Use of fungal luciferase truncates to increase fungal or plant bioluminescence

Abstract

The application discloses application of a fungal luciferase truncated body in improving the bioluminescence intensity of fungi or plants and belongs to the technical field of biotechnology.The genome of the fungi or plants is integrated with a HispS gene, a CPH gene, a H3H gene, an NPGA gene and a fungal luciferase truncated body coding gene, and the amino acid sequence of the fungal luciferase truncated body is shown as SEQ ID NO.1, which is 20 amino acids removed from the N terminal of the amino acid sequence of the fungal luciferase.The fungal luciferase truncated body is used to replace the fungal luciferase and is co-expressed with the HispS, CPH, H3H and NPGA participating in the bioluminescence of fungi or plants, so that the bioluminescence intensity of the fungi and plants can be significantly improved, and the light quantum intensity is increased by more than 1.5 times.The application provides a feasible technical scheme for the construction of sustainable luminescent fungi and the breeding and production of sustainable luminescent plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of truncated fungal luciferase in enhancing the bioluminescence intensity of fungi or plants. Background Technology

[0002] Bioluminescence is a unique life phenomenon in nature, primarily used for courtship, hunting, or deterring predators. Currently, mainstream lighting methods rely heavily on the exploitation of scarce energy sources. If inexpensive carbon sources could be converted into biochemical light energy to replace current lighting methods, it would enable efficient green production and living, realizing the concept of sustainable development.

[0003] Current research reports on the development of self-luminescent plants include: in 2013, Stanford University's "Sustainable Luminescent Plants" project reported the expression of the luciferase gene in Arabidopsis thaliana; in 2014, a bacterial luminescent gene was successfully expressed in tobacco chloroplast DNA, resulting in the cultivation of self-luminescent plants (Fleiss and Sarkisyan, 2019). In 2020, research reported the achievement of fungal self-luminescence in Nicotiana benthamiana, emitting visible light; however, the light intensity is still far from sufficient to replace existing lighting and cannot be put into production applications.

[0004] For a long time, it has been hypothesized that the fungal bioluminescence pathway (FBP) consists of at least four components: molecular oxygen, luciferin 3-hydroxyl hispidin, an NAD(P)H-dependent hydroxylase, and luciferase (Airth and Mc, 1959). Through informatics and functional validation, the enzymes required for the complete FBP have been identified: fungal luciferase (Luz), hispidin synthase (HispS), and hispidin-3-hydroxylase (H3H) (Kotlobay et al., 2018). Another enzyme, caffeoylpyruvate hydrolase (CPH), is not essential for the generation of autoluminescence but significantly prolongs the signal.

[0005] The function of FBP is to convert caffeic acid to hispidin via the activity of HispS, and then, under the catalysis of H3H, convert hispidin to luciferin 3-hydroxyhispidin. Then, in the presence of oxygen, Luz converts luciferin into an unstable high-energy intermediate, releasing light (520 nm) and carbon dioxide, producing caffeoylpyruvate. For regeneration, CPH converts caffeoylpyruvate back to caffeic acid (Kaskova et al., 2017). The key to the complete expression of FBP in plants is the starting material caffeic acid. Caffeic acid is a crucial intermediate in the production of lignin and other key plant metabolites in the vast majority of plants. The relative simplicity of FBP, coupled with the abundance of the precursor molecule caffeic acid, lays the foundation for creating self-luminous plants.

[0006] Two studies published in 2020 investigated the production of bioluminescent plants by FBP using caffeic acid as a precursor from the plant itself (Khakhar et al., 2020; Mitiouchkina et al., 2020). In vector construction, both studies used four genes (NnLuz, NnH3H, NnHispS, NnCPH) from *Neonothopanus nambi*, adding the *AnNPGA* (4'-phosphopantetheinyltransferase) gene from *Aspergillus nidulans*. This gene was then modified post-translational to form the modular polyketide synthase HispS. The target genes were assembled using a plant MoClo cloning kit after codon optimization to obtain the transformation vector (Engler et al., 2014), which was then transiently or stably transformed into tobacco. Although the expression cassette designs differed between the two studies, bioluminescence was observed in both studies using a camera. Crucially, the analysis of stably transformed tobacco in this study showed that the FBP transgenic lines did not exhibit major phenotypic differences from the wild type in terms of plant height, leaf size, chlorophyll content, and carotenoid content (Mitiouchkina et al., 2020). However, current work is limited to constructing FBP systems in tobacco, which exhibit weak luminescence intensity, restricting further research and application.

[0007] Therefore, how to enhance the bioluminescence intensity of FBP systems constructed in plants is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to increase the bioluminescence intensity of fungi or plants by increasing the expression level of fungal luciferase Luz in the fungal bioluminescence pathway (FBP).

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention provides the application of fungal luciferase truncated bodies in improving the bioluminescence intensity of fungi or plants, wherein the genome of the fungi or plant contains a gene encoding the fungal luciferase truncated body, and the amino acid sequence of the fungal luciferase truncated body is shown in SEQ ID NO.1.

[0011] This invention reveals that, compared to self-luminescent fungi or plants obtained by integrating the HispS, CPH, H3H, NPGA, and Luz genes into the fungal or plant genome, replacing the fungal luciferase (Luz) encoding gene with the truncated LuzΔN20 encoding gene significantly increases the self-luminescence intensity of fungi or plants, with a photon intensity of 2.0 equations. 10 Photons(s -1 cm -2 This is more than 1.5 times higher than before.

[0012] The truncated fungal luciferase variant LuzΔN20 is formed by removing 20 amino acids from the N-terminus of the fungal luciferase (Luz) amino acid sequence, as shown in SEQ ID NO.1. Analysis of the Luz protein structure revealed a long α-helix structure at the N-terminus, far from the catalytic active site. Removing 20 amino acids from the N-terminus increases the protein accumulation of fungal luciferase, thereby enhancing the bioluminescence intensity of fungi or plants.

[0013] Furthermore, the nucleotide sequence of the fungal luciferase truncated variant encoding gene Luz△N20 is shown in SEQ ID NO.2.

[0014] The fungal or plant genome also integrates the HispS gene, CPH gene, H3H gene, and NPGA gene. The application includes: using biological techniques to integrate the HispS gene, CPH gene, H3H gene, NPGA gene, and LuzΔN20 into the genome of a caffeic acid-producing fungus or plant to obtain fungi or transgenic plants with enhanced bioluminescence intensity.

