Application of a rice endogenous small peptide in regulating rice resistance
Patent Information
- Application Number
- CN202610818802.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-08
- Publication Date
- 2026-09-15
Smart Images
Figure CN122744331A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of bioprotein technology, and in particular to the application of a rice endogenous small peptide in regulating rice resistance. Background Technology
[0002] Rice is one of the most important food crops, and its yield is directly related to food security. However, rice production is frequently threatened by various diseases such as rice blast, bacterial blight, and sheath blight, which seriously affect the yield and quality of rice. Therefore, it is necessary to control these diseases in rice at present. Currently, control measures mainly include chemical pesticide control and the breeding of disease-resistant varieties. However, chemical pesticide control has problems such as serious environmental pollution and the easy development of pesticide resistance in pathogens, while the breeding of disease-resistant varieties is time-consuming and difficult.
[0003] Plant peptides are an important class of signaling molecules that can be recognized by cell membrane receptors at extremely low concentrations, triggering intercellular signal transduction and thereby regulating plant growth and development, stress responses, and immune defense. Existing research indicates that various endogenous plant peptides can activate ROS bursts and Ca2+ through receptor recognition. 2+ It can influx and MAPK signaling pathways and significantly enhance crop resistance to pathogens. However, current research on the role of rice endogenous small peptide OsSSP31 in regulating rice development, stress response, and immune defense is insufficient, limiting the widespread application of plant endogenous small peptides in green pest control in agriculture. Summary of the Invention
[0004] This application provides an application of rice endogenous small peptides in regulating rice resistance, in order to solve the following technical problem: how to broaden the application scope of rice endogenous small peptides in green pest control in agriculture.
[0005] In a first aspect, embodiments of this application provide an application of rice endogenous small peptide OsSSP31 in regulating rice resistance, the application comprising: using rice endogenous small peptide OsSSP31 in the preparation of a regulator for regulating rice resistance; wherein, the regulator comprises an activator for activating rice immunity and / or a resistance agent for improving rice resistance to rice blast fungus.
[0006] Optionally, the active fragment of the rice endogenous peptide OsSSP31 has the amino acid sequence shown in SEQ ID NO.1, or has an amino acid sequence with ≥90% similarity to the amino acid sequence shown in SEQ ID NO.1.
[0007] Optionally, the molecular formula of the active fragment of the rice endogenous small peptide OsSSP31 is C 336 H 503 N 81 O 87S, the relative molecular mass of the rice endogenous small peptide OsSSP31 is ≥7100 Da.
[0008] Optionally, the instability coefficient of the rice endogenous peptide OsSSP31 is ≤33.0.
[0009] Optionally, the raw material for the regulator includes a fermentation expression product containing the rice endogenous small peptide OsSSP31, and the fermentation expression product includes at least one of engineered bacterial fermentation broth, bacterial cell lysate, purified fermented protein liquid, and freeze-dried fermentation broth preparation.
[0010] Optionally, the raw materials for the regulator may also include at least one of plant immune inducers, biopesticide compositions, and plant disease-resistant agents.
[0011] Secondly, embodiments of this application provide a method for preparing the regulator used in the aforementioned applications, the method comprising: The target gene encoding the rice endogenous small peptide OsSSP31 was ligated into an expression vector to construct a recombinant expression vector; The recombinant expression vector was introduced into host bacterial cells to obtain recombinant bacterial cells; The recombinant bacterial cells were induced to express the fusion protein using an inducer. The fusion protein was purified to obtain a regulator.
[0012] Optionally, a hydrophilic tag is provided at the N-terminus of the nucleotide sequence encoding the fusion protein.
[0013] Optionally, the nucleotide sequence encoding the fusion protein is as shown in SEQ ID NO.2, or has ≥90% similarity to the nucleotide sequence shown in SEQ ID NO.2.
[0014] Optionally, the inducing agent comprises isopropyl-β-D-thiogalactoside, wherein the molar concentration of the inducing agent is from 0.25 mmol / L to 1.00 mmol / L; and / or The induction temperature is between 16°C and 37°C.
[0015] The technical solutions provided in this application have the following advantages compared with the prior art: This application provides an application of rice endogenous peptide OsSSP31 in regulating rice resistance. This application is based on previous research finding that rice endogenous peptide OsSSP31 can induce upregulation of the expression of immune-related genes in rice, activate ROS burst in vivo, induce calcium ion influx in detached leaves, and enhance MAPK phosphorylation, thereby activating core immune signaling pathways and triggering early immune responses in rice. Therefore, rice endogenous peptide OsSSP31 can significantly enhance rice resistance to rice blast fungus. Based on this discovery, the rice endogenous peptide OsSSP31 can be used as an activator to activate rice immunity and / or a resistance agent to enhance rice's resistance to rice blast fungus. At the same time, the rice endogenous peptide OsSSP31 has unparalleled advantages over traditional control technologies in terms of drug resistance, environmental safety, and efficacy stability. It not only specifically solves the key technical problems of green control of rice blast, but also innovates the agricultural application of endogenous peptides, promotes the industrialization process of the entire field of plant peptide immune regulation, and greatly expands the application scope of rice endogenous peptides in green agricultural control. Attached Figure Description
[0016] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.
