Multi-gene optimized plant autofluorescence gene expression cassette and its application in crassulaceae succulent plants

Abstract

The application belongs to the technical field of genetic engineering, and specifically discloses a multi-gene optimized plant self-luminous gene expression cassette and application thereof in Crassulaceae succulent plants. Mycena citricolor Hispidin synthase genes from MchisPS , Luciferase genes from Neonothopanus nambi , NnluZS2 Substrate synthesis genes NnH3HS2 , NnCpHS2 , and Aspergillus nidulans Coenzyme synthesis genes from NPGAS2 are subjected to plant-preferred codon optimization, and under the regulation of a CaMV 35S promoter and a TMV Omega translation enhancer, a high-efficiency expression gene cassette is constructed. Echeveria'Malgan' The gene cassette is introduced into a binary vector, and a Crassulaceae succulent plant (such as Echeveria pulvinata) is transformed by an Agrobacterium-mediated method. The obtained transgenic plant can autonomously and continuously emit stable green light without an external substrate, the luminous intensity is significantly improved, and the transgenic plant is genetically stable. The application first realizes the autonomous bioluminescence of a succulent plant, and provides a new germplasm for ornamental horticulture and biological lighting.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving multi-gene optimized plant bioluminescent gene expression cassettes and their application in Crassulaceae succulents, particularly involving genetic transformation and bioluminescent trait improvement methods for Crassulaceae succulents. Background Technology

[0002] Bioluminescence is a widespread phenomenon in nature, found in everything from fireflies and deep-sea fish to various fungi, and its molecular mechanisms have been elucidated. In recent years, plant bioluminescence technology based on fungal luciferase systems has made groundbreaking progress. In 2017, Kaskova et al. (Proc Natl Acad Sci USA, 2017) first revealed the metabolic cycle of hispidin, derived from caffeic acid, and its oxidation product luminescent substrate in Basidiomycetes fungi, and successfully achieved transient bioluminescence in tobacco. In 2020, Mitiouchkina et al. (Nat Biotechnol, 2020) further... Neonothopanus nambi The co-transformation of tobacco with the derived luciferase gene (nnLuz), substrate synthesis genes (nnHisPS, nnH3Z), and coenzyme synthesis gene (nnFP) achieved, for the first time, autonomous and continuous luminescence in a higher plant without the need for any exogenous substrate addition. This technical approach has since been validated in model plants such as Arabidopsis thaliana and tomato.

[0003] However, the application of fungal luciferase systems to succulents (especially Echeveria succulents in the Crassulaceae family) still faces the following technical obstacles: (1) The expression efficiency of heterologous genes in plants is low. Fungal genes contain a large number of rare codons that plants prefer, resulting in low transcript translation efficiency after direct transfer into plants. In addition, fungal luciferase proteins are prone to misfolding or degradation by proteases in plant cytoplasm, leading to weak or even undetectable luminescent signals.

[0004] (2) Multi-gene co-transformation is difficult. Fungal autoluminescence systems require the coordinated expression of at least 4 to 5 functional genes to form a complete metabolic circuit of "substrate synthesis - luciferase catalysis - coenzyme regeneration". There are technical bottlenecks in the assembly of large-fragment multi-genes, vector capacity limitations, and random integration of multiple copies of T-DNA.

[0005] (3) Genetic transformation of Crassulaceae succulents is difficult. Compared with model plants such as tobacco and Arabidopsis, Crassulaceae succulents (such as Echeveria) have a strong tendency to undergo wound-induced browning. Their leaves and explants are rich in polyphenol oxidase, and large-scale necrosis is easily caused after Agrobacterium infection. At the same time, succulents grow slowly and have a long regeneration cycle. The positive rate of conventional Agrobacterium-mediated transformation is low, making it difficult to obtain stable transgenic lines.

[0006] (4) Existing plant luminescence systems are not bright enough. Although the luminescent tobacco reported in the early period can emit light on its own, it is almost invisible to the naked eye under natural light, and the light intensity gradually decreases as the plant grows, which cannot meet the needs of ornamental horticulture and commercial applications.

