A fungal luciferase mutant and its application in improving the bioluminescence intensity of fungi or plants

Abstract

The present invention belongs to the field of enzyme engineering and biotechnology, and specifically relates to a fungal luciferase mutant and its application in improving the bioluminescence intensity of fungi or plants. The amino acid sequence of the fungal luciferase mutant is shown in SEQ ID NO.1. The present invention obtains a fungal luciferase mutant NnLuz_T138P by mutating the fungal luciferase NnLuz. The co-expression of NnLuz_T138P and other FBP luminescence pathway genes in fungi or plants can significantly improve the bioluminescence intensity of fungi and plants. Compared with the prior art, the light quantum intensity is increased by more than 2 times. The present invention provides a feasible solution for the construction of enhanced sustainable luminescent fungi and the creation of luminescent plants.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering and biotechnology, and specifically relates to a fungal luciferase mutant and application thereof in improving the bioluminescence intensity of fungi or plants. Background Art

[0002] Bioluminescence is a special life phenomenon that exists in nature. It is used by different organisms to achieve a wide range of purposes, such as attracting mates, scaring off predators, and recruiting other organisms to spread spores. Light has a close relationship with humans and plays an extremely important role in human production, life, and reproduction. In addition to using solar energy, the current mainstream lighting methods are overly dependent on the consumption of non-renewable fossil fuels such as coal and natural gas, which has serious adverse effects on human living environment and life and health. By analyzing, utilizing and optimizing the luminescence mechanism existing in nature, efficiently converting cheap biochemical energy into light energy to assist or even replace mainstream lighting methods is of great significance for achieving low-carbon sustainable development.

[0003] As the largest oxygen and organic matter manufacturer on earth, plants provide humans with fresh air and abundant living resources. In the process of modernization, plants will also play an increasingly important role in urban construction and family life. The creation of bioluminescent plants can be used to monitor the dynamic process of plant growth and can also be used as a reporting tool for scientific research. Bioluminescent plants also have aesthetic value and commercial value. In addition, if the luminous intensity can be improved through further research in the future, "luminous trees" can also be developed to replace ordinary street lights, reduce the power loss caused by road lighting, and achieve true green lighting. There have been relevant research reports on the development of luminous plants. As early as 1986, researchers at the University of California, USA, obtained luminous plants by transferring the luciferase gene of fireflies to tobacco for expression and exogenously spraying luciferin substrates (Ow, DW et al., 1986). In 2010, Krichevsky et al. transformed the complete lux operon (luxCDABEG) from marine luminous bacteria into tobacco chloroplasts and cultivated self-luminous plants (Krichevsky, A. et al., 2021). In 2020, there have been reports of creating luminous plants based on the fungal bioluminescence pathway (FBP) (Khakhar et al., 2020; Mitiouchkina et al., 2020). Although the light emitted is visible to the naked eye, its light intensity is far from enough to replace existing lighting and cannot be put into daily use.

[0004] The earliest record of luminous fungi can be traced back to 2,000 years ago. However, it was not until 1962 that the first scientific research report on luminous fungi was published, and the corresponding luminescence mechanism hypothesis was proposed, but the specific substrates and catalytic enzymes were unknown. With the development of science and the continuous deepening of related research, in 2018, Kotlobay et al. (Kotlobay et al., 2018) completely analyzed the mechanism of action of FBP and identified the enzymes required for the complete pathway: fungal luciferase (Luz), hispidin synthase (HispS) and hispidin-3-hydroxylase (H3H). Although caffeoylpyruvate hydrolase (CPH) is not necessary for the production of spontaneous luminescence, it can significantly prolong the luminescence time.

[0005] The mechanism of FBP is: HispS converts caffeic acid into hispidin, and then converts hispidin into the fluorescein substrate 3-hydroxyhispidin under the catalysis of H3H. Subsequently, with the participation of oxygen, Luz converts fluorescein into an unstable high-energy intermediate and releases light with a wavelength of 520 nm and carbon dioxide to produce caffeoylpyruvate. CPH converts caffeoylpyruvate into caffeic acid to achieve substrate regeneration and recycling (Kaskova et al., 2017). Caffeic acid is a key intermediate for most plants to produce lignin and other plant metabolites. The clear mechanism of action of FBP and the abundant precursor caffeic acid in plants have laid an important foundation for creating luminous plants.

