Chimonanthus praecox terpenoid synthetase gene CnTPS1 and application thereof

Abstract

The application discloses the technical field of plant genetic engineering and relates to a CnTPS1 gene of a terpenoid synthetase of Chionanthus retusus and application of the CnTPS1 gene, wherein the nucleotide sequence of the CnTPS1 gene of the terpenoid synthetase of Chionanthus retusus is shown in SEQ ID NO. 1, the protein coded by the CnTPS1 gene of the terpenoid synthetase of Chionanthus retusus is a key enzyme for synthesis of 1,8-cineole of Chionanthus retusus, and the amino acid sequence is shown in SEQ ID NO. 2. In plant genetic engineering, the CnTPS1 gene is a key gene of plant terpenoids, can significantly promote synthesis of plant terpenoids, especially synthesis of 1,8-cineole, and has extremely important significance for breeding of Chionanthus retusus varieties and in-depth development of medicinal values of the Chionanthus retusus varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to a Chimonanthus nitens terpenoid synthetase CnTPS1 gene and application thereof. BACKGROUND

[0002] Chimonanthus nitens is one of six endemic species of Chimonanthus and a typical evergreen shrub. In traditional folk customs, its fresh leaves are often processed into traditional tea drinks named "golden" or "fragrant wind", and mature leaves can be made into granules for relieving various symptoms such as influenza, heatstroke, chronic bronchitis and chest tightness, and have important medicinal value.

[0003] Studies have shown that the essential oil content of Chimonanthus nitens fresh leaves is between 0.5% and 1.5%, and abundant terpenoid volatile compounds constitute its main chemical components, and nearly ten different chemical types are derived. Among them, the eucalyptol type Chimonanthus nitens is the most common. The main advantage component of the essential oil of this type of Chimonanthus nitens leaf is 1,8-eucalyptol, which accounts for about 30% of the total amount, and the total content of the secondary components such as alpha-pinene, beta-pinene, alpha-ophuiene, D-limonene and alpha-terpineol is about 25%. The relative content and composition of these compounds have a decisive influence on the quality of "golden tea". Terpenoid synthetase (TPS) a subfamily members are key enzymes for regulating monoterpenoid compound biosynthesis, however, the TPS regulating Chimonanthus nitens terpenoid component synthesis and the mechanism are still in the blank stage.

[0004] In addition, eucalyptol as a key compound has a wide range of applications in many fields. In the medical field, it has significant antibacterial and anti-inflammatory effects, and is often used to treat skin infections, oral infections and rheumatoid arthritis, and also shows great potential in treating respiratory tract infections. In daily chemical products, eucalyptol can improve skin condition and care hair. In the food field, it is mainly used as an additive for preservatives and antiseptics to prolong the shelf life of food.

[0005] In summary, further exploring the TPS regulating the synthesis of key terpenoid compounds such as 1,8-eucalyptol has extremely important significance for the breeding of Chimonanthus nitens varieties and the in-depth development of its medicinal value. SUMMARY

[0006] The present application provides a Chimonanthus nitens terpenoid synthetase CnTPS1 gene and application thereof, which solves the technical problem that the TPS regulating Chimonanthus nitens terpenoid component synthesis and the mechanism are still in the blank stage, which is not conducive to the breeding of Chimonanthus nitens varieties and the in-depth development of its medicinal value.

[0007] The present application achieves the above-mentioned purpose by the following technical solutions.

[0008] As a first aspect of the present application, a CnTPS1 gene of Chimonanthus nitens terpenoid synthetase is provided, and the nucleotide sequence of the CnTPS1 gene of Chimonanthus nitens terpenoid synthetase is shown as SEQ ID NO. 1.

[0009] As a second aspect of the present application, a protein encoded by the CnTPS1 gene of Chimonanthus nitens terpenoid synthetase is also provided, and the protein is a 1,8-cineole synthetase, and the amino acid sequence is shown as SEQ ID NO. 2.

[0010] As a third aspect of the present application, a specific primer for amplifying the CnTPS1 gene of Chimonanthus nitens terpenoid synthetase is also provided, and the primer includes an upstream primer shown as SEQ ID NO. 3 and a downstream primer shown as SEQ ID NO. 4.

[0011] As a fourth aspect of the present application, a recombinant expression vector containing the CnTPS1 gene of Chimonanthus nitens terpenoid synthetase is also provided, and the recombinant expression vector can correspondingly express the protein.