[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1 This is a schematic flowchart of a method for preparing a regulator used in an application, provided in an embodiment of this application. Figure 2 Phylogenetic analysis of the OsSSP31 homologous family genes provided in the embodiments of this application; Figure 3 A diagram showing the conserved sequence analysis results of the rice endogenous small peptide OsSSP31 provided in the embodiments of this application; Figure 4 The image shows the results of the physicochemical property analysis of the conserved sequence of the rice endogenous small peptide OsSSP31 provided in the embodiments of this application; Figure 5 A SUMO fusion expression design diagram of rice endogenous small peptide OsSSP31 provided in the embodiments of this application; Figure 6 The graph shows the expression and purification results of the fusion protein provided in the embodiments of this application under different induction conditions; Figure 7This is a graph showing the changes in the expression levels of immune-related genes in rice after treatment with the endogenous small peptide OsSSP31, as provided in the embodiments of this application. Figure 8 The image shows the detection results of the activation of rice ROS burst by the endogenous small peptide OsSSP31 in rice, as provided in the embodiments of this application. Figure 9 The image shows the detection results of calcium ion influx in detached rice leaves induced by the endogenous small peptide OsSSP31 provided in the embodiments of this application. Figure 10 The image shows the detection results of enhanced MAPK phosphorylation by the rice endogenous small peptide OsSSP31 provided in the embodiments of this application. Figure 11 The figure shows the experimental results of the rice endogenous small peptide OsSSP31 enhancing the resistance of rice to rice blast fungus, as provided in the embodiments of this application. Detailed Implementation
[0019] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0020] The range descriptions used in this application, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values within that range. For example, the range descriptions of "1 to 6" or "1~6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "comprising" and others used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships involved in this document, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained by purchasing from the market or by existing methods.
[0021] This application provides an application of rice endogenous peptide OsSSP31 in regulating rice resistance. The application includes using rice endogenous peptide OsSSP31 in the preparation of a regulator to regulate rice resistance; wherein the regulator includes an activator that activates rice immunity and / or a resistance agent that enhances rice resistance to rice blast fungus.
[0022] It should be noted that the rice endogenous small peptide OsSSP31 gene ID: LOC_Os01g68500, has a length of 516 bp.
[0023] It should be noted that the regulator prepared from the rice endogenous peptide OsSSP31 can exist in liquid form or powder form; the concentration of rice endogenous peptide OsSSP31 in the regulator can be 1 μmol / L or higher.
[0024] It should be noted that the application of the rice endogenous peptide OsSSP31 in regulating rice resistance provided in this application embodiment is based on the previously discovered unique role and pathway regulation of the rice endogenous peptide OsSSP31 in activating rice immunity and improving rice resistance to rice blast. Therefore, this application systematically broadens the depth and breadth of the application of rice endogenous peptides in agricultural green pest control from four dimensions: molecular mechanism, application form, technological advantages, and industrial extension. It addresses the shortcomings of existing green pest control technologies and provides a new endogenous immune regulation pathway that is both scientific and practical for the green pest control of rice diseases. The specific mechanism is as follows: I. Clarify the molecular mechanism of action and fill the technological gap in the immune regulation of endogenous small peptides.
[0025] Previous research on rice endogenous small peptides has largely remained at the preliminary identification stage, lacking clear molecular pathways and specific immunomodulatory effects, making it difficult to translate rice endogenous small peptides into practical control tools. This application, through a series of experiments, verified the five core functions of the rice endogenous small peptide OsSSP31, clearly elucidated the molecular mechanism by which OsSSP31 regulates rice resistance, filled the technological gap in the field of immune regulation of rice endogenous small peptides, and laid a solid theoretical foundation for its agricultural application.
[0026] 1. Activation of core immune signaling pathways: Rice endogenous peptide OsSSP31 can significantly enhance the phosphorylation level of MAPK (mitogen-activated protein kinase), which is the core hub of plant immune signal transduction. The phosphorylation and activation of MAPK can initiate a series of downstream immune responses, building a multi-layered defense system for rice. At the same time, rice endogenous peptide OsSSP31 can induce the upregulation of the expression of rice immune-related genes (such as PR genes and defense enzyme genes), further enhancing the rice's own immune response capabilities. This elucidates the core mechanism of disease resistance of rice endogenous peptide OsSSP31 at the molecular level.
[0027] 2. Triggering early immune response: Rice endogenous peptide OsSSP31 can rapidly activate the ROS (reactive oxygen species) burst in rice. As an early signaling molecule in plant immune response, ROS can directly inhibit the infection and proliferation of pathogens, mediate the expression of downstream defense genes, initiate hypersensitive necrosis (HR) response, and prevent further spread of pathogens. At the same time, rice endogenous peptide OsSSP31 can induce calcium ion influx in detached rice leaves. Calcium ions, as second messengers, participate in the transmission and amplification of immune signals, accelerate the initiation of immune response, and form a complete immune chain of "signal triggering - response amplification - defense activation".
