Preparation method of fluorescence-enhanced self-luminous plant

Abstract

The present invention relates to a recombinant construct comprising: a first expression cassette comprising a fluorescence enhancing factor; wherein the amino acid sequence of the fluorescence enhancement factor is as shown in SEQ ID NO: 2, or an amino acid sequence which at least has 60%, 65%, 70%, 75%, 80%, 85% or 90% identity with the SEQ ID NO: 2 and is functionally equivalent to the protein as shown in the SEQ ID NO: 2; the first expression cassette is configured to increase the self-luminous intensity of a plant, plant tissue, or cell. According to the application, the LumEnhancer gene is further introduced on the basis of an FBP biological self-luminous system, so that the physiological activity of transgenic plant cells is effectively improved, and the energy metabolism level of the plant is further enhanced, thereby providing a more sufficient energy basis for a bioluminescence reaction and effectively enhancing the luminous intensity of the whole plant.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and in particular to a method for preparing a fluorescence-enhanced self-luminescent plant. Background Technology

[0002] Phytoluminescence technology originated from the observation and exploration of bioluminescence phenomena in nature. Early methods involved the exogenous addition of luciferin and fluorescent proteins, but this required a continuous supply of external chemicals, thus limiting its application. Currently, the luminescence intensity of plants is low; under camera lenses, long exposures are needed to obtain photographs of visible luminescent plants; and under naked-eye conditions, the human eye needs to adapt to darkness before it can observe the faint luminescent phenotypes of plants. Therefore, how to improve the luminescence intensity of plants has become an urgent problem to be solved. Summary of the Invention

[0003] To address the technical problems existing in the prior art, this invention proposes a recombinant construct, comprising: A first expression cassette includes a fluorescence enhancer; wherein the amino acid sequence of the fluorescence enhancer is as shown in SEQ ID NO:2, or has at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity with SEQ ID NO:2, and is functionally equivalent to the amino acid sequence of the protein shown in SEQ ID NO:2; the first expression cassette is configured to enhance the autoluminescence intensity of plants, plant tissues, or cells.

[0004] The recombinant construct described above further includes a fluorescence enhancement factor whose nucleotide sequence is as shown in SEQ ID NO:1; or which has 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity with the protein encoded by the nucleotide sequence shown in SEQ ID NO:1, and is functionally equivalent to the protein encoded by the nucleotide sequence shown in SEQ ID NO:1.

[0005] The recombinant construct as described above further includes: a first promoter that is functional in plants and operatively linked to a fluorescence enhancer.

[0006] The recombinant construct as described above further includes: a second expression cassette comprising a set of fluorescent factors configured to enable the plant to emit light; the set of fluorescent factors comprising: milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene, or alternatives to one or more of these genes; the second expression cassette being configured to enable the plant, plant tissue or cell to emit light.

[0007] The recombinant construct described above, wherein the fluorescent factor group comprises: a combination of milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene, and caffeoylpyruvate hydrolase gene; or a combination of grape polyketide synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene, and caffeoylpyruvate hydrolase gene; or a combination of milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene, caffeoylpyruvate hydrolase gene, TAL gene, and P450 gene.

[0008] The recombinant construct as described above further includes: a second promoter that is functional in plants and operatively linked to each gene in the fluorescent factor group; the first promoter may be the same as or different from the second promoter.

[0009] In the recombinant construct described above, the first promoter and the second promoter are CaMV 35S promoters.

[0010] A carrier comprising any of the recombinant constructs described above.

[0011] The vectors described above, each of which includes one or more recombinant constructs.

[0012] A microorganism comprising any of the recombinant constructs described above, or comprising the vectors described above.

[0013] A fusion protein, which is a recombinant construct as described above, or a vector as described above, or obtained by microbial expression as described above.

[0014] The use of any of the recombinant constructs described above, or the vectors described above, or the microorganisms described above, or the fusion proteins described above in the preparation of self-luminescent plants, self-luminescent plant tissues, or self-luminescent cells.

[0015] As described above, the plant is: algae, bryophytes, ferns, gymnosperms, or angiosperms.

[0016] The plants used in the above-described applications are selected from the group consisting of: Nicotiana benthamiana, Arabidopsis thaliana, tobacco, chrysanthemum, marigold, cosmos, zinnia, coreopsis, echinacea, Shasta daisy, silverleaf chrysanthemum, ageratum, marigold, white chrysanthemum, daisy, asters, cineraria, salvia splendens, sage, lavender, rosemary, mint, patchouli, coleus, rose, rose, tulip, hyacinth, daylily, hosta, spider plant, asparagus fern, aloe vera, liriope, purslane, kale, violet, honesty, carnation, carnation, baby's breath, tall snow lily, dwarf snow lily, jasmine, coral bean, ornamental pepper, tobacco flower, Bauhinia, wisteria, lupin, clover, iris, German iris, butterfly flower, horse chestnut. Rush, 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, Million Bells, and Sweet Alyssum.

[0017] A fluorescence-enhanced, self-luminescent plant tissue or cell whose genome includes: any of the recombinant constructs described above, or containing the vector described above, or the fusion protein described above.

