Application of the Sanghuang polyketide synthase gene SvPKS1 in promoting fungal or plant bioluminescence

Abstract

This invention provides a *Phellinus linteus* polyketide synthase gene. SvPKS1 Applications in promoting bioluminescence in fungi or plants, the SvPKS1 The coding sequence is shown in SEQ ID NO.1, and the fungus is a caffeic acid-producing fungus. The application includes using transgenic technology to... SvPKS1 Other genes in the FBP fungal luminescence pathway ( nnH3H , nnLuz , nnCPH This invention discloses for the first time the *Phellinus linteus* polyketide synthase gene, which is co-expressed in fungi or plants to promote bioluminescence in fungi or plants. SvPKS1 With fungal luminescence pathway genes nnH3H , nnLuz , nnCPH Co-expression promotes bioluminescence in fungi or plants, providing a key candidate enzyme gene for promoting sustainable bioluminescence in fungi or plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the *Phellinus linteus* polyketide synthase gene. SvPKS1 Applications in promoting bioluminescence in fungi or plants. Background Technology

[0002] Bioluminescence is a unique life phenomenon found in approximately 800 genera and 10,000 species, serving functions such as attracting mates, deterring predators, and recruiting other organisms to spread spores. Different bioluminescent systems have been developed into various detection and reporting tools widely used in biomedical applications. Further scientific research to analyze and optimize naturally occurring bioluminescence mechanisms and utilize them to create "luminescent trees" could help convert inexpensive biochemical energy into light energy to assist or even replace mainstream lighting methods, which is of great significance for achieving low-carbon and sustainable development.

[0003] Research on bioluminescent plants has been reported. As early as 1986, scientists at the University of California transferred the luciferase gene from fireflies to tobacco and exogenously sprayed luciferin substrates, obtaining the first bioluminescent plant (Ow, DW et al., 1986). However, the method was limited for several reasons, including the poor permeability and instability of many luciferins across cell membranes, and the complex, multi-step, and expensive process of luciferin synthesis. With the elucidation of the bioluminescent mechanism from marine bioluminescent bacteria, Krichevsky et al. transformed the complete bacterial lux operon (luxCDABEG) into tobacco chloroplasts in 2010, cultivating self-luminescent plants (Krichevsky, A. et al., 021). Although bacterial bioluminescence systems were studied relatively early, their low luminescence intensity and the toxicity of some components to eukaryotic cells limited their development and application in higher organisms. In contrast, fungal bioluminescence has been known for centuries. For a long time, it was hypothesized that the fungal bioluminescent pathway (FBP) consists of at least four components: molecular oxygen, luciferin, an NAD(P)H-dependent hydroxylase, and a luciferase. However, it wasn't until 2015 that Purtov et al. identified the fungal luciferin as 3-hydroxyhispidin, a cell membrane-penetrating metabolite (Angewandte Chemie, 2015, 54:8124-28). Furthermore, experiments have demonstrated the presence of an enzyme in fungal lysates capable of hydroxylating hispidin to form luciferin, but the specific type of this enzyme has not been confirmed. With the deepening of related research, in 2018, Kotlobay et al. (Kotlobay et al., 2018) elucidated the FBP pathway and identified the enzymes required for the complete pathway: hispidin synthase (HispS), hispidin-3-hydroxylase (H3H), fungal luciferase (Luz), and caffeoylpyruvate hydrolase (CPH). The working mechanism of FBP is as follows: HisspS converts caffeic acid to hispidin, and then, under the catalysis of H3H, hispidin is converted into fungal luciferin 3-hydroxyhispidin. Subsequently, Luz converts fungal luciferin into an unstable high-energy intermediate, releasing light at a wavelength of 520 nm during the generation of caffeoylpyruvate. CPH converts caffeoylpyruvate back to caffeic acid to achieve substrate regeneration and recycling (Kaskova et al., 2017). Although caffeoylpyruvate hydrolase (CPH) is not essential for the generation of autoluminescence, it can significantly prolong the luminescence time.