[0015] The proteases encoded by the HispS, CPH, H3H, NPGA, and Luz△N20 genes participate in the caffeic acid cycle in caffeic acid-producing fungi or plants, enabling fungal or plant autoluminescence.

[0016] Furthermore, the coding sequence of the HispS gene is shown in SEQ ID NO.3, the coding sequence of the CPH gene is shown in SEQ ID NO.4, the coding sequence of the H3H gene is shown in SEQ ID NO.5, and the coding sequence of the NPGA gene is shown in SEQ ID NO.6.

[0017] Furthermore, the application includes: using CRISPR / Cas9 gene editing technology to integrate the HispS gene, CPH gene, H3H gene, NPGA gene, and the fungal luciferase truncated gene Luz△N20 into the genome of a caffeic acid-producing fungus to obtain a fungus with enhanced bioluminescence intensity;

[0018] Alternatively, multi-gene assembly technology can be used to integrate the HispS gene, CPH gene, H3H gene, NPGA gene, and the fungal luciferase truncated gene Luz△N20 into the recipient vector to construct a multi-gene vector. Then, the target gene fragment in the multi-gene vector can be introduced into the recipient plant using transgenic technology, and the transgenic plant with enhanced bioluminescence intensity can be obtained through cultivation.

[0019] Furthermore, the caffeic acid-producing fungus is *Saccharomyces cerevisiae* YCA113-2B. This strain is a publicly available biological material; see the literature (Metabolic engineering of *Saccharomyces cerevisiae* for enhanced production of caffeic acid. *Appl Microbiol Biotechnol*. 2021, 105: 5809-5819.).

[0020] Furthermore, when constructing a bioluminescent-intensity-enhanced Saccharomyces cerevisiae, each target gene contains a galactose-induced GAL1 or GAL10 promoter upstream.

[0021] Furthermore, when constructing transgenic plants with enhanced bioluminescence intensity, the TransGene Stacking II system was used for multi-genome assembly. The TransGene Stacking II system is a multi-genome assembly vector system, as described in Chinese Patent Application No. 2017103841977.

[0022] Each target gene in the multi-gene vector contains a 35S promoter sequence upstream. Specifically, the 35S promoter is the CaMV 35S promoter.

[0023] Furthermore, the recipient plant can be, but is not limited to, Phalaenopsis, tobacco, rapeseed, or rice.

[0024] This invention also provides a method for constructing a bioluminescent intensity-enhanced yeast, comprising the following steps:

[0025] (1) The HispS gene fragment, CPH gene fragment, H3H gene fragment, NPGA gene fragment, and fungal luciferase truncated gene fragment Luz△N20 were cloned into the multiple cloning site of plasmid pESC-URA to obtain recombinant plasmids pESC-URA-Luz△N20-CPH, pESC-URA-H3H-NPGA, and pESC-URA-HispS; the nucleotide sequence of the fungal luciferase truncated gene is shown in SEQ ID NO.2.

[0026] (2) Using primers containing a 40bp homologous arm of the ARO10 gene, primers containing a 40bp homologous arm of the Int10 site, and primers containing a 40bp homologous arm of the Int12 site, the recombinant plasmid from step (1) was amplified by PCR to obtain the genome integration donor △Aro10::T with a “promoter-target gene-terminator” expression cassette. ADH1 -Luz△N20-P GAL10 -P GAL1 -CPH-T CYC1 Int10::T ADH1 -NPGA-P GAL10 -P GAL1 -H3H-T CYC1 and Int12::T ADH1 -MCS1-P GAL10 -P GAL1 -HispS-T CYC1 ;

[0027] (3) Using chemical methods to integrate the donor △Aro10::T ADH1 -Luz△N20-P GAL10 -P GAL1 -CPH-T CYC1 The plasmids pRS423-SpSgH-ARO10 and pRS41K-SpCas9 were transformed into caffeic acid-producing yeast cells, and recombinant strain I with successful integration was obtained by screening. Then, Int10::T ADH1 -NPGA-P GAL10 -P GAL1 -H3H-T CYC1 The plasmids pRS426-SpSgH-Int10 and pRS41K-SpCas9 were transformed into recombinant strain I, and recombinant strain II with successful integration was obtained by screening. Then, Int12::T ADH1 -MCS1-P GAL10 -P GAL1 -HispS-TCYC1 The recombinant strain II was transformed with plasmids pRS426-SpSgH-Int12 and pRS41K-SpCas9, and the successfully integrated recombinant strain III was obtained through screening, thus yielding the bioluminescent intensity-enhanced yeast.

[0028] The plasmid pESC-URA is a publicly available biological material. pESC-URA has promoters GAL1 and GAL10 and multiple cloning sites MCS1 and MCS2, which can be inserted into two genes.

[0029] The plasmids pRS423-SpSgH-ARO10, pRS426-SpSgH-Int10, pRS426-SpSgH-Int12, and pRS41K-SpCas9 are all publicly available biological materials. Their construction methods can be found in the literature (Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast. Biotechnol Bioeng, 2022, 119:1314-1326; Chinese patent application number 2021115974834). Using CRISPR / Cas9 gene editing technology, the integration donor is integrated into specific sites ARO10, Int10, and Int12 in the host genome.

[0030] This invention also provides a method for cultivating plants with enhanced self-luminescence intensity, comprising the following steps:

[0031] 1) The HispS gene fragment, H3H gene fragment, and fungal luciferase truncated gene fragment Luz△N20 were inserted into the multiple cloning site of the donor vector pYL322d1 to obtain the donor vectors pYL322d1-HispS, pYL322d1-H3H, and pYL322d1-Luz△N20, respectively; the nucleotide sequence of the fungal luciferase truncated gene is shown in SEQ ID NO.2;

[0032] The CPH gene fragment and NPGA gene fragment were inserted into the multiple cloning site of the donor vector pYL322d2 to obtain the donor vectors pYL322d2-CPH and pYL322d2-NPGA, respectively.

[0033] 2) The donor vector pYL322d1-HispS and the recipient vector pYLTAC380GW were mixed at a ratio of 1:1 to 2:1 and co-transformed into Escherichia coli NS3529 competent cells. The cells were then plated in a double-antibiotic medium containing kanamycin and chloramphenicol and cultured. Plasmids were extracted from positive strains.

