Machilus rubescens TPS synthetase gene MnTPS7 and application thereof
By cloning the MnTPS7 TPS synthase gene of *Lysimachia nummularia*, we achieved efficient biosynthesis of squisabinene and linalool, solving the problems of low synthesis efficiency and environmental pollution in existing technologies, and realizing large-scale production and controllable synthesis.
Patent Information
- Application Number
- CN202610031731.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-12
- Publication Date
- 2026-03-17
AI Technical Summary
Existing technologies struggle to efficiently synthesize high-value terpenoid compounds such as Sesquisabinene and linalool. Natural extraction is inefficient, chemical synthesis causes significant pollution, and existing genetic engineering methods require the construction of two independent expression systems, increasing production complexity and cost.
The MnTPS7 TPS synthase gene was cloned, its bifunctional characteristics were clarified, and the biosynthesis of Sesquisabinene and linalool was achieved through a heterologous expression system. The catalytic properties of the MnTPS7 gene under different substrate conditions were utilized to achieve the efficient synthesis of the target products.
This method enables large-scale, standardized production of sesquisabinene and linalool, avoiding the environmental pollution and unstable yield problems of traditional methods. It provides controllability and efficiency in the synthesis of terpenoid compounds and has good industrial prospects.
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Figure CN121674436A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to, but is not limited to, the field of plant genetic engineering technology, and particularly relates to a TPS synthase gene of *Machilus yunnanensis*. MnTPS7 And its applications. Background Technology
[0002] Sesquisabinene is a natural compound with a unique tricyclic sesquiterpene structure, possessing excellent antibacterial, anti-inflammatory, and antioxidant activities, and has significant application value in pharmaceutical preparations, natural preservatives, and high-end fragrances. Linalool is a monoterpene alcohol widely found in plants, with a refreshing floral scent and antibacterial, sedative, and mosquito-repellent effects, and is widely used in fragrances, cosmetics, pharmaceuticals, and agricultural insecticides. Currently, the acquisition of sesquisabinene and linalool mainly relies on natural plant extraction or chemical synthesis. Natural extraction methods have many limitations: on the one hand, the content of these two compounds in natural plants is extremely low; on the other hand, due to factors such as long plant growth cycles, poor environmental adaptability, and limited resource reserves, it is difficult to achieve large-scale and stable production. Although chemical synthesis can increase yield, the reaction conditions are harsh (high temperature, high pressure, strong acids and alkalis), easily producing toxic and harmful byproducts, polluting the environment, and the purity of the synthesized products is low, making it difficult to meet the high-quality requirements of pharmaceuticals, high-end fragrances, and other fields.
[0003] Terpene synthases (TPSs) are key rate-limiting enzymes in the biosynthetic pathway of terpenoids, and their function determines the types and yields of terpenoid products. Based on the type of catalyzed product, TPSs can be classified into monoterpene synthases (catalyzing GPP to produce monoterpenes) and sesquiterpene synthases (catalyzing FPP to produce sesquiterpenes), etc. In recent years, with the development of genetic engineering technology, cloning TPS genes with defined functions and constructing heterologous expression systems has become a core strategy for achieving green and efficient synthesis of terpenoids. In existing technologies, single-function Sesquisabinene or linalool synthase genes have been cloned from a few plants, but single-function genes can only synthesize one target compound. If two high-value terpenoids need to be obtained simultaneously, two independent expression systems need to be constructed, increasing the complexity and cost of the production process. A bifunctional TPS gene from *Machilus yunnanensis*, possessing both Sesquisabinene and linalool synthesis functions, has not yet been reported. As a traditional and precious timber species in my country, *Machilus chinensis* possesses a unique terpene metabolic pathway. Discovering a bifunctional TPS gene from it is crucial for overcoming the bottlenecks in the large-scale production of these two high-value terpene compounds. Therefore, cloning the bifunctional synthase gene from *Machilus chinensis*, clarifying its function, and developing its applications have significant theoretical value and industrial prospects. Summary of the Invention
[0004] To address the problems existing in the prior art, this invention provides a Runan TPS synthase gene. MnTPS7 And its applications.
