Disease-resistant crop breeding method driven by gene introduction

Abstract

The invention belongs to the technical field of plant breeding, and discloses a gene introduction-driven disease-resistant crop breeding method, which comprises the following steps: collecting pathogen population information in a target crop planting environment, constructing a pathogen dynamic action spectrum, identifying genetic regulation sites for inducing a target crop to generate stress response, and performing gene introduction-driven disease-resistant crop breeding. Carrying out conjoint analysis on transcriptional activation sites under pathogen stress, and outputting a stress expression region of the target crop; designing an inducible disease-resistant factor expression vector based on a stress expression region, and embedding the inducible disease-resistant factor expression vector into a target crop cell through a transformation means to form a transgenic plant capable of inducing and activating disease-resistant expression under pathogen stress; carrying out cross-generation tracking on a gene expression mode of a transgenic plant under pathogen stress and non-stress conditions, identifying whether an introduced gene is gradually replaced by an endogenous gene, and judging whether a target crop realizes endogenous evolutionary convergence of disease-resistant expression; and the sustainability of agricultural production can be improved.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and more specifically, to a gene-introduction-driven method for breeding disease-resistant crops. Background Technology

[0002] Patent CN119391764A discloses a method for high-yield rice breeding by introducing a salt-tolerant gene, comprising the following steps: S1, gene screening and cloning: screening for genes with salt-tolerant capabilities; S2, vector construction: inserting the screened salt-tolerant gene into a suitable expression vector; S3, rice cell transformation: introducing the vector carrying the salt-tolerant gene into rice cells; S4, screening positive plants: confirming successful induction of the salt-tolerant gene into rice cells and selecting positive plants for further cultivation; S5, tissue culture and development: placing the positive plants on a sterile culture medium for tissue culture to develop into complete plants; S6, planting and harvesting: transplanting the obtained transgenic rice plants and harvesting them to obtain rice seeds. This invention, with its salt-tolerant gene, exhibits stronger salt-tolerant performance, and its yield is not affected when planted in saline-alkali land, resulting in high-yield rice.

[0003] Existing methods for breeding disease-resistant crops have the following drawbacks: Existing transgenic disease resistance methods largely rely on continuous, high-expression construction strategies, failing to regulate expression based on the dynamic process of pathogen invasion. This easily leads to wasted plant resources, growth inhibition, or abnormal expression under non-stress conditions. Long-term, high-intensity expression can activate the plant's transgenic silencing mechanism, causing unstable or even inactivated expression of the target disease resistance gene in actual field environments, reducing the persistence of disease resistance. Pathogen distribution and stress during crop growth are dynamic, and traditional methods lack the ability to respond to such spatiotemporal variations, resulting in limited resistance adaptation ranges. Existing breeding methods largely rely on experience and phenotypic selection, lacking mathematical modeling and theoretical guidance for the expression process, making expression system optimization a crude and inefficient process.

[0004] Current transgenic disease resistance breeding relies on the sustained expression of exogenous genes to maintain disease resistance. However, long-term retention of exogenous genes poses potential risks of environmental leakage and raises public acceptance concerns, making it difficult to meet the requirements for non-GMO regulation or market access. Existing technologies mostly employ static expression construction; once transferred into the target plant, they struggle to adjust expression levels in response to crop growth changes or stress conditions. Furthermore, there is no effective mechanism to actively shut down or remove exogenous sequences when disease resistance no longer depends on them. This traditional approach, which relies on exogenous disease resistance mechanisms in the long term, essentially lacks a strategic design for reverting to high-quality, non-GMO germplasm, hindering the deep integration of modern molecular breeding with the goals of green and sustainable agriculture.

[0005] In view of this, the present invention proposes a gene-introduction-driven method for breeding disease-resistant crops to solve the above problems. Summary of the Invention

[0006] To overcome the aforementioned deficiencies of the prior art and to achieve the above objectives, the present invention provides the following technical solution: a gene-introduction-driven method for breeding disease-resistant crops, comprising: S1. Collect pathogen population information in the target crop planting environment, construct the pathogen dynamic action spectrum, identify the genetic regulatory sites that induce stress response in the target crop, jointly analyze the transcriptional activation sites under pathogen stress, and output the stress expression region of the target crop. S2. Based on the stress expression region, design an inducible disease resistance factor expression vector, embed it into the target crop cell through transformation, and form a transgenic plant that can induce and activate disease resistance expression under pathogen stress; S3. Cross-generational tracking of gene expression patterns in transgenic plants under pathogen stress and non-stress conditions, identifying whether introduced genes are gradually replaced by endogenous genes, and determining whether the target crop has achieved endogenous evolutionary convergence in disease resistance expression. S4. After the target crop achieves endogenous evolution convergence, the genetic clearance unit is activated by the pathogen-induced triggering mechanism. The clearance treatment of the chimeric target crop is carried out under the state of disease resistance expression saturation to obtain candidate plants without exogenous expression. S5. Conduct a comprehensive evaluation of the resistance and ecological adaptability of the candidate plants, and retain individuals with a comprehensive score greater than the preset comprehensive score threshold as high-quality disease-resistant germplasm for breeding.

