Chrysanthemum beta-elemene synthase gene McTPS5 and application thereof

Abstract

The application discloses a chamomile beta-elemene synthesis enzyme gene McTPS5 and application thereof. The beta-elemene synthesis enzyme gene McTPS5 is cloned from chamomile, the full-length cDNA sequence of the gene is shown as SEQ ID NO:1, the coding sequence is shown as SEQ ID NO:2, and the amino acid sequence coded by the gene is shown as SEQ ID NO:3. The exogenous recombinant protein of the McTPS5 gene is prepared, and after reaction in a catalytic substrate, the sesquiterpene beta-elemene can be generated, and the beta-elemene can be prepared. Meanwhile, the McTPS5 is transferred into a plant expression vector, and by exogenous transformation of plant materials, the beta-elemene plant or the transgenic material containing the sesquiterpene beta-elemene synthesis enzyme gene can be cultivated.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering. More specifically, it relates to a chamomile β-elemene synthase gene McTPS5 and its application. Background Art

[0002] Chamomile (Matricaria chamomilla L.) is an annual herbaceous plant of the genus Matricaria in the Asteraceae family. It is native to the UK and is now distributed in Europe, northern and western Asia, and is widely cultivated in the western and northern regions of China. Chamomile not only has high ornamental value but is also an important aromatic medicinal plant, with anti-inflammatory, antibacterial, anti-allergic and other effects; among them, terpenoid compounds are the main active ingredients. Terpenoids are the most important volatile compounds in plants. So far, more than 25,000 terpenoid compounds have been detected in plants. According to the C5 isoprene units, terpenoids are divided into hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40), etc.

[0003] Terpenoid synthase genes (TPS) are one of the key structural genes in the terpenoid synthesis pathway. They can catalyze different substrates GPP, FPP and GGPP to form corresponding monoterpenes, sesquiterpenes and diterpenes, etc. Among them, β-elemene is an important sesquiterpenoid compound and also an important anti-cancer drug, with curative effects such as inhibiting the proliferation of tumor cells and inducing apoptosis of tumor cells. β-elemene is usually isolated and extracted from the traditional Chinese medicine Curcuma wenyujin (Curcuma phaeocaulis Valeton) of the Zingiberaceae family. However, due to the long cultivation cycle of this plant, large environmental impact, and the presence of multiple isomers in the extract, the stable supply of β-elemene is severely restricted; and there are few research reports on β-elemene from other plant sources in existing studies. Therefore, it is urgent to develop more methods for efficiently producing β-elemene from different plant sources.

[0004] Currently, domestic research on chamomile terpenoid synthases mainly focuses on chamomile sesquiterpene synthase genes. For example, the chamomile McGDS1 gene can catalyze FPP to generate farnesene; McGDS3 can catalyze FPP to generate germacrene A, and McGDS2 reacts with FPP to generate germacrene D (Ling et al., 2020), and McBBS reacts with FPP to generate α-bisabolol (Guo Chunxiao, 2018), etc. However, there is no research on chamomile in terms of β-elemene synthesis. Therefore, in order to more comprehensively elaborate on the formation and regulation mechanism of chamomile β-elemene components, and at the same time provide a new bioengineering method for the preparation of β-elemene, it is necessary to study the formation mechanism of chamomile β-elemene components. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings in the research on β-elemene, a sesquiterpene in chamomile, and to provide a monofunctional enzyme gene McTPS5 that controls the sesquiterpene component β-elemene in chamomile and its application.

[0006] The first objective of this invention is to provide a chamomile β-elemene synthase gene, McTPS5.

[0007] A second objective of this invention is to provide a chamomile β-elemene synthase, McTPS5.

[0008] A third objective of this invention is to provide the application of the chamomile β-elemene synthase gene McTPS5 and / or chamomile β-elemene synthase McTPS5.

[0009] A fourth objective of this invention is to provide a recombinant vector, a recombinant bacterium containing the recombinant vector, and a cell line containing the recombinant bacterium.

[0010] The fifth object of the present invention is to provide a method for preparing β-elemene.

