Molecular design breeding method for improving functional component content of wheat

Abstract

The invention provides a molecular design breeding method for improving the content of functional components of wheat. The method comprises the following steps: determining one or more target genes; a synthetic biological system is integrated and expressed in a wheat genome, the synthetic biological system comprising: a chemical induction regulation switch for responding to an externally applied chemical inducer; the multi-gene synergistic activation system is used for driving the expression of the target gene after the chemical induction regulation switch is activated; and the delayed negative feedback inhibition loop is used for inhibiting the activity of the multi-gene synergistic activation system after a preset time delay so as to terminate the expression of the target gene. In the expression process of the target gene, the target gene is induced to be started when needed, and can be timely stopped after the preset character function is completed, so that the accumulation of the target functional component is maximized, and the adverse effect on the normal growth and development and agronomic characters of the plant is minimized.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to a molecular design breeding method for increasing the content of functional components in wheat. Background Technology

[0002] Plant genetic engineering, as the core of modern agricultural biotechnology, provides an unprecedentedly powerful tool for crop improvement. By introducing exogenous genes or precisely editing endogenous genes, crop yield, resistance, and nutritional quality can be significantly enhanced. Particularly in major food crops such as wheat, the use of biotechnology to enrich specific functional components, such as anthocyanins, carotenoids, or vitamins, has significant socio-economic implications for improving human dietary structure and increasing the added value of agricultural products.

[0003] Currently, the mainstream technical strategy for enhancing the function of specific genes typically employs constitutive strong promoters to drive the sustained overexpression of target genes. For example, using the maize ubiquitin promoter (pUbi) or the cauliflower mosaic virus 35S promoter (CaMV35S) can maintain high levels of transcriptional activity of the target gene throughout the plant's life cycle and in almost all tissues. However, this unrestrained, continuous expression, given the plant's limited energy and metabolic resources, can place a heavy metabolic burden on the plant, often leading to unintended negative agronomic traits such as stunted growth, decreased fertility, or ultimately reduced yield—the so-called trade-off between growth and metabolism. Furthermore, when the synthesis or accumulation of the target product requires precise regulation, the aforementioned expression methods often fail to meet the needs and may even produce toxic effects due to accumulation in non-target tissues or at incorrect developmental stages.

[0004] To address the aforementioned shortcomings, various spatiotemporally specific expression systems have been developed in the prior art, such as tissue-specific promoter expression systems and chemically inducible promoter expression systems. The expression window in tissue-specific promoter expression systems is fixed, lacking flexibility and unable to be artificially intervened according to actual production needs. While chemically inducible promoter expression systems offer the possibility of artificial regulation of the expression window, their design is often relatively simple, typically only allowing for the switching of a single gene.

[0005] In complex metabolic regulation, the efficient synthesis of target products often requires the synergistic action of multiple genes. For example, it may be necessary to enhance key enzyme genes in downstream synthetic pathways while simultaneously upregulating genes in upstream feedstock supply pathways. Existing expression systems struggle to achieve logical association and synergistic regulation of multiple target genes, leading to metabolic flux imbalances, new metabolic bottlenecks, and ultimately, significantly reduced product accumulation efficiency. Summary of the Invention

[0006] In view of the above problems, this invention is proposed to provide a molecular design breeding method for improving the content of functional components in wheat, which overcomes or at least partially solves the above problems. It can solve the problem that existing expression systems are difficult to achieve logical association and synergistic regulation of multiple target genes, resulting in metabolic flux imbalance and a significant reduction in product accumulation efficiency. It can also bring about the effect of being able to start when needed, and terminate in a timely manner after completing the preset trait function, maximizing the accumulation of target functional components and minimizing the adverse effects on normal plant growth and development and agronomic traits.

[0007] Specifically, according to one aspect of the present invention, a molecular design breeding method for increasing the content of functional components in wheat is provided, characterized by comprising the following steps: S1. Identify one or more target genes, wherein the target genes are genes encoding proteins required in the biosynthesis or transport pathways of one or more functional components; S2. Integrating and expressing a synthetic biology system in the wheat genome, said synthetic biology system comprising: A chemically induced regulatory switch, used to respond to an exogenously applied chemical inducer; A multi-gene synergistic activation system is used to drive the expression of the target gene after the chemically induced regulatory switch is activated; A delayed negative feedback inhibition loop is used to inhibit the activity of the multi-gene synergistic activation system after a preset time delay, so as to terminate the expression of the target gene.

[0008] S3. Insert the multi-gene co-activation system into the endogenous promoter of the target gene, and integrate the other parts of the synthetic biology system into the wheat genome.

[0009] Optionally, the multi-gene co-activation system includes one or more dependent transcription factors and response elements corresponding to the dependent transcription factors; The response element is inserted in situ into the endogenous promoter of the target gene so that the dependent transcription factor corresponding to the response element regulates the expression of the target gene.

[0010] Optionally, the dependent transcription factors are divided into feedforward activators and functional activators; The response element is divided into a feedforward response element corresponding to the feedforward activator and a functional response element corresponding to the functional activator; In step S3, the in-situ insertion of the response element into the endogenous promoter of the target gene is as follows: The feedforward response element is inserted into the endogenous promoter of the feedforward target gene, which is a gene in an upstream metabolic pathway related to the supply of precursors required for the synthesis of the functional component. The functional response element is inserted into the endogenous promoter of the target gene associated with the synthesis of the functional component.

[0011] Optionally, in step S2, after the chemically induced regulatory switch is activated, driving the expression of the target gene specifically involves: The expression of the feedforward activator and the functional activator corresponding to the target gene is driven and binds to the feedforward response element and the functional response element, respectively, thereby synergistically activating the transcription of the feedforward target gene and the target gene.

[0012] Optionally, the delayed negative feedback suppression loop includes a synthetic suppressor and a control element; The synthetic repressor is used to inhibit the activity of the multi-gene co-activation system to terminate the transcription of the target gene; The regulatory element synchronously responds to the transcription of the target gene driven by the multi-gene co-activation system, and drives the expression of the synthetic repressor after a preset time delay.

[0013] Optionally, the chemically induced regulatory switch includes: The master transcription factor has an artificial receptor protein, which has a specific binding ability to the chemical inducer; After the artificial receptor protein binds to the chemical inducer, the transcriptional activity of the master control transcription factor is activated to complete its expression, thereby driving the expression of the target gene.