[0007] To address the aforementioned issues, there is an urgent need in this field to provide a systematically optimized fungal autoluminescent gene expression system and a set of efficient genetic transformation methods suitable for Crassulaceae succulents, so as to obtain transgenic luminescent succulents with high luminescence intensity, genetic stability, and no need for exogenous substrates. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a fungal-derived self-luminescent gene expression cassette modified with codon optimization and regulatory elements, a plant expression vector containing the above-mentioned gene expression cassette, and their applications. The optimized gene expression cassette can efficiently drive the synergistic expression of key genes in the fungal luciferase system in Crassulaceae succulents, achieving autonomous and continuous luminescence without the addition of exogenous substrates. This invention has significant application value in the fields of ornamental horticultural plant development, bioindicators, and indoor green lighting.

[0009] The main technical problems solved by this invention include: (1) Solve the problem of low translation efficiency of luminescence-related genes from fungi in Crassulaceae succulents due to differences in codon preference; (2) Overcome the technical bottlenecks of easy browning of explants of Crassulaceae succulent plants and extremely low positive rate of Agrobacterium-mediated transformation; (3) To achieve coordinated and efficient expression of multiple genes (luciferase, substrate synthase, coenzyme synthase) in plants and enhance luminescence intensity; (4) Obtain new luminescent succulent plant strains that do not require exogenous substrates and can be stably inherited to meet the needs of ornamental and application purposes.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a fungal-derived plant autoluminescent gene expression cassette, the cassette comprising the following sequence optimized with plant-preferred codons: Source Mycena citricolor hispidin synthase gene MchisPSIts nucleotide sequence is shown in SEQ ID NO:1; derived from Neonothopanus nambi luciferase gene NnluZS2 Its nucleotide sequence is shown in SEQ ID NO:2; derived from Neonothopanus nambi Key enzyme genes for substrate synthesis NnH3HS2 and NnCpHS2 Their nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; derived from Aspergillus nidulans Coenzyme synthesis gene NPGAS2 Its nucleotide sequence is shown in SEQ ID NO:5.

[0011] Each of the above genes, individually or in combination, is regulated by the enhanced cauliflower mosaic virus 35S promoter (e35S) and the tobacco mosaic virus TMV Ω translation enhancer, forming the CaMV 35S-TMV Ω-ORF expression unit.

[0012] Preferably, each gene is driven by an independent CaMV 35S-TMV Ω expression unit and shares a plant terminator selected from the caustic acid synthase terminator (NOS terminator).

[0013] Secondly, the present invention provides a plant expression vector comprising the above-mentioned gene expression cassette, wherein the vector is a binary expression vector, preferably selected from the pCAMBIA series.

[0014] The key enzyme sequence of the bioluminescent system was cloned to BamH1 and Sac Between I restriction enzyme sites; the gene expression cassette is preferably cloned into a plant expression vector. HindIII and EcoR Between the I restriction enzyme sites. This invention does not specifically limit the preparation method of the recombinant vector; conventional preparation methods in the art can be used.

[0015] Thirdly, the present invention also provides a recombinant bacterial strain for reconstructing a key enzyme for bioluminescent synthesis, obtained by transforming the recombinant vector into Agrobacterium. In this invention, the recombinant vector is preferably transformed into Agrobacterium via electroporation. The present invention does not specifically limit the operation of the electroporation method; any operation known in the art can be used.

[0016] Fourthly, the present invention provides a breeding method for transgenic succulent plants, wherein the method involves transforming a plant expression vector into Agrobacterium, transforming succulent plant explants using Agrobacterium-mediated transformation, and obtaining transgenic succulent plants integrated with the self-luminescent gene expression cassette through screening and regeneration culture.

[0017] The transformation to Agrobacterium involves creating wounds at the explant bud points, pretreating with acetylsuccinone and using vacuum permeation assistance, preparing an infection solution MGL suitable for succulents, and adding mannitol to the culture medium to prevent browning of the explants.

[0018] More specifically, the above-mentioned transformation to Agrobacterium is as follows: Select explants with buds from the genus Echeveria of the Crassulaceae family (e.g., "wheat straw"), make shallow incisions at the base of the leaf buds until the parenchyma tissue is exposed, air dry at room temperature for 10-30 minutes, and then immerse them in OD. 600 The explants were inoculated in a 0.6-0.8 μg Agrobacterium suspension (containing 200 μmol / L acetylsalicylic acid and 0.1-0.3 mol / L mannitol) at 25°C for 10-20 minutes. The explants were then transferred to MS solid medium supplemented with 2,4-D and 6-BA and cultured in the dark at 20-25°C for 2-3 days. They were then transferred to a selection medium containing carbenicillin and hygromycin or hygromycin for subculture to obtain resistant adventitious shoots. Finally, rooting was induced, and transgenic plants were obtained through molecular identification. The Agrobacterium was Agrobacterium tumefaciens carrying the plant expression vector, with strain EHA105 being the preferred strain.

