The Guangxi Curcuma zedoaria terpene synthase gene CkTPS11, its encoded protein, the preparation method of the expressed protein, and its applications.

Abstract

This invention discloses a *Curcuma zedoaria* terpene synthase gene CkTPS11, its encoded protein, a method for preparing the expressed protein, and its applications, belonging to the field of plant genetic engineering technology. The nucleotide sequence of this gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. This invention also provides a method for preparing the expressed protein, comprising: cloning the CkTPS11 gene from *Curcuma zedoaria* roots, constructing a recombinant expression vector, transforming it into a host bacterium, inducing expression with IPTG, and purifying it to obtain the CkTPS11 protein. This protein can catalyze the production of 8-deoxylactrin from farnesyl pyrophosphate and simultaneously catalyze the production of anethole from geranyl pyrophosphate. This invention provides a novel bifunctional enzyme gene resource for the biosynthesis of terpenoids, which is beneficial for reducing production costs and improving production efficiency, and can be used for the large-scale biomanufacturing of 8-deoxylactrin and anethole.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a Guangxi turmeric terpene synthase gene CkTPS11, its encoded protein, a method for preparing the expressed protein, and its applications. Background Technology

[0002] Guangxi Curcuma zedoaria ( Curcuma kwangsiensis *Curcuma zedoaria* et CFLiang is a plant belonging to the genus *Curcuma* in the family Zingiberaceae. Its dried rhizome and tuberous root are the traditional Chinese medicinal herbs *Curcuma zedoaria* and *Curcuma longa*, respectively. Sesquiterpenes in its volatile oil are its main active substances. Currently, although a few terpene synthase genes have been isolated and functionally characterized from *Curcuma zedoaria*, most of these genes only possess a single catalytic function. No terpene synthase genes capable of simultaneously catalyzing the synthesis of both sesquiterpenes and monoterpenes have been reported. Furthermore, due to the large number and diverse functions of the terpene synthase gene family in *Curcuma zedoaria*, screening and cloning genes with specific catalytic activities remains technically challenging. Therefore, obtaining a novel terpene synthase gene resource with potential multiple catalytic functions is fundamental to enriching the genetic information of this species. Summary of the Invention

[0003] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages described below.

[0004] This invention provides a Guangxi Curcuma zedoaria terpene synthase gene CkTPS11, which encodes a terpene synthase that simultaneously catalyzes the production of 8-deoxylactrin from farnesyl pyrophosphate and the production of anethole from gerany pyrophosphate.

[0005] This invention provides a Guangxi turmeric terpene synthase gene CkTPS11, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] A Guangxi turmeric terpene synthase CkTPS11, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] A method for preparing the CkTPS11 expression protein of Curcuma zedoaria var. zedoaria, comprising the following steps: S1: Using cDNA synthesized by reverse transcription from the root of Curcuma zedoaria in Guangxi as a template, PCR amplification was performed using primers CkTPS11-F and CkTPS11-R to obtain the CkTPS11 gene fragment; the nucleotide sequence of primer CkTPS11-F is shown in SEQ ID NO.3, and the nucleotide sequence of primer CkTPS11-R is shown in SEQ ID NO.4; S2: The CkTPS11 gene fragment and the pET-32a vector were homologously recombinated to construct a recombinant expression vector; S3: Transform the recombinant expression vector into Escherichia coli BL21 host bacteria to obtain recombinant engineered bacteria; S4: Inoculate the recombinant engineered bacteria into LB liquid medium containing ampicillin and culture at 36–38 °C and 210–230 rpm until OD. 600 The value was 0.6–0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium to a final concentration of 0.1–1 mmol / L, and expression was induced at 30–37 °C for 2–6 h. S5: Collect bacterial cells and perform low-temperature ultra-high pressure disruption. Centrifuge at 3-5 ℃ and 3900-4100 rpm for 19-21 min, and collect the supernatant. S6: The protein in the supernatant was purified using Ni-NTA resin, and the target protein was eluted with imidazole elution buffer with a concentration of 60% to 100% to obtain purified CkTPS11 protein.

