Cell division promoting factor FL1 / FL3 specifically interacting with non-light excited state CRY2 and application of cell division promoting factor FL1 / FL3

Abstract

The invention provides a cell division promoting factor FL1 / FL3 capable of specifically interacting with non-light excited state CRY2 and application of the cell division promoting factor FL1 / FL3. The invention discloses an interaction system for the first time, and the system comprises CRY2 and FL1 and / or FL3 which specifically interact with the CRY2 in a non-light excitation state. The invention also provides application of the interaction system as a plant trait regulation target and an optogenetics core element, wherein the trait comprises a cell division related trait or a root length related trait.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, and more specifically, this invention relates to the cell division prokinetic factors FL1 / FL3 that specifically interact with non-photoexcited CRY2 and their applications. Background Technology

[0002] Plants possess a series of photoreceptor proteins that absorb light signals of specific wavelengths and undergo changes in protein levels, thereby regulating plant growth responses upon photoexcitation. Therefore, plant photoreceptor proteins are excellent candidates for photobiological tools. Among them, the blue light receptor Cryptochrome (CRY), with its unique high conservation across animals, plants, and microorganisms, has become a photobiological candidate protein with immense research potential.

[0003] Optogenetics is a comprehensive and cutting-edge scientific technology that combines multiple disciplines such as optics, genetics, and physiology. Its advantages, such as non-invasiveness, high spatiotemporal precision, and reversibility, have enabled it to expand from its initial application in neuroscience research to a wide range of biological research, including botany.

[0004] Previous research has primarily focused on the function of photoreceptors in the photoexcited state, with few reports on their function in the non-photoexcited state (Mechanisms of Cryptochrome-Mediated Photoresponses in Plants, Annual Review of Plant Biology). Studies of CRY as an optogenetic tool have mostly been based on its blue light-dependent interacting protein, and there are no known cases of using proteins that bind to CRY under non-blue light conditions for optogenetic research. This has resulted in a gap in optogenetic research centered on CRY under non-blue light conditions. For example, a previous study on the blue light-induced transcription system in zebrafish (PLoS One) described how blue light excitation of CRY to bind to the interacting protein CIB1 induces gene transcription; however, this study did not address the function and application of CRY under non-blue light conditions.

[0005] In summary, existing technologies have the following shortcomings: 1) Most studies focus on exploring the function of photoreceptors in the photoexcited state, neglecting the functional potential of photoreceptors in the non-photoexcited state. 2) Most optogenetic tools centered on CRY are blue light-dependent "photoinduced" types, lacking the development and research of tools driven by non-blue light conditions or "blue light-inhibited" types. Therefore, this invention aims to solve the above problems by exploring the function of photoreceptors in the non-photoexcited state, screening interacting proteins of photoreceptors in the non-photoexcited state, providing more technical principles for plant trait improvement, and developing CRY-centered optogenetic tools induced by non-blue light conditions or "blue light-inhibited" types, filling the gaps in existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a cell division promoting factor FL1 / FL3 that specifically interacts with non-photoexcited CRY2 and its applications.

[0007] In a first aspect of the invention, a method for regulating plant cell division and / or root length (including taproot length) is provided, comprising: regulating an interaction system or elements thereof in a plant; the interaction system comprising elements: FL1, FL3, and / or CRY2.

[0008] In one or more embodiments, the interaction system is an optogenetic (related) interaction system.

[0009] In one or more embodiments, the interaction system is referred to as CRY2-FL1 / FL3.

[0010] In one or more embodiments, the method includes:

[0011] (a) Upregulating FL1 and / or FL3 to promote cell division and increase root length; preferably, the upregulation of FL1 and FL3 includes: upregulating the expression, abundance or activity of FL1 and / or FL3;

[0012] (b) Downregulating FL1 and / or FL3 to inhibit cell division and reduce root length; preferably, the downregulation of FL1 and FL3 includes: downregulating the expression, abundance or activity of FL1 and / or FL3;

[0013] (c) Downregulating CRY2 (including under blue light or non-blue light conditions) to promote cell division and increase root length; preferably, the downregulation of CRY2 includes: downregulating the expression, abundance or activity of CRY2;

[0014] (d) Upregulating CRY2 (including under blue light or non-blue light conditions) to inhibit cell division and reduce root length; preferably, the upregulation of CRY2 includes: upregulating the expression, abundance or activity of CRY2;

[0015] (e) Weakening (including blocking) the interaction between CRY2 and FL1 and / or FL3, thereby promoting cell division and increasing root length; preferably, including: placing the plant in a blue light environment, thereby weakening the interaction between CRY2 and FL1 and / or FL3, enhancing FL1 and / or FL3 activity, promoting cell division and increasing root length;

[0016] (f) To cause (including promote) the interaction of CRY2 with FL1 and / or FL3, thereby inhibiting cell division and reducing root length; preferably, to place the plant in a non-blue light environment to form a non-photoexcited state of CRY2, thereby the CRY2 interacting with FL1 and / or FL3 to inhibit the activity of FL1 and / or FL3, thereby inhibiting cell division and reducing root length.

[0017] In one or more embodiments, in (a), “upregulating the FL1 and / or FL3” includes: upregulating the function of the FL1 and / or FL3 in plants with low expression, abundance or activity of the FL1 and / or FL3 (e.g., significantly lower than the average level of the same plant).

[0018] In one or more embodiments, in (b), “downregulating FL1 and / or FL3” includes: downregulating the function of FL1 and / or FL3 in plants where the expression, abundance or activity of FL1 and / or FL3 is normal or high (e.g., significantly higher than the average level of the same plant).

[0019] In one or more embodiments, in (c), “downregulating CRY2” includes: downregulating the function of CRY2 in plants where CRY2 expression, abundance, or activity is normal or high (e.g., significantly higher than the average level of the same plant).

[0020] In one or more embodiments, in (d), “upregulating CRY2” includes: upregulating the function of CRY2 in plants with low CRY2 expression, abundance, or activity (e.g., significantly lower than the average level of the same plant).

[0021] In one or more embodiments, the interactive system further includes elements FKD1 and / or FL2, and the method further includes further reducing FKD1 and / or FL2 on the basis of reducing FL1 and FL3.

[0022] In one or more embodiments, (a) the coding genes for FL1 and / or FL3 or expression constructs or vectors containing said coding genes are introduced into plants to upregulate FL1 and / or FL3.

[0023] (b) knocking out or silencing FL1 and / or FL3, or inhibiting the activity of FL1 and / or FL3 in plants; preferably, including: reducing the activity of FL1 and / or FL3 by site-directed mutagenesis, knocking out or down-knocking out FL1 and / or FL3 genes by gene editing with a CRISPR system, knocking out or down-knocking out FL1 and / or FL3 genes by homologous recombination system, or silencing the genes by interfering molecules that specifically interfere with the expression of FL1 and / or FL3 genes.

[0024] In (c), knocking out or silencing CRY2 in plants, or inhibiting CRY2 activity; preferably, this includes: reducing CRY2 activity by site-directed mutagenesis, knocking out or downsing the CRY2 gene by gene editing using a CRISPR system, knocking out or downsing the CRY2 gene by a homologous recombination system, or silencing the gene by interfering molecules that specifically interfere with CRY2 gene expression; or

[0025] In (d), the gene encoding CRY2 or an expression construct or vector containing the gene encoding CRY2 is introduced into plants to upregulate CRY2.

[0026] In one or more embodiments, the "non-blue light environment (condition)" refers to dark conditions or light conditions that do not contain blue light. Treating plants with dark or blue light-free conditions causes the CRY2 in the plants to be in a non-(blue) light excited state.

[0027] In one or more embodiments, the “non-blue light environment (condition)” also includes illumination conditions with extremely small (virtually negligible) amounts of blue light.