[0018] A method for preparing a fluorescence-enhanced, self-luminescent plant, plant tissue, or cell, comprising: The transfer of a recombinant construct comprising any of the above-described recombinant constructs, or a vector as described above, or a microorganism as described above, or a fusion protein as described above, into a plant, plant tissue, or cell.

[0019] The application of fluorescence enhancers in enhancing the fluorescence of self-luminous plants, plant tissues or cells; wherein the amino acid sequence of the fluorescence enhancer is as shown in SEQ ID NO:2, or has at least 60%, 65%, 70%, 75%, 80%, 85% or 90% identity with SEQ ID NO:2, and is functionally equivalent to the amino acid sequence of the protein shown in SEQ ID NO:2.

[0020] The use of any of the recombinant constructs described above, or the vectors described above, or the microorganisms described above, or the fusion proteins described above, in enhancing the fluorescence of self-luminescent plants, self-luminescent plant tissues, or self-luminescent cells.

[0021] In addition, the FBP light-emitting system incorporates LumSelf-luminescent plants obtained through genetic modification can be photographed visually using a mobile phone or camera lens without long exposure; the bioluminescent phenotype of the plant can be easily observed with the naked eye. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Provided according to one embodiment of this application Lum A schematic diagram of the three-dimensional structure of a gene sequence; Figure 2 DNA map of the pCAMBIA2300 vector provided according to one embodiment of this application; Figure 3 is a schematic diagram of the gene structure of a luminescent gene cluster according to an embodiment of this application. Figure 3A for Lum -Luminous gene clusters, Figure 3B For luminescent gene clusters; and Figure 4 is a schematic diagram of the T-DNA map of a luminescent gene cluster provided according to an embodiment of this application; wherein... Figure 4A For 2300- Lum -Luminescent gene cluster vector, Figure 4B It is a 2300-luminescent gene cluster vector. Detailed Implementation

[0024] 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.

[0025] 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 are 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.

[0026] The terms used in this article have the following meanings: The terms "first" and "second" used herein are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0027] The term "optionally" used in this document is for descriptive purposes only and should not be construed as indicating or implying relative importance. Therefore, a feature specified as "optionally" may explicitly or implicitly include or exclude that feature.

[0028] The terms "an embodiment," "some embodiments," "example," "specific example," or "some examples" used herein refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0029] For nucleotides, the terms "homology," "identity," or "similarity" used herein to describe amino acid or nucleic acid sequences relative to a reference sequence refer to the percentage of identical amino acids or nucleotides between two amino acid or nucleic acid sequences 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.

[0030] For amino acids, the terms “(substantial) homology,” “identity,” or “similarity” as used herein are used to describe or compare the degree of amino acid similarity between two or more polypeptides or their specified sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The homology % between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = (number of identical 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.

[0031] 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.

[0032] In this application, "protein functional equivalence" refers to proteins that have substantially the same biological function, even if they differ in amino acid sequence, species of origin, three-dimensional structure, or post-translational modifications. Such equivalent proteins can achieve the same or substantially the same functional performance as the target protein in a given biological system, such as catalyzing the same biochemical reactions, binding the same substrates or ligands, activating the same signaling pathways, or producing phenotypic effects consistent with the target protein in the same experimental model.

[0033] Unless otherwise specified, all self-luminescent plants mentioned in this application are transgenic plants containing the fungal FBP luminescence 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, Shasta daisy, silverleaf chrysanthemum, ageratum, marigold, white chrysanthemum, daisy, asters, cineraria, salvia splendens, sage, lavender, rosemary, mint, patchouli, coleus, rose, rose, tulip, hyacinth, daylily, hosta, spider plant, asparagus fern, aloe vera, liriope, *Gnaphalium affine*, kale, violet, *Orychophragmus violaceus*, carnation, baby's breath, tall snow lily, dwarf snow lily, jasmine, coral bean, ornamental pepper, tobacco flower, *Bauhinia purpurea*, 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.

[0034] For the purposes of this invention, the terms “luminescence,” “autoluminescence,” and “bioluminescence” are used interchangeably and refer to the luminescence phenomenon during a chemical reaction catalyzed by luciferase.

[0035] Unless otherwise specified, the "enhancing factor" or "fluorescence enhancing factor" mentioned in this article has the same meaning. It refers to a factor that can be specifically used to introduce exogenous FBP pathway systems to obtain luminescent plants, plant tissues or cells. This factor can further enhance the luminescence intensity of luminescent plants while maintaining plant growth, reproduction and cell activity.

[0036] In some embodiments, enhancers are DNA regulatory elements that enhance gene transcriptional activity over long distances. Enhancers are located far from the promoter, with some enhancers being up to 1 megabase (1 Mb) from the core promoter. Enhancers can be located upstream, downstream, or even within introns of a gene, and can function regardless of their orientation. Enhancers physically contact the promoter through "DNA circularization." When a specific protein binds to the enhancer, it pulls the DNA to form a circular structure, delivering the enhancer directly to the vicinity of the promoter.