[0011] The sixth objective of this invention is to provide a method for constructing transgenic materials containing β-elemene.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] This invention cloned a 1704 bp chamomile β-elemene synthase gene, McTPS5, from the tubular flowers of chamomile. The full-length cDNA sequence of the McTPS5 gene is shown in SEQ ID NO:1. The coding region (CDS) of the McTPS5 gene is 1680 bp, and its nucleotide sequence is shown in SEQ ID NO:2. It is presumed to encode 559 amino acids, and its amino acid sequence is shown in SEQ ID NO:3. The presumed protein molecular weight is 64.52 kDa, and the isoelectric point (pI) is 4.80. The gene sequence contains a conserved DDXXD sequence.

[0014] This invention shows that the expression level of the chamomile gene McTPS5 is high in the tubular flowers of chamomile, and is positively correlated with the expression level of β-elemene in the tubular flowers. Furthermore, the McTPS5 gene encodes an enzyme that catalyzes the formation of the sesquiterpene β-elemene from ferruginous polyphenols (FPP). The chamomile β-elemene synthase gene McTPS5 can be used to prepare β-elemene through microbial metabolic engineering, which can then be used to prepare plant essential oils or pharmaceuticals. Simultaneously, ligating McTPS5 into a plant transformation vector and then introducing it into chamomile or other plants can yield transgenic plants expressing the McTPS5 gene, which is helpful for cultivating β-elemene plants or constructing transgenic materials containing the sesquiterpene β-elemene synthase gene.

[0015] Based on the McTPS5 gene sequence information provided by the present invention, those skilled in the art can easily obtain a gene equivalent to McTPS5 by the following methods: (1) obtaining it through database retrieval; (2) obtaining it by screening genomic libraries or cDNA libraries of chamomile or other plants using the McTPS5 gene fragment as a probe; (3) obtaining it from the genome, mRNA and cDNA of chamomile or other plants by designing oligonucleotide primers based on the McTPS5 gene sequence information and using PCR amplification; (4) obtaining it by modifying it using genetic engineering methods based on the McTPS5 gene sequence; (5) obtaining the gene by chemical synthesis.

[0016] Therefore, this invention provides the application of the chamomile β-elemene synthase gene McTPS5 or the chamomile β-elemene synthase McTPS5 in the preparation of β-elemene, in the breeding of β-elemene plants, or in the construction of transgenic materials containing β-elemene.

[0017] Specifically, this invention provides the application of the chamomile β-elemene synthase gene McTPS5 or the chamomile β-elemene synthase McTPS5 in the breeding of β-elemene plants. First, in the selection of β-elemene plants, the presence of the gene McTPS5 or the β-elemene synthase McTPS5 in the plant is detected to identify whether the plant contains β-elemene. Second, in the cultivation of β-elemene-containing plants, the expression of the McTPS5 gene in the plant is promoted to increase the β-elemene content in the plant.

[0018] This invention provides a recombinant vector containing the chamomile β-elemene synthase gene McTPS5.

[0019] The present invention provides a recombinant bacterium containing the recombinant vector.

[0020] The present invention provides a cell line comprising the recombinant bacteria.

[0021] This invention provides a method for preparing β-elemene, using farnesyl pyrophosphate (FPP) as a substrate and employing chamomile sesquiterpene synthase McTPS5 or its recombinant protein for catalysis to prepare β-elemene.

[0022] This invention also provides a method for constructing transgenic materials containing β-elemene, wherein the chamomile β-elemene synthase gene McTPS5 is transferred into a plant expression vector, and transgenic materials containing β-elemene are obtained by exogenously transforming plant materials.

[0023] The present invention has the following beneficial effects:

[0024] This invention provides a chamomile β-elemene synthase gene, McTPS5, which exhibits high expression levels in the tubular flowers of chamomile and is positively correlated with the expression level of β-elemene in the tubular flowers. This gene can catalyze the formation of the sesquiterpene compound β-elemene from ferruginous plant propagation (FPP). By promoting the expression of McTPS5 in plants, the content of terpenoid components can be increased, which can be used for β-elemene breeding. Furthermore, β-elemene can be prepared in vitro using a recombinant protein containing the β-elemene synthase McTPS5 gene, which can be further used for the development and preparation of subsequent products, such as essential oils, fragrances, and pharmaceuticals. Simultaneously, by constructing the McTPS5 gene fragment in a plant expression vector and exogenously transforming other plant materials, transgenic materials containing the β-elemene synthase gene can be obtained, providing an effective method for cultivating medicinal plants.