[0005] This invention is achieved as follows: a Sesquisabinene / linalool synthase gene. MnTPS7 The Runnan Sesquisabinene / linalool synthase gene MnTPS7 The nucleotide sequence is shown in SEQ ID NO.1:
[0006] Another objective of this invention is to provide a Sesquisabinene / linalool synthase gene. MnTPS7 The expressed protein is MnTPS7, and the amino acid sequence of the protein is shown in SEQ ID NO.2: .
[0007] Another objective of this invention is to provide a Sesquisabinene / linalool synthase gene. MnTPS7 A method for preparing the expressed protein MnTPS7, comprising: based on the Runnan Sesquisabinene / linalool synthase gene. MnTPS7 A prokaryotic expression vector was constructed, and the resulting expression vector was transformed into a host bacterium. The host bacterium was then cultured to express the MnTPS7 protein.
[0008] Furthermore, the primer pair sequences designed during the construction of the prokaryotic expression vector for this gene are shown in SEQ ID NO.9 and SEQ ID NO.10, and the plasmid vector used is pET-32a; SEQ ID NO.9 GCCATGGCTGATATCGGATCCATGGCATCTGACGGAAGCATG SEQ ID NO.10 CTCGAGTGCGGCCGCAAGCTTTCCCAAGTTGATGGATTCCTTCAGTG.
[0009] Another objective of this invention is to provide a Sesquisabinene / linalool synthase gene. MnTPS7 The application of the expressed protein in the preparation of Sesquisabinene / linalool.
[0010] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: From the perspective of biosynthetic regulation mechanisms, this invention, for the first time, clearly identifies and provides a Sesquisabinene / linalool synthase gene, MnTPS7, derived from *Machilus chinensis*. This gene occupies a key node in the sesquiterpene metabolic pathway and can directly determine the direction and efficiency of Sesquisabinene production. Compared with existing methods that rely on low-abundance accumulation in natural plants or indirect synthesis through complex mixed enzyme systems, the acquisition and functional confirmation of this gene transforms the biosynthesis of Sesquisabinene / linalool from an uncontrollable natural metabolic process into a molecularly regulated process, providing a reliable foundation for the precise regulation of the synthesis of target terpenoid compounds through genetic engineering.
[0011] From the perspective of enzymatic function and catalytic properties, this invention systematically reveals the reaction characteristics of the MnTPS7-encoded protein under different substrate conditions by verifying its in vitro catalytic activity. Experimental results show that when farnesyl pyrophosphate (FPP) is added alone as a substrate, the catalytic product is predominantly Sesquisabinene, while when geranyl pyrophosphate (GPP) is added alone as a substrate, the catalytic product is predominantly Linalool.
[0012] From the perspective of industrial value at the application level, the invention provides... MnTPS7The gene and its expressed protein can be directly applied to the biosynthetic preparation system of Sesquisabinene / Linalool, avoiding the problems of high raw material consumption, long cycle, strong environmental dependence, and unstable yield in traditional plant extraction processes. Functional proteins with stable catalytic activity can be obtained through heterologous expression systems, providing core enzyme elements for constructing efficient Sesquisabinene / Linalool synthetic pathways in microbial or cell factories. This not only facilitates the large-scale, standardized production of Sesquisabinene / Linalool but also has promising prospects for application in the fields of fragrances, fine chemicals, and related functional materials.
[0013] From the perspective of the integrity and scalability of the technical system, this invention revolves around MnTPS7 A complete technology chain has been constructed, from gene acquisition, protein expression, functional verification to synthetic applications. The technical path is clear, the implementation conditions are well-defined, and it is easy for those skilled in the art to directly adopt or further optimize it. At the same time, due to the core position of this gene and protein in the terpene metabolic network, its application is not limited to the preparation of a single product. It can also be used as a regulatory node to expand the biosynthetic research of other related terpene compounds, and has good extension potential and continuous innovation value.
[0014] In summary, this invention provides the Sesquisabinene / Linalool synthase gene from Linalool. MnTPS7 It and its functional proteins have solved the problems of low synthesis efficiency and limited regulation methods of Sesquisabinene / Linalool at the molecular level. In terms of technical effect, it has achieved high efficiency, stability and controllability in the synthesis of target products, with significant technical advantages and positive practical application effects.
