Method for improving the intensity of spontaneous light of plants

Abstract

The present application relates to a system capable of improving the self-luminous intensity of plants, comprising: a nucleus expression vector active in the nucleus of plant cells, the nucleus expression vector comprising a fungal bioluminescence pathway (FBP) gene or an improved fungal bioluminescence pathway (FBP') gene; and a chloroplast expression vector active in the chloroplast of plants, the chloroplast expression vector comprising enzymes for synthesizing 3-aryl acrylic acid. The present application increases the content of coffee acid and milk tree base in plants, provides more sufficient precursor materials for the fungal bioluminescence pathway, and significantly improves the self-luminous intensity of plants. The present application breaks through the bottleneck of the existing precursor supply of self-luminous plants, and provides an innovative solution for creating high-efficiency and stable self-luminous plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for enhancing the intensity of plant autoluminescence. Background Technology

[0002] Plant bioluminescence technology has broad application prospects in urban landscaping, home gardening, and biosensors. Fungal bioluminescence pathway (FBP) is a system that uses caffeic acid as a substrate to produce green fluorescence through a series of enzymatic reactions. However, the supply of endogenous caffeic acid in plants is often a key factor limiting the luminescence efficiency of FBP. Furthermore, the expression levels and stability of transformed genes using existing genetic engineering methods are affected by position effects and gene silencing, thus limiting the accumulation of caffeic acid and the enhancement of luminescence intensity within plant cells. Summary of the Invention

[0003] To address the technical problems existing in the prior art, this invention proposes a system for enhancing the autoluminescence intensity of plants, comprising: a nuclear expression vector active in the plant cell nucleus, the nuclear expression vector comprising a fungal bioluminescence pathway (FBP) gene or a modified fungal bioluminescence pathway (FBP') gene; and a chloroplast expression vector active in the plant chloroplast, the chloroplast expression vector comprising an enzyme for synthesizing 3-arylacrylic acid.

[0004] In the system described above, the enzyme of 3-arylacrylic acid is selected from the group consisting of tyrosine ammonia-lyase (TAL), the HpaB and HpaC components of h4-hydroxyphenylacetate 3-monooxygenase reductase, 3-deoxy-D-arabinohepulose-7-phosphate synthase (AroG), phenylalanine ammonia-lyase (C3H), and enzymes of the cytochrome P450 monooxygenase family; preferably, the enzyme of the cytochrome P450 monooxygenase family is selected from p-coumaroyl-CoA 3-hydroxylase and cytochrome P450 monooxygenase (P450); preferably, the amino acid sequence of P450 is as shown in any of SEQ ID NO. 74-83; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO. 74-83; the amino acid sequence of C3H is as shown in SEQ ID NO. 85; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO. 74-83; The amino acid sequence shown in NO. 85 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology; the amino acid sequence of AroG is shown in any of SEQ ID NO. 87-104; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with any of the amino acid sequences shown in SEQ ID NO. 87-104; the amino acid sequence of TAL is shown in any of SEQ ID NO. 58-72; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with any of the amino acid sequences shown in SEQ ID NO. 58-72.

[0005] In the system described above, the 3-arylacrylic acid is selected from the group consisting of caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

[0006] In the system described above, the FBP or FBP' includes one or more of the following genes: luciferase (LUZ), caffeoylpyruvate hydrolase (CPH), milk alkaloid hydroxylase (H3H), and an enzyme that catalyzes the synthesis of milk alkaloid from caffeic acid; preferably, the amino acid sequence of the CPH is as shown in SEQ ID NO. 22; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO. 22; (the amino acid sequences PKS, LUZ, etc., are to be numbered here and below, and are intended to be included in almost all currently disclosed functional sequences) preferably, the amino acid sequence of the H3H is as shown in any of SEQ ID NO. 44-56; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in any of SEQ ID NO. 44-56.

[0007] In the system described above, the enzyme catalyzing the synthesis of milk alkaloids from caffeic acid is milk alkaloid synthase (Hisps) or polyketide synthase (PKS); preferably, the amino acid sequence of Hisps is as shown in SEQ ID NO. 24; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO. 24; preferably, the amino acid sequence of PKS is as shown in any one of SEQ ID NO. 26-42; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in any one of SEQ ID NO. 26-42.

[0008] In the system described above, the amino acid sequence of the luciferase may or may not be modified; the modified amino acid sequence of the luciferase may be one or more of the following modifications: the amino acid sequence of the luciferase has 1-60 amino acids deleted from its N-terminus; the amino acid sequence of the luciferase has a transmembrane-compatible guide peptide sequence tandemly at its N-terminus; the amino acid sequence of the luciferase has a retardation peptide tandemly at its C-terminus; the amino acid sequence of the luciferase includes one or more site mutations; preferably, the amino acid sequence of the luciferase is as shown in any of SEQ ID NO. 2-20; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequences shown in any of SEQ ID NO. 2-20.

