Method for improving self-luminous intensity of plant organisms by utilizing metabolic pathway redesign

Abstract

The invention discloses a method for improving self-luminous intensity of plants by utilizing metabolic pathway redesign, and belongs to the technical field of biology. The method comprises the following step: introducing TAL, HpaB and HpaC genes into a biological self-luminous plant of which a genome is integrated with a fungal luminous pathway enzyme coding gene by utilizing a biological technical means. According to the method, a metabolic pathway redesign method is utilized for the first time, optimized TAL, HpaB and HpaC genes are introduced, tyrosine is converted into p-coumaric acid through TAL, then p-coumaric acid is converted into caffeic acid under the action of HpaB and HpaC, and the biological self-luminous intensity of plants is remarkably improved by improving the biosynthesis yield of caffeic acid. The invention provides a feasible technical scheme for breeding and production of sustainable luminous plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for improving the intensity of plant bioluminescence by utilizing metabolic pathway redesign. Background Technology

[0002] Bioluminescence, the light produced by living organisms through natural chemical reactions, is widely found in marine and terrestrial species and is one of nature's most amazing phenomena. As a powerful biosignal, bioluminescence has been a focus of research in many scientific fields, including basic and applied biology, chemistry, and medicine (Tsarkova et al., 2016). In the process of modernization, ornamental plants will play an increasingly important role in modern urban construction and home life. Furthermore, plants possess independent energy production and storage mechanisms as well as autonomous self-repair capabilities (Giraldo et al., 2014), making the research and modification of plant bioluminescence and sustainable bioluminescence highly significant.

[0003] As the bioluminescent mechanisms of different luminescent bacteria and fungi have been successively elucidated, researchers have successfully created plants that can emit light without the need for external luciferin by transferring genes from these luminescent pathways into plants for expression. Research on self-luminescent plants has been reported before. In 2014, Krichevsky et al. successfully obtained bioluminescent tobacco by integrating the lux operon of *Photobacterium leiognathi* into the chloroplast genome of tobacco. In 2018, Kotlobay et al. successfully elucidated the FBP (Fungal Bioluminescence Pathway) in a class of bioluminescent mushrooms (Kotlobay et al., 2018). This bioluminescent pathway generates light through a caffeic acid metabolic cycle composed of four enzymes. Specifically, caffeic acid is converted into milkweed alkaloid by Hispidin synthase (Hisps), which is then catalyzed by Hispidin-3-hydroxylase (H3H) to generate 3-hydroxylated milkweed alkaloid (fungal luciferin). Luciferin is oxidized to caffeoylpyruvate by luciferase (Luz) and produces photons. Finally, caffeoylpyruvate hydrolase (CPH) converts caffeoylpyruvate back into caffeic acid, thus initiating a new metabolic cycle to generate light.

[0004] The clear structural pathway of FBP, combined with the fact that caffeic acid, naturally found in plants, serves as its precursor molecule, offers the possibility for the rapid application of FBP in plant modification. In 2020, Mitiouchkina et al. successfully achieved autonomous luminescence in tobacco without the need for exogenous substrates (Mitiouchkina et al., 2020); in 2023, the research team of Professor Du Hao from Zhejiang University significantly enhanced the luminescence intensity in plants using the BnC3H gene from Brassica napus and the NPGA gene from Aspergillus nidulans, creating luminescent eFBP tobacco and luminescent poplar (Peng Zheng et al., 2023).

[0005] Caffeic acid is a widely distributed metabolite produced by the phenylpropane pathway in plants. It is also a key intermediate in the production of lignin and other critical plant metabolites (Deng and Lu, 2017). Caffeoyl-CoA and milk syrup are metabolized by endogenous enzymes in plants and diverted as FBP substrates. Therefore, the insufficient production of caffeic acid and milk syrup remains a limiting factor for enhancing luminescence in FBP systems and a technical problem that needs to be solved in the research of creating luminescent plants. Summary of the Invention

[0006] The purpose of this invention is to utilize synthetic biology techniques to redesign metabolic pathways and provide a method for enhancing bioluminescence by increasing the content of caffeic acid and milk tree alkaloids in plants, and to apply this method to the breeding of sustainable bioluminescent plants.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a method for improving the bioluminescence intensity of plants by metabolic pathway redesign, wherein the bioluminescent plant is a transgenic plant whose genome integrates genes encoding enzymes of fungal luminescence pathways. The method includes: introducing TAL, HpaB, and HpaC genes into the transgenic plant using biological techniques, wherein the coding sequences of the TAL, HpaB, and HpaC genes are as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, or have more than 70% homology with the shown sequences and are functionally equivalent to the encoded proteins.

