A method for realizing continuous self-luminous of plants by using recombinant self-luminous yeast

Abstract

This disclosure belongs to the field of biotechnology, specifically relating to a method for achieving continuous self-luminescence in plants using recombinant self-luminescent yeast. Specifically, this disclosure provides a method for preparing a continuously self-luminescent plant by adding caffeic acid to promote luminescent metabolism in *Saccharomyces cerevisiae* and adjusting the concentration of *Saccharomyces cerevisiae* to increase the luminescence intensity of the recombinant self-luminescent yeast, thereby preparing a self-luminescent plant with high stability, high safety, no need for long-term expression, and high photon intensity.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology, specifically relating to a method for achieving continuous self-luminescence in plants using recombinant self-luminous yeast. Background Technology

[0002] Currently, self-luminescent plants are mainly achieved through three pathways: physical light technology, nanomaterials technology, and biotechnology. The "luminescent plants" commonly seen in municipal greening projects LED lights onto the plant surface to create a visual luminescence effect. However, this physical lighting is not true bioluminescence, resulting in an unnatural appearance, disrupting the plant's circadian rhythm, affecting its normal growth, and causing light pollution. Another method involves injecting fluorescent nanomaterials into the plant, but this method suffers from problems such as material toxicity, easy degradation, short luminescence lifespan, high cost, and potential environmental pollution. Therefore, researchers are attempting to use biotechnology to obtain self-luminescent plants that possess the bioluminescent properties commonly found in nature, such as fireflies, jellyfish, and fungi.

[0003] In 1986, researchers achieved endogenous expression of firefly luciferase in tobacco using genetic engineering techniques. By applying the exogenous substrate of this enzyme, firefly luciferin, they obtained luminescent tobacco for the first time. However, the high cost of the exogenous substrate significantly increased the cost, and the tobacco plant only emitted a faint light barely perceptible to the naked eye. To solve the problem of expensive exogenous substrates, the key to obtaining self-luminescent plants is to modify the plant genome, enabling it to synthesize luciferin and luciferase autonomously without relying on exogenous substrates. In 2010, researchers used *Photobacterium regrowii* (… Photobacterium lion-toothed The complete luminescent metabolic pathway—the lux operon—is integrated into the tobacco chloroplasts. This transgenic tobacco can be observed with the naked eye in the dark (1.3 × 10⁻⁶). 6 (Photons / second), but this technology is difficult to apply to most monocotyledonous plants. Furthermore, high expression of heterologous proteins in chloroplasts reduces plant vigor, thus affecting normal plant growth and development.

[0004] Until 2018, a major breakthrough was achieved in the study of self-luminous plants. Researchers elucidated the mechanism of light-emitting fungi (Gymnophyton floccosum). Neonothopanus nambiThe complete metabolic pathway of bioluminescence (FBP) involves the conversion of endogenous fungal caffeic acid into an unstable high-energy intermediate, catalyzed by hispidin synthase (Hispidin Synthase), histidine-3-hydroxylase (H3H), and fungal luciferase (Luz), thereby releasing 520nm green light. Caffeic acid is widely found in most higher plants and is a major metabolic intermediate in the synthesis of lignin and anthocyanins. The abundant natural caffeic acid in plants provides an important foundation for the cultivation of self-luminescent plants. Based on this, two studies on self-luminescent plants based on FBP were published concurrently in 2020. Researchers optimized the codons of fungal bioluminescence genes and integrated them into the model plant *Nicotiana benthamiana* using transgenic technology, obtaining self-luminescent tobacco with luminescence intensity detectable by consumer cameras and mobile phones.

