A ginger lotus terpene synthase gene CaTPS2 and its application

Abstract

This invention discloses a ginger lotus terpene synthase gene, CaTPS2, and its applications. The coding sequence of the ginger lotus terpene synthase gene CaTPS2 provided by this invention is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO:2. Studies show that the CaTPS2 gene is most highly expressed in flowers, with a small amount expressed in fertile bracts, and almost no expression in sterile bracts and leaves, which is basically consistent with the expression of the ginger lotus terpene fragrance compounds nerolidol and linalool. After the exogenous recombinant protein of CaTPS2 catalyzes the substrates FPP and GPP, the terpene compounds nerolidol and linalool can be generated, respectively, indicating that CaTPS2 is a bifunctional synthase gene controlling the terpene components nerolidol and linalool in ginger lotus. This invention provides technical support for increasing the content of terpene components in ginger lotus and for the preparation of nerolidol and linalool.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. More specifically, it relates to a ginger lotus terpenoid synthase gene CaTPS2 and its applications. Background Technology

[0002] Ginger lily (Curcuma longa) is a perennial bulbous flowering plant belonging to the genus Curcuma in the family Zingiberaceae. It is mainly distributed in South and Southeast Asia, with a few species extending into China. The bracts of ginger lily closely resemble those of lotus flowers, and it is characterized by its vibrant colors, unique flower shape, long flowering period, and long vase life, making it very popular in domestic and international markets. The sesquiterpenes and monoterpenes contained in ginger lily possess strong aroma and biological activity, making them important raw materials for the pharmaceutical, food, and cosmetic industries. For example, the monoterpene linalool can be used to make fragrances, deodorants, and sedatives, and also has excellent antibacterial activity; the sesquiterpene paclitaxel can be used to treat tumors; and the sesquiterpene nerolidol not only has certain anti-inflammatory, antifungal, and antiparasitic effects, but also exhibits good antitumor activity.

[0003] Terpenoid synthases (TPSs) are key enzymes catalyzing the synthesis of terpenoid compounds, playing a crucial role in the structural diversity of terpenoids and being essential for plant growth, development, and resistance regulation. Based on the different products formed by TPS catalysis, TPSs are classified into monoterpene synthases (MonoTPSs), sesquiterpene synthases (SesquiTPSs), and diterpene synthases (DiTPSs), which catalyze the formation of corresponding monoterpenes, sesquiterpenes, and diterpenes from substrates GPPs, FPPs, and GGPPs, respectively. Further research has shown that some TPSs possess bifunctional enzyme properties, reacting with both GPPs and FPPs to produce monoterpenes and sesquiterpenes. For example, 'Siberian' lily LoTPS3 reacts with GPP to produce linalool, and can also react with FPP to produce nerolidol and α-farnesene (Abbas et al., 2019); Clematis CfTPS1 and CfTPS2 can both catalyze GPP to produce linalool, and can also catalyze FPP to produce nerolidol (Jiang et al., 2020); Hosta HsTPS1 catalyzes GPP to produce linalool, citronellol and nerol, and catalyzes FPP to produce small amounts of (E,E)-farnesol and (E,E)-farnesaldehyde (Cui et al., 2022).

[0004] Cloning terpene synthase genes is a prerequisite for studying the formation mechanism of ginger lotus terpenoids. It lays a theoretical foundation for comprehensively elucidating the formation and regulation mechanism of ginger lotus terpenoid fragrance and increasing the content of ginger lotus terpenoids through genetic engineering. However, there are currently no reports in China on the research of ginger lotus terpenoid fragrance substances and their synthases, and their biosynthetic pathways are still unclear. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings in the research on terpene synthase genes in ginger lotus and to provide a synthase gene CaTPS2 that controls the synthesis of terpene components nerolidol and linalool in ginger lotus.

[0006] The first objective of this invention is to provide a ginger lotus terpene synthase gene CaTPS2.

[0007] The second objective of this invention is to provide a ginger lotus terpene synthase CaTPS2.

[0008] A third objective of this invention is to provide the application of the ginger lotus terpene synthase gene CaTPS2 or the ginger lotus terpene synthase CaTPS2.

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

[0010] The fifth objective of this invention is to provide a method for cultivating terpene-containing aromatic plants.

[0011] The sixth object of the present invention is to provide a method for preparing nerolidol and / or linalool.