[0009] This invention optimized the plant codons of the RgTAL (Tyrosine ammonialyase) gene from the genome of *Rhodotorula glutinis*, the PaHpaB (FAD-dependent 4-hydroxyphenylacetate-3-monooxygenase) gene from *Pseudomonas aeruginosa*, and the SeHpaC (NADH-flavin oxidoreductase) gene from *Salmonella enteritidis*, and investigated their application in improving the intensity of plant bioluminescence. TAL can convert tyrosine to p-coumaric acid, and HpaB and HpaC catalyze the production of caffeic acid from p-coumaric acid. The study showed that by introducing genes for the tyrosine synthesis pathway and genes for the synthesis of caffeic acid, the ability of plants to produce caffeic acid and milk alkaloids was significantly enhanced, thereby increasing the intensity of plant bioluminescence.

[0010] In this invention, the coding sequences of the TAL, HpaB, and HpaC genes are not limited to those shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the coding sequences of the genes can be optimized according to the codon preferences of the recipient plant.

[0011] The methods described above for introducing TAL, HpaB, and HpaC genes into transgenic plants can include, but are not limited to, using the TransGene Stacking II system for multi-genome assembly and using Agrobacterium-mediated transformation to introduce exogenous genes into recipient plants.

[0012] The plant may be, but is not limited to, tobacco. Tobacco varieties may include, but are not limited to: Nicotiana abentonhamiana, Nicotiana tabacum, and Nicotiana alata.

[0013] Furthermore, the method also includes: using biological techniques to downregulate the expression of at least one of 4-coumaric acid:coenzyme A ligase, chalcone synthase, caffeic acid-O-methyltransferase, caffeoyl-coenzyme A-methyltransferase, and MYB transcription factor in transgenic plants.

[0014] 4-Coumarate CoA ligase (4CL) is a key branching enzyme in the phenylpropanoid pathway. It can catalyze the entry of caffeic acid and ferulic acid into specific lignin biosynthetic pathways via cinnamyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD). It can also catalyze the entry of coumaric acid into the flavonoid pathway via chalcone synthase (CHS) or chalcone isomerase (CHI) (Harding et al., 2002). CHS is considered a key entry enzyme in the production of polyketide phenylpropanoids in plants. CHS catalyzes the condensation reaction of the CoA ester of cinnamic acid or its derivatives (such as coumaric acid or ferulic acid) with malonyl-CoA. Subsequently, a series of isomerases, reductases, hydroxylases, glycosyltransferases, and acyltransferases appear, modifying the basic flavonoid skeleton to generate various flavonoid chemical subclasses (Ferrer et al., 2008). Caffeic acid O-methyltransferase (COMT) has been shown to catalyze the methoxylation of caffeic acid and 5-hydroxyferulic acid in vitro; caffeoyl-CoA O-methyltransferase (CCoAOMT) catalyzes the conversion of caffeoyl-CoA to feruloyl-CoA. Both pathways enter the lignin synthesis pathway by consuming caffeic acid. MYB11 (MYB family transcription factors) participate in the regulation of flavonoid biosynthesis in plants, regulating the gene expression of enzymes related to L-tyrosine synthesis and indirectly regulating the gene expression of enzymes involved in certain steps of the caffeic acid synthesis pathway.

[0015] This invention reduces the outflow of caffeic acid and milk alkaloids by inhibiting the expression of genes that cause the diversion of caffeic acid and milk alkaloids in plants, allowing more of them to flow into the FBP luminescence pathway. This, combined with the introduction of the aforementioned exogenous genes, achieves "open source and reduce flow".

[0016] Furthermore, the coding sequences of the 4-coumaric acid:coenzyme A ligase, chalcone synthase, caffeic acid-O-methyltransferase, caffeoyl-coenzyme A-methyltransferase, and MYB transcription factor are as shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, or have more than 70% homology with the sequences shown and encode functionally equivalent proteins.

[0017] The aforementioned biological techniques for downregulating gene expression can include, but are not limited to, amiRNA technology.

[0018] In this invention, the enzyme encoding genes of the fungal luminescence pathway include the Hisps gene, CPH gene, H3H gene, NPGA gene, and Luz gene. The proteases encoded by the Hisps, CPH, H3H, and Luz genes participate in the caffeic acid cycle within the plant, enabling autoluminescence. The coding sequences of the above genes can be found in Chinese Patent Application No. 2021112288039.

[0019] Furthermore, a 5' leader sequence is integrated upstream of the Hisps, H3H, and Luz genes, with the nucleotide sequence 5'-ACAATTACCAACAACAACAAACAACAAACAACATTACAATTACTATTTACAATTA-3'. Adding a 5' leader sequence before the coding regions of the luminescent core enzyme genes Hisps, H3H, and Luz enhances translation.