[0037] In some embodiments, the enhancement factor referred to herein is LumEnhance r gene (or simply r gene) Lum(Gene) or the protein LUM encoded by that gene. As described in the examples herein, Lum The amino acid sequence of the gene is as shown in SEQ ID NO:2, or has at least 60%, 65%, 70%, 75%, 80%, 85% or 90% identity with SEQ ID NO:2, and is functionally equivalent to the protein shown in SEQ ID NO:2. Lum The nucleotide sequence of the gene is as shown in SEQ ID NO:1; or a nucleotide sequence that has 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity with the protein encoded by the nucleotide sequence shown in SEQ ID NO:1, and is functionally equivalent to the protein encoded by the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, lum The genes can be synthesized by the applicant according to the needs of the experiment. In other embodiments, lum The gene is directly cloned from the plant, and the protein sequence encoded by it has 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity with the nucleotide sequence shown in SEQ ID NO:2.

[0038] The term "expression cassette" as used herein refers to a modified fusion gene containing a promoter and a specified gene. An expression cassette enables the specified gene to be expressed in a specified cell under the drive of the promoter. In some embodiments, an expression cassette contains one specified gene. In some embodiments, multiple different expression cassettes can be simultaneously transfected into the same plant. In other embodiments, an expression cassette includes multiple specified genes.

[0039] For example, a first expression cassette includes a first promoter and a fluorescence enhancer; wherein the first expression cassette includes only one promoter and one fluorescence enhancer.

[0040] For example, a second expression cassette includes a second promoter and a set of fluorescent factors; wherein the set of fluorescent factors includes a milkweed synthase gene, a milkweed hydroxylase gene, a luciferase gene, and a caffeoylpyruvate hydrolase gene. In some embodiments, each set of fluorescent factors is preceded by a promoter.

[0041] The term "fluorescent factor group" refers to a combination of factors that, when introduced into plant cells, integrate and express, enable the cells to produce autofluorescence. In some embodiments, the fluorescent factor group is a known FBP pathway plant bioluminescence system genome, such as the FBP pathway mentioned in patent application CN119242698A, or a modification of that genome. In some embodiments, the fluorescent factor group genes mentioned in this application are stored in the applicant's laboratory.

[0042] In some embodiments, the fluorescent factor group includes a combination of the milkweed alkaloid synthase gene, the milkweed alkaloid hydroxylase gene, the luciferase gene, and the caffeoylpyruvate hydrolase gene. The sequences of the milkweed alkaloid synthase gene, the milkweed alkaloid hydroxylase gene, the luciferase gene, and the caffeoylpyruvate hydrolase gene can be referenced to the sequences disclosed in the patent applications for the milkweed alkaloid synthase gene, the milkweed alkaloid hydroxylase gene, the luciferase gene, and the caffeoylpyruvate hydrolase gene.

[0043] In other embodiments, the fluorescent factor group includes a combination of grape polyketide synthase gene, milk tree alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene, etc.

[0044] In other embodiments, the fluorescent factor group, in addition to the combination of milk tree alkaloid synthase gene, milk tree alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene, further includes caffeic acid synthesis pathways such as P450.

[0045] The term “milk alkaloid hydroxylase” is used herein to describe an enzyme that catalyzes the reaction of pre-luciferin to fungal luciferin, for example, the synthesis of 3-hydroxymilk alkaloid from milk alkaloid.

[0046] The term “milk alkaloid synthase” is used herein to describe an enzyme capable of catalyzing the synthesis of fungal proluciferin from precursors of proluciferin, such as milk alkaloid from caffeic acid.

[0047] The term "PKS" is used in this document to describe enzymes belonging to the group of type III polyketide synthases capable of catalyzing the synthesis of milk alkaloids from caffeoyl-CoA.

[0048] The term "caffeoylpyruvate hydrolase" 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.

[0049] The term "functional analog" is used in this invention to describe compounds or proteins that perform the same function and / or can be used for the same purpose. For example, all the fungal luciferins listed in Table 1 are functional analogs of each other.

[0050] In some embodiments, the term "substitute" as used herein refers to a gene that has the same or similar function as the gene being replaced in a plant, plant tissue, or plant cell. In some embodiments, the substitute may be a sequence of the replaced gene with one or more amino acids replaced, added, or deleted; in other embodiments, the substitute may be a gene with more than 60% identity to the replaced gene's amino acid sequence, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; in still other embodiments, the substitute may be a gene with less than 60% identity to the replaced gene. For example, the applicant has found that the milk alkaloid synthase gene and the grape polyketide synthase gene have similar functions in the FBP fluorescent pathway system, and that they are interchangeable and substitutes for each other.

[0051] The term "plant" as used in this article refers to a class of multicellular eukaryotic organisms in nature. Their cells have cell walls composed of cellulose, and the vast majority can produce nutrients through photosynthesis. They typically grow sessilely, absorbing water and minerals through their roots. They utilize photosynthetic pigments such as chlorophyll to convert light energy into chemical energy, transforming carbon dioxide and water into organic matter and oxygen to support their growth, development, and reproduction. Plants generally include major groups such as bryophytes, ferns, gymnosperms, and angiosperms.

[0052] In some embodiments, the plant is an ornamental plant, such as, but not limited to, tobacco, rose, crabapple, sunflower, chrysanthemum, dahlia, bellflower, string of pearls, morning glory, and million bells.

[0053] The term "partial tissue" as used in this article refers to the roots, stems, leaves, flowers, fruits, seeds, etc. of a plant. In some embodiments, partial tissues of a plant may also be callus, protocorms, or protocormoids prepared from the aforementioned tissues under specific conditions and through specific culture.