[0034] 3) The plasmid extracted in step 2) was digested with homing enzyme I-Sce I, then transformed into Escherichia coli strain NEB10-β, cultured, screened, and plasmid extracted to obtain a positive clone pYLTAC380GW-HispS containing the target gene HispS.

[0035] 4) Mix the donor vector pYL322d2-CPH and the recipient vector pYLTAC380GW-HispS prepared in step 3) at a ratio of 1:1 to 2:1, and co-transfer them into Escherichia coli NS3529 competent cells. Spread the mixture on a double-antibiotic medium containing kanamycin and ampicillin and culture it. Extract plasmids from positive strains.

[0036] 5) The plasmid extracted in step 4) was digested with homing enzyme PI-Sce I, then transformed into Escherichia coli strain NEB10-β, cultured, screened, and plasmid extracted to obtain a positive clone pYLTAC380GW-HispS-CPH containing the target genes HispS and CPH.

[0037] 6) Repeat steps 2)-5), using the new plasmid containing the target gene obtained in the previous step as the recipient vector, and cross-recombining donor vectors containing different genes until all the target genomes are loaded into the recipient vector. In the final step, the BP recombination reaction is used to ligate the expression cassette element of the removed selection marker gene to construct the multi-gene vector pYLTAC380MF-HispS-CPH-H3H-NPGA-Luz△N20.

[0038] 7) Using Agrobacterium-mediated transformation, the target gene fragment from the multi-gene vector was introduced into the recipient plant, and the plant was cultured to obtain transgenic plants with enhanced bioluminescence intensity.

[0039] The donor carriers pYL322d1 and pYL322d2, and the recipient carrier pYLTAC380GW are all publicly available biological materials, see Chinese Patent Application No. 2017103841977.

[0040] The recipient plant can be, but is not limited to, orchids, tobacco, rapeseed, or rice.

[0041] The beneficial effects of this invention are as follows:

[0042] This invention discloses for the first time that the truncated fungal luciferase LuzΔN20 replaces fungal luciferase in co-expression with HispS, CPH, H3H, and NPGA, which are involved in fungal or plant bioluminescence. This significantly enhances the bioluminescence intensity of fungi and plants, increasing the quantum intensity by more than 1.5. This invention provides a feasible technical solution for the construction of sustainable luminescent fungi and the breeding and production of sustainable luminescent plants. Attached Figure Description

[0043] Figure 1 Predicting cell localization for NnLuz.

[0044] Figure 2 The diagram shows the protein structure prediction and analysis of NnLuz and its variants. A represents the accuracy analysis of the protein structure prediction of NnLuz; B represents the protein structure prediction model of NnLuz; and CH represent the protein structure predictions for truncating the N-terminus of NnLuz by 20, 25, 30, 36, 40, and 49 amino acids, respectively.

[0045] Figure 3 A schematic diagram showing the truncated N-terminus 20, 25, 30, 36, 40, and 49 amino acids of NnLuz.

[0046] Figure 4 To illustrate the optical signals of Saccharomyces cerevisiae with different lengths at the N-terminus of NnLuz, the left image is taken under bright field conditions, and the right image is taken under dark field conditions.

[0047] Figure 5 Comparison of YCA113-FBP and YCA113-FBP (LuzΔN20), where A is the bioluminescence image; B is the photon analysis, and error represents the standard deviation of three replicates. A two-tailed t-test (*P≤0.05) was used to assess statistical significance; C shows images taken under ambient light with an exposure of 1 / 200 second and in darkness with an exposure of 30 seconds.

[0048] Figure 6 This is a schematic diagram of NnH3H-EGFP and vectors expressing different forms of NnLuz-mCherry fusion protein.

[0049] Figure 7 Fluorescence signal diagrams of NnH3H-EGFP and Saccharomyces cerevisiae strains expressing different forms of NnLuz-mCherry fusion protein.

[0050] Figure 8 The effect of N-terminal truncation of NnLuz on protein expression levels is shown, with error representing the standard deviation of three replicates. Statistical significance was assessed using Dunnett's multiple comparison test (P ≤ 0.0001).

[0051] Figure 9 This is a schematic diagram of the FBP(Luz△N20) enhanced self-emissive module.

[0052] Figure 10This study compares the FBP transgenic lines and the FBP(Luz△N20) transgenic lines. A represents independent FBP(Luz△N20) lines screened using a bioluminescence imaging system; B represents bioluminescent images of FBP and FBP(Luz△N20) leaves; C represents bioluminescence intensity analysis of FBP and FBP(Luz△N20) leaves. Error represents the standard deviation of three replicates, and statistical significance was assessed using a two-tailed t-test (*P≤0.05); D represents images of FBP-4 and FBP(Luz△N20)-7 taken under ambient light and in darkness, respectively. Detailed Implementation

[0053] 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.

[0054] 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.

[0055] The plasmids p423-SpSgH, pRS426-SpSgH-Int10, pRS426-SpSgH-Int12, pRS423-SpSgH-Int16, and pRS41K-SpCas9 were provided by the laboratory of Professor Lian Jiachang of Zhejiang University. The construction method can be found in the literature (Liu T, Gou Y, Zhang B, et al. 2022. Construction of ajmalicine and sanguinarine de novobiosynthetic pathways using stable integration sites in yeast. Biotechnol Bioeng[J], 119:1314-1326.; Chinese patent application number 2021115974834).

[0056] The construction method of plasmid pRS423-SpSgH-ARO10 is the same as that of pRS426-SpSgH-Int10. Specifically, the gRNA sequence of ARO10 is designed using the Benchling CRISPR tool. The N20 sequence targeting ARO10 is screened by balancing the On-Target Score and Off-Target Score. The target primers △ARO10-F and △ARO10-R for N20 are synthesized in advance. △ARO10-F: gatcAAAGTGCATGGTTGAAGCAG; △ARO10-R: aaacCTGCTTCAACCATGCA CTTT. These primers are constructed into the sgRNA plasmid p423-SpSgH, transformed into Ecoli. DH5α, cultured overnight, and positive clones are screened by colony PCR. A small amount of plasmid is extracted and sent for sequencing. One copy each of the correctly sequenced strain and plasmid pRS423-SpSgH-ARO10 is stored.

