Artificial agglomerate enhanced plant molecular proximity regulation system and application thereof

Abstract

The present invention is entitled "A Plant Molecular Proximity Regulation System Based on Artificial Conglomerates and Its Application", belonging to the fields of plant synthetic biology, bioengineering and gene circuit design technology. The technical problem to be solved is to develop new plant chemical induction tools and establish a general strategy for enhancing molecular proximity function using artificial conglomerates. The key technical solution is a plant chemical induction proximity regulation system, which includes: (1) a DNA binding module; (2) a chemical induction recognition module; (3) a transcription regulation module; and (4) a response promoter module.

Description

Technical Field

[0001] This invention belongs to the fields of plant synthetic biology, bioengineering and gene circuit design technology, and specifically relates to a plant molecular proximity regulation system based on artificial condensates and its application. Background Technology

[0002] Chemical-induced proximity (CIP) technology is one of the core technologies in synthetic biology and gene regulation. This technology relies on small molecule drugs to mediate specific and reversible interactions between proteins, precisely achieving proximity binding, conformational regulation, and functional activation of target proteins. It can programmably and spatiotemporally specific artificially regulate core life processes such as cellular transcriptional regulation, protein degradation, signal transduction, and metabolic pathway remodeling. It has been widely used in animal cell engineering, gene therapy, and microbial synthetic biology, and is a core tool for constructing precise and controllable gene regulation networks.

[0003] Currently, established CIP-based induction expression systems in the plant field mainly include estradiol induction systems and rapamycin induction systems. These systems can achieve the induction of exogenous gene expression and have been applied to plant gene function research and synthetic biology. However, the variety of chemical induction tools available for plant systems is still relatively limited, and there is still a need for further expansion and optimization in terms of biological orthogonality, regulatory performance, and multi-system compatibility among different induction systems. With the development of complex gene circuits, multi-input logic regulation, and modular gene expression systems in plants, the demand for chemical induction tools with different induction mechanisms that do not interfere with each other is increasing. Therefore, developing new plant chemical induction systems and enriching the plant orthogonal induction tool library is of great significance for plant synthetic biology and the construction of complex gene regulatory networks.

[0004] In recent years, the danoprevir and grazoprevir drug-induced transcriptional regulation system has overcome the technical limitations of traditional induction systems. This system relies on two specific small molecule drugs to mediate a precise and efficient drug-dependent interaction between the NS3a protein and an artificial recognition protein, achieving highly specific regulation and demonstrating excellent regulatory performance and application potential in mammalian cell systems.

[0005] Relevant patent documents retrieved: Publication No. WO2025261380A1, published on December 26, 2025, discloses a trimerization regulatory element, a method for constructing it, and its applications. The trimerization regulatory element includes protein A and protein B, obtained from the resolution of hepatitis C virus protease NS3a, as well as protein C. The trimerization of the trimerization regulatory element can be mediated by a protease NS3a-specific targeting drug (grazoprevir / danoprevir). The trimerization regulatory element provided by this invention exhibits excellent small-molecule-mediated trimerization effects, uses drugs approved by the National Medical Products Administration, and the proteins used are not present in the human body, resulting in better bioorthogonality. Furthermore, it can serve as an effective drug regulatory switch in clinical practice, effectively avoiding serious cytotoxicity and off-target effects.

[0006] However, to date, the danoprevir and grazoprevir-induced transcriptional regulation systems have not yet been systematically constructed, functionally validated, and applied in plant systems. Their adaptability, regulatory efficiency, and application value in plant cells remain undefined, representing a significant technological gap in the field of plant chemical induction and regulation tools.

[0007] Furthermore, in recent years, the synthetic condensate strategy has gradually become an important research direction in the field of synthetic biology. This strategy involves fusing protein domains with condensate-forming capabilities with transcriptional regulatory elements, signaling proteins, or enzyme molecules to create a locally enriched functional microenvironment within the cell. This increases the local concentration and interaction efficiency of related biomolecules, thereby enhancing biological processes such as transcriptional regulation, signal transduction, and metabolic regulation. It has shown great application potential in animal cells and microbial systems.

[0008] Relevant patent documents retrieved: Publication number CN113164622A, published on July 23, 2021, discloses a composition and method for regulating gene regulation by modulating the formation, composition, maintenance, dissolution, and regulation of agglomerates. This invention identifies and constructs natural and synthetic agglomerates, discloses methods for regulating the structure and function of agglomerates, and discloses related compositions and applications for regulating gene expression and cellular function using agglomerates, providing a new technical solution for agglomerate-based gene regulation.

[0009] However, to date, there are no reports on applying synthetic conglomerates to plant CIP-induced transcription systems, particularly lacking technical solutions for enhancing CIP-induced transcription efficiency, expanding the dynamic response range, and improving gene expression performance using synthetic conglomerates. The applicability of synthetic conglomerates in plant cells and their enhancing effect on CIP-induced transcription systems still lack systematic validation. Therefore, it is necessary to establish a conglomerate-enhanced chemically induced transcription regulation system suitable for plant systems, further improving induced expression performance while maintaining the bioorthogonality of the chemically induced system, and providing new technical means for complex gene circuits and precise gene regulation in plants. Summary of the Invention

[0010] The purpose of this invention is to provide: A plant molecular proximity regulation system based on artificial condensates and its application, and related technologies, to solve technical problems such as developing new plant chemical induction tools and establishing a general strategy for enhancing molecular proximity function using artificial condensates, or a combination thereof.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as vector construction and incubation, shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The term “molecular proximity-dependent function” used in this article refers to a molecular action mode in which functions such as intracellular biochemical reactions, transcriptional activation, protein degradation, and signal transduction require the spatial proximity of two or more proteins to occur efficiently.

[0016] The term "Danoprevir" as used in this article refers to a small molecule hepatitis C virus NS3 protease inhibitor.

[0017] The term "NS3a protein" used in this article refers to the NS3 protease domain derived from HCV (hepatitis C virus).

[0018] The term "35S promoter" used in this article refers to the constitutive strong promoter of cauliflower mosaic virus, a universal strong promoter for transient expression in plants.

[0019] The term "IDR" used in this article refers to Intrinsically Disordered Region, a protein sequence without a fixed three-dimensional folded conformation that relies on multivalent weak interactions to mediate phase separation and condensation.