[0008] Preferably, the PCR amplification program is as follows: 32 cycles of holding at 95 ℃ for 3 min, then holding at 94 ℃ for 30–60 s, holding at 60 ℃ for 30–60 s, holding at 72 ℃ for 1–2 min, and finally holding at 72 ℃ for 10 min.

[0009] The application of the protein encoded by the Guangxi Curcuma zedoaria terpene synthase gene CkTPS11 or the Guangxi Curcuma zedoaria terpene synthase CkTPS11 is characterized in that the protein is used to catalyze the production of 8-deoxylactrin from farnesyl pyrophosphate and to catalyze the production of anethole from gerany pyrophosphate.

[0010] This invention offers at least the following advantages: A terpene synthase gene, CkTPS11, was cloned from *Curcuma zedoaria* (Guangxi). This gene encodes an enzyme with dual functions: simultaneously catalyzing the production of 8-deoxylactrin from farnesyl pyrophosphate and anethole from gerany pyrophosphate. This enriches the research on dual-function terpene synthase genes in *Curcuma* species and provides new gene resources for studying the biosynthetic mechanisms of terpenoids. Recombinant proteins prepared using this gene can generate corresponding terpene products from two different substrates. Compared to single-function synthase genes, one enzyme can catalyze the synthesis of two products, which helps reduce biosynthetic costs and improve production efficiency. Furthermore, the microbial fermentation production method based on this gene, compared to traditional plant extraction and chemical synthesis, is environmentally friendly, produces high-purity products, and is not limited by natural conditions, providing a new technical approach for the large-scale production of 8-deoxylactrin and anethole.

[0011] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0012] Figure 1 Image showing electrophoresis detection of PCR-amplified CkTPS11 CDS sequence; Figure 2 The gene expression of CkTPS11 in different tissues of Curcuma zedoaria from Guangxi; Figure 3 Here is a PCR electrophoresis image of colonies of the pET-32a-CkTPS11 recombinant plasmid; Figure 4 The image shows the SDS-PAGE electrophoresis result of the pET-32a-CkTPS11 recombinant protein after in vitro induction; where M: protein marker; 1: pET-32a empty vector control; 2: no IPTG induction; 3, 4, 5: induced at 30 ℃ for 2 h, 4 h, and 6 h, respectively; 6, 7, 8: induced at 37 ℃ for 2 h, 4 h, and 6 h, respectively. Figure 5 SDS-PAGE electrophoresis images for the solubility analysis of recombinant pET-32a-CkTPS11 protein; where M: protein marker; 1: pET-32a empty vector control; 2: lysed and precipitated recombinant plasmid pET-32a-CkTPS11; 3: supernatant of lysed recombinant plasmid pET-32a-CkTPS11; 4: PBS bacterial suspension of pET-32a-CkTPS11. Figure 6 SDS-PAGE electrophoresis image of the purified pET-32a-CkTPS11 soluble protein product; where M: protein marker; 1: pET-32a empty control; 2: overnight elution with mixed Ni medium; 3: elution collected after elution with binding buffer; 4-13 are elutions collected after elution with 10%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% elution buffer, respectively. Figure 7 The image shows the in vitro catalytic reaction of CkTPS11 protein with FPP detected by GC-MS. In the image, Figure A is the chromatogram of the catalytic product; Figure B is the mass spectrum of the catalytic product 8-deoxylactucin. Figure 8 The image shows the in vitro catalytic reaction of CkTPS11 protein with GPP detected by GC-MS. In the image, Figure A is the chromatogram of the catalytic product, and Figure B is the mass spectrum of the catalytic product, anethole. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0014] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0015] Example 1: Cloning of the CkTPS11 gene 1.1 Total RNA extraction and cDNA synthesis from Curcuma zedoaria in Guangxi Five tissues—roots, rhizomes (Curcuma zedoaria), tubers (Curcuma longa), leaves, and flowers—were collected from *Curcuma zedoaria* in Guangxi. Total RNA was extracted from each tissue using the Adley EASYspinPlus Polysaccharide-Polyphenol Complex Plant RNA Rapid Extraction Kit. All consumables used in the extraction process, including mortars and pipette tips, were autoclaved at 121 °C for 20 min, dried in a 65 °C oven, and cooled before use. RNA integrity was assessed using 1% agarose gel electrophoresis, and RNA concentration and purity were determined using a micro spectrophotometer. Subsequently, first-strand cDNA was synthesized using the Thermo Scientific RevertAid RT Kit (catalog number: K1691), and its concentration and purity were determined.