[0004] Caffeic acid is a key intermediate in the synthesis of lignin and other important secondary metabolites in plants, and its natural existence lays an important foundation for the creation of bioluminescent plants. In 2020, two independent research groups published two studies on the creation of bioluminescent plants based on FBP (Khakhar et al., 2020; Mitiouchkina et al., 2020). Although the bioluminescence produced by these two methods was very weak, and the naked eye needed a long period of adaptation to a dark environment to see the bioluminescence, the two studies solved the key problems of enabling plants to continuously produce light without affecting plant growth, representing a major breakthrough in the field of bioluminescent plants. Building on this, researchers used metabolic engineering to reconstruct the endogenous caffeic acid biosynthesis pathway in plants, enhancing the biosynthesis of endogenous caffeic acid while reducing the consumption of caffeic acid by other metabolic pathways, thereby increasing the intensity of bioluminescence to a certain extent (Peng Zheng et al., 2023; Jieyu Ge et al., 2024). Other studies have used a combination of random mutation and rational design to modify key enzymes of FBP, obtaining enzyme mutants with high catalytic performance. These mutants have increased the luminescence intensity of different eukaryotes by 1-2 orders of magnitude, allowing the naked eye to see the plants luminescence without needing to adapt to dark conditions.

[0005] Discovering and verifying functionally equivalent but sequence-different isozymes is key to overcoming existing technological limitations. Summary of the Invention

[0006] Based on the current state of technology, this invention provides a *Phellinus linteus* polyketide synthase gene. SvPKS1 Applications in promoting bioluminescence in fungi or plants.

[0007] The specific technical solution is as follows:

[0008] In one aspect, the present invention provides a *Phellinus linteus* polyketide synthase gene. SvPKS1 Applications in promoting bioluminescence in fungi or plants, the SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1;

[0009] The fungus is a caffeic acid-producing fungus;

[0010] The fungi or plants mentioned contain genes that can encode NnH3H protein, NnLuz protein and NnCPH protein;

[0011] Preferably, the fungus or plant also contains a gene encoding the AnNPGA protein.

[0012] In one aspect, the present invention provides a gene containing *Phellinus linteus* polyketide synthase. SvPKS1 The application of recombinant vectors in promoting bioluminescence in fungi or plants, the SvPKS1The encoded nucleotide sequence is shown in SEQ ID NO.1;

[0013] The fungus is a caffeic acid-producing fungus;

[0014] The fungus or plant contains genes that encode NnH3H protein, NnLuz protein and NnCPH protein; preferably, the fungus or plant also contains genes that encode AnNPGA protein.

[0015] Preferably, the original expression vector of the recombinant vector is the pCAMBIAM1300 vector.

[0016] In one aspect, the present invention provides the application of genetically engineered bacteria in promoting bioluminescence in fungi or plants, said genetically engineered bacteria containing the *Phellinus linteus* polyketide synthase gene. SvPKS1 Or it may contain the pheasant polyketide synthase gene. SvPKS1 The recombinant vector; SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1;

[0017] The fungus is a caffeic acid-producing fungus;

[0018] The fungus or plant contains genes that encode NnH3H protein, NnLuz protein and NnCPH protein; preferably, the fungus or plant also contains genes that encode AnNPGA protein.

[0019] In one aspect, the present invention provides the application of Phellinus linteus polyketide synthase SvPKS1 in promoting bioluminescence in fungi or plants, wherein the amino acid sequence encoded by Phellinus linteus polyketide synthase SvPKS1 is shown in SEQ ID NO.2.

[0020] The fungus is a caffeic acid-producing fungus;

[0021] The fungus or plant contains NnH3H protein, NnLuz protein and NnCPH protein; preferably, the fungus or plant also contains AnNPGA protein.

[0022] In another aspect, the present invention provides a method for constructing a luminescent fungus, comprising: introducing the *Phellinus linteus* polyketide synthase gene... SvPKS1 The fungus was introduced into a caffeic acid-producing fungus to obtain a bioluminescent fungus;

[0023] The Sanghuang polyketide synthase gene SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1;

[0024] The caffeic acid-producing fungus contains genes that can encode NnH3H protein, NnLuz protein and NnCPH protein;

[0025] Preferably, the NnH3H protein has the accession number QJQ48094.1 in the NCBI database;

[0026] Preferably, the NnLuz protein has the accession number QJQ48096.1 in the NCBI database;

[0027] Preferably, the NnCPH protein has the accession number QJQ48093.1 in the NCBI database.

[0028] Further, the caffeic acid-producing fungus is *Saccharomyces cerevisiae*; more preferably, the *Saccharomyces cerevisiae* is YCA113. 2B.

