Application of HYPK gene in plant photomorphogenesis and plant type improvement

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an HYPK gene in plant photomorphogenesis and plant type improvement. Experimental verification on a model plant arabidopsis thaliana shows that the HYPK gene can promote ubiquitination degradation of E3 ligase COP1 on a substrate HY5, and deletion / reduction of HYPK gene expression can make the plant present a constitutive photomorphological phenotype along with COP1 activity reduction and HY5 protein accumulation. The discovery provides an important gene resource for cultivating new varieties of close-planting-resistant and lodging-resistant crops.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to HYPK Application of genes in plant photomorphogenesis and plant architecture improvement. Background Technology

[0002] Arabidopsis thaliana, as a classic model plant, is widely used to elucidate the molecular mechanisms related to plant growth and development. In the field of photomorphogenesis, its molecular pathways have been extensively studied, with the core mechanisms involving a light signaling network primarily composed of photoreceptors, the E3 ubiquitin ligase COP1, and the transcription factor HY5. Photomorphogenesis is a crucial developmental transition in plant seedlings after exposure to light, characterized by inhibited hypocotyl elongation, cotyledon unfolding, and chlorosis, also known as de-epiphyllinization. This process is mediated by multiple photoreceptors, enabling plants to precisely respond to different light qualities and intensities.

[0003] The ubiquitin-proteasome system plays a crucial role in light signal transduction, enabling rapid responses to environmental signals through the specific degradation of target proteins. Among these, the E3 ubiquitin ligase COP1 is a core negative regulator of photomorphogenesis, promoting the degradation of positive light regulators (such as the transcription factor HY5) under dark conditions, thereby inhibiting photomorphogenesis. Light exposure inhibits COP1 activity, leading to the accumulation of proteins such as HY5, which in turn initiates the expression of photomorphogenesis-related genes. Several photoreceptors and regulatory proteins are known to participate in this process; for example, phyB and CRY1 can inhibit COP1 function, while the SPA protein enhances its activity.

[0004] Huntingtin yeast two-hybrid protein K (HYPK) is a small, conserved protein in eukaryotes. Studies have shown that it can serve as a component of protein acetyltransferase complexes, participate in protein acetylation, and influence protein interactions and stability through molecular chaperone-like functions. In human cells, HYPK regulates the stability and subcellular localization of phosphorylated proteins. Preliminary studies in plants suggest that HYPK may participate in the regulatory network of protein acetylation and ubiquitination, but its specific functions and mechanisms in light signaling pathways and photomorphogenesis remain unclear, and there are no reports of its application in plant architecture improvement.

[0005] In current research on the regulatory mechanisms of plant photomorphogenesis, HYPK This invention addresses the gaps in our understanding of gene function. HYPK The novel applications of genes in regulating plant photomorphogenesis and improving plant architecture provide new genetic resources and technological approaches for crop genetic improvement. Summary of the Invention

[0006] This invention is first clearly defined HYPKThe gene positively regulates the biological activity of the E3 ubiquitin ligase COP1. Loss of HYPK function leads to a constitutive photomorphogenesis phenotype in plants, along with decreased COP1 activity and HY5 protein accumulation; conversely, restoration of intact HYPK protein significantly enhances HYPK function. COP1 interaction restores COP1 activity.

[0007] In dense planting, shading between plants easily leads to excessive stem growth and decreased lodging resistance. Therefore, improving crop shade tolerance and light adaptability is crucial for breeding. Research on light signal-regulated plant architecture provides an important approach for this. Studies have shown that using gene resources from the model plant Arabidopsis thaliana to regulate crop plant architecture is a feasible and effective strategy. For example, the phyB-LG1-HB53 module constructed in maize can form a "tight top and loose bottom" plant architecture to increase yield (Wang et al., 2024) (Shi et al., 2024). This strategy is also applicable to rice, where phyB can regulate OsCCA1 splicing through ELD1, thereby improving its tolerance to dense planting (Cai et al., 2025).

[0008] This invention has discovered that, HYPK The loss-of-function mutant exhibited enhanced photomorphogenesis under low light and different light qualities, with shortened hypocotyls and reduced plant height, indicating significantly improved shade tolerance. This gene provides a new genetic resource and breeding tool for the targeted breeding of new crop varieties tolerant of dense planting and resistant to lodging. The results of this invention can provide important theoretical basis for the utilization of light signals and the regulation of growth and development in crops and horticultural plants, thereby providing guidance for breeding and variety improvement of traits such as seed germination, morphogenesis, and plant type optimization. Simultaneously, by regulating the expression and activity of related genes and proteins through genetics, breeding, and genetic engineering, potential technical pathways can be provided for improving plant adaptability to light environments and optimizing important traits such as seedling formation and mature plant type.