[0020] In a first aspect, the present invention provides: a plant chemically induced proximity regulation system, wherein the plant chemically induced proximity regulation system includes a Danoprevir-induced proximity regulation system and a Grazoprevir-induced proximity regulation system.

[0021] Among them are technical features such as Danoprevir-induced neighborhood regulation system and Grazoprevir-induced neighborhood regulation system.

[0022] Specifically, the plant chemically induced proximity regulation system includes: (1) DNA binding module; (2) Chemically induced recognition module; (3) Transcriptional regulation module; (4) Response startup submodule.

[0023] Furthermore, in (1), the DNA binding module is selected from at least one of the following: Gal4 DNA binding domain, LexA DNA binding domain, dCas9, TALE, and zinc finger protein.

[0024] Preferably, the DNA binding module in (1) is selected from the Gal4 DNA binding domain.

[0025] Furthermore, when the plant chemical induction proximity regulation system is induced by Danoprevir, the chemical induction recognition module described in (2) is composed of NS3a protein and DNCR protein.

[0026] Furthermore, the NS3a is fused with the DNA-binding module for expression.

[0027] Furthermore, the DNCR is fused with the transcription activation module for expression.

[0028] According to some embodiments of the present invention, in the presence of Danoprevir, NS3a specifically binds to DNCR, thereby inducing the expression of the target gene.

[0029] According to some embodiments of the present invention, the present invention provides a method for constructing a Danoprevir-induced proximity regulation system, comprising the following steps: (1) Expression vector construction: Gal4-NS3a fusion protein expression vector and DNCR-VP64 fusion protein expression vector were constructed; (2) Preparation of tobacco protoplasts: Tobacco leaves were given an enzymatic hydrolysate, and the reaction was terminated with W5 buffer. The protoplasts were obtained by filtration and centrifugation, and the concentration was adjusted to 1-2 × 10⁻⁶. 6 Cells / mL; (3) PEG transfection: Mix plasmid, protoplast and transformation solution and incubate. After the reaction is complete, add W5 solution, mix well, centrifuge and discard the supernatant, and incubate to express foreign gene; (4) Danoprevir treatment: Add Danoprevir and culture.

[0030] According to some embodiments of the present invention, step (1) includes constructing a Gal4-NS3a fusion protein expression vector and a DNCR-VP64 fusion protein expression vector, which are linked together by a self-cleaved T2A peptide coding sequence to form a polycistronic expression unit in the same expression cassette (SEQ ID NO. 1). A 4×UAS, 6×UAS, or 8×UAS response element is placed upstream of the minimal promoter (SEQ ID NO. 2-4) to drive the expression of firefly luciferase (fLUC) (SEQ ID NO. 5). Renal luciferase (rLUC) is driven by a 35S promoter as an internal control (SEQ ID NO. 6).

[0031] Furthermore, the recombinant plasmid is selected from at least one of pLIP, pEAQ-HT, pEAQ-DEST1, pJL-TRBO, and pCambia.

[0032] Furthermore, when the plant chemical induction proximity regulation system is Grazoprevir induced, the chemical induction recognition module described in (2) is composed of NS3a protein and GNCR protein.

[0033] Furthermore, the NS3a is fused with the DNA-binding module for expression.

[0034] Furthermore, the GNCR is fused with and expressed in a transcriptional activation module.

[0035] According to some embodiments of the present invention, in the presence of Grazoprevir, NS3a specifically binds to GNCR, thereby inducing the expression of the target gene.

[0036] According to some embodiments of the present invention, the present invention provides a method for constructing a Grazoprevir-induced proximity regulation system, comprising the following steps: (1) Expression vector construction: Gal4-NS3a fusion protein expression vector and GNCR-VP64 fusion protein expression vector were constructed; (2) Preparation of tobacco protoplasts: Tobacco leaves were given an enzymatic hydrolysate, and the reaction was terminated with W5 buffer. The protoplasts were obtained by filtration and centrifugation, and the concentration was adjusted to 1-2 × 10⁻⁶. 6 Cells / mL; (3) PEG transfection: Mix plasmid, protoplast and transformation solution and incubate. After the reaction is complete, add W5 solution, mix well, centrifuge and discard the supernatant, and incubate to express foreign gene; (4) Grazoprevir treatment: Grazoprevir was added to the culture.

[0037] According to some embodiments of the present invention, in step (1), the Gal4-NS3a H1 fusion protein expression vector and the GNCR-VP64 fusion protein expression vector are constructed and linked together by self-cleaving of the T2A peptide coding sequence to form a polycistronic expression unit (SEQ ID NO. 15) in the same expression cassette.

[0038] Specifically, the plant chemically induced proximity regulation systems described herein can be used in plant protoplast expression systems, plant leaf transient expression systems, plant suspension cell systems, plant root hair systems, plant stable transformation systems, or cell-free expression systems.

[0039] Furthermore, in (3), the transcriptional regulatory module is selected from at least one of VP16, VP64, VPR, EDLL, SRDX, TPL or their derived regulatory domains.

[0040] Preferably, the transcriptional regulation module in (3) is VP64.

[0041] Furthermore, the response promoter module in (4) includes one or more DNA binding sites and a minimal promoter.

[0042] Furthermore, the response promoter contains 1 to 20 DNA binding sites.

[0043] Preferably, the response promoter contains 4 to 12 DNA binding sites.

[0044] More preferably, the response promoter contains eight DNA binding sites.

[0045] Specifically, the plants include, but are not limited to, Nicotiana benthamiana, cultivated tobacco, Arabidopsis thaliana, rice, corn, wheat, soybean, tomato, potato and other monocotyledonous or dicotyledonous plants.

[0046] Specifically, the plant chemical induction proximity regulation system may further include an aggregate enhancement module.

[0047] Specifically, the aggregate enhancement module includes at least one protein domain capable of promoting the formation of biomacromolecule aggregates.

[0048] Furthermore, the condensate enhancement module is selected from any one or more of the following: (1) AtSSF source: PrLD; (2) AtFCA source PrLD; (3) The N-terminal domain of the FUS protein; (4) LAF-1 source RGG structural domain; (5) Other naturally or artificially designed intrinsically disordered domains (IDRs).

[0049] According to some embodiments of the present invention, the aggregate enhancement module is selected from the RGG domain derived from LAF-1 protein, the N-terminal low-complexity domain (FUSn) derived from human FUS protein, the prion-like domain (AtFCA PrLD) of Arabidopsis FCA protein, and the prion-like domain (AtSSF PrLD) of Arabidopsis SSF protein.