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

[0013] This invention provides a ginger lotus terpene synthase gene CaTPS2, the coding region (CDS) of which is 1737 bp, and its nucleotide sequence is shown in SEQ ID NO:1. The gene encodes 579 amino acids, and its amino acid sequence is shown in SEQ ID NO:2. It is estimated that the protein molecular weight is 66.96 kDa and the isoelectric point (pI) is 6.00. The gene sequence contains a conserved DDXXD sequence.

[0014] Based on the CaTPS2 gene sequence information provided by the present invention, those skilled in the art can easily obtain a gene equivalent to CaTPS2 by the following methods: (1) obtaining it through database retrieval; (2) obtaining it by screening genomic libraries or cDNA libraries of ginger lily or other plants using CaTPS2 gene fragments as probes; (3) obtaining it from the genome, mRNA and cDNA of ginger lily or other plants by designing oligonucleotide primers based on the CaTPS2 gene sequence information and using PCR amplification; (4) obtaining it by modifying it using genetic engineering methods based on the CaTPS2 gene sequence; (5) obtaining the gene by chemical synthesis.

[0015] This invention provides a primer pair for amplifying the ginger lotus terpene synthase gene CaTPS2, the primer sequences of which are shown in SEQ ID NO:3-4.

[0016] This invention shows that the expression of the CaTPS2 gene, a terpene synthase gene in ginger lotus, is related to the flower development process. The CaTPS2 gene is most highly expressed in flowers, with a small amount expressed in fertile bracts, and almost no expression in sterile bracts and leaves. After the exogenous recombinant protein of CaTPS2 catalyzes the substrates FPP and GPP, the terpene compounds nerolidol and linalool can be generated, respectively. This indicates that CaTPS2 belongs to the synthase gene that controls the synthesis of the terpene components nerolidol and linalool in ginger lotus, providing a new bioengineering method for the preparation of nerolidol and linalool.

[0017] Therefore, this invention protects the application of the ginger lotus terpene synthase gene CaTPS2 or the ginger lotus terpene synthase CaTPS2 in the preparation of nerolidol and / or linalool, and in the cultivation of terpene-containing aromatic plants.

[0018] Furthermore, the terpenoid is nerolidol and / or linalool.

[0019] Furthermore, the plant in question is ginger lily.

[0020] This invention provides a recombinant vector containing the ginger lotus terpene synthase gene CaTPS2.

[0021] The present invention provides a recombinant bacterium containing the above-described recombinant vector.

[0022] The present invention provides a cell line comprising the above-mentioned recombinant bacteria.

[0023] This invention also provides a method for cultivating terpenoid-containing aromatic plants, wherein the ginger lotus terpenoid synthase gene CaTPS2 or ginger lotus terpenoid synthase CaTPS2 is ligated into a plant transformation vector, and then introduced into ginger lotus or other plant cells to obtain CaTPS2 gene-transformed varieties.

[0024] The CaTPS2 gene, a terpene synthase gene from ginger lily provided by this invention, has significant application value. One application is to ligase the CaTPS2 gene sequence into any plant transformation vector and introduce the CaTPS2 gene into ginger lily or other plant cells using any transformation method. This yields transgenic plants expressing the gene, which can then be used in production. When the gene described in this invention is constructed into a plant transformation vector, the gene or its regulatory sequence can be appropriately modified. Alternatively, the original promoter of the gene can be replaced with another promoter before its transcription start codon, thereby broadening and enhancing the plant's ability to produce terpenoids such as nerolidol and linalool, and enhancing its resistance.

[0025] Preferably, the recombinant vector or recombinant bacteria is transformed into plants to cultivate terpene-containing floral plants.

[0026] The present invention also provides a method for preparing nerolidol and / or linalool, using farnesyl pyrophosphate (FPP) and geraniyl pyrophosphate (GPP) as substrates, and nerolidol and / or linalool are produced by catalysis of ginger terpene synthase CaTPS2.

