Luciferase mutant and application thereof

Abstract

The invention discloses a luciferase mutant, the amino acid sequence of which is as shown in SEQ ID NO. 1, and the polynucleotide sequence of which is as shown in SEQ ID NO. 2. Compared with a wild type, the luciferase mutant disclosed by the invention has the advantages that the catalytic activity is improved by 3 times, and the luciferase mutant can be industrially prepared, can be used for preparing a luciferase detection method kit and can also be used as a reporter protein or a reporter gene for biological research.

Description

Technical Field

[0001] The invention discloses a protein mutant and belongs to the technical field of polypeptides. Background Art

[0002] Luciferase (Luciferase, Luc) is a general term for enzymes that can produce biological fluorescence in nature. The most representative one is a Photinus pyrali Luciferase in fireflies. The catalytic reaction of luciferase is: in the presence of magnesium ions, it first catalyzes luciferin and ATP to produce luciferin-AMP and pyrophosphate, and then catalyzes luciferin-AMP and oxygen to produce oxidized luciferin, AMP and carbon dioxide, while releasing fluorescence. When the enzyme content added to the luciferase reaction system is consistent and the substrate is in excess, the luminescence intensity is proportional to the luciferase activity. The reaction catalyzed by luciferase emits yellow-green light at a pH of 7.5-8.5, with a wavelength of 562-570 nm. The emitted photons can be detected by light-sensitive elements, such as fluorescence detectors or improved optical microscopes, which makes it possible to observe a variety of life activities.

[0003] Luciferase can be synthesized by genetic engineering and used in a variety of detection experiments. Luciferase can be used as a "reporter protein" in molecular biology research, for example, to detect the transcription of a specific promoter in cells transfected with luciferase or to detect the ATP level in cells; this technology is called reporter gene assay or luciferase assay. Reporter genes or reporter groups are chemical groups, proteins or enzymes that are easily detected by experimental instruments and the genes encoding them, so they are widely used in experiments. For example, the luciferase gene used in luciferase reporter gene experiments and the fluorescent reporter group in qPCR. Reporter genes are widely used in gene expression regulation, signal transduction, promoter analysis, receptor function identification, gene therapy, and drug screening. Luciferase is also a heat-sensitive protein and is often used to study the protective ability of heat shock proteins during protein thermal denaturation.

[0004] Luciferase is an ideal reporter protein due to its excellent sensitivity, ease of use, and quantitative detection. Therefore, providing new luciferases and their applications has great technical applications and market demand.

[0005] The object of the present invention is to provide a luciferase mutant with higher enzyme activity. Summary of the invention

[0006] Based on the above invention objectives, the present invention first provides a luciferase mutant, the amino acid sequence of the luciferase mutant is shown in SEQ ID NO.1.

[0007] Secondly, the present invention provides a polynucleotide encoding the above luciferase mutant. According to the common sense of those skilled in the art, any polynucleotide encoding the above luciferase mutant with the amino acid sequence shown in SEQ ID NO.1 belongs to the polynucleotide of the present invention.

[0008] In a preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO.2.

[0009] Third, the present invention provides an expression vector containing the above polynucleotide. As known to those skilled in the art, the above luciferase mutant provided by the present invention can be prepared by genetic engineering methods, and conventional expression vectors in the art, such as prokaryotic expression vectors or eukaryotic expression vectors, can be used for industrial preparation of the above luciferase mutant.

[0010] Fourth, the present invention provides a host cell containing the above expression vector. As known to those skilled in the art, the above expression vector can be transferred into a host cell, such as a prokaryotic host cell such as Escherichia coli or a eukaryotic host cell such as yeast, to achieve industrial preparation of the above luciferase mutant.