[0028] 3. Targeted control of major diseases: Under the synergistic effect of the above molecular mechanisms, the rice endogenous small peptide OsSSP31 can significantly enhance the resistance of rice to rice blast fungus, specifically solving the problem of controlling rice blast, the number one disease of rice. Traditional green control methods have problems such as rapid loss of resistance and unstable effect. However, the rice endogenous small peptide OsSSP31 achieves disease resistance by regulating the rice's own immune mechanism, filling the application gap of endogenous small peptides in the control of major rice diseases and enriching the rice endogenous immune regulation molecular resources that can be used for agricultural production.
[0029] Second, based on clear effects, innovate the agricultural application forms of endogenous small peptides and break through industrialization bottlenecks.
[0030] Based on the five core effects of rice endogenous small peptide OsSSP31, this application breaks through the limitations of traditional rice endogenous small peptide applications that rely on transgenic technology, and innovatively develops an industrializable application form, transforming molecular research in the laboratory into a green control tool that can be used in agricultural production, thus greatly expanding its application scenarios.
[0031] 1. Pioneering an "exogenous spraying" application model to lower the barriers to promotion: Unlike the traditional transgenic overexpression of small peptide genes for disease resistance breeding, this application utilizes the characteristic of rice endogenous small peptide OsSSP31 to trigger an immune response in rice through exogenous application (without altering the rice genome, it can induce a series of immune responses such as upregulation of immune-related genes and ROS burst). If rice endogenous small peptide OsSSP31 is developed into an immune activator / resistance agent that can be directly sprayed, this product has high safety, and the small peptide molecules of rice endogenous small peptide OsSSP31 are easy to synthesize, easy to degrade, and leave no residue. It can be produced on a large scale and at low cost through chemical synthesis or bio-fermentation, solving the core problem that endogenous small peptides are difficult to industrialize and apply, allowing endogenous small peptides to be quickly applied to field production.
[0032] 2. Achieving dual control through "prevention + treatment" and enhancing application value: Based on the well-defined effects of rice endogenous peptide OsSSP31, regulators containing rice endogenous peptide OsSSP31 can achieve dual control effects: during preventive spraying, it can induce the expression of immune-related genes in advance, activate ROS burst, and initiate calcium ion signaling, thus constructing a basic immune barrier for rice and reducing the probability of rice blast infection; during therapeutic spraying, it can rapidly enhance MAPK phosphorylation levels, amplify immune responses, inhibit pathogen hyphal expansion, and reduce disease severity, significantly improving the practicality of rice endogenous peptide OsSSP31 in field control and broadening its application scenarios.
[0033] Third, based on the core effectiveness advantages, make up for the shortcomings of existing green prevention and control technologies, and enhance application competitiveness.
[0034] The five major effects of rice endogenous peptide OsSSP31 give it unparalleled advantages over traditional control technologies in terms of drug resistance, environmental safety, and efficacy stability. This further expands the application space of rice endogenous peptide OsSSP31 in green control and promotes the upgrading of green control technologies.
[0035] 1. Resistance Risk: Traditional chemical pesticides directly target pathogens, resulting in a very high risk of resistance. Traditional biological pesticides inhibit pathogens through biological pathways, thus having a low to medium risk of resistance. However, rice endogenous small peptide OsSSP31 works by inducing immune-related genes and activating ROS and other autoimmune mechanisms. It does not directly target pathogens, thus avoiding target mutations in pathogens and resulting in a very low risk of resistance.
[0036] 2. Environmental safety: Traditional chemical pesticides are prone to residue pollution and can easily damage the ecological environment, resulting in low environmental safety. Traditional biological pesticides are also easily affected by the environment, making their environmental safety moderate. Rice endogenous small peptide OsSSP31 is derived from rice endogenous substances, can be rapidly degraded, leaves no residue, and is safe for humans, animals, and natural enemies of rice-related pests and diseases, meeting green agriculture standards. Therefore, rice endogenous small peptide OsSSP31 has extremely high environmental safety.
[0037] 3. Efficacy stability: Traditional chemical pesticides have high efficacy stability due to the high stability of molecules. However, traditional biological pesticides are easily affected by environmental factors such as temperature, humidity, and soil properties, resulting in lower efficacy stability. In contrast, the rice endogenous small peptide OsSSP31 acts directly on rice itself, activating the rice's immune pathway through stable signaling pathways such as MAPK phosphorylation and calcium ion influx. It is less affected by the environment and therefore has high efficacy stability.
[0038] 4. Scope of action: Traditional chemical pesticides can only act on pathogens at a single target, resulting in a narrow scope of action. In contrast, traditional biological pesticides can inhibit the growth of pathogens through biological action, which makes their scope of action more moderate. Rice endogenous small peptide OsSSP31 can activate a broad-spectrum immune response to deal with multiple pathogens such as rice blast fungus, and the upregulation of immune-related genes can enhance the overall defense capability, thus having a wide scope of action.
[0039] The rice endogenous peptide OsSSP31 fundamentally avoids the development of pathogen resistance through mechanisms such as inducing the expression of immune-related genes, activating ROS burst, and enhancing MAPK phosphorylation, thus enabling long-term sustainable control of rice blast. At the same time, the endogenous nature of rice endogenous peptide OsSSP31 completely solves the problem of traditional pesticide residue pollution, significantly improves food safety and environmental safety, and further broadens its application scope in organic and green agriculture.