[0012] As a further optimization scheme of the present application, the recombinant expression vector is a recombinant transient overexpression vector pCambia1300-CnTPS1-Flag / C.

[0013] As a fifth aspect of the present application, an application of the CnTPS1 gene of Chimonanthus nitens terpenoid synthetase or any of the recombinant expression vectors described above in promoting synthesis of plant terpenoids is also provided.

[0014] As a further optimization scheme of the present application, the application approach is that overexpression of the CnTPS1 gene of Chimonanthus nitens terpenoid synthetase promotes synthesis of plant terpenoids.

[0015] As a further optimization scheme of the present application, the plant is Chimonanthus nitens or tobacco.

[0016] As a further optimization scheme of the present application, the terpenoid is at least one of 1,8-cineole, α-pinene, β-pinene or α-terpineol.

[0017] As a sixth aspect of the present application, a method for promoting synthesis of 1,8-cineole in plants is also provided, and the method includes the following steps:

[0018] (1) constructing the recombinant expression vector described above by using genetic engineering technology;

[0019] (2) The recombinant expression vector is transiently transformed into plants by Agrobacterium transformation method, and the plant overexpressing CnTPS1 gene of Cneorum tanguense terpenoid synthase is obtained, so as to promote the synthesis of 1,8-cineole in the plant.

[0020] The present application has the following beneficial effects:

[0021] Firstly, the present application screens the candidate gene coding 1,8-cineole synthase by adopting the second-generation sequencing of the tender leaf tissue of three chemotypes of Cneorum tanguense, and names it as CnTPS1, and the fusion protein is obtained by inducing the prokaryotic expression system.

[0022] Further, the present application adds the substrate geranyl pyrophosphate (GPP) to the fusion protein to detect the in-vitro catalytic activity, and detects the components by gas chromatography-mass spectrometry (GCMS), and the catalytic product of CnTPS1 is 51.36% of 1,8-cineole, which indicates that the CnTPS1 provided by the present application is the key enzyme for the synthesis of 1,8-cineole of Cneorum tanguense, and has important application value in the in-vitro catalytic production of 1,8-cineole.

[0023] Finally, the present application uses injection method to transfer the Agrobacterium containing pCambia1300-CnTPS1-Flag / C plant overexpression vector into the tobacco leaf of the receptor material, and after 72 hours of transient transformation, the CnTPS1 gene is expressed in large quantities in the tobacco leaf, and after essential oil extraction and component analysis, it is found that the overexpression of CnTPS1 gene significantly promotes the synthesis of plant terpenoids, especially 1,8-cineole. Therefore, in plant genetic engineering, CnTPS1 as the key gene of plant terpenoids can significantly promote the synthesis of plant terpenoids, especially 1,8-cineole, and has extremely important significance for the breeding of Cneorum tanguense varieties and the in-depth development of its medicinal value. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The electrophoresis map of PCR cloning CnTPS1 gene provided for the present application 1 is shown in the figure, lane 1: DL2000Marker; lane 2: CnTPS1 gene PCR product;

[0025] Figure 2 The electrophoresis map of PCR cloning CnTPS1 gene provided for the present application 1 is shown in the figure, lane 1: DL2000Marker; lane 2: CnTPS1 gene PCR product; SDS-PAGE electrophoretogram of the purified product of the recombinant protein, wherein lane 1: supernatant after ultrasonic disruption; lane 2: suspension of precipitate after ultrasonic disruption; lane 3: flow-through; lane 4: 25 mM imidazole eluate; lane 5: 50 mM imidazole eluate; lane 6: 75 mM imidazole eluate 1; lane 7: 75 mM imidazole eluate 2; lane 8: 75 mM imidazole eluate 3; lane 9: 100 mM imidazole eluate; lane 10: 150 mM imidazole eluate 1; lane 11: 150 mM imidazole eluate 2; lane 12: 200 mM imidazole eluate; lane 13: 250 mM imidazole eluate;

[0026] Figure 3 GC-MS detection provided for Example 3 of the present application Spectrum of the product of the in vitro catalytic reaction, wherein A represents a 1,8-cineole standard; B represents the product of the in vitro catalysis of CnTPS1*; and C represents the product of the catalysis of the empty protein;

[0027] Figure 4 Spectrum of the components of the essential oil of the tobacco leaf after the transient overexpression of CnTPS1 provided for Example 4 of the present application. DETAILED DESCRIPTION

[0028] The following detailed description of the application is made with reference to the accompanying drawings, and it is necessary to point out here that the following detailed description is only used to further illustrate the application, and cannot be understood as limiting the scope of protection of the application, and the skilled in the art can make some non-essential improvements and adjustments to the application according to the above application content.

