Chimonanthus nitens terpene synthase CnTPS1 gene and application thereof

Abstract

The invention discloses a chimonanthus nitens terpene synthase CnTPS1 gene and application thereof in the technical field of plant genetic engineering, the nucleotide sequence of the chimonanthus nitens terpene synthase CnTPS1 gene is as shown in SEQ ID NO.1, the protein coded by the chimonanthus nitens terpene synthase CnTPS1 gene is a key enzyme for synthesis of chimonanthus nitens 1, 8-cineole, and the amino acid sequence of the chimonanthus nitens terpene synthase CnTPS1 gene is as shown in SEQ ID NO.2. In plant genetic engineering, CnTPS1, as a key gene of plant terpenoids, can significantly promote synthesis of plant terpenoids, especially synthesis of 1, 8-cineole, and is of great significance in breeding of chimonanthus nitens varieties and in-depth development of medicinal values of chimonanthus nitens varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a CnTPS1 gene of terpene synthase from wintersweet and its application. Background Technology

[0002] Mountain wintersweet (Chimonanthus nitens) is one of the six endemic species in the genus Chimonanthus, and is a typical evergreen shrub. In traditional folk customs, its tender leaves are often processed into a traditional tea called "Golden" or "Fragrant Breeze," while mature leaves can be made into granules to relieve various symptoms such as influenza, heatstroke, chronic bronchitis, and chest tightness, thus possessing important medicinal value.

[0003] Studies show that the essential oil content of fresh wintersweet leaves ranges from 0.5% to 1.5%, with abundant volatile terpenoids constituting its main chemical components, giving rise to nearly ten different chemical types. Among them, the eucalyptol type is the most common. The main dominant component of the essential oil from this type of wintersweet leaf is 1,8-cineole, accounting for about 30% of the total, while the total content of minor components such as α-pinene, β-pinene, α-phellandrene, D-limonene, and α-terpineol accounts for about 25%. The relative content and composition of these compounds have a decisive influence on the quality of "Golden Tea". Members of the terpene synthase (TPS) a subfamily are key enzymes regulating the biosynthesis of monoterpenoids; however, research on TPS and its mechanism of action in regulating the synthesis of terpenoid components in wintersweet is still in its infancy.

[0004] Furthermore, eucalyptol, as a key compound, has wide applications in numerous fields. In the pharmaceutical field, it possesses significant antibacterial and anti-inflammatory effects, and is commonly used to treat skin infections, oral infections, and rheumatoid arthritis, while also showing great potential in treating respiratory infections. In daily chemical products, eucalyptol can improve skin condition and care for hair. In the food industry, it is primarily used as an additive in preservatives and antiseptics to extend the shelf life of food products.

[0005] In conclusion, in-depth exploration of the TPS that regulates the synthesis of key terpenoid compounds such as 1,8-cineole is of great significance for the breeding of *Hedysarum heterotropoides* varieties and the further development of its medicinal value. Summary of the Invention

[0006] The purpose of this invention is to provide a CnTPS1 gene for synthase of terpenoids in wintersweet and its application. This invention solves the technical problem that the research on TPS, which regulates the synthesis of terpenoid components in wintersweet and its mechanism of action, is relatively lacking, which is not conducive to the breeding of wintersweet varieties and the in-depth development of its medicinal value.

[0007] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, a CnTPS1 gene of terpene synthase is provided, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] As a second aspect of the present invention, a protein encoded by the CnTPS1 gene of the terpene synthase as described above is also provided, said protein being a 1,8-cineole synthase, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] As a third aspect of the present invention, a specific primer for amplifying the CnTPS1 gene of terpene synthase as described above is also provided, comprising an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0010] As a fourth aspect of the present invention, a recombinant expression vector is also provided, containing the CnTPS1 gene of the terpene synthase as described above, which is capable of correspondingly translating and expressing the protein as described above.

[0011] As a further optimization of the present invention, the recombinant expression vector is the recombinant transient overexpression vector pCambia1300-CnTPS1-Flag / C.

