Luciferase variants and uses thereof

Abstract

This invention relates to a luciferase variant having a 35-80 amino acid deletion near the N-terminus of the sequence shown in SEQ ID NO.:1; preferably, the deletion is 35-40, 40-45, or 55-60 amino acids near the N-terminus of the sequence shown in SEQ ID NO.:1; the luciferase variant is configured to increase the luminescence intensity or caffeic acid content of an organism or its tissues or cells. This invention solves the problems of low luminescence efficiency and poor signal stability of the FBP system in organisms. This application systematically designs N-terminal truncated LUZ sequences of different lengths to screen for mutants that improve the luminescence effect of organisms. These mutants can be combined with other elements to further enhance the luminescence intensity of organisms.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a luciferase variant and its applications. Background Technology

[0002] FBP luminescence systems are a type of bioluminescent detection platform based on the reaction between luciferase and substrate, and are widely used in reporter gene analysis, protein-protein interaction studies, and pathogen detection. However, wild-type luciferase (LUZ) in FBP systems suffers from drawbacks such as low luminescence efficiency and rapid signal decay, which limits its application in high-sensitivity detection.

[0003] The N-terminal region of luciferase typically contains protein folding regulatory sequences and potential inhibitory domains. Truncation of this region can effectively eliminate steric hindrance and enhance enzyme-substrate binding efficiency, thereby improving luminescence performance. Currently, systematic research on truncation modifications of the luz sequence is lacking, and the impact of different truncation lengths on the luminescence performance of the FBP system remains unclear. Summary of the Invention

[0004] To address the technical problems existing in the prior art, the present invention proposes a luciferase variant whose amino acid sequence has a deletion of 35-80 amino acids near the N-terminus relative to the sequence shown in SEQ ID NO.:1; preferably, its amino acid sequence has a deletion of 35-40, 40-45, or 55-60 amino acids near the N-terminus relative to the sequence shown in SEQ ID NO.:1; the luciferase variant is configured to increase the luminescence intensity or caffeic acid content of an organism or its tissues or cells.

[0005] The luciferase variant shown above has a deletion of 37, 42, or 58 amino acids near the N-terminus of the sequence shown in SEQ ID NO.:1.

[0006] The luciferase variants described above further include one or more point mutations in their amino acid sequences.

[0007] The luciferase variants described above, wherein the amino acid sequence of the luciferase variant is as shown in SEQ ID NO.:15, 17, or 23; or is at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.:15, 17, or 23; or the nucleotide sequence of the luciferase variant is as shown in SEQ ID NO.:16, 18, or 24; or the translated amino acid sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.:16, 18, or 24.

[0008] A luminescence-enhancing carrier comprising any of the luciferase variants described above.

[0009] An engineered bacterium for gene transformation of organisms, comprising any of the luciferase variants described above, or a luminescence-enhancing vector as described above.

[0010] Applications of any of the luciferase variants described above, or the luminescence enhancement carriers described above, or the engineered bacteria described above, in increasing the biosynthetic content of caffeic acid in an organism or its tissues or cells, or in increasing the luminescence intensity of an organism or its tissues or cells.

[0011] In the applications described above, the organism is selected from plants, animals, cells, or fungi.

[0012] A reagent or kit for increasing the biosynthetic content of caffeic acid or increasing the luminescence intensity of an organism or its tissues or cells, comprising any of the recombinant constructs described above, or the luminescence-enhancing carriers described above, or the engineered bacteria described above.

[0013] A method for enhancing caffeic acid biosynthesis or increasing the luminescence intensity of an organism or its tissues or cells, comprising: obtaining a recombinant construct as described above; and The recombinant construct is transformed into a specified tissue or cell of an organism.

[0014] A self-luminescent organism or its tissue or cell that can express any of the recombinant constructs described above, or the luminescence-enhancing vectors described above, or specific genes in engineered bacteria as described above.