[0007] Preferably, the method for collecting pathogen population information in the target crop planting environment includes: In the planting environment of the target crop, samples were collected from the rhizosphere soil, leaves, stems and fruits of the target crop containing pathogenic microorganisms through field sampling, covering the high incidence period of diseases during the growth of the target crop; The collected samples were processed using sterile cotton swabs, cone samplers, and liquid nitrogen quick-freezing technology to extract total microbial DNA or total RNA from the collected samples; the extracted microbial nucleic acids were amplified in specific regions, including the 16S rRNA gene region of bacteria, the ITS region of fungi, and the dsRNA region of viruses. The amplified products were subjected to high-throughput sequencing, and bioinformatics methods were used for quality control, sequence assembly, OTU clustering, and species annotation to obtain pathogen population information in the target crop planting environment. The pathogen population information includes pathogen species taxonomic composition, relative abundance, community structure, and spatial and temporal dynamic changes.

[0008] Preferably, the method for identifying the genetic regulatory site includes: Based on pathogen population information in the target crop planting environment, a dynamic pathogen interaction spectrum was constructed to identify key spatiotemporal nodes of pathogen stress. Corresponding tissue samples of the target crop were collected and transcriptome sequencing was performed to obtain gene expression profiles under pathogen stress and non-stress conditions. Differential expression analysis was used to screen gene clusters that respond to pathogen stress. Combined with genome-wide association analysis and epigenomics, genetic regulatory sites that induce stress responses in target crops were located. These genetic regulatory sites include transcription factor binding sites and open chromatin regions.

[0009] Preferably, the method for outputting the stress expression region of the target crop includes: The constructed pathogen dynamic action spectrum, gene expression spectrum, and genetic regulatory sites are integrated to form a multidimensional dataset containing the spatiotemporal characteristics of pathogen stress, gene expression changes, and regulatory sites. Based on the multidimensional dataset, the spatiotemporal correspondence between the dynamic changes of the pathogen community and the gene expression and transcriptional activation sites of the target crop is analyzed. By combining the dynamic changes in chromatin openness and transcription factor binding sites, we identified transcriptional activating elements and target genes induced by pathogen stress. We constructed a pathogen stress response gene regulatory network containing target genes and transcriptional activation sites. We defined the genes activated and involved in regulation under pathogen stress and all corresponding transcriptional activation sites as the stress expression regions of the target crop.

[0010] Preferably, the method for designing an inducible disease resistance factor expression vector includes: Screening for functional genes that are upregulated under pathogen stress and are associated with disease resistance from the stress expression regions of target crops. Functional genes include pathogen recognition receptor genes, signal transduction factor genes, and defense effector genes. Inducible regulatory elements that are co-expressed with or have a regulatory relationship with functional genes are extracted. The inducible regulatory elements and the coding sequence of the target crop are used to construct an expression cassette. The expression cassette is a functional module with pathogen-induced expression ability. Screenable marker elements and genetic clearance recognition elements are introduced into the expression vector to form an expression vector containing a complete expression cassette and functional modules; the induction effect of the constructed expression vector under pathogen stress conditions is verified by transient expression or stable transformation experiments.

[0011] Preferably, the method for obtaining the transgenic plant includes: The constructed inducible disease resistance factor expression vector was introduced into plant cells using transformation methods suitable for the target crop, including Agrobacterium-mediated transformation, gene gun method, electroporation method, and protoplast PEG-mediated transformation. The transformed cells were then subjected to resistance screening and tissue culture to obtain transgenic plants containing exogenous expression cassettes. The expression process of exogenous genes in transgenic plants was simulated, and the expression level of exogenous genes over time was controlled by an induction feedback expression regulation function, confirming that the designed inducible disease resistance factor expression vector operated effectively in transgenic plants.

[0012] Preferably, the method for obtaining the gene expression pattern includes: The transgenic plants were propagated through successive generations to obtain m generations of offspring plants; pathogen stress treatment group and non-stress control group were set up in each generation; target tissue samples were collected at the growth stage of each treatment group and total RNA was extracted. The expression levels of exogenous disease resistance factor genes and related regulatory elements were detected using quantitative real-time PCR, transcriptome sequencing, and reporter gene detection. The expression intensity, induction response rate, and temporal changes of exogenous genes in transgenic plants of different generations under pathogen stress and non-stress conditions were analyzed. Trend curves of expression levels with generation were plotted, and gene expression pattern analysis was performed.

[0013] Preferably, the method for determining whether the target crop has achieved endogenous evolutionary convergence in disease resistance expression includes: Based on the trend curve of expression level changes with generation, it is determined whether the expression of exogenous genes shows a continuous decline; based on transcriptome sequencing data, endogenous genes related to the function of exogenous disease resistance genes are screened, and it is analyzed whether the expression level of endogenous genes gradually increases in each generation; a co-expression network of exogenous and endogenous genes is constructed to infer the substitution relationship between endogenous genes and exogenous gene expression. The disease resistance function of endogenous genes is verified by gene knockout or repression techniques, and the ability of endogenous genes to maintain disease resistance under the condition of weakened exogenous gene expression is evaluated. By combining the trend of exogenous gene expression decline and endogenous gene expression enhancement in successive generations, and combining the plant disease resistance phenotype and physiological indicators, it is determined whether disease resistance expression dominated by endogenous genes has been formed, and the endogenous evolutionary convergence of disease resistance expression is achieved.