[0014] Optionally, the transcriptional activation activity of the master transcription factor is controlled by the binding state of the artificial receptor protein to the specific chemical inducer.

[0015] Optionally, the activation of transcriptional activity to complete expression, thereby driving the expression of the target gene, specifically involves: The expression of each dependent transcription factor is driven by a promoter of the master transcription factor’s specific binding site, which contains a specific binding site that can be recognized by the DNA binding domain of the master transcription factor.

[0016] Optionally, the regulatory element is configured to have a low affinity for one of the dependent transcription factors in one or more of the multi-gene co-activation systems; The promoter is effectively activated after the concentration of dependent transcription factors accumulates over time and reaches a predetermined threshold, thereby driving the expression of synthetic repressors.

[0017] Optionally, the functional ingredient is selected from one or more of dietary fiber, anthocyanins, carotenoids, phenolic acids, vitamins, iron, and zinc.

[0018] In a molecular design breeding method for increasing the content of functional components in wheat according to the present invention, by employing a chemical induction regulation switch independent of the wheat endogenous system and combining it with the exogenous application of chemical inducers at specific developmental stages of wheat plants, the transcriptional activation of target genes can be precisely limited to the required time window, avoiding the adverse effects of constitutive expression on metabolic resource competition or growth and development that may occur throughout the wheat's growth cycle.

[0019] Furthermore, in the molecular design breeding method for increasing the content of functional components in wheat according to the present invention, dependent transcription factors are classified into feedforward activators and functional activators, which are then used to regulate the feedforward target genes in the upstream precursor supply pathway and the target genes in the downstream functional component synthesis pathway, respectively. This achieves a simultaneous increase in metabolic flux in both the raw material supply and final product synthesis stages, solving the technical bottleneck of insufficient precursor supply that may result from single pathway modification, and effectively increasing the yield of the target product. Furthermore, in the molecular design breeding method for increasing the content of functional components in wheat according to the present invention, the provided synthetic biology system possesses self-terminating regulatory characteristics, which can reduce the continuous metabolic load on wheat plants. By integrating a delayed negative feedback inhibition loop containing a synthetic repressor and a delayed regulatory element into the system, the system can automatically shut down transcriptional activation of the target gene and the feedforward target gene after being activated and running for a preset time. This avoids excessive accumulation of the target functional components or their metabolic intermediates, helps maintain cellular metabolic homeostasis, and thus ensures the normal physiological state of the engineered plant.

[0020] The above and other objects, advantages and features of the present invention will become more apparent to those skilled in the art from the following detailed description of specific embodiments of the invention in conjunction with the accompanying drawings. Attached Figure Description

[0021] The following sections will describe some specific embodiments of the invention in detail by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings: Figure 1 This is a flowchart illustrating a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Figure 2 This is a schematic diagram of the synthetic biology system structure in a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention; Figure 3This is a schematic diagram of the T-DNA region structure of a multi-gene expression vector in an application example of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Figure 4 This is a schematic diagram of the target metabolic pathway and regulatory target points in an application example of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Figure 5 This is a schematic diagram of an in-situ editing strategy for endogenous promoters in a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Figure 6 This is a flowchart of wheat genetic transformation in an application example of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Figure 7 This is a PCR detection gel electrophoresis image from an application example of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Figure 8 This is a Southern Blot analysis of an application example of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Figure 9 This is a promoter editing and sequencing alignment diagram in an application example of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention; Figure 10 This is an example HPLC chromatogram of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Figure 11 This is a statistical chart of anthocyanin content in an application example of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention; Figure 12 This is a comparative diagram of the main agronomic traits of transgenic plants in an application example of a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention. Detailed Implementation

[0022] Obviously, the accompanying drawings described below are merely some examples or embodiments of this application. Those skilled in the art can apply this application to other similar scenarios based on these drawings without any inventive effort. Furthermore, it is understood that although the efforts made in this development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this application, any changes to design, manufacturing, or production based on the technical content disclosed in this application are merely conventional technical means and should not be construed as insufficient disclosure of the content of this application.

[0023] In this application, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described in this application may be combined with other embodiments without conflict.

[0024] Unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning understood by one of ordinary skill in the art to which this application pertains. The terms “a,” “an,” “an,” “the,” and similar words used in this application do not indicate quantity limitation and may represent singular or plural. The terms “comprising,” “including,” “having,” and any variations thereof used in this application are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that includes a series of steps or modules (units) is not limited to the listed steps or units, but may also include steps or units not listed, or may include other steps or units inherent to such processes, methods, products, or apparatus.

[0025] Figure 1 This is a flowchart illustrating a molecular design breeding method for increasing the content of functional components in wheat according to an embodiment of the present invention, as shown below. Figure 1 As shown, and refer to Figures 2 to 12 This invention provides a molecular design breeding method for increasing the content of functional components in wheat, comprising the following steps: S1. Identify one or more target genes, wherein the target genes are genes encoding proteins required in the biosynthesis or transport pathways of one or more functional components. Specifically, the target genes are used to synthesize the required proteins, thereby synthesizing the biological traits desired by the user.

[0026] S2. Integrate and express a synthetic biology system in the wheat genome, the synthetic biology system comprising: a chemically induced regulatory switch, a multi-gene synergistic activation system, and a delayed negative feedback inhibition loop.

[0027] Chemical induction control switches are used to respond to exogenously applied chemical inducing agents.

[0028] The multi-gene synergistic activation system is used to drive the expression of the target gene after the chemically induced regulatory switch is activated. Specifically, the chemically induced regulatory switch can regulate the initiation of transcription of the target gene, controlling the start time of target gene activation within a desired time window.

[0029] A delayed negative feedback inhibition loop is used to inhibit the activity of the multi-gene co-activation system after a preset time delay, thereby terminating the expression of the target gene. Specifically, this delayed negative feedback inhibition loop inhibits the transcriptional activation of the target gene, responsible for ending the transcription of the target gene. The multi-gene co-activation system functions by receiving signals from chemically induced regulatory switches and distributing them to one or more target regulatory pathways.

[0030] S3. Insert the multi-gene co-activation system into the endogenous promoter of the target gene, and integrate the other parts of the synthetic biology system into the wheat genome.