[0012] As a fifth aspect of the present invention, the application of the *Heliotropium indicum* terpene synthase CnTPS1 gene as described above or the recombinant expression vector as described above in promoting the synthesis of plant terpene compounds is also provided.

[0013] As a further optimization of the present invention, the application method is: overexpressing the CnTPS1 gene of *Hedysarum heterotropoides* to promote the synthesis of plant terpenoid compounds.

[0014] As a further optimization of the present invention, the plant is wintersweet or tobacco.

[0015] As a further optimization of the present invention, the terpene compound is at least one selected from 1,8-cineole, α-pinene, β-pinene, or α-terpineol.

[0016] As a sixth aspect of the present invention, a method for promoting the synthesis of 1,8-cineole in plants is also provided, comprising the following steps: (1) Construct recombinant expression vectors as described above using genetic engineering techniques; (2) The recombinant expression vector was transiently transformed into plants by Agrobacterium transformation to obtain plants overexpressing the CnTPS1 gene of terpene synthase in wintersweet, so as to promote the synthesis of 1,8-cineole in plants.

[0017] The beneficial effects of the present invention are: First, by performing next-generation sequencing on young leaf tissues from three chemical types of wintersweet, this invention screened out a candidate gene encoding 1,8-cineole synthase, named it CnTPS1, and obtained the fusion protein through prokaryotic expression system.

[0018] Furthermore, the present invention conducted in vitro catalytic activity assays on the fusion protein with added substrate gerany pyrophosphate (GPP). The composition was analyzed by gas chromatography-mass spectrometry (GC-MS). 51.36% of the catalytic product of CnTPS1 was 1,8-cineole, indicating that CnTPS1 provided by the present invention is a key enzyme in the synthesis of 1,8-cineole from *Hedysarum heterotropoides*, and has significant application value in the in vitro catalytic production of 1,8-cineole.

[0019] Finally, this invention utilizes an injection method to transform Agrobacterium containing the pCambia1300-CnTPS1-Flag / C plant overexpression vector into the recipient material, tobacco leaves. After 72 hours of transient transformation, the CnTPS1 gene was highly expressed in the tobacco leaves. Essential oil extraction and component analysis revealed that overexpression of the CnTPS1 gene significantly promoted the synthesis of plant terpenoids, especially 1,8-cineole. This indicates that in plant genetic engineering, CnTPS1, as a key gene for plant terpenoids, can significantly promote the synthesis of plant terpenoids, especially 1,8-cineole, which is of great significance for the breeding of *Chimonanthus praecox* varieties and the in-depth development of its medicinal value. Attached Figure Description

[0020] Figure 1 This is an electrophoresis image of the PCR clone CnTPS1 gene provided in Example 1 of the present invention. In the image, lane 1: DL2000 Marker; lane 2: PCR product of CnTPS1 gene. Figure 2The image shows an SDS-PAGE electrophoresis diagram of the purified His-CnTPS1* recombinant protein product provided in Example 2 of this invention. In the diagram, lane 1: supernatant after ultrasonic disruption; lane 2: precipitate suspension after ultrasonic disruption; lane 3: flow-through buffer; lane 4: 25 mM imidazole elution buffer; lane 5: 50 mM imidazole elution buffer; lane 6: 75 mM imidazole elution buffer 1; lane 7: 75 mM imidazole elution buffer 2; lane 8: 75 mM imidazole elution buffer 3; lane 9: 100 mM imidazole elution buffer; lane 10: 150 mM imidazole elution buffer 1; lane 11: 150 mM imidazole elution buffer 2; lane 12: 200 mM imidazole elution buffer; lane 13: 250 mM imidazole elution buffer. Figure 3 The GC-MS detection spectrum of CnTPS1* in vitro catalytic reaction products provided in Example 3 of this invention is shown in the figure. In the figure, A represents 1,8-cineole standard; B represents CnTPS1* in vitro catalytic product; and C represents empty protein catalytic product. Figure 4 This is the essential oil composition spectrum of tobacco leaves after transient overexpression of CnTPS1 provided in Example 4 of the present invention. Detailed Implementation

[0021] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0022] Example 1: Obtaining the candidate gene encoding 1,8-cineole synthase Sample collection and sequencing Young leaf tissues of three chemotypes of *Chimonanthus praecox*—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)—were collected from the germplasm resource bank of *Chimonanthus praecox* at the Shangrao Forestry Research Institute. These tissues were then sent to the IlluminaNovaSeq 6000 platform of Nanjing Jisihuiyuan Biotechnology Co., Ltd. for next-generation transcriptome sequencing, yielding a total of 66.64 Gb of filtered data.