[0029] Example 1: Obtaining of a candidate gene encoding a 1,8-cineole synthase

[0030] 1. Sample collection and sequencing

[0031] Young leaf tissues of 1,8-cineole type (the first dominant component of the leaf essential oil is 1,8-cineole), linalool type (the first dominant component of the leaf essential oil is linalool) and camphor type (the first dominant component of the leaf essential oil is camphor) of three chemotypes of M. mume were collected in the Germplasm Resource Bank of M. mume in the Forestry Science Institute of Shangrao City, and were sent to Nanjing Jisihuiruan Biological Illumina NovaSeq 6000 platform for carrying out second-generation transcriptome sequencing, and a total of 66.64 Gb of filtered data was obtained.

[0032] 2. Data analysis and gene screening

[0033] A total of 85741 Unigenes were obtained by sequence assembly, and 38 TPS-encoding genes were obtained after annotation, of which 14 belonged to the TPS-a subfamily. Expression analysis showed that transcript TRINITY_DN1248 was significantly expressed in eucalyptol-type young leaves compared with linalool-type and camphor-type, and was screened as a candidate gene encoding 1,8-eucalyptol synthase, and was named CnTPS1.

[0034] 3. Primer design

[0035] Based on the transcriptome data, the CnTPS1 cloning primers were designed as follows:

[0036] The upstream primer CnTPS1-F is shown in SEQ ID NO. 3: 5'-AGATGGAGTACAAAGTTACAT-3';

[0037] The downstream primer CnTPS1-R is shown in SEQ ID NO. 4: 5'-GAGAATTTCCCACGACAAG-3'.

[0038] 4. RNA extraction and quality detection

[0039] The total RNA of the eucalyptol-type young leaf tissue of Leptospermum scoparium was extracted by using the universal plant RNA extraction kit (model ER302) of the Beijing Genewiz Biotech Co., Ltd. The quality and concentration of the extracted total RNA were detected by agarose gel electrophoresis and spectrophotometer to ensure that the RNA met the requirements of the subsequent experiments.

[0040] 5. cDNA synthesis, gene amplification and cloning

[0041] 100 ng of qualified total RNA was taken and subjected to reverse transcription by using the EasyScript All-in-One First-Strand cDNA Synthesis Kit of the Beijing Genewiz Biotech Co., Ltd. to synthesize cDNA. The full-length CDS of the CnTPS gene was amplified by PCR with CnTPS1-F and CnTPS1-R primers. The reaction system was: 50 ng of template cDNA, 0.5 µL of forward and reverse primers, 10.0 µL of 2× high-fidelity pfu PCR premix, and ddH2O was added to 20.0 µL. The reaction conditions were: denaturation at 94℃ for 3 min, 35 cycles of 94℃ for 20 s, 55℃ for 20 s, 72℃ for 60 s, and finally extension at 72℃ for 5 min. After PCR amplification, the obtained product was detected and separated by agarose gel electrophoresis, and the specific band conforming to the expectation was recovered by cutting the gel. Figure 1 ).

[0042] Subsequently, the recovered product was inserted into the pEASY®-Blunt cloning vector (CB301-01) produced by Golden Gate Biotechnology, and transformed into E. coli Top10 competent cells (Shanghai Generay Biotech, B528412). It was coated on solid LB medium containing 100 mg / L ampicillin (Amp) and incubated at 37°C for 12 hours. Single colonies were selected by colony PCR screening to obtain positive single clones. Further extraction of plasmid DNA and submission to Shanghai Generay Biotech for Sanger first-generation sequencing analysis to determine the accuracy and integrity of the amplified gene sequence.

[0043] 6. Sequencing result analysis

[0044] The sequencing results revealed that the CDS sequence of the candidate gene CnTPS1 was as shown in SEQ ID NO. 1, with a length of 1764 bp, which could encode a polypeptide chain composed of 587 amino acid residues, and the sequence was as shown in SEQ ID NO. 2.

[0045] Example 2: Obtaining of fusion protein

[0046] The specific steps are as follows:

[0047] 1. Primer design and target gene amplification

[0048] According to the Uniprot (https: / / www.uniprot.org) homologous gene blast prediction, the first 35 amino acid residues of CnTPS1 were the chloroplast localization signal peptide, so they were removed when inducing the mature protein. The protein without the chloroplast localization signal peptide was denoted as .

