Tal protein mutants and uses thereof

Abstract

The present application relates to a TAL H180 The application relates to application of a gene mutant in increasing biosynthesis amount of 3-aryl acrylic acid or increasing light emission intensity of an organism or a cell. The application provides an innovative solution for creating an efficient self-luminous plant, and can be widely applied to the fields of biosensor development, in-vivo imaging, ornamental plant cultivation and the like, and has important scientific value and broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and particularly to TAL protein mutants and their applications. Background Technology

[0002] Bioluminescence is a light phenomenon produced by organisms through natural chemical reactions. With its advantages of requiring no exogenous substrate, allowing for real-time monitoring, and causing minimal damage to organisms, it has broad application prospects in fields such as bioimaging, environmental monitoring, gene expression analysis, and ornamental plant cultivation. The fungal bioluminescence pathway (FBP) is one of the most widely used heterologous light-emitting systems in plants. Its core is based on the caffeic acid metabolic cycle, generating photons through the catalytic action of key enzymes such as luciferase (Luz) and milkweed alkaloid-3-hydroxylase (H3H). Caffeic acid, as the starting substrate and core intermediate of this metabolic cycle, directly determines the luminescence intensity and persistence of the FBP system through its accumulation within the plant.

[0003] In existing technologies, increasing the endogenous caffeic acid content in plants through metabolic engineering is a core strategy for enhancing the luminescence performance of FBP systems. TAL proteins (tyrosine ammonia-lyases) are key regulators of the caffeic acid synthesis pathway, catalyzing the breakdown of tyrosine into p-coumaric acid, thereby promoting the synthesis and accumulation of downstream caffeic acid. However, TAL proteins suffer from limited regulatory efficiency and insufficient stability in plant heterologous expression systems, resulting in insufficient caffeic acid content in plants to meet the high-efficiency luminescence requirements of FBP systems, leading to weak luminescence intensity and short duration.

[0004] To address this issue, existing technologies often employ overexpression of TAL proteins or the introduction of other metabolic pathway genes in combination. However, overexpression can easily lead to metabolic disorders in plants, and the synergistic effect of single gene combinations is limited.

[0005] Therefore, it is still necessary to seek other methods that can significantly improve the intensity of plant bioluminescence. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this invention proposes a TAL H180 Applications of gene mutants in increasing the biosynthesis of 3-arylacrylic acid or improving the luminescence intensity of organisms.

[0007] As described above, TAL H180 The amino acid sequence of the gene is as shown in SEQ ID NO.:1, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:1.

[0008] As described above, TAL H180 The nucleotide sequence of the gene is as shown in SEQ ID NO.:2, or its corresponding amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence corresponding to the sequence shown in SEQ ID NO.:2.

[0009] As described above, TAL H180 The gene mutant is mutated to glutamine, asparagine, phenylalanine, leucine, isoleucine, methionine, valine, serine, proline, threonine, alanine, tyrosine, lysine, aspartic acid, cysteine, tryptophan, glutamic acid, arginine, or glycine; preferably, TAL H180 The gene mutant mutated to glutamine.

[0010] As 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.

[0011] As described above, the organisms are plants, animals, cells, or fungi.

[0012] As described above, TAL H180 The use of gene mutants in combination with enzymes used to synthesize 3-arylacrylic acid can increase the biosynthesis of 3-arylacrylic acid or increase the luminescence intensity of organisms or cells.

[0013] As described above, the enzyme used to synthesize 3-arylacrylic acid is selected from the group consisting of tyrosine ammonia-lyase, the HpaB and HpaC components of h4-hydroxyphenylacetate 3-monooxygenase reductase, phenylalanine ammonia-lyase (C3H), and enzymes of the cytochrome P450 monooxygenase family; preferably, the enzymes of the cytochrome P450 monooxygenase family are selected from p-coumaroyl-CoA 3-hydroxylase and cytochrome P450 monooxygenase (P450).

[0014] In the applications described above, the amino acid sequence of HpaB is as shown in SEQ ID NO.:20, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:20; the amino acid sequence of HpaC is as shown in SEQ ID NO.:21, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:21; the amino acid sequence of C3H is as shown in SEQ ID NO.:19, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:19; and the amino acid sequence of P450 is as shown in SEQ ID NO.:22, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:22.

[0015] This application relates to a recombinant construct, including: TAL H180 Gene mutants, the TAL H180 Gene mutants are engineered to increase the biosynthesis of caffeic acid or to increase the luminescence intensity of an organism or cell.

[0016] The recombinant construct as described above further includes an enzyme for synthesizing 3-arylacrylic acid.

[0017] The recombinant construct described above further includes: a fluorescent factor set configured to enable the plant to emit light; the fluorescent factor set includes: milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene, or alternatives to one or more of these genes; the fluorescent factor set is configured to enable the plant, plant tissue or cell to emit light.