[0009] The system described above is used for converting genes into the nucleus of plant cells using a nuclear expression vector for nuclear localization of genes; preferably, the nuclear expression vector is pCAMBIA2300.

[0010] The system described above includes chloroplast-localizing genes as expression vectors for converting genes into plant chloroplasts.

[0011] Applications of any of the systems described above in the preparation of reagents or kits for enhancing the autoluminescence intensity of plants.

[0012] Methods for enhancing plant autoluminescence intensity using any of the systems described above include: converting a fungal bioluminescence pathway (FBP) gene or a modified fungal bioluminescence pathway (FBP') gene into the plant cell nucleus; and converting an enzyme for the synthesis of 3-arylacrylic acid into the plant cell chloroplast.

[0013] This application increases the content of caffeic acid and milkweed alkaloids in plants, providing more abundant precursor substances for the fungal bioluminescence pathway and significantly enhancing the self-luminescence intensity of plants. This invention overcomes the bottleneck in the supply of precursors for existing self-luminescent plants, providing an innovative solution for creating efficient and stable self-luminescent plants. Attached Figure Description

[0014] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein: Figure 1 This is a schematic diagram of metabolic pathway optimization according to an embodiment of this application; wherein, 1. Plant cell represents a plant cell, 2. Chloroplast represents a chloroplast, 3. Cytosol represents cytosol, 10. CCoAOMT represents caffeoyl coenzyme A, 11. Phenylalanine represents phenylalanine, 12. Tyrosine represents tyrosine, 13. Cinnamic acid represents cinnamic acid, 14. p -Coumaric acid represents p-coumaric acid, 15.Caffeic acid represents caffeic acid, 16.AroG geng represents the Arog gene, 17.P450 gene represents the P450 gene, 18.Fungal bioluminescence pathway represents the fungal bioluminescence pathway, 19.FBP system represents the FBP system, 20.Luciferase represents luciferase, 21.Luciferin represents luciferin, and 22.Light emission represents light emission; Figure 2A This is a schematic diagram of the construction of a nuclear expression vector for the FBP gene according to an embodiment of this application; Figure 2B This is a schematic diagram of the construction of a nuclear expression vector for the eFBP gene according to an embodiment of this application; Figure 3A This is a schematic diagram of the construction of chloroplast expression vectors of Arog, P450 and TAL according to an embodiment of this application; Figure 3B This is a schematic diagram of the construction of chloroplast expression vectors of Arog, C3H and TAL according to an embodiment of this application; Figure 4 These are images of plant bioluminescence after transformation with different carriers according to an embodiment of this application; Figure 5 This is a statistical graph of the luminescence intensity of plants after transformation with different carriers according to an embodiment of this application; Figure 6 This is a statistical chart showing the cumulative caffeic acid in plants after transformation with different carriers according to an embodiment of this application. Detailed Implementation

[0015] 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.

[0016] 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.

[0017] Traditional genetic engineering methods typically introduce all heterologous genes into plants via nuclear transformation. This application breaks with conventional genetic engineering methods by transforming fungal bioluminescence system (FBP)-related genes into the plant cell nuclear genome, while simultaneously introducing key genes in the 3-arylacrylic acid synthesis pathway into the plant chloroplast genome via chloroplast transformation. This significantly increases the accumulation of caffeic acid in plant cells, thereby enhancing the plant's bioluminescence intensity.

[0018] The 3-arylacrylic acid mentioned in this article is selected from the following group: caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferous acid, sinapic acid, and ferulic acid.

[0019] Intracellularly, 3-arylacrylic acid is synthesized via relevant enzymes. The enzymes used to synthesize 3-arylacrylic acid are selected from the group consisting of tyrosine ammonia-lyase, the HpaB and HpaC components of h4-hydroxyphenylacetate 3-monooxygenase reductase, 3-deoxy-D-arabinohepenoyl-7-phosphate synthase (AroG), phenylalanine ammonia-lyase (C3H), and enzymes of the cytochrome P450 monooxygenase family. In some embodiments, enzymes of the cytochrome P450 monooxygenase family are selected from p-coumaroyl-CoA 3-hydroxylase and cytochrome P450 monooxygenase (P450).

[0020] In some embodiments, the enzyme used to synthesize 3-arylacrylic acid is selected from the following combinations: TAL and P450 combination, AroG, TAL and P450 combination, TAL and C3H combination, AroG, TAL, and C3H combination, TAL and C3H combination, P450 and C3H combination, TAL and HpaB, HpaC combination, or other combinations of enzymes that can increase the accumulation of 3-arylacrylic acid in plant cells.

[0021] Those skilled in the art should understand that this application is intended to provide a method for enhancing the luminescence intensity of luminescent plants. The enzymes used to synthesize 3-arylacrylic acid or combinations of enzymes used to synthesize 3-arylacrylic acid are only used to illustrate the technical effects of this application and are not intended to limit the technical solution of this application.