[0020] Furthermore, the BnC3H gene is also integrated into the genome of the transgenic plant. Integration of the BnC3H gene, based on the above, can enhance the luminescence intensity of the plant. The coding sequence of the BnC3H gene can be found in Chinese Patent Application No. 2021112288039.

[0021] Compared to self-luminescent plants obtained by integrating Hisps, CPH, H3H, NPGA, Luz, and BnC3H genes into the plant genome, this invention further redesigns metabolic pathways by introducing genes for tyrosine synthesis and caffeic acid synthesis, and inhibiting the expression of caffeic acid and milk alkaloid efflux genes, thereby significantly improving the intensity of plant self-luminescence.

[0022] This invention also provides a method for creating bioluminescent plants, comprising the following steps:

[0023] (1) Using multi-gene assembly technology, Hisps, CPH, H3H, NPGA, Luz, TAL, HpaB and HpaC genes were integrated into the recipient vector to construct a multi-gene vector;

[0024] (2) Using transgenic technology, the target gene fragment in the multi-gene vector is introduced into the recipient plant and cultivated to obtain transgenic plants with enhanced bioluminescence intensity.

[0025] The recipient plant can be, but is not limited to, tobacco.

[0026] Furthermore, each target gene in the multi-gene vector contains a 35S promoter sequence upstream.

[0027] Furthermore, in step (1), the TransGene Stacking II system is used for multi-genome assembly. The TransGene Stacking II system is a multi-genome assembly vector system, see Chinese Patent Application No. 2017103841977.

[0028] Furthermore, pYL322d1 was used as donor vector I, pYL322d2 as donor vector II, and pYLTAC380GW as recipient vector.

[0029] Furthermore, the method also includes silencing the expression of the 4CL, CHS, COMT, CCoAOMT and MYB11 genes using amiRNA technology.

[0030] Artificial microRNAs are miRNA / miRNA* sequences in the hairpin stem-loop duplex of endogenous miRNAs modified with artificial sequences. These modified molecules can utilize all the key enzymes required for miRNA biogeneration to produce mature amiRNAs. In amiRNA expression elements, the hairpin stem-loop amiRNA precursor is transcribed and then forms an amiRNA dimer under the action of the DCL enzyme. Subsequently, the amiRNA dimer binds to the RISC complex, ultimately triggering the breakage of the target mRNA.

[0031] Furthermore, the target sequence for the 4CL gene is: 5'-CTTTTCCAGTCTGTGCAGCCA-3';

[0032] The target sequence for the CHS gene is: 5'-TTTTGCAGAGGCTTTCCGCAT-3';

[0033] The target sequence for the COMT gene is: 5'-TACTAGCAAACATGTCGGCAC-3';

[0034] The target sequence for the CCoAOMT gene is: 5'-TTCACAAGCTGCATCAGTCTC-3';

[0035] The target sequence for the MYB11 gene is: 5'-TACAAATCAACTACAGCTTGA-3'.

[0036] amiRNAs can silence multiple target genes and tandem with multiple desired repressed genes, achieving multi-gene silencing. Because amiRNAs are designed to accommodate various mismatches, the complementary sites between the amiRNA and each target gene do not necessarily have to be identical. Therefore, amiRNAs can not only silence target genes with completely identical sequences simultaneously, but also silence target genes with only a few base differences in their target sequences, solving the problem of gene family redundancy. This invention utilizes a CSY4 fragment (sequence 5'-CTGCCGTATAGGCAG-3') to tandem multiple amiRNAs to construct an amiRNA array, and introduces the CSY4 encoding gene (CDS sequence shown in SEQ ID NO. 9). CSY4 cleaves the CSY4 fragment to release amiRNAs, which are used to collectively suppress the expression of genes in tobacco that cause the diversion of caffeic acid and milk alkaloids. The amiRNA array constructed in this invention can suppress multiple homologous genes from several closely related species.

[0037] The beneficial effects of this invention are as follows:

[0038] This invention, for the first time, utilizes metabolic pathway redesign to introduce codon-optimized exogenous genes TAL, HpaB, and HpaC. TAL converts tyrosine into p-coumaric acid, which is then converted into caffeic acid under the action of HpaB and HpaC. By increasing the biosynthetic yield of caffeic acid, the bioluminescence intensity of plants is significantly enhanced. This invention provides a feasible technical solution for the breeding and production of sustainably luminescent plants. Attached Figure Description

[0039] Figure 1 A schematic diagram of the target amiRNA-related vector and the empty pCAMBIA1300 vector.