[0023] Data Analysis and Gene Screening 85,741 Unigenes were obtained through sequence assembly, and 38 TPS-encoding genes were obtained after annotation, of which 14 belong to the TPS-a subfamily. Expression analysis showed that the transcript TRINITY_DN1248 was significantly more expressed than the linalool and camphor types in eucalyptol-type young leaves, and was selected as a candidate gene encoding 1,8-cineole synthase, and named CnTPS1.

[0024] Primer design Based on transcriptome data, the following CpTPS1 cloning primers were designed: The upstream primer CnTPS1-F is shown in SEQ ID NO.3: 5'-AGATGGAGTACAAAGTTACAT-3'; The downstream primer CnTPS1-R is shown in SEQ ID NO.4: 5'-GAGAATTTCCCACGACAAG-3'.

[0025] RNA extraction and quality testing Total RNA was extracted from young leaves of *Chimonanthus eucalyptolus* using a universal plant RNA extraction kit (model ER302) manufactured by TransGen Biotech. The quality and concentration of the extracted total RNA were assessed by agarose gel electrophoresis and spectrophotometry to ensure that the RNA met the requirements for subsequent experiments.

[0026] cDNA synthesis, gene amplification and cloning 100 ng of qualified total RNA was reverse transcribed using the EasyScript All-in-One First-Strand cDNA Synthesis Kit from Beijing TransGen Biotechnology Co., Ltd. to synthesize cDNA. The full-length CDS of the CnTPS gene was amplified by PCR using CnTPS1-F and CnTPS1-R primers. The reaction system consisted of: 50 ng template cDNA, 0.5 µL each of forward and reverse primers, 10.0 µL of 2× high-fidelity PFU PCR premix, and ddH2O added to a final volume of 20.0 µL. Reaction conditions were: denaturation at 94℃ for 3 min, 35 cycles (94℃ for 20 s, 55℃ for 20 s, 72℃ for 60 s), and a final extension at 72℃ for 5 min. After PCR amplification, the products were detected and separated by agarose gel electrophoresis, and the expected specific bands were recovered by gel excision. Figure 1 ).

[0027] Subsequently, the recovered product was inserted into the pEASY®-Blunt cloning vector (CB301-01) produced by TransGen Biotech and transformed into *E. coli* Top10 competent cells (Shanghai Sangon Biotech, B528412). The cells were plated on solid LB medium containing 100 mg / L ampicillin (Amp) and incubated at 37°C for 12 hours. Single colonies were selected, and positive clones were obtained through colony PCR screening. Plasmid DNA was further extracted and sent to Shanghai Sangon Biotech for Sanger sequencing analysis to determine the accuracy and integrity of the amplified gene sequence.

[0028] 6. Sequencing Result Analysis Sequencing results revealed that the CDS sequence of the candidate gene CpTPS1, as shown in SEQ ID NO.1, is 1764 bp in length and encodes a polypeptide chain consisting of 587 amino acid residues, as shown in SEQ ID NO.2.

[0029] Example 2: Obtaining the His-CnTPS1* fusion protein The specific steps are as follows: 1. Primer design and target gene amplification According to Uniprot (https: / / www.uniprot.org) homologous gene blast prediction, the first 35 amino acid residues of CnTPS1 are the chloroplast signal peptide. Therefore, it is discarded when inducing the mature protein. The protein with the chloroplast signal peptide discarded is denoted as CnTPS1*.