[0040] IV. Extend the application boundaries of the effects, expand industrial value, and promote the upgrading of the green prevention and control system.
[0041] Based on the five core effects of rice endogenous small peptide OsSSP31, the application of rice endogenous small peptide OsSSP31 is not limited to direct disease control, but also extends to multi-dimensional industrial value, further broadening the application boundaries of rice endogenous small peptides in agricultural green prevention and control, and promoting the construction of a more complete green prevention and control system.
[0042] 1. Core Components of a Comprehensive Green Prevention and Control System: Regulators containing the rice endogenous peptide OsSSP31 can be used synergistically with existing green prevention and control technologies such as disease-resistant varieties, biopesticides, and agricultural practices. When combined with varieties with low resistance to rice blast, it can compensate for insufficient varietal resistance by inducing the expression of immune-related genes and enhancing MAPK phosphorylation. When combined with biocontrol bacteria, it can improve the stability of the control effect of biocontrol bacteria by activating ROS bursts and initiating calcium ion signaling. Simultaneously, the rice endogenous peptide OsSSP31 can be applied to different growth stages of rice, from seedling to heading stage. Especially for difficult-to-control diseases such as neck blast, exogenous spraying can rapidly activate the immune response, achieving full-process control and broadening its application scenarios.
[0043] 2. Providing a paradigm for the application of endogenous small peptides in other crops: This application clarifies the five major effects of the rice endogenous small peptide OsSSP31 and elucidates its molecular mechanism. It establishes a complete technical route of "small peptide identification - functional verification (immune genes, ROS, calcium ions, MAPK, disease resistance) - regulator development - field application". This route can be directly replicated in the research of endogenous small peptides in other food crops such as wheat and corn, promoting the discovery and development of more endogenous small peptides with agricultural application value, and further broadening the overall application scope of plant endogenous small peptides in green pest control.
[0044] 3. Extend the value of the industrial chain: In addition to being directly developed as a spray-type regulator, the effects of rice endogenous small peptide OsSSP31 can also be extended to multiple applications: (1) It can be used as a molecular marker to screen rice disease-resistant varieties that naturally express high levels of rice endogenous small peptide OsSSP31 (i.e., high expression of immune-related genes, strong ROS burst ability, and high MAPK phosphorylation level), thereby accelerating the disease-resistant breeding process; (2) It can be developed as a seed treatment agent, which can induce calcium ion influx and activate immune response during the rice sowing period through seed soaking or coating, thereby achieving "full-process immune control from the seedling stage", further expanding its industrial application value.
[0045] In summary, this application provides an application of the rice endogenous peptide OsSSP31 in regulating rice resistance. This application clarifies five core effects of OsSSP31: inducing upregulation of expression of immune-related genes in rice, activating ROS burst, inducing calcium ion influx, enhancing MAPK phosphorylation, and improving resistance to rice blast fungus. It clearly elucidates the molecular mechanism by which OsSSP31 regulates rice resistance, transforming rice endogenous peptides from "laboratory molecules" in basic research into "green control tools" usable in agricultural production. This not only specifically addresses key technical challenges in the green control of rice blast, but also innovates the agricultural application of endogenous peptides, compensates for existing technological shortcomings, extends industrial value, establishes a standardized paradigm for the agricultural application of endogenous peptides, promotes the industrialization of the entire field of plant peptide immune regulation, and significantly broadens the application scope of rice endogenous peptides in green agricultural control, providing new technical support for reducing chemical pesticide use, ensuring food security, and promoting sustainable agricultural development.
[0046] In some optional embodiments, the active fragment of the rice endogenous peptide OsSSP31 has an amino acid sequence as shown in SEQ ID NO.1, or has an amino acid sequence that is ≥90% similar to the amino acid sequence shown in SEQ ID NO.1.
[0047] In these embodiments, the active fragment of the rice endogenous peptide OsSSP31 is defined to have an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence with ≥90% similarity to the amino acid sequence shown in SEQ ID NO.1. This clarifies the specific composition of the conserved active fragment in the rice endogenous peptide OsSSP31, which is beneficial for the study of the mechanism of action and large-scale production of the rice endogenous peptide OsSSP31.
[0048] In some optional embodiments, the molecular formula of the active fragment of the rice endogenous peptide OsSSP31 is C 336 H 503 N 81 O 87 S, the relative molecular mass of the rice endogenous small peptide OsSSP31 is ≥7100 Da.
[0049] In these embodiments, the molecular formula is C 336 H 503 N 81 O 87The presence of S and rice endogenous peptide OsSSP31 with a relative molecular mass ≥7100 Da clarifies the specific chemical composition and molecular weight of this rice endogenous peptide OsSSP31, which is beneficial for subsequent research on the mechanism of action of rice endogenous peptide OsSSP31. It also clarifies the mechanisms by which rice endogenous peptide OsSSP31 induces upregulation of rice immune-related gene expression, activates in vivo ROS burst, induces calcium ion influx in detached leaves, enhances MAPK phosphorylation, and improves resistance to rice blast fungus.
[0050] In some alternative embodiments, the instability coefficient of the rice endogenous peptide OsSSP31 is ≤33.0.