[0016] 1.2 Amplification and Recovery of the CkTPS11 Gene Fragment Using cDNA synthesized by reverse transcription from the root of Curcuma zedoaria in Guangxi as a template, specific primers CkTPS11-F and CkTPS11-R (synthesized by Wuhan Qingke Biotechnology Co., Ltd.) were designed based on the CDS sequence of the Curcuma zedoaria genome. The primer sequences are as follows: CkTPS11-F: 5'-atggagaagcaatcactagctctt-3' (SEQ ID NO. 3); CkTPS11-R: 5'-ttatatgggaagaggttcaatcaacaacaaa-3' (SEQ ID NO. 4); PCR amplification system (total 50 μL): 2×Phanta Max Buffer, 25 μL; dNTP Mix (10 mmol / L), 1 μL; CkTPS11-F (10 μmol / L), 2 μL; CkTPS11-R (10 μmol / L), 2 μL; Phanta Max Super-Fidelity DNA Polymerase, 1 μL; cDNA, 2 μL; ddH2O 17 μL.

[0017] Amplification program: 95 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 1 min, for a total of 32 cycles; final extension at 72 ℃ for 10 min, and storage at 4 ℃.

[0018] The PCR products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, a specific band appeared between 1000 and 2000 bp, consistent with the expected size of the CkTPS11 gene. The target fragment was recovered using a Novizan gel recovery kit.

[0019] 1.3 Cloning vector ligation and transformation of recombinant products Ligate the recovered target fragment with the pTOPO cloning vector, following the instructions for the pTOPO-Blunt SimpleCloning Kit (catalog number: CV1701). Keep the ligation product on ice and immediately transform DH5α competent cells: Remove competent cells from -80 ℃ and thaw on ice (approximately 1-3 min); add 5 μL of ligation buffer, mix gently, and incubate on ice for 5 min; heat shock at 42 ℃ for 60 s, then quickly return to ice and incubate for 2-3 min; add 500 μL of LB liquid medium (antibiotic-free), and incubate at 37 ℃ with shaking at 200 rpm for 10-20 min; spread 100-200 μL of the bacterial culture onto LB agar plates containing ampicillin (100 μg / mL) and incubate inverted at 37 ℃ overnight.

[0020] 1.4 Positive identification of recombinant products Single colonies were picked using a sterilized pipette tip and placed in a centrifuge tube containing 50 μL of ddH2O. 1 μL of the bacterial culture was used as a template for PCR identification. The PCR system (20 μL) consisted of: 10 μL of 2×PCR Mix, 0.3 μL of primer M13F (10 μmol / L), 0.3 μL of primer M13R (10 μmol / L), 1 μL of template, and 8.4 μL of ddH2O. The primer sequences are as follows: M13F: 5'-tgtaaaacgacggccagt-3' (SEQ ID NO.5); M13R: 5'-caggaaacagctatgacc-3' (SEQ ID NO. 6); PCR program: 94 ℃ pre-denaturation for 10 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for a total of 32 cycles; 72 ℃ extension for 5 min; incubation at 4 ℃. PCR products were detected by 1% agarose gel electrophoresis (100 V, 30 min), and positive clones were sent to a gene company for sequencing. Bacterial cultures with correct sequencing were preserved, and recombinant plasmids were extracted using a plasmid extraction kit.

[0021] Sequencing results showed that the full-length nucleotide sequence of the obtained CkTPS11 gene was 1716 bp (SEQ ID NO.1), encoding 571 amino acids (SEQ ID NO.2).