[0005] However, due to limitations such as genotype, the genetic transformation systems of most flowering plants remain extremely immature, severely restricting the application of this technology in flowering plants. Currently, researchers mostly use Agrobacterium (… Agrobacterium Transient expression technology can achieve a brief luminescent effect in cut roses, lilies, orchids, which are difficult to achieve stable genetic transformation. However, Agrobacterium expression requires 48-72 hours of accumulation time, and the bacterial solution can cause some damage to the petals, resulting in poor plant appearance; at the same time, Agrobacterium itself can also cause some pollution to the environment. Summary of the Invention

[0006] This disclosure aims to overcome the aforementioned deficiencies of the prior art by utilizing food-grade brewing yeast ( Saccharomyces yeast This invention discloses a bioluminescent plant strain YCA113-oFBP, which provides a bioluminescent plant that requires no expensive substrates, exhibits visible luminescence intensity, and is simple and environmentally friendly to prepare. The technical solution adopted in this invention is as follows: On the one hand, this disclosure provides a method for preparing a continuously self-luminous plant, which includes the following steps: (1) Constructing a recombinant self-luminescent Saccharomyces cerevisiae strain: The nucleotide sequence encoding the FBP pathway gene and / or NPGA gene was integrated into the genome of a Saccharomyces cerevisiae strain capable of synthesizing caffeic acid to obtain a Saccharomyces cerevisiae strain; (2) Preparation of bacterial suspension: Activate the brewing yeast strain constructed in step (1), expand the culture until the bacterial suspension is saturated, adjust the bacterial suspension concentration to OD600=2.0-3.0, and add caffeic acid; (3) Introducing into plants: Introducing the bacterial suspension containing caffeic acid prepared in step (2) into the organs or tissues of plants.

[0007] Preferably, the FBP pathway genes include HispS, CPH, H3H, and Luz.

[0008] Preferably, step (1) involves integrating the nucleotide sequences encoding the NPGA gene or its variant, the HispS gene or its variant, the CPH gene or its variant, the H3H gene or its variant, and the Luz gene or its variant into the genome of a Saccharomyces cerevisiae strain capable of synthesizing caffeic acid, thereby obtaining a Saccharomyces cerevisiae strain.

[0009] Preferably, the plant includes cut roses ( Hybrid rose Phalaenopsis orchids Phalaenopsis ),lily( Lily ), succulent plants ( Succulent Five generations under one roof ( Solanum mammosum More preferably, the plant is a cut rose.

[0010] Preferably, the caffeic acid concentration is at least 40 mM, for example 40 mM, 60 mM, 80 mM, 100 mM, or 150 mM; more preferably 40 mM.

[0011] Preferably, the culture medium used for the brewing yeast is yeast extract peptone dextrose medium (YPD).

[0012] Preferably, the method for introducing the plant includes injection or vacuum immersion.

[0013] Preferably, the concentration of the bacterial suspension is OD600 = 2.0.

[0014] Preferably, the vacuum treatment conditions for the vacuum immersion method are 40 kPa pressure for 10 s.

[0015] Preferably, step (3) involves introducing the bacterial suspension into the petals of the plant.

[0016] Preferably, the gene encoding the NPGA gene is derived from Aspergillus nidulans (…). Aspergillus nidulans ).

[0017] Preferably, the gene encoding the HispS gene originates from the genus Mycena (…). Mycenae Fungi.

[0018] Preferably, the gene encoding the CPH gene originates from *Gymnocladus mukorossi* (a type of fungus). Neonothopanus nambi ).

[0019] Preferably, the gene encoding the H3H gene is derived from *Gymnospermum glabra*.

[0020] Preferably, the gene encoding the Luz gene is derived from *Gymnospermum glabra*.

[0021] Preferably, the genome of the Saccharomyces cerevisiae strain contains a nucleotide sequence encoding the NPGA gene as shown in SEQ ID NO: 1.

[0022] Preferably, the genome of the Saccharomyces cerevisiae strain contains a nucleotide sequence encoding the HispS gene as shown in SEQ ID NO: 2.

[0023] Preferably, the genome of the Saccharomyces cerevisiae strain contains a nucleotide sequence encoding the CPH gene as shown in SEQ ID NO: 3.

[0024] Preferably, the genome of the Saccharomyces cerevisiae strain contains a nucleotide sequence encoding the H3H V2 gene as shown in SEQ ID NO: 4.

[0025] Preferably, the genome of the Saccharomyces cerevisiae strain contains a nucleotide sequence encoding the Luz V4 gene as shown in SEQ ID NO: 5.