[0031] In this embodiment, by employing a chemically induced regulatory switch independent of the wheat's endogenous system, and combining this with the exogenous application of chemical inducers at specific developmental stages of the wheat plant, the transcriptional activation of the target gene can be precisely limited to the desired time window. This avoids the potential adverse effects on metabolic resource competition or growth and development caused by constitutive expression throughout the wheat's growth cycle. Simultaneously, the synthetic biology system possesses self-terminating regulatory characteristics, reducing the continuous metabolic load on the wheat plant. By integrating a delayed negative feedback inhibition loop into the synthetic biology system, the system can automatically shut down the transcriptional activation of the target gene after being activated and running for a preset time. This avoids excessive accumulation of the target functional component or its metabolic intermediates, helping to maintain cellular metabolic homeostasis and thus ensuring the normal physiological state of the engineered plant.

[0032] In some embodiments of the present invention, the chemically induced regulatory switch includes a master transcription factor. The master transcription factor has an artificial receptor protein. The artificial receptor protein has a specific binding capacity to the chemical inducer. After the artificial receptor protein binds to the chemical inducer, the transcriptional activity of the master transcription factor is activated to complete expression, thereby driving the expression of the target gene. Specifically, the chemically induced regulatory switch functions to receive a specific chemical inducer signal applied exogenously and convert it into a transcriptional regulatory signal for downstream genes.

[0033] The chemically induced regulation described herein refers to the absence of cross-recognition and mutual interference between the chemical inducer and its artificial receptor protein and any endogenous signaling molecules and receptor pathways in the wheat host, ensuring the high specificity and independence of signal transduction in this synthetic biology system. In addition to the GAL4-ER-VP64 system, other systems with similar characteristics, such as ecdysone receptor (EcR)-based systems or tetracycline-based (Tet-On / Tet-Off) systems, are also suitable for constituting the chemically induced regulatory switch of this invention.

[0034] In some embodiments of the present invention, the multi-gene co-activation system includes one or more dependent transcription factors and corresponding response elements. The response element is inserted in situ into the endogenous promoter of the target gene, so that the dependent transcription factor corresponding to the response element regulates the expression of the target gene. An endogenous promoter refers to a promoter sequence naturally present in the organism's own genome that can drive the normal transcriptional expression of the gene in vivo. The "natural switch" of the target gene in its native genome is formed by the organism's own evolution, rather than an artificially introduced or heterologously inserted sequence. For example, the promoter responsible for regulating the expression of anthocyanin synthesis genes in wheat, if not artificially modified and belonging to the wheat genome itself, is the endogenous promoter of that gene. In this embodiment, the response element corresponding to the transcription factor is inserted into the endogenous promoter of the target gene, so that the transcriptional activation process of the target gene can be controlled by the dependent transcription factor, thereby allowing transcription to begin at the desired time. This avoids the potential adverse effects on metabolic resource competition or growth and development caused by constitutive expression throughout the wheat's growth cycle.

[0035] In some embodiments of the present invention, the dependent transcription factor is divided into a feedforward activator and a functional activator. The response element is divided into a feedforward response element corresponding to the feedforward activator and a functional response element corresponding to the functional activator.

[0036] In step S3, the in-situ insertion of the response element into the endogenous promoter of the target gene is as follows: inserting the feedforward response element into the endogenous promoter of the feedforward target gene, wherein the feedforward target gene is a gene in an upstream metabolic pathway related to the supply of precursors required for the synthesis of the functional component; and inserting the functional response element into the endogenous promoter of the target gene related to the synthesis of the functional component.

[0037] Specifically, the dependent transcription factors are classified into at least two types: feedforward activators and functional activators. Both types of activators are orthogonal transcription factors without endogenous homologs in wheat, for example, formed by the fusion of nuclease-inactivated Cas9 protein (dCas9) or transcription activator-like effector protein (TALE) with a transcription activation domain (such as VPR). Correspondingly, the response elements are also classified into feedforward response elements and functional response elements, which are the DNA sequences that the feedforward activator and functional activator can specifically bind to, respectively.

[0038] This embodiment distinguishes dependent transcription factors into feedforward activators and functional activators, which are used to regulate the feedforward target genes in the upstream precursor supply pathway and the target genes in the downstream functional component synthesis pathway, respectively. This achieves a simultaneous increase in metabolic flux in both the raw material supply and final product synthesis stages, solving the technical bottleneck of insufficient precursor supply that may be caused by single pathway modification, and effectively improving the yield of target products.

[0039] In some embodiments of the present invention, the activation of transcriptional activity to complete expression and thereby drive the expression of the target gene specifically means that the expression of each dependent transcription factor is driven by a promoter of the specific binding site of the master transcription factor, the promoter containing a specific binding site that can be recognized by the DNA binding domain of the master transcription factor.

[0040] Specifically, the master transcription factor is a man-made fusion protein, or artificial receptor protein, containing three functional domains: a ligand-binding domain that specifically binds to a particular chemical inducer, a sequence-specific DNA-binding domain, and a transcriptional activation domain. For example, this master transcription factor can be formed by fusing the ligand-binding domain of the human estrogen receptor (ER), the DNA-binding domain of the yeast GAL4 protein, and the transcriptional activation domain of herpes simplex virus VP64. Its transcriptional activation activity is controlled by its binding state to a specific chemical inducer (such as β-estradiol), and there is no cross-reactivity between the master transcription factor and its responding chemical inducer and endogenous molecular signaling pathways in wheat. The expression of each dependent transcription factor is driven by an independent promoter containing a specific binding site (e.g., the UAS element of GAL4) recognized by the DNA-binding domain of the master transcription factor.

[0041] In some embodiments of the present invention, in step S2, after the chemically induced regulatory switch is activated, driving the expression of the target gene specifically involves: the expression of the feedforward activator and the functional activator corresponding to the target gene being driven, and binding to the feedforward response element and the functional response element respectively, thereby synergistically activating the feedforward target gene and the transcription of the target gene.

[0042] In some embodiments of the present invention, the delayed negative feedback suppression loop includes a synthetic suppressor and a control element.

[0043] The synthetic repressor is used to inhibit the activity of the multi-gene co-activation system to terminate the transcription of the target gene. The regulatory element responds synchronously to the multi-gene co-activation system driving the transcription of the target gene, and after a preset time delay, drives the expression of the synthetic repressor.