[0029] Furthermore, the caffeic acid-producing fungus also contains a gene that can encode the AnNPGA protein;

[0030] Preferably, the AnNPGA protein has the accession number QJQ48097.1 in the NCBI database.

[0031] Furthermore, this invention utilizes CRISPR / Cas9 gene editing technology to insert the *Phellinus linteus* polyketide synthase gene. SvPKS1 Genes encoding NnH3H, NnLuz, NnCPH, and AnNPGA proteins were introduced into caffeic acid-producing fungi to obtain bioluminescent fungi.

[0032] In another aspect, the present invention also provides a method for constructing a luminescent plant, comprising: introducing the *Phellinus linteus* polyketide synthase gene... SvPKS1 By introducing the bioluminescent material into plant cells, a bioluminescent plant can be obtained.

[0033] The Sanghuang polyketide synthase gene SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1;

[0034] The plant genome integrates genes encoding NnH3H protein, NnLuz protein, and NnCPH protein;

[0035] Preferably, the NnH3H protein has the accession number QJQ48094.1 in the NCBI database;

[0036] Preferably, the NnLuz protein has the accession number QJQ48096.1 in the NCBI database;

[0037] Preferably, the NnCPH protein has the accession number QJQ48093.1 in the NCBI database.

[0038] Furthermore, the plant cells also contain a gene encoding the AnNPGA protein.

[0039] Preferably, the AnNPGA protein has the accession number QJQ48097.1 in the NCBI database.

[0040] Furthermore, this invention utilizes multi-gene stacking technology to integrate the *Phellinus linteus* polyketide synthase gene. SvPKS1 Genes encoding NnH3H, NnLuz, NnCPH, and AnNPGA proteins were introduced into a recipient vector to obtain bioluminescent plants; more preferably, the TransGene Stacking II system was used for multi-gene stacking.

[0041] Furthermore, the plant in question is a higher plant.

[0042] Furthermore, the higher plants mentioned are tobacco, Arabidopsis thaliana, or petunia.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention obtained a *Phellinus linteus* polyketide synthase gene by screening and testing NnHispS isoenzymes in the FBP pathway. SvPKS1 ,Will SvPKS1 Co-expression of other FBP luminescence pathway genes in fungi or plants can promote fungal and / or plant luminescence. This invention provides a key candidate enzyme for promoting sustainable luminescence in fungi and / or plants. Attached Figure Description

[0045] Figure 1 The results are the structural domain analysis results of NnHispS and SvPKS1 in Example 1.

[0046] Figure 2 In Example 2, SvPKS1 was expressed in the non-self-luminescent YCA113-2B-LCHH-ΔnnHispS yeast strain, and the function and biological activity of SvPKS1 in the yeast were determined by detecting the luminescence signal.

[0047] Figure 3 In Example 3, NnH3H, NnCPH, NnLuz and SvPKS1 were expressed in wild-type Nicotiana benthamiana leaves, and the function and bioactivity of SvPKS1 in the plant were determined by detecting the luminescence signal.

[0048] Figure 4 This diagram illustrates the T-DNA region of the 380MF-nnH3H-nnCPH-nnLuz-nnNPGA-SvPKS1 vector and its overexpression characterization results; among which, Figure 4 In Example 4, A is a schematic diagram of the transgenic vector 380MF-nnH3H-nnCPH-nnLuz-nnNPGA-SvPKS1T-DNA region. Figure 4In Example 4, B represents the luminescence detection result of 380MF-nnH3H-nnCPH-nnLuz-nnNPGA-SvPKS1 expressed in tobacco leaves three days after its expression.

[0049] Figure 5 The results of the Benedictine smoke luminescence experiment using the 380MF-nnH3H-nnCPH-nnLuz-nnNPGA-SvPKS1 carrier in Example 5 are shown.

[0050] Figure 6 The results of the Arabidopsis thaliana luminescence experiment using the 380MF-nnH3H-nnCPH-nnLuz-nnNPGA-SvPKS1 carrier in Example 6 are shown.