[0050] Preferably, the aggregate enhancement module is selected from the N-terminal low-complexity domain (FUSn) of human FUS protein and the prion-like domain (AtSSF PrLD) of Arabidopsis SSF protein.

[0051] Specifically, the condensate enhancement module is fused with the DNA binding module, the chemically induced recognition module, the transcriptional regulation module, or a combination thereof for expression.

[0052] Preferably, the aggregate enhancement module is fused to one side of the DNA binding module.

[0053] In some embodiments, the aggregate enhancement module promotes the formation of point-like aggregate structures, droplet-like structures, aggregate structures, or other locally enriched structures within cells.

[0054] Specifically, the condensate enhancement module is applicable to a variety of chemical induction systems.

[0055] Furthermore, the chemical induction systems include, but are not limited to, Grazoprevir induction systems and Rapamycin induction systems.

[0056] Furthermore, the Grazoprevir induction system consists of the Gal4-NS3a fusion protein and the GNCR-VP64 fusion protein.

[0057] Furthermore, the Rapamycin induction system is composed of the Gal4-FKBP fusion protein and the FRB-VP64 fusion protein.

[0058] Secondly, the present invention provides a kit comprising the above-described plant chemical induction proximity regulation system.

[0059] Thirdly, the present invention provides a method for enhancing molecular proximity-dependent functions in plant cells, comprising: introducing the aforementioned condensate enhancement module into a molecular proximity system.

[0060] Furthermore, the molecular proximity-dependent function includes any one or more of the following: (1) Transcriptional activation; (2) Transcriptional repression; (3) Regulation of metabolic pathways; (4) Protein recruitment; (5) Signal transduction regulation; (6) Chromatin regulation; (7) Targeted protein degradation; (8) RNA regulation; (9) Epigenetic regulation.

[0061] Preferably, the molecular proximity-dependent function is chemically induced transcriptional activation.

[0062] Fourthly, the present invention provides a method for regulating the synthesis of plant metabolites using the aforementioned system, wherein the method utilizes a chemically induced recognition module to drive the expression of metabolic pathway enzymes.

[0063] In some embodiments, the metabolic pathway includes the betaine synthesis pathway.

[0064] Specifically, the beet pigment synthesis pathway includes CYP76AD1, DODA, and glycosyltransferases.

[0065] Preferably, the metabolic pathway is implemented using the RUBY expression framework.

[0066] According to some embodiments of the present invention, an enhanced Danoprevir induction system (vector sequence SEQ ID NO. 23) of AtSSF PrLD is fused to the N-terminus of Gal4-NS3a, and RUBY expression is driven using the above system.

[0067] Fifthly, the present invention provides a method for enhancing the degradation of plant-targeted proteins using the aforementioned system, the method comprising: The aforementioned system is used to recruit ubiquitin ligase components to the vicinity of the target protein, thereby promoting the degradation of the target protein.

[0068] Specifically, the E3 ubiquitin ligase components include, but are not limited to, NSlmb, TIR1, COI1, CUL1, CUL3 or their functional fragments.

[0069] Furthermore, the E3 ubiquitin ligase component is NSlmb.

[0070] Specifically, the target protein includes endogenous proteins, exogenous proteins, fluorescent proteins, or their fusion proteins.

[0071] Preferably, the target protein is GFP.

[0072] According to some embodiments of the present invention, the method includes the following steps: S1. Construction of expression vector; S2. Agrobacterium infection and transient expression: The exogenous GFP expression recombinant plasmid and the above expression vector were transformed into Agrobacterium competent cells. The cultured bacterial solution was resuspended in the infection buffer and the OD600 was adjusted to 0.8. The exogenous expression recombinant plasmid was mixed with the corresponding degradation system vector and co-infected tobacco leaves.

[0073] Step S1 includes: constructing a plant-targeted protein degradation system based on the VHHGFP4 and NSlmb fusion protein (SEQ ID NO. 24).

[0074] Furthermore, an enhanced degradation system incorporating AtSSF PrLD at the N-terminus or C-terminus of the degradation module (N-terminal fusion vector sequence: SEQ ID NO. 26, C-terminal fusion vector sequence: SEQ ID NO. 27).

[0075] Further, in step S2, the recombinant plasmid is selected from at least one of pLIP, pEAQ-HT, pEAQ-DEST1, pJL-TRBO, and pCambia; preferably pEAQ-HT.

[0076] Further, the Agrobacterium mentioned in step S2 is selected from GV3101, LBA4404, EHA105, K599 or C58; preferably GV3101.

[0077] In a sixth aspect, the present invention provides the application of the aforementioned systems, kits or methods in plant synthetic biology, plant metabolic engineering, plant gene circuit construction, plant protein degradation regulation and plant cell engineering.

[0078] The present invention has at least the following beneficial effects: This invention establishes, for the first time, a Danoprevir and Grazoprevir-induced proximity-dependent regulatory system in plants, adaptable to various plants and expression systems. Furthermore, it utilizes condensation domains to enhance induced transcriptional activation, and the condensate enhancement strategy is universally applicable to multiple chemical induction systems. The system provided by this invention can not only activate gene transcription but also enhance plant metabolic pathway synthesis and improve the efficiency of target protein degradation, extending to various molecular proximity-dependent regulatory processes. Attached Figure Description

[0079] Figure 1 This section describes the establishment of the Danoprevir-induced transcriptional regulatory system in plants. A. Schematic diagram of the Danoprevir induction system vector. B. Performance of the Danoprevir induction system in tobacco protoplasts. C. Performance of the Danoprevir induction system in a transient expression system in tobacco leaves.

[0080] Figure 2 The condensate-forming domain enhances Danoprevir-induced transcriptional activation. A. The performance of the Danoprevir-induced system when the IDR is inserted between DNCR and VP64. B. The performance of the Danoprevir-induced system when the IDR is fused to the N-terminus of Gal4-NS3a.

[0081] Figure 3 This is a diagram showing the performance of the Grazoprevir-induced transcription system in plants.

[0082] Figure 4 This study explores the application of aggregate enhancement strategies in chemically induced systems.

[0083] Figure 5 Figure showing the results of using a condensate-enhanced Danoprevir-induced system to regulate beet pigment synthesis. Comparison with non-condensate designs.