[0044] Specifically, the delayed negative feedback inhibition loop functions by generating an inhibitory signal after a period of system activation. This signal acts on the multi-gene co-activation system or a chemically induced regulatory switch, thereby terminating the transcriptional activation activity of the entire system. This delayed negative feedback inhibition loop includes a synthetic repressor and a regulatory element with a preset time delay. The synthetic repressor is a protein capable of inhibiting transcription, such as a fusion protein of a TALE protein targeting a specific promoter sequence and a KRAB transcriptional repressor domain.

[0045] In this embodiment, the provided synthetic biology system possesses self-terminating regulatory characteristics, which can reduce the continuous metabolic load on wheat plants. By integrating a delayed negative feedback inhibition loop containing a synthetic repressor and a delayed regulatory element into the system, the system can automatically shut down transcriptional activation of the target gene and the feedforward target gene after being activated and running for a preset time. This avoids excessive accumulation of the target functional component or its metabolic intermediates, helps maintain cellular metabolic homeostasis, and thus ensures the normal physiological state of the engineered plant.

[0046] In some embodiments of the invention, the regulatory element is configured to target a promoter with low affinity for one of the dependent transcription factors in one or more of the multi-gene co-activation systems. This promoter is effectively activated after the concentration of the dependent transcription factor accumulates over time and reaches a predetermined threshold, thereby driving the expression of a synthetic repressor.

[0047] Specifically, the regulatory element with a preset time delay is a promoter designed to drive the expression of a synthetic repressor. The promoter is designed to include one or more response elements with low affinity for a specific dependent transcription factor in a multi-gene co-activation system. This structural characteristic ensures that the regulatory element is effectively activated only after the concentration of the dependent transcription factor accumulates over time and reaches a predetermined threshold, thereby driving the expression of the synthetic repressor and achieving delayed repression.

[0048] The dynamic behavior of the above-mentioned regulation process can be quantitatively described by a set of rate equations: Subordinate transcription factors (with (representative) concentration The rate of change with time t can be expressed as: in, This is the activation state function of the chemically induced regulatory switch, and its value is determined by the presence or absence of the exogenous inducer (1 when present, 0 when absent). To be in the chemically induced regulatory switch activation state The maximum synthesis rate; for The degradation rate constant.

[0049] Target gene ( transcription rate It is determined by both the intrinsic activity of its endogenous promoter and the artificially induced signal: in, This represents the maximum transcription rate of the gene. This represents the intrinsic basal activity level of the endogenous promoter of this gene at time t. This describes the dependent transcription factor. The regulatory function of concentration on transcription rate.

[0050] Synthetic repressor concentration The delayed expression and negative feedback process can be represented as: in, It refers to the concentration of specific dependent transcription factors responsible for activating repressor expression; It is the activation concentration threshold for achieving a delayed response, and its value is determined by the affinity of the response element in the regulatory element; n is the Hill coefficient, which characterizes the cooperativeness of regulation; and These are synthetic repressors The maximum synthesis rate and degradation rate constant.

[0051] when When accumulation reaches a level sufficient to inhibit the activity of the multi-gene synergistic activation system, The effective value will decrease, thus shutting down the entire system.

[0052] In some embodiments of the present invention, the functional ingredient is selected from one or more of dietary fiber, anthocyanins, carotenoids, phenolic acids, vitamins, iron, and zinc.

[0053] Below, we will proceed according to each appendix. Figure 3-12 The following describes further examples of practical applications of the present invention.

[0054] See attached document Figure 3 , Figure 3 This is a schematic diagram of the T-DNA region structure of a multi-gene expression vector according to an embodiment of the present invention. This embodiment describes a specific method for integrating a designed synthetic biology system onto a single plant expression vector.

[0055] The construction of multi-gene expression vectors employs a modular cloning strategy based on IIS-type restriction endonucleases (such as BsaI and BpiI), for example, the Golden Gate assembly method. This strategy utilizes pre-designed standardized adapter sequences to seamlessly and directionally link multiple independent DNA fragments (i.e., expression cassettes). The construction process includes three main steps: synthesis of the basic modules, construction of each expression cassette, and final assembly into the target vector.

[0056] First, codon optimization and chemical synthesis of basic DNA modules were performed. The coding sequences of the master control transcription factor of chemically induced regulatory switch 10 (e.g., GAL4-ER-VP64), several dependent transcription factors of the multi-gene co-activation system (e.g., dCas9-VPR and TALE-VP64), and the synthetic repressor of the delayed negative feedback inhibition loop (e.g., TALE-KRAB) were all optimized according to wheat codon preferences to improve their translation efficiency in wheat cells. The optimized sequences were then chemically synthesized by an external service provider and cloned into the Level 0 basic vector.

[0057] Secondly, construct independent expression cassettes. The expression of each functional protein requires a complete expression cassette, the structure of which is usually promoter-coding sequence (CDS)-terminator.

[0058] For the master transcription factor that regulates the chemically induced switch 10, an expression cassette 1 was constructed. This expression cassette 1 uses a promoter that is constitutively active in wheat, such as the maize ubiquitin promoter (pUbi), followed by the coding sequence of the master transcription factor, and terminated by a commonly used transcription terminator, such as the Agrobacterium carmine synthase terminator (tNOS).

[0059] For each dependent transcription factor in the multi-gene co-activation system, expression cassettes II and III were constructed. For each dependent transcription factor (feedforward activator and functional activator), expression cassettes II and III are driven by a minimal promoter (e.g., the mini35S promoter) containing multiple tandem UAS (Upstream Activating Sequence, GAL4 response element) sequences. This design ensures that the expression of these dependent transcription factors is strictly controlled by the master transcription factor of the chemically induced regulatory switch 10. The remaining parts of expression cassettes II and III, namely the coding sequences and terminators, are structurally similar to expression cassette I.

[0060] For the synthetic repressor of the delayed negative feedback inhibition loop, expression cassette four is constructed. This expression cassette four employs a special delayed-response promoter. This promoter contains a response element sequence with low affinity for a dependent transcription factor (e.g., dCas9-VPR) in a multi-gene co-activation system, followed by the coding sequence of the synthetic repressor and a terminator.