[0049] According to the Nanjing Novozyme ClonExpress II One Step Cloning Kit (C112) kit instructions, primers for constructing the prokaryotic expression vector were designed. The sequence of the prokaryotic expression vector pET-32a can be obtained by querying addgene (https: / / www.addgene.org / vector-database / 2582 / ). After inserting the target gene fragment into the multiple cloning site of the vector, a 6His (6 histidines) tag was further added to the N terminus, which facilitated the subsequent separation and purification of the fusion protein. The fusion protein obtained by prokaryotic expression system induction was denoted as .

[0050] The primer sequence design is as follows:

[0051] Upstream primer As shown in SEQ ID NO.5: 5'-gctgatatcggatccgaattcaATGGCCCTTCATCAGCTT-3';

[0052] Downstream primer As shown in SEQ ID NO.6: 5'-tgcggccgcaagcttgtcgacTTACATCTTTGTGGATGAA-3'.

[0053] Using a diluted pEASY®-Blunt-CnTPS1 cloning plasmid as a template, the CnTPS1* gene sequence was obtained by PCR amplification using high-fidelity Pfu enzyme. The amplified product was then analyzed by agarose gel electrophoresis and purified to obtain the recombinant sequence. .

[0054] 2. Construction of prokaryotic expression vectors *

[0055] First, the pET-32a plasmid was linearized by double digestion with EcoRI and SalI restriction enzymes. Then, a solution containing 5×CE II Buffer (2 μL), linearized plasmid DNA (50 ng), and Exnase enzyme (1 μL) was prepared. The enzyme-catalyzed reaction solution containing ddH2O was incubated at 37 °C for 30 min. After the reaction, the product was transformed into *E. coli* Top10 competent cells using a heat shock method and plated on LB agar containing 100 mg / L Amp, then incubated upside down at 37 °C for 12–16 h. Successful construction was confirmed by bacterial PCR screening and Sanger sequencing. Prokaryotic expression vector.

[0056] 3. Recombinant plasmid transformation and protein-induced expression

[0057] Extraction of recombinant plasmids The bacteria were transformed into competent BL21(DE3) protein expression strain cells (Shanghai Sangon Biotech, B528414) using a heat shock method, and plated on LB solid medium containing 100 mg / L Amp, and cultured at 37°C for 12–16 h. Positive single clones were selected and inoculated into LB liquid medium containing the corresponding antibiotics for overnight culture. The next day, the overnight culture was transferred to fresh LB liquid medium at a 1:50 ratio, and cultured until OD (Organic Oxidation) was achieved. 600 When the value reaches about 0.8, add IPTG to a final concentration of 0.5 mM, and culture at 20℃ with shaking for about 16 h to induce protein expression. A blank control was set up by transforming the empty vector pET-32a.

[0058] 4. Preliminary detection of protein expression

[0059] Each bacteria liquid 2 mL, 12,000 rpm centrifugal 10 min to collect bacteria. Resuspend the precipitate with 100 μL PBS buffer, boil for 10 min to lyse the cells, centrifuge again to collect the supernatant, and resuspend the precipitate with PBS buffer. Take 10 μL of supernatant and precipitate suspension, add 2x protein loading buffer, and perform SDS-PAGE electrophoresis analysis to preliminarily detect protein expression and confirm The fusion protein was successfully expressed in the supernatant.

[0060] 5. Protein purification and verification

[0061] Centrifugal collection of residual bacteria, followed by resuspension of bacteria with PBS buffer. Ultrasonic disruption of bacteria on ice, frozen ultracentrifugation (12,000 rpm, 4°C) for 15 min. Purification of fusion protein from the supernatant using a Nanjing Novi Zan Ni Sepharose FF kit, and detection of the purified product by SDS-PAGE electrophoresis (see Figure 2 ). The theoretical size is 62.23 KDa, plus the size of the tag protein is about 14 KDa, the size of the whole fusion protein is about 76 KDa, close to the position of the purified protein band in the figure.

[0062] 6. Protein concentration determination

[0063] The recombinant protein concentration was determined using the Yixing Bio BCA protein concentration test kit, and the specific operation was performed according to the instructions.