[0051] In these embodiments, the rice endogenous peptide OsSSP31 with an instability coefficient ≤33.0 indicates that the rice endogenous peptide OsSSP31 has strong stability and can exist stably and act in rice.
[0052] In some optional embodiments, the raw material for the regulator includes a fermentation expression product containing the rice endogenous small peptide OsSSP31, and the fermentation expression product includes at least one of engineered bacterial fermentation broth, bacterial cell lysate, purified fermented protein liquid, and freeze-dried fermentation broth preparation.
[0053] In these embodiments, the raw materials of the regulator are limited to the fermentation expression product of rice endogenous small peptide OsSSP31, and the fermentation expression product is limited to at least one of engineered bacterial fermentation broth, bacterial cell lysate, purified fermented protein liquid and freeze-dried fermentation broth preparation. This can cover most of the bio-fermentation technologies that can produce rice endogenous small peptide OsSSP31, and rice endogenous small peptide OsSSP31 can be produced on a large scale at a lower cost through bio-fermentation.
[0054] It should be noted that when the regulator is engineered bacterial fermentation broth or bacterial lysate, the engineered bacterial fermentation broth or bacterial lysate can be inactivated and purified to avoid the impact of live bacteria in the fermentation broth or bacterial lysate on rice crops.
[0055] In some alternative embodiments, the raw materials for the regulator may also include at least one of plant immune inducers, biopesticide compositions, and plant disease resistance agents.
[0056] In these embodiments, the raw materials for the limiting regulator may also include at least one of plant immune inducers, biopesticide compositions, and plant disease resistance agents. By combining the plant immune inducers, biopesticide compositions, or plant disease resistance agents with the fermentation expression product containing the rice endogenous small peptide OsSSP31, the disease resistance of rice can be significantly improved.
[0057] It should be noted that the plant immune inducer can be a 5% amino oligosaccharide aqueous solution (Haidaosu); the biological pesticide composition can be a mixture of 24% Jinggang ethoxylate microemulsion (Tianrunhe) and Haidaosu (Zhengye No. 1); and the plant disease resistance agent can be 24% Jinggang ethoxylate microemulsion (Tianrunhe).
[0058] Figure 1 An exemplary schematic diagram of a method flow for preparing a regulator for application provided by an embodiment of this application is shown; Based on a general inventive concept, such as Figure 1 As shown, this application provides a method for preparing the regulator used in the application, the method comprising: S1. The target gene encoding the rice endogenous small peptide OsSSP31 was ligated into an expression vector to construct a recombinant expression vector; S2. The recombinant expression vector is introduced into host bacterial cells to obtain recombinant bacterial cells; S3. The recombinant bacterial cells are induced to express a fusion protein using an inducer; wherein the gene encoding the fusion protein has a nucleotide sequence as shown in SEQ ID NO.2, or has a nucleotide sequence with ≥90% similarity to the nucleotide sequence shown in SEQ ID NO.2; S4. The fusion protein is purified to obtain a regulator.
[0059] It should be noted that traditional methods for obtaining endogenous small peptides from rice mainly rely on chemical synthesis, which suffers from high costs, difficulties in large-scale production, and insufficient engineering applications, thus limiting the widespread application of plant endogenous small peptides in green pest control in agriculture. In contrast, this application presents a novel technical route for the green and efficient preparation of plant endogenous small peptides, using a microbial fermentation expression system based on a recombinant expression vector → recombinant bacterial cells → fusion protein → purification.
[0060] In some alternative embodiments, a hydrophilic tag is provided at the N-terminus of the nucleotide sequence encoding the fusion protein.
[0061] In these embodiments, a hydrophilic tag is set at the N-terminus of the nucleotide sequence encoding the fusion protein. By setting the hydrophilic tag, the rice endogenous small peptide OsSSP31 can be changed from hydrophobic to hydrophilic, which meets the purification requirements for large-scale preparation and is conducive to clarifying the specific mechanism of action of the rice endogenous small peptide OsSSP31.
[0062] In some alternative embodiments, the nucleotide sequence encoding the fusion protein is as shown in SEQ ID NO.2, or has ≥90% similarity to the nucleotide sequence shown in SEQ ID NO.2.
[0063] In these embodiments, for the amino acid sequence of the rice endogenous peptide OsSSP31 determined above, the nucleotide sequence of the fusion protein containing the rice endogenous peptide OsSSP31 or the nucleotide sequence with similarity ≥90% can be deduced by combining the amino acid sequence with information such as functional tags. This is beneficial for constructing a recombinant vector of the rice endogenous peptide OsSSP31 and realizing the mass production of the rice endogenous peptide OsSSP31 through biological methods.
[0064] In some alternative embodiments, the inducer comprises isopropyl-β-D-thiogalactoside, the molar concentration of which is from 0.25 mmol / L to 1.00 mmol / L; and / or the induction temperature is from 16°C to 37°C.
[0065] In these embodiments, isopropyl-β-D-thiogalactoside is used as an inducer, and the molar concentration of the inducer is limited to 0.25 mmol / L to 1.00 mmol / L. This inducer can promote the expression of the rice endogenous peptide OsSSP31 in recombinant bacteria, resulting in a large amount of fusion protein containing the rice endogenous peptide OsSSP31. Furthermore, induction at temperatures between 16°C and 37°C can promote sufficient expression of the rice endogenous peptide OsSSP31 in recombinant bacteria, yielding a large amount of fusion protein containing the rice endogenous peptide OsSSP31.