[0015] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: The gene of this invention MnTPS7 This invention possesses bifunctional catalytic properties, enabling the simultaneous and efficient synthesis of two high-value terpenoid compounds, Sesquisabinene and linalool. From an industrial application perspective, both products are key raw materials in the fields of pharmaceuticals, fragrances, cosmetics, and agricultural pest control. This invention allows for large-scale production through a heterologous expression system, while reducing the environmental costs associated with chemical synthesis, thus offering both economic and social benefits and promising broad commercial application prospects.
[0016] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally: There are currently no reports on bifunctional synthase genes for Sesquisabinene and linalool derived from plants of the genus *Machilus*. This invention is the first to clone from *Machilus*. MnTPS7The gene was identified as having a dual-function characteristic that can simultaneously catalyze the production of linalool from GPP and sesquisabinene from FPP, filling the gap in bifunctional terpene synthase gene resources from *Phoebe zhennan* and also filling the technical gap in "simultaneous synthesis of two high-value terpenes by a single gene". This provides new material for the study of the functional diversity of terpene synthases and the analysis of the terpene metabolic mechanism of *Phoebe zhennan*. Attached Figure Description
[0017] Figure 1 PCR cloning MnTPS7 Gene electrophoresis diagram.
[0018] Figure 2 Schematic diagram of the Pfam domain of the TPS gene family.
[0019] Figure 3 : MnTPS7 A schematic diagram showing gene expression in the roots, stems, and leaves of *Machilus chinensis*.
[0020] Figure 4 SDS-PAGE electrophoresis image of the purified product of pET-32a-MnTPS7 recombinant protein; lane 1 is the precipitate suspension after ultrasonic disruption; lane 2 is the supernatant after ultrasonic disruption; lanes 3-9 are 500 mM imidazole elution buffer.
[0021] Figure 5 : Schematic diagram of the in vitro catalytic reaction products of MnTPS7 protein detected by GC-MS; where Figure A shows the catalytic products of the reaction between MnTPS7 and FPP; Figure B shows the catalytic products of the reaction between MnTPS7 and GPP.
[0022] Figure 6 Mass spectrometry comparison images; where image A is the mass spectrometry comparison image of Sesquisabinene; image B is the mass spectrometry comparison image of Linalool. Detailed Implementation
[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0024] like Figure 1 As shown, this invention proposes a Sesquisabinene / Linalool synthase gene. MnTPS7 The Sesquisabinene / Linalool synthase gene of *Linalool* MnTPS7 The nucleotide sequence is shown in SEQ ID NO.1:
[0025] This invention provides a Sesquisabinene / Linalool synthase gene. MnTPS7 The expressed protein is MnTPS7, and the amino acid sequence of the protein is shown in SEQ ID NO.2: MASDGSMDSMEVRRSANYHPTVWDIARIRSLLARSDCTAALALSYDHHKRLKETIQRRLQDITQPHHLLGLIDAVQHLGVAYQFEQEISDALNGLHSENTEQAIKDSLHHTSLYFRLLRQHGCNLSSDIFNKFKEEGGG FKASLCEDAMGLLSMYEAAHLGVKGEAILEEAKVFSTENLKILMEKEERKLADRIEHALEIPLHWRALRMEARWYIDVYEKEDGRIGDLLDFAKLDFNMAQMVYQTELKELSMWWELLGLPEKMGFFRDVLLESYLLVIA VVVEPQYSQCRIAITKATVLMTAMDDFYDVYGLPDELKVFTDTVNRWDLEGIDQLPEYMKLYYLALYNTTNETAYIILKEKGFNATHYLKKLWAMQSNSYFREAQWFNSGYIPKFDEYLDNALVSVGAPFALGLSYPMIQ QHISKEEIDLIPEDLNLLRWASIIFRLYDDLATSKAEQQRGDVPKSIRCYMHETGCSEEVAANHIRDLISDAWKELNAECLKPTSLSKHYLGVAPNSARSGVLMYHHDYDGFEDPHSRTTAHITSIFFEPVPLKESINLG This invention provides a Sesquisabinene / Linalool synthase gene. MnTPS7 A method for preparing the expressed protein MnTPS7, comprising: based on the Sesquisabinene / Linalool synthase gene of Runnan. MnTPS7 A prokaryotic expression vector was constructed, and the resulting expression vector was transformed into a host bacterium to express the pET-32a-MnTPS7 recombinant protein.