[0084] Figure 6 The results of enhancing plant-targeted protein degradation using aggregates are shown in the figure. Comparison with non-aggregate designs. Detailed Implementation

[0085] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0086] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0087] Example 1: Establishment of the Danoprevir-induced transcriptional regulatory system in plants (1) Construction of expression carrier Gal4-NS3a fusion protein expression vector and DNCR-VP64 fusion protein expression vector were constructed and linked together via a self-cleaved T2A peptide coding sequence to form a polycistronic expression unit in the same expression cassette (SEQ ID NO. 1). 4×UAS, 6×UAS, or 8×UAS response elements were placed upstream of the minimal promoter (SEQ ID NO. 2-4) to drive firefly luciferase (fLUC) expression (SEQ ID NO. 5). Renal luciferase (rLUC) was driven by the 35S promoter as an internal control (SEQ ID NO. 6). All expression vectors were constructed based on the pLIP backbone (ZL202511237782.5), assembled using the Gibson Assembly method, and validated by sequencing.

[0088] SEQ ID NO. 1: MPKKKRKVGGGGSMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPRGGGGGSGGGGSGGGGSGGGGSLDPGGGGSGKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSPGSGSGTAVNIGGGTGPMDLQRPQGGGGEGRGSLLTCGDVEENPGPMSSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNHGGGGGSGGGGSGGGGSGGGGSLDPGGGGSGDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGGSGGSGSPKTRRRPRRSQRKRPPT*。

[0089] SEQ ID NO. 2: cggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGA。

[0090] SEQ ID NO. 3: cggagtactgtcctccgagcggagtactgtcctccgCTTGCATGCCcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGA。

[0091] SEQ ID NO. 4: cggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgCTTGCATGCCcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGA。

[0092] SEQ ID NO. 5:

[0093] SEQ ID NO. 6:

[0094] (2) Preparation of tobacco protoplasts Leafy tobacco leaves at the 4-6 leaf stage were selected, sterilely washed with water, the midrib was removed, and the lower epidermis was peeled off with tape. The exposed tissue was placed face down and floated in an enzymatic hydrolysis solution containing 1% (m / v) cellulase R10 (Yakult L0012) and 0.3% (m / v) cleavage enzyme (Yakult L0021). The solution was shaken at 50 rpm for 2.5 h for enzymatic hydrolysis. The reaction was terminated with W5 buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES (pH 5.7)). Protoplasts were obtained by filtration and centrifugation, and the concentration was adjusted to 1-2 × 10⁻⁶. 6 per mL.

[0095] (3) PEG transfection Prepare a PEG-CaCl2 solution containing 40% (m / v) PEG4000 (Sigma 95904), 0.2 M mannitol (Sigma, M1902), and 100 mM CaCl2 (Sigma, V900266), fresh for each use. Prepare a WI buffer solution containing 0.5 M mannitol, 20 mM KCl, and 4 mM MMES (pH 5.7), for later use.

[0096] Take 5 μL of pLIP test vector at a concentration of 1500 ng / μL and add 50 μL of protoplasts prepared by the tape peeling method to the bottom of each 2 mL round-bottom centrifuge tube. After gently mixing, add 55 μL of PEG-CaCl2 transformation solution (plasmid:protoplast:transformation solution = 1:10:11) and gently tap the bottom of the tube to mix. Incubate at room temperature for 10 min. After the reaction is complete, slowly add 220 μL of W5 solution to each tube, mix well, and centrifuge at 100 × g for 5 min using a refrigerated centrifuge with a horizontal rotor. Discard the supernatant containing the transformation solution, resuspend in 120 μL of WI solution, place in a 48-well plate, and incubate for 12 h to express the exogenous gene.

[0097] (4) Transient expression in tobacco leaves pLIP plasmids were transformed into the GV3101 (pSoup-p19) strain. After resistance selection, the plasmids were amplified and resuspended in injection buffer containing 10 mM MES, 10 mM MgCl2, and 100 μM acetylsyringone. The buffer was adjusted to OD600=1.0 and incubated at room temperature for 2 h before being injected into the abaxial surface of leaves from three independent tobacco plants of appropriate age. Samples were collected 48 h later using a pooled sampling method for luciferase detection.

[0098] (5) Danoprevir treatment Danoprevir was added after tobacco protoplast transfection. Final concentration: 500 nM. DMSO was used as a negative control. Cultured for 12 h.

[0099] Tobacco leaves were injected with Agrobacterium and then with Danoprevir. Final concentration: 500 nM. DMSO was used as a negative control. The mixture was incubated for 48 h.

[0100] (6) Dual-luciferase assay fLUC and rLUC were detected using the Dual-Luciferase Assay Kit (Yisheng Biotechnology, 11402ES60), and the ratio of fLUC to rLUC was calculated. Further, following the previously standardized quantitative method (ZL202511237782.5), with the 35S promoter as the RPU of 1.0, the activities of other promoters were expressed as relative values ​​to obtain the relative activity. The relative activity was calculated as: relative activity = (fLUC / rLUC) / (1.0 RPU / rLUC). 待测 / rLUC 待测 ) / (fLUC 35S / rLUC 35S The fold induction was calculated based on transcriptional activity before and after induction.

[0101] (7) Experimental results The results showed that Danoprevir treatment significantly induced the expression of the target gene. The 4×UAS, 6×UAS, and 8×UAS promoters all responded to Danoprevir induction. Among them, 8×UAS exhibited the highest induction efficiency. Figure 1 This demonstrates that the Danoprevir induction system can achieve controlled transcriptional activation in plant cells.

[0102] Example 2: Condensate-forming domain enhances Danoprevir-induced transcriptional activation (1) Construction of expression carrier To improve the activation efficiency of the Danoprevir-induced transcription system, several intrinsically disordered domains (IDRs) with condensate-forming capabilities were selected for testing, including the RGG domain from LAF-1 protein, the N-terminal low-complexity domain (FUSn) from human FUS protein, the prion-like domain (AtFCA PrLD) from Arabidopsis FCA protein, and the prion-like domain (AtSSF PrLD) from Arabidopsis SSF protein.