[0025] In addition, the present invention can further provide or apply transgenic plants with medicinal value obtained using McTPS5 gene fragments and corresponding seeds, as well as plants transformed by overexpressing the McTPS5 gene or recombinants based on the gene, or seeds obtained from such plants for β-elemene plant breeding, and can also transfer the gene of the present invention into other plants by sexual hybridization. Attached Figure Description

[0026] Figure 1 This invention relates to the cloning of the McTPS5 gene and the analysis of the homology of the McTPS5 amino acid sequence (A: agarose gel electrophoresis of the McTPS5 clone; B: analysis of the homology of the McTPS5 amino acid sequence).

[0027] Figure 2 This is a diagram showing the prokaryotic expression of the McTPS5 gene in this invention (K is the supernatant of the empty vector protein, C is the precipitate after bacterial lysis of the recombinant protein, S is the supernatant of bacterial lysis of the recombinant protein, F is the flow-through solution of the recombinant protein, W is the washing solution of the recombinant protein, E is the elution solution of the recombinant protein, and M is the marker).

[0028] Figure 3 This invention relates to the in vitro enzyme-catalyzed reaction of the McTPS5 gene recombinant protein (in vitro catalysis of FPP to generate β-elemene). Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] Example 1: Obtaining the full-length cDNA of the McTPS5 gene

[0032] 1. Extraction of total RNA from chamomile tubular flowers:

[0033] Fresh chamomile tubular flowers were used as the material for RNA extraction. The pipette tips and Eppendorf tubes used for RNA extraction were sterilized at 121°C for 25 min, repeated twice. Glassware and mortars were wrapped in aluminum foil and subjected to dry heat treatment at 180°C for 3 h, then cooled for later use. Freshly purchased chamomile cut flowers were propagated in water. Approximately 500 mg of fresh chamomile tubular flowers were weighed and rapidly ground into powder in liquid nitrogen. Total RNA was then extracted from the chamomile tubular flowers using a plant RNA extraction kit (Magen). RNA integrity was assessed by 1% agarose gel electrophoresis, and the concentration and purity of total RNA were determined using a micro-spectrophotometer. The extracted RNA was stored at -80°C for later use.

[0034] 2. Synthesis of the first strand of cDNA:

[0035] Single-stranded cDNA was synthesized using total RNA from chamomile tubular flowers as a template and Evo M-MLV reverse transcriptase from Aikerui. In a microcentrifuge tube, 1000 ng Total RNA, 1 μL Oligo d(T)18 Primers, and 1 μL dNTPs (10 mM each) were added, and RNase-free H2O was added to a final volume of 10 μL. The mixture was gently rubbed, centrifuged for a few seconds, and then incubated at 70°C for 10 min, followed immediately by an ice bath for 2 min. Then, 4 μL 5×M-MLV buffer, 0.5 μL RNase Inhitor (40 U / μL), and 1 μL M-MLV reverse transcriptase were added, and RNase-free H2O was added to a final volume of 20 μL. The mixture was centrifuged, incubated at 42°C for 60 min, then incubated at 70°C for 15 min to inactivate the reverse transcriptase. The mixture was then cooled on ice for 2 min and stored at -20°C for later use.

[0036] 3. Full-length cDNA cloning:

[0037] First, primers were designed based on the annotated gene sequences in the chamomile transcriptome database. The upstream primer F1 was 5'-ATGGCAGCGGTTCAAGCTACTA-3' (as shown in SEQ ID NO:4). The downstream primer R1 was 5'-TTACACGGGTAGAGAATCCACAAAC-3' (as shown in SEQ ID NO:5). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Using the synthesized first-strand cDNA as a template, PCR amplification was performed using Phanta high-fidelity enzyme. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 90 s, 35 cycles, and a final extension at 72℃ for 10 min. After the PCR reaction, the presence of the target fragment band in the PCR product was preliminarily detected by 1.0% agarose gel electrophoresis. Then, the gel containing the target fragment was cut out under UV light using a scalpel and recovered using a DNA gel recovery kit (SanPrep Column DNA Gel Recovery Kit, Sangon Biotech). The recovery method was performed according to the kit's instructions. The recovered product was then analyzed by 1% agarose gel electrophoresis to determine its recovery efficiency and approximate concentration. The recovered product was stored at -20°C for later use.