[0019] Fifthly, this invention provides the application of the aforementioned gene expression cassette in the cultivation of self-luminescent succulents. The self-luminescent gene expression cassette is stably integrated into the genome of the cultivated succulent, and can autonomously and continuously emit green light without the addition of exogenous substrates. The luminescence intensity is significantly improved compared to the unoptimized system, and it is genetically stable for at least a 6-month observation period.

[0020] Preferably, the succulent plant belongs to the genus *Echeveria* in the family Crassulaceae, and more preferably is wheat straw (…). Echeveria 'Malgan' The preferred organs in which the genetically modified succulent emits its own light within the organism include roots, stems, and leaves.

[0021] In a sixth aspect, the present invention provides the application of a fungal-derived plant self-luminescent system in ornamental horticulture, bioindication, or indoor green lighting.

[0022] In addition, this invention also provides a specific method for transfecting succulents with recombinant strains, including the following steps: 1) Selecting a 501 bp 35S-Ω constitutive promoter and a 253 bp NOS terminator, and constructing a recombinant strain containing... MchisPS , NnluZS2 , NnH3HS2 , NnCpHS2 and NPGAS2The five gene autoluminescence synthesis pathways in plants were used to form five independent expression cassettes: SmchisPS, SNnluZS2, SNnH3HS2, SNnCpHS2, and SNPGAS2, respectively; 2) the gene expression cassettes were tandemly linked into the plant expression vector pCAMBIA1301 to obtain a recombinant vector; 3) the recombinant vector was transformed into a host bacterium to obtain a recombinant strain; 4) the recombinant strain was used to infect and transform plants, and the gene expression status was determined by directly observing whether the transgenic plants emitted autoluminescence. Beneficial effects

[0023] Significantly improved expression efficiency: The plant-preferred codon optimization combined with the TMV Ω translation enhancer provided by this invention significantly improves the translation efficiency of fungal bioluminescent genes in Crassulaceae plants, and the bioluminescence intensity is increased several times compared with the unoptimized sequence, solving the technical problem of weak expression of heterologous genes.

[0024] Breakthrough in transformation success rate: The synergistic effect of acetylsyringone activating the vir gene, mannitol osmotic regulation protecting the explant, and bud wound targeting dividing cells effectively overcomes the obstacles of easy browning and necrosis and difficult regeneration in Crassulaceae plants, and for the first time achieved stable genetic transformation of succulent plants and obtained luminescent lines.

[0025] Autonomous and continuous luminescence: The multi-gene optimized expression cassette achieves a complete self-sustaining cycle of substrate synthesis, luciferase catalysis, and coenzyme regeneration. The plant can emit light continuously day and night without the need for any exogenous addition of substrates, and its genetic stability lasts for more than 6 months.

[0026] Original application value: This invention is the first to successfully apply a fungal self-luminous system to Crassulaceae succulents, providing new germplasm resources and technical support for the development of ornamental horticultural plants, bioindicators and indoor green lighting. Attached Figure Description

[0027] Figure 1 In Embodiment 1 of the present invention, AE respectively represent SmchisPS , SNnluZS2 , SNnH3HS2 , SNnCpHS2 and SNPGAS2 A schematic diagram of the structure of five independent expression boxes.

[0028] Figure 2 The self-luminescent synthesis pathway represented in Embodiment 1 of this invention SmchisPS SNnluZS2 SNnH3HS2 , SNnCpHS2 and SNPGAS2 A schematic diagram of the expression frame structure formed by the fusion of five independent expression boxes.