[0103] First, the aforementioned IDRs were inserted between DNCR and VP64 to construct DNCR-IDR-VP64 fusion proteins (DNCR-RGG-VP64: SEQ ID NO. 7, DNCR-FUSn-VP64: SEQ ID NO. 8, DNCR-AtFCA PrLD-VP64: SEQ ID NO. 9, DNCR-AtSSF PrLD-VP64: SEQ ID NO. 10); then, the same IDRs were fused to the N-terminus of the Gal4-NS3a fusion protein to construct IDR-Gal4-NS3a fusion proteins (RGG-Gal4BD-NS3a: SEQ ID NO. 11, FUSn-Gal4BD-NS3a: SEQ ID NO. 12, At FCA PrLD-Gal4BD-NS3a: SEQ ID NO. 13, At SSF PrLD-Gal4BD-NS3a: SEQ ID NO. 14). All expression vectors were constructed using the Gibson Assembly method and validated by sequencing. SEQ ID NO. 7: PKTRRRPRRSQRKRPPTGGGGSSSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEA AEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNH GGGGGSGGGGSMESNQSNNGGSGNAALNRGGRYVPPHLRGGDGGAAAAASAGGDDRRGGAGGGGYRRGGGNSGGGGGGGYDRGYNDNRDDRDNRGGSGGYGRDRNYEDRGYNGGGGGGGNRGYNN NRGGGGGGYNRQDRGDGGSSNFSRGGYNNRDEGSDNRGSGRSYNNDRRDNGGDGLEGGGGSGGGGSLDPGGGGSGDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML.

[0104] SEQ ID NO. 8: PKTRRRPRRSQRKRPPTGGGGSSSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNHGGGGGSGGGGSASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGGGGGGSGGGGSLDPGGGGSGDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML。

[0105] SEQ ID NO. 9: PKTRRRPRRSQRKRPPTGGGGSSSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNHGGGGGSGGGGSPSNTGIRGAGSDFSPKPGQATLPSNQGGPLGGYGVPPLNPLPVPGVSSSATLQQQNRAAGQHITPLKKPLHSPQGLPLPLRPQTNFPGAQAPLQNPYAYSSQLPTSQLPPQQNISRATAPQTPLNINLRPTTVSSATVQFPPRSQQQPLQKMQHPPSELAQLLSQQTQSLQATFQSSQQAISQLQQQVQSMQQPNQNLPLSQNGRAGKQQWAGSAIPRVASTTGSTPVSYVQTAAPAVSQSVGSVKCTWTEHTSPDGFKYYYNGLTGESKWEKPEEMIVFEREQQKQQQHQEKPTIQQSQTQLQPLQQQPQQVQQQYQGQQLQQPFYSSLYPTPGASHNTQYPSLPVGQNSQFPMSGIGQNAQDYARTHIPVGAASMNDISRTQQSRQSPQELMWKNKAGGGGSGGGGSLDPGGGGSGDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML。

[0106] SEQ ID NO. 10: PKTRRRPRRSQRKRPPTGGGGSSSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNHGGGGGSGGGGSAFPGAPPPVPYYHNNYNNPPHHQIHPPPPPHHHIAAVGFHKYPQNDNRDQRFNQPHYSGQQQNMIVDQSNNAPPPFGGGGSGGGGSLDPGGGGSGDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML。

[0107] SEQ ID NO. 11: PKKKRKVGGGGSGGGGSMESNQSNNGGSGNAALNRGGRYVPPHLRGGDGGAAAAASAGGDDRRGGAGGGGYRRGGGNSGGGGGGGYDRGYNDNRDDRDNRGGSGGYGRDRNYEDRGYNGGGGGGGNRGYNNNRGGGGGGYNRQDRGDGGSSNFSRGGYNNRDEGSDNRGSGRSYNNDRRDNGGDGLEGGGGSGGGGSLDPGGGGSGMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPRGGGGGSGGGGSGGGGSGGGGSLDPGGGGSGKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSP。

[0108] SEQ ID NO. 12: PKKKRKVGGGGSGGGGSASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGGGGGGSGGGGSLDPGGGGSGMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPRGGGGGSGGGGSGGGGSGGGGSLDPGGGGSGKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSP。

[0109] SEQ ID NO. 13: PKKKRKVGGGGSPSNTGIRGAGSDFSPKPGQATLPSNQGGPLGGYGVPPLNPLPVPGVSSSATLQQQNRAAGQHITPLKKPLHSPQGLPLPLRPQTNFPGAQAPLQNPYAYSSQLPTSQLPPQQNISRATAPQTPLNINLRPTTVSSATVQFPPRSQQQPLQKMQHPPSELAQLLSQQTQSLQATFQSSQQAISQLQQQVQSMQQPNQNLPLSQNGRAGKQQWAGSAIPRVASTTGSTPVSYVQTAAPAVSQSVGSVKCTWTEHTSPDGFKYYYNGLTGESKWEKPEEMIVFEREQQKQQQHQEKPTIQQSQTQLQPLQQQPQQVQQQYQGQQLQQPFYSSLYPTPGASHNTQYPSLPVGQNSQFPMSGIGQNAQDYARTHIPVGAASMNDISRTQQSRQSPQELMWKNKAGGGGSGGGGSLDPGGGGSGMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPRGGGGGSGGGGSGGGGSGGGGSGGGGSGKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSP。

[0110] SEQ ID NO. 14: PKKKRKVGGGGSAFPGAPPPVPYYHNNYNNPPHHQIHPPPPPHHHIAAVGFHKYPQNDNRDQRFNQPHYSGQQQNMIVDQSNNAPPPFGGGGSGGGGSLDPGGGGSGMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPRGGGGGSGGGGSGGGGSGGGGSLD PGGGGSGKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSP.

[0111] (2) Protoplast transfection and Danoprevir induction Protoplasts of *Nicotiana benthamiana* were prepared according to the method described in Example 1 and transiently transfected using a PEG-mediated method. Each transfection system was transfected with a dual-luciferase reporter vector containing (1) a DNA-binding module; (2) a chemically induced recognition module; (3) a transcriptional regulation module; and (4) a response promoter module, with a total DNA amount controlled at 15 μg. After transfection, Danoprevir was added to the culture system to a final concentration of 500 nM; an equal volume of DMSO was added to the control group. Protoplasts were collected after 12 h of treatment for subsequent analysis.

[0112] (3) Dual-luciferase detection fLUC and rLUC were detected using a dual-luciferase assay kit (Yisheng Biotechnology, 11402ES60). The ratio of fLUC to rLUC was calculated. Further, using the 35S promoter as a 1.0 RPU, the activities of other promoters were expressed according to their relative values ​​to obtain relative activity. Relative activity = (fLUC / rLUC) 待测 / rLUC 待测 ) / (fLUC 35S / rLUC 35S All experiments were performed in at least three biological replicates, with the group not treated with Danoprevir serving as a background control for calculating the fold induction.