[0028] In one or more embodiments, the "blue light-containing environment (condition)" refers to illumination conditions containing a significant amount of blue light higher than "trace amounts," such as 5, 10, 20, or 30 μmol photons m -2 s -1 Or the above blue light illumination conditions.

[0029] In one or more embodiments, downregulating the FL1 and / or FL3 and / or CRY2 genes includes: modifying the FL1 and / or FL3 and / or CRY2 genes to produce a terminator earlier (the protein encoded by the terminator provides termination).

[0030] In another aspect of the invention, therein is provided the use of an interaction system in a plant, elements thereof, or regulators thereof, for regulating plant cell division or root length, or for preparing a construct / composition (or formulation, such as a pesticide composition or pesticide formulation) for regulating plant cell division or root length; said interaction system includes elements: FL1, FL3, and / or CRY2.

[0031] In one or more embodiments, the regulator comprises: a cell division and / or root growth promoter, comprising selected from: (i) the encoding gene of FL1 and / or FL3 or an expression construct or vector containing said encoding gene; (ii) a loss-of-function site-directed mutagen for CRY2, a CRISPR system for knocking out or knocking down the CRY2 gene, a homologous recombination system for knocking out or knocking down the CRY2 gene, or an interfering molecule that specifically interferes with the expression of the CRY2 gene; (iii) weakening (including blocking) (e.g., in a blue light-containing environment) the interaction between CRY2 and FL1 and / or FL3. The reagents; or, cell division and / or root growth inhibitors, including those selected from: (i) loss-of-function site-directed mutagenesis reagents for FL1 and / or FL3, CRISPR systems that knock out or knock down the FL1 and / or FL3 genes, homologous recombination systems that knock out or knock down the FL1 and / or FL3 genes, and interfering molecules that specifically interfere with the expression of the FL1 and / or FL3 genes; (ii) the gene encoding CRY2 or an expression construct or vector containing said gene; and (iii) reagents that enable (promote) the interaction of CRY2 with FL1 and / or FL3 (e.g., in a non-blue light environment).

[0032] In one or more embodiments, FL1 and / or FL3 include their Auxin canalization domain (including bioactive fragments containing their Auxin canalization domain); preferably, FL1 includes: a bioactive fragment containing FL1 1-324 aa, a bioactive fragment containing FL1 22-307 aa, and a bioactive fragment containing FL1 1-307 aa; preferably, FL3 includes: a bioactive fragment containing FL3 1-340 aa, a bioactive fragment containing FL3 18-317 aa, and a bioactive fragment containing FL3 1-317 aa; or, CRY2 includes its PHR domain (including bioactive fragments containing its PHR domain); preferably, CRY2 includes: a bioactive fragment containing CRY2 1-489 aa; a bioactive fragment containing CRY2 1-486 aa; and a bioactive fragment containing CRY2 1-565 aa.

[0033] In another aspect of the invention, an interaction system for use in optogenetics is provided, the interaction system comprising: elements CRY2 and FL1 and / or FL3.

[0034] In one or more embodiments, under blue light conditions, the interaction between CRY2 and FL1 and / or FL3 in the interaction system is weakened, and the activity of FL1 and / or FL3 is enhanced, thereby promoting plant cell division and increasing root length.

[0035] In one or more embodiments, under non-blue light conditions, CRY2 in the interaction system interacts with FL1 and / or FL3, and the activity of FL1 and / or FL3 is inhibited, thereby inhibiting plant cell division and increasing root length.

[0036] In one or more embodiments, the interaction system is present in isolated plant cells, tissues, or organs. Preferably, the plant cells, tissues, or organs recombinantly express the various elements of the interaction system or recombinantly express the regulators that regulate the interaction system; the interaction system or its regulators are recombinantly expressed in the cells (including high expression, for example, through artificially constructed expression constructs).

[0037] In another aspect of the invention, there is provided the use of an optogenetic interaction system for use as a molecular marker for identifying plant cell division or root length; said interaction system includes elements: FL1, FL3, and / or CRY2.

[0038] In another aspect of the invention, a method for identifying plants with varying degrees of cell division or root length is provided, comprising: analyzing the operation of an interaction system in the plant; the interaction system comprising elements: FL1, FL3, and / or CRY2; wherein, if FL1 and / or FL3 are highly expressed or active, CRY2 is lowly expressed or active, and the interaction between CRY2 and FL1 and / or FL3 is weak, then the plant is a plant with long roots or high cell division activity; if FL1 and / or FL3 are lowly expressed or active, CRY2 is highly expressed or active, and the interaction between CRY2 and FL1 and / or FL3 is strong, then the plant is a plant with short roots or low cell division activity.

[0039] In one or more embodiments, the high expression or activity, or strong interaction, is defined as a statistically significant or marked increase or enhancement, such as an increase or enhancement of 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90%, or higher. Preferably, the significance is defined as a comparison with the average expression or activity in control plants (including plants of the same type or species).

[0040] In one or more embodiments, the low expression or activity, or weak interaction, is a statistically or significantly reduced or weakened level, such as a reduction or weakening of 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90%, or higher.

[0041] In one or more embodiments, the recombinant expression includes: artificially created, or artificially modified.

[0042] In one or more embodiments, the recombinant expression includes introducing a foreign or heterologous gene into the cells, tissues, or organs of a plant.

[0043] In one or more embodiments, the plant cells, tissues, or organs do not directly generate living plants or serve as plant propagation material.

[0044] In one or more embodiments, the plant includes plants selected from the group consisting of the interaction system; the interaction system includes elements: FL1, FL3, and / or CRY2.

[0045] In one or more embodiments, the plant includes: a dicotyledonous plant.

[0046] In one or more embodiments, the plant includes: a monocotyledonous plant.

[0047] In one or more embodiments, the plant includes (but is not limited to): cruciferous plants.

[0048] In one or more embodiments, the cruciferous plants include (but are not limited to): *Mucor*, *Brassica*, *Radix*, *Isatis*, *Gnaphalium*, *Salix*, *Salix*, *Fragrance*, *Violet*, *Gnaphalium*, *Capsella*, *Nymphoides*, *Ipomoea*, etc.

[0049] The cruciferous plants mentioned include (but are not limited to): Arabidopsis thaliana, Chinese cabbage, kale, mustard greens, radish, rapeseed, etc.

[0050] In one or more embodiments, the FL1 comprises a protein selected from the group consisting of: (a1) a protein having the amino acid sequence of SEQ ID NO:1; (b1) a protein derived from (a1) having the function of the (a1) protein, formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO:1; (c1) a protein derived from (a1) having the function of the (a1) protein, having at least 80% (preferably 85%; more preferably 90%; more preferably 95%, such as 98%, 99%) homology to the protein sequence defined by (a1); or (d1) a protein active fragment defined by (a1), or a protein formed by adding a tag sequence, an enzyme digestion sequence, or a reporter protein to both ends thereof. The FL1 also includes the aforementioned bioactive fragments.

[0051] In one or more embodiments, the FL3 comprises a protein selected from the group consisting of: (a2) a protein having the amino acid sequence of SEQ ID NO:2; (b2) a protein derived from (a2) having the protein function of (a2) formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO:2; (c2) a protein derived from (a2) having the protein function of (a2) and having 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more, such as 98%, 99%) homology to the protein sequence defined by (a2); or (d2) a protein active fragment defined by (a2), or a protein formed by adding a tag sequence, an enzyme digestion sequence, or a reporter protein to both ends thereof. The FL3 also includes the aforementioned bioactive fragments.

[0052] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0053] Figure 1 CRY2 can interact with FL1 / FL3 and is suppressed by blue light.

[0054] (AB) The results of the yeast two-hybrid auxotrophic growth experiment under five light conditions showed that the interaction between CRY2 and FL1 and FL3 was inhibited by white light (A) and blue light (B).