[0038] 4. Construction of recombinant plasmids:

[0039] Based on the size and concentration of the recovered target fragment, an appropriate amount of the purified product was ligated to the cloning vector. The TaKaRa pMD19-T vector was used, and the molar ratio of target DNA to cloning vector was controlled at approximately 3:1. Specific procedures were performed according to the manufacturer's instructions. Ligation was carried out at 16°C for 3–6 hours. The competent DH5α (TaKaRa) cells were removed from the -80°C freezer beforehand and placed in an ice box to thaw naturally. The ligated recombinant vector was added to the competent cells, and transformation was performed on ice for 30 minutes. A heat shock at 42°C for 90 seconds was then performed, followed by immediate ice placement for 2–5 minutes. Then, 1 mL of LB liquid medium was added, mixed well, and cultured at 37°C with shaking at 180 rpm for 1 hour. 30 μL of X-gal (20 mg / mL) and 30 μL of IPTG (20 mg / mL) were spread on the surface of an LB solid medium plate containing 100 μg / mL ampicillin. An appropriate amount of transformation buffer was then spread on top. After complete absorption of the transformation buffer, the plate was inverted and cultured overnight at 37°C. The results were observed after approximately 16 hours.

[0040] Subsequently, white colonies were screened using X-gal / IPTG blue-white screening, and recombinant plasmids were preliminarily identified. The plates were stored at 4°C. After preliminary screening using blue-white screening, single white colonies were picked from LB agar plates using sterile pipette tips and inoculated into LB liquid medium containing 100 μg / mL ampicillin. The culture was then incubated at 37°C and 180 rpm for 3–6 h in a temperature-controlled shaking incubator. PCR was then performed on the bacterial culture using universal primers M13-47 and M13-48 for the pMD19-T vector, following the instructions. Finally, the PCR products were detected by 1% agarose gel electrophoresis.

[0041] The agarose gel electrophoresis results of the cloned gene are as follows: Figure 1 As shown in Figure A, the result showed a single band of the predicted size. After ligation into the pMD19-T vector, bacterial culture containing the target fragment was selected for DNA sequencing. The sequencing work was performed by Guangzhou Aiji Biotechnology Co., Ltd. The obtained sequence was compared with the original genome sequence information. The comparison results showed that this invention cloned a gene with a full length of 1704 bp from the tubular flowers of chamomile, named the McTPS5 gene. The nucleotide sequence of this gene is shown in SEQ ID NO:1; the coding region (CDS) is 1680 bp, and its nucleotide sequence is shown in SEQ ID NO:2; based on the cDNA sequence, it is inferred to encode 559 amino acids, and its amino acid sequence is shown in SEQ ID NO:3. The inferred protein molecular weight is 64.52 kDa, and the isoelectric point (pI) is 4.80. Amino acid sequence alignment and homology analysis were performed, as shown in... Figure 1 As shown in B, the McTPS5 gene sequence contains a conserved DDXXD sequence. Furthermore, comparison and homology analysis were performed at NCBI, and it was preliminarily identified as a member of the TPS gene family.

[0042] Further analysis of the McTPS5 gene expression showed that the McTPS5 gene was highly expressed in the tubular flowers of chamomile and was positively correlated with the expression level of β-elemene in the tubular flowers. Promoting the expression of the McTPS5 gene in plants can increase the content of the terpene compound β-elemene.