[0006] In 2020, two studies on the creation of self-luminous plants based on FBP were published (Khakhar et al., 2020; Mitiouchkina et al., 2020). Both studies used Neonothopanus nambi The four FBP genes ( NnLuz , H3H , NnHisP , NcPH ) to construct a plant expression vector, Khakhar et al. also added a gene from Aspergillus nidulans ( Aspergillus nidulans )of AnNPGA(4'-phosphopantetheinyltransferase) gene to increase the activity of HispS. Although the two studies differ in the design of the expression cassette, the luminescence phenomenon can be observed with the naked eye through transient transformation or stable transformation into tobacco and other plants. Unfortunately, the luminescence intensity of plants produced by these two methods is very weak, and the naked eye needs to adapt to the dark environment for a long time to see the self-luminescence of plants. Subsequent studies have found that metabolic engineering is used to reconstruct the endogenous caffeic acid synthesis metabolic pathway of plants, strengthen the biosynthesis of endogenous caffeic acid, and weaken the consumption of caffeic acid by other branch metabolic pathways, so that more caffeic acid flows to the luminescence pathway. This transformation can improve the intensity of plant self-luminescence to a certain extent (Peng Zheng et al., 2023; Jieyu Ge et al., 2024). It is worth noting that some studies have modified the key enzymes of FBP by combining random mutations and rational design to obtain enzyme mutants with high catalytic performance, which increased the luminescence intensity of different eukaryotic organisms by 1-2 orders of magnitude, and the naked eye does not need to adapt to dark conditions to see plant luminescence.

[0007] Although the luminous intensity of plants has been significantly improved through various studies, the luminous intensity is still very weak, and it is still a long way from the goal of truly using luminous plants for low-brightness lighting. Therefore, how to further optimize the FBP system to enhance the intensity of plant bioluminescence is a technical problem that technicians in this field need to solve. Summary of the invention

[0008] In view of the need for optimizing the FBP system and enhancing the bioluminescence intensity of plants in the prior art, the present invention provides a fungal luciferase mutant and its application in improving the bioluminescence intensity of fungi or plants. The specific technical scheme is as follows:

[0009] In a first aspect, the present invention provides a fungal luciferase mutant, wherein the amino acid sequence of the fungal luciferase mutant is shown in SEQ ID NO.1.

[0010] In a second aspect, the present invention provides a gene encoding the above-mentioned fungal luciferase mutant.

[0011] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned gene.

[0012] In a fourth aspect, the present invention provides a genetically engineered bacterium comprising the above-mentioned gene.

[0013] In a fifth aspect, the present invention provides the use of the above-mentioned fungal luciferase mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, and the above-mentioned genetically engineered bacteria in improving the bioluminescence intensity of fungi or plants.

[0014] In a sixth aspect, the present invention provides a construction method for enhancing the bioluminescence intensity of fungi, comprising: introducing a gene encoding a fungal luciferase mutant into a caffeic acid-producing fungus to obtain a fungus with enhanced bioluminescence intensity;

[0015] The amino acid sequence of the fungal luciferase mutant is shown in SEQ ID NO.1;

[0016] The fungal genome is integrated with the coding genes of NnHisps protein, NnCPH protein, NnH3H protein and AnNPGA protein; the accession number of NnHispS protein in the NCBI library is QJQ48095.1, the accession number of NnCPH protein in the NCBI library is QJQ48093.1, the accession number of NnH3H protein in the NCBI library is QJQ48094.1, and the accession number of AnNPGA protein in the NCBI library is QJQ48097.1.

[0017] Furthermore, the caffeic acid-producing fungus is Saccharomyces cerevisiae YCA113-2B.

[0018] Furthermore, the present invention utilizes CRISPR / Cas9 gene editing-assisted efficient homologous recombination technology to NnHisP , NcPH , H3H , AnNPGA and fungal luciferase mutants NnLuz_T138P The gene expression cassette is integrated into the fungal genome to obtain a fungus with enhanced bioluminescence intensity.