[0022] TAL, as used in this application, refers to tyrosine ammonia-lyase, a key regulator of the caffeic acid synthesis pathway. It catalyzes the breakdown of tyrosine into p-coumaric acid, thereby promoting the synthesis and accumulation of downstream caffeic acid. In some embodiments, the amino acid sequence of TAL is as shown in any of SEQ ID NO. 58-72; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequences shown in any of SEQ ID NO. 58-72. In some embodiments, the nucleotide sequence of TAL is as shown in SEQ ID NO. 57.

[0023] The P450 mentioned in this application refers to a superfamily of oxidases with heme (iron protoporphyrin IX) as a cofactor, named for the characteristic absorption peak at 450 nm that occurs when the reduced state binds to CO₂. In fungal bioluminescent systems (FBP), specific P450 enzymes are responsible for the key hydroxylation step in the caffeic acid cycle. In some embodiments, the amino acid sequence of P450 is as shown in any of SEQ ID NO. 74-83; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequences shown in any of SEQ ID NO. 74-83. In some embodiments, the nucleotide sequence of P450 is as shown in SEQ ID NO. 73.

[0024] The C3H described in this application belongs to the cytochrome P450 monooxygenase superfamily and is a key hydroxylase in the plant phenylpropanoid metabolic pathway. In the presence of NADPH and molecular oxygen, it specifically catalyzes the hydroxylation of the benzene ring at position 3 of coumaric acid (4-hydroxycinnamic acid) to generate caffeic acid (3,4-dihydroxycinnamic acid). In some embodiments, the amino acid sequence of C3H is as shown in SEQ ID NO. 85; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence shown in SEQ ID NO. 85. In some embodiments, the nucleotide sequence of C3H is as shown in SEQ ID NO. 84.

[0025] In this application, AroG refers to 3-deoxy-D-arabinohepenosyl-7-phosphate synthase (DAHP synthase). In some embodiments, the amino acid sequence of AroG is as shown in any of SEQ ID NO. 87-104, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequences shown in any of SEQ ID NO. 87-104. In some embodiments, the nucleotide sequence of AroG is as shown in SEQ ID NO. 86.

[0026] The chloroplast genome has advantages such as multiple copies, high expression levels, no gene silencing, and maternal inheritance, making it an ideal target for plant genetic engineering. Figure 1 This is a schematic diagram of metabolic pathway optimization according to an embodiment of this application, illustrating the phenylpropane metabolic pathway and the fungal bioluminescence pathway, and highlighting how the expression of AroG, TAL, and P450 genes in chloroplasts promotes caffeic acid synthesis, thereby enhancing the luminescence efficiency of the FBP system. Figure 1As shown, introducing key genes of the caffeic acid synthesis pathway into chloroplasts is expected to achieve efficient expression of these genes, thereby significantly increasing the accumulation of caffeic acid and enhancing the bioluminescence intensity of plants.

[0027] In this application, FBP refers to a fungal bioluminescent system, which is a set of enzymatic reaction modules derived from luminescent fungi. These luminescent fungi include species such as *Armillaria* and *Schefflera* within the Agaricales class. FBP utilizes caffeic acid as a substrate and, under the catalysis of fungal luciferase (LUZ), involves NAD(P)H and oxygen. Molecular oxygen is activated through the histidine-tyrosine active site, forming a high-energy peroxide intermediate. This intermediate is ultimately oxidatively cleaved to generate 3-hydroxyhistidine, releasing green light (peak wavelength approximately 520–530 nm). This complete bioluminescent mechanism distinguishes it from bacterial luciferase systems. Product synthesis does not depend on long-chain aldehydes, and the milk tree hydroxylase (Hispidin-3-hydroxylase, H3H) involved in the caffeic acid cycle works synergistically with luciferase to ensure continuous substrate regeneration.

[0028] In some embodiments, FBP includes luciferase (LUZ), caffeoyl pyruvate hydrolase (CPH), hispidin-3-Hydroxylase (H3H), and an enzyme that catalyzes the synthesis of milk alkaloids from caffeic acid. In some embodiments, the enzyme that catalyzes the synthesis of milk alkaloids from caffeic acid is hispidin synthase (Hisps) or polyketide synthases (PKS).

[0029] nFBP refers to a vector carrying FBP-related genes that can be transformed into the cell nucleus. Here, n represents the cell nucleus.

[0030] cFBP refers to a vector carrying FBP-related genes that can be transformed into chloroplasts. Here, 'e' represents chloroplast.

[0031] In this application, eFBP refers to an improved FBP system that enhances caffeic acid accumulation in plant cells and increases plant luminescence intensity. Here, 'e' stands for enhancement.