[0014] Preferably, the method for obtaining candidate plants without exogenous expression includes: After confirming through cross-generational tracking and expression pattern analysis that the target crop has achieved endogenous evolutionary convergence in disease resistance expression, the genetic scavenging unit is activated using a pathogen-induced triggering mechanism. The genetic scavenging unit includes promoters that can respond to pathogen stress and genes that perform scavenging functions. When the expression of disease resistance in the target crop reaches saturation and the pathogen stress signal is sensed, the expression of the genetic clearing unit is activated through the activation function of the genetic clearing unit, thereby initiating the clearing program; the activated genetic clearing unit triggers programmed cell clearing, and performs clearing treatment on the chimeric target crop under the state of saturated disease resistance expression, to obtain candidate plants without exogenous expression.

[0015] Preferably, the method for breeding as disease-resistant high-quality germplasm includes: Candidate plants were evaluated using a comprehensive assessment of their resistance and ecological adaptability, which included disease resistance indicators, agronomic trait indicators, and ecological adaptability indicators. Relevant disease resistance indicators, agronomic trait indicators, and ecological adaptability indicators were collected through field multi-environment experiments and molecular detection methods. Each indicator is quantified and weighted to calculate the comprehensive score of the candidate plants. Based on the preset comprehensive score threshold, candidate plants with a comprehensive score greater than or equal to the preset comprehensive score threshold are selected as disease-resistant and high-quality germplasm for breeding.

[0016] The technical effects and advantages of the gene-introduction driven disease-resistant crop breeding method of the present invention are as follows: By controlling the expression of disease resistance factors through pathogen-inducible promoters and regulatory elements, gene expression is activated only under pathogen stress and remains quiescent under non-stress conditions, thus avoiding energy waste and growth inhibition and improving resource utilization efficiency. A mathematical expression model is constructed by setting inducible input functions and expression feedback inhibition functions to predict and regulate transgenic expression behavior, reducing the risk of uncontrolled gene expression and systemic disorder.

[0017] By precisely controlling the expression intensity and duration, the risk of silencing caused by sustained high expression is avoided, improving the effective operating time and performance stability of exogenous genes in practical applications. It enables adaptive expression responses to different pathogen pressures, enhancing the disease resistance system's ability to recognize and respond to spatial and temporal variations in pathogen populations. Through induced feedback expression regulation functions, a quantitative correlation is established between biological signals and expression results, providing data support and modeling tools for screening disease resistance genes, designing expression vectors, and optimizing breeding materials, thus improving the accuracy and scientific rigor of the breeding process. It achieves a complete technical closed loop from pathogen identification, regulatory construction, transformation screening to expression modeling, providing a novel solution for intelligent and highly controllable disease-resistant crop breeding.

[0018] By precisely activating genetic clearance units using pathogen-induced signals to automatically remove exogenous disease resistance factors under the premise that crop disease resistance expression no longer depends on exogenous genes, candidate plants without exogenous expression are obtained. This reduces the potential risks of transgenic products to the environment and ecosystems, meeting the current urgent needs for green agriculture and sustainable breeding. Through the coupling of induced expression and saturation feedback, not only is on-demand expression of disease resistance factors achieved, but the clearance module is also triggered after expression reaches a threshold, effectively addressing the negative effects of overexpression. The clearance program is initiated through an induced trigger mechanism, achieving a natural transition from exogenous function introduction to endogenous expression replacement, constructing a dynamic evolutionary breeding pathway of introduction, adaptation, and release. Obtaining candidate plants ultimately without exogenous expression facilitates the market promotion and consumer acceptance of new varieties, while also reducing regulatory barriers for varieties entering the international market and enhancing the commercial application potential of germplasm resources. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of a gene-introduction-driven method for breeding disease-resistant crops. Figure 2 This is a schematic diagram of a gene-introduction-driven disease-resistant crop breeding system. Figure 3 The flowchart illustrates the method for breeding disease-resistant and high-quality germplasm provided by this invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0021] Please see Figure 1 and Figure 3 As shown in Example 1, a gene-introduction-driven disease-resistant crop breeding method proposed in this invention is further illustrated, including: With the increasing global demand for crop disease resistance in agricultural production, transgenic technology, as an important molecular breeding method, is widely used to improve crop disease resistance. However, existing transgenic disease resistance breeding methods generally suffer from several technical bottlenecks and shortcomings. Traditional transgenic disease resistance strategies often employ continuous, high-expression construction methods, which cannot regulate the expression of disease resistance genes in real time according to the dynamic changes of pathogen invasion, leading to waste of plant resources, growth inhibition, and abnormal expression under non-stress conditions. In addition, long-term high-intensity expression of exogenous genes can easily activate the transgenic silencing mechanism in plants, causing unstable expression or even inactivation of disease resistance genes in actual field environments, significantly reducing the durability and reliability of disease resistance.

[0022] The spatial distribution of pathogens and stress during crop growth exhibit significant dynamic and spatiotemporal variations. However, current technologies lack the ability to effectively respond to these spatiotemporal variations, thus limiting the adaptability of disease resistance. Furthermore, existing breeding methods primarily rely on experience and phenotypic selection, lacking mathematical modeling and systematic theoretical guidance for gene expression processes. This results in inefficient and extensive optimization of expression systems, making it difficult to achieve precise regulation of disease resistance.

[0023] More importantly, existing transgenic disease-resistant breeding methods generally rely on the continuous expression of exogenous genes to maintain disease resistance. This not only brings potential risks of environmental leakage but also faces limitations in public acceptance of transgenic products, making it difficult to meet the stringent requirements of non-GMO regulatory policies and market access. Traditional expression systems are mostly statically constructed. Once exogenous genes are introduced into plants, it is difficult to flexibly adjust the expression level according to the crop's own growth changes or pathogen stress. Furthermore, there is a lack of effective mechanisms to actively shut down or remove exogenous genes after disease resistance has been stably established.