[0055] The (CD) yeast two-hybrid Liquid-Assay results showed that the interaction between CRY2 and FL1(C) and FL3(D) is inhibited by blue light.

[0056] (E)Pull-down experiments show that the interaction between CRY2 and FL1 and FL3 is suppressed by blue light.

[0057] Experimental results of (FG)co-IP show that the interaction between CRY2 and FL1(F) and FL3(G) is suppressed by blue light.

[0058] (H) The results of the yeast two-hybrid Liquid-Assay experiment showed that FL1 and FL3 were only associated with CRY2. D387A Interaction rather than CRY2 W374A .

[0059] (I) The tobacco Split-LUC results showed that CRY2 interacts with FL1 and FL3 under dark conditions.

[0060] (J) Tobacco BiFC results showed that CRY2 interacts with FL1 and FL3 under dark conditions. Scale bar: 20 μm.

[0061] (K) Tobacco BiFC results showed that FL1 and FL3 were only similar to CRY2. D387A Interaction rather than CRY2 W374A Scale bar: 20μm.

[0062] Figure 2 CRY2 interacts with the N-terminus of FL1 and FL3 through its N-terminus.

[0063] (A) Schematic diagram of CRY2 segmented protein.

[0064] (B) The results of the yeast two-hybrid auxotrophic growth experiment showed that CRY2 interacts with FL1 and FL3 through its N-terminus.

[0065] (C) Schematic diagram of FL1 and FL3 segmented proteins.

[0066] (D) The results of the yeast two-hybrid auxotrophic growth experiment showed that CRY2 interacts with the N-terminus of FL1 and FL3; among them, "-LWHA" indicates a deficiency of Leu, Trp, His and adenine (Ade); "-LW" indicates a deficiency of Leu and Trp.

[0067] Figure 3 FL1 and FL3 proteins are conserved in plants and promote root elongation.

[0068] (A) FL1 and FL3 proteins are conserved in plants.

[0069] (BC)fl1 sd (B) and fl3 sd (C) Schematic diagram of fragment deletion mutant.

[0070] (DE) The root length phenotypes (D) and statistical results (E) of FL1 and FL3 related mutants indicate that FL1 and FL3 can promote the elongation of the taproot of Arabidopsis thaliana.

[0071] (F) The results of the yeast two-hybrid auxotrophic growth experiment showed that FL1 and FL3 can interact with FKD1 and FL2.

[0072] (GH) Root length phenotypes (G) and statistical results (H) of FL1 and FL3 related overexpression materials indicate that FL1 and FL3 can restore the root length defect of flΔ4 and promote the elongation of Arabidopsis taproot.

[0073] (IK) Morphology of root tip meristem and statistical results of meristem endodermal cell number under dark (I) and blue light (J) conditions for FL1 and FL3 related overexpression materials (K).

[0074] Figure 4In regulating cell division in the meristematic zone of Arabidopsis thaliana's primary root, CRY2 is located upstream of FL1 and FL3.

[0075] (AB) Root length phenotypes of etiolated seedlings of each genotype (A) and statistical results (B).

[0076] (CD) Morphology of root tip meristem of etiolated seedlings of each genotype (C) and statistical results of the number of endodermal cells in the meristem (D).

[0077] (EF) Root length phenotype of seedlings of each genotype (E) and statistical results (F).

[0078] (GI) Statistical results of root tip meristem morphology and meristem cell number in seedlings of different genotypes under dark (G) and blue light (H) conditions (I). Detailed Implementation

[0079] This invention discloses that photoreceptors possess biological functional activity in the non-photoexcited state. Through in-depth analysis and research, it reveals for the first time an optogenetic-related interaction system, comprising elements FL1, FL3, and / or CRY2. This invention also provides applications of the aforementioned interaction system as targets for regulating cell division or plant root length traits, including root-related traits or cell division-related traits.

[0080] Interactive System

[0081] As used in this article, the “non-blue light condition” refers to the illumination conditions that maintain CRY2 in a “non-blue light excited state”.

[0082] As used herein, the term "non-blue light excited state" includes "continuous non-blue light excited state".

[0083] As used herein, “non-blue light conditions” include dark conditions and light conditions that do not contain blue light.

[0084] As used herein, the term "interacting system" includes interacting systems in dark environments, interacting systems in non-blue light environments, or interacting systems in environments containing blue light. According to embodiments of the present invention, FL exhibits stronger modulation under blue light, while CRY2 exhibits stronger modulation in darkness.

[0085] As used herein, the term "plant (crop)" refers to a plant containing the aforementioned interaction system; preferably containing CRY2 or its homologs, and FL1 and / or FL3 or their homologs that interact with CRY2 under non-blue light conditions. The plant includes monocotyledonous or dicotyledonous plants. For example, the plant may include plants of the Brassicaceae, Solanaceae, Poaceae, Euphorbiaceae, etc. More preferably, the plant is a Brassicaceae plant. More preferably, the plant is a crop (such as a cash crop). For example, the term "plant" includes, but is not limited to, Arabidopsis thaliana, Chinese cabbage, kale, mustard greens, radish, rapeseed, etc.

[0086] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0087] It should be understood that, guided by the technical solutions of this invention, those skilled in the art can achieve the same or similar technical effects by changing the types of various plants, and these variations are also included in this invention.

[0088] In this invention, the interaction system includes CRY2, and FL1 and / or FL3 that interact with CRY2 under non-blue light conditions. Each molecule in the interaction system includes genes or proteins from Arabidopsis thaliana (At), genes or proteins homologous to those from Arabidopsis thaliana, containing substantially the same structural domains, and having substantially the same functions.

[0089] As used herein, the protein amino acid sequence of “FL1”, for example as shown in SEQ ID NO:1, may also be its homolog (homologous protein or homologous gene).

[0090] As used herein, the protein amino acid sequence of “FL2”, for example as shown in SEQ ID NO:2, may also be its homolog (homologous protein or homologous gene).

[0091] As used herein, the protein amino acid sequence of “CRY2” may be, for example, the GenBank accession number (NCBI number: 839529, geneID in Tair database: AT1G04400) or its homolog (homologous protein or homologous gene).

[0092] In this invention, the molecules (proteins) in the said interaction system also include their fragments, derivatives, and analogs. As used herein, the terms "fragment," "derivative," and "analyte" refer to protein fragments that substantially retain the same biological function or activity as the protein, and may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing additional amino acid sequences to the protein sequence; or (iv) proteins with one or more amino acid residues (or domains) removed (or truncated), for example, by removing inactive sequence regions. These fragments, derivatives, and analogs are within the scope known to those skilled in the art as defined herein. Biologically active fragments of the molecules in the said interaction system can all be used in this invention.

[0093] As used herein, "variant," "mutant," "mutant protein," "mutant form," "Mt," or "Mt" refers to a peptide or protein whose amino acid sequence has been altered compared to a reference sequence (wild-type sequence) through the insertion, deletion, or substitution of one or more amino acids, but which retains at least one biological activity; at the nucleic acid level, the corresponding coding site has changed compared to the corresponding reference sequence. In this invention, "mutation" generally refers to a mutation closely related to the occurrence of disease. The "inserted or substituted amino acid" can be a natural amino acid or a non-natural amino acid.

[0094] It should be understood that while the molecules (genes or proteins) in the interaction system described in this invention are preferably derived from cruciferous plants, particularly Arabidopsis thaliana, genes (such as those degenerate with the corresponding Arabidopsis genes) derived from other plants that are highly homologous (e.g., having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) to the corresponding Arabidopsis genes or proteins are also within the scope of this invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0095] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or protein-coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0096] The host cell is usually a plant cell. Transformation of plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, with Agrobacterium-mediated transformation being preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0097] When used as targets for artificial regulation or in the creation of screening systems, the proteins or encoding genes mentioned above can be naturally occurring, such as those purified and isolated from mammals or plants; or they can be recombinantly prepared, for example, recombinant proteins can be produced using conventional gene recombination techniques. Furthermore, any variations that do not affect the biological activity of these proteins are acceptable, such as derivatives or variants whose function remains unchanged.