[0051] Figure 7 The results of the experiment in Example 7 on promoting petunia luminescence using the 380MF-nnH3H-nnCPH-nnLuz-nnNPGA-SvPKS1 carrier. Detailed Implementation

[0052] The technical solution of the present invention will be described below with reference to specific embodiments, so that those skilled in the art can better understand the present invention. It should be noted that the following descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0053] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0054] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions were performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0055] The yeast-associated plasmids p423-SpSgH, pRS426-SpSgH-Int10, and pRS41K-SpCas9 were provided by the laboratory of Professor Lian Jiachang at 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 novo biosynthetic pathways using stable integration sites in yeast. Biotechnol Bioeng [J], 119:1314-1326.) and Chinese patent application number 2021115974834.

[0056] The caffeic acid-producing yeast strain YCA113-2B was donated by Associate Professor Ye Lidan's laboratory at Zhejiang University. This yeast genome integrates... nnHispS , nnCPH , nnH3H and nnLuz The expression cassettes of four genes were used to obtain the YCA113-2B-LCHH luminescent yeast. The construction method is described in Chinese Patent Application No. 2025100915776.

[0057] pYL322d1, pYL322d2, pYLTAC380GW, and pYLMF-H 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.

[0058] The coding sequences of NnH3H, NnLuz, NnCPH, and AnNPGA proteins were chemically synthesized after optimization based on host codon preferences. The NCBI accession number for NnH3H is QJQ48094.1, for NnLuz it is QJQ48096.1, for NnCPH it is QJQ48093.1, and for AnNPGA it is QJQ48097.1.

[0059] SvPKS1 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0060] SEQ ID NO.1:

[0061]

[0062] SEQ ID NO.2:

[0063]

[0064] Example 1 SvPKS1 Cloning and Sequence Analysis

[0065] Using combined metabolomics and transcriptomics analysis of *Phellinus linteus*, along with expression pattern studies and molecular docking prediction, several PKS genes potentially involved in hispidin synthesis were screened. Further analysis of PKS functional domains using the online website antSMASH (https: / / fungismash.secondarymetabolites.org / ) revealed that one enzyme, SvPKS1, possesses an AMP-ACP-KS-AT-DH-KR-ACP-ACP structure, typical of fungal type I PKS genes. SvPKS1 encodes a 2573-amino acid protein, which, compared to the 1698-amino acid NnHispS, has three additional domains at its C-terminus: DH, KR, and ACP. Figure 1 This suggests that SvPKS1 may catalyze the generation of a polyketide intermediate with a higher reduction degree than the NnHispS product, and its final luminescent properties may differ from those of the classical pathway.

[0066] Sequence alignment using BLASTP (default parameters) showed that the amino acid sequence similarity between SvPKS1 and NnHispS was less than 40% (Table 1), which is lower than the 45% similarity limit for milk tree alkaloid synthase in claim 201980056875.4. Therefore, the utilization of this enzyme in promoting bioluminescence will not be restricted by the aforementioned patent.

[0067] Table 1. Results of amino acid sequence similarity comparison between NnHispS and SvPKS1

[0068]

[0069] Example 2 SvPKS1 In YCA113-2B-LCHH_Δ nnHispS Activity analysis in yeast

[0070] First, CRISPR / Cas9 gene editing technology was used to edit the YCA113-2B-LCHH yeast constructed in patent ZL202510091577.6. nnHispS Gene knockout is performed. The method is as follows:

[0071] 1) Designing knockouts using Benchling CRISPR tools nnHispS After determining the optimal target site based on the gRNA sequence, the target primer Δ for N20 was synthesized. nnHispS -F (SEQ ID NO.3) and Δ nnHispS-R (SEQ ID NO.4), and ligate the gRNA into the p423-SpSgH plasmid, transform it into E. coli DH5α, culture overnight, and screen positive single clones by colony PCR for sequencing. Plasmids with correct sequencing results are then used. The target primer sequences are as follows:

[0072] SEQ ID NO.3:

[0073] 5'-gatcCAGCTCTCGCTCAACATCTT-3';

[0074] SEQ ID NO.4:

[0075] 5'-aaacAAGATGTTGAGCGAGAGCTG-3'.

[0076] 2) p423-SpSgH-Δ nnHispS The plasmids pRS41K-SpCas9 were chemically transformed into yeast cells YCA113-2B-LCHH and plated on SD-His / G418 plates with corresponding amino acid auxotropes. The luminescence of the transformants on the plates was detected using an in vivo imaging system; non-luminescent transformants were selected and further processed. nnHispS Sequencing followed by PCR amplification with specific primers nnHispS The strain with the frameshift mutation in the coding sequence is YCA113-2B-LCHH-Δ nnHispS .