[0113] (4) Experimental results The results showed that when the IDR was inserted between DNCR and VP64, the transcriptional activation ability induced by Danoprevir was significantly reduced, and some constructs almost completely lost their induction activity. In contrast, when the same IDR was fused to the N-terminus of the Gal4-NS3a protein, it significantly increased the transcriptional activation level induced by Danoprevir. Among them, FUSn, AtSSF PrLD, AtFCA PrLD, and LAF1-RGG all showed enhancing effects, with induction folds increasing to 8.9–12.2 times, significantly higher than the control system without IDR. Figure 2 The above results indicate that the condensate-forming domain can effectively enhance the output capacity of the Danoprevir-induced transcription system, and that this enhancement effect is significantly position-dependent.

[0114] Example 3: Establishment of the Grazoprevir-induced transcription system in plants (1) Construction of expression carrier To establish a Grazoprevir-induced transcriptional regulation system that can function in plant cells, Gal4-NS3a H1 fusion protein expression vector and GNCR-VP64 fusion protein expression vector were constructed. The two vectors were linked by self-cleaving of the T2A peptide coding sequence to form a polycistronic expression unit in the same expression cassette (SEQ ID NO. 15).

[0115] SEQ ID NO. 15: *

[0116] NS3a H1 is the NS3 protease domain derived from hepatitis C virus (HCV), and GNCR is an engineered binding protein that specifically recognizes the NS3a-Grazoprevir complex. Upon the addition of Grazoprevir, GNCR specifically interacts with Grazoprevir-bound NS3a H1, thereby recruiting the VP64 transcriptional activation domain to the vicinity of the Gal4 binding site, thus activating downstream gene transcription.

[0117] Concurrently, a dual-luciferase reporter vector containing an 8×UAS response element was constructed (the 8×UAS response promoter is shown in SEQ ID NO. 4, firefly luciferase (fLUC) is shown in SEQ ID NO. 5, and Renilla luciferase (rLUC) is driven by a 35S promoter as an internal reference SEQ ID NO. 6), with firefly luciferase (fLUC) serving as the reporter gene and Renilla luciferase (rLUC) serving as the internal reference gene. All expression vectors were assembled using the Gibson Assembly method and verified by sequencing.

[0118] (2) Protoplast transfection and Grazoprevir induction Protoplasts of *Nicotiana benthamiana* were prepared according to the method described in Example 1 and transiently transfected using a PEG-mediated method. Each transfection system was transfected with a dual-luciferase reporter vector containing (1) a DNA-binding module; (2) a chemically induced recognition module; (3) a transcriptional regulation module; and (4) a response promoter module, with a total DNA amount controlled at 15 μg. After transfection, Grazoprevir was added to the culture system to a final concentration of 500 nM; an equal volume of DMSO was added to the control group. Protoplasts were collected after 12 h of treatment for subsequent analysis.

[0119] (3) Dual-luciferase detection fLUC and rLUC were detected using a dual-luciferase assay kit. The ratio of fLUC to rLUC was calculated. Further, using the 35S promoter as a 1.0 RPU, the activities of other promoters were expressed relative to their values ​​to obtain relative activity. Relative activity = (fLUC / rLUC) 待测 / rLUC 待测 ) / (fLUC 35S / rLUC 35S All experiments were performed in at least three biological replicates, with the group not treated with Danoprevir serving as a background control for calculating the fold induction.

[0120] (4) Experimental results The results showed that without Grazoprevir, the system exhibited only a low basal expression level; after the addition of Grazoprevir, reporter gene expression was significantly increased, indicating that Grazoprevir can effectively mediate the specific interaction between NS3a H1 and GNCR, and further drive downstream transcriptional activation. Figure 3The above results demonstrate that the Grazoprevir induction system based on NS3a H1-GNCR can function normally in plant cells, achieving chemically induced expression of the target gene. This result is the first validation of the feasibility of the Grazoprevir-induced neighbor system in plants, providing a new technical solution for the development of plant chemical induction regulation tools.

[0121] Example 4: Application of aggregate enhancement strategies in various chemically induced systems (1) Construction of expression carrier To verify whether the aggregate enhancement strategy constructed in this invention has universality, aggregate forming domains FUSn and AtSSF PrLD were introduced into the Grazoprevir-induced system and the Rapamycin-induced system, respectively.

[0122] The Grazoprevir induction system consists of the Gal4-NS3a H1 fusion protein and the GNCR-VP64 fusion protein. The Rapamycin induction system consists of the Gal4-FKBP fusion protein and the FRB-VP64 fusion protein, which are linked by a self-cleaved T2A peptide coding sequence to form a polycistronic expression unit in the same expression cassette (SEQ ID NO. 16). SEQ ID NO. 16: MPKKKRKVGGGGSMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPRGGGGGSGGGGSGGGGSGGGGSLDPGGGGSGG VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGSGSGTAVNIGG GTGPMDLQRPQGGGGEGRGSLLTCGDVEENPGPASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWD LYYHVFRRISKGGGGGSGGGGSGGGGSGGGGSLDPGGGGSGDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGGSGGSGSPKTRRRPRRSQRKRPPT*.

[0123] For the two systems mentioned above, control vectors without IDR and expression vectors fused FUSn or AtSSF PrLD to the N-terminus of the Gal4BD-NS3a H1 and Gal4-FKBP modules were constructed respectively (FUSn-Gal4BD-NS3a H1: SEQ ID NO. 17, AtSSF PrLD-Gal4-NS3a H1: SEQ ID NO. 18, FUSn-Gal4-FKBP: SEQ ID NO. 19, AtSSFPrLD-Gal4-FKBP: SEQ ID NO. 20). All vectors were constructed based on the pLIP backbone and were verified by sequencing.

[0124] SEQ ID NO. 17: MPKKKRKVGGGGSASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQS YNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGGGGGGSGGGGSLDPGGGGSGMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPR.

[0125] SEQ ID NO. 18: MPKKKRKVGGGGSAFPGAPPPVPYYHNNYNNPPHHQIHPPPPPHHHIAAVGFHKYPQNDNRDQRFNQPHYSGQQQNMIVDQSNNAPPPFGGGGSGGGGSLDPGGGGSGMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPR.