[0057] YCA113-2B was donated by Associate Professor Ye Lidan's laboratory at Zhejiang University. Its construction method can be found in the literature (Zhou P, Yue C, Shen B, et al. 2021. Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid. Appl Microbiol Biotechnol[J], 105: 5809-5819.).

[0058] pYL322d1, pYL322d2, pYLTAC380GW, and PYLMFH-Bnmlpro 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.

[0059] Example 1: Construction process of self-luminous brewing yeast

[0060] This study used CRISPR / Cas9 gene editing technology to integrate the genome of Saccharomyces cerevisiae.

[0061] 1. Gene fragments were synthesized by codon optimization of the coding sequences of the following genes: NPGA (4'-phosphopantetheinyl transferase) gene from the Aspergillus nidulans genome, H3H (hispidin-3-hydroxylase) gene, HispidS (Hispidin synthase) gene, CPH (Caffey pyruvatehydrolase) gene, and fungal luciferase (Luz) gene from the Neonothopanus nambi genome, based on the host's codon preference.

[0062] The sequence information for HispS can be found in the gene accession number: QJQ48095.1; the sequence information for CPH can be found in the gene accession number: QJQ48093.1; the sequence information for H3H can be found in the gene accession number: QJQ48094.1; the sequence information for NPGA can be found in the gene accession number: QJQ48097.1; and the sequence information for Luz can be found in the gene accession number: QJQ48096.1.

[0063] The coding sequences of the HispS gene are shown in SEQ ID NO.3, the CPH gene in SEQ ID NO.4, the H3H gene in SEQ ID NO.5, the NPGA gene in SEQ ID NO.6, and the Luz gene in SEQ ID NO.7.

[0064] 2. The specific construction process of self-luminous brewing yeast is as follows:

[0065] (1) Construct a donor vector and clone the target gene into a vector containing a galactose promoter (P). GAL1 Or P GAL10 Recombinant plasmids pESC-URA-NnLuz-NnCPH, pESC-URA-NnH3H-AnNPGA, and pESC-URA-NnHispS containing the target gene were obtained by inserting the target gene into the MCS1 or MCS2 of the universal plasmid pESC (Lian et al. 2015). The specific gene insertion sites are shown in Table 1.

[0066] Table 1. Main plasmids used in this study

[0067]

[0068] (2) Using primers containing a 40bp homologous arm of the ARO10 gene, PCR was performed on the recombinant plasmid pESC-URA-NnLuz-NnCPH to obtain the genome integration donor of "promoter-target gene-terminator": △Aro10::T ADH1 -NnLuz-P GAL10 -P GAL1 -NnCPH-T CYC1 PCR was performed on the recombinant plasmid pESC-URA-NnH3H-AnNPGA using primers containing a 40bp homologous arm with the Int10 site to obtain the integration donor: Int10::T ADH1 -AnNPGA-P GAL10 -P GAL1 -NnH3H-T CYC1 PCR was performed on the recombinant plasmid pESC-URA-NnHispS using primers containing a 40bp homologous arm at the Int12 site to obtain the integration donor: Int12::T ADH1 -MCS1-P GAL10 -P GAL1 -NnHispS-T CYC1 The primers are shown in Table 2.

[0069] Table 2. Primers used for heterologous gene integration into yeast genome

[0070]

[0071] Note: The entire text uses T. ADH1 -MCS1-P GAL10 -P GAL1 -MCS2-T CYC1 The expression box is thus consistent with the primers of the integrated donor.

[0072] (3) Based on the CRISPR / Cas9 genome editing tool in Saccharomyces cerevisiae (Lian, J., Hamedi Rad, M., Hu, S. & Zhao, H. Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nature Communications 8, 2017, 1688-1696.), the integration donor △Aro10::T ADH1 -NnLuz-P GAL10 -P GAL1 -NnCPH-T CYC1The plasmids pRS423-SpSgH-ARO10 and pRS41K-SpCas9, along with their corresponding single guide RNA coding sequences, were chemically transformed into yeast cells YCA113-2B. The cells were then screened on SED-G418 plates with corresponding amino acid deficiencies to obtain YCA113-3B. The integration donor Int10::T was then used... ADH1 -AnNPGA-P GAL10 -P GAL1 -NnH3H-T CYC1 pRS426-SpSgH-Int10 and pRS41K-SpCas9 were transformed into yeast cells YCA113-3B, and YCA113-4B was obtained by screening. Then Int12::T ADH1 -MCS1-P GAL10 -P GAL1 -NnHispS-T CYC1 pRS426-SpSgH-Int12 and pRS41K-SpCas9 were transformed into yeast cells YCA113-4B, and YCA113-FBP-N was obtained by screening, as shown in Table 3.

[0073] Table 3. Recombinant Saccharomyces cerevisiae strains

[0074]

[0075] In the above screening steps, transformants on the plate were picked, cells were ruptured, and primers were designed using the target sequence on the genome to verify whether the target gene had been integrated into the genome using PCR. If the band size was correct and the sequencing results were accurate, it indicated that the genome integration was successful, yielding YCA113-FBP-N. The autoluminescence intensity of the Saccharomyces cerevisiae was detected using a fully intelligent gel imaging system (CCD, Charge-Coupled Device).

[0076] Example 2: Predicting NnLuz localization and simulating the protein structure of NnLuz

[0077] 1. Predict the signal peptide and cellular localization of the NnLuz protein using Philius Transmembrane Prediction Server and Phobius.

[0078] The prediction results from the Philius Transmembrane Prediction Server show that the first 20 amino acids of NnLuz are predicted to be in the cytoplasm with a confidence level of 49%; amino acids 20 to 36 are predicted to be transmembrane domains with a confidence level of 53%; and amino acids after the 36th amino acid are predicted to be on a cell membrane with the highest confidence level of 95%.

[0079] Phobius predictions are almost identical to those of the Philius Transmembrane Prediction Server, except that the transmembrane domain is located in the 20th to 40th amino acid region. Figure 1 As shown.

[0080] Based on the above analysis, most of NnLuz is located in non-cytoplasmic regions. This paper predicts that the protein of NnLuz may be a membrane protein with a redundant sequence at the N-terminus.