[0015] A method for preparing a self-luminescent organism or its tissues or cells includes: transforming any of the recombinant constructs described above, or the luminescence-enhancing carriers described above, or the engineered bacteria described above into specific tissues or cells of the organism.

[0016] A material kit for preparing self-luminescent organisms or cells, comprising any of the recombinant constructs described above, or the luminescence-enhancing carriers described above, or the engineered bacteria described above.

[0017] This invention addresses the problems of low luminescence efficiency and poor signal stability of FBP systems in organisms. This application systematically designs N-terminal truncated LUZ sequences of different lengths to screen for mutants that improve luminescence performance in organisms. These mutants can be combined with other components to further enhance the luminescence intensity of organisms. Attached Figure Description

[0018] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein: Figure 1 This is a schematic diagram of an FBP-LUN carrier according to an embodiment of the present invention, wherein the LUNs have different lengths; Figure 2 This is a statistical graph of the luminescence intensity of plants after transformation with fluorescent factor groups containing different lengths of LUZ according to an embodiment of the present invention; Figure 3 These are bioluminescent images of plants transformed with fluorescent factor groups containing different lengths of LUZ according to an embodiment of the present invention; and Figure 4 This is a statistical chart of caffeic acid content in plants after transformation with fluorescent factor groups containing different lengths of LUZ, according to an embodiment of the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] In the following detailed description, reference can be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application have been described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that other embodiments or modifications to the embodiments of the present application may also be utilized.

[0021] The proper nouns mentioned in this article have the following meanings: For nucleotides, the term "homology" as used herein refers to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined using conventional methods such as NCBI Blast v2.0 to calculate the degree of sequence identity between two or more nucleotide sequences. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity are referenced in WO04 / 037999, EP0967284, EP1085089, WO00 / 55318, WO00 / 78972, WO98 / 49185, and GB2357768-A, etc.

[0022] For amino acids, the term "homology" as used herein describes or compares the degree of amino acid similarity between two or more polypeptides or their specified sequences at optimal alignment and comparison. The homology % between two sequences varies with the number of common positions shared by these sequences at optimal alignment (i.e., homology % = (number of common positions / total number of positions) × 100)%, where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy.

[0023] In this application, without substantially affecting antibody activity (retaining at least 95% activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain variants of the antibody or its functional fragment sequence. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequence described in this application can have at least 80% identity (or homology) with the reference sequence, meaning at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with each reference sequence. The sequence consistency described in this application can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences mentioned in this application are shown from the N-terminus to the C-terminus.

[0024] Unless otherwise specified, all bioluminescent plants mentioned in this application are transgenic plants containing the fungal FBP bioluminescence system. This application does not specifically limit the types of plants, including, but is not limited to, *Nicotiana benthamiana*, *Arabidopsis thaliana*, tobacco, chrysanthemum, marigold, cosmos, zinnia, coreopsis, echinacea, *Symplocos davidii*, silverleaf daisy, *Age of Osmanthus fragrans*, *Mallotus patulum*, *Tetrapanax papyriferus*, daisy, *Aster tataricus*, *Cinnamomum cassia*, *Salvia splendens*, sage, lavender, rosemary, mint, patchouli, coleus, rose, rose, tulip, hyacinth, daylily, hosta, spider plant, asparagus fern, aloe vera, *Liriope muscari*, *Gnaphalium affine*, kale, violet, *Orychophragmus violaceus*, carnation, baby's breath, tall snow lily, dwarf snow lily, jasmine, coral bean, ornamental pepper, tobacco flower, *Cercis chinensis*, wisteria, lupin, clover, iris, German iris, butterfly flower, and *Iris tectorum*. Anthurium, Peace Lily, Monstera deliciosa, Epipremnum aureum, Alocasia macrorrhiza, Calla Lily, Tomato, Chili Pepper, Eggplant, Pumpkin, Rapeseed, Colored Chili Pepper, Colored Rapeseed, Cabbage, Broccoli, Cauliflower, Cotton, Osmanthus, Jasmine, Lilac, Forsythia suspensa, Jasminum nudiflorum, Forsythia suspensa, Poinsettia, Crown of Thorns, Croton, Jade Plant, Kalanchoe blossfeldiana, Echeveria, Echeveria elegans, Sedum lineare, Cactus, Gymnocalycium mihanovichii, Epiphyllum oxypetalum, Christmas Cactus, Tradescantia zebrina, Tradescantia zebrina, Commelina communis, Amaryllis, Cyclamen, Gloxinia, Oxalis, Bellflower, Lobelia chinensis, Verbena officinalis, Geranium, Nasturtium, Gazania latifolia, Marguerite daisy, Bleeding Heart, Foxglove, Ranunculus asiaticus, Columbine, Plumbago aurantium, Vinca minor, Bells dwarf, and Alyssum fusiforme, etc.

