs containing mycelial agar blocks using a 4mm punch. The mycelial side of each disc was then placed in contact with the center of each leaf and cultured at room temperature for 48 hours. (7) Observe the infection of soybean leaves by mycelial blocks with the naked eye, take pictures, process the pictures with Adobe Photoshop 2021 software, and process and count the size of lesions with ImageJ.

[0029] like Figure 2 Results A show that the white line is a ruler, representing a length of 2 cm. Compared with the control group, soybeans pretreated with small peptides GmSubPEP3 and GmSubPEP4 showed enhanced resistance to damping-off caused by Rhizoctonia solani. This indicates that small peptides GmSubPEP3 and GmSubPEP4 can be used as biological pesticides to improve resistance to soybean damping-off.

[0030] Example 3 GmSubPEPs, a small peptide, can be used as a biopesticide to enhance plants' resistance to Phytophthora root rot. Detection of small peptide pretreatment to improve soybean resistance to Phytophthora root rot strains Phytophthora sojae (P. sojae) The specific experimental steps for determining the resistance to P7076 are as follows: (1) Planting soybean etiolated seedlings: Soybean cultivar Wm82 was planted in 100% vermiculite and cultured in the dark for about 3 days; (2) Preparation of 10% V8 medium: After preparing V8 medium according to the formula shown in Table 2, dispense it into small Erlenmeyer flasks, sterilize at 121℃ for 15 min, and store in a 4℃ cold room for later use. Table 2 Formulation of 10% V8 medium (1L)

[0031] (3) Phytophthora root rot strains Phytophthora sojae (P. sojae) Activation of P7076: While waiting for soybean seedlings to grow, strain P7076 should be activated. The specific method is as follows: Take a culture plate of Pythium spp. strain P7076 stored in a 10℃ incubator, gently poke a little mycelium with a pipette tip, spread it on a new 10% V8 medium without antibiotics, and place it at room temperature for about 5 days. (4) Clean the soybean roots: Three days after the soybeans have grown, gently wash the vermiculite off the soybean roots with water; (5) Prepare pretreatment peptide solution and control solution and treat soybean etiolated seedlings: Prepare 10 mL of 1 μM GmSubPEPs solution and ddH2O as control solution. Soak soybean etiolated seedlings in the peptide solution and control solution for 24 h, with about 20 etiolated seedlings in each treatment. (6) Infection of the hypocotyl of soybean etiolated seedlings by Phytophthora root rot strain P7076: Soybean etiolated seedlings were laid flat in a round dish, and the whole seedling was in contact with sterile filter paper moistened with ddH2O; Phytophthora root rot strain P7076, which had been cultured for about 5 days, was made into small round discs containing mycelial agar blocks using a 4mm punch. The mycelial side of each small disc was placed in contact with the hypocotyl of each etiolated seedling, and cultured at room temperature in the dark for 48 hours. (7) Observe the infection of mycelial blocks on soybean hypocotyls with the naked eye, take pictures, and process the images using Adobe Photoshop 2021 software. At the same time, take a 2cm hypocotyl with a blade, extract DNA, and perform qPCR experiments to determine the DNA content. Phytophthora sojae (P. sojae) Biomass of P7076 and soybean was calculated, and the results were plotted as bar charts using Excel and Prism 9.

[0032] like Figure 2 Results B show that the black line is a ruler, representing a length of 2 cm. Compared with the control group, soybeans pretreated with small peptides GmSubPEP1, GmSubPEP2, and GmSubPEP4 showed enhanced resistance to Phytophthora root rot. This indicates that small peptides GmSubPEP1, GmSubPEP2, and GmSubPEP4 can be used as biological pesticides to improve resistance to soybean Phytophthora root rot.

[0033] Example 4 The specific experimental steps for detecting the ROS burst induced by GmSubPEPs in tobacco with transiently expressed receptors are as follows: (1) The plasmid containing the receptor GSPRs nucleotide sequence (P1300 plasmid with C-terminal flag tag, as shown in the figure) Figure 3 (As shown) transiently expressed in tobacco for 3 days; (2) Add 200 μL ddH2O to each well of a 96-well white microplate, and use a punch to take tobacco leaves after transient expression. Take 8 replicates for each treatment (i.e., take 8 small round leaves of 4 mm). (3) Gently hold the small round leaf with tweezers and place it into a 96-well white microplate. Cover the microplate with a piece of white paper and place it under a fluorescent light for overnight incubation. (4) Add 200 μL of detection solution to each well of the microplate. 1600 μL of detection solution needs to be prepared for each of the 8 replicates. The detection solution is divided into control solution and small peptide solution. The ROS reaction detection solution is prepared by adding 1600 μL of ddH2O, 1.6 μL of peroxidase and 1.6 μL of substrate luminol to a 2 mL centrifuge tube and mixing well to obtain the control solution. Add small peptide (final concentration of 1 μM, small peptide is small peptide GmSubPEPs or small peptide flg22) to the control solution and mix well to obtain the small peptide solution. (5) After adding the detection solution, the ROS burst level was detected by an enzyme-linked immunosorbent assay (ELISA) reader after different treatment times (0~160 min). (6) Use Excel and Prism 9 to draw a curve graph.