[0032] In some embodiments, the eFBP includes optimized LUZ, CPH, Hisps or PKS, and H3H. In some embodiments, the eFBP includes the guide peptide-LUZ', CPH, Hisps or PKS, and H3H. In some embodiments, the eFBP includes optimized or unoptimized LUZ, P450 peptide-LUZ, CPH, Hisps or PKS, and H3H. In some embodiments, the eFBP includes a mutant of any of the genes LUZ, CPH, Hisps or PKS, and H3H. In some embodiments, the eFBP includes P450 peptide-LUZ, PKS, CPH, H3H, and LUZ. In some specific embodiments, the eFBP includes P450 peptide-Δ39nnLUZ_v2, PKS, CPH, H3H, and Δ19nnLUZ.

[0033] neFBP refers to a vector that carries eFBP-related genes and can transform these genes into the cell nucleus.

[0034] ceFBP refers to a vector that carries eFBP-related genes and can transform these genes into chloroplasts.

[0035] LUZ refers to a monooxygenase with a unique folding structure. 3-hydroxymilk alkaloids are oxidized to luciferin under the catalysis of luciferase, producing luciferin and emitting light. In this application, LUZ is further improved by truncating, replacing parts of its amino acid sequence, incorporating other gene sequences, or mutating key sites. For ease of distinction, the modified or unmodified luciferase is designated as LUZ, and the modified luciferase is designated as LUZ'. In some embodiments, truncating LUZ typically involves removing 1-50 amino acids from the N-terminus of the LUZ amino acid sequence. In some embodiments, the LUZ sequence includes one or more site mutations. In some embodiments, the amino acid sequence of LUZ is as shown in any of SEQ ID NO. 2-15, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO. 2-15. In some embodiments, the nucleotide sequence of LUZ is as shown in SEQ ID NO. 1.

[0036] In some embodiments, the N-terminal truncated form of LUZ is linked to a guide peptide. In some embodiments, the amino acid sequence of the N-terminal truncated form of LUZ linked to the guide peptide, LUZ'-1, is as shown in any of SEQ ID NO. 16-20, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequences shown in any of SEQ ID NO. 16-20.

[0037] In this context, CPH refers to an enzyme capable of catalyzing the oxidation of luciferin by fungi into simpler compounds, such as precursors for proluciferin. For example, it can catalyze the conversion of caffeoylpyruvate to caffeic acid. Under the catalysis of CPH, caffeoylpyruvate is converted into caffeic acid, thereby regenerating caffeic acid and achieving cyclic luminescence. In some embodiments, the amino acid sequence of CPH is as shown in SEQ ID NO. 22, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO. 22. In some embodiments, the nucleotide sequence of CPH is as shown in SEQ ID NO. 21.

[0038] In this paper, PKS is used to describe enzymes belonging to the group of type III polyketide synthases capable of catalyzing the synthesis of hispidin from caffeoyl-CoA. Caffeic acid, under the catalysis of Hisps or PKS, combines with propionyl-CoA to generate hispidin.

[0039] In some embodiments, the amino acid sequence of Hisps is as shown in SEQ ID NO. 24, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO. 24. In some embodiments, the nucleotide sequence of Hisps is as shown in SEQ ID NO. 23.

[0040] In some embodiments, the amino acid sequence of PKS is as shown in any of SEQ ID NO. 26-42, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO. 26-42. In some embodiments, the nucleotide sequence of PKS is as shown in SEQ ID NO. 25.

[0041] In this document, H3H refers to an enzyme that catalyzes the conversion of pre-luciferin to fungal luciferin. Milkweed alkaloids undergo hydroxylation under the action of H3H to generate 3-hydroxymilkweed alkaloids. In some embodiments, the amino acid sequence of H3H is as shown in any of SEQ ID NO. 44-56, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO. 44-56. In some embodiments, the nucleotide sequence of H3H is as shown in SEQ ID NO. 43.

[0042] Those skilled in the art should understand that this application is intended to provide a method for enhancing the luminescence intensity of luminescent plants. The above FBP or eFBP is only used to illustrate the technical effects of this application and is not intended to limit the technical solution of this application.

[0043] The organisms referred to in this article are cellular organisms that can absorb foreign genes and express them in their cells. In some embodiments, the organisms are plants, animals, bacteria, fungi, etc.

[0044] The technical solution of this application will be described below through specific embodiments. The following embodiments are merely one implementation method for illustrating the technical solution and do not limit the scope of protection of this application.

[0045] Example 1: Construction of plant expression vectors This application does not limit the construction method of plant expression vectors; any construction method that can achieve the technical effects of this application can be used. In this embodiment, the constructed vectors include nuclear expression vectors and chloroplast expression vectors. The nuclear expression vector is constructed based on pCAMBIA2300, with a 35S promoter, and the vector is as follows: Figures 2A-2B As shown; the chloroplast expression vector was constructed based on standard tobacco ( Nicotiana tabacum Chloroplast homologous recombination transformation vectors, such as Figures 3A-3B As shown, its left arm includes the rps14-trnfM-rps14A region, and its right arm includes the trnG-3' partpsbZ region, with the promoter being PrrnG10L.