[0022]

[0023] The sequence of SEQ ID NO.2 is shown below:

[0024] Example 2: Tissue expression analysis of the CkTPS11 gene qRT-PCR was performed using the Novizan Real-Time PCR kit. Using cDNA extracted from five tissues of *Curcuma zedoaria* (root, rhizome, tuber, leaf, and flower) in Example 1 as templates, qRT-PCR-specific primers CkTPS11-qPCR-F and CkTPS11-qPCR-R were designed based on the *Curcuma zedoaria* genome CDS sequence. The CkActin gene was used as an internal control, and three biological replicates were set up for each sample. The primer sequences are as follows: CkTPS11-qPCR-F:ccgcgcaagtcactcttttc (SEQ ID NO.7); CkTPS11-qPCR-R:ctttccacaactcggtcacg (SEQ ID NO.8); CkActin-F: tgtgcttgattctggtgatggt (SEQ ID NO.9); CkActin-R: agcaaggtccagacgaagaatag (SEQ ID NO. 10); qRT-PCR reaction system (10 μL): ChamQ Universal SYBR qPCR Master Mix 5 μL, forward and reverse primers (10 μmol / L) 0.3 μL each, cDNA 0.5 μL, ddH2O 3.9 μL. Reaction program: 95 ℃ pre-denaturation for 10 min; 95 ℃ denaturation for 10 s, 60 ℃ annealing for 10 s, 72 ℃ extension for 20 s, for a total of 40 cycles. 2 −ΔΔCt The relative expression levels of CkTPS11 in different tissues were calculated using the method described above. The results are as follows: Figure 2 As shown, CkTPS11 was expressed at the highest level in roots, followed by rhizomes and tubers, and at very low levels in leaves and flowers.

[0025] Example 3: Prokaryotic expression and purification of CkTPS11 protein 3.1 Construction of prokaryotic expression recombinant vectors Using the correctly sequenced positive plasmid from Example 1 as a template, homologous recombination primers pET-32a-CkTPS11-F and pET-32a-CkTPS11-R with EcoRI and SalI restriction sites were designed for PCR amplification. The primer sequences are as follows: pET-32a-CkTPS11-F: 5'-ggatccgatatcagccagaattcatggagaagcaatcactagctctt-3' (SEQ ID NO. 11); pET-32a-CkTPS11-R: 5'-aagcttgcggccgcagtcgacttatatgggaagaggttcaatcaaca-3' (SEQ ID NO. 12); PCR system (50 μL): 2×Phanta Max Buffer, 25 μL; dNTP Mix (10 mmol / L), 1 μL; forward and reverse primers (10 μmol / L), 2 μL each; Phanta Max Super-Fidelity DNA Polymerase, 1 μL; cDNA, 2 μL; ddH2O, 17 μL.

[0026] Amplification program: 95 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s; 55 ℃ annealing for 30 s; 72 ℃ extension for 1 min; 32 cycles in total; 72 ℃ extension for 10 min; storage at 4 ℃. The target fragment was recovered after electrophoresis detection of the PCR product.

[0027] pET-32a vector linearization: Double digestion with EcoRI and SalI restriction endonucleases was performed. The reaction system (10 μL) was as follows: 10×H Buffer, 2 μL; pET-32a vector, 1 μL; EcoRI, 1 μL; SalI, 1 μL; ddH2O, 5 μL; digestion at 37 ℃ for 16 h. Homologous recombination of the amplified target fragment with the linearized vector was performed using the Novizan ClonExpress II One Step Cloning Kit, following the manufacturer's instructions. The ligation product was transformed into DH5α competent cells. Colony PCR identification and sequencing comparison confirmed the correct recombinant prokaryotic expression vector, named pET-32a-CkTPS11. Figure 3 ).