[0029] Figure 3This is a flowchart of the genetic transformation process of transgenic succulents in Example 2 of the present invention, where A and D represent wheat straw (… Echeveria 'Malgan' The stages of co-culture, callus induction, differentiation, and regeneration of plants are as follows:

[0030] Figure 4 Wild-type and the genetically modified wheat straw of this invention ( Echeveria 'Malgan' Phenotypic images under light, where A is the whole plant phenotype of wild-type (WT) succulents; B is the whole plant phenotype of wild-type succulents under dark conditions; C is the whole plant phenotype of transgenic wheat straw (…). Echeveria 'Malgan' Phenotypic diagram under light; D represents transgenic wheat straw ( Echeveria 'Malgan' Phenotypic diagram under dark conditions. Detailed Implementation

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1: Construction of a multi-gene plant expression vector 1. Optimized gene synthesis Optimization is carried out according to the following principles: (i) Optimize gene codons according to the codon preference of succulents to improve gene translation efficiency; (ii) Eliminate the recognition sites of commonly used restriction endonucleases inside the gene to facilitate expression cassette construction; (iii) Eliminate inverted repeat sequences, stem-loop structures and transcription termination signals to balance GC / AT inside the gene and improve RNA stability; (iv) Make the gene-encoded protein conform to the N-terminal principle to improve the stability of the translated protein; (v) Optimize the mRNA secondary structure free energy to improve gene expression efficiency.

[0033] This invention relates to the source Mycena citricolor hispidin synthase gene MchisPS Its nucleotide sequence is shown in SEQ ID NO:1; derived from Neonothopanus nambi luciferase gene NnluZS2 Its nucleotide sequence is shown in SEQ ID NO:2; derived from Neonothopanus nambi Key enzyme genes for substrate synthesis NnH3HS2 and NnCpHS2 Their nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; derived from Aspergillus nidulans Coenzyme synthesis gene NPGAS2The nucleotide sequence of the gene, as shown in SEQ ID NO:5, was optimized, and each gene was regulated by a combination of the enhanced cauliflower mosaic virus 35S promoter (e35S) and the tobacco mosaic virus TMV Ω translation enhancer, forming a CaMV 35S-TMV Ω-ORF expression unit. This significantly improved the catalytic efficiency of luciferase in plant cytoplasm, increasing the luminescence intensity several times compared to the wild type, providing a key molecular modification strategy for optimizing plant autoluminescence systems. It contains the following components: nnHisPS This gene encodes hispidin synthase, which catalyzes the synthesis of hispidin (a fungal luciferin precursor) using caffeic acid as a substrate. This enzyme is a key rate-limiting enzyme in the fungal bioluminescence pathway. The hispidin synthase gene is optimized based on plant-preferred codons. MchisPS Its nucleotide sequence is shown in SEQ ID NO: 1.

[0034] This invention utilizes the wild-type luciferase gene NnluZ By rationally designing and performing site-directed mutagenesis at specific sites, mutants can be obtained. NnluZS2 Specifically, replacing isoleucine at position 3 with serine (I3S) enhances the hydrophilicity of the N-terminal region of the protein, which is beneficial for correct post-translational folding; replacing phenylalanine at position 11 with leucine (F11L) reduces the probability of the protein being recognized and degraded by proteases in plant cells without changing the stability of the hydrophobic core; and replacing isoleucine at position 63 with threonine (I63T) introduces a polar side chain, optimizing the microenvironment near the active site and thus improving the affinity of the enzyme for the substrate 3-hydroxyhispidin. The synergistic mutations at these three sites significantly improve the catalytic efficiency of luciferase in plant cytoplasm, increasing the luminescence intensity several times compared to the wild type, providing a key molecular modification strategy for optimizing plant autoluminescence systems. Its nucleotide sequence is shown in SEQ ID NO: 2.

[0035] This invention further involved the rational design and site-directed mutagenesis of wild-type hispidin-3-hydroxylase (NnH3H) to obtain mutants. NnH3HS2 Specifically, replacing the aspartic acid at position 37 with glutamic acid (D37E) lengthens the acidic side chain and enhances its affinity for ferrous ions (Fe). 2+ The coordination ability of the cofactor is improved, thereby enhancing the catalytic efficiency of the active site of the hydroxylase; replacing valine at position 181 with isoleucine (V181I) fine-tunes the conformation of the substrate channel without significantly altering the hydrophobic core volume, facilitating the smooth entry of the substrate hispidin and the release of the product 3-hydroxyhispidin. The co-mutation at these two sites... NnH3HS2The hydroxylase activity in succulent plant cells was significantly enhanced compared to the wild type, providing a key guarantee for optimizing the efficiency of substrate synthesis pathways. Its nucleotide sequence is shown in SEQ ID NO: 3. NnCpHS (caffeoylpyruvate hydrolase) is responsible for catalyzing the hydrolysis of caffeoylpyruvate to regenerate caffeic acid in fungal bioluminescence systems, thereby achieving the recycling of luminescent substrates. It is a key gene for maintaining autonomous and continuous luminescence in plants. [The following sentence appears to be incomplete and requires further context: "Optimized based on plant-preferred codons..."] NnCpHS2 The gene, whose nucleotide sequence is shown in SEQ ID NO: 4.