[0046] Construction of vector 1 (hereinafter referred to as nFBP): The genes nnLUZ (as shown in SEQ ID NO.1), Hisps (as shown in SEQ ID NO.23), CPH (as shown in SEQ ID NO.21), and H3H (as shown in SEQ ID NO.43) were cloned into the plant binary expression vector pCAMBIA2300 to construct an expression cassette containing a 35S promoter and a NOS terminator, as shown in the figure. Figure 2A As shown.

[0047] Vector 2 (hereinafter referred to as cFBP) construction: Based on the characteristics of plant chloroplast genomes, a chloroplast transformation vector was designed. The nnLUZ (as shown in SEQ ID NO.1), Hisps (as shown in SEQ ID NO.23), CPH (as shown in SEQ ID NO.21), and H3H (as shown in SEQ ID NO.43) genes were cloned into the chloroplast transformation vector to ensure their efficient expression in chloroplasts.

[0048] Vector 3 (hereinafter referred to as neFBP) construction: The P450 peptide-Δ39 nnLUZ_v2, PKS (as shown in SEQ ID NO.25), CPH (as shown in SEQ ID NO.21), H3H (as shown in SEQ ID NO.43), and Δ19 nnLUZ gene were cloned into the plant binary expression vector pCAMBIA2300 to construct an expression cassette containing a 35S promoter and a NOS terminator, as shown in the figure. Figure 2B As shown.

[0049] Vector 4 (hereinafter referred to as ceFBP): Based on the characteristics of the plant chloroplast genome, a chloroplast transformation vector was designed. The P450 peptide-Δ39 nnLUZ_v2, PKS (as shown in SEQ ID NO.25), CPH (as shown in SEQ ID NO.21), H3H (as shown in SEQ ID NO.43), and Δ19 nnLUZ genes were cloned into the chloroplast transformation vector to ensure their efficient expression in chloroplasts.

[0050] Vector 5 (hereinafter referred to as nAPT) construction: The plant codon-optimized AroG gene (as shown in SEQ ID NO. 86), P450 gene (as shown in SEQ ID NO. 73), and TAL gene from Rhodotorula glutinis (as shown in SEQ ID NO. 57) were cloned into the plant binary expression vector pCAMBIA2300 to construct an expression cassette containing a 35S promoter and a NOS terminator, as shown in the figure. Figure 3A As shown.

[0051] Vector 6 (hereinafter referred to as eAPT) construction: Based on the characteristics of the plant chloroplast genome, a chloroplast transformation vector was designed. The plant codon-optimized AroG gene (as shown in SEQ ID NO. 86), P450 gene (as shown in SEQ ID NO. 73), and TAL gene from Rhodotorula glutinis (as shown in SEQ ID NO. 57) were cloned into the chloroplast transformation vector to ensure their efficient expression in chloroplasts.

[0052] Construction of vector 7 (hereinafter referred to as nATC): The plant codon-optimized AroG gene (as shown in SEQ ID NO. 86), TAL gene (as shown in SEQ ID NO. 57), and C3H gene (as shown in SEQ ID NO. 84) were cloned into the plant binary expression vector pCAMBIA2300 to construct an expression cassette containing a 35S promoter and a NOS terminator, as shown in the figure. Figure 3B As shown.

[0053] Vector 8 (hereinafter referred to as eATC): Based on the characteristics of the plant chloroplast genome, a chloroplast transformation vector was designed. The plant codon-optimized AroG gene (as shown in SEQ ID NO.86), TAL gene (as shown in SEQ ID NO.57), and C3H gene (as shown in SEQ ID NO.84) were cloned into the chloroplast transformation vector to ensure their efficient expression in chloroplasts.

[0054] Example 2: Plant Transformation and Screening Nuclear transformation: The nuclear transformation vector constructed in Example 1 is introduced into Agrobacterium (e.g., EHA105), and higher plants such as tobacco (Nicotiana benthamiana) are transiently or stably transformed using Agrobacterium-mediated transformation. This application does not limit the method of nuclear transformation; the target gene can be transformed into the plant cell nucleus.

[0055] The nuclear transformation method is as follows: Preparation and transformation of Agrobacterium competent cells: Agrobacterium EHA105 competent cells stored at -80℃ were thawed on ice, and 1 μg of the nuclear transformation recombinant plasmid constructed in Example 1 was added. The mixture was gently mixed and then incubated on ice for 30 minutes. The cells were then flash-frozen in liquid nitrogen for 5 minutes, immediately placed in a 37℃ water bath for heat shock for 5 minutes, and then immediately incubated on ice for 2 minutes. 800 μL of antibiotic-free LB broth was added, and the cells were cultured at 28℃ with shaking at 200 rpm for 4-6 hours. 200 μL of the bacterial culture was spread onto LB solid medium containing 50 mg / L kanamycin and 50 mg / L rifampin, and incubated upside down at 28℃ for 2-3 days. Single colonies were picked for PCR identification to obtain positive transformed Agrobacterium.