[0025] For the purposes of this invention, the terms "luminescence" and "bioluminescence" are used interchangeably and refer to the luminescence phenomenon during a chemical reaction catalyzed by luciferase.

[0026] The term "fluorescent factor set" refers to a combination of factors that, when introduced into a somatic cell, integrate and express, induce autofluorescence in the cell. In some embodiments, the fluorescent factor set is the FBP system or its variations. The FBP system includes genes such as milkweed alkaloid synthase, milkweed hydroxylase, luciferase, and caffeoylpyruvate hydrolase. Variations include replacing milkweed alkaloid synthase with polyketide synthase; mutants of each gene; and homologous gene substitution.

[0027] The term "organism" refers to a living organism with cellular activity that can absorb foreign genes and express them in its cells. In some embodiments, an organism is a plant, animal, bacteria, fungus, etc.

[0028] The term "milk alkaloid hydroxylase (H3H)" is used herein to describe an enzyme that catalyzes the conversion of proluciferin to fungal luciferin, for example, the synthesis of 3-hydroxymilk alkaloid from milk alkaloid. According to one embodiment of this application, other enzymes that catalyze the conversion of proluciferin to fungal luciferin may also be used in this application, and this application does not limit the specific enzyme used.

[0029] The terms "milk alkaloid synthase (Hisps)" or "polyketide synthase (PKS)" are used herein to describe enzymes capable of catalyzing the synthesis of fungal proluciferin from precursors of proluciferin, such as the synthesis of milk alkaloids from caffeic acid. According to one embodiment of this application, other enzymes used to describe enzymes capable of catalyzing the synthesis of fungal proluciferin from precursors of proluciferin may also be used in this application, and are not limited to milk alkaloid synthase or polyketide synthase.

[0030] The term "caffeoylpyruvate hydrolase (CPH)" is used herein to describe an enzyme capable of catalyzing the oxidation of luciferin by fungi into simpler compounds, such as precursors for the formation of proluciferin. For example, it can catalyze the conversion of caffeoylpyruvate to caffeic acid.

[0031] In this application, unless otherwise specified, the fungal bioluminescent system (FBP) relies on a series of enzymatic reactions to convert common plant metabolites into luminescent compounds. This system is encoded by a fungal bioluminescent gene cluster, including: polyketide synthase (KPS), hispidin-3-hydroxylase (H3H), luciferase (Luz), and caffeoyl pyruvate hydrolase (CPH).

[0032] Unless otherwise specified in this application, the amino acid sequence of LUZ is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO.:1.

[0033] In some embodiments, the amino acid sequence of LUZ is selected from one or more of the following groups: the sequence shown in SEQ ID NO.:1 with 1-100 amino acids deleted near the N-terminus; preferably, 8-70 amino acids deleted; more preferably, 20-65 amino acids deleted; more preferably, 35-60 amino acids deleted; more preferably, 35-55 amino acids deleted; more preferably, 35-50 amino acids deleted; more preferably, 35-45 amino acids deleted; more preferably, 35-40 amino acids deleted.