[0028] 3.2 Induced expression of recombinant proteins The correctly sequenced recombinant plasmid pET-32a-CkTPS11 was transformed into BL21(DE3) competent cells. Single colonies were picked, and after positive identification by bacterial culture PCR, 5 μL of the positive bacterial culture was inoculated into 5 mL of LB liquid medium containing ampicillin (100 μg / mL) and cultured overnight at 37 ℃ and 220 r / min. 400 μL of the revived bacterial culture was then transferred to fresh LB medium containing ampicillin and cultured at 37 ℃ and 220 r / min for approximately 2 h until OD (digestive growth) was reached. 600 The value reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mmol / L, and expression was induced at 30 ℃ (2 h, 4 h, 6 h) and 37 ℃ (2 h, 4 h, 6 h) respectively. After induction, the bacterial culture was centrifuged at 12000 r / min for 2 min at room temperature, the supernatant was discarded, and the precipitate was washed once with 10 mL PBS. 1 mL of the resuspended bacterial culture was boiled at 100 ℃ for 10 min, and electrophoresis was performed on an 8% SDS-PAGE gel (GenScript, Nanjing) at 120 V for 40 min. The gel was stained with Coomassie Brilliant Blue for 2 h, then destained with water (changing the water every 2 h) until the background was clear, and photographed for storage. The results are as follows: Figure 4 As shown, the recombinant CkTPS11 protein was successfully expressed under all induction conditions. Its molecular weight is approximately 80 kDa. The expression level was highest when induced at 37 ℃ for 6 h, which was determined to be the optimal induction condition (37 ℃, 6 h).

[0029] 3.3 High-level expression and solubility analysis of recombinant proteins Expand the culture under optimal induction conditions: Transfer the activated bacterial solution to LB medium containing ampicillin at a ratio of 1:100, and incubate at 37 ℃ and 220 r / min until OD.600 The concentration was 0.6–0.8. IPTG was added to a final concentration of 0.5 mmol / L, and induction was performed at 37 ℃ and 220 r / min for 6 h. The bacterial culture was collected, centrifuged at 12000 r / min for 2 min, the supernatant was discarded, and the precipitate was washed once with 10 mL PBS. Finally, the precipitate was resuspended in 50 mL Binding Buffer (containing 14.61 g NaCl, 1.21 g Tris-base, and 0.1702 g imidazole, diluted to 500 mL water) and kept on ice. The bacterial cells were lysed using a low-temperature ultra-high pressure cell disruptor (4 ℃), and the lysate was collected. A small amount of the lysate was centrifuged, and the supernatant and precipitate were analyzed by SDS-PAGE. The results are shown below. Figure 5 As shown, the target protein band was present in both the supernatant and the precipitate, indicating that the CkTPS11 protein is a soluble protein.

[0030] 3.4 Purification of recombinant proteins Centrifuge a large amount of the disrupted bacterial cells at 4 °C and 4000 r / min for 20 min, collect the supernatant, and filter through a 0.45 μm filter membrane. First, pack 2 mL of ProteinIsoNi-NTA Resin medium onto a column, and equilibrate the Ni with 10 column volumes of 20% ethanol and 10 column volumes of Binding Buffer (containing 0.1702 g imidazole, 14.61 g NaCl, and 1.21 g Tris-base, adjusted to 500 mL of water). 2+ Column. The filtered supernatant was added to the column and incubated at 4 °C for 2 h. Gradient elution was then performed with different concentrations of Elution Buffer (10%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100%), and the eluates were collected for SDS-PAGE analysis. Results are as follows: Figure 6 As shown, elution buffers with 60% or higher imidazole concentrations yielded single target protein bands around 80 kDa. The 60% imidazole elution buffer was concentrated and desalted, and the protein concentration was determined for subsequent enzyme activity analysis.

[0031] Example 4: In vitro enzyme activity verification of CkTPS11 protein Take 60 μg of purified CkTPS11 protein, add MgCl2 (final concentration 25 mM), DTT (dithiothreitol, final concentration 5 mM), Mops buffer (final concentration 30 mM, pH 7.4), and substrate farnesyl pyrophosphate (FPP) or geranyyl pyrophosphate (GPP) (both final concentration 5 mM), and bring the volume to 1000 μL with ddH2O. Incubate at 30 ℃ for 1 h, then continue incubation at 45 ℃ for 15 min to terminate the reaction. Add 500 μL of n-hexane, shake vigorously for 5 min, and centrifuge at 12000 rpm for 10 min to separate the oil and water phases, extracting the organic phase; repeat the extraction once, combine the two organic phases, filter through a 0.22 μm filter membrane, and transfer to a sample vial for GC-MS analysis.