[0061] Finally, all constructed expression cassettes 1, 2, 3, and 4 were assembled into a target vector (Level 2 vector) suitable for Agrobacterium-mediated plant transformation. In this embodiment, the pCAMBIA series vector was selected as the backbone. This vector contains a left (LB) and right (RB) boundary of T-DNA, with a multiple cloning site for inserting foreign genes between the boundaries. Furthermore, the outside of the T-DNA region contains resistance genes for Agrobacterium selection (such as spectinomycin resistance genes), and the inside of the T-DNA region contains resistance genes for plant selection (such as hygromycin resistance genes).

[0062] Multiple expression cassettes (expression cassette 1, 2, 3, and 4) were simultaneously cloned into the multiple cloning site of the T-DNA region of the pCAMBIA vector using a one-step Golden Gate reaction, following a pre-defined order and orientation. The reaction products were transformed into *E. coli* for amplification, and initial screening was performed using colony PCR and plasmid restriction enzyme digestion electrophoresis to select clones of the correct size. Finally, positive clones were subjected to Sanger sequencing to verify the ligation sites and key sequences of all expression cassettes, ensuring that the final multi-gene expression vector structure was completely correct and free of any mutations.

[0063] The resulting vector was named pWheat-SynBio-Regulator and was used for subsequent wheat genetic transformation.

[0064] See attached document Figure 4 , Figure 4This is a simplified metabolic pathway diagram of the target functional component, anthocyanin synthesis, selected according to an embodiment of the present invention. This embodiment describes how to determine the target genes and feedforward target genes to be regulated based on the preset functional goal of increasing the anthocyanin content of wheat grains.

[0065] First, the target functional component was identified and its biosynthetic network in wheat was analyzed. In this embodiment, the target functional component was identified as anthocyanin. By consulting publicly available bioinformatics databases (such as KEGG, Plant Cyc) and relevant literature, it was determined that the biosynthetic pathway of anthocyanin in wheat belongs to a branch of the phenylpropane metabolic pathway. This pathway uses phenylalanine as a starting substrate, and through a series of enzymatic reactions, ultimately produces pigments such as anthocyanin glycosides.

[0066] Secondly, based on metabolic network analysis, genes directly involved in anthocyanin synthesis and considered key rate-limiting steps were identified as target genes. In this embodiment, two key target genes were identified: Chalcone synthase gene (TaCHS): This gene encodes chalcone synthase, the first key enzyme in the flavonoid synthesis pathway, which catalyzes the condensation of 4-coumaroyl-CoA and malonyl-CoA to produce chalcone.

[0067] The dihydroflavonol 4-reductase gene (TaDFR) encodes dihydroflavonol 4-reductase, a key enzyme downstream of the anthocyanin synthesis pathway. It catalyzes the conversion of dihydroflavonols into colorless anthocyanins, a crucial step in determining flower color type and anthocyanin accumulation. Homologous sequence information for these two genes can be obtained through BLAST alignment of known CHS and DFR protein sequences in the wheat genome database.

[0068] Furthermore, to ensure a sufficient substrate supply when the target gene is activated, it is necessary to identify key genes in its upstream metabolic pathways as feedforward target genes. The purpose of feedforward regulation is to avoid substrate depletion caused by the enhancement of downstream pathways by simultaneously upregulating the raw material supply pathway. In this embodiment, a key feedforward target gene was identified: Phenylalanine ammonia-lyase gene (TaPAL): This gene encodes phenylalanine ammonia-lyase, the first enzyme in the entire phenylpropanoid metabolic pathway, which catalyzes the deamination of phenylalanine to cinnamic acid. This step is the main valve controlling the metabolic flux into the entire phenylpropanoid network, and its activity directly affects the synthetic flux of all downstream products, including anthocyanins. The sequence information of this gene was also obtained through database alignment.

[0069] Finally, sequence analysis was performed on the endogenous promoter regions of the selected target genes (TaCHS, TaDFR) and the feedforward target gene (TaPAL). Bioinformatics tools were used to predict their core promoter regions (e.g., TATA-box elements) and upstream regulatory regions. Their expression patterns in different wheat tissues and developmental stages were analyzed (e.g., by querying publicly available transcriptome data), confirming that they possess basal expression activity during the grain-filling stage, thus providing a basis for subsequent enhanced regulation through the insertion of response elements. This analysis provides precise target information for the aforementioned design of gRNAs and editing templates. This invention is not limited to increasing anthocyanin content. The method is also applicable to regulating the synthesis of other functional components that are not essential for plant growth but have important economic or nutritional value. For example, if the target functional component is a carotenoid, the target gene can be selected from phytoene synthase (TaPSY) and β-carotene hydroxylase (TaBCH), and the feedforward target gene can be selected from 1-deoxy-D-xylitol-5-phosphate synthase (TaDXS) in the upstream methyl erythritol phosphate pathway (MEP). As another example, if the target functional component is vitamin E (tocopherol), the target gene can be selected from urobilinogen geraniol-geraniol transferase (TaHGGT), and the pre-frescoes target gene can be selected from tyrosine aminotransferase (TaTAT). Those skilled in the art can determine the corresponding target genes and feedforward target genes based on the target metabolic pathway.

[0070] See attached document Figure 5 , Figure 5 This is a schematic diagram illustrating a strategy for in-situ insertion of response elements into the endogenous promoter of a target gene using the CRISPR / Cas9 system. This embodiment describes a specific method for precisely inserting the designed response element into the defined target gene and the endogenous promoter region of the feedforward target gene.

[0071] First, single-guide RNAs (gRNAs) were designed and screened. Multiple gRNAs were designed for the endogenous promoter sequences of each target gene (TaCHS, TaDFR) and feedforward target gene (TaPAL). The selection principle for gRNA targets was to choose non-critical cis-acting element regions located in the core region of the promoter (e.g., upstream of the TATA-box to -0 bp), to introduce new regulatory elements without disrupting the basic promoter activity. Target prediction was performed using online gRNA design tools (such as CRISPR-P2.0), and their potential off-target risks were assessed. Two to three gRNA target sequences with high scores and low off-target risks were selected for subsequent activity validation. Protoplast validation experiments were used to screen for gRNAs with the highest cleavage efficiency for each promoter target.