[0019] Furthermore, the coding genes of the NnHisps protein, NnCPH protein, NnH3H protein and AnNPGA protein are driven to express by the galactose-induced GAL1 or GAL10 promoter.

[0020] In a seventh aspect, the present invention provides a construction method for enhancing the bioluminescence intensity of plants, comprising: integrating genes encoding NnHispS protein, NnCPH protein, NnH3H protein, AnNPGA protein and fungal luciferase mutant into the plant genome to obtain a plant with enhanced bioluminescence intensity;

[0021] The amino acid sequence of the fungal luciferase mutant is shown in SEQ ID NO.1, the accession number of NnHispS protein in the NCBI library is QJQ48095.1, the accession number of NnCPH protein in the NCBI library is QJQ48093.1, the accession number of NnH3H protein in the NCBI library is QJQ48094.1, and the accession number of AnNPGA protein in the NCBI library is QJQ48097.1.

[0022] Furthermore, in the present invention, multi-gene stacking technology is used to NnHisP , NcPH , H3H , AnNPGA and NnLuz_T138P The gene is integrated into a receptor vector to construct a plant expression vector containing the target gene. The target gene is introduced into the receptor plant using transgenic technology to obtain a transgenic plant with enhanced bioluminescence intensity.

[0023] Furthermore, the plant is a higher plant.

[0024] Furthermore, the higher plant is chrysanthemum, petunia, rapeseed, phalaenopsis or tobacco.

[0025] Furthermore, transgenic plants with enhanced bioluminescence intensity were constructed, and multi-gene stacking was performed using the TransGene Stacking II system.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention obtains a fungal luciferase mutant NnLuz_T138P by mutating the fungal luciferase NnLuz. The co-expression of NnLuz_T138P and other FBP luminescence pathway genes in fungi or plants can significantly enhance the bioluminescence intensity of fungi and plants. Compared with the prior art, the light quantum intensity is increased by more than 2 times. The present invention provides a feasible solution for the construction of enhanced sustainable luminescent fungi and the creation of luminescent plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Luminescence assay for YCA113-2B-LCHH and YCA113-2B yeast strains.

[0029] Figure 2 Luminescence detection and rotation verification for the YCA113-2B-LCHH-ΔNnLuz yeast strain; Figure 2 In A, 1, 2, and 3 represent three monoclonal bacterial liquids of the YCA113-2B-LCHH-ΔNnLuz strain, and P represents the YCA113-2B-LCHH bacterial liquid; B is the detection of the luminescence effect of free expression of NnLuz in the YCA113-2B-LCHH-ΔNnLuz strain without self-luminescence.

[0030] Figure 3 Schematic diagram of the beneficial mutation sites of NnLuz predicted by the software; A represents all single-site mutation modes; B is a schematic diagram of the transformation method used to test different mutants.

[0031] Figure 4The results of luminescence intensity detection of YCA113-2B-LCHH-ΔNnLuz strain expressing different NnLuz mutants in isolation; A is a CCD photograph of strains expressing different NnLuz mutants after streaking and growing on a plate for two days; B is the relative bioluminescence intensity analysis of strains expressing different NnLuz mutants after spotting and growing on a plate for two days, with the luminescence intensity of NnLuz_WT as 1 for standardization, and the error represents the standard deviation of three replicates; C is the bioluminescence intensity analysis of strains expressing different NnLuz combination mutants and NnLuz_V4, and the error represents the standard deviation of three replicates. The Dunnett's multiple comparison test (***P≤0.001) was used to evaluate the statistical significance.

[0032] Figure 5 Comparison of luminescence intensity of NnLuz wild type or mutant in Nicotiana benthamiana leaves after 4 days of transient expression; A is the phenotype obtained by taking pictures with the Teca-Plant system; B is the relative bioluminescence intensity analysis based on A photon, which is standardized with the luminescence intensity of NnLuz_WT as 1, and the error represents the standard deviation of three replicates.