[0054] The term "cell" as used in this article refers to plant somatic cells cultured in three dimensions. These cells can originate from any plant tissue.

[0055] This application relates to a recombinant construct comprising: a first expression cassette including a fluorescence enhancer; wherein the amino acid sequence of the fluorescence enhancer is as shown in SEQ ID NO:2, or has at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity with SEQ ID NO:2, and is functionally equivalent to the amino acid sequence of the protein shown in SEQ ID NO:2; and a second expression cassette including a set of fluorescent factors configured to enable the plant to emit its own light; wherein the set of fluorescent factors includes: milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene, and caffeoylpyruvate hydrolase gene, or alternatives to one or more of these genes.

[0056] In some embodiments, the nucleotide sequence of the fluorescence enhancement factor is as shown in SEQ ID NO:1; or a nucleotide sequence that has 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity with the protein encoded by the nucleotide sequence shown in SEQ ID NO:1, and is functionally equivalent to the protein encoded by the nucleotide sequence shown in SEQ ID NO:1.

[0057] In some embodiments, the fluorescent factor group includes: a combination of milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene; or a combination of grape polyketide synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene.

[0058] In some embodiments, the recombinant construct further includes: a first promoter that is functional in a plant and operatively linked to a fluorescence enhancer; and a second promoter that is functional in a plant and operatively linked to each gene in the fluorescence enhancer set; the first promoter may be the same as or different from the second promoter. In some embodiments, the first promoter and the second promoter are CaMV 35S promoters.

[0059] This application relates to a carrier comprising the above-described recombinant constructs; in some embodiments, each of the carriers includes one or more recombinant constructs.

[0060] This application relates to microorganisms comprising the aforementioned recombinant constructs or vectors. In some embodiments, the microorganism can be any microorganism capable of transferring specific genes or proteins, vectors, etc., into plants and expressing them therein to achieve their functions. In some embodiments, the microorganism can be Agrobacterium.

[0061] This application relates to a fusion protein, which is obtained by the aforementioned recombinant construct, vector, or microbial expression.

[0062] The aforementioned recombinant constructs, vectors, microorganisms, or fusion proteins are used in the preparation of self-luminous plants, self-luminous plant tissues, or self-luminous cells.

[0063] In some embodiments, the plant is an algae, bryophyte, fern, gymnosperm, or angiosperm; in some embodiments, specifically in some embodiments of this application, the plant cell is a dicotyledonous plant cell or a monocotyledonous plant cell. Preferably, the plants are selected from the group consisting of: Nicotiana benthamiana, Arabidopsis thaliana, tobacco, chrysanthemum, marigold, cosmos, zinnia, coreopsis, echinacea, Shasta daisy, silverleaf chrysanthemum, ageratum, marigold, white chrysanthemum, daisy, asters, cineraria, salvia splendens, sage, lavender, rosemary, mint, patchouli, coleus, rose, rose, tulip, hyacinth, daylily, hosta, spider plant, asparagus fern, aloe vera, liriope, purslane, kale, violet, honesty, carnation, carnation, baby's breath, tall snow lily, dwarf snow lily, jasmine, coral bean, ornamental pepper, tobacco flower, Bauhinia, wisteria, lupin, clover, iris, German iris, butterfly flower, iris, iris chinensis. 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, Million Bells, and Sweet Alyssum.

[0064] This application relates to the application of fluorescence enhancement factors in enhancing the fluorescence of self-luminous plants, plant tissues or cells; wherein the amino acid sequence of the fluorescence enhancement factor is as shown in SEQ ID NO:2, or has at least 60%, 65%, 70%, 75%, 80%, 85% or 90% identity with SEQ ID NO:2, and is functionally equivalent to the amino acid sequence of the protein shown in SEQ ID NO:2.

[0065] This application relates to a fluorescence-enhanced, self-luminescent plant tissue or cell whose genome includes: the aforementioned recombinant construct, or the aforementioned vector, or the aforementioned fusion protein.

[0066] This application relates to a method for preparing a fluorescence-enhanced, self-luminescent plant, plant tissue, or cell, comprising: transferring the aforementioned recombinant construct, or the aforementioned vector, or the aforementioned fusion protein, or the aforementioned vector into the plant, plant tissue, or cell.

[0067] Therefore, in the first aspect of this application, a method is provided. Lum The use of genes in enhancing plant luminescence intensity. Based on results from embodiments of this application, it was found that through FBP bioluminescent systems (including...)NnLuz , H3H , CPH and Hisps Further introduction based on genes Lum The gene effectively enhanced the physiological activity of transgenic plant cells and suppressed cell damage caused by the accumulation of intermediate products of the FBP pathway (such as milk alkaloids and caffeoylpyruvate). The enhanced cell activity also led to increased energy metabolism, providing a more sufficient energy base for the bioluminescent reaction and effectively increasing the overall luminescence intensity of the plant. Furthermore, the introduction of [a specific gene] into the FBP luminescence system... Lum Genes that produce self-luminous plants can be photographed with a mobile phone or camera lens without long exposure; the luminescent phenotype of the plant can be easily observed with the naked eye.