[0024] To effectively address the above problems, this invention proposes a gene-introduction-driven method for breeding disease-resistant crops, comprising: S1. Collect pathogen population information in the target crop planting environment, construct the pathogen dynamic action spectrum, identify the genetic regulatory sites that induce stress response in the target crop, jointly analyze the transcriptional activation sites under pathogen stress, and output the stress expression region of the target crop. S2. Based on the stress expression region, design an inducible disease resistance factor expression vector, embed it into the target crop cell through transformation, and form a transgenic plant that can induce and activate disease resistance expression under pathogen stress; S3. Cross-generational tracking of gene expression patterns in transgenic plants under pathogen stress and non-stress conditions, identifying whether introduced genes are gradually replaced by endogenous genes, and determining whether the target crop has achieved endogenous evolutionary convergence in disease resistance expression. S4. After the target crop achieves endogenous evolution convergence, the genetic clearance unit is activated by the pathogen-induced triggering mechanism. The clearance treatment of the chimeric target crop is carried out under the state of disease resistance expression saturation to obtain candidate plants without exogenous expression. S5. Conduct a comprehensive evaluation of the resistance and ecological adaptability of the candidate plants, and retain individuals with a comprehensive score greater than the preset comprehensive score threshold as high-quality disease-resistant germplasm for breeding.

[0025] Methods for collecting pathogen population information in the target crop planting environment include: In the planting environment of the target crop, samples were collected from the rhizosphere soil, leaves, stems and fruits of the target crop where pathogenic microorganisms exist through field sampling. The sampling covered the high incidence period of diseases in the growth of the target crop (such as the greening period and the jointing period). The collected samples were processed using sterile cotton swabs, cone samplers, and liquid nitrogen quick-freezing technology to extract total microbial DNA or total RNA from the collected samples; the extracted microbial nucleic acids were amplified in specific regions, including the 16S rRNA gene region of bacteria, the ITS region of fungi, and the dsRNA region of viruses. The amplified products were subjected to high-throughput sequencing, and bioinformatics methods were used for quality control, sequence assembly, OTU clustering, and species annotation to obtain pathogen population information in the target crop planting environment. The pathogen population information includes pathogen species taxonomic composition, relative abundance, community structure, and spatial and temporal dynamic changes.

[0026] Methods for identifying genetic regulatory sites include: Based on pathogen population information in the target crop planting environment, a dynamic pathogen interaction spectrum was constructed to identify key spatiotemporal nodes of pathogen stress. Corresponding tissue samples of the target crop were collected and transcriptome sequencing was performed to obtain gene expression profiles under pathogen stress and non-stress conditions. Differential expression analysis was used to screen gene clusters that respond to pathogen stress. Combined with genome-wide association analysis and epigenomics, genetic regulatory sites that induce stress responses in target crops were located. These genetic regulatory sites include transcription factor binding sites and open chromatin regions.

[0027] Methods for outputting the stress expression region of the target crop include: The constructed pathogen dynamic action spectrum, gene expression spectrum, and genetic regulatory sites are integrated to form a multidimensional dataset containing the spatiotemporal characteristics of pathogen stress, gene expression changes, and regulatory sites. Based on the multidimensional dataset, the spatiotemporal correspondence between the dynamic changes of the pathogen community and the gene expression and transcriptional activation sites of the target crop is analyzed. By combining the dynamic changes in chromatin openness and transcription factor binding sites, we identified transcriptional activating elements and target genes induced by pathogen stress. We constructed a pathogen stress response gene regulatory network containing target genes and transcriptional activation sites. We defined the genes activated and involved in regulation under pathogen stress and all corresponding transcriptional activation sites as the stress expression regions of the target crop.

[0028] Methods for designing vectors for inducible disease resistance factor expression include: Screening for functional genes that are upregulated under pathogen stress and are associated with disease resistance from the stress expression regions of target crops. Functional genes include pathogen recognition receptor genes, signal transduction factor genes, and defense effector genes. Inducible regulatory elements that are co-expressed with or have a regulatory relationship with functional genes are extracted. The inducible regulatory elements and the coding sequence of the target crop are used to construct an expression cassette. The expression cassette is a functional module with pathogen-induced expression ability. The expression cassette further includes a terminator sequence to form a functional module that can induce the expression of the target gene under pathogen stress conditions.

[0029] Screenable marker elements and genetic clearance recognition elements are introduced into the expression vector to form an expression vector containing a complete expression cassette and functional modules; the induction effect of the constructed expression vector under pathogen stress conditions is verified by transient expression or stable transformation experiments.

[0030] Methods for obtaining transgenic plants include: The constructed inducible disease resistance factor expression vector was introduced into plant cells using transformation methods suitable for the target crop, including Agrobacterium-mediated transformation, gene gun method, electroporation method, and protoplast PEG-mediated transformation. The transformed cells were then subjected to resistance screening and tissue culture to obtain transgenic plants containing exogenous expression cassettes. The expression process of exogenous genes in transgenic plants was simulated, and the expression level of exogenous genes over time was controlled by an induction feedback expression regulation function, confirming that the designed inducible disease resistance factor expression vector operated effectively in transgenic plants.