[0011] Fifth, the present invention provides a method for screening luciferase mutants, the method comprising the following steps: (1) The first vector containing the nCas9 encoding gene and the cytosine deaminase encoding gene is transferred into yeast, and the nCas9 encoding gene and the cytosine deaminase encoding gene are integrated into the GS115 genome by homologous recombination to obtain a recombinant yeast strain containing the nCas9 encoding gene and the cytosine deaminase encoding gene in the genome; (2) transferring the second vector containing the luciferase encoding gene into the recombinant yeast obtained in step (1), integrating the luciferase encoding gene into the genome of the recombinant strain by homologous recombination, and screening the recombinant yeast strain having a single copy of the luciferase encoding gene, wherein the second vector carries an inducible promoter element; (3) transferring the gRNA plasmid library targeting the luciferase encoding gene into the recombinant yeast strain obtained in step (2) to mutate the luciferase encoding gene, wherein the gRNA plasmid library contains 51 sequences targeting different segments of the luciferase encoding gene, as shown in SEQ ID NOs. 3-53, and the gRNA is constructed on a third vector; (4) inducing the yeast strain obtained in step (3) to express the luciferase gene; (5) Screening luciferase mutants based on the enzyme activity displayed by the luciferase expression product described in step (4).

[0012] In a preferred technical solution, the yeast strain is GS115.

[0013] In a specific embodiment of the present invention, the first vector is pGAP, and the first vector containing the nCas9 encoding gene and the cytosine deaminase encoding gene in step (1) is named "pGAP-nCas9-(GGGGS)" in the present invention. 10 -PmCDA1", and the recombinant yeast strain whose genome contains the nCas9 encoding gene and the cytosine deaminase encoding gene was named "GS115-nCas9 -PmCDA1".

[0014] In a specific embodiment of the present invention, the second vector is pPIC9K, and the pPIC9K contains an AOX1 promoter, which can be induced to transcribe and express exogenous coding genes under strict control of methanol. The second vector containing the luciferase coding gene in step (2) is named "pPIC9K-Luc" in the present invention, and the recombinant yeast strain with a single copy of the luciferase coding gene is named "GS115-nCas9 -PmCDA1-Luc" in the present invention.

[0015] In a specific embodiment of the present invention, the third vector is pTEF, which contains a Sh ble expression frame and can be used for screening recombinant strains. The third vector containing 51 sequences of different segments of the target luciferase encoding gene in step (3) is shown in SEQ ID NO. 3-53, and the gRNA is named "pTEF-gRNA" in the present invention.

[0016] Sixth, the use of the luciferase mutant in the preparation of a luciferase detection kit. The luciferase detection method of the present invention generates fluorescence intensity through the action of luciferase and substrate, such as ATP, so as to detect the substrate and other targets associated therewith.

[0017] Seventh, the present invention provides the use of the above-mentioned luciferase mutant as a reporter protein. By associating luciferase with a target molecule (high sensitivity and rapid luminescence reaction), its luminescence signal can be used to evaluate the activity of compounds in a compound library or the effect of inhibitors to achieve high-throughput screening. Luciferase can also be used to construct a biosensor for detecting the presence and concentration changes of a specific substance. By combining luciferase with an appropriate sensor, quantitative or localized detection of target molecules in a biological sample can be achieved.

[0018] Finally, the present invention provides the use of the polynucleotide encoding the luciferase mutant as a luciferase reporter gene, wherein the reporter gene also contains a promoter and / or a polynucleotide encoding a gene. For example, luciferase is often used as a bioluminescent reporter gene to study biological processes such as gene expression, transcriptional regulation, and protein interaction. By connecting the luciferase gene to a gene or promoter of interest, their expression level or activity can be monitored in real time. In addition, luciferase can be used as a marker for biomolecule imaging. By introducing the luciferase gene into a target cell or tissue, when a specific substrate (such as luciferin) is present, the luciferase will catalyze a reaction that emits a visible light signal, thereby achieving real-time observation of a specific location or process, such as tumor cells labeled with luciferase can show changes in the size of tumors in vivo.