[0026] Preferably, the nucleotide sequences encoding the NPGA gene, HispS gene, CPH gene, H3H gene, and Luz gene are expressed by inducible promoters. Preferably, the inducible promoters include galactose-induced promoters, doxycycline-induced promoters, estrogen-induced promoters, etc. Specifically, the galactose-induced promoters include GAL1 and GAL10.

[0027] On the other hand, this disclosure provides self-luminous plants prepared using the aforementioned method.

[0028] On the other hand, this disclosure provides the application of the aforementioned self-luminous plants in nighttime landscape design, bio-art creation, environmental beautification, and science education.

[0029] On the other hand, this disclosure provides the Saccharomyces cerevisiae strain used in the aforementioned methods.

[0030] On the other hand, this disclosure provides the application of Saccharomyces cerevisiae strains in the preparation of self-luminous plants or as bioluminescent indicators.

[0031] Compared with the prior art, this disclosure has the following beneficial effects: This disclosure enhances fungal luminescence intensity by adding different concentrations of caffeic acid to promote a stably integrated luminescent metabolic pathway in fungi (preferably Saccharomyces cerevisiae). Simultaneously, it adjusts the concentration of the fungal strain to further increase luminescence intensity. This innovatively injects enhanced self-luminescent fungi into living plants (e.g., flowers, especially petals of cut roses), allowing for rapid observation of visible light emitted from the plant in the dark. Compared to Agrobacterium-mediated transient expression technology, this method is more stable, safer, requires less time, and increases quantum intensity by two times. Attached Figure Description

[0032] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this specification and, together with the description, serve to explain the principles of this specification.

[0033] Figure 1 The luminescence characteristics of strains YCA113-2B and YCA113-oFBP are shown.

[0034] Figure 2 The images show bioluminescence of the YCA113-oFBP yeast strain under different concentrations of caffeic acid.

[0035] Figure 3 The performance of the YCA113-oFBP yeast strain in different plants is shown.

[0036] Figure 4 The comparison of luminescence intensity after injecting roses with different concentrations of YCA113-oFBP yeast culture is shown.

[0037] Figure 5 The comparison of luminescence intensity of roses treated by vacuum immersion under different pressure conditions is shown.

[0038] Figure 6 The performance of the YCA113-oFBP yeast strain in roses of different colors is shown.

[0039] Figure 7 The comparison of luminescence intensity of the YCA113-oFBP yeast strain in roses of different colors is shown.

[0040] Figure 8 The comparison of luminescence intensity between YCA113-oFBP yeast and EHA105-oFBP Agrobacterium in rose is shown. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided through specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of this disclosure. All other implementations obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure.

[0042] This disclosure may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this disclosure may be combined with technical features of any or more other embodiments to obtain further embodiments. This disclosure includes such further embodiments obtained through combination. (I) Definitions and Explanations

[0043] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. In this disclosure, unless otherwise stated, the scientific and technical terms used have meanings commonly understood by those skilled in the art. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this disclosure are all widely used terms and routine procedures in their respective fields. It should also be understood that the terminology used in this disclosure is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0044] In this disclosure, the term "injection method" refers to a procedure in which exogenous substances (such as bacterial solutions) are injected into the interior or interstitial space of a target plant tissue through artificial injection.

[0045] In this disclosure, the term "vacuum immersion method" refers to a method of immersing plant material in a bacterial solution, then creating a vacuum negative pressure environment to expel air from the plant tissue and intercellular spaces, and finally using the pressure difference to fully force the bacterial solution into the tissue after the pressure returns to normal.

[0046] In this disclosure, the term "YPD" or "YPD medium" refers to Yeast Extract Peptone Dextrose Medium, a commonly used microbial culture medium that mainly contains yeast extract, peptone, glucose, and other components, and is suitable for the culture and proliferation of yeast and some fungi.

[0047] In this disclosure, the term "caffeic acid" refers to 3,4-dihydroxycinnamic acid, a natural phenolic acid compound that can serve as a precursor or metabolic substrate in fungal bioluminescence pathways. This application describes the ability of *Saccharomyces cerevisiae* to heterologously synthesize caffeic acid.

[0048] In this disclosure, the term "nesting Aspergillus" refers to a fungus belonging to the genus Aspergillus in the phylum Ascomycota.