[0018] This application relates to a reagent or kit for increasing the biosynthetic content of caffeic acid or increasing the luminescence intensity of organisms or cells, including any of the recombinant constructs described above.

[0019] This application relates to a method for improving caffeic acid biosynthesis or increasing the luminescence intensity of organisms or cells. It includes: obtaining TAL (talc). H180 The mutated encoding gene; constructing a TAL-containing gene. H180 The mutated gene encoding plant expression vector; constructing expression vectors containing FBP system genes and genes for an enzyme that synthesizes 3-arylacrylic acid; transforming the expression vectors into engineered bacteria that can be stably expressed in plants; preferably, the engineered bacteria are Agrobacterium; and transforming the engineered bacteria into plant cells.

[0020] In some embodiments, the TAL obtained by the present invention through site-directed mutagenesis H180Q The mutant significantly improved the regulatory efficiency of the plant caffeic acid synthesis pathway, and the caffeic acid content increased significantly, providing sufficient substrate for the FBP system and enhancing the material basis of bioluminescence from the source.

[0021] This application relates to a self-luminescent organism or cell that can express any of the recombinant constructs described above.

[0022] This application relates to a method for preparing a self-luminescent organism or cell, comprising: converting the recombinant construct as described above into a somatic cell.

[0023] TAL H180Q When the mutant is combined with the HpaB+HpaC dual-gene combination, the luminescence enhancement effect is optimal. Compared with the FBP system and the TAL combination system alone, the luminescence intensity is significantly improved, breaking through the limitations of single gene modification.

[0024] This mutant and related methods are applicable to a variety of higher plants such as tobacco, with a wide range of applications. Furthermore, the Agrobacterium-mediated transient transformation method is simple to operate and highly efficient. This invention provides an innovative solution for creating highly efficient self-luminous plants, which can be widely used in fields such as biosensor development, in vivo imaging, and ornamental plant cultivation. It has significant scientific value and broad application prospects. Attached Figure Description

[0025] 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 gene element combination of a recombinant vector according to an embodiment of the present invention; Figure 2 This is a statistical chart of caffeic acid content in tobacco transiently transformed by different multiplex gene vectors according to an embodiment of the present invention; Figure 3 This is a statistical graph of the luminescence intensity of tobacco transiently transformed by different multi-gene recombinant vectors according to an embodiment of the present invention; Figure 4 This is an image of the transient conversion luminescence intensity of *Nicotiana benthamiana* mediated by *Agrobacterium* according to an embodiment of the present invention; and Figure 5 This is a multiple sequence alignment diagram of the amino acid sequences of the TAL gene in different species according to an embodiment of the present invention. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] In recent years, site-directed mutagenesis of core enzymes in bioluminescence systems through rational design has become a research hotspot. For example, modifying the Luz and H3H enzymes in the FBP system can significantly improve their catalytic efficiency. However, there are no reports on the targeted modification of TAL, a key regulatory protein in the caffeic acid synthesis pathway, for enhancing plant bioluminescence intensity. Therefore, rationally designing and modifying TAL proteins, screening for efficient mutants, and optimizing gene combinations are crucial to overcoming current technological bottlenecks and significantly improving plant bioluminescence intensity.

[0029] This invention discloses a TAL protein mutant and its application in enhancing plant bioluminescence intensity. This invention optimizes the key regulatory protein TAL in the caffeic acid synthesis pathway through site-directed mutagenesis to obtain TAL. H180Q The mutant significantly improved the substrate supply efficiency and luminescence intensity of the fungal bioluminescence system (FBP). Experiments demonstrated that: 1) TAL H180Q Compared to the TAL protein, the mutant significantly increases the caffeic acid content in plants, providing sufficient substrate for the FBP system; 2) TAL H180Q When co-expressed with the FBP system and functional genes (P450, C3H or HpaB+HpaC), the luminescence intensity is significantly better than the combination system corresponding to TAL; 3) This mutant can play a stable role in tobacco plants, creating a self-luminous plant with significantly enhanced luminescence intensity.

[0030] This invention overcomes the defects and shortcomings of existing technologies by optimizing the TAL protein through site-directed mutagenesis to obtain TAL. H180Q The mutant significantly improved its regulatory efficiency on the caffeic acid synthesis pathway; at the same time, the optimal combination of functional genes was screened and worked synergistically with the FBP system to achieve a significant enhancement of plant bioluminescence intensity.

[0031] This invention modifies TAL proteins using site-directed mutagenesis and combines this with functional gene combination screening to achieve a significant enhancement of plant bioluminescence intensity. The specific technical solution is as follows: Improvement of TAL: The luminescence intensity of the FBP system directly depends on the supply of caffeic acid. As a key enzyme in the caffeic acid synthesis pathway, the insufficient activity of TAL protein leads to low caffeic acid accumulation in the plant, which cannot meet the needs of efficient luminescence of the FBP system. Therefore, improving the catalytic activity of TAL protein is the core breakthrough for brightening.