[0009] In this regard, the present invention includes, but is not limited to, the following: In one aspect, the present invention provides HYPK The application of genes in enabling constitutive photomorphogenesis and / or plant architecture improvement in plants is characterized by reducing / inactivating the photomorphogenesis of plants. HYPK Gene expression.

[0010] In another aspect, the present invention provides for reducing / deactivating HYPK Application of gene expression in constitutive photomorphogenesis and / or plant architecture improvement in plants.

[0011] In another aspect, the present invention provides a way to reduce / inactivate the plant's... HYPKApplication of recombinant vectors for gene expression in constitutive photomorphogenesis and / or plant architecture improvement in plants.

[0012] In another aspect, the present invention provides the application of recombinant engineered bacteria in improving constitutive photomorphogenesis and / or plant architecture in plants, characterized in that the recombinant engineered bacteria contains bacteria that reduce / inactivate certain plant components. HYPK Recombinant vectors for gene expression.

[0013] In another aspect, the present invention provides a method for improving the constitutive photomorphogenesis and / or plant form of plants, characterized in that the method comprises: introducing into the plant a substance that reduces / inactivates certain plant components. HYPK A recombinant vector for gene expression or a recombinant engineered bacterium containing the recombinant vector.

[0014] In another aspect, the present invention provides a method for obtaining plants with improved traits, characterized in that the method comprises: introducing into the plant a substance that reduces / inactivates certain plant components. HYPK A recombinant vector for gene expression or a recombinant engineered bacterium containing the recombinant vector.

[0015] In one aspect, the application or method of the present invention includes the following steps: (1) Design HYPK The target sequence of the gene is used to construct a CRISPR-Cas9 vector. Preferably, the target sequence is as shown in SEQ ID NO: 3. (2) The vector obtained in step (1) is transferred into engineered bacteria; and (3) Infect plant tissues with the engineered bacteria obtained in step (2).

[0016] In one aspect, the target sequence of the present invention is shown in SEQ ID NO: 3.

[0017] In one aspect, the plant described in this invention is Arabidopsis thaliana.

[0018] In one aspect, the CRISPR-Cas9 vector / recombinant vector of the present invention is a vector pCambia1300-UBQ:Cas9-P2A-GFP-rbcS-E9t containing the target sequence.

[0019] In one aspect, the engineered bacteria of the present invention is Agrobacterium. Preferably, it is Agrobacterium GV3101.

[0020] In one aspect, the present invention describes HYPK The amino acid sequence encoded by the gene is shown in SEQ ID NO: 2.

[0021] In one aspect, the present invention describes HYPKThe nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 1.

[0022] In one aspect, the plant type improvement / improved trait described in this invention is a reduced plant height.

[0023] In one aspect, the plant described in this invention is Arabidopsis thaliana.

[0024] In this invention, there are no particular limitations on the plants suitable for gene transformation, as long as they are suitable for gene transformation operations, such as various crops, flowering plants, or forestry plants. The plants may be (but are not limited to): dicotyledons, monocotyledons, or gymnosperms.

[0025] As a preferred approach, the term "plant" includes, but is not limited to, Arabidopsis thaliana; any plant possessing the gene or a gene homologous to it is applicable.

[0026] The term "plant" as used in this invention includes the whole plant, its parent and offspring plants, and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues, and organs, all of which contain our target gene or nucleic acid. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores; similarly, each of these objects contains the target gene / nucleic acid.

[0027] This invention includes any plant cell, or any plant obtained or obtainable by the methods described herein, as well as all plant parts and their propagules. This patent also includes transfected cells, tissues, organs, or whole plants obtained by any of the foregoing methods. The only requirement is that the offspring exhibit the same genotype or phenotypic characteristics, and that offspring obtained using the methods of this patent have identical characteristics.

[0028] The invention also extends to the harvestable parts of the plants as described above, but is not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers, and bulbs. Furthermore, it relates to other derivatives of the plant after harvest, such as dried granules or powders, oils, fats and fatty acids, starches, or proteins.

[0029] In one aspect, the Arabidopsis thaliana of the present invention AtHYPK The gene (AT3G06610) has a gene sequence number (Gene ID) of 819840 in NCBI. AtHYPK The messenger RNA (mRNA) sequence (NM_111537) of the gene is 878 bp in length, and its nucleotide sequence contains the sequence shown in SEQ ID NO: 1. AtHYPKThe gene's coding sequence is 348 bp in length, and the encoded amino acid sequence (NP_566288) is 115 aa in length, containing the sequence shown in SEQ ID NO: 2.