[0049] In this disclosure, the term "spotted fungus" refers to fungi belonging to the Basidiomycota phylum.

[0050] In this disclosure, the term "NPGA" or "NPGA gene" refers to the gene encoding 4'-phosphopantetheinyl transferase, whose expression product can catalyze the 4'-phosphopantetheinyl aminoylation modification of acyl carrier proteins and participate in metabolic pathways such as fatty acids and polyketides.

[0051] In this disclosure, the term "HispS" or "HispS gene" refers to the gene encoding hispidin synthase, whose expression product catalyzes the synthesis of hispidin, a key metabolic step in the fungal bioluminescence pathway.

[0052] In this disclosure, the term "CPH" or "CPH gene" refers to a gene encoding caffeoylpyruvate hydrolase, the expression product of which can participate in the hydrolytic metabolism of caffeoylpyruvate-like substances and provide substrates or intermediate metabolites for fungal bioluminescence pathways.

[0053] In this disclosure, the term "H3H" or "H3H gene" refers to the gene encoding hispidin-3-hydroxylase, whose expression product can catalyze the hydroxylation modification of hispidin, which is a key metabolic step in the fungal bioluminescence pathway.

[0054] In this disclosure, the term "Luz" or "Luz gene" refers to the gene encoding luciferase, whose expression product can catalyze the oxidation and luminescence reaction of luciferin substrates, and is a core functional protein in the fungal bioluminescence pathway.

[0055] In this disclosure, the term "self-luminescent plant" refers to a plant whose tissues possess the characteristic of continuous self-luminescence. It can produce self-luminescence visible to the naked eye in dark environments, and luminescent photographs can be taken using a consumer-grade camera (e.g., ISO set to 800-1600, exposure 5-30 seconds). Preferably, the self-luminescence lasts for at least 24 hours; more preferably, it lasts for at least 48 hours, at least 72 hours, or longer.

[0056] In this disclosure, the term "plant" should be understood as a differentiated multicellular organism capable of photosynthesis, encompassing crops at all stages of maturity or development, including monocotyledons ( Monocotyledons ), dicotyledons ( Dicotyledons More specifically, vegetable crops (asparagus) Asparagus officinalis ,lettuce Lettuce green Melons Cucurbitaceae rapeseed crop Brassica napus ,carrot Carrot ,onion Garlic onion (etc.), fruits and vine crops (apples) Domestic apple ,Tangerine Citrus spp., blueberries Vaccinium spp. ,Grape Wine grapevine etc.), field crops (corn) Corn Rice White rice Small grains Grasses Leguminosae Fabaceae Oil-bearing plants, etc., and trees (broadleaf trees) Broad-leaved angiosperms Fruit trees, ornamental trees, etc.). The plants described in this disclosure particularly refer to various cut flowers. The cut flowers include, but are not limited to: Rosaceae cut flowers (…). Rosaceae Such as cut roses Hybrid rose etc.), Gentianaceae cut flowers ( Gentianaceae such as lisianthus Eustoma grandiflorum etc.), Amaryllidaceae cut flowers ( Amaryllidaceae ,lily Lily spp., amaryllis Hippeastrum ruby daffodils Narcissus tazetta etc.), Liliaceae cut flowers ( Liliaceae ,tulip Tulipa gesneriana etc.), orchid cut flowers ( Orchidaceae Phalaenopsis orchid Phalaenopsis Cymbidium orchids Cymbidium hybrid Cattelan Cattleya spp., Dendrobium Dendrobium spp., etc.), fresh cut flowers of the Asteraceae family ( Asteraceae ,chrysanthemum Chrysanthemum morifolium Gerbera Gerbera jamesonii Cosmos Cosmos two-winged Chamomile Matricaria chamomilla etc.), Araceae cut flowers ( Araceae Anthurium Anthurium andraeanum White palm Spathiphyllum kochii Monstera deliciosa Delicious monstera Guanyin (Avalokitesvara) Alocasia odorata etc.), Ranunculaceae cut flowers ( Buttercups Ranunculus asiaticus Buttercup Asian etc.), Campanulaceae cut flowers ( Campanula Platycodon grandiflorus Platycodon grandiflorum Bellflower Bellflower Solanaceae fruit-bearing cut flowers (spp., etc.) Solanaceae Such as nipple / five generations under one roof Solanum mammosum (etc.), and other types (such as succulents) Succulent Forget-me-not Forget-me-not Starry Sky Gypsophila paniculate Lover's grass Limonium broadleaf Eucalyptus leaves Eucalyptus spp., silverleaf chrysanthemum Senecio ashtray Areca palm Yellow-flowered Dypsis wait).