[0033] Figure 6 The figure is a comparison of the luminescence effects of FBP(NnLuz_T138P) transgenic lines and FBP transgenic lines; A is an independent strain of FBP(NnLuz_T138P) screened by luminescence imaging; B is the bioluminescence image of FBP and FBP(NnLuz_T138P) flowers; C is the bioluminescence intensity analysis of FBP and FBP(NnLuz_T138P) flowers, the error represents the standard deviation of 6 replicates, and the two-tailed t-test (**P≤0.01) was used to evaluate the statistical significance. DETAILED DESCRIPTION

[0034] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0035] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. The experimental methods without specifying the detailed conditions were carried out in accordance with conventional experimental methods or the operating instructions recommended by the supplier.

[0037] Plasmids p423-SpSgH, pRS426-SpSgH-Int10, and pRS41K-SpCas9 were donated by the laboratory of Researcher Lian Jiachang of Zhejiang University. The construction methods 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).

[0038] The construction method of plasmid pRS423-SpSgH-ARO10 was similar to that of pRS426-SpSgH-Int10, that is, the Benchling CRISPR tool was used to design the gRNA sequence of ARO10. After the optimal target was determined by the On-Target Score and Off-TargetScore parameters, the target primers ΔARO10-F (5'-gatcAAAGTGCATGGTTGAAGCAG -3') and ΔARO10-R (5'- aaacCTGCTTCAACCATGCACTTT -3') of N20 were synthesized and constructed into the p423-SpSgH plasmid, transformed into Escherichia coli DH5α, cultured overnight, and sent for sequencing after screening positive clones by colony PCR. A copy of the correctly sequenced strain and plasmid pRS423-SpSgH-ARO10 was saved for backup.

[0039] The caffeic acid-producing yeast strain YCA113-2B was donated by the laboratory of Associate Professor Ye Lidan of Zhejiang University. The construction method of the strain 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], 105:58095819.).

[0040] pYL322d1, pYL322d2, pYLTAC380GW and pYLMF-H were donated by Professor Liu Yaoguang's laboratory at South China Agricultural University. The construction methods thereof refer to Chinese patent application number 2017103841977.

[0041] The coding sequences of NnHispS, NnCPH, NnH3H, NnLuz and AnNPGA were optimized according to the codon preference of the host and then chemically synthesized. The sequence information of NnHispS can be found in the NCBI library with the gene accession number QJQ48095.1; the sequence information of NnCPH can be found in the NCBI library with the gene accession number QJQ48093.1; the sequence information of NnH3H can be found in the NCBI library with the gene accession number QJQ48094.1; the sequence information of NnLuz can be found in the NCBI library with the gene accession number QJQ48096.1; the sequence information of AnNPGA can be found in the NCBI library with the gene accession number QJQ48097.1.

[0042] The amino acid sequence of the mutant NnLuz_T138P is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding NnLuz_T138P is shown in SEQ ID NO.2.

[0043] SEQ ID NO.1:

[0044] MRINISLSSLFERLSKLSSRSIAITCGVVLASAIAFPIIRRDYQTFLEVGPSYAPQNFRGYIIVCVLSLFRQEQKGLAIYDRLPEKRRWLADLPFREGTRPSITSHIIQRQRTQLVDQEFATRELIDKVIPRVQARHPDKTFLSTSKFEFHAKAIFLLPSIPINDPLNIPSHDTVRRTKREIAHMHDYHDCTLHLALAAQDGKEVLKKGWGQRHPLAGPGVPGPPTEWTFLYAPRNEEEARVVEMIVEASIGYMTNDPAGKIVENAK。

[0045] SEQ ID NO.2:

[0046] ATGAGAATCAACATCTCACTCTCTTCTCTTTTCGAGAGATTGTCAAAGTTGTCATCTAGGTCAATCGCTATCACTTGTGGTGTGGTTTTGGCTTCTGCTATCGCTTTCCCAATCATCAGAAGGGATTACCAGACTTTCTTAGAAGTTGGACCTTCTTACGCTCCACAGAACTTCAGGGGTTACATCATCGTGTGTGTTTTGTCATTGTTCAGACAGGAGCAGAAGGGTTTAGCTATCTACGATAGATTGCCTGAGAAGAGAAGATGGCTTGCTGATTTGCCATTCAGGGAGGGTACTAGGCCTTCAATCACTTCACATATCATCCAGAGACAGAGAACTCAGCTCGTGGATCAGGAGTTCGCTACTAGGGAGCTTATCGATAAGGTTATCCCTAGAGTGCAGGCTAGGCATCCGGATAAGACTTTCTTGTCTACTTCAAAGTTCGAGTTCCATGCTAAGGCTATCTTCCTCCTCCCTTCAATCCCAATCAACGATCCTCTTAACATCCCATCTCATGATACTGTTAGAAGGACTAAGAGGGAGATCGCTCACATGCATGATTACCATGATTGTACTCTCCATTTGGCTCTTGCTGCTCAGGATGGTAAGGAGGTGCTTAAGAAGGGATGGGGACAGAGGCATCCACTCGCTGGACCTGGTGTGCCTGGACCACCAACTGAGTGGACTTTCCTTTACGCTCCTAGGAACGAGGAGGAGGCTAGAGTGGTTGAGATGATCGTTGAGGCTTCTATCGGATACATGACTAACGATCCAGCTGGAAAGATCGTTGAGAACGCTAAGTAA。

[0047] Example 1 Construction of Bioluminescent Saccharomyces cerevisiae

[0048] (1) Clone the target genes (NnHispS, NnCPH, NnH3H, NnLuz) into a vector containing a galactose-inducible promoter (P GAL1 or P GAL10) at MCS1 or MCS2 of the universal plasmid pESC-URA to obtain the recombinant plasmids pESC-URA-NnLuZ-NnCPH and pESC-URA-NnH3H-NnHispS with the target gene. The specific information is shown in Table 1.

[0049] Table 1

[0050]

[0051] The coding sequence of the NnHispS gene after codon optimization is shown in SEQ ID NO.4, the coding sequence of the NnCPH gene is shown in SEQ ID NO.6, the coding sequence of the NnH3H gene is shown in SEQ ID NO.8, and the coding sequence of the NnLuz gene is shown in SEQ ID NO.10.

[0052] (2) The recombinant plasmids pESC-URA-NnLuz-NnCPH and pESC-URA-NnH3H-NnHispS were amplified by PCR using primers with a 40 bp homology arm near the ARO10 gene target site and a primer with a 40 bp homology arm near the Int10 site, respectively, to obtain the genomic integration donor: ΔAro10::T ADH1 -NnLuz-P GAL10 -P GAL1 -NnCPH-T CYC and Int10::T ADH1 -NnH3H-P GAL10 -P GAL1 -NnHispS-T CYC , primers are shown in Table 2.

[0053] Table 2

[0054]

[0055] (3) Use CRISPR / Cas9 to integrate the target gene and integrate the donor ΔARO10::T ADH1 -NnLuz-P GAL10 -P GAL1 -NnCPH-T CYC The plasmids pRS423-SpSgH-ARO10 and pRS41K-SpCas9 were chemically transformed into yeast cells YCA113-2B, and then plated on SD-His / G418 plates with corresponding amino acid deficiencies to obtain YCA113-2B-LC. ADH1 -NnH3H-P GAL10 -P GAL1 -NnHispS-TCYC It was transformed into yeast cells YCA113-2B-LC with pRS426-SpSgH-Int10 and pRS41K-SpCas9, and the YCA113-2B-LCHH self-luminous yeast strain was obtained by screening.

[0056] The screening process of recombinant yeast is as follows: select the transformants on the plate and perform colony PCR amplification using the target sequence primers on the genome. If the band size is correct and the sequencing results are correct, it means that the genome integration is successful. The resulting YCA113-2B-LCHH strain is tested for self-luminescence by the fully intelligent gel imaging system CCD (Charge-Coupled Device) (the results are shown in the figure). Figure 1 as shown).