[0031] The induced feedback expression regulation function is: ; where represents the rate of change of gene expression level at time point ; represents the induction efficiency constant, meaning the magnitude of the expression increase caused by a unit of induced input energy; represents the induced input function; represents the intensity coefficient of expression feedback inhibition, which, according to expert experience, ranges from 0 to 1; represents the expression feedback inhibition function; represents the index of the time point; This approach addresses the following problems in existing technologies: Traditional transgenic disease resistance methods rely heavily on continuous, high-expression construction strategies, failing to regulate expression based on the dynamic process of pathogen invasion. This can easily lead to wasted plant resources, growth inhibition, or abnormal expression under non-stress conditions. Long-term, high-intensity expression can activate the plant's transgenic silencing mechanism, causing unstable or even inactivated expression of the target disease resistance gene in actual field environments, reducing the persistence of disease resistance. Pathogen distribution and stress during crop growth are dynamic, and traditional methods lack the ability to respond to such spatiotemporal variations, resulting in limited resistance adaptation ranges. Existing breeding methods largely rely on experience and phenotypic selection, lacking mathematical modeling and theoretical guidance for the expression process, making expression system optimization a crude and inefficient process.

[0032] Compared to existing technologies, the advantages include: controlling the expression of disease resistance factors through pathogen-inducible promoters and regulatory elements ensures that gene expression is activated only under pathogen stress and remains quiescent under non-stress conditions, thus avoiding energy waste and growth inhibition and improving resource utilization efficiency. Furthermore, by constructing mathematical expression models using inducible input functions and expression feedback inhibition functions, the expression behavior of transgenic organisms can be predicted and regulated, reducing the risk of uncontrolled gene expression and systemic disorder.

[0033] By precisely controlling the expression intensity and duration, the risk of silencing caused by sustained high expression is avoided, improving the effective operating time and performance stability of exogenous genes in practical applications. It enables adaptive expression responses to different pathogen pressures, enhancing the disease resistance system's ability to recognize and respond to spatial and temporal variations in pathogen populations. Through induced feedback expression regulation functions, a quantitative correlation is established between biological signals and expression results, providing data support and modeling tools for screening disease resistance genes, designing expression vectors, and optimizing breeding materials, thus improving the accuracy and scientific rigor of the breeding process. It achieves a complete technical closed loop from pathogen identification, regulatory construction, transformation screening to expression modeling, providing a novel solution for intelligent and highly controllable disease-resistant crop breeding.

[0034] Methods for obtaining gene expression patterns include: The transgenic plants were propagated through successive generations to obtain m generations of offspring plants; pathogen stress treatment group and non-stress control group were set up in each generation; target tissue samples were collected at the growth stage of each treatment group and total RNA was extracted. The expression levels of exogenous disease resistance factor genes and related regulatory elements were detected using quantitative real-time PCR, transcriptome sequencing, and reporter gene detection. The expression intensity, induction response rate, and temporal changes of exogenous genes in transgenic plants of different generations under pathogen stress and non-stress conditions were analyzed. Trend curves of expression levels with generation were plotted, and gene expression pattern analysis was performed.

[0035] Methods for determining whether a target crop has achieved endogenous evolutionary convergence in disease resistance expression include: Based on the trend curve of expression level changes with generation, it is determined whether the expression of exogenous genes shows a continuous decline; based on transcriptome sequencing data, endogenous genes related to the function of exogenous disease resistance genes are screened, and it is analyzed whether the expression level of endogenous genes gradually increases in each generation; a co-expression network of exogenous and endogenous genes is constructed to infer the substitution relationship between endogenous genes and exogenous gene expression. The disease resistance function of endogenous genes is verified by gene knockout or repression techniques, and the ability of endogenous genes to maintain disease resistance under the condition of weakened exogenous gene expression is evaluated. By combining the trend of exogenous gene expression decline and endogenous gene expression enhancement in successive generations, and combining the plant disease resistance phenotype and physiological indicators, it is determined whether disease resistance expression dominated by endogenous genes has been formed, and the endogenous evolutionary convergence of disease resistance expression is achieved.

[0036] Methods for obtaining candidate plants without exogenous expression include: After confirming through cross-generational tracking and expression pattern analysis that the target crop has achieved endogenous evolutionary convergence in disease resistance expression, the genetic scavenging unit is activated using a pathogen-induced triggering mechanism. The genetic scavenging unit includes promoters that can respond to pathogen stress and genes that perform scavenging functions. When the expression of disease resistance in the target crop reaches saturation and the pathogen stress signal is sensed, the expression of the genetic clearing unit is activated through the activation function of the genetic clearing unit, thereby initiating the clearing program; the activated genetic clearing unit triggers programmed cell clearing, and performs clearing treatment on the chimeric target crop under the state of saturated disease resistance expression, to obtain candidate plants without exogenous expression.

[0037] The activation function of the genetic scavenging unit is: ; where represents the activation intensity of the genetic scavenging unit at time point ; represents the expression level of the exogenous gene at time point ; represents the preset saturation threshold for disease resistance expression, that is, when the expression of the exogenous gene reaches or exceeds this value, the plant is considered to be in a state of saturated disease resistance expression; represents the scavenging response coefficient, which refers to the activation intensity of the genetic scavenging unit caused by each unit of exogenous expression exceeding the threshold, and is a dimensionless constant; This approach addresses the following issues in existing technologies: Traditional transgenic disease-resistant breeding relies on the continuous expression of exogenous genes to maintain disease resistance. However, long-term retention of exogenous genes carries potential risks of environmental leakage (such as gene drift and ecological invasion) and public acceptance, making it difficult to meet the requirements for non-GMO regulation or market access. Existing technologies mostly involve static expression construction; once transferred into the target plant, they struggle to adjust expression levels in response to crop growth changes or stress conditions. Furthermore, there is no effective mechanism to actively shut down or remove exogenous sequences when disease resistance no longer depends on them. The traditional approach perpetuates crop dependence on exogenous disease resistance mechanisms, essentially lacking a strategic design for reverting to high-quality non-GMO germplasm, thus hindering the deep integration of modern molecular breeding with the goals of green and sustainable agriculture.