[0057] In this disclosure, the terms "cut rose" or "rose" refer to the collective name of rose varieties that have been selected and are suitable for cultivation as cut flowers. They belong to one of the core categories of cut flowers and specifically include varieties such as red rose, pink rose, white rose, champagne rose, yellow rose, Elsa rose, cappuccino rose, Freud rose, Roselle rose, and Manta rose. (II) Detailed Technical Solution

[0058] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described. Example 1: Construction of an enhanced self-luminescent brewing yeast

[0059] Aspergillus nidus ( Aspergillus nidulans ) in the genome NPGA (4'-phosphopantetheinyltransferase) gene, Mycena genus ( Mycena Fungal origin HispS Hispidin synthase gene and *Gymnospermum glabra* ( Neonothopanus nambi ) in the genome H3H Variant H3H V2 of the (hispidin-3-hydroxylase) gene CPHThe coding sequences of the (caffeoylpyruvate hydrolase) gene and the variant Luz V4 of the fungal luciferase (Luz) gene were optimized according to the host's codon preference before synthesizing the corresponding gene fragments. The sequence information for NPGA is available at accession number QJQ48097.1; the sequence information for H3H is available at accession number QJQ48094.1; the sequence information for CPH is available at accession number QJQ48093.1; and the sequence information for Luz is available at accession number QJQ48096.1.

[0060] The construction steps of the recombinant self-luminescent Saccharomyces cerevisiae strain YCA113-oFBP are as follows: (1) Constructing a donor carrier Table 1. Nucleotide sequence of the target gene

[0061] The nucleotide sequence of the target gene shown in Table 1 was cloned into a gene containing a galactose promoter ( P GAL1 or P GAL10 By modifying the universal plasmid pESC at MCS1 or MCS2, a recombinant plasmid pESC-URA- containing the target gene is obtained. NnLuz V4 - NnCPH pESC-URA- NnH3H V2 - AnNPGA pESC-URA- mciHispS The gene insertion sites in the plasmids are shown in Table 2. Table 2. Plasmids used in this study

[0062] (2) Constructing a genome integration donor

[0063] Using with ARO10 Primer pair for the 40 bp homologous arm of the gene in the recombinant plasmid pESC-URA- mciHispS PCR was performed to obtain a genome integration donor consisting of a promoter, target gene, and terminator. △Aro10::TADH1-MCS1-PGAL10- PGAL1-mciHispS-TCYC1 Primers containing a 40 bp homologous arm at the Int10 site were used to target the recombinant plasmid pESC-URA- NnH3H V2 - AnNPGA PCR was performed to obtain integration donors: Int10:: T ADH1 -AnNPGA-PGAL10 -P GAL1 -NnH3H V2- T CYC1 Primers containing a 40 bp homologous arm at the Int12 site were used to target the recombinant plasmid pESC-URA- NnLuz V4 - NnCPH PCR was performed to obtain the integration donor: Int12:: T ADH1 -NnLuz V4-P GAL10 -P GAL1 -NnCPH-T CYC1 Primers are shown in Table 3. Table 3. Primers used for heterologous gene integration into the yeast genome