[0068] In a second aspect, this application provides a method for increasing the luminescence intensity of plants. According to embodiments of this application, the method includes... Lum Gene sequence introduced into plant cells, or expressed in plant cells. Lum Gene-encoded proteins. The method according to embodiments of this application can enhance the intensity of plant luminescence and improve the efficiency of luminescence inheritance, thereby enabling the acquisition of visible photos of luminescent plants through a mobile phone or camera lens without long exposure, and allowing the luminescent phenotype of the plant to be easily observed with the naked eye.

[0069] In some specific embodiments of this application, it further includes... NnLuz Gene, H3H Gene, CPH Gene, Hisps Gene sequence introduced into plant cells, or expressed in plant cells. NnLuz Gene, H3H Gene, CPH The protein encoded by the Hisps gene. According to embodiments of this application, NnLuz Gene, H3H Gene, CPH Gene, Hisps Gene-encoded proteases participate in the caffeic acid cycle within plants, enabling them to emit their own light; Lum When the gene and the aforementioned gene are combined to construct a vector and introduced into the plant genome, the intensity of the plant's autoluminescence can be increased.

[0070] In some specific embodiments of this application, the NnLuz gene nucleotide sequence is as shown in SEQ ID NO: 3, or has more than 70% identity with SEQ ID NO: 3; the H3H gene nucleotide sequence is as shown in SEQ ID NO: 4, or has more than 70% identity with SEQ ID NO: 4; the CPH gene nucleotide sequence is as shown in SEQ ID NO: 5, or has more than 70% identity with SEQ ID NO: 5; and the Hisps gene nucleotide sequence is as shown in SEQ ID NO: 6, or has more than 70% identity with SEQ ID NO: 6.

[0071] In a third aspect, this application provides Lum Use of genes in the construction of multi-gene vectors, wherein the multi-gene vectors further include NnLuz Gene, H3H Gene, CPH Gene, Hisps Genes. Will contain NnLuz , H3H , CPH , Hisps and Lum The introduction of expression vectors for multiple target genes into plants enables the stable integration and long-term expression of multiple functional genes in the plant genome, improving the efficiency of obtaining transgenic plants and their genetic stability. This ensures the protection of the luminescent system and enhancing factors (such as...) Lum Effective expression of luminescence in recipient plants can maintain the normal growth and development of recipient plants, which is beneficial for obtaining self-luminescent plant materials with high luminescence intensity and stable genetics.

[0072] In some specific embodiments of this application, the multi-gene vector is selected from the pCAMBIA2300 vector.

[0073] According to one embodiment of this application, NnLuz Gene, H3H Gene, CPH Gene, Hisps Gene-encoded proteases participate in the caffeic acid cycle within plants, enabling them to emit their own light; Lum The gene, along with the aforementioned gene, was used to construct a vector and introduced into the plant genome. This effectively enhanced the physiological activity of transgenic plant cells and suppressed cell damage caused by the accumulation of FBP pathway intermediates (such as milk alkaloids and caffeoylpyruvic acid). The increased cell activity also led to enhanced energy metabolism, providing a more sufficient energy base for bioluminescence reactions and effectively increasing the overall luminescence intensity of the plant. In some embodiments, the gene can be replaced or modified. NnLuz Gene, H3H Gene, CPH Gene,Hisps Any one or more genes.

[0074] In some embodiments of this application, each target gene in the multi-gene vector contains a 35S promoter sequence upstream, and each target gene in the multi-gene vector contains a CaMV poly(A) terminator downstream. Specifically, the 35S promoter is selected from the CaMV 35S promoter. By configuring 35S promoters and CaMV poly(A) terminators for each target gene in the multi-gene vector, efficient and stable expression of each gene in the plant is ensured, autoluminescence intensity is enhanced, cell damage is reduced, and the overall performance of the transgenic plant is improved.

[0075] In some examples of this application, the construction of a multi-gene vector includes: introducing a target gene fragment from the multi-gene vector into a recipient plant using Agrobacterium-mediated transformation. The Agrobacterium-mediated transformation involves introducing a target gene fragment from the multi-gene vector into a recipient plant. NnLuz , H3H , CPH , Hisps and Lum The introduction of expression vectors for multiple target genes into plants enables the stable integration and long-term expression of multiple functional genes in the plant genome, improving the efficiency of obtaining transgenic plants and their genetic stability. This ensures the protection of the luminescent system and enhancing factors (such as...) Lum Effective expression of luminescence in recipient plants can maintain the normal growth and development of recipient plants, which is beneficial for obtaining self-luminescent plant materials with high luminescence intensity and high cell activity.

[0076] In a fourth aspect, this application provides a method for detecting the luminescence intensity of plants. According to an embodiment of this application, the method includes detecting the amount of photon accumulation in plants treated by the method described in the second aspect of this application.

[0077] In some specific embodiments of this application, the detection of the accumulated photon quantity is performed in a dark environment, and the detection time is not less than 1 minute.

[0078] In a fifth aspect, this application provides a method for targeted selection or identification of bioluminescent plants. According to embodiments of this application, the method includes identifying bioluminescent plants in the test plant. Lum The expression of a gene or the protein it encodes, said Lum The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2. This was determined by detection... Lum The expression level of genes or their encoded proteins can efficiently identify plant materials with excellent luminescence capabilities, effectively improving the screening efficiency and success rate of bioluminescent plants.