[0081] 2. AlphaFold 2.0 was used to predict the possible protein structures of five NnLuz molecules. The calculation results were almost identical, such as... Figure 2 As shown in Figure A, the confidence levels for the N-terminus and C-terminus are between 40% and 80%, the confidence level near the 175th amino acid is between 50% and 80%, and the confidence levels for the remaining regions are almost all above 80%. The NnLuz protein structure with the highest pLDDT score was selected as the research object from the five predictions. It was found that NnLuz has a long α-helix structure at its N-terminus that is far from the catalytic active center, as shown in Figure A. Figure 2 As shown in B, based on the aforementioned prediction results, it is speculated that it may contain a transmembrane structural domain.

[0082] To investigate the effects of the N-terminal cytoplasmic and transmembrane regions on the expression and localization of NnLuz, this study considered gradient truncation of its N-terminus, such as... Figure 3 As shown, simulations on AlphaFold 2.0 simultaneously truncated the first 20, 25, 30, 36, 40, and 49 amino acids at the N-terminus, and the structure of its active region was not significantly affected, as shown in the figure. Figure 2 As shown in CH.

[0083] Example 3: Knocking out NnLuz in autoluminescent Saccharomyces cerevisiae and integrating NnLuz truncated variants of different lengths

[0084] This study used CRISPR / Cas9 gene editing technology to knock out the NnLuz gene in the luminescent yeast constructed in Example 1, and then integrated an N-terminal truncated variant to verify the activity of the N-terminal truncated variant. The method is as follows:

[0085] 1) Benchling CRISPR tools were used to design the gRNA sequence of NnLuz. The N20 sequence targeting NnLuz was screened by balancing the On-Target Score and Off-Target Score. The N20 target primers △NnLuz-F and △NnLuz-R were synthesized in advance and constructed into the sgRNA plasmid p423-SpSgH. The plasmid was transformed into Ecoli.DH5α, cultured overnight, and positive clones were screened by colony PCR. The plasmid was extracted in small quantities and sent for sequencing. One copy each of the correctly sequenced strain and the plasmid p423-SpSgH-△NnLuz was stored.

[0086] △NnLuz-F:gatcATGTAACCCCTGAAGTTCTG;

[0087] △NnLuz-R:aaacCAGAACTTCAGGGGTTACAT.

[0088] 2) The plasmids p423-SpSgH-△NnLuz and pRS41K-SpCas9 were chemically transformed into yeast cells YCA113-FBP-N and plated on SED-G418 plates with the corresponding amino acid auxotropes. Transformants on the plates were picked, and the cells were lysed. Two primers from the genome were selected for PCR to verify whether the target gene had been integrated into the genome. If the band size was correct and the sequencing results were accurate, the genome integration was successful, yielding YCA113-FBP-N-△NnLuz.

[0089] 3) Construct a donor vector and clone the target gene into a vector containing a galactose promoter (P). GAL1 Or P GAL10 Using the universal plasmid pESC, recombinant plasmids pESC-URA-NnLuz△N20, pESC-URA-NnLuz△N25, pESC-URA-NnLuz△N30, pESC-URA-NnLuz△N36, pESC-URA-NnLuz△N40, and pESC-URA-NnLuz△N49 containing the target gene were obtained, as shown in Table 4. The truncation positions of each truncated variant are as follows: Figure 3 As shown, the coding sequence was optimized using codons to synthesize the corresponding gene fragment. The nucleotide sequence of Luz△N20 is shown in SEQ ID NO.2.

[0090] Table 4. Vector construction of truncated NnLuz N-terminal signal peptide

[0091]

[0092] 4) Using primers containing a 40bp homologous arm of the Int16 gene, the recombinant plasmid obtained in step 3) was subjected to PCR to obtain the genome integration donor of "promoter-target gene-terminator": T ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz△N20-T CYC1 T ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz△N 25-T CYC1 T ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz△N 30-T CYC1 T ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz△N 36-T CYC1 T ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz△N 40-T CYC1 T ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz△N 49-T CYC1 .

[0093] 5) Using the CRISPR / Cas9 genome editing tool in Saccharomyces cerevisiae, the plasmids pRS423-SpSgH-Int16 and pRS41K-SpCas9, which contain the integration donor and the corresponding single guide RNA coding sequences, were chemically transformed into yeast cells YCA113-FBP-N-△NnLuz and plated on SED-G418 plates with the corresponding amino acid auxotypes.

[0094] 6) For the transformants on the plate, pick spots to lyse the cells, and design primers to select the target sequence on the genome. Use PCR to verify whether the target gene has been integrated into the genome. If the band size is correct and the sequencing results are accurate, it indicates that the genome integration was successful, and the *Saccharomyces cerevisiae* strain shown in Table 5 is obtained.

[0095] Table 5. Screening of recombinant Saccharomyces cerevisiae strains with truncated N-terminal signal peptides of NnLuz

[0096]

[0097] 7) Pick three single clones of the strain constructed above and put them into test tubes containing 5 mL of liquid YPD. Incubate at 220 rpm for 18 h. Then take 200 μL of the bacterial solution from each strain into a 24-well plate containing 5 mL of liquid YPD medium and ferment at 800 rpm for 36 h. Then use a CCD imaging system to detect the luminescence of each strain.

[0098] CCD imaging also revealed that, compared to the control YCA113-FBP, only the yeast strain YCA113-FBP (Luz△N20) integrating NnLuz20 showed significant luminescence with a stronger signal. This suggests that the redundant amino acid fragment at the N-terminus in the cytoplasm was truncated, promoting better folding and localization of the target protein. Figure 4 As shown, the YCA113-FBP(Luz△N25) strain could no longer be detected for luminescence, possibly because the truncated transmembrane domain of amino acids 21-40 would affect the localization of NnLuz, preventing its functional expression.

[0099] 8) Take 8 μL of each of the three groups of fermented YCA113-FBP-△NnLuz, YCA113-FBP, YCA113-FBP(Luz△N20), and YCA113-FBP(Luz△N25) bacterial cultures, spot them on YPD solid medium, and invert them in a constant temperature incubator at 30℃ for overnight culture.

[0100] Analysis using a NIGHTSHADE-LB985 imaging and photon dosimeter revealed that the yeast strain YCA113-FBP (Luz△N20) integrating the NnLuz△20 variant had a photon intensity 1.5 times higher than the original control YCA113-FBP. Figure 5 As shown.