[0098] Application of Interactive Systems

[0099] In this invention, by setting non-blue light conditions (such as dark conditions), the biological functional activity of photoreceptors in non-photoexcited states was discovered for the first time, filling a gap in the study of photoreceptor function and providing new research directions and approaches for optical signal transduction and optogenetics.

[0100] In this invention, by setting non-blue light conditions (such as dark conditions), the interacting proteins of photoreceptors in the non-photoexcited state were obtained for the first time through screening.

[0101] In this invention, by employing a point-mutated form of photoreceptor in a continuously non-photoexcited state during the screening process, and combining in vivo and in vitro strategies, the protein screening operations of this invention enhance the screening depth while also improving the reliability and biological significance of the screening results. The screening method can be used to verify or evaluate the interaction characteristics between the screened interacting proteins and the photoreceptor, providing reliable and novel protein complex selection for optogenetics.

[0102] This invention marks the first discovery and confirmation of two proteins, FL1 and FL3, that bind to CRY2 under non-blue light conditions. The binding of these two proteins to CRY2 is inhibited by blue light, whereas previously reported interacting proteins exhibit blue light-promoted binding. This allows for the selection of novel protein complexes in optogenetics, distinct from previous "light-induced" methods. CRY2, FL1, and FL3 can serve as candidate proteins for developing novel optogenetic tools (characterized by protein interactions contrary to those in previous CRY-based optogenetic tools).

[0103] FL1 and FL3 are the first discovered cell division and root length regulators that can interact with CRY2. They are expected to be used to develop related technologies for regulating cell division and root length by light signals, thereby achieving the regulation of cell division and root length by light signals.

[0104] The CRY2-FL1 / FL3 interaction proposed in this invention is a novel interaction form in the field. Previous tools were based on "blue light promoting the interaction between CRY2 and interacting proteins," while the "CRY-FL1 / FL3" of this invention can achieve "blue light inhibition" or "non-blue light promotion," which has good application prospects.

[0105] Once the function of the interacting system (preferably including its upstream and downstream genes) is known, various methods well-known to those skilled in the art can be used to regulate the expression or activity of the interacting system or its molecules, or to regulate its upstream or downstream genes. For example, various methods well-known to those skilled in the art can be used for recombinant expression or overexpression. These methods are all included in this invention.

[0106] In this invention, regulation of the interaction system or molecules within the system includes upregulation or downregulation. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein activity or protein expression, and these are statistically significant. The upregulators include promoters, agonists, activators, etc. Any substance that can increase the activity of the interaction system or molecules within the system (including upstream and downstream proteins), improve the stability of the interaction system or molecules within the system, upregulate the expression of the interaction system or molecules within the system, increase the effective duration of action of the interaction system or molecules within the system, or increase the activation level of individual molecules can be used in this invention as a substance useful for upregulating the interaction system or molecules within the system. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0107] The terms "downregulation" and "inhibition" include "downregulation" and "inhibition" of protein activity or protein expression, and must be statistically significant. Downregulators include inhibitors, retardants, etc. Any substance that can reduce the activity of the interaction system or molecules within it (including upstream and downstream proteins), reduce the stability of the interaction system or molecules within it, downregulate the expression of the interaction system or molecules within it, reduce the effective duration of action of the interaction system or molecules within it, or reduce the activation level of individual molecules can be used in this invention as a substance useful for downregulating the interaction system or molecules within it. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0108] As used herein, downregulators of the interacting system or molecules in the system (including upstream and downstream proteins) include inhibitors, antagonists, blockers, and other terms that are used interchangeably. The term "downregulator" refers to any substance that can reduce the activity of the interacting system or molecules in the system, reduce the stability of the interacting system or molecules in the system, downregulate the expression of the interacting system or molecules in the system, reduce the effective duration of action of the interacting system or molecules in the system, or inhibit the transcription and translation of the interacting system or molecules in the system; all such substances can be used in this invention.

[0109] The interaction system of the present invention can also be applied to screening substances (including potential substances) that regulate plant cell division or root length. The screening method includes: (1) adding candidate substances to a system with an interaction system; the interaction system includes: CRY2, and FL1 and / or FL3 that interact with CRY2 under non-blue light conditions; (2) detecting the system and observing the working condition of the interaction system therein. If the interaction between CRY2 and FL1 and / or FL3 under non-blue light conditions is weakened or the interaction time is reduced, the expression or activity of FL1 and / or FL3 is increased, and the expression or activity of CRY2 is reduced, then the candidate substance is a substance that can be used to increase root length and promote cell division; if the interaction between CRY2 and FL1 and / or FL3 under low blue light conditions is promoted or the interaction time is increased, the expression or activity of FL1 and / or FL3 is reduced, and the expression or activity of CRY2 is increased, then the candidate substance is a substance that can be used to reduce root length and reduce cell division.

[0110] Screening for substances that act on proteins or genes as targets is a method well-known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Those skilled in the art understand how to select appropriate screening methods based on the type of substance to be screened. Through large-scale screening, a class of potential substances that specifically regulate the interaction system and thus have a regulatory effect on plant cell division or root length can be obtained.

[0111] According to the new findings of this invention, it can also be applied to: 1) studying the function of all photoreceptors in the biological world (including plants) under non-photoexcited conditions by setting dark conditions; 2) screening interacting proteins of all photoreceptor proteins in the biological world under non-photoexcited conditions by setting dark conditions; 3) screening interacting proteins by using non-photoresponsive point mutants of all photoreceptor proteins in the biological world, thereby increasing the depth of screening for interacting proteins in the non-photoexcited state; 4) modifying part of the protein sequence according to the characteristics of photoreceptor proteins in the biological world (e.g., designing truncated proteins) can improve the photoreceptor protein... 5) Use all photoreceptors in the biological world and their interacting proteins in the non-photoexcited state as candidate proteins for developing optogenetic tools based on photoreceptors, and flexibly truncate or modify the proteins according to the interaction segments to develop more efficient optogenetic tools; 6) Use CRY2-FL1 and CRY2-FL3 modules (including using homologous proteins of CRY2, FL1 and FL3 in other organisms, as well as proteins containing homologous domains) to study the regulation of light signals on cell division (including plant cell division and root elongation) across species.

[0112] According to embodiments of the present invention, a point mutant of the non-photoresponsive form of the blue light receptor CRY2 can also be used. D387A By screening interacting proteins, we can also obtain the interacting proteins of Arabidopsis thaliana blue light receptor CRY2 in the non-photoexcited state.

[0113] According to an embodiment of the present invention, the truncated form of the blue light receptor CRY2 is CRY2. N489 It interacts more strongly with FL1, and CRY2 N489 The interaction with FL1 is still suppressed by blue light, hence CRY2 N489 It also has the potential to screen non-photoexcited CRY2 interacting proteins.

[0114] CRY2, FL1, and FL3 can be used as candidate proteins for developing optogenetic tools centered around CRY2. The proteins can be flexibly truncated or modified according to the interaction segments to develop more efficient optogenetic tools.

[0115] The CRY2-FL1 and CRY2-FL3 modules in Arabidopsis thaliana can be used to regulate the cell division and root elongation of Arabidopsis thaliana by light signals.

[0116] Technical effect

[0117] Compared to existing research schemes that explore the function of photoreceptors in the photoexcited state, this invention explores the function of photoreceptors in the non-photoexcited state from the opposite direction, providing a new perspective for the study and understanding of plant photoreceptors and confirming the research potential of this research direction.