[0019] The present invention uses Pichia pastoris as a host to use CRISPR-mediated base editors to mutate the luciferase sequence and perform high-throughput screening to obtain a mutant that is highly sensitive to ATP. The luciferase mutant has a higher enzyme catalytic activity, which is 3 times higher than that of the wild-type luciferase, and the luciferase mutant can be prepared by industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . pPIC9K-Luc plasmid map; Figure 2 . Schematic diagram of the distribution of 51 gRNAs in pPIC9K-Luc; Figure 3 . Comparison of fluorescence intensity of 9 mutant strains and wild-type luciferase; Figure 4 . Comparison of fluorescence intensity of luciferase between 4 mutants and wild-type luciferase; Figure 5 . Sequence alignment of Luc-3 mutant and wild-type luciferase; Figure 6 . Comparison of fluorescence intensity of Luc-3 mutant and wild-type luciferase. Figure 7 . Comparison of fluorescence intensity between Luc-3 mutants and control group. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0022] The experimental reagents involved in the present invention include: peptone, yeast extract (Oxoid Company), agar powder (Novon Company), glucose, sodium chloride, potassium dihydrogen phosphate (Xilong Science Co., Ltd.), glycerol, sodium hydroxide (Jiangsu Qiangsheng Functional Chemical Co., Ltd.), methanol (Sinopharm Group Chemical Reagent Co., Ltd.), potassium dihydrogen phosphate trihydrate (Chengdu Kelong Chemical Co., Ltd.), plasmid extraction kit (Qiagen Company), SDS-PAGE gel configuration kit (GenScript Biotech Co., Ltd.), gel recovery kit (Omega Company), YNB (Yeast Nitrogen Base w / o AminnoAcids), sorbitol (Sigma Company), XhoI, EcoRI, SacI, AflII restriction endonuclease and Quick Ligase enzyme (New England Biolabs Company), 2*Phanta Flash Master Mix, ClonExpress Multis OneStep Cloning Kit (Vazyme Company), KOD enzyme (TOYOBO Company), Zeocin (Z + , bleomycin, Invitrogen), G418 (Thermo Fisher Scientific), TOP10 competent cells (Tiangen Biochemical Technology Co., Ltd.), pGAP vector (Invitrogen), pPIC9K vector (Invitrogen), pTEF vector (Addgene), luciferase detection kit (Shandong Yuntang Intelligent Technology Co., Ltd.).

[0023] The culture medium involved in the present invention includes: YPD culture medium (1% yeast extract, 2% peptone, 2% glucose), YPDS Z + Culture medium (1% yeast extract, 2% peptone, 2% glucose, 0.1% Z + Resistance, 1 M sorbitol), MD medium (1.34% YNB, 2% glucose), YPD G418 medium (1% yeast extract, 2% peptone, 2% glucose, 2% (V / V) G418), LB medium (0.5% yeast extract, 1% peptone, 1% sodium chloride), LLB medium (0.5% yeast extract, 1% peptone, 0.5% sodium chloride), MGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1 M potassium phosphate and 2% glycerol), MMY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1 M potassium phosphate, 0.5% methanol). All media were prepared into solid plates with 2% agar powder.

[0024] The solutions involved in the present invention include: 10× glucose (200 g glucose is diluted to 1 L and sterilized at 115°C for 15 min), 10× glycerol (100 g glycerol is diluted to 1 L and sterilized by suction filtration using a 0.22 μm filter membrane), 10× YNB (34 g YNB and 100 g ammonium sulfate are diluted to 1 L and sterilized by suction filtration using a 0.22 μm filter membrane), 1 M sorbitol (182.17 g sorbitol is diluted to 1 L and sterilized by suction filtration using a 0.22 μm microfiltration membrane), and 1 M phosphate buffer (118.12 g potassium dihydrogen phosphate and 30.12 g potassium dihydrogen phosphate trihydrate, with potassium hydroxide used to adjust the pH value to 6.0±0.1).

[0025] Example 1. Screening of luciferase mutants 1. Construction of a single clone stably expressing luciferase (1) Construction of the recombinant strain GS115-nCas9-PmCDA1 The gene encoding cytosine deaminase PmCDA1 (GenBank: ABO15149.1) was fused to the C-terminus of the point (D10A) mutant (nCas9) of Cas9 (GenBank: KT031982.1), and the two were connected by a connecting peptide (GGGGS). 10 Connect, synthesized by the company nCas9-(GGGGS) 10 -PmCDA1 fragment, constructed by homologous recombination pGAP-nCas9-(GGGGS) 10 -PmCDA1 recombinant plasmid, which contains the HIS4 expression cassette and can be used for yeast transformation screening. The linearized recombinant plasmid obtained by Nhe I restriction endonuclease was electroporated into GS115 yeast, and the electroporation parameters were 2000 Voltage, 25 Capacitance (μF), 200 Resistance (Ω), and 20 Cuvette (mm), so that it was integrated into the GS115 genome through homologous recombination. Positive clones were screened on MD plates, and colony PCR was performed using primers GAP1-F / CYC1-R (5'-CGTCGCTGGCAATAATAGCG-3' / 5'-CCTTCCTTTTCGGTTAGAGC-3') to identify the recombinant strain GS115-nCas9 -PmCDA1. After sequencing confirmation, the recombinant strain GS115-nCas9 -PmCDA1 was obtained.