[0026] The primer pairs designed during the construction of the prokaryotic expression vector for this gene are shown in SEQ ID NO.9 and SEQ ID NO.10, and the plasmid vector used is pET-32a; SEQ ID NO.9 GCCATGGCTGATATCGGATCCATGGCATCTGACGGAAGCATG SEQ ID NO.10 CTCGAGTGCGGCCGCAAGCTTTCCCAAGTTGATGGATTCCTTCAGTG.
[0027] This invention provides a Sesquisabinene / Linalool synthase gene. MnTPS7 The application of the expressed protein in the preparation of Sesquisabinene / Linalool.
[0028] 1. Extraction of total RNA from *Machilus yunnanensis* leaves Freshly collected leaves of *Machilus yunnanensis* were used as material, and a reagent kit was used. Total RNA was extracted using the Universal PlantTotal RNA Kit (ER302-01, TRAN). The pipette tips and mortar used for RNA extraction were placed in sterile sealed bags, autoclaved at 121°C for 20 minutes, and then dried in a 60°C oven. After cooling, the RNA was used for processing. RNA integrity was assessed by 1% agarose gel electrophoresis, and concentration and purity were determined using a micro-spectrophotometer.
[0029] 2. RCR amplification and purification Single-stranded cDNA was synthesized using total RNA from *Machilus yunnanensis* leaves as a template and the TIANScript II RT Kit. Primers were designed based on the second-generation transcriptome sequencing sequence of *Machilus yunnanensis*: upstream primer F1: 5'-ATGGCATCTGACGGAAGCAT-3' (as shown in SEQ ID NO: 3), and downstream primer R1: 5'-TCATCCCAAGTTGATGGATTCC-3' (as shown in SEQ ID NO: 4). The cDNA was synthesized by Beijing Qingke Biotechnology Co., Ltd. Using the above cDNA as a template, single-stranded cDNA was synthesized using… PCR amplification was performed using Max DNA Polymerase polymerase, following the instructions in the manufacturer's manual.
[0030] After the PCR reaction, the products were detected by 1% agarose gel electrophoresis. The gel containing the target fragment band was cut out under UV light using a scalpel and recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit, following the manufacturer's instructions. The recovered products were then detected by 1% agarose gel electrophoresis to ensure successful recovery.
[0031] 3. Cloning vector ligation Using the Tiangen Lethal Based Simple Fast Cloning Kit, according to the size and concentration of the target fragment, take an appropriate amount of the recovered product obtained in the above steps and ligate it with the cloning vector pLB, following the instructions. After ligation, remove DH5α E. coli competent cells (Novizan) from the -80℃ ultra-low temperature freezer and thaw them on ice; add 4μL of the ligation solution to 50μL of E. coli competent cells and incubate on ice for 30min; heat shock in a 42℃ water bath for 90s, then place on ice for 2min; add 800μL of antibiotic-free LB liquid medium, incubate at 37℃ and 225rpm for 1h; centrifuge at 12000rpm for 30s, collect the cells, discard the supernatant, and spread them on LB solid medium containing 100μg / ml ampicillin; seal the plates with sealing film and incubate upside down at 37℃ for 12-16h.
[0032] Recombinant plasmids were screened using a blue-white screening method, with the selected white single colonies used as templates. The universal primers for the pLB vector, pLB-F and pLB-R, were used, and the method was performed according to the manufacturer's instructions. Finally, the products were detected by 1% agarose gel electrophoresis. Colonies containing the target fragment size were selected as candidate bacteria, picked up with a pipette tip, and placed in LB liquid medium containing 100 μg / ml ampicillin. These samples were then sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing.
[0033] The sequence obtained from sequencing was compared with the original transcriptome sequence, and domain prediction was performed using HMMER. This confirmed that the gene sequence obtained in this invention is the full-length sequence of the TPS family, and the gene was named... MnTPS7 At the same time, its protein sequence was obtained.
[0034] MnTPS7 The results of 1% agarose gel electrophoresis of the gene are as follows: Figure 1 As shown, the clone yielded a single band that matched the estimated size. Sequencing alignment yielded... MnTPS7 The coding sequence (CDS) of the gene is shown in SEQ ID: 1, totaling 1680 bp and encoding 559 amino acids. The amino acid sequence is shown in SEQ ID: 2. The protein molecular weight is 64.3 kDa, and the isoelectric point is 5.26. HMMER analysis of the conserved domains of MnTPS7 showed that it contains two conserved N-terminal and C-terminal domains, PF01397 and PF03936. Figure 2 Therefore, it is believed that MnTPS7 It is a TPS terpene synthase.