[0030] Arabidopsis AtHYPK The nucleotide sequence contains SEQ ID NO: The sequence shown in 1 is as follows: ATGGAGGGAGCAGAGGAAGCTGGGGCTGAGATAGCGGTTGATTCAAAGGACTTGCAGCAACAAAGCAAAGCTTTCGACAAGCTCACCGATCGTGTCGAGGATCGCCAGCTCGATTCCTCTCGTGTTCAATCGGCTATGGCTTCGATTGCTGCTTCTAGGGAGGCTGATCT GAATGCTAAGAGGTTGAGGGAGAAAGAACTGGCCTCTGTGAAGATCAATCCTGCAGATGTTGAGTTTATTGTAAACGAACTTGAGATAGAAAAGAATGTGGCGGAAAGAACTCTAAGGGAGCACAAAGGTGATGCGGTTGCTGCGACTAGGCAATTGCTTTCACGATATCCTCTATGA (SEQ ID NO: 1).

[0031] Arabidopsis AtHYPK The amino acid sequence encoded by the gene (NP_566288) contains the sequence shown in SEQ ID NO: 2, specifically as follows: MEGAEEAGAEIAVDSKDLQQQSKAFDKLTDRVEDRQLDSSRVQSAMASIAASREADLNAKRLREKELASVKINPADVEFIVNELEIEKNVAERTLREHKGDAVAATRQLLSRYPL* (SEQ ID NO: 2).

[0032] The beneficial technical effects of the present invention include, but are not limited to, the following: (1) By comprehensively applying genetic and molecular biological methods, the study systematically demonstrated the effects of Arabidopsis thaliana. HYPK The positive regulatory effect on photomorphogenesis. This argument includes: constructing... hypk Mutants and verify their usability; utilizing hypk The mutant was confirmed to be involved in constitutive photomorphogenesis and photoresponsiveness; it was verified that HYPK can promote the degradation of the classical substrate of light signaling, HY5 protein.

[0033] (2) This invention provides a practical approach to crop improvement: by using gene editing technology to target and modify the HYPK gene of plants, functionally modified plants with finely regulated COP1 activity can be obtained, laying a technical foundation for directly cultivating new varieties with excellent traits such as tolerance to dense planting and lodging resistance. Attached Figure Description

[0034] Figure 1 It is built with CRISPR-Cas9 hypk A schematic diagram of the mutant; Figure 2 These are the Coomassie Brilliant Blue staining results after purification of HYPK-pCold protein; Figure 3 These are the results of immunoblotting detection of HYPK protein in wild-type and various mutant lines; Figure 4 Wild type and hypk Schematic diagram of mutant growth under dark conditions and various colors of light (4A) and statistical diagram of hypocotyl length of 4-week-old seedlings (4B). Figure 5 Wild type and hypk Statistical results of growth phenotype and plant height of mutants grown under long-day conditions for one month; Figure 6 The results show the protein content of HY5 and HYPK in wild-type and mutant strains grown under full sunlight for 5 days. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0036] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0038] Unless otherwise stated, the implementation of this invention will utilize conventional botanical techniques, microbiological techniques, tissue culture techniques, molecular biology techniques, chemical techniques, biochemical techniques, DNA recombination techniques, and bioinformatics techniques that are readily apparent to those skilled in the art. These techniques have been fully explained in published literature. Furthermore, the methods employed in this invention, including DNA extraction, phylogenetic tree construction, gene editing methods, gene editing vector construction, and the acquisition of gene-edited plants, except for those used in the examples below, can all be implemented using methods already disclosed in existing literature.

[0039] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “nucleotide,” “nucleic acid molecule,” or “polynucleotide” mean, but are not limited to, isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), naturally occurring, mutant, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, and single-stranded or double-stranded structures. These nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. “Gene” or “gene sequence” is broadly used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include coding sequences in cDNA, and / or include cDNA and its regulatory sequences. In particular embodiments, such as concerning isolated nucleic acid sequences, cDNA is preferred by default.

[0040] 1. Biomaterials Arabidopsis thaliana Col-0 and HY5-GFP seeds were preserved in the laboratory; hy5-215 and cop1-4 The mutant was preserved in the laboratory; hypk-594 and hypk-54 The mutant was obtained through CRISPR-Cas9 gene editing.

[0041] The CRISPR-Cas9 vector pATU6M / pCam1300-UBQ-Cas9 and the E. coli expression vector pCold were preserved in the laboratory. Escherichia coli clone competent cells Trans1-T1 (TransGen Biotech), Escherichia coli expression competent cells Transetta BL21(DE3) (TransGen Biotech), and Agrobacterium GV3101 were preserved in the laboratory; Primer synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd. Gene sequencing was performed by Beijing Qingke Biotechnology Co., Ltd.

[0042] Arabidopsis thaliana, Col-0 ecotype, is a commonly used model plant for research. The cultivation conditions are: temperature 22 ± 1℃, light-dark cycle 16 h / 8 h, and light intensity 50 μmol•m. -2 •s -1 .