[0072] Secondly, a DNA repair template (Donor Template) for homology-directed repair (HDR) was designed. A separate repair template was designed for each promoter editing event. This repair template is a linear or circular double-stranded DNA segment, and its structure includes three parts: 5' Homologous arm: A DNA sequence that is completely homologous to the promoter region upstream of the gRNA cleavage site, with a length of 500-1000 bp.

[0073] The sequence to be inserted is located between two homologous arms. This sequence contains a specific response element designed according to the gene type. For the feedforward target gene (TaPAL), the sequence to be inserted contains a feedforward response element (i.e., the binding sequence of the feedforward activator TALE-VP64). For the target gene (TaCHS, TaDFR), the sequence to be inserted contains a functional response element (i.e., the binding sequence of the functional activator dCas9-VPR). To improve activation efficiency, the sequence to be inserted may contain multiple tandem copies of the response element (e.g., 3 to 5 copies), each separated by a short linker sequence.

[0074] 3' Homologous arm: A DNA sequence that is completely homologous to the promoter region downstream of the gRNA cleavage site, and is about the same length as the 5' homologous arm.

[0075] Next, a CRISPR / Cas9 expression vector for promoter editing was constructed. To achieve simultaneous editing of multiple gene promoters, a polycistronic editing vector containing multiple gRNA expression cassettes was constructed. This vector uses the wheat U6 promoter to drive the expression of selected gRNAs targeting the TaPAL, TaCHS, and TaDFR promoters, respectively. Simultaneously, the vector uses a strong promoter (such as pUbi) to drive the expression of the Cas9 nuclease. Furthermore, repair template DNA fragments designed for the three genes were cloned into the T-DNA region of this vector, or co-transformed with the Cas9 / gRNA expression vector plasmid.

[0076] This embodiment uses the CRISPR / Cas9 system for genome editing. However, the present invention is not limited thereto. Other genome editing tools capable of sequence-specific DNA cutting, such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or CRISPR / Cas12a (Cpf1)-based systems, can all be used to achieve in situ modification of endogenous promoters within the framework of this invention.

[0077] Finally, the promoter editing vector was introduced into wheat immature embryos via Agrobacterium-mediated transformation. Within the plant cells, the Cas9 protein, guided by gRNA, precisely cleaved the three target promoter sequences, generating double-strand breaks (DSBs). The cell's own HDR repair mechanism utilized the co-transformed repair template DNA as a template for repair, thereby precisely integrating the insert sequence containing the responsive element into the predetermined cleavage site of the promoter. This operation completed the artificial regulatory interface modification of endogenous genes, enabling their transcriptional activation to be controlled by subordinate transcription factors in a multi-gene co-activation system. Cells modified in this step will be used for subsequent plant regeneration and selection.

[0078] Reference Figure 6 After the multi-gene expression vector (pWheat-SynBio-Regulator) and promoter editing vector were constructed, they were introduced into wheat cells using Agrobacterium-mediated transformation and regenerated to obtain transgenic plants. The specific process is as follows: Preparation of Agrobacterium strains: The validated pWheat-SynBio-Regulator vector and promoter editing vector were introduced into Agrobacterium strain AGL1, either separately or jointly, via electroporation. Positive colonies were picked and streaked onto YEP solid medium containing the appropriate antibiotics (e.g., rifampin, carbenicillin, and spectinomycin) for activation. Activated single colonies were inoculated into YEP liquid medium and cultured at 28°C with shaking at 2 rpm until the logarithmic growth phase (OD600 value 0.6–0.8). The cells were collected by centrifugation, resuspended in infection buffer (e.g., liquid MS medium containing 0 μM acetylsylphenone) to adjust the OD600 value to 0.8–1.0, and incubated at room temperature for 2–4 hours to activate the expression of the Agrobacterium Vir gene.

[0079] Preparation and infection of wheat explants: Mature seeds of wheat varieties 'Kenong 199' or 'Bobwhite' were selected and germinated under aseptic conditions. Immature embryos 12-15 days after pollination were used as explants. The isolated embryos were placed in the prepared Agrobacterium suspension for co-infection. The infection process was carried out under vacuum (e.g., -0.08 MPa, 10 minutes), followed by standing at normal pressure for minutes to improve the efficiency of Agrobacterium entry into plant tissues.

[0080] Co-cultivation and screening: After infection, the embryos were removed, excess bacterial culture was blotted off with sterile filter paper, and placed on a co-culture solid medium. The medium also contained 0 μM acetylsyringone to maintain the activation of the Vir gene. Co-culture was carried out at 23-25°C in the dark for 2-3 days. After co-culture, the embryos were transferred to a selection medium containing a selection agent (e.g., 50 mg / L hygromycin for screening integration events of the pWheat-SynBio-Regulator vector) and an antibacterial agent (e.g., 250 mg / L carbenicillin for inhibiting Agrobacterium growth).

[0081] Callus induction, differentiation, and plant regeneration: On the selection medium, only cells that successfully integrate T-DNA and express the hygromycin resistance gene can survive and proliferate, forming resistant callus. This process typically takes 4-6 weeks, with subculture every two weeks to fresh selection medium. Robust resistant callus is then transferred to differentiation medium and cultured under light to induce the differentiation of green buds. When the buds reach 2-3 cm in length, they are cut and transferred to rooting medium to induce rooting, eventually forming complete plantlets (T0 generation). The regenerated plantlets are transplanted into nutrient soil and acclimatized and cultured in a greenhouse until they flower and bear fruit, yielding T1 generation seeds. This process completes the integration of the designed synthetic biology system and editing system into the wheat genome.

[0082] In addition to Agrobacterium-mediated transformation, the expression vectors and editing vectors described in this invention can also be introduced using other genetic transformation methods suitable for wheat, such as gene gun method (particle bombardment method), which involves injecting gold or tungsten powder particles containing plasmid DNA at high speed into wheat embryos or other suitable recipient tissues.