[0079] In some specific embodiments of this application, the...Lum The genes come from Arabidopsis thaliana, wheat, rice, maize, etc.

[0080] In some specific embodiments of this application, the method employs sequencing and PCR amplification for nucleotide sequence identification. Based on the aforementioned method, it is possible to rapidly confirm whether successful integration and expression have occurred in transgenic plants. Lum Genes; can also be quantitatively assessed Lum Correlation between gene expression levels and plant luminescence properties.

[0081] 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: hispidin synthase (Hisps), hispidin-3-hydroxylase (H3H), luciferase (Luz, i.e., the NnLuz gene), and caffeoyl pyruvate hydrolase (CPH), as detailed below: The process of catalytically generating luciferin using caffeic acid as a substrate is as follows: Step 1: Caffeic acid reacts with propionyl-CoA to produce milk alkaloids. Catalytic enzyme: Hispidin Synthase (Hisps) Reaction description: Caffeic acid, catalyzed by milk alkaloid synthase, combines with propionyl-CoA to form milk alkaloid (hispidin). Caffeic acid + 2 propionyl-CoA + 2ATP → milk alkaloids + 2CO2 + H2O + 2AMP Step 2: Hydroxylation of milk alkaloids to generate 3-hydroxymilk alkaloids Catalytic enzyme: Hispidin hydroxylase (H3H) Reaction Description: Milk alkaloids undergo a hydroxylation reaction under the action of milk alkaloid hydroxylase to generate 3-hydroxymilk alkaloids. Milkweed alkaloids + O2 + NADPH + H+ → 3-hydroxymilkweed alkaloids + H2O + NADP+ Step 3: Oxidation of 3-hydroxymilk alkaloids to produce caffeoylpyruvic acid Catalytic enzyme: Luciferase (Luz) Reaction description: 3-hydroxymilk alkaloids are oxidized to luciferin under the catalysis of luciferase, and luminescence is produced.

[0082] 3-Hydroxymilk alkaloid + O2 → Caffeoylpyruvic acid + CO2 + Light Through these three steps, caffeic acid and pyruvate are converted into caffeoylpyruvate via an enzymatic reaction, thus achieving bioluminescence.

[0083] Step 4: Oxidation of 3-hydroxymilk alkaloids to produce caffeoylpyruvic acid Catalytic enzyme: caffeoyl pyruvate hydrolase (CPH) Reaction description: Caffeoylpyruvate is catalyzed by caffeoylpyruvate hydrolase to generate caffeic acid, thereby regenerating caffeic acid and achieving the purpose of cyclic luminescence.

[0084] Caffeoylpyruvic acid + H2O → Pyruvic acid + Caffeic acid.

[0085] The sequences involved in this application are shown in Table 1: Table 1. Nucleotide or amino acid sequences of various genes

[0086] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0087] Example 1: This embodiment focuses on the model plant common wheat (Wheat simonii) Triticum aestivum L. (Source) Lum Genetic modification followed by plant transformation. Plant types included are: tomato, black nightshade, potato, marigold, sunflower, petunia, columbine, staghorn sumac, haworthia, rapeseed, lettuce, and carnation. Specific steps are as follows: 1. Gene cloning and vector construction Lum -Luminescent gene clusters: containing Lum Gene (SEQ ID NO: 1), NnLuz Gene (SEQ ID NO: 3), H3H Gene (SEQ ID NO: 4), CPH Gene (SEQ ID NO: 5) and Hisps A multi-gene vector containing the gene (SEQ ID NO: 6), with the gene structure as follows: Figure 3A As shown, the synthesized Lum- The luminescent gene cluster, with its nucleotide sequence shown in SEQ ID NO:8. The constructed 2300- Lum - The T-DNA sequence of the luminescent gene cluster vector is shown in SEQ ID NO:10, and the map is shown in... Figure 4A As shown. The gene sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. The vector was pCAMBIA2300 (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., DNA map as shown). Figure 2 As shown), the insertion position is: pCAMBIA2300[35S promoter]-XbaI / SacI-NOS terminator.

[0088] luminescent gene clusters: containing NnLuz Gene (SEQ ID NO: 3), H3H Gene (SEQ ID NO: 4), CPH Gene (SEQ ID NO: 5) and Hisps A multi-gene vector containing the gene (SEQ ID NO: 6). The gene sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the gene structure is as follows. Figure 3B As shown, the T-DNA map is as follows: Figure 4B As shown. Each gene contains an enhanced CaMV 35S promoter and a CaMV poly A terminator. The promoter + gene + terminator constitutes a gene expression cassette. The applicant concatenates the expression cassettes of all genes required for luminescence to form the sequence synthesized by the applicant. The insertion position is: pCAMBIA2300[35S promoter]-XbaI / SacI-NOS terminator.

[0089] 1.1 PCR Amplification: The target genes in the LumEnhancer luminescent gene cluster and the luminescent gene cluster were amplified using the gene cloning reaction system and reaction program shown in Table 2. The 2× high-fidelity PCR mix was from Nanjing Novizan Biotechnology Co., Ltd., catalog number: P520-01.