[0057] Example 2 Knockout of NnLuz gene in self-luminescent Saccharomyces cerevisiae YCA113-2B-LCHH

[0058] The NnLuz gene of the YCA113-2B-LCHH yeast constructed in Example 1 was knocked out using CRISPR / Cas9 gene editing technology. The method is as follows:

[0059] 1) The Benchling CRISPR tool was used to design the gRNA sequence of NnLuz. After determining the optimal target through the On-Target Score and Off-Target Score parameters, the target primers ΔNnLuz-F (5'-gatcATGTAACCCCTGAAGTTCTG-3') and NnLuz-R (5'- aaacCAGAACTTCAGGGGTTACAT -3') of N20 were synthesized and constructed into the p423-SpSgH plasmid, transformed into Escherichia coli DH5α, cultured overnight, and sent for sequencing after screening positive clones by colony PCR. The correctly sequenced strain and plasmid pRS423-SpSgH-ΔNnLuz were saved for backup.

[0060] 2) The plasmids of p423-SpSgH-ΔNnLuz and pRS41K-SpCas9 were chemically transformed into yeast cells YCA113-2B-LCHH and plated on SD-His / G418 plates with corresponding amino acid deficiencies. The luminescence of the transformants on the plates was detected by the CCD system, and the non-luminescent transformants were selected and amplified by PCR using NnLuz-specific primers and then sequenced. The strain with frameshift mutation in the NnLuz coding sequence was YCA113-2B-LCHH-ΔNnLuz.

[0061] The constructed strain was chemically transferred into the pESC-URA-NnLuz plasmid and grown on the plate for 3 days. NnLuzThe gene can complement the missing luminescence phenotype of the YCA113-2B-LCHH-ΔNnLuz strain (e.g. Figure 2 A and B in the figure).

[0062] Example 3 YCA113-2B-LCHH-ΔNnLuz yeast free expression of different NnLuz mutants to screen for favorable mutations

[0063] 1) Use the consensus sequence online analysis website Consensus Finder (http: / / kazlab.umn.edu / ) to search the consensus sequence of NnLuz and predict potential favorable mutation sites (such as Figure 3 ).

[0064] 2) Design a point-in-situ PCR primer for each site, and use the pESC-URA-NnLuZ circular plasmid as a template for PCR amplification. The PCR system is as follows: 0.5 μL template; 1 μL each of 10 μM primer F and primer R; 1 μL dNTP (2.5 mM); 5 μL 10× high-fidelity enzyme buffer; 1 μL high-fidelity enzyme; add water to a reaction system of 50 μL. The PCR thermal cycle conditions are as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 60 ℃ annealing for 20 s; 72 ℃ extension for 2 min; 4 ℃ insulation; PCR amplification for 32 cycles. Combinatorial mutagenesis was performed by sequentially mutating a single site. The amplified product was DpN After digestion, 5 μL was added to 50 μL of DH5α competent cells for transformation. Incubate on ice for 20 min; heat shock at 42 °C for 45 s; place on ice for 3 min; add 800 μL LB medium, activate at 37 °C, 220 rpm for 1 h, spread on the plate, culture for 14 h-16 h until a single colony grows, pick the bacteria for sequencing verification, and obtain different NnLuz mutant plasmids.

[0065] 3) The pESC-URA recombinant plasmid containing different NnLuz mutants was transformed into yeast cells YCA113-2B-LCHH-ΔNnLuz by chemical method (PEG / LiAc method) and spread on SD-URA plates (such as Figure 3 (as shown in B in the figure).

[0066] After mixing the monoclonal clones on each plate, streak or dot smear on new SD-URA medium, take pictures and analyze the photon number using the Teca-Plant system. Figure 4From A and B in Figure 3, we can see that among all the single-point mutants of NnLuz, four forms of mutations, T138P, T122Y, T192S and K207E, can improve the luminescence intensity of YCA113-2B-LCHH-ΔNnLuz. The primers for the four key sites are shown in Table 3. Among them, the NnLuz_T138P mutant has a stronger ability to induce yeast luminescence than the reported NnLuz_V4 mutant (Shakhova, ES, Karataeva, TA, Markina, NM et al. 2024. An improved pathway for autonomous bioluminescence imaging in eukaryotes. Nat Methods [J], 21: 406–410.). Combination mutations were performed on the basis of the NnLuz_T138P mutant, and it was found that the combination of the NnLuz_T138P mutant with other mutation forms had no significant effect on the luminescence performance (such as Figure 4 as shown in C in the figure).