[0040] Figure 2 A design diagram of amiRNAs for multiple homologous genes in the genus *Nicotiana*.

[0041] Figure 3 The results show the RT-qPCR expression levels of amiRNA target genes after transient transformation of tobacco leaves, where A is 4CL, B is CCoAOMT, C is COMT, D is CHS, and E is MYB11.

[0042] Figure 4 The contents of caffeic acid (CA) and hispidin in amiRNA samples were analyzed by LC-MS / MS, where A is caffeic acid and B is hispidin.

[0043] Figure 5 This diagram illustrates the amiRNAs array vector and the in vivo processing of endogenous Csy4.

[0044] Figure 6 The values ​​of caffeic acid (CA) and hispidin were determined after transient expression of amiRNAs in eFBP tobacco, where A represents caffeic acid and B represents hispidin.

[0045] Figure 7 This is a schematic diagram of the enhanced bioluminescent modules eFBP1 and eFBP2 vectors. The 5' leader sequence Omega(Ω) is used to enhance translation.

[0046] Figure 8 The diagram shows the Not I enzyme digestion verification process for the final vector construction, where A is a schematic diagram of the plasmid and B is a gel electrophoresis image.

[0047] Figure 9 The comparison shows the luminescence intensity of FBP, eFBP1, and eFBP2 after transient expression in tobacco, where A is a physical image and B is a photon statistics graph.

[0048] Figure 10 The values ​​of caffeic acid (CA) and hispidin after transient expression of FBP, eFBP1, and eFBP2 in tobacco are given, where A represents caffeic acid and B represents hispidin.

[0049] Figure 11 The comparison shows the luminescence intensity of FBP and eFBP2 after transient expression in N. alata and N. tabacum, where A is a physical image and B is a photon count graph.

[0050] Figure 12 The values ​​represent the caffeic acid (CA) and hispidin content after transient expression of FBP and eFBP2 in N. alata and N. tabacum, where A represents caffeic acid and B represents hispidin.

[0051] Figure 13 Preliminary identification and photon comparison of eFBP2 transgenic tobacco, where A is the screening process of transgenic luminescent tobacco, B is a physical image of eFBP2 transgenic tobacco under natural light and dark conditions, C is a comparison of photons of flowers of eFBP and eFBP2 strains, and D is a photon statistical graph.

[0052] Figure 14 To detect the expression level of eFBP2 in transgenic tobacco.

[0053] Figure 15 The content of caffeic acid (CA) and hispidin in eFBP2 genetically modified tobacco. Detailed Implementation

[0054] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0056] The plasmids pYL322d1, pYL322d2, pYLMF-H, and pYLTAC380GW were donated by Professor Liu Yaoguang's laboratory at South China Agricultural University. The construction method can be found in Chinese Patent Application No. 2017103841977.

[0057] Example 1: Design and Construction of amiRNAs

[0058] 1. Using the Web microRNA designer 3 (WMD3) website published by Weigel et al., we targeted Nt4CL1 (Gene symbol: LOC107803673, CDS sequence as shown in SEQ ID NO.4), NtCHS (Gene symbol: LOC107813613, CDS sequence as shown in SEQ ID NO.5), NtCOMT (Gene symbol: LOC107788038, CDS sequence as shown in SEQ ID NO.6), NtCCoAOMT (Gene symbol: LOC107792528, CDS sequence as shown in SEQ ID NO.7), NtMYB11 (Gene symbol: LOC107801493, CDS sequence as shown in SEQ ID NO.8), NtANY (Gene symbol: LOC107829462), and NtGlyA (Gene symbol: LOC107829462). The symbol: LOC107820931) automatically designed four suitable primer sequences (see Table 1), and the corresponding amiRNA can be artificially synthesized using these four primers.

[0059] Table 1. Primer sequences

[0060]

[0061]

[0062] Specifically, in the first round, pRS300 was used as a template to amplify products a, b, and c via PCR. In the second round, overlap PCR was used, with the first-round amplification products a, b, and c as templates to amplify the final product d (Table 2). The resulting final product was then constructed into the pCAMBIA1300-P35S vector digested with Xba I / Sac I using a traditional restriction ligation method. Finally, the following vectors were constructed: pCAMBIA1300-P35S-amiR(4CL1), pCAMBIA1300-P35S-amiR(COMT), pCAMBIA1300-P35S-amiR(CCoAOMT), pCAMBIA1300-P35S-amiR(CHS), pCAMBIA1300-P35S-amiR(MYB11), pCAMBIA1300-P35S-amiR(ANY), and pCAMBIA1300-P35S-amiR(GlyA). Figure 1 ).