[0035] Example 2 MnTPS7 Gene expression analysis Total RNA was extracted from the roots, stems, and leaves of *Machilus chinensis* using the TIANGEN RNA Easy Fast PlantTissue Kit, following the manufacturer's instructions. Reverse transcription was performed using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2, following the manufacturer's instructions. qRT-PCR was performed using the SYBRPRIME qPCR kit (Fast HS) from Baoguang Biotech. qRT-PCR primers were designed using NCBI: F: 5'-AGAGAACACGGAGCAAGCAA-3' (as shown in SEQ ID NO: 5), R: 5'-GCTTCACCTTTCACGCCAAG-3' (as shown in SEQ ID NO: 6). The internal reference gene was *Cinnamomum camphora* ACTIN (ACT, KM086738.1), F: 5'-CCTCGACACACAGGCGTTAT-3' (as shown in SEQ ID NO: 7), R: 5'-CCATGCTCGATGGGATATTTCA-3' (as shown in SEQ ID NO: 8). The reaction mixture consisted of 1 μL cDNA, 0.5 μL (10 μM) F primer, 0.5 μL (10 μM) R primer, 3 μL ddH2O, and 5 μL SYBRPRIME qPCR kit (Fast HS). The assay program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, 39 cycles of denaturation and annealing, extension at 65°C for 5 s, and final extension at 95°C for 5 s. The reaction was performed using a BIO-RAD CFX ConnectReal-Time System (Bio-Rad), with three biological replicates and three technical replicates per sample, employing 2... -ΔΔCt The method processes the data and calculates Runnan. MnTPS7 The expression in different organizations. The results are as follows: Figure 3 As shown, MnTPS7 It is expressed at the highest level in the roots and at a lower level in the stems and leaves.
[0036] Example 3: MnTPS7 Prokaryotic Expression Analysis 1. Carrier Construction according to MnTPS7The coding region of the gene was amplified by PCR using homologous recombination primers containing BamHI and HindIII restriction sites: F: 5'-GCCATGGCTGATATCGGATCCATGGCATCTGACGGAAGCATG-3' (as shown in SEQ ID NO: 9) and R: 5'-CTCGAGTGCGGCCGCAAGCTTTCCCAAGTTGATGGATTCCTTCAGTG-3' (as shown in SEQ ID NO: 10).
[0037] Linearization of the pET-32a prokaryotic expression vector was performed using BamHI and HindIII restriction enzymes at 37°C for 30 min, followed by 80°C for 10 min, and then with novizan. The II One Step Cloning Kit performs homologous recombination between the linearized vector and the target fragment, following the manufacturer's instructions. The ligation product is transformed into DH5α E. coli competent cells (Novizan), and the recombinant prokaryotic expression vector is obtained by colony PCR identification and sequencing.
[0038] 2. Recombinant protein expression The identified recombinant plasmid DNA was transformed into BL21(DE3) competent cells (Novizan). Single colonies were picked and inoculated into 50 ml of LB liquid medium containing 100 μg / ml ampicillin, and cultured overnight at 37°C and 225 rpm. The culture was expanded at a 1:100 ratio; when OD600 ≈ 0.5, 0.1 mM IPTG was added, and the cells were induced at 16°C and 140 rpm for 20 h; the cells were collected by centrifugation at 4000 rpm for 20 min; the supernatant was discarded, and the cells were resuspended in 30 ml of PBS buffer; the cells were sonicated and centrifuged at 12000 rpm and 4°C for 20 min. The supernatant was transferred to a new centrifuge tube to obtain the precipitate and supernatant.
[0039] 3. Recombinant protein purification Equilibrate the Ni column with 3 column volumes of PBS buffer. Slowly pass the supernatant through the Ni column. Remove contaminating proteins with 3 column volumes of PBS buffer containing 20 mM imidazole. Elute the target protein with 14 ml of PBS buffer containing 500 mM imidazole, using 2 ml tubes per elution. Prepare samples from the precipitate, supernatant, and eluent, and analyze using SDS-PAGE. Desalt the eluent containing the target protein, and then determine the protein concentration.