[0036] Plant-preferred codon-optimized coenzyme synthesis genes NPGAS2 Its nucleotide sequence is shown in SEQ ID NO: 5.

[0037] 2. Construction of gene expression cassettes by Mycena citricolor of MchisPS Using the gene as a template, the DNA sequence shown in SEQ ID NO.1 was synthesized, cloned into a plasmid vector, and sequenced to determine its sequence; NnluZS2, NnH3HS2 of Neonothopanus nambi and NnCpHS2 Using the gene as a template, and based on the codon preference of succulents, the DNA sequences shown in SEQ ID NO. 2~4 were synthesized, cloned into a plasmid vector, and sequenced to determine their sequences; Aspergillus nidulans of NPGAS2 Using the gene as a template, and based on the codon preference of succulents, the DNA sequence shown in SEQ ID NO.5 was synthesized, cloned into a plasmid vector, and sequenced to confirm its sequence. A 501 bp constitutive strong promoter of 35SOmega [CaMV35S promoter + Omega translation enhancer] and a 253 bp NOS terminator of the carmine synthase gene were selected to control the expression of the exogenous gene. A five-gene autoluminescent synthetic pathway for reconstructing fungi in succulents was designed and constructed using our improved "overlap extension PCR technology." Five gene expression cassettes were thus constructed: SmchisPS, SNnluZS2, SNnH3HS2, SNnCpHS2, and SNPGAS2. The lengths of the expression cassettes were 5791 bp, 1558 bp, 2023 bp, 1624 bp, and 1789 bp, respectively (e.g., ...). Figure 1 ).

[0038] 3. Construction of multi-gene transformation vectors The five multi-gene expression cassettes obtained above were sequentially inserted between the EcoRI and HindIII restriction sites of the multiple cloning site of the pCAMBIA1301 vector. After each successful insertion, the restriction sites between the expression cassettes were removed, ultimately forming a five-valent gene plant expression vector. Figure 2This yields the recombinant plasmid pCAMBIA1301-FBP5.

[0039] Example 2 Genetic transformation of succulent plants 1. Acquisition and Identification of Transgenic Succulents 1.1 Preparation and electrocution of Agrobacterium 1) Select a single Agrobacterium tumefaciens EHA105 and inoculate it into 5 mL of LB liquid medium (50 μg / mL rifampicin and 50 μg / mL kanamycin), and incubate at 28℃ and 250 rpm for 20 h.

[0040] 2) Transfer 1 mL of bacterial culture to 30 mL of LB liquid medium (rifampicin 50 μg / mL, kanamycin 50 μg / mL), incubate at 28℃ and 250 rpm for 12 h, and measure OD. 600 ≈1.5.

[0041] 3) Collect bacterial cells by centrifugation at 8000 rpm, 4°C, and 10 min. Resuspend the cells in Agrobacterium-mediated transformation osmosis buffer (5 wt% sucrose, 0.05 wt% Silwet L-77) and dilute to OD200. 600 ≈0.8.

[0042] 4) Resuspend the bacterial cells thoroughly in 0.5 times the volume of sterile water pre-cooled at 4°C, centrifuge at 3500g for 10 minutes, and carefully discard the supernatant. Resuspend the bacterial cells thoroughly in 2 mL of sterile 10% glycerol pre-cooled at 4°C. This bacterial solution can be used immediately or frozen at -80°C for storage.

[0043] 5) Add 40 μL of bacterial culture and 100 ng of recombinant plasmid to 1.5 mL of Eppendorf solution, mix well in a 0.2 cm diameter electroporation cuvette, and incubate on ice for 1 minute. Adjust the electroporation parameters to 25 μF, 2.5 kV / cm, and 400 Ω. Electroporate for 4–5 msec.

[0044] 6) After electroporation, 1.0 mL of expression culture medium was added to the bacterial cells, and the mixture was incubated at 29°C for 1 hour. The culture was then plated onto YEB plates containing antibiotics (rifampicin 50 μg / mL + kanamycin 50 μg / mL). Using the above transformation procedure, the plant expression vector pCAMBIA1301-FBP5 was electroporated into Agrobacterium tumefaciens EHA105 to obtain Agrobacterium tumefaciens strain EHA105 (pCAMBIA1301-FBP5).