[0063] Table 2. PCR amplification steps

[0064]

[0065] 2. A homology search was performed on the predicted target sequences in the NCBI database to identify homologous proteins in different species of the genus Nicotiana that are highly similar to these gene sequences. A series of homologous protein sequences were ultimately identified. Figure 2 ).

[0066] Example 2: Validation of the silencing effect of amiRNAs-related vectors on transient expression in tobacco

[0067] The above-mentioned amiRNA-related vector was transformed into Agrobacterium tumefaciens EHA105. After verifying its correctness by transiently expressing the vector in tobacco, transient expression was then performed in tobacco. The specific method for transient expression is as follows:

[0068] 1) The EHA105 Agrobacterium transformed with the amiRNA-related vector was activated in LB medium containing Kana+Rif and cultured for 1 day at 200 rpm in a shaker at 28°C.

[0069] 2) Add 1 mL of bacterial culture to 20 mL of LB medium containing Kana + Rif + 15 μM acetylsyringone for expansion culture. Incubate at 28 °C and 200 rpm until OD reaches 100%. 600 =0.8-1.0;

[0070] 3) Centrifuge at 5000 rpm for 10 min to collect bacteria. Wash Agrobacterium with infiltration buffer (10 mM MES, 10 mM MgCl2, 150 μM AS) and resuspend at OD. 600 =0.8-1.0, let stand at room temperature for 2-3 hours;

[0071] 4) Use a 1mL syringe with the needle removed to draw up the bacterial solution. Gently poke a small hole (without piercing the leaf) on the lower epidermis of a tender ZY100 tobacco leaf that is about 3 weeks old. Press the back with your thumb and slowly inject Agrobacterium into the tobacco leaf with a syringe. Mark the water-soaked area of ​​the tobacco leaf.

[0072] 5) Cultivate the plant at 25℃ under normal growth conditions for about 72 hours, cut off the marked leaf area, put it into liquid nitrogen for quick freezing, and store it in a -80℃ freezer.

[0073] 6) Perform routine RNA extraction, reverse transcription, real-time quantitative PCR (RT-qPCR) analysis, and LC-MS / MS (liquid chromatography-mass spectrometry) analysis on the samples. Primer sequences are shown in Table 3.

[0074] The results showed that amiRNAs associated with 4CL1, COMT, CCoAOMT, CHS, and MYB11 all had a silencing effect on their homologous family genes in tobacco. Figure 3 The results of metabolite analysis showed that the five amiRNA vectors mentioned above could, to some extent, enhance the content of the luminescent substrates caffeic acid and milk alkaloids, consistent with expectations. Figure 4 ).

[0075] Table 3. RT-qPCR primer sequences

[0076]

[0077]

[0078] Example 3: Construction and effect verification of amiRNA array

[0079] After verification in Example 2 above, amiRNAs with inhibitory effects and the ability to enhance caffeic acid and milk alkaloid content were selected. Inspired by the Csy4 treatment system in tomato protoplasts, which efficiently generates multiple Cas9-related guide RNAs (ermáket al., 2017), this invention uses a similar method to generate an amiRNA array to collectively downregulate the caffeic acid synthesis gene family (Nt4CLs, NtCOMTs, NtCCoAOMTs, NtCHSs, and NtMYB11s) in tobacco. Multiple amiRNAs were artificially synthesized using a tandem sequence of the CSY4 fragment (sequence CTGCCGTATAGGCAG), and constructed into pYLMF-H via reverse PCR (inserted after the CmYLCV promoter and before the NOS terminator). The CSY4 encoding gene was inserted after the 35S promoter, becoming the pYLMF-H-amiRNA array. Figure 5 ).

[0080] The constructed pYLMF-H-amiRNAs array was transiently expressed and validated in eFBP tobacco (containing Hisps, CPH, H3H, NPGA, Luz, and BnC3H; the construction method is described in Chinese Patent Application No. 2021112288039), as described in Example 2. The results showed that the sample injected with the pYLMF-H-amiRNAs array vector exhibited a significant increase in caffeic acid and milk alkaloid content. Figure 6 ).