[0126] SEQ ID NO. 19: MPKKKRKVGGGGSASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQSGGGGSGGYGQQDRGGGGGGSGGGGSLDPGGGGSGMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPRGGGGGSGGGGSGGGGSGGGGSLDPGGGGSGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE.

[0127] SEQ ID NO. 20: MPKKKRKVGGGGSAFPGAPPPVPYYHNNYNNPPHHQIHPPPPPHHHIAAVGFHKYPQNDNRDQRFNQPHYSGQQQNMIVDQSNNAPPPFGGGGSGGGGSLDPGGGGSGMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPRGGGGGSGGGGSGGGGSGGGGSLDPGGGGSGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE.

[0128] (2) Protoplast transfection and chemical induction Protoplasts of *Nicotiana benthamiana* were prepared according to the method described in Example 1, and transient transfection was performed using a PEG-mediated method. Each transfection system was supplemented with the corresponding expression vector and dual-luciferase reporter vector, with the total DNA amount controlled at 15 μg. After transfection, Grazoprevir and Rapamycin were added to the culture system to final concentrations of 500 nM and 100 nM, respectively; an equal volume of DMSO was added to the control group. Protoplasts were collected after 12 h of treatment for subsequent analysis.

[0129] (3) Dual-luciferase detection fLUC and rLUC were detected using a dual-luciferase assay kit. The ratio of fLUC to rLUC was calculated. Further, using the 35S promoter as a 1.0 RPU, the activities of other promoters were expressed relative to their values ​​to obtain relative activity. Relative activity = (fLUC / rLUC) 待测 / rLUC 待测 ) / (fLUC 35S / rLUC 35S All experiments were performed in at least three biological replicates, with the group not treated with Danoprevir serving as a background control for calculating the fold induction.

[0130] (4) Experimental results The results showed that ( Figure 4 In both Grazoprevir and Rapamycin induction systems, the introduction of FUSn or AtSSF PrLD enhanced transcriptional activation levels after chemical induction. Further analysis revealed that AtSSF PrLD not only increased expression intensity after induction but also significantly increased the fold increase in induction. In contrast, while FUSn increased expression levels after induction, it also increased background expression, resulting in a relatively limited increase in fold increase. These results demonstrate that the condensate enhancement strategy proposed in this invention is applicable to various chemically induced transcription systems and exhibits good scalability.

[0131] Example 5: Aggregate-enhanced Danoprevir-induced system regulates beet pigment synthesis (1) Construction of expression carrier To verify whether the condensate enhancement strategy constructed in this invention can be applied to the regulation of plant metabolic pathways, a Danoprevir-induced RUBY expression system was constructed. The RUBY expression framework contains three coding sequences (SEQ ID NO. 21) required for betaine biosynthesis: CYP76AD1, DODA, and glucosyltransferase, and can drive the accumulation of betaine in plant tissues through coordinated expression.

[0132] SEQ ID NO. 21:

[0133] A Danoprevir induction system without an aggregate-forming domain (vector sequence SEQ ID NO. 22) and an enhanced Danoprevir induction system fused with AtSSF PrLD at the N-terminus of Gal4-NS3a (vector sequence SEQ ID NO. 23) were constructed, and RUBY expression was driven using these systems. All vectors were assembled using the Gibson Assembly method and verified by sequencing.

[0134] SEQ ID NO. 22:

[0135] Vector sequence SEQ ID NO. 23:

[0136] (2) Agrobacterium infection and Danoprevir treatment Each expression vector was transformed into Agrobacterium GV3101 competent cells. Positive clones were selected, cultured, and resuspended in infection buffer, with the bacterial concentration adjusted to an OD600 of 0.8. Agrobacterium strains corresponding to different constructs were mixed and injected into the abaxial surface of Tobacco Benzovia leaves. After infection, the plants were cultured in the dark for 16 h, then transferred to normal culture conditions for continued growth. Once the exogenous gene was fully expressed, Danoprevir solution was injected into the infected area to achieve a final local concentration of 500 nM on the leaves; the control group received an equal volume of DMSO.

[0137] (3) Detection and quantitative analysis of beet pigments After treatment with Danoprevir, the leaves were cultured for 72 h, and the leaf phenotype was photographed and recorded. Subsequently, leaf tissue from the infected area was collected for beet pigment extraction: infected leaves were immersed in anhydrous ethanol at 65°C for approximately 30 min for decolorization, then leaf discs were prepared using a perforator and extracted with beet pigment in deionized water at room temperature in the dark. The extract was centrifuged, and the supernatant was collected. The absorbance was measured at 538 nm, and the pigment accumulation level was expressed as A538 / g FW. The systematic induction fold was calculated based on the difference between the treated and uninduced groups. All experiments were performed in at least three biological replicates.

[0138] (4) Experimental results The results showed that no significant betaine accumulation was observed in any treatment group without Danoprevir. After Danoprevir was added, RUBY expression was effectively activated, resulting in significant betaine production. Further comparison revealed that the introduction of AtSSF PrLD significantly increased betaine accumulation in leaves. Quantitative analysis showed that the Danoprevir-induced system without AtSSF PrLD achieved approximately 27-fold metabolite accumulation, while the induction fold increased to approximately 54-fold after the introduction of AtSSF PrLD. Figure 5 The above results demonstrate that the condensate enhancement strategy proposed in this invention can not only enhance transcriptional activation output, but also further improve the regulatory efficiency of downstream metabolic pathways and the level of metabolite synthesis.

[0139] Example 6: Conglomerate-enhanced plant-targeted protein degradation system (1) Construction of expression carrier To verify whether the condensate enhancement strategy proposed in this invention can be applied to other proximity-dependent cellular functions besides transcriptional regulation, a plant-targeted protein degradation system based on the VHHGFP4 and NSlmb fusion protein (SEQ ID NO. 24) was constructed. VHHGFP4 specifically recognizes GFP protein, and NSlmb recruits the plant's endogenous SCF ubiquitin ligase complex, thereby promoting the ubiquitination of target proteins and their degradation via the 26S proteasome.

[0140] SEQ ID NO. 24: MMKMETDKIMDETNSNAQAFTTTMLYDPVRKKDSSPTYQTERELCFQYFTQWSESGQVDFVEHLLSRMCHYQHGQINAYLKPMLQRDFITLLPIKGLDHIAENILSYLDAESLKSSELVCKEWLRVISEGMLWKKLIERKVRTDSLWRGLAERRNWMQY LFKPRPGQTQRPHSFHRELFPKIMNDIDSIENNWRTGRHLEMDQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS*.