[0027] The present invention has the following beneficial effects:

[0028] This invention marks the first cloning of a bifunctional enzyme gene, CaTPS2, from *Nelumbo nucifera* that controls the formation of terpenoid compounds nerolidol and linalool. This gene catalyzes the formation of terpenoid compounds nerolidol and linalool from FPP and GPP, playing a crucial role in increasing the content of terpenoid components and enhancing plant resistance. The terpenoid synthase gene CaTPS2 developed in this invention can be used to prepare nerolidol and linalool, and further to prepare essential oils, fragrances, and pharmaceuticals containing these compounds. Constructing the CaTPS2 gene fragment into a plant expression vector allows for the exogenous transformation of other plant materials, thereby obtaining transgenic materials containing terpenoid fragrance genes, providing an effective method for cultivating aromatic and medicinal plants. Attached Figure Description

[0029] Figure 1 This invention relates to the cloning of the CaTPS2 gene and the analysis of the homology of the CaTPS2 amino acid sequence (A: agarose gel electrophoresis of the CaTPS2 clone; B: analysis of the homology of the CaTPS2 amino acid sequence; C: analysis of the conserved domains of CaTPS2).

[0030] Figure 2 This invention illustrates the specificity of CaTPS2 gene expression in ginger lily (A: Phenotypic characteristics of ginger lily; B: CaTPS2 expression level in different parts of ginger lily).

[0031] Figure 3 This invention presents the prokaryotic expression of the CaTPS2 gene and the in vitro enzymatic reaction of the recombinant protein (A: SDS-PAGE gel electrophoresis of the CaTPS2 recombinant protein, M is the marker, C is the precipitate after bacterial lysis of the recombinant protein, S is the supernatant after bacterial lysis of the recombinant protein, E is the elution buffer of the recombinant protein; B: in vitro catalysis of FPP and GPP to generate nerolidol and linalool). Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0033] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0034] The ginger lily samples used in the following examples were all collected from the ginger lily germplasm resource garden of the Flower Research Center of South China Agricultural University.

[0035] Example 1: Cloning of the CaTPS2 gene

[0036] 1. Extraction of total RNA from ginger lily flowers

[0037] Ginger lily flowers preserved in an ultra-low temperature freezer were used as the material for RNA extraction. The pipette tips and Eppendorf tubes used for RNA extraction were soaked in 0.1% DEPC at 37°C overnight, then sterilized at 121°C for 25 min. Glassware and mortars were wrapped in aluminum foil and subjected to dry heat treatment at 180°C for 3 h, then cooled for later use. Total RNA was extracted from the ginger lily rhizomes using the Trizol method according to the Trizol (TaKaRa) manufacturer's instructions. RNA integrity was assessed by 1% agarose gel electrophoresis, and its concentration and purity were determined using a micro-spectrophotometer. The extracted RNA was stored at -80°C for later use.

[0038] 2. PCR amplification and purification

[0039] Total RNA from *Liriope muscari* flowers was used as a template to synthesize single-stranded cDNA using Evo M-MLV reverse transcriptase from Aikerui. Primers were designed based on the annotated gene sequences in the *Liriope muscari* transcriptome database: upstream primer F1: 5'-ATGGCTCCCGCCTCTGCT-3' (as shown in SEQ ID NO:3); downstream primer R1: 5'-TTGTTTAAACAACAATAAGTTTATATACTCCTT-3' (as shown in SEQ ID NO:4). These primers were synthesized by Shanghai Bioengineering Co., Ltd. The synthesized cDNA was then used as a template for PCR amplification using Phanta high-fidelity enzyme, following the manufacturer's instructions. The PCR cycle consisted of 94℃ pre-denaturation for 2 min, 94℃ denaturation for 15 s, 60℃ annealing for 15 s, and 72℃ extension for 90 s, for 35 cycles, followed by a final extension at 72℃ for 5 min. The cDNA was stored at -20℃ for later use.

[0040] After the PCR reaction, a preliminary detection of the PCR product for the target fragment band was performed using 1.0% agarose gel electrophoresis. Following the 1% agarose gel electrophoresis, the gel containing the target fragment was cut off under UV light using a scalpel and recovered using a DNA gel recovery kit (SanPrep Column DNA Gel Recovery Kit, Sangon Biotech). The recovery method was largely in accordance with the kit's instructions. The recovered product was then subjected to 1% agarose gel electrophoresis to assess the recovery efficiency and approximate concentration, ensuring the accuracy of subsequent experiments.