[0032] Selection of mutation sites and construction of mutants: Targeting potential key sites within the functional domain of the TAL protein, four sites were randomly selected for mutation: phenylalanine (Phe) at position 116, glycine (Gly) at position 167, histidine (His) at position 180, and serine (Ser) at position 212. Four mutants were constructed: F116L (Phe→Leu), G167D (Gly→Asp), H180Q (His→Gln), and S212R (Ser→Arg). The coding genes of each mutant were prepared using overlap extension PCR to ensure the accuracy of the mutation sites.

[0033] Mutant functional screening and core mutant identification: Four mutants and TAL were used to construct recombinant vectors, each coupled with the FBP system and the C3H, P450, and HpaB+HpaC dual genes. Tobacco leaves were transiently transformed using the Agrobacterium-mediated transformation method. With the FBP system alone as a control, three biological replicates were set up for each group. Highly efficient mutants were screened by measuring luminescence intensity and caffeic acid content. Results are as follows: Figure 2-4 As shown, only the H180Q mutant can significantly increase the luminescence intensity and caffeic acid content of plants, while the other three mutants are not significantly different from the unmutated TAL control group (P>0.05). Therefore, H180Q was determined to be the optimal mutation site.

[0034] This invention will TAL H180Q The mutant was combined with different functional genes to further enhance the coupling between the caffeic acid synthesis pathway and the FBP system through multi-gene co-expression, thereby achieving a superimposed increase in luminescence intensity.

[0035] The proper nouns mentioned in this article have the following meanings: For nucleotides, the term "homology" as used herein refers to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined using conventional methods such as NCBI Blast v2.0 to calculate the degree of sequence identity between two or more nucleotide sequences. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity are referenced in WO04 / 037999, EP0967284, EP1085089, WO00 / 55318, WO00 / 78972, WO98 / 49185, and GB2357768-A, etc.

[0036] For amino acids, the term "homology" as used herein describes or compares the degree of amino acid similarity between two or more polypeptides or their specified sequences at optimal alignment and comparison. The homology % between two sequences varies with the number of common positions shared by these sequences at optimal alignment (i.e., homology % = (number of common positions / total number of positions) × 100)%, where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy.

[0037] In this application, without substantially affecting antibody activity (retaining at least 95% activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain variants of the antibody or its functional fragment sequence. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequence described in this application can have at least 80% identity (or homology) with the reference sequence, meaning at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with each reference sequence. The sequence consistency described in this application can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences mentioned in this application are shown from the N-terminus to the C-terminus.

[0038] Unless otherwise specified, all bioluminescent plants mentioned in this application are transgenic plants containing the fungal FBP bioluminescence system. This application does not specifically limit the types of plants, including, but is not limited to, *Nicotiana benthamiana*, *Arabidopsis thaliana*, tobacco, chrysanthemum, marigold, cosmos, zinnia, coreopsis, echinacea, *Symplocos davidii*, silverleaf daisy, *Age of Osmanthus fragrans*, *Mallotus patulum*, *Tetrapanax papyriferus*, daisy, *Aster tataricus*, *Cinnamomum cassia*, *Salvia splendens*, sage, lavender, rosemary, mint, patchouli, coleus, rose, rose, tulip, hyacinth, daylily, hosta, spider plant, asparagus fern, aloe vera, *Liriope muscari*, *Gnaphalium affine*, kale, violet, *Orychophragmus violaceus*, carnation, baby's breath, tall snow lily, dwarf snow lily, jasmine, coral bean, ornamental pepper, tobacco flower, *Cercis chinensis*, wisteria, lupin, clover, iris, German iris, butterfly flower, and *Iris tectorum*. Anthurium, Peace Lily, Monstera deliciosa, Epipremnum aureum, Alocasia macrorrhiza, Calla Lily, Tomato, Chili Pepper, Eggplant, Pumpkin, Rapeseed, Colored Chili Pepper, Colored Rapeseed, Cabbage, Broccoli, Cauliflower, Cotton, Osmanthus, Jasmine, Lilac, Forsythia suspensa, Jasminum nudiflorum, Forsythia suspensa, Poinsettia, Crown of Thorns, Croton, Jade Plant, Kalanchoe blossfeldiana, Echeveria, Echeveria elegans, Sedum lineare, Cactus, Gymnocalycium mihanovichii, Epiphyllum oxypetalum, Christmas Cactus, Tradescantia zebrina, Tradescantia zebrina, Commelina communis, Amaryllis, Cyclamen, Gloxinia, Oxalis, Bellflower, Lobelia chinensis, Verbena officinalis, Geranium, Nasturtium, Gazania latifolia, Marguerite daisy, Bleeding Heart, Foxglove, Ranunculus asiaticus, Columbine, Plumbago aurantium, Vinca minor, Bells dwarf, and Alyssum fusiforme, etc.