[0043] 2. Experimental reagents The RNA extraction kit was purchased from Aikerui Biotechnology Co., Ltd. Commonly used reagents such as NaCl were purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd. Various endonucleases and ligases were purchased from Thermo Scientific. KOD enzyme was purchased from TOYOBO; reverse transcriptase MLV was purchased from Invitrogen. The seamless cloning kit was purchased from CloneSmart. The HRP chemiluminescence assay kit was purchased from Tiangen Biotech and ThermoScientific. The plasmid mini-extraction kit and gel recovery kit were purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd. In vitro ubiquitin assay reagents E1 and E2 were purchased from R&D Systems. Anti-HY5 and Anti-HYPK antibodies were prepared in-house. The protein antigens were purified in the laboratory, and the antibody production was completed by Beijing BGI Protein Research Center Co., Ltd. Anti-Actin was purchased from EASYBIO. Anti-Rabbit IgG-H&L (HRP) and anti-Mouse IgG-H&L (HRP) were purchased from Kangwei Century Co., Ltd.

[0044] 3. Experimental equipment The T100 PCR instrument was purchased from Bio-Rad. The ambient temperature centrifuges and refrigerated centrifuges were purchased from Eppendorf. The qRT-PCR instrument was purchased from Roche. The laser confocal microscope was purchased from Zeiss. The spectrophotometer was purchased from EMC. The live imaging system was purchased from Berthold. The digital camera was purchased from Olympus.

[0045] Example 1 hypk Acquisition of mutant materials In order to obtain hypk We constructed and obtained the mutant using CRISPR-Cas9. hypk -594 and hypk -54. hypk -594 has a 9bp deletion at the front end of the gene, resulting in a mutant form of the protein that encodes HYPKΔN3aa. hypk -54 terminates prematurely due to a 1bp deletion and cannot encode the protein. Figure 1 ).

[0046] The specific procedures for gene editing using the CRISPR-Cas9 system are as follows: (1) Selection of sgRNA The principle for selecting target gene sgRNAs is high efficiency and specificity. An efficiency scoring list was designed using the sgRNA design website (http: / / crispr.dfci.harvard.edu / SSC / ), and several sgRNAs with high scores were selected for vector construction. The sequence we selected was: HYPK-sgRNA1 (GAGGGAGCAGAGGAAGCTG, SEQ ID NO: 3), positive strand, with a score of 1.1683.

[0047] (2) Construction The construction mainly consists of two steps. The first step is to tandem the sgRNA into the pAtU6-M (ampicillin resistance) vector. The second step is to cut out the ligated AtU6-sgRNAs and ligate them into the final plant binary vector pCambia1300-UBQ:Cas9-P2A-GFP-rbcS-E9t (this vector provides plant resistance with hygromycin and bacterial resistance with kanamycin).

[0048] a. Design primers for sgRNA sgRNA-F: 5'-GATTGN 19 -3'(GN 19 This is the designed sgRNA sequence. Since the first molecule of the AtU6 promoter requires G, the first molecule of the sgRNA should ideally be G. sgRNA-R: 5'-AAACN 19 C-3'(N) 19 C is the reverse complementary sequence of the designed sgRNA sequence.

[0049] b. Mix equal amounts of primers sgRNA-F and sgRNA-R (100 uM) and anneal.

[0050] c. Digest the pAtU6-M vector with BbsI, recover it, and ligate it with the annealed sgRNA.

[0051] d. Digest the ligated pAtU6-sgRNAs vector with KpnI and SalI, and recover the AtU6-sgRNAs fragment; at the same time, digest the final vector pCambia1300-UBQ:Cas9-P2A-GFP-rbcS-E9t with KpnI and SalI restriction endonucleases, and ligate it.

[0052] (3) Transform the above-constructed vector into Agrobacterium GV3101 and infect Arabidopsis thaliana.

[0053] (4) Screen positive seedlings (50~100 μg / mL hygromycin) and harvest T1 generation.

[0054] (5) Select T1 generation plants with single copy insertion (hygromycin resistance ratio 3:1) and transplant them into the soil. After they grow, take leaves to identify the editing status of the target site.

[0055] (6) Spread the seeds of the above-mentioned T1 generation T2 onto one portion each of resistant and non-resistant MS plates, select plants without hygromycin resistance (i.e., Cas9-free plants), transplant these plants into the soil, and take leaves after they grow to identify whether the target site is homozygous.

[0056] Example 2 hypk Usability test of mutants To determine the usability of the mutant plants, the genome sequencing results after Cas9 editing need to be validated. We cloned... HYPK Genes were used to construct and purify the HYPK-pCold tag protein, which was then used as an antigen to produce the polyclonal antibody Anti-HYPK.