[0066] The molar concentration of the inducer can be 0.25 mmol / L, 0.30 mmol / L, 0.35 mmol / L, 0.40 mmol / L, 0.45 mmol / L, 0.50 mmol / L, 0.60 mmol / L, 0.70 mmol / L, 0.80 mmol / L, 0.90 mmol / L, or 1.00 mmol / L.
[0067] The induction temperature can be 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 30℃, 35℃ or 37℃.
[0068] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national / industry standards; if there is no corresponding national / industry standard, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.
[0069] Example 1
[0070] I. Identification of OsSSP31, an endogenous small peptide in rice: 1. Homologous protein alignment and conserved active sequence determination of the rice endogenous small peptide OsSSP31 family: BLAST search was performed on the rice protein database using the rice endogenous small peptide OsSSP31 protein sequence, followed by phylogenetic tree construction using MEGA7. The results are as follows: Figure 2 As shown, the amino acid sequence conservation of the rice endogenous small peptide OsSSP31 was analyzed using online software such as (ESPript https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi) and (WebLogo https: / / weblogo.threeplusone.com / create.cgi). Highly conserved sites were highlighted, and the results are as follows: Figure 3 As shown, the amino acid sequence of the conserved active fragment of the rice endogenous small peptide OsSSP31 was determined accordingly, and the results are as follows: YNATVAGVISYGRIASLSGVSAQDLFLWFPVRGIRVDVPSSGVIYFDVGVVFKHFPLAVFEAPPPC (SEQ ID NO. 1).
[0071] The amino acid sequence of this conserved active fragment is used as the base sequence for subsequent functional verification and recombinant expression of candidate target gene fragments.
[0072] 2. Physicochemical analysis of the conserved active region of the rice endogenous small peptide OsSSP31 and construction of its expression vector: The amino acid sequence of the conserved active region was predicted using the ProtParam online software, and the results are as follows: Figure 4 As shown, the results indicate that the conserved active region of the rice endogenous peptide OsSSP31 has the molecular formula C. 336 H 503 N 81 O 87 S has a relative molecular mass of 7101.26 Da and an isoelectric point pI = 5.02.
[0073] Furthermore, the instability coefficient of the rice endogenous peptide OsSSP31 is 32.65, indicating that this rice endogenous peptide OsSSP31 can exist stably. The average total hydrophilicity (GRAVY) of the rice endogenous peptide OsSSP31 is 0.489, and the GRAVY value > 0, indicating that the rice endogenous peptide OsSSP31 has strong hydrophobicity and is not easily dissolved in water. Therefore, the rice endogenous peptide OsSSP31 is a hydrophobic protein. Hydrophobic proteins are not conducive to subsequent protein purification. Therefore, when constructing recombinant plasmids, a hydrophilic SUMO tag can be added to the N-terminus of the hydrophobic protein, ultimately forming a... Figure 5The fusion protein shown contains the rice endogenous small peptide OsSSP31. The target gene fragment encoding this fusion protein is: (SEQ ID NO.2).
[0074] Using PET28a-SUMO as the expression vector, the recombinant plasmid pET28a(+)-SUMO-OsSSP31 was constructed by artificially synthesizing the OsSSP31 DNA fragment.
[0075] 3. Induction and purification of fusion protein: The recombinant plasmid obtained above was transformed into BL21 Escherichia coli. After screening and culturing in LB solid medium with kanamycin resistance for 12 h, single colonies that could grow normally were picked and inoculated in LB liquid medium with kanamycin resistance and cultured at 37 ℃ and 220 rpm for 12 h. The glycerol bacteria were then frozen at -80 ℃.
[0076] The transformed and revived BL21 bacterial suspension was spread onto kanamycin-resistant LB agar plates and incubated overnight at 37°C. Single colonies were then picked and transferred to LB liquid medium and incubated at 37°C and 220 rpm for 2 h. Then, different concentrations of isopropyl-β-D-thiogalactoside (IPTG, 0.25 mmol / L, 0.50 mmol / L, and 1.00 mmol / L) were added, and the induction temperatures were set at 16°C, 28°C, and 37°C, respectively. The plates were then shaken at 100 rpm for 12 h to induce expression of the recombinant bacteria. During the shaking culture process, 1 mL of bacterial suspension was collected every hour as a sample. The sample was then centrifuged at 12,000 rpm for 1 min to remove the supernatant and collect the bacterial cells. The bacterial cells were then disrupted by ultrasonic disruption under ice bath conditions, and the disrupted bacterial cell suspension was centrifuged at 10,000 rpm for 30 min at 4°C to obtain a supernatant containing recombinant protein. The supernatant containing recombinant protein was then transferred to a gravity column containing Ni-NTA, and the supernatant before and after purification was collected. The purification status was monitored by SDS-PAGE. The results are as follows: Figure 6 As shown, the theoretical molecular weight of the fusion protein obtained after purification is approximately 30 kDa.