[0039] For the purposes of this invention, the terms "luminescence" and "bioluminescence" are used interchangeably and refer to the luminescence phenomenon during a chemical reaction catalyzed by luciferase.

[0040] The term "fluorescent factor group" refers to a combination of factors that, when introduced into a somatic cell, integrate and express, enable the cell to produce autofluorescence. In some embodiments, the fluorescent factor group includes milk alkaloid synthase gene, milk alkaloid hydroxylase gene, luciferase gene, and caffeoylpyruvate hydrolase gene, etc.

[0041] The term "organism" refers to a living organism with cellular activity that can absorb foreign genes and express them in its cells. In some embodiments, an organism is a plant, animal, bacteria, fungus, etc.

[0042] The term “milk alkaloid hydroxylase” is used herein to describe an enzyme that catalyzes the reaction of pre-luciferin to fungal luciferin, for example, the synthesis of 3-hydroxymilk alkaloid from milk alkaloid.

[0043] The term “milk alkaloid synthase” is used herein to describe an enzyme capable of catalyzing the synthesis of fungal proluciferin from precursors of proluciferin, such as milk alkaloid from caffeic acid.

[0044] The term "PKS" is used in this document to describe enzymes belonging to the group of type III polyketide synthases capable of catalyzing the synthesis of milk alkaloids from caffeoyl-CoA.

[0045] The term "caffeoylpyruvate hydrolase" is used herein to describe an enzyme capable of catalyzing the oxidation of luciferin by fungi into simpler compounds, such as precursors for the formation of proluciferin. For example, it can catalyze the conversion of caffeoylpyruvate to caffeic acid.

[0046] In this application, unless otherwise specified, the fungal bioluminescent system (FBP) relies on a series of enzymatic reactions to convert common plant metabolites into luminescent compounds. This system is encoded by a fungal bioluminescent gene cluster, including: hispidin synthase (Hisps), hispidin-3-hydroxylase (H3H), luciferase (Luz), and caffeoyl pyruvate hydrolase (CPH). The amino acid sequences of the genes mentioned in the FBP system in this application are provided in CN119242698A and will not be repeated here.

[0047] The term "mutant" refers to a protein disclosed in this invention in which one or more amino acids are added, and / or substituted, and / or removed (deleted), and / or incorporated (inserted) into the N-terminus, and / or C-terminus, and / or native amino acid sequence of the protein of this invention. As used herein, the term "mutant" refers to the nucleic acid portion encoding a mutant protein. Furthermore, as used herein, the term "mutant" refers to any variant that is shorter or longer than the protein or nucleic acid disclosed in this invention.

[0048] The term "transformation" refers to the process of introducing heterologous nucleic acids into a host cell or organism. In particular, "transformation" refers to the stable integration of DNA molecules into the genome of a target organism.

[0049] The term "recombination" refers to a host organism, such as bacteria, plants, fungi, or animals, modified by introducing a heterologous nucleic acid molecule. This nucleic acid molecule can be stably integrated into the host genome or exist as an extrachromosomal molecule. This extrachromosomal molecule is capable of self-replication. It should be understood that transgenic or stably transformed cells, tissues, or organisms include both the end products of the transformation process and the transgenic progeny.

[0050] The term "TAL" refers to tyrosine ammonia-lyase, a key regulator of the caffeic acid synthesis pathway. It can catalyze the breakdown of tyrosine into p-coumaric acid, thereby promoting the synthesis and accumulation of downstream caffeic acid.

[0051] The term "TAL" H180 A "gene mutant" refers to a mutation in the amino acid sequence of the TAL gene at histidine residue position 180. In some embodiments, TAL... H180 The histidine at position 180 of the gene is mutated to glutamine, asparagine, phenylalanine, leucine, isoleucine, methionine, valine, serine, proline, threonine, alanine, tyrosine, lysine, aspartic acid, cysteine, tryptophan, glutamic acid, arginine, or glycine. In some embodiments, TAL H180 The histidine at position 180 of the gene is mutated to glutamine, asparagine, or alanine.

[0052] This application proposes a TAL H180 Applications of gene mutants in increasing the biosynthesis of 3-arylacrylic acid or improving the luminescence intensity of organisms.

[0053] In some embodiments, TAL H180 The amino acid sequence of the gene is as shown in SEQ ID NO.:1, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:1.

[0054] In some embodiments, TAL H180 The nucleotide sequence of the gene is as shown in SEQ ID NO.:2, or its corresponding amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence corresponding to the sequence shown in SEQ ID NO.:2.