[0067] Table 3 Primer sequences of four key amino acid sites of NnLuz

[0068]

[0069] Example 4 Construction of plant bioluminescent vector FBP (NnLuz_T138P)

[0070] The TransGene Stacking II system was used to construct the vector FBP (NnLuz_T138P). The construction method of this vector is similar to that of the basic vector FBP (pYLTAC380GW-5G), except that the NnLuz gene on the FBP vector was replaced by NnLuz_T138P. The amino acid sequence of NnLuz_T138P is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. The construction method of pYLTAC380GW-5G refers to the Chinese patent application number 202111228803.9.

[0071] Example 5 Comparison of the luminescence intensity of tobacco leaves with different NnLuz involved in instantaneous transformation

[0072] pYLTAC380GW-4G and vectors carrying different forms of NnLuz genes were co-expressed in wild-type Nicotiana benthamiana leaves by Agrobacterium-mediated transient transformation of tobacco leaves to compare the effects of different NnLuz genes on luminescence intensity.

[0073] pYLTAC380GW-4G is a vector material derived from the Chinese patent application number 202111228803.9, which expresses four genes: NnHispS, NnCPH, NnH3H and AnNPGA; NnLuz_WT, NnLuz_v4 or NnLuz_T138P are cloned into the pCAMBIAM1300 vector, and its 3' end is fused with a 6×His tag and driven by the StUbipro promoter. The steps of Agrobacterium infection are as follows:

[0074] 1) pYLTAC380GW-4G and pCAMBIAM1300 plasmids containing different NnLuz were transformed into Agrobacterium strain EHA105, and single clone positive strains were selected and inoculated into LB liquid medium containing Kana 50 μg / mL and Rif 25 μg / mL, and placed at 28°C and 200 r·min -1 Incubate in a shaker until OD 600 = about 1.0;

[0075] 2) Transfer the bacterial solution to a centrifuge tube and centrifuge at 8000 × r·min -1 Centrifuge for 1 min, discard the supernatant, and collect the bacteria;

[0076] 3) Resuspend the Agrobacterium in a suspension containing 10 mmol / L MES (pH = 5.7), 50 mmol / L MgCl2, and 200 μmol / L AS, and adjust the OD 600 is 1.0;

[0077] 4) After standing at room temperature for 2 h, the three strains containing different NnLuz were mixed with the strain containing pYLTAC380GW-4G in equal volumes;

[0078] 5) Slowly inject the mixed bacterial solution into the lower epidermal veins of tobacco leaves with a sterile syringe without a needle. Inject the three mixed bacterial solutions mentioned above into different parts of each leaf and keep them in the dark for 12 hours after injection;

[0079] 6) The dark-treated tobacco was placed in a light incubator at 25°C, with a photoperiod of 16 hours light / 8 hours dark and an illumination of 2000 Lx for further cultivation.

[0080] The leaves were photographed 3-4 days after injection using the Teca-Plant system and the photon number was analyzed. Figure 5 As shown in A and B, compared with the part expressing NnLuz_WT, the expression of NnLuz_T138P can significantly increase the luminescence intensity of leaves, with the increase reaching 4 times that of the former.

[0081] Example 6 Agrobacterium-mediated genetic transformation of tobacco with FBP (NnLuz_T138P) vector and analysis of luminescence intensity

[0082] 1) Transform the FBP (NnLuz_T138P) vector plasmid and FBP (NnLuz) vector plasmid into Agrobacterium strain EHA105, culture on LA+Rif+Kana plates at 28 ℃ for 36 h, pick a single clone into 3-5 mL LB medium, culture at 200 rpm, 28 ℃ overnight, expand the culture at a ratio of 1:100-1:50 to OD=0.6, centrifuge the bacterial solution, and suspend the bacteria in MS0 liquid medium (MS+3%Sucrose+pH5.8, 50 mL) to OD=0.6 for infection.