[0041] 3. Cloning vector ligation

[0042] Based on the size and effective concentration of the recovered target fragment, take an appropriate amount of the purified product and ligate it with the cloning vector. The TaKaRa pMD19-T vector is selected, and the molar ratio of target DNA to cloning vector is controlled at approximately 3:1. Follow the instructions in the manual for specific procedures. Incubate at 16℃ for 3–6 hours, depending on the length of the target fragment. Beforehand, remove competent DH5α (TaKaRa) cells from the -80℃ freezer and place them in an ice box to allow them to thaw naturally. Add 10 μL of ligation solution to the centrifuge tube containing the competent cells, incubate on ice for 30 minutes, then heat shock at 42℃ for 90 seconds, and immediately place on ice for 2–5 minutes. Then add 1 mL of LB liquid medium, mix well, and incubate at 37℃ with shaking at 180 rpm for 1 hour. Spread 30 μL of X-gal (20 mg / mL) and 30 μL of IPTG (20 mg / mL) on the surface of an LB solid medium plate containing 100 μg / mL ampicillin. Then spread an appropriate amount of transformation solution. After the transformation solution is completely absorbed, invert the plate and incubate overnight at 37°C. Observe the results after about 16 hours. White colonies are screened by X-gal / IPTG blue-white screening, and recombinant plasmids are preliminarily identified. Store the plates at 4°C.

[0043] After initial screening using blue-white screening, single white colonies were picked from LB agar plates using sterilized pipette tips and inoculated into LB liquid medium containing 100 μg / mL ampicillin. The cultures were then incubated at 37°C and 180 rpm for 3–6 hours in a temperature-controlled shaking incubator. PCR was then performed on the bacterial culture using universal primers M13-47 and M13-48 for the pMD19-T vector, following the manufacturer's instructions. Finally, the PCR products were detected by 1% agarose gel electrophoresis. DNA sequencing was performed on bacterial cultures containing the target fragment, and the sequencing was completed by Shanghai Sangon Biotech Co., Ltd.

[0044] The sequence obtained from sequencing was compared and analyzed with the original transcriptome sequence information, and comparison and homology analysis were performed in NCBI to determine that the gene sequence obtained in this invention is the full-length sequence of the TPS family, and it was named CaTPS2 gene. At the same time, its protein sequence was inferred based on the gene coding sequence.

[0045] The cloning agarose gel electrophoresis results of the CaTPS2 gene are as follows: Figure 1 As shown in Figure A, a single band of the predicted size was obtained from cloning. Sequencing alignment revealed the coding sequence (CDS) of the CaTPS2 gene, as shown in SEQ ID NO:1, totaling 1737 bp. It is inferred to encode 579 amino acids, with the amino acid sequence shown in SEQ ID NO:2. The inferred protein molecular weight is 66.96 kDa, and the isoelectric point (pI) is 6.00. Homology analysis of the CaTPS2 amino acid sequence is shown below. Figure 1As shown in Figure B, the gene sequence contains a conserved DDXXD sequence. SMART analysis of the conserved domains of CaTPS2 revealed that it contains two conserved sequences: an N-terminal domain (PF01397) and a C-terminal active domain (PF03936). Figure 1 C), therefore, CaTPS2 is identified as a member of the terpene synthase family.

[0046] Example 2: Expression analysis of the CaTPS2 gene

[0047] Select different organs, different developmental stages of the flower, and different parts of the flower from the ginger lily flower. Figure 2 Total RNA was extracted using the Trizol method (TaKaRa) for A) and the Hieff qPCR SYBR Green Master Mix (low Rox) method. The specific principles of the method are detailed in the instruction manual. Primers for real-time quantitative PCR were designed using Primer Premier 5.0 software. Following the principles of quantitative PCR primer design, primers were designed using Primer Premier 5.0. The primers were then tested for mismatches, primer dimers, and amplification efficiency using quantitative PCR. The optimal primer pair was selected: P1: 5'-ATGGCTCCCGCCTCTGCTTATCAAAC-3' (as shown in SEQ ID NO:5). P2: 5'-TTGCTTCTGCAACTGAAGCGGCCT-3' (as shown in SEQ ID NO:6). The internal reference gene Actin was designed using Primer Premier 5.0 according to the design principles of Real-time PCR primers. The primers were: Actin-P1: 5'-GAGCATGGAATTGTCAGCAA-3' (as shown in SEQ ID NO:7) and Actin-P2: 5'-AGGGGCTTCAGTGAGCAATA-3' (as shown in SEQ ID NO:8).