[0056] Sterile seedling culture: Disinfect tobacco seeds with 75% ethanol for 30 seconds, rinse 3 times with sterile water, then disinfect with 0.1% mercuric chloride solution for 8 minutes, rinse 5-6 times with sterile water, inoculate on MS solid medium, and culture for 4-6 weeks at 25℃ under 16h light / 8h dark conditions to obtain sterile tobacco seedlings.

[0057] Preparation of Agrobacterium infection solution: Select a single positive Agrobacterium colony and inoculate it into LB liquid medium containing the corresponding antibiotic, and incubate at 28°C with shaking at 200 rpm until OD. 600 =0.6-0.8. Centrifuge at 4000 rpm for 10 minutes to collect bacterial cells, resuspend in MS liquid medium containing 100 μmol / L acetylsuccine, and adjust OD. 600 Reduce the concentration to 0.4-0.5 and let it stand at room temperature for 30 minutes before use.

[0058] Leaf disc infection and co-culture: Take fully expanded leaves from sterile tobacco seedlings and punch leaf discs with a diameter of about 0.5 cm. Immerse the leaf discs in the above-mentioned Agrobacterium infection solution for 10-15 minutes, gently shaking them during the process. Remove the leaf discs, blot off excess bacterial solution with sterile filter paper, and inoculate them with the underside of the leaves onto a co-culture medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 100 μmol / L acetylsylphenone, pH 5.8) lined with sterile filter paper. Incubate in the dark at 25°C for 2-3 days.

[0059] Desiccation and selection culture: The co-cultured leaf discs were transferred to desiccation and selection medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 500 mg / L cefotaxime sodium + 20 mg / L hygromycin, pH 5.8) and cultured at 25°C under 16 h light / 8 h dark conditions, and subcultured every 14 days.

[0060] Rooting Culture and Transplanting: When the resistant callus tissue differentiates into adventitious buds about 2-3 cm long, cut off the adventitious buds and transfer them to rooting medium (1 / 2 MS + 0.1 mg / L NAA + 200 mg / L cefotaxime sodium + 10 mg / L hygromycin, pH 5.8) to induce rooting. After the root system is well developed, remove the sealing film of the culture bottle and harden off for 3-5 days, then transplant them into flowerpots filled with sterilized nutrient soil and cultivate under greenhouse conditions.

[0061] Tobacco transient transformation: Prepare Agrobacterium infection solution according to the first two steps, and adjust OD. 600 To a concentration of 0.8-1.0. Using a 1 mL syringe, slowly inject the Agrobacterium infection solution from the underside of the tobacco leaf, ensuring the bacterial solution is evenly distributed throughout the leaf. Incubate the injected tobacco plants at 25°C under 16h light / 8h dark conditions for 48-72 hours before use for subsequent testing.

[0062] Chloroplast transformation: For tobacco plants that have undergone nuclear transformation or not, the chloroplast transformation vector constructed in Example 1 is introduced into their chloroplast genome using the gene gun method (particle bombardment method) or other chloroplast transformation techniques. This application does not limit the method of chloroplast transformation; the target gene can be transformed into the chloroplasts of plant cells.

[0063] This embodiment uses a gene gun (particle bombardment method) to introduce a chloroplast transformation vector into the tobacco chloroplast genome. This method is currently the most mature and widely used method for chloroplast transformation. The chloroplast transformation method is as follows: Aseptic seedling preparation: Select young leaves of aseptic seedlings that have grown for 4-6 weeks and are fully expanded as conversion materials.

[0064] Preparation of gold powder microspheres: Weigh 50 mg of gold powder with a diameter of 0.6 μm and place it in a 1.5 mL centrifuge tube. Add 1 mL of anhydrous ethanol, vortex for 3-5 minutes, centrifuge at 12000 rpm for 1 minute, and discard the supernatant. Wash twice with anhydrous ethanol, resuspend in 1 mL of sterile water, and store at -20℃ for later use.

[0065] Plasmid DNA encapsulation of gold powder: Take 50 μL of the above gold powder suspension, add 10 μL of chloroplast transformation plasmid DNA (constructed in Example 1 at a concentration of 1 μg / μL), 50 μL of 2.5 mol / L CaCl2, and 20 μL of 0.1 mol / L spermidine. Vortex for 3-5 minutes and let stand at room temperature for 10 minutes. Centrifuge at 12000 rpm for 1 minute, discard the supernatant, wash twice with anhydrous ethanol, and finally resuspend in 50 μL of anhydrous ethanol. Keep on ice for later use.

[0066] Gene gun bombardment: The Bio-Rad PDS-1000 / He gene gun was used for bombardment. Tobacco leaves were laid flat in the center of MS solid medium containing 0.4 mol / L mannitol with the underside facing up. The bombardment parameters were set as follows: ruptured membrane pressure 1100 psi, bombardment distance 6 cm, and vacuum degree 28 inches of mercury. Each leaf was bombarded 1-2 times.