[0057] The specific experimental steps are as follows: (1) Design primers Plant RNA was extracted using an RNA extraction kit and reverse transcribed into cDNA. The primer sequences required for cloning AtHYPK using cDNA are as follows: HYPK-OX-F ATGGAGGGAGCAGAGGAAGC (SEQ ID NO: 4) HYPK-OX-R TAGAGGATATCGTGAAAGCAATT (SEQ ID NO: 5) Gene cloning: Using cDNA as a template, the target gene was amplified using high-fidelity DNA polymerase KOD FX and gene-specific primers. The PCR reaction system was: 25 μL 2× KOD mix, 1.5 μL 10 μM forward primer F, 1.5 μL 10 μM reverse primer R, 2 μL template, and 20 μL ddH2O. The PCR reaction program was: pre-denaturation (95℃, 3 min), denaturation (95℃, 10 s), annealing (58℃, 30 s), extension (68℃, 30 s-60 s), with 30-35 cycles.

[0058] Purification and recovery of the target fragment: After amplification, the final product was analyzed by 1% agarose gel electrophoresis to verify the specificity of the amplified fragment. The target fragment was excised using a UV imager, and nucleic acid fragment purification was performed according to the instructions of the Zhongke Ruitai DNA Recovery Kit. The specific procedure is as follows: Place the excised target fragment in a 1.5 mL centrifuge tube, add 500 μL of sol-gel buffer, and incubate at 65 °C for 10 min to completely dissolve the agarose matrix. After the mixture cools to room temperature, transfer it to a nucleic acid adsorption column. Centrifuge at 12,000 rpm for 1 min at room temperature and discard the waste liquid. Add 700 μL of washing buffer to the adsorption column and repeat the centrifugation operation to ensure the removal of impurities from the silica membrane. Centrifuge at 12,000 rpm for 2 min and discard the residual solution in the adsorption column. Transfer the adsorption column to a clean EP tube, add 35 μL of ddH2O suspended in the center of the adsorption column membrane, and incubate at room temperature for 2 min to promote nucleic acid elution. Centrifuge at 12,000 rpm for 2 min to obtain the purified fragment.

[0059] (2) Constructing a carrier Enzyme digestion and ligation: A one-step double digestion was performed using the FastDigest enzyme system (Thermo Scientific) to simultaneously linearize the target DNA fragment and plasmid vector. The digestion reaction mixture consisted of: 5 μL 10× FastDigest Buffer, 6 μL recovered product or 2 μL vector fragment, 1 μL restriction endonuclease A, 1 μL restriction endonuclease B, and ddH2O added to a final volume of 50 μL.

[0060] The enzyme digestion reaction was incubated in a metal bath at 37°C for 1 h. After the enzyme digestion reaction was completed, the linearized vector fragment was purified and recovered according to the above nucleic acid gel recovery steps.

[0061] Due to different vector construction methods, two methods are generally used: T4 restriction enzyme ligation (T4-DNA ligase) and seamless cloning homologous recombination ligation. The ligation reaction was performed using T4 DNA ligase (Thermo Scientific). The reaction system consisted of: 1 μL 10× T4 DNA Ligase Buffer, 6 μL of digested fragment, 2 μL of linearized vector, and 1 μL of T4 DNA ligase. The ligation reaction was carried out at 22℃ for 30 min.

[0062] In vitro recombination ligation was performed using the CloneSmarter Seamless Cloning Kit, following the instructions: the target gene fragment was obtained by PCR amplification; the target gene fragment and the linearized vector were mixed at a 3:1 molar ratio, and 5 μL of 2× recombination premix (Seamless Master Mix) was added to a final volume of 10 μL. The mixture was then gently mixed; the recombination reaction was completed by incubation at 50°C for 15 min; and the product was introduced into Trans1-T1 competent cells using the heat shock transformation method.

[0063] (3) Bacterial transformation E. coli transformation (heat shock transformation): Trans1-T1 competent cells frozen at -80℃ were thawed on ice and aliquoted into 1.5 mL centrifuge tubes (30 μL). Ligation product (5 μL) was added and gently mixed by pipetting. The cells were then equilibrated on ice for 30 min. After a brief heat shock at 42℃ for 60 seconds, the cells were immediately quenched on ice for 3 min. 1 mL of LB recovery medium (antibiotic-free) was added, and the cells were incubated on a shaker at 37℃ (180 rpm) for 60 min. The cells were centrifuged at 4800 rpm for 3 min to obtain a bacterial pellet. The pellet was resuspended in the residual supernatant (approximately 100 μL) and plated onto LB agar plates containing the appropriate antibiotics. The pellets were incubated at 37℃ for 16-18 h. The next day, single colonies were selected for PCR verification.