[0043] Example 2: Induction and purification of McTPS5 protein

[0044] 1. Construction of prokaryotic expression vectors:

[0045] The pET-32a prokaryotic expression vector was used to induce recombinant protein expression. Based on the coding region of the obtained McTPS5 gene (SEQ ID NO:2) and the restriction enzyme sites contained in the pET-32a prokaryotic expression vector, PCR amplification was performed using homologous recombination primers containing BamHI and HindIII restriction sites: F: 5'-gccatggctgatatcggatccATGGCAGCGGTTCAAGCT-3' (as shown in SEQ ID NO:6); R: 5'-ctcgagtgcggccgcaagcttCACGGGTAGAGAATCCAC-3' (as shown in SEQ ID NO:7). The pET-32a prokaryotic expression vector was double-digested with BamHI and HindIII restriction enzymes at 37°C for 3 h, followed by restriction enzyme inactivation at 65°C for 15 min. After 1% agarose gel electrophoresis, the product was purified using a purification kit (SanPrep column PCR product kit, Sangon Biotech) and stored at -20°C for later use. II. Homologous recombination of the gene fragment and the vector was performed, adjusting the vector dosage to 0.03 pmol and the insert dosage to 0.06 pmol, following the instructions. The ligation product was transformed into *E. coli* DH5α competent cells, and the recombinant prokaryotic expression vector was obtained after identification by bacterial PCR and sequencing.

[0046] 2. Recombinant protein induction:

[0047] Rosetta(DE3) competent cells were transformed with the identified recombinant plasmid. Single colonies were picked and inoculated into 5 mL of fresh LB broth (containing 100 mg / L Amp) and cultured overnight at 37°C and 180 rpm. 1000 μL of the seed culture was then transferred to 100 mL of fresh LB broth (containing 100 mg / L Amp) and cultured at 37°C and 180 rpm until OD500. 600The pH value was 0.4-0.6. 10 μL of 1M IPTG was added, and the cells were induced at 16℃ and 100 rpm for 20-24 h. A control group without IPTG induction was also included. Cells were collected by centrifugation at 4℃ and 5000 rpm. Cells were resuspended in 5 mL of lysis buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole) and lysed on ice using an ultrasonic cell disruptor. The cells were centrifuged at 12000 rpm and 4℃ for 20 min. The supernatant was transferred to a new centrifuge tube, the precipitate was washed once with double-distilled water, and then resuspended in 5 mL of lysis buffer. 16 μL of both supernatant and precipitate were taken for SDS-PAGE electrophoresis analysis. A 12.5% ​​SDS-PAGE gel was prepared and loaded sequentially. Electrophoresis was performed on the stacking and separating gels at 80 V and 130 V, respectively. After electrophoresis, the sample was stained with Coomassie Brilliant Blue for 40 minutes, then destained with destaining solution for 24 hours. The experimental results were observed and recorded.

[0048] 3. Purification of recombinant proteins:

[0049] Pack 0.5 mL of Ni-NTA resin into the chromatography column. After the resin has precipitated, drain the internal liquid, add 5 mL of ddH2O, and wash three times. Then add 5 mL of Wash buffer (300 mM NaCl, 50 mM NaH2PO4, 20 mM imidazole), and wash three times. Pre-chill on ice. Add 5 mL of cell lysis supernatant to the pre-chilled chromatography column, mix thoroughly, and incubate at 4°C on a low-speed shaker for 1 h. Flow through the column on ice, collect the liquid in a centrifuge tube, and label it. Take 16 μL of the flow-through for SDS-PAGE analysis. Add 5 mL of Wash buffer, wash the chromatography column three times, and collect 16 μL of each wash buffer (W1, W2, W3) for SDS-PAGE analysis. Then, elute four times with 1 mL of Elution buffer (300 mM NaCl, 50 mM NaH2PO4, 250 mM imidazole), collecting 16 μL of each eluent fraction for SDS-PAGE analysis. Add the eluted protein to an ultrafiltration tube and centrifuge at 5000 rpm for 10 min at 4 °C. Then add 2 mL of Reaction buffer (100 mM HEPES, 200 mM KCl, 200 mM MgCl2, 10% glycerol; 10 mM DTT), centrifuge at 5000 rpm for 10 min at 4 °C, repeating three times. Transfer the ultrafiltered solution to pre-chilled centrifuge tubes, add an equal volume of pure glycerol, mix thoroughly, and aliquot into 200 μL tubes. Perform SDS-PAGE analysis on 16 μL of each aliquot, and store the remainder at -80 °C for later use.

[0050] The prokaryotic expression results of the McTPS5 gene are as follows: Figure 2As shown, the McTPS5 recombinant protein can be expressed in the supernatant after IPTG induction, with a size of approximately 65 kDa, which is close to the expected size and can be used for subsequent experiments.