[0030] Primers for constructing the prokaryotic expression vector pET-32a-CnTPS1* were designed according to the instructions of the Nanjing Novizan ClonExpress II One Step Cloning Kit (C112). The sequence of the prokaryotic expression vector pET-32a can be obtained by consulting AddGene (https: / / www.addgene.org / vector-database / 2582 / ). After the target gene fragment was inserted into the multiple cloning site of the vector, a 6His (6 histidine) tag was added to its N-terminus to facilitate subsequent isolation and purification of the fusion protein. The fusion protein obtained by induction through the prokaryotic expression system is denoted as His-CnTPS1*.

[0031] The primer sequences are designed as follows: The upstream primer pET-32a-CnTPS1*-F is shown in SEQ ID NO.5: 5'-gctgatatcggatccgaattcaATGGCCCTTCATCAGCTT-3'; The downstream primer pET-32a-CnTPS1*-R is shown in SEQ ID NO.6: 5'-tgcggccgcaagcttgtcgacTTACATCTTTGTGGATGAA-3'.

[0032] 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 detected by agarose gel electrophoresis and then recovered from the gel to obtain purified CnTPS1*-DNA carrying the recombinant sequence.

[0033] 2. Construction of the prokaryotic expression vector pET-32a-CnTPS1* First, the pET-32a plasmid was double-digested with EcoRI and SalI restriction enzymes to linearize the plasmid DNA. Then, an enzymatic reaction solution containing 5×CE II Buffer (2 μL), linearized plasmid DNA (50 ng), Exnase enzyme (1 μL), CnTPS1*-DNA (100 ng), and ddH2O was prepared and 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, and cultured upside down at 37 °C for 12–16 h. The pET-32a-CnTPS1* prokaryotic expression vector was successfully constructed, confirmed by bacterial PCR screening and Sanger sequencing.

[0034] 3. Recombinant plasmid transformation and protein-induced expression Recombinant plasmid pET-32a-CnTPS1* was extracted and transformed into competent cells of the protein-expressing strain BL21(DE3) (Shanghai Sangon Biotech, B528414) using a heat shock method. The transformed cells were 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 appropriate antibiotic 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.

[0035] 4. Preliminary detection of protein expression Collect 2 mL of each bacterial culture, centrifuge at 12,000 rpm for 10 min to collect the bacterial cells. Resuspend the precipitate in 100 μL of PBS buffer, boil for 10 min to lyse the cells, centrifuge again to collect the supernatant, and resuspend the precipitate in PBS buffer. Take 10 μL of the supernatant and precipitate suspension respectively, add 2× protein loading buffer, and perform SDS-PAGE electrophoresis analysis to preliminarily detect protein expression and confirm that the His-CnTPS1* fusion protein was successfully expressed in the supernatant.

[0036] 5. Protein purification and validation The remaining bacterial cells were collected by centrifugation and then resuspended in PBS buffer. The cells were then sonicated on ice and subjected to cryogenic ultracentrifugation (12,000 rpm, 4°C) for 15 min. The His-CnTPS1* fusion protein was purified from the supernatant using the Nanjing Novizan Ni Sepharose FF kit, and the purified product was detected by SDS-PAGE electrophoresis (see [link to product details]). Figure 2The theoretical size of CnTPS1* is 62.23 kDa. With the tag protein size of approximately 14 kDa, the overall size of the fusion protein is approximately 76 kDa, which is close to the position of the purified protein band in the figure.

[0037] 6. Protein concentration determination The recombinant protein concentration was determined using the Yisheng Bio BCA Protein Concentration Assay Kit. The specific operation was performed according to the instructions.

[0038] Example 3: In vitro catalytic reaction to verify the function of CnTPS1 In vitro catalytic reaction In a 50 mmol / L Bis-Tris (pH 7.2) buffer system, 1 μg of His-CnTPS1* recombinant protein, 10 μmol / L geranyyl pyrophosphate (GPP, Sigma-Aldrich), 10 mM MgCl2, 10 mM MnCl2, 10% glycerol, and 5 mM DTT were added sequentially to prepare the reaction solution, which was then transferred to a 20 mL clamp-top flask. After sealing, the flask was placed in a 30 ℃ constant-temperature shaker at 100 r / min for 3 h. Simultaneously, a control group was set up, with all conditions identical to the experimental group except for the addition of an equal amount of empty protein, and the in vitro catalytic reaction was carried out concurrently.