[0026] (2) Construction of recombinant plasmid pPIC9K-Luc Synthesized at Sino-US Taihe Biotechnology (Beijing) Co., Ltd. Allobacillus saliphilusThe nucleotide sequence of luciferase from the source (GenBank: AOR57259.1) was used to construct the recombinant plasmid pPIC9K-Luc by restriction ligation (see the plasmid map for details). Figure 1 ), which contains the AOX1 promoter, and the induction of transcription can be strictly controlled by methanol. The recombinant plasmid was transferred into TOP10 competent cells by heat shock method and screened using LB solid plates containing ampicillin to obtain single clones. The specific operation was to take 5 μg of synthetic plasmid containing Luc and pPIC9K vector, respectively, and double-digested with 2 μL XhoI / EcoRI restriction endonucleases for 3 h, and then recovered by gel into 20 μL ultrapure water. The recovered target fragment and linearized vector were connected with Quick Ligase enzyme for 30 min, heat-shocked at 42°C and transformed into TOP10 competent cells, and then 900 μL LB was added and placed in a 37°C shaker. After culturing at 220 rpm for 45 min, the entire bacterial solution was coated with LB A + Screening plate, incubate at 37℃ for 14-16 h to obtain single clones. Randomly select 10 single clones for colony PCR and sequencing to obtain single clones containing the correct recombinant plasmid. Select a correct single clone for culture, and use QIAGEN plasmid extraction kit to obtain about 25 μg of recombinant plasmid.

[0027] (3) Screening of single-copy positive strain GS115-nCas9-PmCDA1-Luc Take 50 μL of GS115-nCas9-PmCDA1 yeast solution and add it to 5 mL of YPD liquid medium. Shake at 30℃ until the culture state is over. Add 2 mL of over-cultured yeast to 200 mL of YPD liquid medium and culture it in a constant temperature shaker at 30℃ for 8 h until the OD600 value is slightly greater than 1.0. Collect the bacteria by centrifugation, wash twice with sterile water, wash once with 1M sorbitol, resuspend the yeast in 1M sorbitol, and adjust the volume to 1.5 mL (ice bath) to complete the yeast competent preparation. Linearize the recombinant plasmid pPIC9K-Luc with restriction endonuclease Sac I, and then electrotransform it into the genome of the competent cell. The electrotransformation parameters are: 2000 Voltage, 25 Capacitance (μF), 200 Resistance (Ω), 20 Cuvette (mm). G418 plates were coated for screening, and 5 single clones were randomly selected for colony PCR. After culturing the single clones containing the target sequence for 24 h, they were induced with 0.5% methanol, and a single clone that could successfully express luciferase in the secretion fluid and react with the substrate luciferin to emit fluorescence was selected for shaking, preservation, and retention as a strain.

[0028] 2. Construction of gRNA plasmid library According to the nucleotide sequence of luciferase, 51 targeting gRNAs were designed (SEQ ID NO.3-53, see Figure 2 ), which was integrated into the pTEF vector by homologous recombination to obtain the recombinant plasmid pTEF-gRNA, which contains the Sh ble expression frame and can be selected by zeocin (Z+) for screening recombinant strains. The recombinant plasmid was transferred into TOP10 competent cells by heat shock method and cultured with 0.1% Z + The single clone was screened on the resistant LLB solid plate. The specific operation was to add homologous fragments (5'-AGGACGAAACGAGTAAGCTCGTCTCAGATC-3' / 5'-ATTTTAACTTGCTATTTCTAGCTCTAAAAC-3') to the two primers of 51 gRNAs to form gRNA primers, and PCR amplification was used to obtain the target fragment containing gRNA. Take 5 μg of pTEF vector and add 4 μL restriction endonuclease Afl II, incubate at 37℃ for 2 h to complete the linearization of the vector, and then recover the gel into 30 μL ultrapure water. Take 1 μL of amplified product and 1 μL of linear vector respectively for homologous recombination using Exnase Multis. Heat shock transformation was performed into TOP10 competent cells at 42℃, and then 900 μL LLB was added and placed in a 37℃ shaker. After culturing at 220 rpm for 45 min, the entire bacterial solution was coated with LLB Zeocin (Z + ) resistance plate, and incubate at 37°C for 14-16 h to obtain single clones. Ten single clones were randomly selected for colony PCR and sequencing to obtain single clones containing the correct recombinant plasmid.