[0055] In some embodiments, TAL H180 The gene mutant is mutated to glutamine, asparagine, phenylalanine, leucine, isoleucine, methionine, valine, serine, proline, threonine, alanine, tyrosine, lysine, aspartic acid, cysteine, tryptophan, glutamic acid, arginine, or glycine; preferably, TAL H180 The gene mutant mutated to glutamine.

[0056] In some embodiments, 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.

[0057] In some embodiments, the organism is a plant, animal, cell, or fungus.

[0058] In some embodiments, TAL H180The use of gene mutants in combination with enzymes used to synthesize 3-arylacrylic acid can increase the biosynthesis of 3-arylacrylic acid or increase the luminescence intensity of organisms or cells.

[0059] In some embodiments, the enzyme used to synthesize 3-arylacrylic acid is selected from the group consisting of tyrosine ammonia-lyase, the HpaB and HpaC components of h4-hydroxyphenylacetate 3-monooxygenase reductase, phenylalanine ammonia-lyase (C3H), and enzymes of the cytochrome P450 monooxygenase family; in some embodiments, the enzymes of the cytochrome P450 monooxygenase family are selected from p-coumaroyl-CoA 3-hydroxylase and cytochrome P450 monooxygenase (P450).

[0060] In some embodiments, the amino acid sequence of HpaB is as shown in SEQ ID NO.:20, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:20; the amino acid sequence of HpaC is as shown in SEQ ID NO.:21, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:21; the amino acid sequence of C3H is as shown in SEQ ID NO.:19, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:19; the amino acid sequence of P450 is as shown in SEQ ID NO.:22, or is at least, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:22.

[0061] This application proposes a recombinant construct, including: TAL H180 Gene mutants, the TAL H180 Gene mutants are engineered to increase the biosynthesis of caffeic acid or to increase the luminescence intensity of an organism or cell.

[0062] In some embodiments, the recombinant construct further includes an enzyme for synthesizing 3-arylacrylic acid.

[0063] In some embodiments, the recombinant construct further includes: a fluorescent factor set configured to enable the plant to emit light; the fluorescent factor set includes: milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene, or alternatives to one or more of these genes; the fluorescent factor set is configured to enable the plant, plant tissue or cell to emit light.

[0064] This application proposes a reagent or kit for increasing the biosynthetic content of caffeic acid or increasing the luminescence intensity of organisms or cells, including the recombinant constructs as described above.

[0065] This application proposes a method for improving caffeic acid biosynthesis or increasing the luminescence intensity of organisms or cells. This includes: obtaining TAL (talc). H180 The mutated encoding gene; constructing a TAL-containing gene. H180 The mutated gene encoding plant expression vector; constructing expression vectors containing FBP system genes and genes for an enzyme that synthesizes 3-arylacrylic acid; transforming the expression vectors into engineered bacteria that can be stably expressed in plants; preferably, the engineered bacteria are Agrobacterium; and transforming the engineered bacteria into plant cells.

[0066] According to one embodiment of this application, a method for enhancing plant bioluminescence intensity includes using TAL H180Q After codon optimization, the coding gene of the mutant was simultaneously and transiently transformed into plants along with the FBP gene cluster and functional genes (P450, C3H, or HpaB+HpaC) for co-expression. The specific steps are as follows: Construct containing TAL H180Q The plant expression vector encoding the gene has a vector backbone of pCAMBIA2300; Using fusion PCR technology, the FBP gene cluster and TAL were combined. H180Q The coding genes and functional genes are spliced ​​together according to a preset combination to construct a multi-gene recombination vector, wherein the multi-gene combination is selected from: FBP+TAL. H180Q +P450, FBP+TAL H180Q +C3H、FBP+TAL H180Q +HpaB+ HpaC; The multi-gene recombinant vector was transformed into Agrobacterium GV3101 competent cells to obtain engineered Agrobacterium bacteria. After activation and expansion culture, the engineered Agrobacterium bacteria were resuspended in infection buffer to OD600 = 0.6-0.8, acetylsyl syringone (final concentration 200 μmol / L) was added, and the bacteria were incubated in the dark at 28℃ for 2-3 h. Select healthy plant leaves that have grown for 4-6 weeks, immerse them in the dye solution for 30-60 seconds, blot off any residual liquid on the surface, and co-culture them at 25℃ in the dark for 2-3 days. Then transfer them to MS solid medium containing cephalosporin and culture them at 25℃ under 16 h light / 8 h dark conditions for 3-5 days to obtain self-luminescent plants with significantly enhanced bioluminescence intensity.

[0067] This application proposes a self-luminescent organism or cell that can express the recombinant constructs as described in any of the preceding ones.

[0068] This application proposes a method for preparing a self-luminescent organism or cell, comprising: converting the recombinant construct as described above into a somatic cell.