[0101] Example 4: Expression analysis of NnLuz and its variants

[0102] 1. Using a (GGGGS) 3-linker fusion to express mCherry at the C-terminus of Luz and its variants, pESC-URA-NnLuz-mCherry, pESC-URA-NnLuz△N20-mCherry, and pESC-URA-NnLuz△N25-mCherry were constructed. EGFP was fused to the C-terminus of H3H to construct pESC-URA-NnH3H-EGFP. Figure 6 As shown, the amount of protein accumulation was indirectly detected by the fluorescence intensity of mCherry.

[0103] 2. Using primers containing a 40bp homologous arm of the ARO10 gene, the recombinant plasmid obtained in step 1 was subjected to PCR to obtain the genome integration donor: T, which consists of a promoter, target gene, and terminator.ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz-mCherry-T CYC1 T ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz△N20-mCherry-T CYC1 T ADH1 -MCS1-P GAL10 -P GAL1 -NnLuz△N25-mCherry-T CYC1 T ADH1 -MCS1-P GAL10 -P GAL1 -NnH3H-EGFP-T CYC1 .

[0104] 3. Using the CRISPR / Cas9 genome editing tool in Saccharomyces cerevisiae, the plasmids pRS423-SpSgH-ARO10 and pRS41K-SpCas9, which contain the integration donor and the corresponding single guide RNA coding sequences, were chemically transformed into yeast strain BY4741 and plated on SED-G418 plates with the corresponding amino acid auxotypes.

[0105] 4. For the transformants on the plate, pick spots to lyse the cells, and design primers to select the target sequence on the genome. Use PCR to verify whether the target gene has been integrated into the genome. If the band size is correct and the sequencing results are accurate, it indicates that the genome integration was successful, and the *Saccharomyces cerevisiae* strain shown in Table 6 is obtained.

[0106] Table 6. Saccharomyces cerevisiae strains expressing NnH3H-eGFP and different forms of NnLuz–mCherry fusion protein

[0107]

[0108] 5. After expressing BY4741-Lm, BY4741-L20m, BY4741-L25m, and BY4741-HE in 5 mL of YPD for 36 h, take 1 mL of bacterial culture, wash twice with PBS, centrifuge at 8000 rpm for 1 min, resuspend the cells in 1 mL of PBS, and transfer 1.5 μL to a glass slide for fluorescence observation under a fluorescence confocal microscope.

[0109] The mCherry fluorescence signal intensity of NnLuz molecules truncated to different lengths was investigated using fluorescence confocal microscopy under the same conditions. The results showed that the mCherry fluorescence signal of NnLuzΔN20 was stronger than that of NnLuz, indicating that truncation can increase the protein accumulation of NnLuz to a certain extent. Figure 7 As shown, the fluorescence of NnLuz△N25 is almost invisible, possibly because truncating the 20th amino acid may affect the correct folding of NnLuz and thus its expression. Therefore, it can be concluded that truncating 20 amino acids may increase the protein accumulation of NnLuz, thereby increasing the fluorescence intensity.

[0110] 6. After expressing BY4741-Lm, BY4741-L20m, BY4741-L25m, and BY4741-HE for 20 h, 100 μL of the culture was diluted 50 times to obtain 500 μL of culture. The culture was centrifuged at 5000 rpm for 30 s and the supernatant was discarded. 1 mL of YPD culture medium was added and the culture was expressed on a shaker at 30 °C for 6 h. 200 μL of the culture was placed in a Corning Costar 96-well plate and the luciferase activity was detected using a microplate reader.

[0111] Yeast strains BY4741-Lm, BY4741-Lm20, and BY4741-Lm25 were detected by an ELISA reader with excitation light at 575 nm. They were found to receive emission light at 610 nm. The mCherry fluorescence signal of strain BY4741-Lm20 was 1.5 times higher than that of strain BY4741-Lm, while the mCherry fluorescence signal of strain BY4741-Lm25 was significantly weaker, consistent with the results of laser confocal microscopy. Figure 8 As shown.

[0112] Example 5: Construction of a large-fragment bioluminescent module DNA vector FBP(Luz△N20)

[0113] The basic vector FBP, namely pYLTAC380GW-5G, contains the genes required for the fungal autoluminescence system: HispS gene, CPH gene, H3H gene, NPGA gene, and Luz gene. The construction method can be found in Chinese patent application number 202111228803.9.

[0114] FBP(Luz△N20) replaces the Luz gene in the aforementioned basic vector pYLTAC380GW-5G with a truncated N-terminal variant encoding a fungal luciferase gene. The nucleotide sequence of this gene is shown in SEQ ID NO.2, and the amino acid sequence of the truncated variant is shown in SEQ ID NO.1.

[0115] This study used the TransGene Stacking II system for multi-genome assembly to construct the vector FBP(Luz△N20). The specific construction process is as follows:

[0116] 1) Construct donor vectors pYL322d1-NnHispS, pYL322d2-NnCPH, pYL322d1-NnH3H, pYL322d2-AnNPGA, and pYL322d1-NnLuz△N20;

[0117] 2) The donor vector pYL322d1-NnHispS and the recipient vector pYLTAC380GW were mixed in a ratio of 1:1 to 2:1 and co-transformed in NS3529 competent cells using the heat shock method. The cells were then placed in an ice bath for 30 min, followed by a heat shock for 90 s and an immediate ice bath for 2-3 min.

[0118] 3) Add 700 μL of antibiotic-free LB to the above mixture, mix well, and incubate at 37°C and 200 rpm for 2-3 hours. Spread the mixture onto a solid LB agar plate containing 25 mg / L Kan and 15 mg / L Cm, invert the plate, and incubate at 37°C for about 18 hours. Then, wash all single clones on the plate into a tube with 5 mL ddH2O and extract a small amount of the mixed plasmid.

[0119] 4) In a 10 μL reaction system, digest 50-100 ng of the mixed plasmid with 0.5 μL of I-Sce I enzyme. After 4-5 hours of reaction, transform it into E. coli NEB10-β. Spread it on a solid LB agar plate containing 25 mg / L Kan and incubate it upside down at 37°C for about 15 hours.