[0081] Example 4: Construction process of large-fragment bioluminescent module DNA vectors eFBP1 and eFBP2

[0082] This invention utilizes the TransGene Stacking II system for multi-genome assembly. The H3H (hispidin-3-hydroxylase) and Hisps (hispidinsynthase) genes, CPH (Caffey pyruvate hydrolase) gene from the *Neonothopanus nambi* genome, the NPGA (4'-phosphopantetheinyl transferase) gene from the *Aspergillus nidulans* genome, the fungal luciferase Luz gene, the HpaB gene from *Pseudomonas aeruginosa*, the HpaC gene from *Salmonella enteritidis*, and the TAL (tyrosine ammonia lyase) gene from *Rhodotorula glutinis* were integrated into the pYLTAC380GW plasmid. Finally, a hygromycin selection marker was added via a one-step BP recombination reaction to construct the eFBP1 vector. Figure 7 Simultaneously, based on the above, the CSY4 gene is added, and in the final step, a hygromycin selection marker and an amiRNA array silencing Nt4CL, NtCHS, NtCOMT, NtCCoAOMT, and NtMYB11 expression are ligated into the vector via a BP recombination reaction, thus constructing the eFBP2 vector. Figure 7 ).

[0083] The sequence information for HispS is available in accession number QJQ48095.1; the sequence information for CPH is available in accession number QJQ48093.1; the sequence information for H3H is available in accession number QJQ48094.1; the sequence information for NPGA is available in accession number QJQ48097.1; and the sequence information for Luz is available in accession number QJQ48096.1. Codon optimization was performed on the coding sequences of RgTAL, CSY4, PaHpaB, and SeHpaC to synthesize the corresponding gene fragments. Specifically, the CDS sequence of the RgTAL gene is shown in SEQ ID NO.1, the CDS sequence of the PaHpaB gene is shown in SEQ ID NO.2, the CDS sequence of the SeHpaC gene is shown in SEQ ID NO.3, and the CDS sequence of the CSY4 gene is shown in SEQ ID NO.9.

[0084] The specific construction process of the eFBP1 and eFBP2 vectors is as follows:

[0085] (1) By using overlap PCR, PaHpaB and SeHpaC, RgTAL and CSY4 were linked together to form the fusion fragment PaHpaB-p2A-SeHpaC, RgTAL-p2A-Csy4 using the P2A fragment (sequence 5'-GCAACAAATTTCTCACTCCTTAAGCAGGCAGGAGATGTTGAAGAGAACCCCGGTCCT-3').

[0086] P2A is a self-cleaving peptide commonly used in the construction of vectors for multi-gene co-expression. P2A peptides can release fusion proteins through their self-cleavage activity without the need for exogenous cleavage enzymes, reducing the complexity of experiments and the size of vectors.

[0087] (2) Construct donor vectors: pYL322d1-HispS, pYL322d2-CPH, pYL322d1-H3H, pYL322d2-NPGA, pYL322d1-Luz, pYL322d2-RgTAL or pYL322d2-RgTAL-P2A-CSY4, pYL322d1-PaHpaB-P2A-SeHpaC;

[0088] (3) The donor vector pYL322d1-Hisps and the recipient vector pYLTAC380GW (at a ratio of 1:1 to 2:1) were mixed in NS3529 competent cells for cotransformation. The heat shock method was used: ice bath for 30 min, heat shock for 90 s, ice bath for 2-3 min, and revive in LB without antibiotics at 37°C and 200 rpm for 2 h. The revival was then spread on LA plates containing kanamycin (Km, 25 mg / L) and chloramphenicol (Chl, 15 mg / L). After about 18 h, single clones grew. All single clones were washed into tubes with ddH2O and the mixed plasmid was extracted.

[0089] (4) Take about 50-100 ng of the mixed plasmid and digest it with 0.5 uL I-Sce I (NEB) in a 10 uL system for 4-5 h. Transform it into Escherichia coli strain NEB10-β (Bomaide Biotechnology Co., Ltd.), plate it on LA plate containing kanamycin (Km, 25 mg / L), incubate at 37℃ for 15 h, then pick single clones and culture them in LB (containing 25 mg / L Km and 0.5 mM IPTG). Perform bacterial PCR identification. Using Green Taq Mix, further extract plasmids that can amplify bright bands, take 200 ng of each and digest them with 0.2 uL Not I in a 20 uL reaction system. If four bands appear, the band containing the target gene of about 6.2 kJ is the desired positive clone pYLTAC380GW-HispS.

[0090] (5) The donor vector pYL322d2-CPH and the recipient vector pYLTAC380GW-Hisps from (4) (at a ratio of 1:1 to 2:1) were mixed in NS3529 competent cells for cotransformation. The transformation was carried out according to the method in (3), and the mixture was plated on LA plates containing kanamycin (Km, 25 mg / L) and ampicillin (Amp, 70 mg / L). After about 18 hours, single colonies grew. All single colonies were washed into tubes with ddH2O and the mixed plasmid was extracted.

[0091] (6) Take about 40-90 ng of mixed plasmid and digest it with 0.5 uL PI-Sce I (NEB) and 0.5 uL BSA in a 10 uL system for 4-5 h. Then transform and verify according to the method in (4). If five bands appear and the target gene fragment is present, it is a positive clone pYLTAC380GW-HispS-CPH.