[0034] In some embodiments, the amino acid sequence of LUZ is selected from one or more of the sequences shown in SEQ ID NO.:5, 7, 9, 11, 13, 15, 17, 19, 21, 23; or it is selected from the amino acid sequences obtained by modifying, deleting, replacing, or truncating the sequences shown in SEQ ID NO.:5, 7, 9, 11, 13, 15, 17, 19, 21, 23.

[0035] In some embodiments, the nucleotide sequence of LUZ is selected from one or more of the sequences shown in SEQ ID NO.:6, 8, 10, 12, 14, 16, 18, 20, 22, 24.

[0036] The term "transformation" refers to the process of introducing heterologous nucleic acids into a host cell or organism. In particular, "transformation" refers to the stable or transient integration of DNA molecules into the genome of a target organism.

[0037] The term "recombination" refers to a host organism, such as bacteria, plants, fungi, or animals, modified by introducing a heterologous nucleic acid molecule. This nucleic acid molecule can be stably integrated into the host genome or exist as an extrachromosomal molecule. This extrachromosomal molecule is capable of self-replication. It should be understood that transgenic or stably transformed cells, tissues, or organisms include both the end products of the transformation process and the transgenic progeny.

[0038] The term "consumables" in this article refers to consumables used in experimental procedures, such as disposable pipettes, syringes, instruments related to gene transformation of organisms, and reagents used for gene transformation.

[0039] This application relates to a luciferase variant having an amino acid sequence that has a deletion of 35-80 amino acids near the N-terminus relative to the sequence shown in SEQ ID NO.:1; preferably, the amino acid sequence has a deletion of 35-40, 40-45, or 55-60 amino acids near the N-terminus relative to the sequence shown in SEQ ID NO.:1; the luciferase variant is configured to increase the luminescence intensity or caffeic acid content of an organism or its tissues or cells.

[0040] In some embodiments, the amino acid sequence of the luciferase variant has a deletion of 37, 42, or 58 amino acids near the N-terminus of the sequence shown in SEQ ID NO.:1.

[0041] In some embodiments, the amino acid sequence of the luciferase variant further includes one or more point mutations.

[0042] In some embodiments, the amino acid sequence of the luciferase variant is as shown in SEQ ID NO.:15, 17, or 23; or is at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.:15, 17, or 23.

[0043] In some embodiments, the nucleotide sequence of the luciferase variant is as shown in SEQ ID NO.:16, 18, or 24; or the translated amino acid sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.:16, 18, or 24.

[0044] This application relates to a luminescence-enhancing carrier comprising the aforementioned luciferase variant.

[0045] This application relates to an engineered bacterium for gene transformation of organisms, comprising the aforementioned luciferase variant or the aforementioned luminescence-enhancing vector.

[0046] The aforementioned luciferase variants, or the aforementioned luminescence-enhancing carriers, or the aforementioned engineered bacteria, are used to increase the biosynthetic content of caffeic acid in organisms or their tissues or cells, or to increase the luminescence intensity of organisms or their tissues or cells.

[0047] In some embodiments, the organism is selected from plants, animals, cells, or fungi.

[0048] This application relates to a reagent or kit for increasing the biosynthetic content of caffeic acid or increasing the luminescence intensity of an organism or its tissues or cells, including the aforementioned recombinant construct, or the aforementioned luminescence-enhancing carrier, or the aforementioned engineered bacteria.

[0049] This application relates to a method for improving the biosynthesis of caffeic acid or increasing the luminescence intensity of an organism or its tissues or cells, comprising: obtaining the aforementioned recombinant construct; and converting the recombinant construct into a specified tissue or cell of an organism.

[0050] This application relates to a self-luminescent organism or its tissue or cells, which can express the aforementioned recombinant construct, or the aforementioned luminescence-enhancing vector, or a specific gene in the aforementioned engineered bacteria.