[0120] 5) Select single clones and culture them in liquid LB (containing 25 mg / L Kan and 0.5 mM IPTG), and use 2x Green TaqMix for bacterial PCR identification. After gel running, expand the bacterial culture that can amplify the target band and extract plasmids.

[0121] 6) In a 20 μL reaction system, 0.2 μL of Not I enzyme was used to digest 200 ng of plasmid for verification. If one of the four bands contains the target gene of size 6.2 kbp, it indicates that a positive clone pYLTAC380GW-NnHispS has been successfully obtained;

[0122] 7) The donor vector pYL322d2-NnCPH and the recipient vector pYLTAC380GW-NnHispS from 6) were mixed in a ratio of 1:1 to 2:1 and co-transformed in NS3529 competent cells using the heat shock method. The transformation was performed according to step 2), and the mixture was plated on a solid LB agar plate containing 25 mg / L Kan and 70 mg / L Apm. After incubation at 37°C for about 18 h, all single clones on the plate were washed into a tube with 5 mL ddH2O, and a small amount of the mixed plasmid was extracted.

[0123] 8) In a 10 μL reaction system, digest 50-100 ng of the mixed plasmid with 0.5 μL of PI-Sce I enzyme. After 4-5 hours of reaction, transform and verify according to the methods in (4)-(6). If 5 bands appear, 2 of which contain the target gene NnHispS with a size of 6.2 kbp and NnCPH with a size of 1.7 kbp, it means that the positive plasmid pYLTAC380GW-NnHispS-NnCPH has been successfully obtained.

[0124] 9) Perform more rounds of recombination and cross-transform donor vectors carrying different genes with recipient vectors constructed in the previous round to construct pYLTAC380GW-NnHispS-NnCPH--NnH3H-AnNPGA-NnLuz△N20, i.e. pYLTAC380GW-5G(Luz△N20);

[0125] 10) Mix 200 ng of pYLTAC380GW-5G(Luz△N20) and 100 ng of PYLMFH-Bnmlpro, add 1 μL of 5×BP enzyme mixture, react for 5 hours, and finally add 1 μL of proteinase K solution. Stop the reaction at 37℃ for 10 minutes to obtain pYLTAC380MF-5G(Luz△N20), i.e., FBP(Luz△N20);

[0126] 11) Transform into NEB10-β competent cells and select single clones for identification. Use Not I restriction enzyme digestion to detect the presence of the corresponding target DNA band in the correct positive final vector. Select positive clones for whole-plasmid sequencing analysis and choose the correct vector for subsequent experiments, such as... Figure 9 .

[0127] Example 6: Analysis of luminescence intensity in transgenic tobacco plants mediated by Agrobacterium-mediated bioluminescence module

[0128] 1) EHA105 bacterial suspension containing hygromycin-labeled vector plasmid FBP or FBP(Luz△N20) was streaked onto solid LB plates containing 25 mg / L Kan and 20 μL / mL rif antibiotics, respectively, and incubated at 28°C for 36 h.

[0129] 2) Pre-culture: Select 4-5 week old fully expanded sterile tobacco healthy leaves in a clean room, cut them into 0.5 cm squares, avoid the midrib and cut off the leaf edge, and incubate the leaves with the upper surface facing down on MS1 ​​solid medium at 25℃ in the dark for 2 days.

[0130] 3) Infection: Pick single colonies from the activated plates and transfer them to 3-5 mL of LB medium at 200 rpm and 28°C for 36 h. Expand the culture to 50 mL at a ratio of 1:100-1:50 and culture for 3-5 h until OD. 600 =0.6, then centrifuge the bacterial culture and resuspend the cells in MSO liquid medium until OD200 is reached. 600 =0.6, add the pretreated tobacco leaves to the bacterial solution, and then gently vortex to ensure that the cut ends of the leaves are completely submerged in the bacterial solution. Let it stand for 5-30 minutes, and then use sterile filter paper to absorb the attached bacterial solution.

[0131] 4) Co-culture: After infection, place the leaves face down on MS1 ​​solid medium and incubate in the dark at 28°C for 2 days.

[0132] 5) Selection culture: Place the leaves with the upper surface facing up on MS1 ​​selection medium containing Timentin(TM)Hygromycin(Hyg) and culture at 25°C under light.

[0133] 6) Rooting culture: When buds of more than 1 cm grow from the leaf margin and can be separated, cut off the buds and transfer them to MS2 rooting medium containing antibiotics.

[0134] 7) Hardening off the seedlings: After two weeks, when roots have grown, open the lid of the seedling box and harden off the seedlings for one week. Then, transfer them to the planting soil for cultivation. At the same time, take three groups of leaves and take pictures for analysis under the fully automatic light-emitting detection system.

[0135] Table 7. Culture medium composition and culture time at each stage

[0136]

[0137] Leaves from positive plants of FBP (Luz△N20) Zhongyan 100 (Lines 5, 7, and 11) and FBP Zhongyan 100 (Lines 1, 2, and 4) with strong light signals were simultaneously detected using a photoluminescence analyzer. The average value showed that the luminescence intensity of FBP (Luz△N20) Zhongyan 100 leaves was approximately twice that of FBP Zhongyan 100 leaves. The results are as follows... Figure 10 .

[0138] Those skilled in the art can make appropriate improvements based on the content of this document. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Modifications or appropriate changes and combinations made by those skilled in the art to the methods and applications described herein without departing from the content, spirit, and scope of this invention are all within the protection scope of this invention.

Claims

1. The application of truncated fungal luciferase in enhancing the bioluminescence intensity of fungi or plants, characterized in that, The fungal or plant genome integrates a gene encoding a truncated fungal luciferase, the amino acid sequence of which is shown in SEQ ID NO.1; the fungus is *Saccharomyces cerevisiae* YCA113-2B, and the plant is tobacco.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the truncated fungal luciferase is shown in SEQ ID NO.

2.

3. The application as described in claim 1, characterized in that, The fungal or plant genome also integrates the HispS gene, CPH gene, H3H gene, and NPGA gene. The coding sequence of the HispS gene is shown in SEQ ID NO.3, the coding sequence of the CPH gene is shown in SEQ ID NO.4, the coding sequence of the H3H gene is shown in SEQ ID NO.5, and the coding sequence of the NPGA gene is shown in SEQ ID NO.

6.