[0064] (3) Screening of recombinant Saccharomyces cerevisiae strains

[0065] Based on the CRISPR / Cas9 genome editing tool in Saccharomyces cerevisiae (Lian, J., HamediRad, M., Hu, S. & Zhao, H. Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nature Communications 8, 2017, 1688-1696.), the integrated donor will be... △Aro10::T ADH1 -NnLuz-P GAL10 -P GAL1 -NnCPH-T CYC1 Along with the corresponding single guide RNA coding sequence, the plasmid pRS423-SpSgH- ARO10 The plasmids pRS41K-SpCas9 were chemically transformed into yeast cells YCA113-2B. Single clones were picked and their integration into the genome was verified by PCR. YCA113-3B cells were obtained through screening, and then the integration donors were... Int10::TADH1-AnNPGA-PGAL10- PGAL1-NnH3H V2-TCYC1 With pRS426-SpSgH- Int10 The cells were transformed into yeast cells YCA113-3B, and YCA113-4B was obtained through PCR verification and screening. Int12::T ADH1 -NnLuz V4-PGAL10 -P GAL1 -NnCPH-T CYC1 With pRS426-SpSgH- Int12 The transformants were transformed into yeast cells YCA113-4B. Single clones were picked from the plates to break down the cells, and two primers from the genome were used for PCR to verify whether the target gene had been integrated into the genome. The genotypes of the strains at each step are described in Table 4.

[0066] If the band size is correct and the sequencing results are accurate, it indicates that the genome integration was successful, resulting in YCA113-oFBP. Table 4. Recombinant Saccharomyces cerevisiae strains

[0067] The autoluminescence intensity of Saccharomyces cerevisiae YCA113-2B and YCA113-oFBP was compared using a fully intelligent gel imaging system CCD (Charge-Coupled Device). Figure 1 As shown. Example 2: Metabolic optimization of YCA113-oFBP luminescence intensity Experimental steps:

[0068] (1) Activation and induction of strains: YCA113-oFBP yeast glycerol strain was taken out from the -80℃ ultra-low temperature freezer, streaked on YPD medium plate for activation, and cultured in the dark at 30℃ for 2 days; single colonies of YCA113-oFBP were picked from YPD plate and inoculated into 5mL YPD liquid medium, and cultured at 30°C and 200 rpm for 16 hours until saturation.

[0069] (2) Metabolic Optimizer Addition Experiment: The bacterial culture obtained in step (1) was centrifuged and collected, resuspended in PBS buffer to OD600 = 1.0, and dispensed into 24-well black microplates, 8 μL per well. At the same time, caffeic acid was added to the experimental wells at concentrations of 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, and 150 mM, respectively. Then, the luminescence intensity of each well at a wavelength of 520 nm was immediately measured using a chemiluminescence detector (unit: photons / second / colony forming unit, ph / s / CFU), and the monitoring was continued for 2 hours. result:

[0070] Compared with the control group, the experimental group with added caffeic acid showed a significant increase in luminescence intensity, with an average increase of 2 times. The 40 mM group showed the best improvement, indicating that caffeic acid flux is the main factor limiting luminescence intensity. Figure 2 As shown. Example 3: Preparation and Observation of Luminescent Flowers (1) Injection method

[0071] Commonly used cut roses, lilies, potted orchids, and succulents were selected as recipient plants. Yeast glycerol strain YCA113-oFBP was removed from a -80℃ ultra-low temperature freezer, streaked onto YPD solid medium plates for activation, and cultured at 30℃ in the dark for 2 days. A single colony of YCA113-oFBP was picked from the YPD plate and inoculated into 5 mL of YPD liquid medium containing 5% glucose. The culture was then incubated at 30℃ and 200 rpm with shaking for 16 hours until saturation. The culture was then expanded by inoculating into Erlenmeyer flasks at a 1:100 ratio and incubated at 30℃ and 200 rpm with shaking for 48 hours until saturation. Finally, 40 mM caffeic acid was added.

[0072] Fill a 1 mL sterile syringe with the bacterial suspension. Carefully insert the needle (a fine-bore insulin needle is recommended) into the base of the petal at approximately a 30-degree angle, avoiding puncture. Slowly inject the bacterial suspension; a slight water-soaked area will be visible around the injection site.

[0073] After injection, the cut flowers were placed in a greenhouse at 25°C and 60% humidity and hydroponically cultured as usual. After 3 seconds of dark adaptation, the flowers emitted a clear yellow-green fluorescence that could be observed with the naked eye in a completely dark environment. The glowing flowers could be easily photographed using a consumer-grade camera (ISO set to 800, exposure time 5-30 seconds).