[0048] Real-time PCR was used for detection, and a standard curve was constructed to screen samples based on their amplification efficiency (E) within the range of 90-110%. Using cDNA from each sample as a template, real-time PCR was performed on an ABI 7500 instrument. Each sample had three biological replicates and three technical replicates, with ddH2O as a negative control. The reaction mixture consisted of 10.0 μL Hieff qPCR SYBR Green Master Mix (Low Rox), 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), 2.0 μL cDNA, and 7.2 μL ddH2O. The detection program was 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 55℃ annealing for 20 s, and 72℃ extension for 20 s, for 40 cycles. After the reaction, 2... －△△Ct The method (Livak et al., 2001) was used to analyze the data and calculate the expression of CaTPS2 in different samples of ginger lotus.

[0049] Gene expression analysis results as follows Figure 2 As shown in Figure B, it can be seen that CaTPS2 expression is highest in the flower, with a small amount expressed in fertile bracts, and almost no expression in sterile bracts and leaves, consistent with the release levels of nerolidol and linalool. In different parts of the flower, CaTPS2 expression is highest in the petals, followed by the lip, and extremely low in the lateral petals and column, largely consistent with the release pattern of linalool, but slightly different from the release pattern of nerolidol in the column. At different developmental stages of the flower, CaTPS2 expression is highest in full bloom, followed by half-open, and lower in the bud and senescence stages, consistent with the release patterns of linalool and nerolidol. These results indicate that CaTPS2 is a gene involved in regulating the synthesis of terpenoids in ginger lily.

[0050] Example 3: CaTPS2 prokaryotic expression and in vitro functional analysis

[0051] 1. Carrier Construction

[0052] Based on the obtained coding region of the CaTPS2 gene, PCR amplification was performed using homologous recombination primers containing BamHI and HindIII restriction sites: F: 5'-gccatggctgatatcggatccATGGCTCCCGCCTCT GCT-3' (as shown in SEQ ID NO: 9); R: 5'-ctcgagtgcggccgcaagcttTTGTTTAAACAA CAATAAGTTTATATACTCCTT-3' (as shown in SEQ ID NO: 10).

[0053] The pET-32a prokaryotic expression vector was double-digested with BamHI and HindIII restriction enzymes, and the large fragment was recovered by 1% agarose gel extraction. II. Homologous recombination of the gene fragment and the vector was performed, adjusting the vector amount to 0.03 pmol and the insert amount to 0.06 pmol, following the instructions. The ligation product was transformed into E. coli DH5α competent cells, and the recombinant prokaryotic expression vector was obtained after identification by bacterial PCR and sequencing.

[0054] 2. Recombinant protein expression

[0055] Rosetta (DE3) competent cells were transformed with identified recombinant plasmid DNA. Single colonies were picked and inoculated into 5 mL of fresh LB broth (containing 100 mg / L Apm) and cultured overnight at 37°C and 180 rpm. 100 μL of the seed culture was then transferred to 100 mL of fresh LB broth (containing 100 mg / L Apm) and cultured at 37°C and 180 rpm until OD500. 600 The pH value was 0.4–0.6. 10 μL of IPTG (1M) was added, and the cells were induced at 16℃ for 20 h. A control group without IPTG induction was also included. Cells were collected by centrifugation, resuspended in 5 mL of lysis buffer, cooled, and then sonicated on ice. The cells were centrifuged at 12000 rpm at 4℃ for 10 min. The supernatant was transferred to a new centrifuge tube, the precipitate was washed once with double-distilled water, and then resuspended in 5 mL of lysis buffer. 16 μL of both supernatant and precipitate were collected and stored at -20℃ for SDS-PAGE electrophoresis analysis. A 12.5% ​​SDS-PAGE gel was prepared and loaded sequentially. Electrophoresis was performed on the stacking and separating gels at 80V and 130V respectively. After electrophoresis, Coomassie brilliant blue staining was performed for 30 min, followed by destaining with destaining solution for 24 h. The results were observed and recorded.