[0118] Compared to all existing CRY-interacting proteins, this invention is the first to demonstrate from multiple perspectives the existence of proteins in plants that interact with CRY under non-blue light conditions but do not interact under blue light, providing a completely opposite approach to optogenetic research centered on CRY.

[0119] In contrast to setting different light conditions to screen the interacting proteins of plant photoreceptors in the photoexcited state, this invention sets dark conditions to screen the interacting proteins of photoreceptors in the non-photoexcited state. Since any photoreceptor is in the non-photoexcited state under dark conditions, this screening condition is universal.

[0120] This invention employs a dual in vivo and in vitro strategy to screen for interacting proteins of the blue light receptor CRY2 in its non-photoexcited state. Furthermore, in an in vitro yeast two-hybrid screening library, CRY2 is used... D387A Assisting in protein screening can yield more candidate proteins and enhance the biological significance of the screening results.

[0121] Compared to all previously discovered CRY interacting proteins, FL1 and FL3, screened in this invention, are two proteins that can regulate cell division in the meristematic zone of Arabidopsis thaliana's primary root.

[0122] Compared to existing optogenetic tools centered around CRY, the FL1 and FL3 screened in this invention allow for research under non-blue light conditions (e.g., red or green light), avoiding the inconvenience of conducting research in darkness and providing candidates for the development of "dark-on" (excitation) optogenetic tools.

[0123] Compared to the blue light "on" (excited) type CRY2-CIB1 tool currently used in optogenetics, the CRY2-FL1 and CRY2-FL3 modules discovered in this invention provide two novel candidate proteins for developing blue light "off" (inhibited) type optogenetic tools.

[0124] CRY2, FL1, and FL3 are highly conserved in plants, so molecular tools developed based on these three proteins that regulate cell division and root elongation through light signals have the potential for wide application in other plants, including major economic crops.

[0125] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0126] Materials and methods

[0127] plant materials

[0128] The Arabidopsis thaliana wild-type material (WT) used in this invention is Col-0 (Columbia), and the overexpression and mutant materials constructed are based on Col-0, cry2-1 (cry2) or fkd1 fl1-2 fl2 fl3 (flΔ4); wherein fl1-2 is a fl1 mutant line.

[0129] The tobacco material used in this invention is Nicotiana benthamiana.

[0130] The mutant fkd1 fl1-2 fl2 fl3 (flΔ4) is characterized by the deletion of four genes: FKD1, FL1, FL2 and FL3. The knockout of FKD1 is mediated by EMS mutagenesis, while the knockout of FL1, FL2 and FL3 is mediated by T-DNA insertion.

[0131] The mutant cry2-1 (cry2) is a gene knockout mutant mediated by fast neutron bombardment that is already in use in the field. For details, see (Regulation of Flowering Time by Arabidopsis Photoreceptors, Science).

[0132] Fragment missing type fl1 sd fl3 sd Establishment of the double mutant: First, fl1 was constructed using CRISPR-Cas9 technology. sd and fl3 sd A fragment deletion single mutant was subsequently obtained by hybridization to obtain fl1. sd fl3 sd Double mutants. In fl1, the FL1 gene has a deletion of positions 572-1299 of the CDS sequence; in fl3, the FL3 gene has a deletion of positions 479-1076 of the CDS sequence.

[0133] Establishment of the cry2 flΔ4 five-mutant: cry2 flΔ4 was obtained by crossing cry2 with flΔ4.

[0134] Establishment of FL1-GFP expression vector: The CDS sequence of FL1 without the stop codon was ligated to the multiple cloning site of the pCambia2302 vector.

[0135] Establishment of FL3-GFP expression vector: The CDS sequence of FL3 without the stop codon was ligated to the multiple cloning site of the pCambia2302 vector.

[0136] Establishment of the FL1-Flag expression vector: The CDS sequence of FL1 without the stop codon was ligated to the multiple cloning site of the pCambia1306 vector.

[0137] Establishment of the FL3-Flag expression vector: The CDS sequence of FL3 without the stop codon was ligated to the multiple cloning site of the pCambia1306 vector.

[0138] Stable transgenic overexpression materials were constructed using Agrobacterium-mediated transformation in the context of the corresponding genetic material.

[0139] Yeast Two Hybrid

[0140] (1) Yeast auxotrophic plate growth experiment (mainly using strain AH109)

[0141] Select a suitable AH109 strain according to the experimental objectives (ensure that both positive and negative controls are set up simultaneously), and prepare LTHA yeast solid culture medium.

[0142] Strains from each experimental and control group were selected and resuspended in 100 μL of sterile ultrapure water. Subsequently, three concentration gradients were set up in parallel and serially diluted at a dilution ratio of 1 / 10.

[0143] Use a pipette (a multi-stage pipette is recommended) to neatly spot bacterial suspensions of various concentration gradients onto the surface of a solid culture medium to form bacterial plaques.

[0144] Dry the culture dish in a clean bench until there are no obvious liquid traces on the surface. Seal the culture dish with breathable tape and place it under conditions suitable for the experimental purpose. After 3-7 days of incubation, determine the interactions based on the growth.

[0145] (2) Liquid-Assay for determining the strength of protein-protein interactions (mainly using Y190 strain, CPRG method)

[0146] According to the experimental requirements, the corresponding yeast strains were inoculated into 5 mL of the corresponding auxotrophic liquid culture medium and cultured overnight at 22°C and 200 rpm.

[0147] Take 2 mL of overnight culture medium and transfer it to 8 mL of YPD medium. Incubate at 28 °C and 200 rpm until the OD600 of the medium reaches 0.5-0.8. Record the OD600 value.

[0148] Take 1.5 mL of bacterial culture into an EP tube, centrifuge at 14,000 rpm for 30 seconds, discard the supernatant, and resuspend in 1 mL of buffer 1. Centrifuge again at 14,000 rpm for 30 seconds, and resuspend the bacterial cells in 300 μL of buffer 1.

[0149] Dispense 300 μL of the resuspension into three new EP tubes and immediately place them in liquid nitrogen for flash freezing.

[0150] Depending on the experimental requirements, multiple experimental groups corresponding to yeast growth time can be set up, and steps 3 to 4 can be repeated.

[0151] All EP tubes were subjected to five freeze-thaw cycles in a 37°C water bath and liquid nitrogen to ensure complete cell disruption.

[0152] Add 700 μL of buffer 2, mix thoroughly, and record this as the initial time of the colorimetric reaction. Simultaneously, add 1 mL of buffer 2 to a new EP tube as a blank control.

[0153] When the color of the experimental or control group changes from yellow to red, immediately add 500 μL of ZnCl2 to each EP tube to terminate the colorimetric reaction.

[0154] After centrifugation at 14,000 rpm, the supernatant was transferred to a new EP tube. The OD578 zero point was set using the blank control liquid. The OD578 values ​​of each experimental group and control group were measured and recorded.

[0155] Formula for calculating β-galactosidase activity based on interaction strength:

[0156] β-galactosidase units=1000×OD578 / (t×V×OD600)

[0157] Where t is the colorimetric reaction time; V is the ratio of the volume of bacterial culture taken in the reaction step to the volume of buffer 1 added (5 in this experiment).

[0158] Buffer 1: Add 2.38g HEPES, 0.9g NaCl, 0.065g L-aspartic acid, 1g BSA and 50μL Tween 20 to 100mL of solution (pH 7.25-7.3).

[0159] Buffer 2: Add 27.1 mg CPRG to 20 mL of Buffer 1 and dissolve thoroughly.

[0160] BiFC

[0161] Based on the protein interaction combination to be verified, CRY2, FL1, FL3, and CRY2 were combined. W374A CRY2 D387A CIS1 and U2AF35B were fused with the C-terminus of CFP (amino acids 155–238) or the N-terminus of YFP (amino acids 1–172) to construct the corresponding vectors, which were then transformed into Agrobacterium tumefaciens GV3101, with CIS1 and U2AF35B serving as controls.