[0083] 2) Select healthy leaves of Zhongyan 100 tobacco grown on sterile MS medium for 4-5 weeks until fully expanded, cut them into 0.5 cm squares with a scalpel (cut off the leaf edges to avoid the main veins), and culture them with the upper surface facing down on MS1 ​​solid medium (MS+0.5 mg / L IAA+2.0 mg / L BA+3% sucrose+0.6-0.8% Phytagel, pH=5.8) at 25°C in the dark for 2-3 days.

[0084] 3) Add the pre-cultured tobacco leaves to the bacterial solution, vortex to ensure that the leaf cut is immersed in the bacterial solution, let it stand for 20-30 min, and use sterile filter paper to absorb the attached bacterial solution; place the infected leaves with the upper surface facing down on MS1 ​​solid culture medium and culture in the dark at 28°C for 2 days; place the leaves with the upper surface facing up on MS1 ​​screening culture medium containing Timentin (TM) and Hygromycin (Hyg) and culture in the light at 25°C; when buds grow on the leaf edge and can be separated (more than 1 cm), cut off the buds and transfer them to MS2 (MS+0.5 mg / L IAA+3% sucrose+0.6-0.8% Phytagel, pH=5.8) solid culture medium containing antibiotics (TM+Hyg). Roots will grow after two weeks. Open the lid of the seedling box and train the seedlings for two days before planting them in the soil.

[0085] Take -2 and -4 of FBP (NnLuz_T138P) respectively (such as Figure 6 The flowers of FBP-23 positive plants with strong FBP light signals were simultaneously detected by a photon analyzer. The results are shown in Figure 6 As shown in B and C, the average value was found that the luminescence intensity of FBP (NnLuz_T138P) was about 3 times higher than that of the flowers of FBP in Chinese tobacco.

Claims

1. A fungal luciferase mutant, characterized in that: The amino acid sequence of the fungal luciferase mutant is shown in SEQ ID NO.

1.

2. A gene encoding the fungal luciferase mutant as claimed in claim 1.

3. A recombinant vector, characterized in that: The recombinant vector comprises the gene according to claim 2.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria comprises the gene as claimed in claim 2.

5. Use of the fungal luciferase mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the genetically engineered bacteria according to claim 4 in improving the bioluminescence intensity of fungi or plants; the fungi are caffeic acid-producing fungi.

6. A method for enhancing the bioluminescence intensity of fungi, characterized in that: include: The gene encoding the fungal luciferase mutant was introduced into the caffeic acid-producing fungus to obtain a fungus with enhanced bioluminescence intensity; The amino acid sequence of the fungal luciferase mutant is shown in SEQ ID NO.1; The fungal genome is integrated with the coding genes of NnHisps protein, NnCPH protein, NnH3H protein and AnNPGA protein; the accession number of NnHispS protein in the NCBI library is QJQ48095.1, the accession number of NnCPH protein in the NCBI library is QJQ48093.1, the accession number of NnH3H protein in the NCBI library is QJQ48094.1, and the accession number of AnNPGA protein in the NCBI library is QJQ48097.

1.

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

8. A method for enhancing the bioluminescence intensity of plants, characterized in that: The following steps are involved: Integrate genes encoding NnHispS protein, NnCPH protein, NnH3H protein, AnNPGA protein and fungal luciferase mutant into plant genome to obtain plants with enhanced bioluminescence intensity; The amino acid sequence of the fungal luciferase mutant is shown in SEQ ID NO.1, the accession number of NnHispS protein in the NCBI library is QJQ48095.1, the accession number of NnCPH protein in the NCBI library is QJQ48093.1, the accession number of NnH3H protein in the NCBI library is QJQ48094.1, and the accession number of AnNPGA protein in the NCBI library is QJQ48097.

1.

9. The method according to claim 8, characterized in that The plant is a higher plant.

10. The method according to claim 9, characterized in that The higher plant is chrysanthemum, petunia, rape, phalaenopsis or tobacco.

Images