[0064] Example 3: In vitro catalytic reaction verification of CnTPS1 function

[0065] In vitro catalytic reaction

[0066] In a 50 mmol / L Bis-Tris (pH 7.2) buffer system, add recombinant protein, 10 μmol / L geranyl pyrophosphate (GPP, Sigma-Aldrich product), 10 mM MgCl2, 10 mM MnCl2, 10 % glycerol and 5 mM DTT, configure into a reaction solution and transfer to a 20 mL beaker. After sealing, place in a 30°C constant temperature shaker, shake at 100 r / min for 3 h. At the same time, set up a control group, except for adding an equal amount of empty protein, the rest of the conditions are exactly the same as the experimental group, and the in vitro catalytic reaction is carried out synchronously.

[0067] 2. Product extraction

[0068] After the reaction was terminated, the solid-phase microextraction fiber probe (Supelco 100 μm PDMS, Fused Silica 24Ga, Manual Holder, 3pk (Red)) was immediately inserted into the beaker, and precise extraction was performed at 60 °C for 30 min to ensure that the product was fully adsorbed on the extraction fiber probe.

[0069] 3. GC-MS detection of product components

[0070] The product components were analyzed in depth using a Shimadzu gas chromatograph-mass spectrometer (GC-MS), and the specific parameter settings were as follows:

[0071] (1) Carrier gas: high-purity helium, flow rate constant at 1.0 mL / min, using split injection mode, split ratio of 20:1.

[0072] (2) Gas phase conditions (GC): injection port temperature 280 °C; column temperature initially set to 50 °C, maintained for 2 min, then increased at a rate of 3 °C / min to 180 °C and maintained for 2 min, then increased at a rate of 8 °C / min to 240 °C, maintained for 5 min, total run time 60 min.

[0073] (3) Mass spectrometry conditions (MS): interface temperature 260 °C, ion source temperature 180 °C, scan range covering 50-620 m / z.

[0074] (4) Data analysis: using the NIST standard spectral library to match and identify the product components, screening out components with a matching degree higher than 85%, and precisely comparing with the caryophyllene standard product chromatogram provided by Sigma-Aldrich company, determining the relative content of each product according to the peak area ratio.

[0075] 4. Results analysis

[0076] As Figure 3 The results showed that when GPP was used as the substrate, the catalytic products of CnTPS1 mainly included 1,8-cineole (51.36 %), α-pinene (7.26 %), β-pinene (24.89 %), D-limonene (2.80 %), γ-terpinene (1.63 %), and α-terpineol (12.06 %).

[0077] The above results confirmed that CnTPS1 has the function of efficiently synthesizing 1,8-cineole.

[0078] Example 4: Verification of CnTPS1 function by transient expression in Nicotiana benthamiana

[0079] In order to further explore the function of CnTPS1 gene in plants, the present application selects Nicotiana benthamiana as a model plant to carry out transient overexpression experiment, and the specific test procedure is as follows:

[0080] 1. Primer design and PCR amplification

[0081] According to the instructions of the cloning kit exv06 produced by Biogle, the primers used to construct the overexpression vector pCambia1300-CnTPS1-Flag / C are designed. The overexpression vector pCambia1300-Flag / C sequence can be referred to Biogle (http: / / biogle.cn / exclone / index / vector / vid / 40). When the target gene is inserted, the Flag tag is introduced at the C terminal of the vector.

[0082] The primer sequence is designed as follows:

[0083] The upstream primer pCambia1300-CnTPS1-F is shown in SEQ ID NO. 7: 5'-tcagcagtcgaagagcATGGAGTACAAAGTTACA-3';

[0084] The downstream primer pCambia1300-CnTPS1-R is shown in SEQ ID NO. 8: 5'-ttagcgtgtgaagagcTTACATCTTTGTGGATGAA-3'.

[0085] The full-length CDS sequence of CnTPS1 gene is obtained by PCR amplification using high-fidelity PrimeSTAR enzyme with pEASY-Blunt-CnTPS1 cloning plasmid as the template. The PCR product is detected by agarose gel electrophoresis, and the purified CnTPS1-DNA is obtained by gel recovery.

[0086] 2. Construction of overexpression vector pCambia1300-CnTPS1-Flag / C

[0087] Prepare the enzyme reaction system: 5×EX II Buffer 2 μL, linearized pCambia1300-Flag / C carrier DNA 50 ng, Exclonase enzyme 1 μL, CnTPS1-DNA 100 ng, ddH2O to 10 μL. After incubation at 37°C for 30 min, it is moved to room temperature for 15 min. The reaction product is transformed into E. coli Top10 competent cells by heat shock method, and is coated on LB solid medium containing 50 mg / L kanamycin and cultured at 37°C for 12-16 h. The positive clones are screened by bacterial liquid PCR, and the vector construction is confirmed by Sanger sequencing.