[0064] The recombinant plasmid (1 μL) was then transformed into BL21(DE3) chemocompetent cells (10 μL) using the heat shock transformation method, and colony PCR was performed using specific primers. Validated single colonies were selected, inoculated into LB seed medium, and placed in a constant temperature shaking incubator (37℃, 180 rpm) for primary amplification culture (12-16 h).

[0065] (4) Prokaryotic protein expression and purification Large-scale induction culture: The seed culture was transferred to 400 mL of sterile LB medium at a 1% inoculation ratio and cultured in a constant temperature shaking incubator (37℃, 180 rpm) for 4-5 h until the OD600 value reached 0.8-1.0. 0.5 mM isopropyl thio-β-D-galactopyranoside (IPTG) was added to the culture system, and the mixture was placed in a constant temperature shaking incubator at 16℃ and 150 rpm for 12-16 h to induce expression. After induction, the cells were collected by centrifugation at 5,000 rpm for 10 min.

[0066] Bacterial resuspension: 25 mL of a pre-equilibrated bacterial lysis buffer system (25 mM Tris-HCl pH 7.5, 150 mM NaCl) at 4℃ was quantitatively injected into the centrifuged bacterial precipitate. The bacterial cells were then uniformly dispersed by vortexing to obtain a milky white homogeneous suspension.

[0067] Cell disruption by ultrasound is performed as follows: The bacterial suspension is placed in an ice-water bath, and cell lysis is carried out using an ultrasonic disruptor (probe diameter 6 mm). Ultrasonic parameters are set as follows: output power 200 W, cycle time 3 s, interval time 8 s, total processing time 20-40 min. The turbidity of the bacterial suspension is monitored in real time until it becomes clear and transparent. Centrifuge at 4°C, 9,000 rpm for 30 min. Filter the supernatant through a filter cloth into a new 50 mL conical centrifuge tube, and add 200-300 μL of equilibrated agarose beads.

[0068] Incubate the mixture at 4°C for at least 2 hours using a rotary mixer. Slowly inject the incubation system of the complex into a pre-equilibrated affinity chromatography column. After the flow-through fraction has completely flowed out, equilibrate the column bed using lysis buffer pre-cooled at 4°C. Allow any remaining liquid to be completely drained by gravity before proceeding to subsequent operations.

[0069] Specific protein elution: His-tag recombinant proteins were eluted using gradient imidazole buffer (200 mM imidazole, Tris-NaCl, pH 8.0). The elution buffer was pre-chilled on ice before stepwise elution: an initial 300 μL of static binding was performed (incubated on ice for 10 min), followed by stepwise elution with 200 μL each time (repeated 3 times). The elution endpoint was monitored (OD595 < 0.1 as determined by the Bradford method).

[0070] Recombinant protein expression detection (Coomassie Brilliant Blue method): The eluent was mixed with an equal volume of 2× loading buffer, and the protein complex was dissociated by heat shock at 95℃ for 5 min. After separation by SDS-PAGE electrophoresis, the gel was placed in a Coomassie Brilliant Blue R-250 staining system (0.1% (w / v) R-250, 30% (v / v) Methanol, 10% (v / v) Acetic acid), and stained by constant shaking at 60 rpm for 5 min. Subsequently, the gel was transferred to deionized water for dynamic destaining at room temperature. The gel background was made transparent by real-time observation. Figure 2 ).

[0071] The purified protein was handed over to BGI Genomics for subsequent antibody immunization serum preparation. The serum was then used for protein immunoblotting experiments.

[0072] (5) Extracting total plant protein Arabidopsis seedlings cultured under continuous white light for 5 days were then transferred to darkness for 6 hours. Samples were taken and placed in 1.5 mL centrifuge tubes containing steel balls and thoroughly ground in a plant shredder (50 Hz, 60 s). The shredder module needed to be pre-cooled in liquid nitrogen beforehand.

[0073] Add a pre-cooled protein extraction buffer solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v / v) NP-40, 1% Triton X-100, 1× protease inhibitor cocktail, 10 mM cysteine ​​protease inhibitor NEM (N-ethylmaleimide), 2 mM PMSF, 50 μM MG132) to the powdered sample at a 1:3 (m / v) ratio. Vortex at high speed until homogenized, then incubate on ice for 10–15 min.

[0074] Cell debris was separated by low-temperature centrifugation (12,000 rpm, 10 min / 4℃), and the clear lysate was collected and transferred to a pre-chilled collection tube (1.5 mL, pre-equilibrated on ice).

[0075] Repeat the previous step, retaining the supernatant protein. Pipette 2 μL of the sample to be tested and inject it into pre-chilled Bio-Rad Bradford working solution (1×, catalog number: 5000205), resulting in a final volume of 1 mL. Perform the colorimetric reaction under light-protected conditions (25℃ / 5 min). Measure the absorbance at the characteristic wavelength of 595 nm using a UV spectrophotometer (UV-2600, EMC). After quantifying the protein according to the standard curve, adjust the sample concentration using the original extraction buffer.