[0034] like Figure 4 As shown, GmSubPEP1 and GmSubPEP2 reduce the ROS of tobacco plants expressing the receptor protein GSPR1, GmSubPEP3 reduces the ROS of tobacco plants expressing the receptor protein GSPR2, and GmSubPEP4 reduces the ROS of tobacco plants expressing the receptor protein GSPR3.

[0035] The amino acid sequence of receptor protein GSPR1 is shown in SEQ ID NO.15, and the nucleotide sequence encoding receptor protein GSPR1 is shown in SEQ ID NO.26; the amino acid sequence of receptor protein GSPR2 is shown in SEQ ID NO.16, and the nucleotide sequence encoding receptor protein GSPR2 is shown in SEQ ID NO.27; the amino acid sequence of receptor protein GSPR3 is shown in SEQ ID NO.17, and the nucleotide sequence encoding receptor protein GSPR3 is shown in SEQ ID NO.28.

[0036] The nucleotide sequence shown in SEQ ID NO.26 contains introns at positions 2894 to 3156, the nucleotide sequence shown in SEQ ID NO.27 contains introns at positions 2858 to 3139, and the nucleotide sequence shown in SEQ ID NO.28 contains introns at positions 2864 to 3145.

[0037] Example 5 GSPR2, as a GmSubPEP3 receptor, enhances plant resistance to damping-off. Detection of small peptide pretreatment to improve wild-type soybean resistance to Rhizoctonia solani, the soybean damping-off disease. Rhizoctonia solani Kuhn Resistance, and to gspr2 The mutant was invalid. Soybean cultivar Wm82 and... gspr2 The mutants were seeded in 100% vermiculite and placed in a 25°C light incubator (60% humidity) for about 14 days under normal light conditions; other experimental steps were the same as in Example 2.

[0038] The results are as follows Figure 5 As shown, the white line is a ruler, indicating a length of 2 cm; compared to the wild group, pretreatment with the small peptide GmSubPEP3 resulted in a shorter length. gspr2 The mutant showed no resistance to damping-off caused by Rhizoctonia solani; indicating that the receptor GSPR2 can act as a receptor for the small peptide GmSubPEP3 to enhance the plant's resistance to damping-off.

[0039] Example 6 GSPR1, acting as a receptor for the small peptide GmSubPEP2, enhances the plant's resistance to Phytophthora root rot. Detection of small peptide pretreatment to improve soybean resistance to Phytophthora root rot strains Phytophthora sojae (P. sojae) P7076 resistance; planting yellow soybean seedlings: soybean cultivar Wm82 and mutants gspr1 Planted in 100% vermiculite and cultured in the dark for about 3 days; other experimental steps are the same as in Example 3.

[0040] The results are as follows Figure 6 As shown (#9-14 and #9-18 are two plants) gspr1 (Mutant number), the black line is a scale, indicating a length of 2 cm; compared to the control group, pretreatment with the small peptide GmSubPEP2... gspr1 Soybeans lost their resistance to Phytophthora root rot; however, pretreatment with the small peptide GmSubPEP4 resulted in the loss of resistance. gspr1 Soybeans still exhibit resistance to Phytophthora root rot; this indicates that the receptor GSPR1 can act as a receptor for the small peptide GmSubPEP2 to enhance the plant's ability to resist soybean Phytophthora root rot.

[0041] Example 7 Using the method described in Example 4, the ROS burst induced by SubPEPs from other legume species in tobacco, which transiently expresses its potential receptor, was detected.

[0042] The results are as follows Figures 7-9 As shown: (1) LcuSubPEP1 and LerSubPEP1 can cause tobacco burst ROS that express the receptor protein LcuGSPR (Lcu.2RBY.6g040140); (2) CaSubPEP1 can cause the ROS of tobacco bursts that express the receptor protein CaGSPR1 (Cicar.04G141200) or the receptor CaGSPR2 (Cicar.04G141400), and CaSubPEP2 can cause the ROS of tobacco bursts that express the receptor protein CaGSPR1 (Cicar.04G141200). (3) MtSubPEP2 can cause ROS in tobacco expressing receptor protein MtGSPR1 (Medtr7g081410); MtSubPEP3 can cause ROS in tobacco expressing receptor protein MtGSPR2 (Medtr7g081780) or receptor protein MtGSPR3 (Medtr7g081480); MtSubPEP4 can cause ROS in tobacco expressing receptor protein MtGSPR2 (Medtr7g081780); MtSubPEP5 can cause ROS in tobacco expressing receptor protein MtGSPR4 (Medtr7g081570) or MtGSPR5 (Medtr7g081720); MtSubPEP6 can cause ROS in tobacco expressing receptor protein MtGSPR5 (Medtr7g081720).