[0092] (7) Multiple rounds of recombination were performed, using d1 and d2 donor vectors containing different genes in cross-transformation with the recipient vectors constructed in the previous round. Finally, the constructed vectors were used with pYLMF-H (100 ng) and pYLMF-H-amiRNAsarray respectively via BP reaction to construct eFBP1 and eFBP2 vectors. The reaction system consisted of 5 μL of 1 μL of 5×BP enzyme mixture incubated at 25°C for 5 hours, followed by the addition of 1 μL of proteinase K solution to terminate the reaction at 37°C for 10 minutes. The mixture was then transformed into NEB10-β (Bomaide Biotechnology Co., Ltd.) competent cells, and single clones were selected for identification. Not I restriction enzyme digestion was used to detect that the correct positive final vector eFBP2 had 13 DNA bands (…). Figure 8 ).

[0093] Example 5: Verification of the transient conversion of Nicotiana tabacum, Nicotiana benthamiana, and Nicotiana alata using eFBP1 and eFBP2 bioluminescent modules.

[0094] EHA105 bacterial cultures containing verified FBPs (containing only the five basic luminescent genes Hisps, CPH, H3H, NPGA, and Luz; construction method described in Chinese patent application number 2021112288039), eFBP1, and eFBP2 modules were streaked onto LA+Kana+Rif plates for activation and cultured at 28°C for 36 hours. Colonies were picked from each plate and transferred to LB+Kana+Rif+15μM As medium and cultured at 28°C and 200rpm until OD200. 600=0.8-1.0, 4000 rpm, 10 min to collect bacterial cells, suspend Agrobacterium cells in infection solution (containing 10 mM MgCl, 10 mM MES, 150 μM As) and let stand at room temperature for 2-3 h.

[0095] Transient expression verification and comparison of luminescence intensity of FBP, eFBP1, and eFBP2 were performed in *Nicotiana benthamiana*. The results showed that eFBP1 and eFBP2 exhibited stronger luminescence compared to FBP, with eFBP2 showing twice the luminescence intensity of eFBP1. Figure 9 Furthermore, LC-MS / MS data showed that eFBP1 produced 2.6 times and 1.2 times more caffeic acid and milk alkaloids than FBP, respectively. Figure 10 The accumulation of caffeic acid and milk alkaloids in eFBP2 was 1.3 times and 1.5 times that of eFBP1, respectively. Figure 10 These results further demonstrate that the combination of microbial-derived tyrosine-caffeic acid synthesis pathway and amiRNA has a significant effect on enhancing plant bioluminescence.

[0096] Furthermore, transient expression was validated in *Nicotiana tabacum* and *Nicotiana alata*, comparing the luminescence intensity of FBP and eFBP2 in these two *Nicotiana* species and detecting their expression levels. The results showed that the luminescence intensity of eFBP2 was significantly higher than that of FBP in both *Nicotiana tabacum* and *Nicotiana alata*. Figure 11 This study also indicated that injecting a vector containing amiRNA arrays could suppress multiple homologous genes from several closely related species. Metabolic results showed that the caffeic acid and galactoside content of eFBP2 was significantly higher than that of FBP2 in both common tobacco and flowering tobacco. Figure 12 ).

[0097] Example 6: Analysis of luminescence intensity in transgenic tobacco plants mediated by Agrobacterium-mediated eFBP2 bioluminescence module

[0098] (1) EHA105 bacterial suspensions containing verified eFBP (containing Hisps, CPH, H3H, NPGA, Luz, and BnC3H; construction method as described in Chinese Patent Application No. 2021112288039) and eFBP2 module were streaked onto LA+Rif+Kana plates and cultured at 28°C for 36 h. Single colonies were picked and transferred to 3-5 mL of LB medium at 200 rpm and cultured at 28°C for 36 h. The culture was then expanded to 50 mL at a ratio of 1:100-1:50 and cultured for 3-5 h until OD was reached. 600=0.6, then centrifuge the bacterial culture and resuspend the cells in MS0 liquid medium (MS + 3% Sucrose + pH 5.8, 50 mL) until OD. 600 =0.6 is used for infection;

[0099] (2) Select fully expanded healthy tobacco leaves (4-5 weeks old), cut them into 0.5cm square pieces with a scalpel (cut off the leaf edge and avoid the midrib), and place the leaves with the upper surface facing down on MS1 ​​solid medium (MS + 0.5mg / L IAA + 2.0mg / L BA + 3% sucrose + 0.6-0.8% Phytagel, pH = 5.8) and incubate in the dark at 25℃ for 2-3 days;