[0162] Agrobacterium cultured overnight was collected by centrifugation and resuspended in transient transformation buffer (10 mM MES, pH 5.8, 10 mM MgCl2, 0.1 mM acetylsylcholine), adjusting the OD600 nm to 0.8. The resuspended solution was mixed with GV3101 strain expressing pSoup-P19 and incubated in the dark at room temperature for 2 hours, followed by infiltration. The Agrobacterium suspension was used to infiltrate intact leaves of 3-week-old *N. benthamiana* using a needle-free 1 mL syringe. After infiltration, the plants were subjected to 16 hours of darkness and then transferred to long-day white light conditions for 3 days of growth. Before imaging, the plants were again subjected to 16 hours of darkness to ensure that CRY2 was in an unexcited state. Fluorescence images were acquired using a Leica SP8 confocal microscope. The excitation and emission wavelengths were 488 nm and 530 nm, respectively, for GFP.

[0163] split-LUC

[0164] Based on the protein-protein interaction combinations to be verified, CRY2, FL1, and FL3 were cloned into pCAMBIA 1300-nLUC or pCAMBIA 1300-cLUC vectors, respectively, and then transformed into Agrobacterium tumefaciens GV3101 strain. Tobacco (N. benthamiana) transformation was performed using a method similar to that used in the BiFC experiment. For imaging, the soaked leaves were treated with a fluorescein solution (1 mM fluorescein containing 0.01% v / v Triton X-100) 2 minutes before imaging, and images were taken using a cooled CCD camera.

[0165] pull-down

[0166] Pre-purified target proteins A and B (concentration range 500 ng to 2 μg) were mixed with corresponding agarose beads and added to EP tubes. Binding buffer was added and the tubes were incubated at 4°C for 1 hour. After incubation, the agarose beads were washed five times with rinsing buffer. The rinsed agarose beads can be directly used for Western blotting analysis to detect protein-protein interactions.

[0167] The binding buffer formulation is as follows: 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, 10% glycerol, and 0.5 mM PMSF.

[0168] Rinse buffer formulation: 50mM Tris (pH 8.0), 150mM NaCl, 0.1% Triton X-100, 1mM EDTA.

[0169] co-IP

[0170] The seeds of the experimental group and the control group were sown on 1 / 2 MS solid medium and grown under the same culture conditions.

[0171] Take 0.5g to 1.5g of 10-day-old plant material and place it in the appropriate crushing mold. Freeze the sample thoroughly with liquid nitrogen. Crush the sample twice using a crusher (50Hz, 60s), then unscrew the mold, add liquid nitrogen, and transfer the powder to a 50mL centrifuge tube (scrape off as much residual powder as possible from the mold wall). Further transfer the sample powder to a mortar (be careful not to miss any samples).

[0172] Prepare Co-IP incubation working buffer: Generally, 0.1g of sample corresponds to 100μL of Co-IP incubation working buffer.

[0173] Rinsing beads: The beads required for rinsing (generally 30 μL of beads per sample). The rinsing procedure is as follows: Rinse the beads 3 times with Co-IP incubation working buffer, centrifuging at 1500 rpm for 2 minutes each time and then discarding the supernatant.

[0174] Add Co-IP incubation working buffer to the mortar and grind thoroughly with a mortar and pestle at least 100 times. Use a syringe with a needle to transfer the sample into a 2 mL specially designed pointed-bottom EP tube (first draw up the sample without the needle, then add the needle to transfer the sample), and incubate at 4°C for 5 minutes.

[0175] Take 30–50 μL of sample as input, and centrifuge the remaining sample at 14,000 rpm for 7 minutes at 4 °C.

[0176] Take equal volumes of supernatant from each experimental group and control group, and transfer them to another 1.5 mL conical EP tube. Add the rinsed beads and incubate at 4°C for 20 minutes (using a Ferris wheel, 15–20 rpm).

[0177] Centrifuge to remove the supernatant, and wash twice with Co-IP buffer, using 1 mL each time, centrifuging at 2000 rpm for 1.5 minutes each time.

[0178] The washed beads can be used for subsequent Western blotting to verify protein interactions.

[0179] Co-IP standard buffer formulation: 50mM Tris-HCl (pH 7.6), 150mM NaCl, 5mM MgCl2, 10% glycerol, 0.1% NP40.

[0180] Co-IP incubation working buffer formulation: 5mM DTT, 1mM PMSF, 1×cocktail.

[0181] Analysis of cell division

[0182] Root cell mitotic activity was assessed by counting the number of meristematic cells in the cortex. For counting meristematic cells, root cell outlines were stained with 10 μg / mL propidium iodide (PI). Root cell morphology was then recorded using a Leica SP8 confocal microscope. The number of cells in the endodermal meristematic zone was analyzed and counted using ImageJ software.

[0183] FL1 amino acid sequence information (SEQ ID NO:1)

[0184] 001 MDQKSKTIGE QWWRSNPTFK PPETPLDSME FLSRTWSASA TEVSRAVVAS

[0185] 051 PPTSQPPQMR FSEIQNGSSD VTLVPEDEEN GIVLGNTFSF ASSETSLMVM

[0186] 101 ERIMAQSPEI SSPRTSGRLS HSSFTDSPPI SPSD IDDFKQ FYRVSPSFNG

[0187] 151 HIRGSSAIPG TAGGSKTVGR WLKDRREKKR EETRAQNAQL HAAVSVAGVA

[0188] 201 AAVAAIAAAT ASQSSSSGTDE QVAKNDSAVA SAATLVAAKC VEAAEIMGAD

[0189] 251 REHLASVVSS AVNVRSAGDI MTLTAAAATA LRGAAQLKAR ALKEVWNIAA

[0190] 301 VIPVDKGTPK GGGGGYRGGE LAPVDNFLGI CSKELLAKGC ELLKRTRKGD

[0191] 351 LHWKVVSIYI NRTKQVILKT KSKHVAGTIT KKKKNVVVGL VKGLPAWPGR

[0192] 401 EMLEGGENLR YFGLKTVEKR VI EFECKSQR EYDLWTQGVS MLLS IASDRK

[0193] 451 HKC

[0194] FL3 amino acid sequence information (SEQ ID NO:2)

[0195] 001 MEKLMVPTWR PDPVYRPPET PLEPMEFLAR SWSVSALEVS KALTPPNPQI

[0196] 051 LLSKTEEEEE EEPISSVVDG DGDTEDTGLV TGNPFSFACS ETSQMVMDRI

[0197] 101 LSHSQEVSPR TSGRLSHSSG PLNGSLTDSP PVSPPESDDI KQFCRANKNS

[0198] 151 LNSVNSQFRS TAATPGPITA TATQSKTVGR WLKDRREKKK EETRAHNAQI

[0199] 201 HAAVSVAGVA AAVAAIAAAT AASSSCGKDE QMAKTDMAVA SAATLVAAQC

[0200] 251 VEAAEVMGAE REYLASVVSS AVNVRSAGDI MTLTAGAATA LRGVQTLKAR

[0201] 301 AMKEVWNIAS VIPMDKGLTS TGGSSNNVNG SNGSSSSSHS GELVQQENFL

[0202] 351 GTCSREWLAR GCELLKRTRK GDLHWKIVSV YINKMNQVML KMKSRHVGGT

[0203] 401 FTKKKKNIVL DVIKNVPAWP GRHLLEGGDD LRYFGLKTVM RGDVEFEVKS

[0204] 451 QREYEMWTQG VSRLLVLAAE RKFRM

[0205] Example 1: CRY2 binding proteins FL1 and FL3 obtained under dark conditions by screening Arabidopsis cDNA libraries using yeast two-hybrid screening and IP-MS.