[0069] The embodiments of this application will be described below through specific examples. Those skilled in the art should understand that the following examples are only for illustrating the technical solutions of this application, and are not intended to limit the technical solutions of this application.

[0070] Example 1: Preparation of the TAL mutant encoding gene In this embodiment, the TAL used for genetic engineering is derived from Flavobacterium johnsonii ( Flavobacterium johnsoniae ).

[0071] Design and preparation of TAL protein mutants: A random mutation combined with functional validation strategy was used to determine the TAL protein modification scheme. First, the three-dimensional structure of the TAL protein was predicted using AlphaFold2, and four potential functional sites at positions 116, 167, 180, and 212 were screened to construct four random mutants: F116L, G167D, H180Q, and S212R, respectively. Subsequently, the coding genes of each mutant were prepared using overlap extension PCR, and upstream and downstream primers containing the corresponding mutation sites were designed. The primers and the sequences of the mutated and unmutated TAL genes are shown in Table 1. Table 1

[0072] With TAL H180Q Taking the coding gene as an example, the preparation method of the coding gene of the TAL mutant is as follows: Primer design: Based on the TAL gene sequence, upstream and downstream primers containing the mutation site at position 180 (histidine → glutamine) and outer primers were designed according to the principle of overlap extension PCR. The primer length was controlled at 18-25 bp, the Tm value was 55-65℃, and the mutation site was centered with at least 10 bp at both ends complementary to the template.

[0073] First round of PCR: Using a plasmid containing the TAL gene as a template, gene fragments containing the 5' and 3' ends with the mutation site were amplified in two separate systems. PCR reaction system (50 μL): Novizan 2× high-fidelity PCR mix 25 μL, forward and reverse primers 1 μL each, template plasmid 1 μL, double-distilled water 22 μL. Reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 5 min. Target fragments were recovered by electrophoresis.

[0074] Second round PCR: The two recovered fragments were mixed in an equimolar ratio and used as templates for complementary extension. Five cycles were performed without the outer primer (95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1.5 min). Then, the outer primer was added, and 30 more cycles were performed under the same conditions as the first round PCR. The full-length mutant gene (TAL) was recovered by electrophoresis. H180Q The coding gene was sequenced and verified to ensure the accuracy of the mutation site. The verification results showed no unexpected mutations.

[0075] The preparation methods for TAL mutant encoding genes, including those with other mutation sites, are similar to those in this embodiment and will not be repeated below.

[0076] Example 2 Construction of a multi-gene recombinant vector Gene synthesis and amplification: The FBP system gene cluster (Luz, H3H, CPH, HispS genes), TAL gene, P450 gene, C3H gene, HpaB gene, and HpaC gene were synthesized commercially. Based on the gene element combinations of the vector, the corresponding gene fragments were amplified using Novizan 2× high-fidelity PCR mix to ensure amplification specificity and fragment integrity. The amino acid sequences of the P450 gene, C3H gene, HpaB gene, and HpaC gene are shown in SEQ ID NO.:22, SEQ ID NO.:19, SEQ ID NO.:20, and SEQ ID NO.:21, respectively.

[0077] Fragment assembly: Single-gene element vectors (FBP system only) directly amplified the FBP gene cluster; multi-gene element vectors were assembled into gene fragments in corresponding order via fusion PCR. During assembly, each gene was configured with an independent 35S promoter and CaMVpoly(A) terminator. Long fragments (containing a four-gene combination of HpaB+HpaC) had extended PCR extension time (adjusted by 1 kb / min). All amplification products were subjected to agarose gel electrophoresis, then recovered using a Novizan gel extraction kit. Sequencing of the recovered products confirmed the gene sequences were accurate.

[0078] Vector linearization: The pCAMBIA2300 vector was double-digested with EcoRI and HindIII. The digestion system (50 μL) consisted of: 40 μL pCAMBIA2300 vector, 1 μL EcoRI, 1 μL HindIII, 5 μL 10×Cutsmart buffer, and 3 μL double-distilled water. Reaction conditions: Incubation at 37℃ for 10 h, followed by treatment at 80℃ for 20 min to terminate the digestion. The linearized vector was recovered by electrophoresis.

[0079] Seamless cloning ligation: The linearized vector and the target gene fragment verified by sequencing are mixed at a molar ratio of 1:3 to 1:5, and the reagents of the Beijing Bomed Seamless Cloning Kit are added. The mixture is incubated under the conditions set in the kit instructions to complete the ligation reaction.

[0080] Transformation and Screening Validation: The ligation product was transformed into *E. coli* DH5α competent cells using the heat shock method. The cells were plated on LB agar plates containing 50 mg / L antibiotic and incubated at 37°C for 12–16 h. Single colonies were picked for colony PCR identification. Plasmids were extracted from positive clones and Sanger sequencing was performed again to verify gene insertion direction, sequence integrity, and the accuracy of mutation sites (for vectors containing mutation sites, such as TAL). H180Q The key step is to verify the 180-base sequence. If the sequencing is successful, the target vector has been successfully constructed.