[0074] like Figure 3 As shown, all the commonly used cut flowers selected can achieve self-illumination, among which roses have a better visual effect due to their double petals and can maintain their effect for 2-3 days. (2) Optimization of the concentration of the injection suspension

[0075] Take the YCA113-oFBP bacterial suspension prepared by the aforementioned (1) injection method, centrifuge it, and resuspend it in sterile YPD liquid medium. Use YPD medium to serially dilute the bacterial suspension to prepare a series of bacterial suspensions with OD600 values ​​of 0, 0.5, 1.0, 2.0, and 3.0. Add 40 mM caffeic acid to each bacterial suspension and inject it into rose petals.

[0076] Result: As Figure 4The results showed that the highest and most stable luminescence intensity per unit volume was observed when the OD600 was between 2.0 and 3.0, and this intensity could be maintained for 72 hours. Excessively high OD600 (e.g., 3.0) may lead to nutrient and oxygen restriction due to bacterial accumulation, thus negatively impacting luminescence efficiency. Furthermore, excessively high bacterial concentrations can affect the plant's osmotic pressure, making the petals more prone to wilting. Therefore, an OD600 of 2.0 was chosen as the optimal injection concentration; excessively high concentrations can negatively affect the plant's condition. (3) Vacuum immersion method

[0077] Take the YCA113-oFBP bacterial suspension with OD600=2.0 prepared as described in (2) above, add 40 mM caffeic acid to it, immerse the rose in the bacterial suspension and place it in a plant in vivo transformation instrument, and treat it according to the grouping and experimental conditions shown in Table 5. Each group is repeated 3 times. Table 5. Grouping and Experimental Conditions

[0078] Results: The experimental results are as follows Figure 5 This indicates that the vacuum conditions in Group D maintained the light intensity without excessively damaging the rose's condition, and the luminescence intensity could be sustained for 72 hours. Example 4: Obtaining luminescent roses of different colors

[0079] Commonly used fresh-cut roses include red roses, pink roses, Elsa roses, white roses, champagne roses, and yellow roses. A YCA113-oFBP bacterial suspension with an OD600 of 2.0 and containing 40 mM caffeic acid, prepared using the aforementioned method, was used. The fresh-cut roses were immersed in the bacterial suspension and placed in a plant in vivo conversion instrument, with the parameters set to 40 kPa for 10 s.

[0080] After injection, fresh-cut roses were placed in a greenhouse at 25°C and 60% humidity for normal hydroponic cultivation. After 3 seconds of dark adaptation, the flowers emitted different colors of fluorescence visible to the naked eye in complete darkness. Photographs of the glowing roses can be taken using a consumer-grade SLR camera (ISO set to 1600, exposure 5-30 seconds).

[0081] Results: The experimental results are as follows Figure 6 and 7 The luminescence phenomenon can last for at least 2-3 days. The experimental results show that red and even dark-colored roses, due to their rich color, can mask the fluorescence released by the luminescent yeast, while other light-colored roses can fully release the fluorescence of the luminescent yeast. Among them, white roses have the brightest light intensity. At the same time, champagne roses and yellow roses can change the color of the luminescent yeast, making it emit a warm yellow light. This provides ideas for the subsequent research and development of flowers with different luminescent colors. Example 5: Comparison with Agrobacterium injection method

[0082] Based on existing technologies (e.g., Mitiouchkina, et al. 2020), Agrobacterium-mediated bacterium containing the same oFBP gene (MciHispS, NnCPH, AnNPGA, H3HV2, LuzV4 as shown in Table 1) was constructed, and the Agrobacterium-mediated bacterium was injected into white roses of the same size.

[0083] The results showed that a weak luminescence signal could only be detected 48-72 hours after injection of the Agrobacterium engineered strain. The luminescence intensity (measured) was barely observable to the naked eye, and the camera could not capture the light signal. Petals injected with the Agrobacterium suspension withered after three days of expression, while white roses treated with the method provided in this application exhibited significant and prolonged luminescence. The petal condition and luminescence were compared... Figure 8 As shown.