[0043] The amino acid sequence of receptor protein MtGSPR1 is shown in SEQ ID NO.18, and the nucleotide sequence encoding receptor protein MtGSPR1 is shown in SEQ ID NO.29; the amino acid sequence of receptor protein MtGSPR2 is shown in SEQ ID NO.19, and the nucleotide sequence encoding receptor protein MtGSPR2 is shown in SEQ ID NO.30; the amino acid sequence of receptor protein MtGSPR3 is shown in SEQ ID NO.20, and the nucleotide sequence encoding receptor protein MtGSPR3 is shown in SEQ ID NO.31; the amino acid sequence of receptor protein MtGSPR4 is shown in SEQ ID NO.21, and the nucleotide sequence encoding receptor protein MtGSPR4 is shown in SEQ ID NO.32; the amino acid sequence of receptor protein MtGSPR5 is shown in SEQ ID NO.22, and the nucleotide sequence encoding receptor protein MtGSPR5 is shown in SEQ ID NO.33.

[0044] The amino acid sequence of receptor protein CaGSPR1 is shown in SEQ ID NO.23, and the nucleotide sequence encoding receptor protein CaGSPR1 is shown in SEQ ID NO.34; the amino acid sequence of receptor protein CaGSPR2 is shown in SEQ ID NO.24, and the nucleotide sequence encoding receptor protein CaGSPR2 is shown in SEQ ID NO.35.

[0045] The amino acid sequence of the receptor protein LcuGSPR is shown in SEQ ID NO.25, and the nucleotide sequence encoding the receptor protein LcuGSPR is shown in SEQ ID NO.36.

[0046] In the nucleotide sequence shown in SEQ ID NO. 29, positions 2822 to 2936 are introns; in the nucleotide sequence shown in SEQ ID NO. 30, positions 2825 to 2960 are introns; in the nucleotide sequence shown in SEQ ID NO. 31, positions 2831 to 3065 are introns; in the nucleotide sequence shown in SEQ ID NO. 32, positions 2801 to 3112 are introns; in the nucleotide sequence shown in SEQ ID NO. 33, positions 2876 to 3262 are introns; and in the nucleotide sequence shown in SEQ ID NO. 36, positions 2843 to 2922 are introns.

[0047] Example 8 The specific experimental steps for the ROS reaction initiated by the small peptide AhySubPEP in peanuts are as follows: (1) Plant red-skinned peanuts in 100% vermiculite and place them in a 25℃ light incubator (60% humidity) for about 14 days under normal light conditions; (2) Add 200 μL ddH2O to each well of a 96-well white microplate, and use a punch to take leaves. Take 8 replicates for each treatment (i.e., take 8 small round leaves of 4 mm). (3) Gently hold the small round leaf with tweezers and place it into a 96-well white microplate. Cover the microplate with a piece of white paper and place it under a fluorescent light for overnight incubation. (4) Add 200 μL of detection solution to each well of the microplate. 1600 μL of detection solution needs to be prepared for each of the 8 replicates. The detection solution is divided into control solution and small peptide solution. The ROS reaction detection solution is prepared by adding 1600 μL ddH2O, 1.6 μL peroxidase and 1.6 μL substrate luminol to a 2 mL centrifuge tube and mixing well to obtain the control solution. Add small peptide (final concentration of 1 μM, small peptide is AhySubPEP) to the control solution and mix well to obtain the small peptide solution. (5) After adding the detection solution, the ROS burst level was detected by an enzyme-linked immunosorbent assay (ELISA) reader after different treatment times (0~160 min). (6) Use Excel and Prism 9 to draw a curve graph.

[0048] like Figure 10 As shown in the figure, the amino acid sequence of AhySubPEP-2 is NYPNVHPPPIPP (SEQ ID NO.39), which is another small peptide sequence in its homology family, serving as a negative control; indicating that the small peptide AhySubPEP can induce ROS burst in peanut leaves.

[0049] This invention utilizes a simple and easy-to-operate method to discover novel small peptides and extend their application to other species, ultimately achieving a novel pesticide with specific and broad-spectrum disease resistance within the legume family. Using soybean as the primary research subject, the receptors for soybean small peptides (GSPRs) were identified, and their receptors were extended to other legume species, validating the potential receptors for these small peptides. This ensures that different legume species have corresponding small peptide molecules that can enhance basic immunity levels and achieve early disease resistance.

[0050] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. A legume-derived small peptide, characterized in that: The amino acid sequence of the legume small peptide is any one of SEQ ID NO. 1-14.

2. The receptor for small legume peptides according to claim 1, characterized in that: The amino acid sequence of the receptor of the legume small peptide is any one of SEQ ID NO. 15-25.

3. The receptor for small legume peptides according to claim 2, characterized in that: The nucleotide sequence encoding the receptor of the legume small peptide is any one of SEQ ID NO. 26-36.

4. Use of the legume small peptide of claim 1 or / and the receptor of the legume small peptide of claim 2 or 3 in the preparation of a biopesticide.

5. Use according to claim 4, characterized in that: The biopesticide is applied to legume plants.

6. Use according to claim 4, characterized in that: The biopesticide is an anti-wilt or anti-phytophthora root rot biopesticide.

7. Use according to claim 6, characterized in that: The concentration of the legume small peptide in the biopesticide is 0.5-1.5 μM.

8. Use according to claim 7, characterized in that: The concentration of the legume small peptide in the biopesticide is 1 μM.

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