[0029] The correctly constructed single clone was cultured, and the recombinant plasmid was obtained using the QIAGEN plasmid extraction kit. 200 ng of each recombinant gRNA plasmid was taken to form the gRNA plasmid library.

[0030] 3. Construction and Screening of Mutant Library 1 μg of the plasmid from the gRNA library was electroporated into the GS115-nCas9-Luc competent cell using YPDS Z + 1632 single clones were obtained by plate screening. All single clones were first cultured in MGY medium for 24 h, then switched to MMY medium for induction for 24 h, and 0.5% methanol was added every 24 h for a total of 72 h.

[0031] Add 10 μL of induced supernatant and 90 μL of reaction system with substrate luciferin to a 96-well plate, pipette evenly and place in the Glomax instrument, set the integration time to 4 s, and detect the chemiluminescence intensity. The luciferase reaction system was cited from the literature (doi: 10.7546 / ijba.2019.23.3.000720) and simplified, see Table 1 for details.

[0032] Table 1. Luciferase reaction system

[0033] The 1632 induced supernatants were screened by chemiluminescence with the luciferase reaction system. Using the wild-type strain as the control, a total of 9 single clones with chemiluminescence values ​​1.2 times or more of the control group were obtained in the preliminary screening (see Figure 3 ), among which the chemiluminescence values ​​of the two mutant strains LE30002-H10 and LF23001-F7 were 1.2-1.5 times that of the wild-type luciferase, the chemiluminescence values ​​of the four mutant strains LE30001-C12, LF23001-D11, LF23002-C12 and LF23002-D11 were 1.5-2.0 times that of the wild-type luciferase, and the chemiluminescence values ​​of the three mutant strains LF23001-C11, LF23001-C12 and LF23002-G11 were more than 2.0 times that of the wild-type luciferase.

[0034] The above 9 monoclones were sequenced, and the results showed that the 9 monoclones were 4 mutants, among which LE30001-C12 was a mutant named Luc-1, LE30002-H10 was a mutant named Luc-2, LF23001-C11 and LF23001-C12 were the same mutant named Luc-3, and the remaining five mutants LF23001-D11, LF23001-F7, LF23002-C12, LF23002-D11 and LF23002-G11 were the same mutant named Luc-4.

[0035] The four mutants were re-incubated and the luminescence intensity of the luciferase reaction system catalyzed by their induced supernatants was detected. The results showed that the enzyme activity of the Luc-3 mutant was significantly enhanced, about 2.5 times that of the wild-type luciferase; the enzyme activities of the other three mutants were also enhanced, among which the Luc-1 mutant was about 1.3 times that of the wild-type luciferase, the Luc-2 mutant was about 1.4 times that of the wild-type luciferase, and the Luc-4 mutant was about 1.6 times that of the wild-type luciferase (see Figure 4 ). Sequencing results showed that the 1142nd base of the Luc-3 mutant mutated from cytosine (C) to thymine (T) (see Figure 5), causing the 381st amino acid to mutate from threonine (T) to isoleucine (I), and the corresponding gRNA is gRNA21 with a sequence as shown in SEQ ID NO.23.

[0036] The luciferase encoding gene of the Luc-3 mutant was sequenced. The nucleic acid sequence of the Luc-3 mutant is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1. Example 2 Activity evaluation of luciferase mutants To verify the stability of luciferase secreted by Luc-3 monoclonal, three biological replicates were performed, including bacterial culture, induction and enzyme activity detection.