[0090] Table 2: Gene Cloning Reaction System

[0091] After the PCR reaction, the DNA was extracted and recovered using a gel according to the instructions of the Novizan gel extraction kit. The recovered product was sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing to determine the gene sequence. The primer sequences are shown in Table 3.

[0092] Table 3: Primer List

[0093] 1.2 Double digestion: The pCAMBIA2300 vector was double-digested with restriction endonucleases EcoRI and HindIII using the reaction system and conditions shown in Table 4 (the endonucleases were purchased from New England Biolabs).

[0094] Table 4: pCAMBIA2300 vector double enzyme digestion

[0095] 1.3 Ligation reaction: The amplified genes were ligated with the enzyme-digested vectors using a seamless cloning kit (purchased from Beijing Bomed Gene Technology Co., Ltd.). Each gene had a 35S promoter and a CaMV poly(A) terminator.

[0096] 1.4 Transformation of Escherichia coli with vector: The ligation product was transformed into competent Escherichia coli DH5α (purchased from Beijing Bomed Gene Technology Co., Ltd.) by heat shock, and positive clones were screened on LB plates containing 50 mg / L Kan.

[0097] 2. Conversion and Screening 2.1 Escherichia coli culture: Select positive clones and culture them in 10 mL of LB containing 50 mg / L kan.

[0098] 2.2 Plasmid Extraction: Recombinant plasmids were extracted using a plasmid mini-prep kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). Plasmid Preservation: The plasmids were frozen and stored at -20°C for later use.

[0099] 3. Microbial detection and Agrobacterium-mediated transformation 3.1 Transformation of Agrobacterium: The plasmid was transformed into Agrobacterium GV3101 competent cells (purchased from Beijing Bomaide Gene Technology Co., Ltd.). The transformation method was performed according to the instructions.

[0100] 3.2 Screening for transformed Agrobacterium: Transformed Agrobacterium was screened on YEP plates containing antibiotics (50 mg / L Kan + 50 mg / L Gen + 25 mg / LRif). (Kan, Kanamycin, Gen, Gentamicin, Rif, and Rif were all purchased from Beijing Coolerbot Technology Co., Ltd.)

[0101] 3.3 Agrobacterium culture and suspension preparation: Positive Agrobacterium clones were selected for liquid culture. The concentration of the suspension was adjusted to OD using MS infection medium (pH 5.8, containing 100 μM acetylsylgenone) as needed. 600 =0.6-0.8.

[0102] 4. Plant transformation 4.1 Plant material preparation: Select suitable tobacco leaves for conversion and cut the leaves into 0.5 cm pieces. Size: 0.5 cm.

[0103] 4.2 Leaf disc transformation: Plant leaf discs were soaked in Agrobacterium suspension at 28°C for 30 min and then transferred to co-culture medium (MS + 1 mg / L 6-BA + 30 g / L sucrose + 6 g / L agar powder + 100 μM acetylsalicylic acid, pH 5.8).

[0104] 4.3 Co-culture and screening: The samples were co-cultured in the dark at 28°C for 2 days, and then transferred to selective medium containing selective antibiotics (MS + 1 mg / L 6-BA + 30 g / L sucrose + 6 g / L agar powder + 100 mg / L Kan, pH 5.8).

[0105] 4.4 Regeneration and rooting: After about 2-3 weeks, resistant shoots were selected and induced to root in rooting medium (MS + 0.1 mg / L NAA + 30 g / L sucrose + 6 g / L agar powder + 100 mg / L Kan, pH 5.8).

[0106] 4.5 Transplanting and growth of tobacco: After rooting, the tobacco plants were transplanted into soil (peat:vermiculite:perlite = 5:3:1) and managed in a greenhouse with a light intensity of 6000 lux (Lx) for 16 h / day.

[0107] 4.6 Transgenic identification: Samples of transplanted tobacco were taken and DNA was extracted using the CTAB method. The reaction system and procedure shown in Table 5 and PCR positive identification were performed.

[0108] Table 5: Reaction System and Reaction Procedure

[0109] Based on the above experiments, we obtained [the following]: Lum -Luminescent gene clusters and transgenic plants containing luminescent gene clusters.

[0110] Test Example 1: This embodiment is used to analyze the contents obtained in Example 1. Lum - The luminescence intensity of luminescent gene clusters and transgenic plants containing luminescent gene clusters was measured. The specific steps are as follows: 1. Luminous intensity detection Using a LightScout full-spectrum quantum meter (Shanghai Zequan Technology), the detection port was aimed directly at the leaf of the plant under test in a dark environment (close to the leaf), and the amount of photons accumulated at different times was measured. The detection results are shown in Table 6.

[0111] As can be seen from Table 6 below, the luminous brightness of the conventional FBP luminous system did not decrease. Lum The light-emitting system is much brighter. The light-emitting intensity of the replaced system is approximately four times that of the FBP system, representing a significant improvement in light intensity.

[0112] Table 6: Luminous Intensity of Different Plants

[0113] 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 recombinant construct, comprising: The first expression cassette includes a fluorescence enhancer; The amino acid sequence of the fluorescence enhancement factor is as shown in SEQ ID NO:2, or has at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity with SEQ ID NO:2 and is functionally equivalent to the protein shown in SEQ ID NO:2; the first expression cassette is configured to enhance the autoluminescence intensity of plants, plant tissues, or cells.