[0051] This application relates to a method for preparing a self-luminescent organism or its tissues or cells, comprising: transforming the aforementioned recombinant construct, or the aforementioned luminescence-enhancing carrier, or the aforementioned engineered bacteria into a specific tissue or cell of the organism.

[0052] This application relates to a material box for preparing self-luminescent organisms or cells, which includes the aforementioned recombinant construct, or the aforementioned luminescence-enhancing carrier, or the aforementioned engineered bacteria.

[0053] Systematic design and synthesis of truncated LUZ sequences: Using the wild-type LUZ gene as a template, I was mutated to T at position 103 and L to K at position 167, resulting in LUZ-v2. A gradient truncated mutant library covering amino acids 1-60 from the N-terminus was designed, containing 10 truncated lengths: LUZΔN6-v2, LUZΔN10-v2, LUZΔN23-v2, LUZΔN29-v2, LUZΔN33-v2, LUZΔN37-v2, LUZΔN42-v2, LUZΔN47-v2, LUZΔN54-v2, and LUZΔN58-v2.

[0054] Specific primers were designed for each truncated fragment, and AsiI and AgeI restriction sites were introduced. The target fragment was obtained by high-fidelity PCR amplification.

[0055] The amplified product was cloned into the pMD19-T vector, and the sequence accuracy was verified by Sanger sequencing. A standard plasmid library of the truncated luz gene was constructed.

[0056] Precise construction of recombinant expression vectors: A modular cloning strategy was employed to directionally insert each truncated luz fragment into the FBP system backbone vector: The backbone vector and standard plasmid were digested with AsiI and AgeI, and the linear vector backbone and truncated luz fragment were recovered. Ligation was performed using T4 DNA ligase at 16°C for 12 hours, followed by transformation into *E. coli* DH5α competent cells. The recombinant expression plasmid was obtained after HygR resistance selection, colony PCR, and enzyme digestion verification.

[0057] FBP luminous intensity performance testing: Experimental system: 50 ng of purified truncated luz proteins, 100 μM luciferin substrate and 2 mM ATP were added to a 200 μL reaction system, and the luminescence intensity (RLU) was detected at 37 °C.

[0058] The results showed that the luminescence intensity of the ΔN37-luz mutant was 3.47 times that of the wild type, which was significantly better than other truncated mutants, making it the best-performing mutant.

[0059] Beneficial effects: Performance breakthrough: The ΔN37-luz mutant significantly improves the luminescence intensity of the FBP system.

[0060] High-sensitivity detection: The optimized FBP system can achieve highly sensitive quantitative detection of caffeic acid, with a wide linear range and low detection limit.

[0061] High application value: This system can be directly applied to the quantitative analysis of caffeic acid in food and medicine, providing an efficient detection tool for related fields.

[0062] The embodiments of this application will be described below through specific examples. Those skilled in the art should understand that the following examples are only for illustrating the technical solutions of this application, and are not intended to limit the technical solutions of this application.

[0063] Example 1: Construction of FBP luminescent carrier Materials Preparation: The vector backbone pCAMBIA1300, Agrobacterium strain GV3101, and Escherichia coli DH5α competent cells were all purchased commercially. The CPH, H3H, and PKS genes were obtained through gene synthesis to ensure sequence accuracy. Different combinations of optimized CPH, H3H, Hisps, and PKS genes with LUZ gene variants resulted in consistent effects on luminescence intensity; therefore, this application does not impose any limitations on the sequences of the CPH, H3H, and PKS genes. In some embodiments, the sequences of the CPH, H3H, and PKS genes involved in this application are referenced in patent application CN119082071A.

[0064] Construction of recombinant intermediate plasmids: CPH, H3H, and PKS gene fragments were assembled into the corresponding multiple cloning sites of the pCAMBIA1300 vector using Gibson Assembly, transformed into DH5α competent cells, plated on LB plates containing 50 mg / L Kan + 50 mg / L LRif, cultured at 28°C for 2 days, and positive clones were picked for sequencing verification to obtain the recombinant intermediate plasmids corresponding to each combination.