[0206] To screen for photoreceptor-binding proteins in the non-photon excited state, two methods were used: in vitro screening of Arabidopsis thaliana cDNA libraries and in vivo IP-MS screening of CRY2 interacting proteins under non-blue light conditions.

[0207] The screening process requires that the photoreceptors be in a non-photoexcited state, so aluminum foil is used to wrap the culture dish to ensure that the screening process is carried out in the dark.

[0208] In the yeast two-hybrid screening library, the inventors selected strain Y190, with pBridge-CRY2 and pBridge-CRY2 as the two strains. D387A A cDNA library of Arabidopsis thaliana (carried on the pACT2 plasmid) was screened using a protein with a mutation at amino acid position 387 of CRY2, mimicking the continuous non-photoexcited state of CRY2. Yeast colony PCR was performed on the selected positive clones (which could grow in a triple-light-excited yeast medium containing 30 mM 3AT and -LWH) using vector primers flanking the library sequence on pACT2, and the resulting colonies were sequenced. All sequencing results were combined and BLASTed, and all results were counted as candidate CRY2-binding proteins under dark conditions (the number of times a protein was selected was recorded; the higher the number of times, the higher the reliability of the result).

[0209] In Arabidopsis thaliana, using GFP-CRY2 transgenic overexpression material driven by the 35S promoter, a complex of GFP-CRY2 interacting proteins was obtained by affinity chromatography (using GFP-specific agarose beads). The complex was then analyzed by mass spectrometry to obtain candidate CRY2 interacting proteins in Arabidopsis thaliana under dark conditions.

[0210] Extensive screening revealed that FL1 (AT5G43870) and FL3 (AT4G14740) appeared in both assays, suggesting that FL1 and FL3 are CRY2-binding proteins under dark conditions.

[0211] Example 2: Verifying the interaction between CRY2 and FL1 / FL3 under non-blue light conditions and confirming that this binding is only suppressed by blue light.

[0212] Based on the library screening, further experimental methods such as yeast two-hybrid, BiFC, split-LUC, pull-down, and co-IP were used to verify the interaction of full-length CRY2, FL1, and FL3 proteins, and to confirm whether the interaction is regulated by blue light.

[0213] In some experimental procedures, it is necessary to ensure that CRY2 is in a non-blue light excited state. Therefore, the experimental system needs to be shielded from light to ensure dark conditions. Specifically, in the tobacco BiFC and split-LUC experiments, the tobacco was placed in an opaque cardboard box and kept in the dark for 12 hours before recording the experimental results. In the pull-down and co-IP experiments, the experimental system (e.g., centrifuge tubes, culture medium, etc.) was wrapped in aluminum foil. Some operations that need to be performed under light (e.g., sample addition) were performed under red or green light conditions.

[0214] The interaction between CRY2 and FL1 and FL3 under dark conditions, and the fact that this interaction is suppressed by blue light, were verified in all five experimental methods mentioned above. Figure 1 ).

[0215] In the yeast two-hybrid experiment, five light conditions were set: white light, red light, green light, blue light, and darkness. The results showed that the binding of CRY2 to FL1 and FL3 was only weakened by blue light. Figure 1 (A and B).

[0216] The interaction strength between CRY2 and FL1 and FL3 was quantitatively determined using a yeast two-hybrid liquid-assay. The inventors set up two lighting conditions: continuous blue light and blue light shifting towards darkness. The results showed that the interaction strength only increased when blue light shifted towards darkness. Figure 1 (C and D).

[0217] In the pull-down and co-IP experiments, the inventors set up dark and blue light conditions to verify the interaction. The results again showed that the binding of CRY2 to FL1 and FL3 is inhibited by blue light. Figure 1 E, F, and G).

[0218] In addition to setting the light conditions, the inventors also used CRY2. W374A (A protein with a W-to-A mutation at amino acid residue 374 of CRY2, mimicking the continuous photoexcitation state of CRY2) and CRY2 D387A Interaction verification was performed. The results showed that in both tobacco BiFC and yeast two-hybrid experiments, FL1 and FL3 were only associated with CRY2. D387A Interacting, but different from CRY2 W374A Interaction ( Figure 1 H, I, and K).

[0219] The above experimental results jointly demonstrate from multiple perspectives that FL1 and FL3 are two proteins that interact only with CRY2 under non-blue light conditions.

[0220] Example 3: CRY2 interacts with the N-termini of FL1 and FL3 through its N-terminus.

[0221] Three fragments were designed based on the N-terminal PHR domain of CRY2: CRY2 N375 (Contains amino acid residues 1-375 of CRY2), CRY2 N489 (Contains amino acid residues 1-489 of CRY2) and CRY2 N565 (Contains amino acid residues 1-565 of CRY2). The interaction of these three fragments with FL1 was verified. The results show that CRY2 containing the complete PHR domain can bind to FL1. Furthermore, the interacting CRY2 fragments interact with FL1 in a manner that is weakened by blue light. Figure 2 (A and 2B).

[0222] Four segments were designed based on the auxin channeling domain and PH domain of FL1 and FL3: FL1 N (Contains amino acid residues 1-324 of FL1), FL1 C (Contains amino acid residues 325-453 of FL1), FL3 N (containing amino acid residues 1-340 of FL3) and FL3 C (Contains amino acid residues 341-475 of FL3). The interaction of these four fragments with CRY2 was then verified. The results showed that the fragment containing the N-terminus of FL (containing the Auxin canalization domain) can interact with CRY2. Figure 2 (C and 2D), but this interaction loses the property of being weakened by blue light.

[0223] Example 4: FL1 and FL3 proteins are conserved in plants and promote root elongation.

[0224] FL1 and FL3 are members of the Arabidopsis FL family (containing nine proteins, FKD1, FL1–8), a family that is conserved from bryophytes to seed plants. Figure 3 A).

[0225] Through observation, the inventors used CRISPR-Cas9 technology and constructed a fragment deletion type fl1 through hybridization. sd fl3 sd Double mutant ( Figure 3 The root phenotypes of B and 3C) and fkd1 fl1-2 fl2 fl3 (flΔ4) were observed. The results showed that fl1 sd fl3 sd FL1 and FL3 exhibit root length deficiency phenotypes, with flΔ4 showing a more pronounced root length deficiency phenotype, indicating that FL1 and FL3 can promote taproot elongation in plants. Figure 3 (D and 3E), and FL family members exhibit a dose-response effect on root length regulation.

[0226] The significant defects in root meristem cell division and root length in flΔ4 could be restored by overexpression of FL1-GFP and FL3-GFP, indicating that FL1 and FL3 can promote meristem cell division and taproot elongation. Figure 3 G-3K). fl1 sd fl3 sd The same applies to double mutants.

[0227] Furthermore, FL1 and FL3 can interact with FKD1 and FL2, indicating that FKD1 and FL2 can also participate in CRY2-FL-mediated cell division and root length regulation. Figure 3 F).

[0228] Example 5: In regulating cell division in the meristematic zone of Arabidopsis thaliana primary root, the non-photoexcited state CRY2 is functionally active and located upstream of FL1 and FL3.

[0229] The cry2 flΔ4 five mutants were constructed by hybridization, and the root length and cell division phenotype of each genotype were observed under dark conditions.

[0230] according to Figure 4 The root length of the A and 4B cry2 single mutants was significantly increased, while the root length of the flΔ4 tetra mutant and the cry2 flΔ4 penta mutant was significantly lower than that of the wild type. This indicates that FL1 and FL3 play a role in increasing root length in plants, and that CRY2 is an upstream molecule of these mutants.

[0231] according to Figure 4 In C and 4D, the cry2 single mutant showed significantly increased cell division (increased number of endodermal cells in the root tip meristem), while the flΔ4 tetra mutant and the cry2 flΔ4 penta mutant showed significantly lower cell division than the wild type. This indicates that FL1 and FL3 play a role in promoting cell division in roots, and that CRY2 is an upstream molecule of these mutants.