[0040] Example 4: In vitro enzyme catalytic function analysis of MnTPS7 Add 50 μg of protein, 10 mM MgCl2, 10 mM DTT, 5 μg FPP / GPP / FPP+GPP, and 25 mM HEPES (pH 7.4) to a final volume of 100 μL. Incubate at 30 °C for 3 h. After the reaction is complete, add 150 μL of n-hexane, shake vigorously for 5 min, and centrifuge at 12000 rpm for 10 min to separate the oil and water phases. Pipette the organic phase and filter it through a 0.22 μm filter into a sample vial for GC-MS analysis.
[0041] A TRACE 1300 gas chromatography-ISQ 7000 mass spectrometry (GC-MS) detector and an HP-5MS column (30 m × 250 μm × 0.25 μm film thickness) were used with helium as the carrier gas at a flow rate of 1.5 mL / min. The initial column temperature was set at 40 °C, equilibrated for 5 min, and then increased to 300 °C at a rate of 8 °C / min for 30 min. Mass spectrometry conditions were: ion transfer line temperature 290 °C, solvent delay 3 min, ion scan range 40 m / z to 600 m / z, with other parameters set to system defaults. An EI source was used as the ion source for the mass spectrometer. The acquired mass spectra were analyzed using the NIST mass library.
[0042] MnTPS7 The results of gene expression in prokaryotes are as follows Figure 4 As shown, the recombinant MnTPS7 protein pET-32a-MnTPS7 is approximately 83.5 kDa. SDS-PAGE gel electrophoresis results show that the size of the pET-32a-MnTPS7 recombinant protein is close to the expected size. In vitro enzyme activity identification is as follows... Figure 5 As shown, when farnesyl pyrophosphate (FPP) is used as a substrate, the main product generated by pET-32a-MnTPS7 catalysis is the sesquisabinene compound. Figure 5 When geraniol (GPP) is used as a substrate, the main product generated by catalysis is the monoterpene Linalool. Figure 5 B, 6B).
[0043] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A Gmelina hainnensis Sesquisabinene / Linalool synthase gene MnTPS7 characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the gene can encode a terpene synthase for synthesizing Sesquisabinene / Linalool.
2. The Gmelina hainanensis Sesquisabinene / Linalool synthase gene according to claim 1 MnTPS7 characterized in that, The gene is derived from a plant tissue of Machilus nuka.
3. The Gmelina hainnensis Sesquisabinene / Linalool synthase gene according to claim 2 MnTPS7 characterized in that, The gene can stably transcribe and express the target protein with catalytic activity in a heterologous expression system.
4. A MnTPS7 protein of Gmelina hainnsii Sesquisabinene / Linalool synthase, characterized in that, The protein is encoded by the Sesquisabinene / Linalool synthase gene of Machilus brevifolia according to claim 1 MnTPS7 encoding an amino acid sequence as set forth in SEQ ID NO. 2, said protein being capable of catalyzing the generation of Sesquisabinene and Linalool from a terpenoid precursor.
5. The MnTPS7 of claim 4, wherein the MnTPS7 is a sesquisabinene / linalool synthase protein. The protein contains a conserved terpene synthase catalytic domain, which is involved in the cooperative completion of substrate binding and cyclization reaction.
6. The MnTPS7 of claim 5, wherein the MnTPS7 is a sesquisabinene / linalool synthase protein. The protein has detectable Sesquisabinene / Linalool synthesis catalytic activity in a prokaryotic expression system.
7. Use of a MnTPS7 protein of a Sesquisabinene / Linalool synthase in the preparation of Sesquisabinene / Linalool, characterized in that, The Machilus nuka Sesquisabinene / Linalool synthase protein of any one of claims 4 to 6 is used to catalyze the conversion of terpene precursors into Sesquisabinene and Linalool.
8. Use according to claim 7, characterized in that, The Machilus nuka Sesquisabinene / Linalool synthase protein is obtained by a heterologous expression method.
9. Use according to claim 8, characterized in that, The heterologous expression method comprises constructing an expression vector containing the gene of claim 1 and expressing it in a host cell.
10. Use according to claim 9, characterized in that, The expression vector is constructed after the Machilus nuka Sesquisabinene / Linalool synthase gene is obtained by amplification with specific primers.