[0083] Reference Figure 7 , Figure 8 and Figure 9 After obtaining regenerated plants (T0 generation) and their offspring, molecular biological tests are required to screen for positive plants that have integrated a complete synthetic biology system and completed the expected endogenous promoter editing. The following is a systematic screening method: First, preliminary screening for transgenic integration was conducted. Genomic DNA was extracted from the young leaves of T0 generation regenerated plants. Polymerase chain reaction (PCR) was used for detection. Multiple primer pairs were designed to target key components within the T-DNA region of the pWheat-SynBio-Regulator vector, such as the coding sequences for the hygromycin resistance gene, the master transcription factor (GAL4-ER-VP64), and at least one dependent transcription factor (e.g., dCas9-VPR). Wild-type wheat DNA was used as a negative control, and plasmid DNA as a positive control. Only plants that amplified bands of the expected size for all target fragments were preliminarily identified as positive transformants containing a complete synthetic biology system.

[0084] Next, the copy number of the exogenous gene was analyzed. Southern blotting analysis was performed on T0 generation plants that tested positive in the initial PCR screening. A digoxigenin (DIG)-labeled probe, complementary to a single-copy gene (such as a hygromycin resistance gene) in the T-DNA region, was used. The plant genomic DNA was completely digested with a restriction endonuclease (such as HindIII) without a cleavage site in the T-DNA region, followed by gel electrophoresis, membrane transfer, and hybridization. The number of hybridization bands determined the integration copy number of the exogenous T-DNA in the wheat genome. Plants with single-copy or low-copy (2-3 copy) insertions were selected for further analysis and propagation to reduce the risk of gene silencing.

[0085] Next, precise identification of endogenous promoter editing events is performed. Specific primers are designed for positive plants with edited promoters. These primers are located on the genomic sequence inside the inserted response element sequence in the repair template and outside the homologous arm region, respectively. Only plants that have undergone precise homologous recombination can amplify fragments of a specific size using these primers. The PCR product is then subjected to Sanger sequencing, and the sequencing results are compared with the expected edited sequence to confirm that the response element has been precisely and accurately inserted into the endogenous promoters of the TaPAL, TaCHS, and TaDFR genes as designed. This step is crucial for verifying the success of the core technology operation of this invention.

[0086] Finally, preliminary validation of the system's functionality was conducted. Homozygous positive plants from the T1 or T2 generations were selected for induction experiments at the seedling stage. The experimental group was treated with a solution containing a specific chemical inducer (such as β-estradiol), while the control group was treated with an equal volume of solution without the inducer. Samples were taken at different time points after treatment (e.g., 0h, 6h, 12h, 24h), and total RNA was extracted and reverse transcribed into cDNA. Real-time quantitative PCR (qRT-PCR) was used to detect the transcriptional levels of each component and target gene in the system. The expected results showed that, compared with the control group, the transcriptional levels of dependent transcription factors (dCas9-VPR, TALE-VP64), synthetic repressors (TALE-KRAB, with a time delay), and the edited target genes (TaPAL, TaCHS, TaDFR) were significantly upregulated in the experimental group. This result demonstrates that the constructed synthetic biology system possesses the function of responding to exogenous induction and activating downstream gene expression.

[0087] See attached document Figure 10 and attached Figure 11 , Figure 10 This is an example of a high-performance liquid chromatography (HPLC) chromatogram for anthocyanin content determination. Figure 11 This is a statistical chart of anthocyanin content. This example describes a specific method for inducing homozygous positive plants selected during key wheat developmental stages and quantitatively detecting changes in the content of target functional components.

[0088] First, the experimental materials were prepared and cultivated. Homozygous transgenic wheat lines of generation T3 or higher, confirmed by molecular identification, carrying a complete synthetic biology system and with the target promoter successfully edited, were selected. Wild-type wheat (WT) and transgenic wheat transformed only with an empty vector were used as control groups. All materials were cultivated under identical field or greenhouse conditions, employing standard field management practices to ensure consistent growth conditions for all plants.

[0089] Secondly, induction treatment was implemented during the wheat grain-filling stage. This stage is crucial for the accumulation of functional components (such as anthocyanins). Approximately 15 days after flowering, when the grains begin to rapidly accumulate dry matter, the transgenic experimental group plants were induced. A specific chemical inducer (β-estradiol) was dissolved in deionized water containing 0.1% Tween- to prepare a 10 μM working solution. Using a handheld sprayer, the working solution was evenly sprayed onto the entire spike and flag leaf of the plant until the liquid began to drip. For the transgenic control group and the wild-type control group, an equal volume of the solvent without the inducer (0.1% Tween- aqueous solution) was sprayed. To ensure the induction effect, the spraying was repeated every 3 days, for a total of 3 treatments.

[0090] Next, sample collection and pretreatment were performed. Wheat ears were randomly collected from plants in each treatment group at different time points after the initial induction treatment (e.g., day 0, day 7, and day 14 after treatment) and after grain maturity. The grains were harvested, immediately flash-frozen in liquid nitrogen, and stored at -80°C for later use. Before analysis, the frozen grain samples were freeze-dried in a freeze dryer until constant weight was achieved. The dried grains were then ground into a uniform fine powder using a high-throughput tissue homogenizer.

[0091] Finally, anthocyanin content was extracted and quantitatively analyzed. 50 mg of wheat seed powder was accurately weighed and added to 1 mL of methanol solution containing 1% hydrochloric acid as the extraction solvent. Extraction was performed in the dark at 4°C with shaking for 24 hours. After centrifugation (12,000 rpm, 10 min), the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the sample to be tested. Quantitative analysis was performed using a high-performance liquid chromatography (HPLC) system. The chromatographic column was a C18 reversed-phase column, and the mobile phase was a gradient elution of acetonitrile and 0.1% formic acid aqueous solution. The detection wavelength was set to 5 nm. The total anthocyanin content in the sample was calculated and determined by comparing with a standard curve plotted using a known concentration of cyanidin-3-O-glucoside standard. Statistical analysis was performed on the anthocyanin content of different treatment groups and at different time points to verify the actual effect of the method of this invention on increasing the content of the target functional components in wheat seeds.

[0092] See attached document Figure 12 , Figure 12 This is a comparative diagram of the main agronomic traits of transgenic plants according to an embodiment of the present invention. This embodiment describes a method for comprehensively evaluating the agronomic traits of transgenic wheat plants carrying synthetic biology systems and their control groups, in order to determine the impact of the technical solution described in this invention on the normal growth and development and yield composition of wheat.