[0076] (6) Immunoblotting experiment Prepare a suitable concentration of polyacrylamide gel according to experimental requirements. Using the Bio-Rad electrophoresis tank equipment, set up the gel and fill the inner and outer tanks with 1× SDS electrophoresis buffer. Use a pipette to add 10-40 μL of sample and 3 μL of protein pre-stained marker to the sample wells. The formulation of 10× SDS-PAGE electrophoresis buffer is: 0.25 M Tris, 2.5 M glycine, and 1% SDS.

[0077] Start electrophoresis at a constant voltage of 90 V. Once the sample has migrated to the separating gel interface, adjust the voltage to 130 V and continue electrophoresis until the pre-stained protein marker shows that the target band has reached 70% of the migration distance of the separating gel, at which point the program is terminated. Constant current transfer: After electrophoresis, gently remove the gel containing the target protein from the gel plate and immerse it in distilled water. Simultaneously, cut a polyvinylidene fluoride (PVDF) transfer membrane (0.45 μm) and a double layer of filter paper according to the gel size (8.5 × 6.5 cm). Activate the PVDF membrane by immersing it in 100% methanol (3 min / RT). Stack the membrane in a sandwich configuration on the negative electrode side (black side) of the transfer clamp in the following order: wetted sponge pad - pre-equilibrated filter paper - SDS-PAGE gel to terminate electrophoresis - activated PVDF membrane with the contact side facing the positive electrode - second layer of filter paper - positive electrode side sponge pad (red side). Use a glass rod to roll between layers to remove air bubbles and ensure tight contact at the interface. Close the transfer clamp and inject 1× transfer buffer (0.025 M Tris, 0.192 M Glycine, 20% (v / v) methanol) into the transfer tank. Maintain a constant current of 300 mA for approximately 1 h.

[0078] Blocking: After the PVDF membrane was transferred, it was blocked at room temperature with 5% (w / v) skim emulsion-TTBS blocking solution (20 mM Tris-HCl / pH7.5, 150 mM NaCl, 0.1% (v / v) Tween-20) and continuously shaken at 60 rpm for 60 min to effectively block non-specific binding sites.

[0079] Primary antibody incubation: Immerse the blocked membrane in an incubation system containing primary antibody (rabbit / mouse IgG, diluted 1:1000 in 3% skim milk-TTBS) and incubate at room temperature for 2 h on a horizontal shaker (60 rpm) or at 4°C overnight (12-16 h) to complete antigen-antibody binding.

[0080] Secondary antibody incubation: After the primary antibody incubation, the membrane was washed using the TTBS washing system (3×5 min), and then immersed in the secondary antibody reaction solution (2% skim milk / TTBS, HRP-labeled secondary antibody 1:10,000) and incubated on a constant temperature shaker (60 rpm / 25℃) for 60 min to complete signal amplification.

[0081] Chemiluminescence detection: After final washing (TTBS, 3 × 5 min), the membrane was transferred to clean plastic wrap and 200 μL of ECL chemiluminescence substrate (Tiangen, catalog number: EC105) was evenly spread on it. The signal was acquired using a chemiluminescence imaging system (Celvin S 320) to detect the target protein.

[0082] Detection of wild type and hypk The abundance of HYPK protein in mutants can be observed. hypk-594 In the mutant, the HYPK protein is smaller than WT, and the protein band is located lower. hypk-54 HYPK protein was not detected in the mutant. Figure 3 This result is consistent with the sequencing results, validating... hypk Availability of mutants.

[0083] Example 3: Functional verification of HYPK in light-regulated plant seedling morphogenesis To investigate whether HYPK participates in photomorphogenesis, experiments were conducted in darkness and under various colors of light (red light RL, 10 μmol m). -2 s -1 Blue light BL, 14 μmol m -2 s -1 Far-red light (FR), 12 μmol m -2 s -1 White light WL, 50 μmol m -2 s -1 Observing the hypocotyl phenotype under these conditions, we found hypk The mutants were all significantly shorter than the wild-type Col-0, indicating the presence of a constitutive photomorphogenesis phenotype. Figure 4 Transplant the seedlings into soil and allow them to continue growing for one month. Observe the phenotype of the one-month-old seedlings; you can observe that the plants exhibit the same growth trend. hypk The mutant plants were all significantly shorter than the wild-type Col-0 ( Figure 5 ).

[0084] Hybridization experiment: Arabidopsis thaliana parental materials (in this experiment) cop1-4 and hypk-594The plants were cultured until they reached peak reproductive growth. Unopened or slightly open flower buds from the maternal plant were selected, and the perianth segments and stamens were removed using hybridization forceps, ensuring the pistil structure remained intact. Simultaneously, floral organs at the early flowering stage were collected from the paternal plant, and pollen from the mature anthers was evenly applied to the stigma of the maternal plant. After artificial pollination, the inflorescences were labeled. Once the siliques matured, the hybrid seeds were collected to obtain the F1 generation. The F1 seeds were sown on MS medium for propagation and subsequent molecular identification and phenotypic screening analysis.