[0081] A schematic diagram of the obtained multi-gene recombinant vector fragment is shown below. Figure 1 As shown in the figure. TAL represents the wild-type TAL gene and the point-mutated TAL gene.

[0082] Example 3: Agrobacterium-mediated transformation and transient plant transformation Agrobacterium transformation: The sequenced recombinant vectors were transformed into Agrobacterium GV3101 competent cells by freeze-thaw method. After transformation, the cells were plated on LB plates containing rifampicin (50 mg / L) and kanamycin (50 mg / L) and incubated upside down at 28°C for 48 h. Single colonies were picked and transferred to LB liquid medium containing the corresponding antibiotics and cultured at 28°C and 200 rpm for 16-20 h to obtain seed culture.

[0083] Agrobacterium expansion culture: The seed culture was inoculated into fresh LB liquid medium (containing the same antibiotic) at a volume ratio of 1:100, and cultured at 28℃ and 200 rpm with shaking until OD600=0.8-1.0. The cells were collected by centrifugation at 4℃ and 5000 rpm for 10 min.

[0084] Preparation of infection solution: The bacterial cells were resuspended in infection buffer (4.43 g MS basal medium powder, 100 g sucrose, deionized water to a final volume of 1 L, pH 5.6-5.8, sterilized and then acetylsuccinone to a final concentration of 200 μmol / L) and the OD600 was adjusted to 0.6-0.8. The cells were then incubated in the dark at 28℃ for 2-3 h.

[0085] Plant infection: Select healthy, disease-free tobacco plants that have grown for 4-6 weeks, immerse the leaves in the infection solution, and gently shake for 30-60 seconds to ensure that both the front and back of the leaves are in contact with the infection solution.

[0086] Co-culture and incubation: After infection, the leaves were blotted dry with sterile filter paper to remove residual liquid, and then spread evenly in a petri dish lined with moist filter paper. They were co-cultured at 25°C in the dark for 2-3 days. Subsequently, they were transferred to MS solid medium containing cephalosporin (500 mg / L) and incubated at 25°C under 16 h light / 8 h dark conditions for 3-5 days.

[0087] Example 4: Detection of Caffeic Acid Content Sample preparation: Leaves of plants and transiently transformed plants were collected and immediately frozen in liquid nitrogen and ground. About 1 g of frozen tissue was placed in a 50 ml centrifuge tube and freeze-dried. The freeze-dried material was stored at -20℃. Each group of samples was prepared and analyzed three times.

[0088] 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).

[0089] 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.

[0090] Figure 2 The caffeic acid content in leaves transformed with different multi-gene recombinant vectors is shown. For example... Figure 2 As shown, the conversion contains TAL H180Q After the mutated vector was introduced, the caffeic acid content in the leaves was significantly higher than that in the corresponding transformation of leaves containing the unmutated TAL vector. The transformation of TAL... F116L TAL G167D TAL S212R The caffeic acid content in the leaves of the mutant group was not significantly different from that of the transformed wild-type TAL group (P>0.05), indicating that the histidine mutation at position 180 of TAL can enhance the regulatory ability of the TAL protein on the downstream caffeic acid synthesis pathway, thereby increasing the accumulation of caffeic acid.

[0091] When the multiplex genome vector of this embodiment is transformed into other plants, such as petunias and calendula, the caffeic acid content is sometimes similar to that of tobacco transformed in this application. Therefore, it can be seen that TAL… H180QThe mutant's effect of increasing caffeic acid accumulation is universal across hosts and can stably function in a variety of higher plants.

[0092] Example 5: Luminescence Detection After the plants were cultured to the target stage, the LightScout full-spectrum quantum meter (Shanghai Zequan Technology) was used in a dark environment. The detection port was placed close to the leaf of the plant to be tested, and the amount of light quantum accumulation at different times was detected (shooting conditions: Huawei P30 Pro, ISO 6400, time-lapse photography 2s, aperture 1.6 mm). The luminous intensity data was recorded and analyzed.

[0093] Figure 3 The luminescence intensity of leaves transformed with different multi-gene recombinant vectors is shown. Figure 4 Photographs of leaf fluorescence after transformation with different multi-gene recombinant vectors are shown. Figure 3-4 As shown, the conversion contains TAL H180Q Following the mutation of the vector, the plants exhibited higher luminescence intensity than those transformed with the unmutated TAL vector, while the TAL-transformed plants showed higher luminescence intensity. F116L TAL G167D TAL S212R The luminescence intensity of the mutant group was not significantly different from that of the non-mutated TAL group (P>0.05). The experimental results indicate that TAL... H180Q The mutant can effectively enhance the bioluminescence intensity of plants. When the multiplex genomic vector of this embodiment is transformed into other plants, such as petunias and calendula, the resulting plants often exhibit similar bioluminescence intensities to those transformed into tobacco in this application. This demonstrates that TAL... H180Q The mutant's effect of enhancing bioluminescence intensity in plants has host universality and can stably function in a variety of higher plants.