[0045] 1.2 Agrobacterium-mediated genetic transformation of succulents 1) Collection of Agrobacterium tumefaciens Agrobacterium EHA105 carrying the plant expression vector (pCAMBIA1301-FBP5) was picked from YEB plates and inoculated into 5 mL of YEB liquid medium containing 50 mg / L kanamycin. The medium was then cultured on a shaker at 200 rpm at 28°C until OD (out of control) was reached. 600 =0.5, then re-inoculated into fresh YEB medium at a ratio of 1:100 and cultured with shaking. The antibiotics in the YEB medium and the culture conditions were the same as before. When OD 600 When the concentration of bacteria reaches 0.5, the bacterial culture is centrifuged at a relative centrifugal force of 6000×g and 4℃ for 10 minutes. The Agrobacterium cells are collected and resuspended in MGL liquid to OD. 600 =0.5, to prepare Agrobacterium suspension for subsequent infection.

[0046] The YEB culture medium formula is as follows: The MGL infection solution is formulated as follows: 5 g / L tryptone + 0.1~0.3 mol / L mannitol + 5 g / L NaCl + 1 g / L glycine + 0.1 g / L MgSO4·7H2O + KH2PO4, with the remainder being water, and the pH adjusted to 5.8.

[0047] 2) Agrobacterium infection of succulent leaves Use tweezers to take sterile, healthy wheat straw ( Echeveria 'Malgan' Mature leaves were used as explants, with the leaf base buds intact. One or two shallow incisions were made at the base of the axillary bud / terminal bud with a sterile scalpel until the thin-walled tissue was exposed, without penetrating the explant. The leaves were then air-dried at room temperature for 15 minutes.

[0048] The activated Agrobacterium containing pCAMBIA1301-FBP5 was resuspended in the infection solution (MGL, OD). 600 =0.6-0.8, containing 200 μmol / L acetylsyringone + 0.1~0.3 mol / L mannitol), immerse the explants, gently shake for 10-20 min at 25℃, then use vacuum infiltration at 0.05~0.1 MPa for 3-5 minutes, then discard the bacterial solution. Blot off excess bacterial solution from the succulent surface with sterile filter paper, transfer to a co-culture medium (with a sterile filter paper placed on top to prevent excessive growth of Agrobacterium), with the underside of the leaves facing down, and incubate in the dark at 25℃ for 3 days (avoid light to prevent inhibition of Agrobacterium activity).

[0049] After co-culturing, the explants were transferred to sterile water containing carbenicillin (250 mg / L) and washed three times with shaking (3-5 min each time) to thoroughly remove residual Agrobacterium. The explants were then transferred to culture dishes containing sterile filter paper, excess water was blotted out, and the explants were transferred to selection medium and incubated at 25°C in a 16-h light / 8-h dark incubator, with subculturing every two weeks. During this period, non-transformed cells gradually yellowed and died, while transformed cells (containing resistance genes) at the base of the succulent formed green callus or buds.

[0050] Once obvious buds appear, the explants are transferred to differentiation medium to induce bud differentiation (25℃, light culture). When the buds grow to 2-3 cm, individual buds are cut off and transferred to rooting medium to induce rooting. After 2-3 weeks, complete regenerated plants are formed. Finally, these seedlings are cultured in soil for subsequent experiments.

[0051] Co-culture medium: MSB + mannitol 18g / L + 6-BA 0.5mg / L + NAA 0.1mg / L + acetylsyringone (AS) 100 μmol / L + 8g / L agar powder, pH adjusted to 5.8; Screening medium: MSB + mannitol 18g / L + 6-BA 1.0mg / L + NAA 10mg / L + KT 1.0mg / L + hygromycin (Hyg) 35 mg / L + carbenicillin (cb) 500 mg / L + 8g / L agar powder, pH adjusted to 5.8; Differentiation medium: MSB (remove ammonium nitrate, increase potassium nitrate) + mannitol 18g / L + 6-BA 0.5mg / L + NAA 4mg / L + hygromycin (Hyg) 25 mg / L + carbenicillin (cb) 500 mg / L + 8g / L agar powder, pH adjusted to 5.8; Rooting medium: 1 / 2 MS medium + 0.1 mg / L NAA + 8 g / L agar powder, pH adjusted to 5.8.