[0067] Dark culture and callus induction: After bombardment, the leaves were cultured in the dark at 25°C for 16-24 hours. Then, the leaves were cut into small pieces of about 0.5 cm × 0.5 cm and inoculated on callus induction medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA, pH 5.8) and cultured in the dark at 25°C for 2 weeks.

[0068] Selection culture: Callus tissue was transferred to selection medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 500 mg / L spectinomycin, pH 5.8) and cultured at 25°C under 16h light / 8h dark conditions, with subculture every 14 days. After 3-4 rounds of selection, resistant callus tissue was obtained.

[0069] Differentiation and rooting culture: Resistant callus was transferred to differentiation medium (MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 500 mg / L spectinomycin, pH 5.8) to induce the differentiation of adventitious shoots. When the adventitious shoots grew to 2-3 cm, they were cut off and transferred to rooting medium (1 / 2 MS + 500 mg / L spectinomycin, pH 5.8) to induce rooting.

[0070] Homogenization identification and transplanting: Total DNA was extracted from transformed plants, and the degree of homogenization of the chloroplast genome was identified by PCR and Southern blot. For plants that were not fully homogenized, multiple rounds of selection and culture were continued until fully homogenized plants were obtained. After hardening off, the homogenized plants were transplanted to a greenhouse for further cultivation.

[0071] Screening and identification of transformed plants Screening of nuclear-transformed plants: During nuclear transformation, 20 mg / L hygromycin was used as the selection pressure. Plants that survived and differentiated were considered preliminary positive plants. Genomic DNA was extracted from the preliminary positive plants, and the target gene fragment was amplified by PCR for identification, thus obtaining nuclear-transformed positive plants.

[0072] Screening of chloroplast-transformed plants: During chloroplast transformation, 500 mg / L spectinomycin was used as a selection pressure. Plants that survived and differentiated were considered preliminary positive plants. The integration and homogenization of the target gene were identified by PCR, Southern blot, and real-time quantitative PCR to obtain fully homogenized chloroplast-transformed positive plants.

[0073] Obtaining dual-transformed plants: The nuclear-transformed positive plants obtained above were used as recipient materials, and chloroplast transformation was performed according to the method described above. After screening with spectinomycin and molecular identification, dual-transformed positive plants that simultaneously integrated nuclear transformation vector and chloroplast transformation vector were obtained. Example 3: Luminous Intensity Detection The method for detecting luminescence intensity includes culturing tobacco transformed with the gene from Example 2 for 24-48 hours, and in this application, culturing for 36 hours. The cultured tobacco leaves are placed in a dark environment, and a LightScout full-spectrum quantum meter (Shanghai Zequan Technology) is used with the detection port pressed tightly against the leaf surface to detect the amount of photons accumulated (shooting conditions: Huawei P30 Pro, ISO 6400, time-lapse photography 2s, aperture 1.6 mm). Three biological replicates are set up for each group, and the luminescence intensity data are recorded and statistically analyzed.

[0075] Figure 4 Images of plant bioluminescence after transformation with different vectors according to an embodiment of this application are shown. Figure 5 The graph shows the statistical distribution of plant luminescence intensity after transformation with different carriers according to an embodiment of this application.

[0076] Example 4: Detection of Caffeic Acid Content Sample preparation: Tobacco leaves that had been transformed in Example 2 and cultured for 36 hours were immediately frozen and ground in liquid nitrogen. About 1 g of the frozen tissue was placed in a 50 ml centrifuge tube and freeze-dried. The freeze-dried material was stored at -20°C. Each sample group was prepared and analyzed three times.

[0077] Extraction and filtration: Weigh approximately 50 mg of lyophilized powder, treat with 7 ml of 70% methanol aqueous solution in an ultrasonic bath for 30 minutes, centrifuge at 4,000 rpm for 10 minutes, collect the supernatant, and filter with a Phenex GF / PVDF syringe filter (30 mm in diameter, 0.45 μm in pore size).

[0078] LCMS Analysis: Analysis was performed using a Shimadzu 8030 system (HPLC-DAD-ESI-TQ MS). The column was a Discovery C18 column (4.6 × 150 mm, 5 μm). Mobile phase A was 0.3% aqueous acetic acid (v / v), and mobile phase B was acetonitrile. Gradient run: 0–4 min 10–40% B, 4–5 min 40–80% B, 5–10.5 min 100% B isocratic elution, followed by restoration to initial conditions. Column temperature: 40℃, flow rate: 1 ml / min, injection volume: 20 μl.

[0079] Figure 6 This is a statistical chart showing the cumulative caffeic acid in plants after transformation with different carriers according to an embodiment of this application.