[0088] 3. Transform the recombinant plasmid into Agrobacterium tumefaciens

[0089] After extracting the recombinant plasmid pCambia1300-CnTPS1-Flag / C, heat shock method was used to transform the competent cells of Agrobacterium tumefaciens GV3101, which was then coated on LB solid medium containing 50 mg / L kanamycin, 100 mg / L rifampicin and 50 mg / L gentamicin, and cultured at 37°C for 12-16 hours. Positive monoclonal was selected and inoculated into 50 mL LB liquid medium containing the same antibiotics, and cultured at 37°C until the OD 600 value was about 0.8. The bacterial cells were collected by centrifugation and rinsed twice with 10 mM MgCl2 to remove residual rifampicin.

[0090] 4. Preparation of infection solution and infection of tobacco leaves

[0091] An infection solution containing 200 mM acetosyringone, 0.5 mM MES and 10 mM MgCl2 was prepared, and the bacterial cells were suspended and adjusted to an OD 600 value of about 1.0, and cultured at room temperature in the dark for 2-3 hours. The growing leaves of Nicotiana benthamiana were selected and evenly punctured on the back. The infection solution was sucked with a 1 mL syringe and injected from the lower epidermis to the surface of the leaves until the leaves were wet. The GV3101 infection solution carrying the empty vector pCambia1300-Flag / C was set as a control. After injection, the leaves were cultured in the dark for 1 day and under normal light for 2 days.

[0092] 5. Essential oil extraction and component analysis

[0093] After 1 g of sample was ground, 10 mL of n-hexane was added to extract the essential oil at room temperature for 3 hours. Then, centrifugation was performed at 12,000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. GC-MS was used to analyze the essential oil components, and the operating conditions were the same as those in Example 3.

[0094] As Figure 4 The results showed that the contents of 1,8-cineole, α-pinene, β-pinene and α-terpineol in the tobacco sample after transient overexpression of the CnTPS1 gene were significantly higher than those in the control group.

[0095] The above examples only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application.

Claims

1. A gene for a montana terpenoid synthase CnTPS1 characterized in that The montana terpenoid synthase CnTPS1 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. A monoterpene synthase of Rhododendron anthopogis according to claim 1, wherein the amino acid sequence of the protein encoded by the gene is represented by SEQ ID NO:

1. CnTPS1 a protein encoded by the gene, characterized in that, The protein is 1,8-cineole synthase, and the amino acid sequence is shown as SEQ ID NO.

2.

3. A specific primer combination for amplifying the P. ginseng terpenoid synthase gene of claim 1. CnTPS1 characterized in that, The specific primer combination comprises an upstream primer shown as SEQ ID NO. 3, and a downstream primer shown as SEQ ID NO.

4.

4. A recombinant expression vector, characterized in that, A gene comprising the montana terpene synthase of claim 1 CnTPS1 capable of corresponding to the translation expression of the protein of claim 2.

5. The recombinant expression vector of claim 4, wherein, The recombinant expression vector is the repeated transient overexpression vector pCambia 1300 -CnTPS1 - Flag / C.

6. A monoterpene synthase of Rhododendron anthopogis as claimed in claim 1. CnTPS1 Use of the gene of claim 4 or 5 or the recombinant expression vector of any one of claims 4-5 in promoting the synthesis of terpenoids in tobacco, characterized in that, The application approach is: overexpressing Wintersweet terpenoid synthetase CnTPS1 The gene promotes the synthesis of tobacco terpenoids, which are at least one of 1,8-cineole, α-pinene, β-pinene or α-terpineol.

7. A method of promoting the synthesis of tobacco 1,8-cineole, characterized in that, The method comprises the following steps: (1) constructing the recombinant expression vector as claimed in any one of claims 4-5 by using genetic engineering technology; (2) The recombinant expression vector is transiently transformed into tobacco by Agrobacterium transformation method to obtain the terpene synthase gene of P. przewalskii Maxim. overexpressed in tobacco to promote the synthesis of 1,8-cineole in tobacco. CnTPS1 The recombinant expression vector is transiently transformed into tobacco by Agrobacterium transformation method to obtain the terpene synthase gene of P. przewalskii Maxim. overexpressed in tobacco to promote the synthesis of 1,8-cineole in tobacco.

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