[0094] Example 6TAL H180 Conserved sites in different species In this embodiment, TAL homologous genes from different species were further cloned, and the amino acid sequences of the homologous genes were subjected to multiple sequence alignment. The alignment results are as follows: Figure 5 As shown.

[0095] Depend on Figure 5 It can be seen that the TAL H180 The site has not undergone natural mutations in different species, thus it is a conserved site. Referring to the experimental data of this application, this site is a key site for increasing caffeic acid content and consequently increasing plant luminescence intensity. Therefore, to improve plant luminescence intensity, TAL from any bacterial strain can be transformed into the plant. H180 mutated genes.

[0096] 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 type of TAL H180 Applications of gene mutants in increasing the biosynthesis of 3-arylacrylic acid or improving the luminescence intensity of organisms.

2. The application according to claim 1, wherein, TAL H180 The amino acid sequence of the gene is as shown in SEQ ID NO.:1, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:1; or TAL H180 The nucleotide sequence of the gene is as shown in SEQ ID NO.:2, or its corresponding amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence corresponding to the sequence shown in SEQ ID NO.:

2.

3. The application according to claim 1, TAL H180 The gene mutant is mutated to glutamine, asparagine, phenylalanine, leucine, isoleucine, methionine, valine, serine, proline, threonine, alanine, tyrosine, lysine, aspartic acid, cysteine, tryptophan, glutamic acid, arginine, or glycine; preferably, TAL H180 The gene mutant mutated to glutamine.

4. The application 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.

5. In the application according to claim 1, the organism is a plant, animal, cell, or fungus.

6. The application according to claim 1, TAL H180 The use of gene mutants in combination with enzymes used to synthesize 3-arylacrylic acid can increase the biosynthesis of 3-arylacrylic acid or increase the luminescence intensity of organisms or cells.

7. The application according to claim 6, wherein the enzyme for synthesizing 3-arylacrylic acid is selected from the group consisting of: Tyrosine ammonia-lyase, HpaB and HpaC components of h4-hydroxyphenylacetate 3-monooxygenase reductase, phenylalanine ammonia-lyase (C3H), and enzymes of the cytochrome P450 monooxygenase family; Preferably, the enzymes of the cytochrome P450 monooxygenase family are selected from p-coumaroyl-CoA3-hydroxylase and cytochrome P450 monooxygenase (P450).

8. The application according to claim 7, wherein, The amino acid sequence of HpaB is as shown in SEQ ID NO.:20, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:20; The amino acid sequence of HpaC is as shown in SEQ ID NO.:21, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:21; The amino acid sequence of C3H is as shown in SEQ ID NO.:19, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence shown in SEQ ID NO.:19; The amino acid sequence of P450 is as shown in SEQ ID NO.:22, or is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%70% identical to the sequence shown in SEQ ID NO.:

22.

9. A recombinant construct, comprising: TAL H180 Gene mutants, the TAL H180 Gene mutants are engineered to increase the biosynthesis of caffeic acid or to increase the luminescence intensity of an organism or cell.

10. The recombinant construct according to claim 9, further comprising an enzyme for synthesizing 3-arylacrylic acid.

11. The recombinant construct of claim 9, further comprising: Fluorescent factor group; It is configured to enable the plant to emit its own light; the fluorescent factor group includes: milkweed alkaloid synthase gene, milkweed alkaloid hydroxylase gene, luciferase gene and caffeoylpyruvate hydrolase gene, or alternatives to one or more of these genes; the fluorescent factor group is configured to enable the plant, plant tissue or cell to emit its own light.

12. A reagent or kit for increasing the biosynthetic content of caffeic acid or increasing the luminescence intensity of an organism or cell, comprising the recombinant construct as described in any one of claims 9-11.

13. A method for enhancing caffeic acid biosynthesis or increasing the luminescence intensity of an organism or cell. This includes: Get TAL H180 The mutated encoding gene; Construct containing TAL H180 Plant expression vectors for mutated coding genes; Expression vectors containing FBP system genes and genes for an enzyme used to synthesize 3-arylacrylic acid were constructed, respectively. The expression vector is transformed into an engineered bacterium that can be stably expressed in plants; preferably, the engineered bacterium is Agrobacterium; and Transform engineered bacteria into plant cells.

14. A self-luminescent organism or cell that can express the recombinant construct as described in any one of claims 9-11.

15. A method for preparing a self-luminous organism or cell, comprising: Transform the recombinant construct as described in any one of claims 9-11 into a biological cell.

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