[0051] Example 3: In vitro enzymatic reaction of McTPS5 protein

[0052] Add 20 μL each of 300 mM HEPES (pH 7.5), 50 mM DTT and 25 mM MgCl2, 20 μL of the protein extract prepared in Example 2, 1 μL of farnesyl pyrophosphate (FPP), and 119 μL of ddH2O to a final volume of 200 μL. Seal the sample vial and react at 28 °C for 1 h. Insert a 75 μm polydimethyloxane (PMDS) extraction fiber into the glass vial and perform headspace solid-phase microextraction for 1 h. After the reaction, place the extraction fiber into a high-performance gas chromatograph-mass spectrometer for analysis.

[0053] Gas chromatography conditions were as follows: HP-1NNOWAX column (30m × 0.25mm); high-purity helium as carrier gas, split ratio 20:1, column inlet pressure 50Pa, flow rate 1mL / min; sampling time 2min; temperature program: initial column temperature 45℃, hold for 2min, increase to 80℃ at 5℃ / min, hold for 1min, then increase to 250℃ at 10℃ / min, hold for 5min. Mass spectrometry conditions were as follows: GC-MS interface temperature 220℃, electron impact source EI, 350V; ion source temperature 170℃; electron energy 70eV; scan mass range 35–335 aum. The acquired mass spectra were analyzed using the WILLEY / MAINLIB library.

[0054] In vitro enzyme activity identification results as follows Figure 3 As shown, when FPP is used as a substrate, the product of the in vitro enzymatic reaction catalyzed by pET-32a-McTPS5 is identified by mass spectrometry as the sesquiterpene β-elemene. This indicates that the enzyme encoded by the McTPS5 gene is a functional enzyme gene that can catalyze the production of the sesquiterpene β-elemene from FPP, and can be used for the in vitro preparation of β-elemene.

[0055] In summary, this invention provides a novel β-elemene synthase gene, McTPS5, which exhibits high expression levels in the tubular flowers of chamomile and is positively correlated with the expression level of β-elemene in the tubular flowers. Furthermore, it can catalyze the formation of sesquiterpenoid compounds β-elemene by FPP (fiber-dependent polyphenol oxidase). By promoting the expression of McTPS5 in plants, the content of terpenoid components in plants can be increased, which can be used for β-elemene plant breeding. Additionally, β-elemene can be prepared in vitro using recombinant proteins containing the β-elemene synthase McTPS5, and further used for the development and preparation of subsequent products, such as essential oils, fragrances, and pharmaceuticals. Simultaneously, this invention provides an effective method for cultivating medicinal plants. By transferring this gene into plants through plant vectors, it is beneficial for cultivating β-elemene plants or constructing transgenic materials containing the sesquiterpenoid β-elemene synthase gene. In addition, the present invention can further provide or apply transgenic plants with medicinal value obtained using McTPS5 gene fragments and corresponding seeds, as well as plants transformed by overexpressing the McTPS5 gene or recombinants based on the gene, or seeds obtained from such plants for β-elemene plant breeding, and can also transfer the gene of the present invention into other plants by sexual hybridization.

[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A gene for synthase of chamomile β-elemene McTPS5 Or the application of chamomile β-elemene synthase McTPS5 in the preparation of β-elemene, characterized in that, The full-length cDNA sequence of the gene is shown in SEQ ID NO:1, and the gene coding sequence is shown in SEQ ID NO:2; the amino acid sequence of the synthase is shown in SEQ ID NO:

3.

2. A gene for synthase of chamomile β-elemene McTPS5 The application of chamomile β-elemene synthase McTPS5 in the construction of transgenic materials containing β-elemene, characterized in that... The full-length cDNA sequence of the gene is shown in SEQ ID NO:1, and the gene coding sequence is shown in SEQ ID NO:2; the amino acid sequence of the synthase is shown in SEQ ID NO:

3.

3. A method for preparing β-elemene, characterized in that, β-elemene was prepared by using farnesyl pyrophosphate (FPP) as a substrate and chamomile sesquiterpene synthase McTPS5 or its recombinant protein as a catalyst; the amino acid sequence of the synthase is shown in SEQ ID NO:3.

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