[0038] Compared to existing technologies, the advantages are as follows: By precisely activating the genetic clearance unit using pathogen-induced signals under the premise that crop disease resistance expression no longer depends on exogenous genes, the automatic clearance of exogenous disease resistance factors is achieved, thereby obtaining candidate plants without exogenous expression. This reduces the potential risks of transgenic products to the environment and ecosystems, meeting the current urgent needs for green agriculture and sustainable breeding. Through the coupling of induced expression and saturation feedback, not only is on-demand expression of disease resistance factors achieved, but the clearance module is also triggered after the expression reaches a threshold, effectively addressing the negative effects caused by overexpression. The clearance program is initiated through an induced trigger mechanism, achieving a natural transition from the introduction of exogenous functions to endogenous expression replacement, constructing a dynamic evolutionary breeding pathway of introduction, adaptation, and release, avoiding the unidirectional dependence defects of traditional transgenic technology pathways. By obtaining candidate plants ultimately without exogenous expression, the transgenic label associated with exogenous genes is avoided, which is beneficial for the market promotion and consumer acceptance of new varieties. It also lowers regulatory barriers for varieties entering the international market and enhances the commercial application potential of germplasm resources.

[0039] Methods for breeding disease-resistant and high-quality germplasm include: Candidate plants were evaluated using a comprehensive assessment of their resistance and ecological adaptability, which included disease resistance indicators, agronomic trait indicators, and ecological adaptability indicators. Relevant disease resistance indicators, agronomic trait indicators, and ecological adaptability indicators were collected through field multi-environment experiments and molecular detection methods. Disease resistance indicators are used to evaluate the strength, broad spectrum, and response speed of candidate plants to major pathogens; agronomic trait indicators are used to evaluate important agronomic traits such as yield, quality, plant type, and maturity period; ecological adaptability indicators are used to evaluate the drought tolerance, salt tolerance, temperature tolerance, and other stress resistance of candidate plants, as well as the stability of their growth cycle. Each indicator is quantified and weighted to calculate the comprehensive score of the candidate plants. Based on the preset comprehensive score threshold, candidate plants with a comprehensive score greater than or equal to the preset comprehensive score threshold are selected as disease-resistant and high-quality germplasm for breeding.

[0040] The preset comprehensive score threshold is set by staff. By collecting different comprehensive scores, the average of multiple comprehensive scores is taken as the preset comprehensive score threshold.

[0041] This embodiment controls the expression of disease resistance factors through pathogen-inducible promoters and regulatory elements, ensuring that gene expression is activated only under pathogen stress and remains quiescent under non-stress conditions. This avoids energy waste and growth inhibition, improving resource utilization efficiency. A mathematical expression model is constructed by setting induction input functions and expression feedback inhibition functions to predict and regulate transgenic expression behavior, reducing the risk of uncontrolled gene expression and systemic disorder.

[0042] By precisely controlling the expression intensity and duration, the risk of silencing caused by sustained high expression is avoided, improving the effective operating time and performance stability of exogenous genes in practical applications. It enables adaptive expression responses to different pathogen pressures, enhancing the disease resistance system's ability to recognize and respond to spatial and temporal variations in pathogen populations. Through induced feedback expression regulation functions, a quantitative correlation is established between biological signals and expression results, providing data support and modeling tools for screening disease resistance genes, designing expression vectors, and optimizing breeding materials, thus improving the accuracy and scientific rigor of the breeding process. It achieves a complete technical closed loop from pathogen identification, regulatory construction, transformation screening to expression modeling, providing a novel solution for intelligent and highly controllable disease-resistant crop breeding.

[0043] By precisely activating genetic clearance units using pathogen-induced signals to automatically remove exogenous disease resistance factors under the premise that crop disease resistance expression no longer depends on exogenous genes, candidate plants without exogenous expression are obtained. This reduces the potential risks of transgenic products to the environment and ecosystems, meeting the current urgent needs for green agriculture and sustainable breeding. Through the coupling of induced expression and saturation feedback, not only is on-demand expression of disease resistance factors achieved, but the clearance module is also triggered after expression reaches a threshold, effectively addressing the negative effects of overexpression. The clearance program is initiated through an induced trigger mechanism, achieving a natural transition from exogenous function introduction to endogenous expression replacement, constructing a dynamic evolutionary breeding pathway of introduction, adaptation, and release. Obtaining candidate plants ultimately without exogenous expression facilitates the market promotion and consumer acceptance of new varieties, while also reducing regulatory barriers for varieties entering the international market and enhancing the commercial application potential of germplasm resources. Example