[0077] Example 2
[0078] Based on the fusion protein obtained in Example 1, the following experiments were further conducted: 4. Induction of Rice Immune-Related Gene Expression by Rice Endogenous Small Peptide OsSSP31: 15-day-old rice seedlings were treated with flg22 (Flagellin 22-amino-acid peptide, a conserved 22-peptide fragment of bacterial flagellin, belonging to pathogen-associated molecules that can trigger innate immune responses in plants) and OsSSP31 liquid (a fusion protein containing the rice endogenous small peptide OsSSP31 at a concentration of 1 μmol / L) for 3 hours respectively. Immediately afterwards, the plant samples were frozen in liquid nitrogen and ground into fine powder. Equal masses of the powder were placed in pre-chilled 1.5 mL centrifuge tubes. Total RNA was extracted from the powder using the Jianshi Biotechnology Plant Total RNA Extraction Kit, and cDNA was synthesized using the Takara PrimeScript™ Reverse Transcription Kit to obtain rice cDNA. Finally, based on qRT-PCR technology, rice cDNA was detected using the SYBR® Premix Ex Taq™ kit (Takara) to detect immune-related genes OsNAC4, OsPAL1, OsPR3, OsPR1a, and OsWRKY45. The reaction system and procedure were prepared according to the manufacturer's instructions. After the reaction, the number of cycles (Ct value) required for the fluorescence signal in each reaction tube to reach the set fluorescence threshold was recorded. Actin (LOC_Os03g50885) was used as an internal control to calibrate the Ct values of all detected rice cDNAs. Each rice cDNA was tested three times, and the average of the three replicates was taken for each data set. The results are as follows: Figure 7 As shown, by Figure 7 It can be seen that treatment with the rice endogenous small peptide OsSSP31 can induce upregulation of the expression of immune-related genes in rice, indicating that the rice endogenous small peptide OsSSP31 can activate the innate immune response in rice.
[0079] 5. Rice Endogenous Small Peptide OsSSP31 Activates ROS Burst in Rice: Quantitative Analysis of Reactive Oxygen Spectroscopy (ROS) Levels in Rice Leaves Based on Luminol Chemiluminescence. Two-week-old rice seedlings were harvested, and 3mm leaf segments were cut from the leaves to obtain small leaf samples. These leaf segments were immersed in sterile water for 12 hours, then the sterile water was removed, and the leaf segments were transferred to 96-well plates. A solution containing 10 mmol / L luminol and 5 mg / mL peroxidase was added to each well. Finally, flg22 and OsSSP31 were added to a final concentration of 1 μmol / L. The 96-well plates were immediately placed in a microplate reader to detect fluorescence signals. The fluorescence signals in each well of the 96-well plate were continuously monitored at a frequency of once per minute for 60 minutes, and the relative optical units (RLU) values were recorded to track the dynamic generation of ROS. Results are as follows: Figure 8As shown, the rice endogenous small peptide OsSSP31 can significantly activate the ROS burst in rice.
[0080] Rice endogenous peptide OsSSP31 induces calcium ion influx in rice: This study investigated the effect of rice endogenous peptide OsSSP31 on calcium ion influx from detached rice leaves using non-invasive microseismic (NMT) technology. 2+ The induction effect of influx. Wild-type rice leaves were treated with flg22 and OsSSP31 at a final concentration of 1 μmol / L for 8 min and the results were recorded. Then, the treated wild-type rice leaves were fixed in an NMT detection device, and Ca2+ in the epidermal cells of wild-type rice leaves was detected in real time. 2+ The flow rate, in units of pmol / (cm). 2 •s), recording the dynamic changes in ion current once per minute. Simultaneously, untreated wild-type rice leaves were used as a negative control group and a positive control group treated only with flg22. The Ca2+ levels in different groups were compared. 2+ Peak influx rate, cumulative amount, and time-series characteristics were analyzed to clarify the ability of the rice endogenous small peptide OsSSP31 to specifically activate calcium signaling. Results are as follows: Figure 9 As shown, the rice endogenous peptide OsSSP31 can induce calcium ion influx in rice leaf epidermal cells. In summary, the above experiments indicate that the rice endogenous peptide OsSSP31 can activate a typical immune response in the early immune signaling stage.
[0081] 6. Rice endogenous small peptide OsSSP31 enhances MAPK phosphorylation: Normally cultured rice seedlings were used, and leaf samples of 3mm to 5mm length were cut. The cut leaf samples were placed in sterile water overnight, then the sterile water was discarded. The leaf samples were treated with 1μmol / L flg22 and OsSSP31 solution for 0min, 5min, 15min, and 30min respectively. After treatment, the leaf samples were thoroughly frozen in liquid nitrogen and then ground into powder. Equal masses of leaf powder were placed in 1.5mL pre-chilled centrifuge tubes, and MAPK extraction buffer was added to extract proteins from the leaf powder, yielding protein detection samples. After mixing the protein sample, it was placed on ice and incubated for 30 min. Then, it was centrifuged at 12000 rpm for 15 min at 4°C. After centrifugation, the supernatant was weighed, and 4×SDS-PAGE loading buffer was added according to the specified ratio and mixed thoroughly. The mixture was then boiled at 95°C for 10 min to denature the protein sample, yielding the test protein sample. A suitable amount of the test protein sample was used for Western blotting validation. The phosphorylation intensity of MPK6 / 3 was detected using a phospho-p44 / 42 MAPK antibody, and the amount of Actin protein in the sample was used as an internal control for calibration. Results are as follows: Figure 10As shown, the rice endogenous peptide OsSSP31 can enhance the phosphorylation level of MAPK in rice, indicating that the rice endogenous peptide OsSSP31 can trigger the rice immune-related signal transduction pathway.