[0039] 2. Product Extraction After the reaction was terminated, the solid phase microextraction fiber probe (Supelco 100 μm PDMS, Fused Silica24Ga, Manual Holder, 3pk (Red)) was immediately inserted into the clamp bottle and extracted precisely for 30 min at 60 °C to ensure that the product was fully adsorbed onto the extraction fiber probe.

[0040] 3. GC-MS detection of product composition The product components were analyzed in depth using a Shimadzu gas chromatography-mass spectrometry (GC-MS) system, with the specific parameters set as follows: (1) Carrier gas: high-purity helium, with a constant flow rate of 1.0 mL / min, using a split injection mode with a split ratio of 20:1.

[0041] (2) Gas phase conditions (GC): Inlet temperature 280 ℃; column temperature initially set to 50 ℃, held for 2 min, then increased to 180 ℃ at a rate of 3 ℃ / min and held for 2 min, then increased to 240 ℃ at a rate of 8 ℃ / min and held for 5 min, for a total running time of 60 min.

[0042] (3) Mass spectrometry conditions (MS): interface temperature 260 ℃, ion source temperature 180 ℃, scanning range covering 50-620 m / z.

[0043] (4) Data analysis: The components of the products were matched and identified using the NIST standard spectral library. Components with a matching degree of more than 85% were screened out and accurately compared with the chromatogram of caryophyllene standard provided by Sigma-Aldrich. The relative content of each product was determined based on the peak area ratio.

[0044] 4. Results Analysis like Figure 3 The results showed that when GPP was used as a substrate, the catalytic products of CpTPS1 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%).

[0045] The above results confirm that CnTPS1 has the function of efficiently synthesizing 1,8-cineole.

[0046] Example 4: Transient expression of CnTPS1 in Nicotiana benthamiana to verify its function To further investigate the function of the CnTPS1 gene in plants, this invention selected Nicotiana benthamiana as a model plant and conducted a transient overexpression experiment. The specific experimental procedure is as follows: 1. Primer design and PCR amplification Primers for constructing the overexpression vector pCambia1300-CnTPS1-Flag / C were designed according to the instructions of the exv06 cloning kit produced by Biogle Biotechnology. The sequence of the overexpression vector pCambia1300-Flag / C can be found at Biogle Biotechnology (http: / / biogle.cn / exclone / index / vector / vid / 40). This vector introduces a Flag tag at the C-terminus when inserting the target gene.

[0047] The primer sequences are designed as follows: The upstream primer pCambia1300-CnTPS1-F is shown in SEQ ID NO.7: 5'-tcagcagtcgaagagcATGGAGTACAAAGTTACA-3'; The downstream primer pCambia1300-CnTPS1-R is shown in SEQ ID NO.8: 5'-ttagcgtgtgaagagcTTACATCTTTGTGGATGAA-3'.

[0048] Using the pEASY®-Blunt-CpTPS1 cloning plasmid as a template, PCR amplification was performed using the high-fidelity PrimeSTAR enzyme to obtain the full-length CDS sequence of the CpTPS1 gene. The PCR products were detected by agarose gel electrophoresis and purified CnTPS1-DNA was obtained by gel recovery.

[0049] 2. Construct the overexpression vector pCambia1300-CnTPS1-Flag / C The enzymatic reaction system was prepared as follows: 2 μL of 5×EX II Buffer, 50 ng of linearized pCambia1300-Flag / C vector DNA, 1 μL of Exclonase enzyme, 100 ng of CnTPS1-DNA, and ddH2O to a final volume of 10 μL. The mixture was incubated at 37°C for 30 min, then allowed to stand at room temperature for 15 min. The reaction product was transformed into *E. coli* Top10 competent cells using a heat shock method, plated on LB agar containing 50 mg / L kanamycin, and incubated at 37°C for 12–16 h. Positive clones were screened by colony PCR, and the successful vector construction was confirmed by Sanger sequencing.