[0080] Compared with the prior art, the present invention has the following beneficial effects: Significantly improves luminescence intensity: By efficiently expressing key genes (AroG, TAL, P450) in the caffeic acid synthesis pathway in chloroplasts, the content of luminescent precursors such as caffeic acid and milk alkaloids in plants is greatly increased, thereby significantly improving the luminescence intensity of the fungal bioluminescence system, which is superior to the traditional method of nuclear transformation of all genes.

[0081] Overcoming the precursor supply bottleneck: The high copy number and high expression characteristics of the chloroplast genome ensure the stable and high-level expression of the caffeic acid synthesis gene, effectively solving the limitation of insufficient precursor supply in fungal bioluminescence systems.

[0082] Improving stability and safety: Chloroplast transformation has the characteristics of no gene silencing and maternal inheritance, which helps to maintain the stability of traits and biosafety of transgenic plants.

[0083] Innovative Solution: This invention combines the advantages of nuclear transformation and chloroplast transformation, providing a novel strategy and technical approach for creating efficient and stable self-luminescent plants.

[0084] 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 system for enhancing the intensity of plant autoluminescence, comprising: A nuclear expression vector that is active in the plant cell nucleus, the nuclear expression vector comprising a fungal bioluminescent pathway (FBP) gene or a modified fungal bioluminescent pathway (FBP') gene; as well as A chloroplast expression vector active in plant chloroplasts, the chloroplast expression vector comprising an enzyme for synthesizing 3-arylacrylic acid.

2. The system according to claim 1, wherein the enzyme of 3-arylacrylic acid is selected from the group consisting of: tyrosine ammonia-lyase (TAL), HpaB and HpaC components of h4-hydroxyphenylacetate 3-monooxygenase reductase, 3-deoxy-D-arabinohepulose-7-phosphate synthase (AroG), phenylalanine ammonia-lyase (C3H), and enzymes of the cytochrome P450 monooxygenase family; preferably, the enzyme of the cytochrome P450 monooxygenase family is selected from p-coumaroyl-CoA 3-hydroxylase and cytochrome P450 monooxygenase (P450). Preferably, the amino acid sequence of P450 is as shown in any of SEQ ID NO.74-83; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO.74-83. The amino acid sequence of C3H is shown in SEQ ID NO. 85; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO.

85. The amino acid sequence of AroG is as shown in any of SEQ ID NO. 87-104; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO. 87-104. The amino acid sequence of the TAL is as shown in any of SEQ ID NO. 58-72; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO. 58-72.

3. The system according to claim 1, wherein the 3-arylacrylic acid is selected from the group consisting of: caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

4. The system according to claim 1, wherein the FBP or FBP' comprises one or more of the following genes: luciferase (LUZ), caffeoylpyruvate hydrolase (CPH), milk alkaloid hydroxylase (H3H), and an enzyme that catalyzes the synthesis of milk alkaloid from caffeic acid; Preferably, the amino acid sequence of the CPH is as shown in SEQ ID NO.22; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO.22; (the amino acid sequences such as PKS and LUZ are to be numbered here and below, and are intended to be included in almost all currently disclosed functional sequences) Preferably, the amino acid sequence of H3H is as shown in any of SEQ ID NO.44-56; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO.44-56.

5. In the system according to claim 4, the enzyme catalyzing the synthesis of milk alkaloids from caffeic acid is milk alkaloid synthase (Hisps) or polyketide synthase (PKS). Preferably, the amino acid sequence of the Hisps is as shown in SEQ ID NO.24; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO.

24. Preferably, the amino acid sequence of the PKS is as shown in any of SEQ ID NO.26-42; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with any of the amino acid sequences shown in SEQ ID NO.26-42.

6. The system according to claim 4, wherein the amino acid sequence of the luciferase is modified or not modified; The modified luciferase has an amino acid sequence that is one or more of the following modifications: The amino acid sequence of the luciferase has 1-60 amino acids deleted from its N-terminus; The amino acid sequence of the luciferase has a transmembrane-bound guide peptide sequence at the N-terminus. The amino acid sequence of the luciferase is linked to a retention peptide at the C-terminus. The amino acid sequence of the luciferase includes one or more site mutations; Preferably, the amino acid sequence of the luciferase is as shown in any one of SEQ ID NO.2-20; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in any one of SEQ ID NO.2-20.

7. The system according to claim 1, a nuclear expression vector for converting genes into plant cell nuclei, wherein the nuclear expression vector is pCAMBIA2300.

8. The system according to claim 1, wherein the expression vector for converting genes into plant chloroplasts includes chloroplast localization genes.

9. The use of the system as described in any one of claims 1-8 in the preparation of reagents or kits for enhancing the autoluminescence intensity of plants.

10. A method for enhancing the autoluminescence intensity of plants using the system as described in any one of claims 1-8, comprising: Transform the fungal bioluminescence pathway (FBP) gene or the modified fungal bioluminescence pathway (FBP') gene into the plant cell nucleus; as well as An enzyme that is converted into chloroplasts in plant cells for the synthesis of 3-arylacrylic acid.

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