4. The application as described in any one of claims 1-3, characterized in that, The applications include: using CRISPR / Cas9 gene editing technology to integrate the HispS gene, CPH gene, H3H gene, NPGA gene, and fungal luciferase truncated gene into the genome of a caffeic acid-producing fungus to obtain a fungus with enhanced bioluminescence intensity; or, using multi-gene assembly technology to integrate the HispS gene, CPH gene, H3H gene, NPGA gene, and fungal luciferase truncated gene into a recipient vector to construct a multi-gene vector, and using transgenic technology to introduce the target gene fragment from the multi-gene vector into a recipient plant, and then cultivating it to obtain a transgenic plant with enhanced bioluminescence intensity.

5. The application as described in claim 4, characterized in that, When constructing a bioluminescence-enhanced Saccharomyces cerevisiae, each target gene contains a galactose-induced GAL1 or GAL10 promoter upstream.

6. The application as described in claim 4, characterized in that, The TransGeneStackingII system was used for multi-gene assembly, and each target gene in the multi-gene vector contained a 35S promoter sequence upstream.

7. A method for constructing a bioluminescence-enhanced yeast, characterized in that, Includes the following steps: (1) The HispS gene fragment, CPH gene fragment, H3H gene fragment, NPGA gene fragment, and fungal luciferase truncated gene fragment Luz△N20 were cloned into the multiple cloning site of plasmid pESC-URA to obtain recombinant plasmids pESC-URA-Luz△N20-CPH, pESC-URA-H3H-NPGA, and pESC-URA-HispS; the nucleotide sequence of the fungal luciferase truncated gene is shown in SEQ ID NO.2; the coding sequence of the HispS gene fragment is shown in SEQ ID NO.3; the coding sequence of the CPH gene fragment is shown in SEQ ID NO.4; the coding sequence of the H3H gene fragment is shown in SEQ ID NO.5; and the coding sequence of the NPGA gene fragment is shown in SEQ ID NO.

6. (2) The recombinant plasmid from step (1) was amplified by PCR using primers with a 40bp homologous arm of the ARO10 gene, primers with a 40bp homologous arm of the Int10 site, and primers with a 40bp homologous arm of the Int12 site, respectively, to obtain genome integration donors △Aro10::TADH1-Luz△N20-PGAL10-PGAL1-CPH-TCYC1, Int10::TADH1-NPGA-PGAL10-PGAL1-H3H-TCYC1 and Int12::TADH1-MCS1-PGAL10-PGAL1-HispS-TCYC1; (3) Using chemical methods, the integration donor △Aro10::TADH1-Luz△N20-PGAL10-PGAL1-CPH-TCYC1, along with plasmids pRS423-SpSgH-ARO10 and pRS41K-SpCas9, was transformed into caffeic acid-producing yeast cells. Successfully integrated recombinant strain I was obtained through screening. Then, Int10::TADH1-NPGA-PGAL10-PGAL1-H3H-TCYC1 and plasmid pRS426-SpSgH-I were combined. nt10 and pRS41K-SpCas9 were transformed into recombinant strain I, and recombinant strain II with successful integration was obtained by screening. Then, Int12::TADH1-MCS1-PGAL10-PGAL1-HispS-TCYC1 and plasmids pRS426-SpSgH-Int12 and pRS41K-SpCas9 were transformed into recombinant strain II, and recombinant strain III with successful integration was obtained by screening, which is the bioluminescent intensity-enhanced yeast; the fungus is Saccharomyces cerevisiae YCA113-2B.

8. A method for cultivating a bioluminescent intensity-enhanced plant, characterized in that, Includes the following steps: 1) The HispS gene fragment, H3H gene fragment, and fungal luciferase truncated gene fragment were inserted into the multiple cloning site of donor vector pYL322d1 to obtain donor vectors pYL322d1-HispS, pYL322d1-H3H, and pYL322d1-Luz△N20; the nucleotide sequence of the fungal luciferase truncated gene is shown in SEQ ID NO.

2. The CPH gene fragment and NPGA gene fragment were inserted into the multiple cloning site of donor vector pYL322d2 to obtain donor vectors pYL322d2-CPH and pYL322d2-NPGA; the coding sequence of the HispS gene fragment is shown in SEQ ID NO.3, the coding sequence of the CPH gene fragment is shown in SEQ ID NO.4, the coding sequence of the H3H gene fragment is shown in SEQ ID NO.5, and the coding sequence of the NPGA gene fragment is shown in SEQ ID NO.

6. 2) The donor vector pYL322d1-HispS and the recipient vector pYLTAC380GW were mixed at a ratio of 1:1 to 2:1 and co-transformed into Escherichia coli NS3529 competent cells. The cells were then plated in a double-antibiotic medium containing kanamycin and chloramphenicol and cultured. Plasmids were extracted from positive strains. 3) The plasmid extracted in step 2) was digested with homing enzyme I-Sce I, then transformed into Escherichia coli strain NEB10-β, cultured, screened, and plasmid extracted to obtain a positive clone pYLTAC380GW-HispS containing the target gene HispS. 4) Mix the donor vector pYL322d2-CPH and the recipient vector pYLTAC380GW-HispS prepared in step 3) at a ratio of 1:1 to 2:1, and co-transfer them into Escherichia coli NS3529 competent cells. Spread the mixture on a double-antibiotic medium containing kanamycin and ampicillin and culture it. Extract plasmids from positive strains. 5) The plasmid extracted in step 4) was digested with the homing enzyme PI-Sce I, then transformed into Escherichia coli strain NEB10-β, cultured, screened, and plasmid extracted to obtain a positive clone pYLTAC380GW-HispS-CPH containing the target genes HispS and CPH. 6) Repeat steps 2)-5), using the new plasmid containing the target gene obtained in the previous step as the recipient vector, and cross-recombining donor vectors containing different genes until all the target genomes are loaded into the recipient vector. In the final step, the BP recombination reaction is used to ligate the expression cassette element of the removed selection marker gene to construct the multi-gene vector pYLTAC380MF-HispS-CPH-H3H-NPGA-Luz△N20. 7) Using Agrobacterium-mediated transformation, the target gene fragment from a multi-gene vector was introduced into a recipient plant, and the plant was cultured to obtain a transgenic plant with enhanced bioluminescence intensity; the plant was tobacco.

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