Claims

1. A method for preparing a continuously self-luminous plant, comprising the following steps: (1) Construction of recombinant self-luminescent brewing yeast ( Saccharomyces cerevisiae Strain: The nucleotide sequence encoding the FBP pathway gene and / or NPGA gene is integrated into the genome of a Saccharomyces cerevisiae strain capable of synthesizing caffeic acid to obtain a Saccharomyces cerevisiae strain; (2) Preparation of bacterial suspension: Activate the brewing yeast strain constructed in step (1), expand the culture until the bacterial suspension is saturated, adjust the bacterial suspension concentration to OD600=2.0-3.0, and add caffeic acid; (3) Introducing into plants: Introducing the bacterial suspension containing caffeic acid prepared in step (2) into the organs or tissues of plants.

2. The preparation method according to claim 1, wherein step (1) is to integrate the nucleotide sequences encoding the NPGA gene or its variant, the HispS gene or its variant, the CPH gene or its variant, the H3H gene or its variant, and the Luz gene or its variant into the genome of a Saccharomyces cerevisiae strain capable of synthesizing caffeic acid, thereby obtaining a Saccharomyces cerevisiae strain; Preferably, the caffeic acid concentration is at least 40 mM; Preferably, the brewer's yeast is cultured using yeast peptone glucose medium.

3. The preparation method according to claim 1 or 2, wherein the method of introducing the plant includes injection or vacuum immersion; Preferably, the concentration of the bacterial suspension in step (2) is OD600 = 2.0; Preferably, the vacuum treatment conditions for the vacuum immersion method are 40 kPa pressure for 10 s.

4. The preparation method according to any one of claims 1-3, wherein the plant includes cut roses ( Rosa hybrida Phalaenopsis orchids Phalaenopsis ),lily( Lilium ), succulent plants ( Suculenta Five generations under one roof ( Solanum mammosum More preferably, the plant is a cut rose; More preferably, step (3) involves introducing the bacterial suspension into the petals of the plant.

5. As claimed in any one of claims 1-4, the gene encoding the NPGA gene is derived from Aspergillus nidulans (… Aspergillus nidulans ); Preferably, the gene encoding the HispS gene originates from the genus Mycena (…). Mycena Fungi; Preferably, the gene encoding the CPH gene originates from *Gymnocladus mukorossi* (a type of fungus). Neonothopanus nambi ); Preferably, the gene encoding the H3H gene originates from *Gymnospermum glabra*. Preferably, the gene encoding the Luz gene is derived from *Gymnotrichum candida*. Preferably, the genome of the *Saccharomyces cerevisiae* strain contains a nucleotide sequence encoding the NPGA gene as shown in SEQ ID NO: 1; Preferably, the genome of the *Saccharomyces cerevisiae* strain contains a nucleotide sequence encoding the HispS gene as shown in SEQ ID NO: 2; Preferably, the genome of the *Saccharomyces cerevisiae* strain contains a nucleotide sequence encoding the CPH gene as shown in SEQ ID NO: 3; Preferably, the genome of the *Saccharomyces cerevisiae* strain contains a nucleotide sequence encoding the H3H V2 gene as shown in SEQ ID NO: 4; Preferably, the genome of the Saccharomyces cerevisiae strain contains a nucleotide sequence encoding the Luz V4 gene as shown in SEQ ID NO:

5.

6. The preparation method according to any one of claims 1-5, wherein the nucleotide sequences encoding the NPGA gene, HispS gene, CPH gene, H3H gene and / or Luz gene are expressed by an inducible promoter; preferably, the inducible promoter includes a galactose-induced promoter, a doxycycline-induced promoter, an estrogen-induced promoter, etc.; specifically, the galactose-induced promoter includes GAL1 and GAL10.

7. A self-luminous plant prepared by any one of claims 1-6.

8. The application of the self-luminous plant as described in claim 7 in nighttime landscape design, bio-art creation, environmental beautification, and science education.

9. The Saccharomyces cerevisiae strain used in the preparation method according to any one of claims 1-6.

10. The use of the Saccharomyces cerevisiae strain of claim 9 in the preparation of self-luminous plants or as a bioluminescent indicator.

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