[0077] Secondly, the target gene SvPKS1 The recombinant plasmid pESC-URA containing the galactose-inducible promoters (PGAL1 and PGAL10) was cloned into the MCS1 region (restriction site BamHI / XhoI) of the plasmid pESC-URA-. SvPKS1 .

[0078] Finally, pESC-URA- was chemically synthesized. SvPKS1 plasmid transformed into YCA113-2B-LCHH-Δ nnHispS The luminescence was detected in yeast cells and examined after 3 days of growth on plates. Results showed expression... SvPKS1 and positive control nnHispS Genes can be reintroduced to YCA113-2B-LCHH-Δ nnHispS The strain lacked the luminescent phenotype, while the negative control empty vector and the SvPKS1 truncated variant (Trun_SvPKS1) did not, indicating that... SvPKS1 It possesses bioactivity for luminescence in yeast. Figure 2 ).

[0079] Example 3: Activity analysis of SvPKS1 in plants

[0080] Agrobacterium-mediated transient transformation of tobacco leaves co-expressed in wild-type Nicotiana benthamiana leaves SvPKS1 and nnH3H-nnCPH-nnLuz Three days later, luminescence detection was performed on the injection site of the leaf to determine the outcome. SvPKS1 Activity within the plant. SvPKS1 The clone was inserted into the pCAMBIAM1300 vector and expressed via a 35S promoter. The pYLTAC380GW- vector was constructed using the TransGeneStacking II system. nnH3H-nnCPH-nnLuz The vector, constructed using a method similar to that of the basic vector FBP (pYLTAC380GW-5G) in Chinese Patent Application No. ZL202111228803.9, involves the following gene stacking order: nnH3H , nnCPH , nnLuz pYLTAC380GW-5G injection was used as a positive control. nnH3H-nnCPH-nnLuz and pCAMBIAM130- SvPKS1 Separate injections were used as negative controls. The steps for Agrobacterium infection are as follows:

[0081] 1) pYLTAC380GW- nnH3H-nnCPH-nnLuz and pCAMBIAM1300- SvPKS1 The plasmids were transformed into Agrobacterium strain EHA105. Single-clone positive strains were selected and inoculated into LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL rifampin. The medium was then cultured in a shaker at 28 ℃ and 200 r / min until the OD600 was approximately 1.0.

[0082] 2) Transfer the bacterial culture to a centrifuge tube, centrifuge at 8000×r / min for 1 min, discard the supernatant, and collect the bacterial cells;

[0083] 3) Resuspend Agrobacterium in a resuspension containing 10 mmol / L MES (pH=5.7), 50 mmol / L MgCl2, and 200 μmol / L Acetosyringone (AS), and adjust OD600 to 0.5;

[0084] 4) After the resuspension has been left to stand at room temperature for 2 hours, the two strains are mixed in equal volumes;

[0085] 5) Slowly inject the mixed bacterial solution into the leaf between the veins on the lower epidermis of the tobacco leaf using a sterile syringe without the needle, while injecting the control strain into other parts of the leaf at the same time. After injection, treat the leaf in the dark for 12 hours.

[0086] 6) Place the dark-treated tobacco in a light incubator at 25°C with a photoperiod of 16 hours of light / 8 hours of darkness and an illuminance of 2000 Lx for further cultivation.

[0087] Three days after injection, the leaflets were photographed using the Teca-Plant system to examine the luminescence at the injection site. The results are as follows: Figure 3 As shown, compared with the site expressing the positive control vector, co-expression SvPKS1 and nnH3H-nnCPH-nnLuz It can also make the injection site glow, while expression alone cannot make the leaves glow, indicating that SvPKS1 can replace the function of NnHispS in plants, successfully participate in and reconstruct the fungal bioluminescence pathway, thus confirming that it has the corresponding biological function.