[0141] Degradation systems without condensate-forming domains were constructed (vector sequence: SEQ ID NO. 25), and enhanced degradation systems with AtSSF PrLD fused to the N-terminus or C-terminus of the degradation module were constructed (N-terminal fusion vector sequence: SEQ ID NO. 26, C-terminal fusion vector sequence: SEQ ID NO. 27). All expression vectors were assembled using Gibson Assembly and validated by sequencing.

[0142] SEQ ID NO. 25:

[0143] SEQ ID NO. 26:

[0144] SEQ ID NO. 27:

[0145] (2) Agrobacterium infection and transient expression The GFP expression vector (pEAQ-HT-eGFP: SEQ ID NO. 28) and expression vectors with different degradation systems were transformed into Agrobacterium GV3101 competent cells, respectively. The cultured bacterial suspension was resuspended in infection buffer, and the OD600 was adjusted to 0.8. Subsequently, the GFP expression vector and the corresponding degradation system vector were mixed in equal proportions and co-infected with Nicotiana benthamiana leaves. After infection, the plants were cultured in the dark for 24 h, and then cultured under normal conditions for another 24–72 h.

[0146] SEQ ID NO. 28:

[0147] (3) GFP fluorescence detection and quantitative analysis After the expression of the exogenous protein reached a stable level, the infected area was photographed and recorded using the Dynaplant in vivo fluorescence imaging system. The fluorescence intensity of GFP was quantitatively analyzed using the accompanying software, with the GFP-only treatment group used as the 100% fluorescence reference value. All experiments were performed in at least three biological replicates.

[0148] (4) Experimental results The results showed that a strong green fluorescence signal was detectable in the control group expressing only GFP. When the degradation system without AtSSF PrLD was co-expressed, the GFP fluorescence intensity decreased to about 70%, indicating that the target protein could be effectively degraded. Further introduction of AtSSF PrLD, whether fused to the N-terminus or C-terminus of the degradation module, further enhanced the degradation effect, with the GFP fluorescence intensity decreasing to about 40%. Figure 6 The above results demonstrate that the condensate enhancement strategy proposed in this invention is not only applicable to chemically induced transcriptional activation systems, but can also improve the degradation efficiency of plant target proteins, thereby extending to the engineered regulation of various proximity-dependent cell functions.

[0149] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A plant chemically induced proximity regulation system, characterized in that, The plant chemically induced proximity regulation system includes: (1) DNA binding module; (2) Chemically induced recognition module; (3) Transcriptional regulation module; (4) Response startup submodule; The DNA binding module is selected from at least one of the following: Gal4 DNA binding domain, LexA DNA binding domain, dCas9, TALE, and zinc finger protein; The transcriptional regulatory module is selected from at least one of VP16, VP64, VPR, EDLL, SRDX, TPL or their derived regulatory domains.

2. The plant chemical induction proximity regulation system according to claim 1, characterized in that, This includes Danoprevir-induced neighborhood regulatory systems and Rapamycin-induced neighborhood regulatory systems.

3. The plant chemical induction proximity regulation system according to claim 2, characterized in that, When Danoprevir induces the proximity regulatory system, the chemically induced recognition module consists of the NS3a protein and the DNCR protein.

4. The plant chemical induction proximity regulation system according to claim 3, characterized in that, The NS3a protein is fused with the DNA binding module for expression, and the DNCR protein is fused with the transcription activation module for expression.

5. The plant chemical induction proximity regulation system according to claim 2, characterized in that, When Grazoprevir induces the proximity regulatory system, the chemically induced recognition module consists of the NS3a protein and the GNCR protein.

6. The plant chemical induction proximity regulation system according to claim 5, characterized in that, The NS3a protein is fused with the DNA binding module for expression, and the GNCR protein is fused with the transcription activation module for expression.

7. The plant chemical induction proximity regulation system according to claim 1, characterized in that, The response promoter module includes 1 to 20 DNA binding sites.

8. The plant chemical induction proximity regulation system according to claim 1, characterized in that, The plant chemically induced proximity regulation system can be used in plant protoplast expression systems, plant leaf transient expression systems, plant suspension cell systems, plant root hair systems, plant stable transformation systems, or cell-free expression systems.

9. The plant chemical induction proximity regulation system according to claim 1, characterized in that, The plants include monocotyledonous plants or dicotyledonous plants.

10. The plant chemical induction proximity regulation system according to any one of claims 1-9, characterized in that, It may also include a cohesive enhancement module.

11. The plant chemical induction proximity regulation system according to claim 1, characterized in that, The condensate enhancement module is selected from any one or more of the following: (1) AtSSF source: PrLD; (2) AtFCA source PrLD; (3) The N-terminal domain of the FUS protein; (4) LAF-1 source RGG structural domain; (5) Other naturally or artificially designed inherent disordered structural domains.

12. The plant chemical induction proximity regulation system according to claim 10, characterized in that, The condensate enhancement module is fused to one side of the DNA binding module.

13. A reagent kit, characterized in that, The kit includes the plant chemical induction proximity regulation system according to any one of claims 1-12.

14. A method for enhancing molecular proximity-dependent functions in plant cells, characterized in that, include: The condensation enhancement module of any one of claims 10-12 is introduced into the molecular proximity system.

15. The method according to claim 14, characterized in that, The molecular proximity-dependent function includes any one or more of the following: (1) Transcriptional activation; (2) Transcriptional repression; (3) Regulation of metabolic pathways; (4) Protein recruitment; (5) Signal transduction and regulation; (6) Chromatin regulation; (7) Targeted protein degradation; (8) RNA regulation; (9) Epigenetic regulation.

16. A method for regulating the synthesis of plant metabolites using the system according to any one of claims 1-12, characterized in that, The method described above utilizes a chemically induced recognition module to drive the expression of metabolic pathway enzymes.

17. A method for enhancing the degradation of plant-targeted proteins using the system according to any one of claims 1-12, characterized in that, The method includes: The system described in any one of claims 1-12 recruits ubiquitin ligase components to the vicinity of the target protein, thereby promoting the degradation of the target protein.

18. The application of the system according to any one of claims 1-12, the kit according to claim 13, or the method according to any one of claims 14-17 in plant synthetic biology, plant metabolic engineering, plant gene circuit construction, plant protein degradation regulation, and plant cell engineering.

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