[0085] Protein abundance of HY5 and HYPK was detected in 5-day-old plant seedlings grown under full sun conditions. It was observed that, compared with the wild type, the HYPK protein band was significantly higher. hypk-594 and hypk-594 cop1-4 The smaller-than-average size indicates that the hybridization was successful and the double mutant is usable; meanwhile, compared with the wild type, HY5... hypk-594 There is some accumulation in mutants. cop1-4 The accumulation of mutants is enhanced, while hypk-594 cop1-4 This accumulation is further enhanced in the double mutant, indicating that HYPK can promote the degradation of HY5 protein, a classical transcription factor substrate for light signals. Figure 6 ).

[0086] In summary, this invention discloses the application of Arabidopsis protein HYPK in constitutive photomorphogenesis in plants. Firstly, the constructed HYPK protein was identified through HYPK antibody detection. hypk Availability of mutants. Through observation... hypk The mutant phenotype confirmed that HYPK is involved in regulating photomorphogenesis in plants. In vivo experiments further confirmed that HYPK can promote the degradation of the classical light signaling substrate HY5 protein.

[0087] This technology has discovered a novel regulatory component, HYPK, in the light signaling pathway and developed a novel molecular tool based on HYPK to regulate light signal transduction. This tool provides an important genetic resource basis for breeding new crop varieties with high-density planting tolerance and lodging resistance using molecular genetic breeding techniques.

[0088] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

[0089] References Cai, L., Hao, B., Xu, Z., Cui, S., Wu, Q., Lee, J., . . . Wan, J.(2025). ELD1 mediates photoperiodic flowering via OsCCA1 alternative splicingand interacts with phytochrome signaling in rice. Nat Commun, 16 (1), 5329.doi:10.1038 / s41467-025-60839-6 Shi, Q., Xia, Y., Wang, Q., Lv, K., Yang, H., Cui, L., . . . Li, G.(2024). Phytochrome B interacts with LIGULELESS1 to control plantarchitecture and density tolerance in maize. Mol Plant, 17 (8), 1255-1271. doi:10.1016 / j.molp.2024.06.014 Wang, Z., Wang, W., Zhao, D., Song, Y., Lin, X., Shen, M., . . .Wang, J. (2024). Light-induced remodeling of phytochrome B enables signaltransduction by phytochrome-interacting factor. Cell, 187 (22), 6235-6250.e6219. doi:10.1016 / j.cell.2024.09.005。

Claims

1. HYPK The application of genes in constitutive photomorphogenesis and / or plant architecture improvement in plants is characterized by, Reduce / inactivate plants HYPK Gene expression.

2. Application of reducing / inactivating HYPK gene expression in improving constitutive photomorphogenesis and / or plant architecture in plants.

3. Reduce / inactivate in plants HYPK Application of recombinant vectors for gene expression in constitutive photomorphogenesis and / or plant architecture improvement in plants.

4. The application of a recombinant engineered bacterium in the formation of constitutive photomorphogenesis and / or improvement of plant architecture, characterized in that, The recombinant engineered bacteria contain substances that reduce / inactivate plants. HYPK The recombinant vector for gene expression, preferably, is Agrobacterium.

5. A method for improving the constitutive photomorphogenesis and / or plant architecture of plants, characterized in that, The method includes: introducing into the plant a substance that reduces / inactivates the plant. HYPK A recombinant vector for gene expression or a recombinant engineered bacterium containing the recombinant vector.

6. A method for obtaining plants with improved traits, characterized in that, The method includes: introducing into the plant a substance that reduces / inactivates the plant. HYPK A recombinant vector for gene expression or a recombinant engineered bacterium containing the recombinant vector.

7. The application according to any one of claims 1-4 or the method according to claim 5 or 6, characterized in that, The application or method includes the following steps: (1) Design HYPK The target sequence of the gene is used to construct a CRISPR-Cas9 vector. Preferably, the target sequence is as shown in SEQ ID NO:

3. (2) The vector obtained in step (1) is transferred into engineered bacteria; and (3) Infect the callus or seeds with the engineered bacteria obtained in step (2).

8. The application according to any one of claims 1-4 or the method according to claim 5 or 6, characterized in that, The plant in question is Arabidopsis thaliana.

9. The application or method according to claim 8, characterized in that, The HYPK The amino acid sequence encoded by the gene is shown in SEQ ID NO:

1.

10. The application or method according to claim 8, characterized in that, The HYPK The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 2.

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