[0050] 3. Transformation of recombinant plasmids into Agrobacterium rhizogenes After extracting the recombinant plasmid pCambia1300-CnTPS1-Flag / C, it was transformed into Agrobacterium rhizogenes GV3101 competent cells using the heat shock method. The cells were then plated on LB solid medium containing 50 mg / L kanamycin, 100 mg / L rifampin, and 50 mg / L gentamicin, and cultured at 37°C for 12–16 hours. Positive single clones were selected and inoculated into 50 mL of LB liquid medium containing the same antibiotics, and cultured at 37°C until the bacterial culture reached OD. 600 The value was approximately 0.8. The bacterial cells were collected by centrifugation and washed twice with 10 mM MgCl2 to remove residual rifampin.

[0051] 4. Preparation of dyeing solution and dyeing of tobacco leaves Prepare an infection solution containing 200 mM acetylsyleugenol, 0.5 mM MES, and 10 mM MgCl2, suspend the bacterial cells, and adjust the OD value to 0.5 mM. 600 The value was approximately 1.0. The cells were incubated at room temperature in the dark for 2-3 hours. Leaves of *Nicotiana benthamiana* during their growth period were selected, and holes were evenly punched on the underside of the leaves. 1 mL of the infection solution was drawn up using a syringe and injected into the leaf surface from the lower epidermis to moisten it. A control was prepared using GV3101 infection solution carrying the empty vector pCambia1300-Flag / C. The cells were incubated in the dark for 1 day after injection, followed by 2 days of normal light incubation.

[0052] 5. Essential oil extraction and component analysis After grinding 1g of sample, 10 mL of n-hexane was added, and the essential oil was extracted at room temperature for 3 hours. Subsequently, the sample was centrifuged at 12,000 rpm for 30 min, and the supernatant was collected and filtered through a 0.22 μm organic filter membrane. The essential oil components were analyzed using GC-MS under the same operating conditions as in Example 3.

[0053] like Figure 4 The results showed that after transient overexpression of the CnTPS1 gene, the contents of 1,8-cineole, α-pinene, β-pinene and α-terpineol in tobacco samples were significantly higher than those in the control group.

[0054] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A terpene synthase from *Heliotropium indicum* CnTPS1 Genes, characterized by, The terpene synthase of *Hericium erinaceus* CnTPS1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A terpene synthase as described in claim 1 CnTPS1 Gene-encoded proteins are characterized by, The protein is a 1,8-cineole synthase, and its amino acid sequence is shown in SEQ ID NO.

2.

3. An enzyme for amplifying the terpene synthase described in claim 1. CnTPS1 Gene-specific primers, characterized in that, This includes the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.

4.

4. A recombinant expression vector, characterized in that, Contains the terpene synthase as described in claim 1 CnTPS1 The gene is capable of being translated and expressed to produce the protein described in claim 2.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector is the repetitive transient overexpression vector pCambia1300. -CnTPS1 -Flag / C.

6. A terpene synthase as described in claim 1 CnTPS1 The application of genes or recombinant expression vectors as described in any one of claims 4-5 in promoting the synthesis of plant terpenoids.

7. The application according to claim 6, characterized in that, The application pathway is as follows: overexpression of *Hericium erinaceus* terpene synthase. CnTPS1 Genes promote the synthesis of terpenoids in plants.

8. The application according to claim 6, characterized in that, The plant in question is either wintersweet or tobacco.

9. The application according to any one of claims 6-8, characterized in that, The terpene compound is at least one of 1,8-cineole, α-pinene, β-pinene, or α-terpineol.

10. A method for promoting the synthesis of 1,8-cineole in plants, characterized in that, Includes the following steps: (1) Constructing the recombinant expression vector as described in any one of claims 4-5 using genetic engineering techniques; (2) The recombinant expression vector was transiently transformed into plants by Agrobacterium-mediated transformation to obtain the terpene synthase of *Heliotropium indicum*. CnTPS1 Plants that overexpress the gene to promote the synthesis of 1,8-cineole in the plant.

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