[0088] Example 4, luminescent carrier 380MF- nnH3H-nnCPH-nnLuz-anNPGA-SvPKS1 Construction and functional verification

[0089] Example 3 confirmed that SvPKS1 possesses biological activity in plants and can combine with nnH3H-nnCPH-nnLuz to achieve basic luminescence. To further improve the performance of the luminescence system and promote higher luminescence intensity and greater application value, the vector was optimized. Studies have shown that anNPGA can enhance plant luminescence intensity. Therefore, when designing a transgenic vector containing the SvPKS1 gene, anNPGA was simultaneously assembled on the vector to promote plant luminescence. The final vector contained five key genes and was named 380MF-nnH3H-nnCPH-nnLuz-anNPGA-SvPKS1 (…). Figure 4 (A) The vector was constructed using the TransGene Stacking II system, and the method was similar to that used to construct the basic vector FBP (pYLTAC380GW-5G). The construction method for pYLTAC380GW-5G is described in Chinese Patent Application No. 202111228803.9.

[0090] The effectiveness of the vectors was tested before stable transformation into plants. pYLTAC380GW-5G and 380MF-nnH3H-nnCPH-nnLuz-anNPGA-SvPKS1 were expressed in wild-type Nicotiana benthamiana leaves via Agrobacterium-mediated transient transformation. The transient expression method was the same as that used in Example 3.

[0091] The luminescence signal was detected by photographing leaves 3 days after injection using the Teca-Plant system. Consistent with expectations, both leaf regions expressing pYLTAC380GW-5G and 380MF-nnH3H-nnCPH-nnLuz-anNPGA-SvPKS1 exhibited a luminescent phenotype. Figure 4 The B in the figure indicates that the carrier is effective and can be used for the subsequent creation of luminescent plants.

[0092] Example 5: Agrobacterium-mediated transgenic promotion of luminescence in Tobacco Benzoinus.

[0093] 1) Agrobacterium strain EHA105 containing the vector plasmid 380MF-nnH3H-nnCPH-nnLuz-anNPGA-SvPKS1 was streaked on LA+Rif+Kana plates and incubated overnight at 28°C. Single colonies were picked and transferred to 3-5 mL of LB medium and incubated overnight at 200 rpm at 28°C. The culture was expanded at a ratio of 1:100-1:50 until OD=0.6. After centrifugation, the bacterial culture was resuspended in MS0 liquid medium (MS + 3% sucrose + pH 5.8, 50 mL) until OD=0.6 for infection.

[0094] 2) Select healthy tobacco leaves of Zhongyan 100 that have been planted on sterile MS medium for 4-5 weeks until fully expanded. Cut them into 0.5cm square pieces with a scalpel (cut off the leaf edge and avoid the midrib). Place the leaves with the upper surface facing down on MS1 ​​solid medium (MS + 0.5mg / L indoleacetic acid (IAA) + 2.0mg / L 6-benzyladenine (6-BA) + 3% sucrose + 0.6-0.8% plant gel (Phytagel), pH=5.8) and incubate in the dark at 25℃ for 2-3 days.

[0095] 3) Add pre-cultured tobacco leaves to the bacterial solution, vortex to ensure the leaf cut is submerged, let stand for 20-30 minutes, and then blot away the adhering bacterial solution with sterile filter paper. Place the infected leaves, top-side down, on MS1 ​​solid medium and incubate in the dark at 28°C for 2 days. Place the leaves, top-side up, on MS1 ​​selection medium containing Timentin (TM) and the Bar gene and incubate in the light at 25°C. When buds emerge from the leaf margins and can be separated (over 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 + Bar). Roots will develop after two weeks. Open the seedling tray lid and harden the seedlings for two days before transplanting them into soil. Positive transgenic seedlings are detected using a plant in vivo imaging system and photographed.

[0096] like Figure 5As shown, a visually visible and stable bioluminescent signal was detected in tobacco plants (T1 and T2 generation whole plants), indicating that the optimized luminescent carrier system containing SvPKS1 can be successfully and stably expressed in the model plant tobacco and produce a luminescent phenotype, laying a solid foundation for promoting the application of plant luminescence.

[0097] Example 6: Agrobacterium-mediated transgenic promotion of Arabidopsis thaliana luminescence

[0098] 1) Agrobacterium strain EHA105 containing the vector plasmid 380MF-nnH3H-nnCPH-nnLuz-anNPGA-SvPKS1 was streaked on LA+Rif+Kana plates and incubated overnight at 28°C. Single colonies were picked and transferred to 3-5 mL of LB medium and incubated overnight at 28°C at 200 rpm. The culture was expanded at a ratio of 1:100-1:50 until OD=0.6. After centrifugation, the bacterial suspension was resuspended in infiltration buffer (5% Sucrose and 100µL / L PEG-8 trisiloxane (Silwet L-77), 300 mL) until OD=0.8. The Agrobacterium suspension was then transferred to an open container.