[0065] Luminescent vector assembly: Seamless cloning primers were designed to amplify the expression cassette regions (containing the CaMV 35S promoter, gene fragments, and NOS terminator) of the CPH, H3H, and PKS genes; the pCAMBIA1300 vector was digested with restriction endonucleases such as HindIII and EcoRI to remove redundant sequences; homologous recombination technology was used to assemble the above four expression cassette fragments into the digested vector backbone, which was then transformed into DH5α competent cells. Positive clones were screened, and after sequencing verification, a preliminary luminescent vector was obtained.

[0066] Example 2: Synthesis of truncated luz sequences The CPH gene, H3H gene, PKS gene, and LUZ coding genes of different lengths were obtained. The fluorescent factor genome genes were tandemly linked with the LUZ gene to obtain LUZ tandem sequences of different lengths. The lengths of the LUZ sequences were determined by deletions of 6, 10, 23, 29, 33, 37, 42, 47, 54, and 58 amino acids near the N-terminus of the LUZ amino acid sequence.

[0067] The LUZ sequences of different lengths are shown in Table 1: Table 1. LUZ sequences of different lengths

[0068] Example 3 Construction of the luminescence enhancement carrier 1. Material Preparation The vector backbone pCAMBIA1300, Agrobacterium strain GV3101, and Escherichia coli DH5α competent cells were purchased from commercial sources. The coding sequences of LUZ, CPH, H3H, and PKS genes of different lengths were obtained through gene synthesis, and the sequences were validated to ensure accuracy. The homologous recombination kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. The restriction endonuclease HindIII, EcoRI, and PCR-related reagents were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0069] Figure 1 This is a schematic diagram of an FBP-LUN carrier according to an embodiment of the present invention, wherein the LUN has different lengths.

[0070] Example 4: Agrobacterium-mediated transformation and transient plant transformation Agrobacterium transformation: The sequenced recombinant vectors were transformed into Agrobacterium GV3101 competent cells by freeze-thaw method. After transformation, the cells were plated on LB plates containing rifampicin (50 mg / L) and kanamycin (50 mg / L) and incubated upside down at 28°C for 48 h. Single colonies were picked and transferred to LB liquid medium containing the corresponding antibiotics to obtain seed culture.

[0071] Agrobacterium expansion culture: Inoculate the seed culture into fresh LB liquid medium (containing the same antibiotic) at a volume ratio of 1:100, and collect the bacterial cells by centrifugation at 5000 rpm for 10 min.

[0072] Preparation of infection solution: Resuspend the bacterial cells in infection buffer and adjust OD. 600 =0.6-0.8, incubate in the dark at 28℃ for 2-3 h.

[0073] Plant infection: Select healthy, disease-free tobacco plants that have grown for 4-6 weeks and have robust leaves. Immerse the leaves in the infection solution, ensuring that both the front and back of the leaves are in contact with the infection solution.

[0074] Co-culture and incubation: After infection, the leaves were blotted dry with sterile filter paper to remove residual liquid on the surface, and then laid flat in a petri dish lined with wet filter paper. They were co-cultured at 25°C in the dark for 2-3 days.

[0075] Example 5: Luminescence Detection After the plants were cultured to the target stage, the LightScout full-spectrum quantum meter (Shanghai Zequan Technology) was used in a dark environment. The detection port was placed close to the leaf of the plant to be tested to detect the amount of light quantum accumulation at different times (shooting conditions: Huawei P30 Pro, ISO 6400, time-lapse photography 2s, aperture 1.6 mm).