[0052] Example 3: Identification of Genetically Modified Succulents This invention enables multi-level identification of the obtained transgenic succulent plants. After successfully expressing the optimized and modified recombinant plasmid of the bioluminescence system into the succulent plants, the gene expression status can be determined by directly observing the presence and location of bioluminescence under dark conditions. Furthermore, the bioluminescence phenotype of the plants is detected by high-sensitivity CCD imaging after dark adaptation, screening out positive lines that emit visible green light. Subsequently, genomic DNA is extracted for PCR amplification to verify the exogenous gene. MchisPS , NnluZS2 , NnH3HS2 , NnCpHS2 and NPGAS2The expression of five genes was analyzed. The results showed that the wild control did not produce any self-luminescence under dark conditions, while the positive transgenic succulents showed a strong green glow throughout the entire plant, especially in the growing point and young tissues where the luminescence signal was strongest.

[0053] As demonstrated by the above embodiments, this invention successfully reconstructed a fungal-derived autoluminescent synthesis pathway in succulent plants of the genus *Echeveria* in the Crassulaceae family using a combined regulatory element of the enhanced CaMV 35S promoter and the TMV Ω translation enhancer. The expression performance of this optimized system in heterologous hosts was also systematically identified. The results showed that the FBP5 gene system (…) optimized with plant-preferred codons and mutated at key sites… MchisPS , NnluZS2 , NnH3HS2 , NnCpHS2 , NPGAS2 This invention achieved functional expression in transgenic succulents, enabling the plants to autonomously and continuously emit green fluorescence without the addition of exogenous substrates. The luminescence intensity was significantly higher than that of the unoptimized system, and the fluorescence remained genetically stable for at least a 6-month observation period. The resulting transgenic succulents exhibited a clearly visible green luminescence phenotype after dark adaptation, with the strongest luminescence signal observed particularly in the growing points and young tissues, demonstrating significant ornamental horticultural value. This invention not only creates a novel germplasm of autonomously luminescent succulents with stable inheritance, providing entirely new materials for plant bioluminescence and ornamental horticulture, but also verifies the effectiveness of codon optimization combined with the TMVΩ translation enhancer in overcoming the expression barriers of fungal heterologous genes in plants, providing a technical reference for the reconstruction of other fungal secondary metabolic pathways in plants.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multi-gene optimized plant autoluminescent gene expression cassette, characterized in that, The gene expression cassette contains the following element: a plant-preferred codon-optimized hispidin synthase gene. MchisPS Its nucleotide sequence is shown in SEQ ID NO: 1; Plant-preferred codon-optimized luciferase gene NnluZS2 Its nucleotide sequence is shown in SEQ ID NO:2; Substrate synthesis genes with plant-preferred codon optimization NnH3HS2 and NnCpHS2 Their nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; Plant-preferred codon-optimized coenzyme synthesis genes NPGAS2 Its nucleotide sequence is shown in SEQ ID NO: 5; Each of the above genes is regulated by a combination of the cauliflower mosaic virus CaMV 35S promoter and the tobacco mosaic virus TMVΩ translation enhancer.

2. The gene expression cassette according to claim 1, characterized in that, The CaMV 35S promoter is an enhanced double-copy 35S promoter, and the TMVΩ translation enhancer is the 5′-untranslated region sequence of the TMV U1 strain, inserted between the promoter and the ATG start codon of the target gene.

3. The gene expression cassette according to claim 1, characterized in that, Each gene in the gene expression cassette is driven by an independent CaMV35S–TMVΩ expression unit and shares a plant terminator selected from the carmine synthase terminator.

4. A plant expression vector comprising the self-luminescent gene expression cassette according to any one of claims 1 to 3, characterized in that, The vector is a binary expression vector.

5. A breeding method for transgenic succulent plants, characterized in that, The plant expression vector described in claim 4 is transformed into Agrobacterium, and the explants of succulent plants are transformed using the Agrobacterium-mediated transformation method. After screening and regeneration culture, transgenic succulent plants integrating the gene expression cassette are obtained.

6. The method according to claim 5, characterized in that, The succulent plant in question is *Sedum morganianum*, belonging to the Crassulaceae family.

7. The application of the gene expression cassette according to any one of claims 1 to 3 in the cultivation of self-luminous succulents.

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