[0100] (3) Add the pre-cultured tobacco leaves to the bacterial solution, vortex to ensure that the leaf cut is submerged in the bacterial solution, let stand for 5-30 minutes, and use sterile filter paper to absorb the attached bacterial solution; place the infected leaves with the upper surface facing down on MS1 ​​solid medium and incubate in the dark at 28°C for 2 days; place the leaves with the upper surface facing up on MS1 ​​selection medium containing Timentin and hygromycin and incubate in the light at 25°C; when buds grow from the leaf margin and can be separated (more than 1 cm), cut off the buds and transfer them to MS2 solid medium (MS + 0.5 mg / L IAA + 3% sucrose + 0.6-0.8% Phytagel, pH = 5.8) containing antibiotics (TM + HygB). Roots will grow after two weeks. Open the lid of the seedling box and harden the seedlings for one week before transferring them to the planting soil for cultivation. At the same time, cut off a corner of the leaf and take a picture for analysis under a fully automatic luminescence detection system.

[0101] The result is shown in the figure. Figure 13 As shown in the figure, the luminescence intensity of transgenic tobacco plants stably overexpressing the eFBP2 vector module was significantly higher than that of the original eFBP vector module. Expression level detection of the eFBP2 transgenic plants showed that all exogenous genes were expressed, and the expression levels of amiRNA repressor genes were significantly lower compared to eFBP. Figure 14 The content of caffeic acid and milk alkaloids was significantly higher in the plant than in the eFBP plant. Figure 15 ).

Claims

1. A method for improving the intensity of plant bioluminescence by metabolic pathway redesign, wherein the bioluminescent plant is a transgenic plant whose genome integrates a gene encoding a fungal bioluminescent pathway enzyme, characterized in that, The method includes: introducing TAL, HpaB, and HpaC genes into the transgenic plant using biological techniques, wherein the coding sequences of the TAL, HpaB, and HpaC genes are as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, or have more than 70% homology with the shown sequences and are functionally equivalent to the encoded proteins.

2. The method as described in claim 1, characterized in that, The method further includes: using biological techniques to downregulate the expression of at least one of 4-coumaric acid:coenzyme A ligase, chalcone synthase, caffeic acid-O-methyltransferase, caffeoyl-coenzyme A-methyltransferase, and MYB transcription factor in transgenic plants.

3. The method as described in claim 2, characterized in that, The coding sequences of the 4-coumaric acid:coenzyme A ligase, chalcone synthase, caffeic acid-O-methyltransferase, caffeoyl-CoA-methyltransferase, and MYB transcription factor are as shown in SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, or have more than 70% homology with the sequences shown and encode functionally equivalent proteins.

4. The method as described in claim 1, characterized in that, The enzyme encoding genes of the fungal luminescent pathway include the Hisps gene, CPH gene, H3H gene, NPGA gene, and Luz gene.

5. The method as described in claim 4, characterized in that, The Hisps, H3H, and Luz genes have a 5' leader sequence integrated upstream of them, and the nucleotide sequence of the 5' leader sequence is 5'-ACAATTACCAACAACAACAAACAACAAACAACATTACAATTACTATTTACAATTA-3'.

6. The method as described in claim 4 or 5, characterized in that, The transgenic plant genome also integrates the BnC3H gene.

7. A method for creating bioluminescent plants, characterized in that, Includes the following steps: (1) Using multi-gene assembly technology, Hisps, CPH, H3H, NPGA, Luz, TAL, HpaB and HpaC genes were integrated into the recipient vector to construct a multi-gene vector; (2) Using transgenic technology, the target gene fragment in the multi-gene vector is introduced into the recipient plant and cultivated to obtain transgenic plants with enhanced bioluminescence intensity.

8. The method for creating bioluminescent plants as described in claim 7, characterized in that, Multi-genome assembly was performed using the TransGeneStacking II system.

9. The method for creating bioluminescent plants as described in claim 7, characterized in that, The method also includes using amiRNA technology to silence the expression of the 4CL, CHS, COMT, CCoAOMT and MYB11 genes.

10. The method for creating bioluminescent plants as described in claim 9, characterized in that, The target sequence for the 4CL gene is: 5'-CTTTTCCAGTCTGTGCAGCCA; The target sequence for the CHS gene is: 5'-TTTTGCAGAGGCTTTCCGCAT-3'; The target sequence for the COMT gene is: 5'-TACTAGCAAACATGTCGGCAC-3'; The target sequence for the CCoAOMT gene is 5'-TTCACAAGCTGCATCAGTCTC-3'; the target sequence for the MYB11 gene is 5'-TACAAATCAACTACAGCTTGA-3'.

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