[0037] Take 10 μL of Luc-3 and wild-type luciferase yeast culture medium respectively and add them to 1 mL of MGY medium. Culture them at 30℃ and 800 rpm for 24 h. Remove the medium by centrifugation and replace it with MMY medium for induction for 24 h. Add 0.5% methanol every 24 h for a total of 2 times. Induce expression for 72 h and take the supernatant by centrifugation. At this time, the secreted luciferase is still in the supernatant.

[0038] Add 10 μL of induction supernatant and 90 μL of luciferase reaction system to a 96-well plate, pipet evenly and place in a Glomax instrument, set the integration time to 4 s, and detect the chemiluminescence intensity.

[0039] The statistical results of three biological replicates showed that the enzyme activity of the Luc-3 mutant was significantly enhanced, about 3 times that of the control group (see Figure 6 ), which is consistent with the screening results. The specific RLU values ​​of the three biological replicates were 12093, 11760 and 11050 for the wild-type luciferase, and 34000, 34333 and 35844 for the Luc-3 mutant. The results show that the Luc-3 mutant has high stability, good reproducibility and stable enzyme activity. Example 3 Application of luciferase mutants in detection In order to explore the possibility of using luciferase mutant (Luc-3) in detection, we purchased a domestic luciferase detection kit for comparison. The purchased kit was used as the control group, and the luciferase in the Luc-3 replacement kit was used as the experimental group. The results showed that the fluorescence value of Luc-3 was about 1.5 times that of the control group (see Figure 7 ), indicating that Luc-3 has the potential to be developed into a test kit. The specific operation is as follows: Take 10 μL of Luc-3 glycerol bacteria and transfer them to 1 mL of MGY medium. Use a shaker at 30℃ 800 rpm for 24h, centrifuge at 2000 Íg for 5 min to remove the supernatant, resuspend with 1 mL of MMY medium, use a shaker at 28℃ 800 rpm for 72h, and add 5 μL of methanol every 24h. After the induction, centrifuge at 2000 Íg for 5 min to take the supernatant and place it at 4℃ for later use. Take 90 μL of commercial reagent without adding the original luciferase, add 10 μL of induction supernatant containing 0.48 μg Luc-3; add 0.52 μg of the original luciferase to 90 μL of commercial reagent, add 10 μL of ultrapure water, add 10 μL of 1 μM ATP respectively, mix in a black 96-well plate, and immediately use a TECAN multifunctional microplate reader to detect the chemiluminescence value and record the data.

Claims

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

1.

2. A polynucleotide encoding the luciferase mutant according to claim 1.

3. The polynucleotide according to claim 2, characterized in that The sequence of the polynucleotide is shown as SEQ ID NO.

2.

4. An expression vector containing the polynucleotide according to claim 2 or 3.

5. A host cell containing the expression vector according to claim 4.

6. A method for screening luciferase mutants, characterized in that: The method comprises the following steps: (1) The first vector containing the nCas9 encoding gene and the cytosine deaminase encoding gene is transferred into yeast, and the nCas9 encoding gene and the cytosine deaminase encoding gene are integrated into the GS115 genome by homologous recombination to obtain a recombinant yeast strain containing the nCas9 encoding gene and the cytosine deaminase encoding gene in the genome; (2) transferring the second vector containing the luciferase encoding gene into the recombinant yeast obtained in step (1), integrating the luciferase encoding gene into the genome of the recombinant strain by homologous recombination, and screening the recombinant yeast strain having a single copy of the luciferase encoding gene, wherein the second vector carries an inducible promoter element; (3) transferring the gRNA plasmid library targeting the luciferase encoding gene into the recombinant yeast strain obtained in step (2) to mutate the luciferase encoding gene, wherein the gRNA plasmid library contains 51 sequences targeting different segments of the luciferase encoding gene, as shown in SEQ ID NOs. 3-53, and the gRNA is constructed on a third vector; (4) inducing the yeast strain obtained in step (3) to express the luciferase gene; (5) Screening luciferase mutants based on the enzyme activity displayed by the luciferase expression product described in step (4).

7. The method according to claim 6, characterized in that The yeast strain is GS115.

8. Use of the luciferase mutant according to claim 1 in preparing a luciferase detection kit.

9. Use of the luciferase mutant according to claim 1 as a reporter protein.

10. Use of the polynucleotide according to claim 2 or 3 as a luciferase reporter gene.

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