[0076] Figure 2 This is a statistical graph of the luminescence intensity of plants after transformation with fluorescent factor groups containing different lengths of LUZ according to an embodiment of the present invention; Figure 3 This is a photograph of a plant transformed with fluorescent factor groups containing different lengths of LUZ according to an embodiment of the present invention. Figures 2-3 As shown, when there are 37, 42, and 58 deletions near the N end of LUZ, the leaf brightness of Nicotiana benthamiana is significantly improved.

[0077] Example 6: Detection of Caffeic Acid Content Sample preparation: Leaves of plants and transiently transformed plants were collected and immediately frozen in liquid nitrogen and ground. About 1 g of frozen tissue was placed in a 50 ml centrifuge tube and freeze-dried. The freeze-dried material was stored at -20℃. Each group of samples was prepared and analyzed three times.

[0078] Extraction and filtration: Weigh approximately 50 mg of lyophilized powder and filter it with 7 ml of 70% methanol solution using a Phenex GF / PVDF syringe filter (30 mm in diameter, 0.45 μm in pore size).

[0079] LCMS Analysis: Analysis was performed using a Shimadzu 8030 system (HPLC-DAD-ESI-TQ MS). The column was a Discovery C18 column (4.6 × 150 mm, 5 μm). Mobile phase A was 0.3% acetic acid aqueous solution (v / v), and mobile phase B was acetonitrile. Gradient run: 0–4 min 10–40% B, 4–5 min 40–80% B, 5–10.5 min 100% B isocratic elution, followed by restoration to initial conditions. Column temperature: 40℃, flow rate: 1 ml / min, injection volume: 20 μl.

[0080] Figure 4 This is a statistical graph showing the caffeic acid content in plants transformed with fluorescent factor groups containing different lengths of LUZ, according to an embodiment of the present invention. Figure 4 As shown, when there are 37, 42, and 58 deletions near the N-terminus of LUZ, the caffeic acid content in the leaves of Nicotiana benthamiana is significantly increased.

[0081] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. A luciferase variant having a deletion of 35-80 amino acids near the N-terminus relative to the sequence shown in SEQ ID NO.:1; preferably, having a deletion of 35-40, 40-45, or 55-60 amino acids near the N-terminus relative to the sequence shown in SEQ ID NO.:

1. The luciferase variant is configured to increase the luminescence intensity or caffeic acid content of an organism or its tissues or cells.

2. The luciferase variant according to claim 1, wherein the amino acid sequence has a deletion of 37, 42, or 58 amino acids near the N-terminus of the sequence shown in SEQ ID NO.:

1.

3. The luciferase variant according to claim 1, wherein the amino acid sequence further comprises one or more point mutations.

4. The luciferase variant according to claim 1, wherein, The amino acid sequence of the luciferase variant is as shown in SEQ ID NO.:15, 17, or 23; or is at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.:15, 17, or 23; or The nucleotide sequence of the luciferase variant is as shown in SEQ ID NO.:16, 18, or 24; or the translated amino acid sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO.:16, 18, or 24.

5. The use of the luciferase variant as described in any one of claims 1-4 in increasing the biosynthetic content of caffeic acid in an organism or its tissues or cells, or in increasing the luminescence intensity of an organism or its tissues or cells.

6. A reagent or kit for increasing the biosynthetic content of caffeic acid or increasing the luminescence intensity of an organism or its tissues or cells, comprising the recombinant construct as described in any one of claims 1-4.

7. A method for improving caffeic acid biosynthesis or increasing the luminescence intensity of an organism or its tissues or cells, comprising: Obtain the recombinant construct as described in any one of claims 1-4; as well as The recombinant construct is transformed into a specified tissue or cell of an organism.

8. A self-luminescent organism or its tissue or cells, which can express a specific gene in the recombinant construct as described in any one of claims 1-4.

9. A method for preparing a self-luminous organism or its tissues or cells, comprising: Transform the recombinant construct as described in any one of claims 1-4 into a specific tissue or cell of an organism.

10. A material box for preparing self-luminous organisms or cells, comprising the recombinant construct as described in any one of claims 1-4.

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