[0082] Example 3
[0083] Based on the experiments in Example 2, the following further experiments were conducted: 7. Rice endogenous peptide OsSSP31 enhances rice resistance to rice blast fungus: The fermentation expression product of rice endogenous peptide OsSSP31 (purified protein solution, containing a fusion protein concentration of 1 μmol / L) was sprayed onto the surface of normally growing rice plants. Subsequently, rice blast fungus spores (numbered 70-15) were inoculated. Disease incidence on rice leaves was observed and photographed. Rice leaves were then cut, and the area of blast fungus lesions was measured. The disease evaluation method for rice blast fungus was based on "DB43 / T 2032—2021 Technical Specification for Indoor Comprehensive Evaluation of Rice Blast Resistance Characteristics". Results are as follows: Figure 11 As shown, by Figure 11 It is known that treatment with the endogenous small peptide OsSSP31 in rice can enhance the resistance of rice to rice blast fungus, with a disease resistance improvement of 3 levels compared to rice, which can reduce the severity of disease occurrence.
[0084] In summary, the application of the rice endogenous peptide OsSSP31 in regulating rice resistance provided in this application embodiment clarifies five core effects of the rice endogenous peptide OsSSP31: inducing upregulation of expression of immune-related genes in rice, activating ROS burst in vivo, inducing calcium ion influx in detached leaves, enhancing MAPK phosphorylation, and improving resistance to rice blast fungus. This systematically broadens the depth and breadth of the application of rice endogenous peptides in green pest control in agriculture, overcomes the shortcomings of existing green pest control technologies, and provides a new endogenous immune regulation pathway that is both scientific and practical for green pest control of rice diseases.
[0085] In addition, the embodiments of this application provide a method for preparing regulators for application. This method, by constructing a microbial fermentation expression system, replaces the traditional chemical synthesis of plant endogenous peptides, providing a new technical path for the green and efficient preparation of rice endogenous small peptide OsSSP31. At the same time, it also provides a new technical approach for novel immune inducers containing rice endogenous small peptide OsSSP31, and provides a potential technical solution for expanding the application of plant endogenous small peptides in green agricultural control.
[0086] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed in this application.
Claims
1. The application of a rice endogenous small peptide OsSSP31 in regulating rice resistance, characterized in that, The application includes: using the rice endogenous small peptide OsSSP31 in the preparation of a regulator to regulate rice resistance; wherein the regulator includes an activator that activates rice immunity and / or a resistance agent that enhances rice resistance to rice blast fungus.
2. The application according to claim 1, characterized in that, The active fragment of the rice endogenous small peptide OsSSP31 has an amino acid sequence as shown in SEQ ID NO.1, or has an amino acid sequence with ≥90% similarity to the amino acid sequence shown in SEQ ID NO.
1.
3. The application according to claim 1, characterized in that, The molecular formula of the active fragment of the rice endogenous peptide OsSSP31 is C 336 H 503 N 81 O 87 S, the relative molecular mass of the active fragment of the rice endogenous small peptide OsSSP31 is ≥7100 Da.
4. The application according to claim 1, characterized in that, The instability coefficient of the rice endogenous peptide OsSSP31 is ≤33.
0.
5. The application according to claim 1, characterized in that, The raw material for the regulator includes a fermentation expression product containing the rice endogenous small peptide OsSSP31, and the fermentation expression product includes at least one of engineered bacterial fermentation broth, bacterial cell lysate, purified fermented protein liquid, and freeze-dried fermentation broth preparation.
6. The application according to claim 1, characterized in that, The raw materials for the regulator also include at least one of plant immune inducers, biopesticide compositions, and plant disease-resistant agents.
7. A method for preparing the regulator for use in any one of claims 1 to 6, characterized in that, The method includes: The target gene encoding the rice endogenous small peptide OsSSP31 was ligated into an expression vector to construct a recombinant expression vector; The recombinant expression vector was introduced into host bacterial cells to obtain recombinant bacterial cells; The recombinant bacterial cells were induced to express the fusion protein using an inducer. The fusion protein was purified to obtain a regulator.
8. The method according to claim 7, characterized in that, The nucleotide sequence encoding the fusion protein has a hydrophilic tag at its N-terminus.
9. The method according to claim 7 or 8, characterized in that, The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO.2, or has a similarity of ≥90% to the nucleotide sequence shown in SEQ ID NO.
2.
10. The method according to claim 7, characterized in that, The inducing agent comprises isopropyl-β-D-thiogalactoside, and the molar concentration of the inducing agent is from 0.25 mmol / L to 1.00 mmol / L; and / or The induction temperature is between 16°C and 37°C.