[0044] Please see Figure 2 As shown, for parts not described in detail in this embodiment, please refer to the description in Embodiment 1. A gene-introduction-driven disease-resistant crop breeding system is provided, comprising: The pathogen stress characterization module collects pathogen population information in the target crop planting environment, constructs a dynamic pathogen action spectrum, identifies genetic regulatory sites that induce stress responses in target crops, jointly analyzes transcriptional activation sites under pathogen stress, and outputs the stress expression region of the target crop. A reversible chimeric construction module was designed based on the stress expression region to create an inducible disease resistance factor expression vector. This vector was then embedded into the target crop cells through transformation to form transgenic plants that can induce and activate disease resistance expression under pathogen stress. The gene chimerism tracking module tracks the gene expression patterns of transgenic plants under pathogen stress and non-stress conditions across generations, identifies whether the introduced gene is gradually replaced by endogenous genes, and determines whether the target crop has achieved endogenous evolutionary convergence in disease resistance expression. The genetic elimination breeding module activates the genetic elimination unit by using a pathogen-induced triggering mechanism after the target crop achieves endogenous evolution convergence. Under the state of saturated disease resistance expression, it performs elimination treatment on the chimeric target crop to obtain candidate plants without exogenous expression. The disease-resistant germplasm evaluation and breeding module comprehensively scores candidate plants for resistance and ecological adaptability, retaining individuals with a comprehensive score greater than a preset comprehensive score threshold as high-quality disease-resistant germplasm for breeding.

[0045] Since the electronic device described in this embodiment is used to implement the gene-introduction-driven disease-resistant crop breeding method described in this application embodiment, those skilled in the art can understand the specific implementation and various variations of the electronic device in this embodiment based on the gene-introduction-driven disease-resistant crop breeding method described in this application embodiment. Therefore, how the electronic device implements the method in this application embodiment will not be described in detail here. Any electronic device used by those skilled in the art to implement the gene-introduction-driven disease-resistant crop breeding method described in this application embodiment falls within the scope of protection of this application.

[0046] The above formulas are all dimensionless calculations. The formulas are derived from software simulations based on a large amount of collected data to obtain the most recent real-world results. The preset parameters and thresholds in the formulas are set by those skilled in the art according to the actual situation.

[0047] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for users of ordinary technical skills, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for breeding disease resistant crops driven by gene introduction, characterized by, Comprise: S1, collect pathogen population information in the planting environment of target crops, construct pathogen dynamic action spectrum, identify genetic regulation sites inducing target crops to produce stress response, jointly analyze transcription activation sites under pathogen stress, and output stress expression region of target crops; S2, based on the stress expression region, design inducible disease resistance factor expression vector, embed it into target crop cells through transformation means, and form transgenic plants that can induce and activate disease resistance expression under pathogen stress; S3, track gene expression pattern of transgenic plants under pathogen stress and non-stress conditions across generations, identify whether the introduced genes are gradually replaced by endogenous genes, and judge whether the target crops achieve endogenous evolutionary convergence of disease resistance expression; S4, after the target crops achieve endogenous evolutionary convergence, use pathogen induction trigger mechanism to activate genetic removal unit, perform removal processing on chimeric target crops under the saturation state of disease resistance expression, and obtain candidate plants without exogenous expression; S5, comprehensive score of resistance and ecological adaptability of candidate plants, retain individuals with comprehensive score greater than preset comprehensive score threshold as disease-resistant high-quality germplasm for breeding.

2. The method for breeding a disease-resistant crop driven by gene introduction according to claim 1, wherein The method for collecting pathogen population information in the planting environment of target crops comprises: In the planting environment of target crops, through field sampling method, collect samples of pathogenic microorganisms in rhizosphere soil, leaves, stems and fruits of target crops, and sampling covers the high incidence period of target crops growth; Use sterile cotton swab, cone tube sampler and liquid nitrogen quick freezing technology to standardize the collected samples, and extract total DNA or total RNA from the collected samples; specific region amplification is performed on the extracted microbial nucleic acids, and the specific region includes 16S rRNA gene region of bacteria, ITS region of fungi and dsRNA region of virus; High-throughput sequencing is performed on the amplification products, and bioinformatics methods are used for quality control, sequence splicing, OTU clustering and species annotation to obtain pathogen population information in the planting environment of target crops; the pathogen population information includes pathogen species classification composition, relative abundance, community structure, spatial and temporal dynamic change.

3. The method of claim 2, wherein the method is a method for breeding a disease-resistant crop driven by gene introduction. The identification method of genetic regulation site comprises: According to the pathogen population information in the planting environment of target crops, construct the pathogen dynamic action spectrum, determine the key space-time node of pathogen stress, collect the corresponding tissue samples of target crops, perform transcriptome sequencing, and obtain the gene expression profile under pathogen stress and non-stress conditions; Through differential expression analysis, the gene group responding to pathogen stress is screened out, combined with whole genome association analysis and epigenomics technology, the genetic regulation site inducing target crops to produce stress response is located, and the genetic regulation site includes transcription factor binding site and chromatin opening region.

4. The method of claim 3, wherein the method is a method for breeding a disease-resistant crop driven by gene introduction. The method for outputting the stress expression region of target crops comprises: Integrate the constructed pathogen dynamic action spectrum, gene expression profile and genetic regulation site to form a multidimensional data set containing pathogen stress space-time characteristics, gene expression changes and regulation sites; based on the multidimensional data set, analyze the dynamic change of pathogen community and the space-time corresponding relationship of target crop gene expression and transcription activation site; The dynamic changes of chromatin openness and transcription factor binding sites are combined to determine the transcriptional activation elements and target genes induced and activated under pathogen stress conditions; a pathogen stress response gene regulatory network is constructed, which comprises the target genes and transcriptional activation sites; and the genes activated and involved in regulation under pathogen stress and all the corresponding transcriptional activation sites are defined as stress expression regions of the target crops.