[0232] The results indicate that the absence of FL1 and FL3 resulted in significantly shorter root lengths compared to the wild type. Furthermore, the non-photoexcited state CRY2, located upstream of FL1 and FL3, regulates cell division and consequently controls the elongation of the Arabidopsis taproot.

[0233] according to Figure 4 In E and 4F, overexpression of FL1 or FL3 in the wild type significantly promoted taproot elongation. In the cry2 mutant, overexpression of FL1 or FL3 (downregulation of CRY2 and upregulation of FL1 or FL3) further promoted taproot elongation.

[0234] according to Figure 4G–4I, in wild type, significantly increases cell division by overexpression of FL1 or FL3. In cry2 mutants, overexpression of FL1 or FL3 (downregulation of CRY2 and upregulation of FL1 or FL3) further increases cell division.

[0235] By comparing the statistical results of root length and cell division under blue light and dark conditions, it was found that under blue light conditions, overexpression of FL1 or FL3 in the wild-type background significantly promoted the elongation of the primary root and cell division compared with dark conditions. This characteristic was weakened in the overexpression of FL1 or FL3 in the cry2 background, indicating that the inhibition of FL1 or FL3 by CRY2 can be relieved by blue light.

[0236] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for regulating plant cell division and / or root length, comprising: Regulating interaction systems or elements thereof in plants; said interaction system includes elements: FL1, FL3, and / or CRY2.

2. The method as described in claim 1, characterized in that, The method includes: (a) Upregulating FL1 and / or FL3 to promote cell division and increase root length; preferably, the upregulation of FL1 and FL3 includes: upregulating the expression, abundance or activity of FL1 and / or FL3; (b) Downregulating FL1 and / or FL3 to inhibit cell division and reduce root length; preferably, the downregulation of FL1 and FL3 includes: downregulating the expression, abundance or activity of FL1 and / or FL3; (c) Downregulating CRY2 to promote cell division and increase root length; preferably, the downregulation of CRY2 includes: downregulating the expression, abundance or activity of CRY2; (d) Upregulating CRY2, thereby inhibiting cell division and reducing root length; preferably, the upregulation of CRY2 includes: upregulating the expression, abundance or activity of CRY2; (e) Weakening the interaction between CRY2 and FL1 and / or FL3, thereby promoting cell division and increasing root length; preferably, including: placing the plant in a blue light environment, thereby weakening the interaction between CRY2 and FL1 and / or FL3, enhancing the activity of FL1 and / or FL3, promoting cell division and increasing root length; (f) Allowing CRY2 to interact with FL1 and / or FL3, thereby inhibiting cell division and reducing root length; preferably, including: placing the plant in a non-blue light environment to form a non-photoexcited state of CRY2, thereby allowing the CRY2 to interact with FL1 and / or FL3, inhibiting FL1 and / or FL3 activity, inhibiting cell division and reducing root length.

3. The method as described in claim 1, characterized in that, In (a), the coding genes of FL1 and / or FL3 or expression constructs or vectors containing the coding genes are introduced into plants to upregulate FL1 and / or FL3. (b) knocking out or silencing FL1 and / or FL3, or inhibiting the activity of FL1 and / or FL3 in plants; preferably, including: reducing the activity of FL1 and / or FL3 by site-directed mutagenesis, knocking out or down-knocking out FL1 and / or FL3 genes by gene editing with a CRISPR system, knocking out or down-knocking out FL1 and / or FL3 genes by homologous recombination system, or silencing the genes by interfering molecules that specifically interfere with the expression of FL1 and / or FL3 genes. In (c), knocking out or silencing CRY2 in plants, or inhibiting CRY2 activity; preferably, this includes: reducing CRY2 activity by site-directed mutagenesis, knocking out or downsing the CRY2 gene by gene editing using a CRISPR system, knocking out or downsing the CRY2 gene by a homologous recombination system, or silencing the gene by interfering molecules that specifically interfere with CRY2 gene expression; or In (d), the gene encoding CRY2 or an expression construct or vector containing the gene encoding CRY2 is introduced into plants to upregulate CRY2.

4. Use of an interaction system in a plant, elements therein, or regulators thereof for regulating plant cell division or root length, or for preparing a construct / composition for regulating plant cell division or root length; said interaction system comprising elements: FL1, FL3, and / or CRY2.

5. The use as described in claim 4, characterized in that, The regulator includes: Cell division and / or root growth promoters, including those selected from: (i) genes encoding FL1 and / or FL3 or expression constructs or vectors containing said genes; (ii) loss-of-function site-directed mutagenesis agents for CRY2, CRISPR systems for knocking out or knocking down the CRY2 gene, homologous recombination systems for knocking out or knocking down the CRY2 gene, and interfering molecules that specifically interfere with the expression of the CRY2 gene; (iii) agents that weaken the interaction between CRY2 and FL1 and / or FL3; or Cell division and / or root growth inhibitors, including those selected from: (i) loss-of-function site-directed mutagen agents for FL1 and / or FL3, CRISPR systems that knock out or knock down FL1 and / or FL3 genes, homologous recombination systems that knock out or knock down FL1 and / or FL3 genes, and interfering molecules that specifically interfere with the expression of FL1 and / or FL3 genes; (ii) the gene encoding CRY2 or an expression construct or vector containing said gene; and (iii) agents that enable CRY2 to interact with FL1 and / or FL3.

6. The method as described in any one of claims 1 to 3 or the use as described in any one of claims 4 to 5, characterized in that, The FL1 and / or FL3 include their Auxin canalization domains; preferably, the FL1 includes: a bioactive fragment containing FL1 1-324 aa, a bioactive fragment containing FL1 22-307 aa, and a bioactive fragment containing FL1 1-307 aa; preferably, the FL3 includes: a bioactive fragment containing FL3 1-340 aa, a bioactive fragment containing FL3 18-317 aa, and a bioactive fragment containing FL3 1-317 aa; or The CRY2 includes its PHR domain; preferably, the CRY2 includes: a bioactive fragment containing CRY2 1-489aa; a bioactive fragment containing CRY2 1-486aa; and a bioactive fragment containing CRY2 1-565aa.

7. An interaction system for use in optogenetics, the interaction system comprising: The interaction system includes components: CRY2 and FL1 and / or FL3; Preferably, in a blue light environment, the interaction between CRY2 and FL1 and / or FL3 in the interaction system is weakened, and the activity of FL1 and / or FL3 is enhanced, thereby promoting plant cell division and increasing root length. Preferably, in a non-blue light environment, CRY2 interacts with FL1 and / or FL3 in the interaction system, and the activity of FL1 and / or FL3 is inhibited, thereby inhibiting plant cell division and increasing root length.

8. Use of an optogenetic interaction system for use as a molecular marker for identifying plant cell division or root length; said interaction system comprising elements: FL1, FL3, and / or CRY2.

9. A method for identifying plant cell division or root length, comprising: Analyze the operation of interaction systems in plants; the interaction system includes elements: FL1, FL3, and / or CRY2; wherein, if FL1 and / or FL3 are highly expressed or have high activity, CRY2 is lowly expressed or has low activity, and the interaction between CRY2 and FL1 and / or FL3 is weak, then the plant has long roots or high cell division activity; if FL1 and / or FL3 are lowly expressed or have low activity, CRY2 is highly expressed or has high activity, and the interaction between CRY2 and FL1 and / or FL3 is strong, then the plant has short roots or low cell division activity.

10. The method or use as described in any one of claims 1-9, characterized in that, The plants mentioned include those selected from the group consisting of plants containing the interaction system; the interaction system includes elements: FL1, FL3, and / or CRY2.

Images