[0099] 2) Select healthy plants in the early fruiting stage, cut off the pods and open flowers, and place the pot, along with the inflorescence, upside down on top of a container containing Agrobacterium suspension. Immerse the entire inflorescence in the Agrobacterium suspension for about 20-30 seconds, taking care to avoid contact between the leaves and the suspension. During this process, avoid pouring vermiculite into the Agrobacterium suspension. Remove the pot and place it horizontally in a dark box, maintaining a certain level of humidity. After 24 hours, place the treated Arabidopsis plants under light conditions of 22-25℃ to allow them to grow normally. Harvest mature seeds after approximately three weeks.

[0100] 3) The received seeds were planted on 1 / 2 MS solid medium containing 10 mM Bar to screen for resistant seedlings. The seedlings were then transferred to soil and the luminescent phenotype was detected using the Teca-Plant system.

[0101] like Figure 6 As shown, bioluminescent signals were successfully observed in mature plants (T1 and T2 generations).

[0102] Example 7: Agrobacterium-mediated transgenic promotion of petunia luminescence

[0103] 1) Seed sterilization: Take 100 seeds and place them in a 1.5 mL sterilized centrifuge tube. Add 1 mL of sterile sodium hypochlorite solution (containing 1% available chlorine), shake to mix, and sterilize for 7-8 minutes, shaking occasionally. Discard the sodium hypochlorite solution, add 1 mL of sterile water to wash, and repeat 3 times. Discard the waste liquid, sow the seeds on 1 / 2 MS medium, and incubate at 25℃ under light for later use.

[0104] 2) Culture of sterile seedlings: 10-14 days after germination, the hypocotyl is cut off at the middle, and the terminal bud with cotyledons and hypocotyl is transferred to a new 1 / 2 MS medium (containing 0.075 mg / L paclobutrazol) for culture. After 4 weeks of continued culture, leaves of suitable size are selected for genetic transformation.

[0105] 3) Agrobacterium infection: Petunia seedling leaves were cut into 0.5cm squares (a larger leaf disc is beneficial for regeneration) and placed on MS1 ​​medium (4.63g / L MS + 6-BA 3.0 mg / L + IAA 0.2mg / L + 30g / L Sucrose + 8g / L agar) and cultured for 2 days. Agrobacterium single clones were added to 100mL of YEB medium (containing 50mg / L kanamycin and rifampin) at a 1:100 ratio and cultured at 200rpm and 28℃ with shaking for 15h, until the OD600 reached approximately 1.0. The bacterial cells were collected by centrifugation (7000rpm / min, 10min) and resuspended in 100mL of MS3 medium (4.63g / L MS + 30g / L Sucrose + MES 50mg / L + AS 50mg / L), with a final bacterial concentration of OD=0.2 (infection solution). Place the pre-cultured material into the bacterial solution prepared in the previous step for 5 minutes, shaking occasionally to ensure full contact between the bacterial solution and the leaf disc. Remove the material, blot the bacterial solution off the surface of the material with sterile filter paper, and place it on a solid MS2 medium (4.63 g / L MS + 6-BA 3.0 mg / L + IAA 0.2 mg / L + AS 50 mg / L + 30 g / L Sucrose + 8 g / L Agar) with sterile filter paper placed on the surface, leaf surface upwards, and incubate in the dark at 25°C for 2 days.

[0106] 4) Screening of transformed plants: Materials co-cultured for 2 days were removed, and the bacterial solution on the surface of the materials was blotted dry with sterile filter paper. They were then placed on MS4 medium (MS1 + 5 mg / L glufosinate (Basta) + 500 mg / L carbenicillin (Cb)) and cultured for 3 weeks. The medium was changed every 3 weeks, using MS5 (4.63 g / L MS + 30 g / L Sucrose + 8 g / L Agar + 2 mg / L 6-BA + 0.1 mg / L IAA + 5 mg / L Basta + 500 mg / L Cb) and MS6 (4.63 g / L MS + 30 g / L Sucrose + 8 g / L Agar + 1 mg / L 6-BA + 5 mg / L Basta + 500 mg / L Cb). The callus tissues that had sprouted were numbered, and seedlings were cut (all buds on each individual callus tissue were considered as an independent genetic transformation line), and cultured on 1 / 2 MS medium with the corresponding resistance to induce rooting. The rooted seedlings were then tested using the Teca-Plant system to detect luminescent plants.