[0032] GC-MS analysis conditions: A TSQ 8000 EVO tandem mass spectrometer was used, with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm); high-purity helium was used as the carrier gas at a constant flow rate of 1.2 mL / min; the injection port temperature was 250 ℃, and the solvent delay was 3.5 min. Temperature program: 40 ℃ held for 3.5 min, then increased to 100 ℃ at 10 ℃ / min, then to 180 ℃ at 7 ℃ / min, and finally to 280 ℃ at 25 ℃ / min, held for 5 min. Mass spectrometry conditions: Electron impact ionization (EI) source, ion source temperature 230 ℃, quadrupole temperature 150 ℃, mass spectrometer interface temperature 280 ℃, selected ion detection mode (SIM) was used, and the acquired mass spectra were compared with the NIST mass spectrum library.

[0033] The results are as follows Figure 7 As shown: When FPP is used as a substrate, the major product catalyzed by CkTPS11 is the sesquiterpene compound 8-deoxylactucin. For example... Figure 8 As shown, when GPP is used as a substrate, the main product catalyzed is the monoterpene compound anethole. These results indicate that the CkTPS11 protein has a dual function of simultaneously catalyzing the formation of two different types of terpenoid products from both FPP and GPP.

[0034] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Other modifications can be readily made by those skilled in the art; therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A Guangxi Curcuma zedoaria terpene synthase gene CkTPS11, characterized in that, The nucleotide sequence of the gene CkTPS11 is shown in SEQ ID NO.

1.

2. A Guangxi Curcuma zedoaria terpene synthase CkTPS11, characterized in that, The amino acid sequence of the enzyme CkTPS11 is shown in SEQ ID NO.

2.

3. A method for preparing the Guangxi Curcuma zedoaria terpene synthase CkTPS11 expression protein, characterized in that, Includes the following steps: S1: Using cDNA synthesized by reverse transcription from the root of Curcuma zedoaria in Guangxi as a template, PCR amplification was performed using primers CkTPS11-F and CkTPS11-R to obtain the CkTPS11 gene fragment; the nucleotide sequence of primer CkTPS11-F is shown in SEQ ID NO.3, and the nucleotide sequence of primer CkTPS11-R is shown in SEQ ID NO.4; S2: The CkTPS11 gene fragment and the pET-32a vector were homologously recombinated to construct a recombinant expression vector; S3: Transform the recombinant expression vector into the BL21 host bacterium to obtain the recombinant engineered bacterium; S4: Inoculate the recombinant engineered bacteria into LB liquid medium containing ampicillin and culture at 36–38 °C and 210–230 rpm until OD. 600 The value was 0.6–0.

8. IPTG was added to the culture medium to a final concentration of 0.1–1 mmol / L, and expression was induced at 30–37 °C for 2–6 h. S5: Collect bacterial cells and perform low-temperature ultra-high pressure disruption. Centrifuge at 3-5 ℃ and 3900-4100 rpm for 19-21 min, and collect the supernatant. S6: The protein in the supernatant was purified using Ni-NTA resin, and the target protein was eluted with imidazole elution buffer with a concentration of 60% to 100% to obtain purified CkTPS11 protein.

4. The method for preparing the Guangxi Curcuma zedoaria terpene synthase CkTPS11 expression protein according to claim 3, characterized in that, The PCR amplification program was as follows: 32 cycles of holding at 95 ℃ for 3 min, then at 94 ℃ for 30–60 s, at 60 ℃ for 30–60 s, at 72 ℃ for 1–2 min, and finally at 72 ℃ for 10 min.

5. The application of the protein encoded by the Guangxi Curcuma zedoaria terpene synthase gene CkTPS11 as described in claim 1, or the Guangxi Curcuma zedoaria terpene synthase CkTPS11 as described in claim 2, characterized in that, This protein is used to catalyze the production of 8-deoxylactrin from farnesyl pyrophosphate and the production of anethole from gerany pyrophosphate.

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