5. The method of claim 4, wherein the method is a method of breeding a disease resistant crop driven by gene introduction. The method for designing the inducible disease resistance factor expression vector comprises the following steps: functional genes related to disease resistance are screened from the stress expression regions of the target crops under pathogen stress conditions, and the functional genes include pathogen recognition receptor genes, signal transduction factor genes and defense effector genes; inducible regulatory elements co-expressed or having a regulatory relationship with the functional genes are extracted, and the inducible regulatory elements and the coding sequences of the target crops are jointly constructed into an expression cassette, which is a functional module having pathogen-induced expression capability; screenable marker elements and genetic elimination recognition elements are introduced into the expression vector to form an expression vector comprising the complete expression cassette and the functional module; and the inducible expression effect of the constructed expression vector under pathogen stress conditions is verified through transient expression or stable transformation experiments.

6. The method of claim 5, wherein the method is a method for breeding a disease-resistant crop driven by gene introduction. The method for obtaining the transgenic plants comprises the following steps: the constructed inducible disease resistance factor expression vector is introduced into plant cells through a transformation method suitable for the target crops, the transformation method includes Agrobacterium-mediated transformation, gene gun method, electroporation method and protoplast PEG-mediated method; and the cells after transformation are subjected to resistance screening and tissue culture to obtain transgenic plants containing the exogenous expression cassette; the expression process of the exogenous genes in the transgenic plants is simulated, the expression amount of the exogenous genes changes over time and is controlled by an inducible feedback expression control function, and it is confirmed that the designed inducible disease resistance factor expression vector is effectively operated in the transgenic plants.

7. The method of claim 6, wherein the method is a method for breeding a disease-resistant crop driven by gene introduction. The method for obtaining the gene expression pattern comprises the following steps: m generations of offspring plants are obtained through continuous generation breeding of the obtained transgenic plants; pathogen stress treatment groups and non-stress control groups are respectively set in each generation; target tissue samples are collected at the growth stages of each treatment group, and total RNA is extracted; fluorescent quantitative PCR, transcriptome sequencing and reporter gene detection methods are used to detect the expression levels of the exogenous disease resistance factor genes and related regulatory elements; the expression intensity, inducible response rate and time sequence change of the exogenous genes in the transgenic plants of different generations under pathogen stress and non-stress conditions are analyzed, a trend curve of the expression amount changing with the generations is drawn, and gene expression pattern analysis is performed.

8. The method of claim 7, wherein the method is a method of breeding a disease resistant crop driven by gene introduction. The method for judging whether the target crops achieve endogenous evolutionary convergence of disease resistance expression comprises the following steps: whether the expression of the exogenous genes appears sustained attenuation is judged according to the trend curve of the expression amount changing with the generations; based on the transcriptome sequencing data, endogenous genes functionally related to the exogenous disease resistance genes are screened, and whether the expression levels of the endogenous genes gradually increase in each generation is analyzed; a co-expression network of the exogenous genes and the endogenous genes is constructed, and a substitution relationship of the endogenous genes to the exogenous genes is inferred. The anti-disease function of the endogenous gene is verified by gene knockout or inhibition technology, and the ability of the endogenous gene to maintain the anti-disease property in the case of weakened expression of the exogenous gene is evaluated; the trend of attenuated expression of the exogenous gene and enhanced expression of the endogenous gene in successive generations is comprehensively analyzed, and the anti-disease phenotype and physiological indexes of the plants are combined to determine whether the anti-disease expression is dominated by the endogenous gene, so as to realize the endogenous evolutionary convergence of the anti-disease expression.

9. The method of claim 8, wherein the method is a method of breeding a disease resistant crop driven by gene introduction. The method for obtaining the candidate plant without exogenous expression comprises the following steps: After it is confirmed by the cross-generation tracking and expression mode analysis that the target crop has realized the endogenous evolutionary convergence of the anti-disease expression, a pathogen-induced triggering mechanism is used to activate a genetic elimination unit, and the genetic elimination unit comprises a promoter that can respond to pathogen stress and a gene that performs the elimination function; When the anti-disease expression of the target crop in vivo reaches a saturated state and the pathogen stress signal is sensed, the genetic elimination unit is activated by an activation function, so that the genetic elimination unit is expressed, thereby starting the elimination program; the activated genetic elimination unit triggers programmed cell elimination, and performs the elimination treatment on the chimeric target crop in the anti-disease expression saturated state, so as to obtain the candidate plant without exogenous expression.

10. The method of claim 9, wherein the method is a method of breeding a disease resistant crop driven by gene introduction. The method for breeding the anti-disease high-quality germplasm comprises the following steps: The candidate plants are comprehensively scored in terms of resistance and ecological adaptability, the comprehensive score comprises disease resistance indexes, agronomic trait indexes and ecological adaptability indexes; the disease resistance indexes, the agronomic trait indexes and the ecological adaptability indexes are collected by field multi-environment tests and molecular detection means; Each index is quantified and weighted, and the comprehensive score of the candidate plant is calculated; according to a preset comprehensive score threshold, the candidate plant with a comprehensive score greater than or equal to the preset comprehensive score threshold is screened out as the anti-disease high-quality germplasm for breeding.

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