[0107] like Figure 7 As shown, the rooted seedlings obtained after resistance screening and tissue culture successfully exhibited stable and continuous luminescence signals, indicating that the optimized luminescent vector system containing SvPKS1 can be successfully and stably expressed in the horticultural ornamental plant petunia, demonstrating a promising new germplasm of luminescent flowers.

Claims

1. Phellinus linteus polyketide synthase gene SvPKS1 Its application in promoting bioluminescence in fungi or plants is characterized by, The SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1; The fungus is a caffeic acid-producing fungus; The fungi or plants mentioned contain genes that encode NnH3H, NnLuz, and NnCPH proteins.

2. The application as described in claim 1, characterized in that, The fungus or plant also contains a gene encoding the AnNPGA protein.

3. The application of recombinant vectors in promoting bioluminescence in fungi or plants, characterized in that, The recombinant vector contains the *Phellinus linteus* polyketide synthase gene. SvPKS1 The SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1; The fungus is a caffeic acid-producing fungus; The fungi or plants mentioned contain genes that can encode NnH3H protein, NnLuz protein and NnCPH protein; The fungus or plant also contains a gene encoding the AnNPGA protein.

4. The application of genetically engineered bacteria in promoting bioluminescence in fungi or plants, characterized in that, The genetically engineered bacteria contain the *Phellinus linteus* polyketide synthase gene. SvPKS1 Or it may contain the pheasant polyketide synthase gene. SvPKS1 The recombinant vector; SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1; The fungus is a caffeic acid-producing fungus; The fungi or plants mentioned contain genes that can encode NnH3H protein, NnLuz protein and NnCPH protein; The fungus or plant also contains a gene encoding the AnNPGA protein.

5. The application of Sanghuang polyketide synthase SvPKS1 in promoting bioluminescence in fungi or plants, characterized in that, The amino acid sequence encoded by the SvPKS1 polyketide synthase from Phellinus linteus is shown in SEQ ID NO.2; The fungus is a caffeic acid-producing fungus; The fungi or plants mentioned contain NnH3H protein, NnLuz protein and NnCPH protein; The fungus or plant also contains AnNPGA protein.

6. A method for constructing a luminescent fungus, characterized in that, include: Phellinus linteus polyketide synthase gene SvPKS1 The fungus was introduced into a caffeic acid-producing fungus to obtain a bioluminescent fungus; The Sanghuang polyketide synthase gene SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1; The caffeic acid-producing fungus contains genes that can encode NnH3H protein, NnLuz protein and NnCPH protein; The NnH3H protein has the accession number QJQ48094.1 in the NCBI database; The NnLuz protein has the accession number QJQ48096.1 in the NCBI database; The NnCPH protein has the accession number QJQ48093.1 in the NCBI database.

7. The construction method according to claim 6, characterized in that, The caffeic acid-producing fungus is *Saccharomyces cerevisiae*; the *Saccharomyces cerevisiae* is YCA113-2B.

8. The construction method according to claim 6, characterized in that, The caffeic acid-producing fungus also contains a gene that can encode the AnNPGA protein. The AnNPGA protein has the accession number QJQ48097.1 in the NCBI database.

9. A method for constructing a luminescent plant, characterized in that, include: Phellinus linteus polyketide synthase gene SvPKS1 By introducing the bioluminescent material into plant cells, a bioluminescent plant can be obtained. The Sanghuang polyketide synthase gene SvPKS1 The encoded nucleotide sequence is shown in SEQ ID NO.1; The plant genome integrates genes encoding NnH3H protein, NnLuz protein, and NnCPH protein; The NnH3H protein has the accession number QJQ48094.1 in the NCBI database; The NnLuz protein has the accession number QJQ48096.1 in the NCBI database; The NnCPH protein has the accession number QJQ48093.1 in the NCBI database.

10. The method according to claim 9, characterized in that, The plant cells also contain a gene encoding the AnNPGA protein; the AnNPGA protein has the accession number QJQ48097.1 in the NCBI database.

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