[0093] To conduct this evaluation, a field plot experiment was performed. Three treatment groups were set up: Group A was the wild-type control group (WT); Group B consisted of uninduced homozygous transgenic plants (TG-NI), which were sprayed with a solvent (0.1% Tween-water solution) without an inducer at the same time points as Group C; Group C consisted of induced homozygous transgenic plants (TG-I), which were sprayed with an inducer during the grain-filling stage as described above. A randomized block design was used, with three replicate plots for each treatment group. Each plot was 6 square meters, and planting density and routine field management practices were kept consistent.

[0094] During the wheat waxy ripening stage, a representative main stem plant was randomly selected from each plot for index measurement. Key agronomic traits measured included: Plant height: The vertical distance from the ground surface to the top of the ear (excluding the awn).

[0095] Ear length: The length measured from the neck node to the top of the ear.

[0096] Number of grains per ear: The actual number of grains per main ear is counted after manual threshing.

[0097] 1000-grain weight: After all the samples from the plots have been harvested, threshed, and impurities removed, a random sample is taken from the mixed sample of each plot, and 1000 grains are counted using an automatic grain counter and then weighed. This operation is repeated three times for each sample and the average value is taken.

[0098] For a more comprehensive evaluation, in addition to the above indicators, other agronomic traits can be measured, including: the number of effective tillers per plant, heading time (the number of days from sowing to 50% of the plants heading), and maturity time (the number of days from sowing to 90% of the plants turning yellow).

[0099] All collected data were processed using statistical analysis software. One-way ANOVA and Tukey's multiple comparison test were used to analyze the significance of differences in various agronomic traits among different treatment groups, with a significance level set at p<0.05.

[0100] The analysis results showed that, compared with the wild-type control group (Group A), the uninduced transgenic genome (Group B) and the induced transgenic genome (Group C) did not exhibit statistically significant differences in key agronomic traits such as plant height, spike length, number of grains per spike, and thousand-grain weight (p>0.05). This data indicates that the integration of the synthetic biology system in this invention, and its induction and activation at specific times, did not have any observable negative impact on the individual growth, development, or final yield components of wheat plants.

[0101] Therefore, those skilled in the art should recognize that although numerous exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications conforming to the principles of the present invention can be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the invention. Thus, the scope of the present invention should be understood and construed as covering all such other variations or modifications.

Claims

1. A molecular design breeding method for increasing the content of functional components in wheat, characterized in that, Includes the following steps: S1. Identify one or more target genes, wherein the target genes are genes encoding proteins required in the biosynthesis or transport pathways of one or more functional components; S2. Integrating and expressing a synthetic biology system in the wheat genome, said synthetic biology system comprising: A chemically induced regulatory switch, used to respond to an exogenously applied chemical inducer; A multi-gene synergistic activation system is used to drive the expression of the target gene after the chemically induced regulatory switch is activated; A delayed negative feedback inhibition loop is used to inhibit the activity of the multi-gene co-activation system after a preset time delay, so as to terminate the expression of the target gene. S3. Insert the multi-gene co-activation system into the endogenous promoter of the target gene, and integrate the other parts of the synthetic biology system into the wheat genome.

2. The molecular design breeding method for increasing the content of functional components in wheat according to claim 1, characterized in that, The multi-gene co-activation system includes one or more dependent transcription factors and response elements corresponding to the dependent transcription factors; The response element is inserted in situ into the endogenous promoter of the target gene so that the dependent transcription factor corresponding to the response element regulates the expression of the target gene.

3. The molecular design breeding method for increasing the content of functional components in wheat according to claim 2, characterized in that, The dependent transcription factors are divided into feedforward activators and functional activators; The response element is divided into a feedforward response element corresponding to the feedforward activator and a functional response element corresponding to the functional activator; In step S3, the in-situ insertion of the response element into the endogenous promoter of the target gene is as follows: The feedforward response element is inserted into the endogenous promoter of the feedforward target gene, which is a gene in an upstream metabolic pathway related to the supply of precursors required for the synthesis of the functional component. The functional response element is inserted into the endogenous promoter of the target gene associated with the synthesis of the functional component.

4. The molecular design breeding method for increasing the content of functional components in wheat according to claim 3, characterized in that, In step S2, after the chemically induced regulatory switch is activated, the expression of the target gene is driven specifically as follows: The expression of the feedforward activator and the functional activator corresponding to the target gene is driven and binds to the feedforward response element and the functional response element, respectively, thereby synergistically activating the transcription of the feedforward target gene and the target gene.

5. The molecular design breeding method for increasing the content of functional components in wheat according to claim 4, characterized in that, The delayed negative feedback suppression loop includes a synthetic suppressor and a control element; The synthetic repressor is used to inhibit the activity of the multi-gene co-activation system to terminate the transcription of the target gene; The regulatory element synchronously responds to the transcription of the target gene driven by the multi-gene co-activation system, and drives the expression of the synthetic repressor after a preset time delay.

6. The molecular design breeding method for increasing the content of functional components in wheat according to claim 5, characterized in that, The chemically induced regulatory switch includes: The master transcription factor has an artificial receptor protein, which has a specific binding ability to the chemical inducer; After the artificial receptor protein binds to the chemical inducer, the transcriptional activity of the master control transcription factor is activated to complete its expression, thereby driving the expression of the target gene.

7. The molecular design breeding method for increasing the content of functional components in wheat according to claim 6, characterized in that, The transcriptional activation activity of the master transcription factor is controlled by the binding state of the artificial receptor protein to the specific chemical inducer.

8. The molecular design breeding method for increasing the content of functional components in wheat according to claim 6, characterized in that, The activation of transcriptional activity to complete expression, thereby driving the expression of the target gene, specifically involves: The expression of each dependent transcription factor is driven by a promoter of the master transcription factor’s specific binding site, which contains a specific binding site that can be recognized by the DNA binding domain of the master transcription factor.

9. The molecular design breeding method for increasing the content of functional components in wheat according to claim 5, characterized in that, The regulatory element is configured to target a promoter with low affinity for one of the dependent transcription factors in one or more of the multi-gene co-activation systems; The promoter is effectively activated after the concentration of dependent transcription factors accumulates over time and reaches a predetermined threshold, thereby driving the expression of synthetic repressors.

10. The molecular design breeding method for increasing the content of functional components in wheat according to claim 1, characterized in that, The functional ingredients are selected from one or more of dietary fiber, anthocyanins, carotenoids, phenolic acids, vitamins, iron, and zinc.

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