2. The recombinant construct according to claim 1, further, The nucleotide sequence of the fluorescence enhancement factor is as shown in SEQ ID NO:1; or it has 60%, 65%, 70%, 75%, 80%, 85% or 90% identity with the protein encoded by the nucleotide sequence shown in SEQ ID NO:1, and is functionally equivalent to the protein encoded by the nucleotide sequence shown in SEQ ID NO:

1.

3. The recombinant construct according to claim 1, further comprising: The first promoter that is functional in plants and operatively linked to a fluorescence enhancer.

4. The recombinant construct according to any one of claims 1-3, further comprising: The second expression cassette includes a group of fluorescent factors configured to enable the plant to emit its own light. The fluorescent factor group includes: milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene, or alternatives to one or more of these genes; the second expression cassette is configured to enable the plant, plant tissue or cell to emit light.

5. The recombinant construct according to claim 4, wherein the fluorescent factor group comprises: A combination of milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene, and caffeoylpyruvate hydrolase gene; or A combination of the grape polyketide synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene, and caffeoylpyruvate hydrolase gene; or A combination of milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene, caffeoylpyruvate hydrolase gene, TAL gene, and P450 gene.

6. The recombinant construct according to claim 4, further comprising: A second promoter that is functional in plants and operatively linked to each gene in the fluorescent factor group; The first promoter may be the same as or different from the second promoter.

7. The recombinant construct according to claim 6, wherein the first promoter and the second promoter are CaMV 35S promoters.

8. A vector comprising the recombinant construct according to any one of claims 1-7.

9. The vector according to claim 8, wherein each of the vectors comprises one or more recombinant constructs.

10. A microorganism comprising any one of the recombinant constructs of claims 1-7, or comprising the vector of claim 8 or 9.

11. A fusion protein, which is a recombinant construct as described in any one of claims 1-7, or a vector as described in claim 8 or 9, or obtained by microbial expression as described in claim 10.

12. The use of the recombinant construct as described in any one of claims 1-7, or the vector as described in claim 8 or 9, or the microorganism as described in claim 10, or the fusion protein as described in claim 11, in the preparation of self-luminous plants, self-luminous plant tissues, or self-luminous cells.

13. The application according to claim 12, wherein the plant is: algae, bryophytes, ferns, gymnosperms, or angiosperms.

14. The application according to claim 13, wherein the plant is selected from the group consisting of: Nicotiana benthamiana, Arabidopsis thaliana, tobacco, chrysanthemum, marigold, cosmos, zinnia, coreopsis, echinacea, Shasta daisy, silverleaf chrysanthemum, ageratum, marigold, white chrysanthemum, daisy, asters, cineraria, salvia splendens, sage, lavender, rosemary, mint, patchouli, coleus, rose, rose, tulip, hyacinth, daylily, hosta, spider plant, asparagus fern, aloe vera, liriope, purslane, kale, violet, honesty, carnation, carnation, baby's breath, tall snow lily, dwarf snow lily, jasmine, coral bean, ornamental pepper, tobacco flower, Bauhinia, wisteria, lupin, clover, iris, German iris, butterfly Butterfly flower, Iris, 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, Winter Jasmine, Forsythia suspensa, Poinsettia, Crown of Thorns, Croton, Jade Plant, Kalanchoe, Echeveria, Echeveria elegans, Sedum lineare, Cactus, Gymnocalycium mihanovichii, Epiphyllum oxypetalum, Christmas cactus, Tradescantia zebrina, Tradescantia zebrina, Commelina communis, Amaryllis, Cyclamen, Gloxinia, Oxalis, Bellflower, Lobelia, Verbena officinalis, Geranium, Nasturtium, Gazania latifolia, Marguerite daisy, Bleeding heart, Foxglove, Ranunculus asiaticus, Columbine, Plumbago aurantium, Vinca minor, Million Bells and Sweet Alyssum.

15. A fluorescence-enhanced, self-luminescent plant tissue or cell, the genome of which includes: The recombinant construct according to any one of claims 1-7, or comprising the vector according to claim 8 or 9, or the fusion protein according to claim 11.

16. A method for preparing a fluorescence-enhanced, self-luminescent plant, plant tissue, or cell, comprising: The recombinant construct comprising any one of the claims 1-7, or the vector comprising the claim 8 or 9, or the microorganism comprising the claim 10, or the fusion protein comprising the claim 11, is transferred into a plant, plant tissue, or cell.

17. The application of fluorescence enhancement factors in enhancing the fluorescence of self-luminous plants, plant tissues or cells; wherein the amino acid sequence of the fluorescence enhancement factor is as shown in SEQ ID NO:2, or has at least 60%, 65%, 70%, 75%, 80%, 85% or 90% identity with SEQ ID NO:2, and is functionally equivalent to the amino acid sequence of the protein shown in SEQ ID NO:

2.

18. The use of the recombinant construct as described in any one of claims 1-7, or the vector as described in claim 8 or 9, or the microorganism as described in claim 10, or the fusion protein as described in claim 11, in enhancing the fluorescence of self-luminescent plants, self-luminescent plant tissues, or self-luminescent cells.

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