[0056] 3. Purification of recombinant proteins

[0057] Pack 0.5 mL of Ni-NTA resin into the chromatography column. After the resin has precipitated, drain the internal liquid, add 5 mL of ddH2O, and wash three times repeatedly. Then add 5 mL of Wash buffer and wash three times repeatedly. Pre-cool at 4°C. Add 5 mL of cell lysis supernatant to the pre-cooled chromatography column, mix thoroughly, and incubate at 4°C on a low-speed shaker for 1 h. Collect the eluent in a centrifuge tube and label it. Take 20 μL of the eluent for SDS-PAGE analysis. Add 2 mL of Wash buffer and elute the chromatography column three times, collecting 16 μL of each eluent fraction for SDS-PAGE analysis. Wash the column four times with 0.5 mL of Elution buffer, collecting each eluent fraction sequentially in different collection tubes, and then take 16 μL of each for SDS-PAGE analysis. Add the eluted protein to an ultrafiltration tube and centrifuge at 5000 rpm for 10 min at 4°C. Then add 2 mL of reaction buffer, centrifuge at 5000 rpm for 10 min at 4 °C, and repeat three times. Collect the ultrafiltration solution and transfer it to a pre-cooled centrifuge tube, add an equal volume of pure glycerol and mix well by pipetting. Aliquot the solution into 200 μL tubes, take 16 μL for SDS-PAGE analysis, and store the remainder at -80 °C for later use.

[0058] 4. Identification of enzyme catalytic function

[0059] Add 20 μL each of 30 mM HEPES (pH 7.5), 50 mM DTT, and 25 mM MgCl2, along with 20 μL of protein extraction buffer and 1 μL of GPP / FPP, and ddH2O. 119 μL was added to make a final volume of 200 μL and sealed in a sample vial. After sealing, the sample was reacted at 28 °C for 1 h. A 75 μm polydimethyloxane (PMDS) extraction fiber was then inserted into the glass vial, and headspace solid-phase microextraction was performed for 1 h. After the reaction, the extraction fiber was placed in a gas chromatograph-mass spectrometer for analysis. The gas chromatographic conditions were as follows: HP-1NNOWAX column (30 m × 0.25 mm); high-purity helium as carrier gas, split ratio 20:1, column inlet pressure 50 Pa, flow rate 1 mL / min; sampling time 2 min; temperature program: column initial temperature 45 °C, held for 2 min, increased to 80 °C at a rate of 5 °C / min, held for 1 min, and then increased to 250 °C at a rate of 10 °C / min, held for 5 min. The mass spectrometry conditions were as follows: GC-MS interface temperature 220℃, electron impact source EI, 350V; ion source temperature 170℃; electron energy 70eV; scan mass range 35–335 aum. The acquired mass spectra were analyzed using the WILLEY / MAINLIB library.

[0060] CaTPS2 gene prokaryotic expression results and in vitro enzyme activity identification, as follows: Figure 3As shown, the SDS-PAGE gel electrophoresis results of the CaTPS2 recombinant protein showed that the expression size of the CaTPS2 recombinant protein was approximately 67 kDa, which is close to the expected size. Figure 3 A). When FPP was used as a substrate, the product of the in vitro enzyme-catalyzed reaction of pET-32a-CaTPS2 was identified by mass spectrometry as the sesquiterpene nerolidol, and when GPP was used as a substrate, it catalyzed the formation of the monoterpene linalool. Figure 3 B). This indicates that the CaTPS2 gene encodes an enzyme that catalyzes the synthesis of terpenoids nerolidol and linalool from FPP and GPP.

[0061] In summary, this invention is the first to clone a novel terpene synthase gene, CaTPS2, from *Nelumbo nucifera*. This gene catalyzes the formation of terpene compounds nerolidol and linalool from FPP and GPP, playing a crucial role in increasing the terpene content and resistance of plants. The terpene synthase gene CaTPS2 studied in this invention can be used to prepare nerolidol and linalool, and further to prepare essential oils, fragrances, and pharmaceuticals containing nerolidol and linalool. Constructing the CaTPS2 gene fragment into a plant expression vector allows for the exogenous transformation of other plant materials, thereby obtaining transgenic materials containing terpene fragrance genes, providing an effective method for cultivating aromatic and medicinal plants.

[0062] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A ginger lotus terpene synthase gene CaTPS2 Its characteristics are, The coding sequence of the gene is shown in SEQ ID NO:

1.

2. A ginger lotus terpene synthase CaTPS2, characterized in that, The amino acid sequence of the synthase is shown in SEQ ID NO:

2.

3. The ginger lotus terpene synthase gene according to claim 1 CaTPS2 Or the application of the ginger terpene synthase CaTPS2 as described in claim 2 in the preparation of nerolidol and / or linalool, characterized in that, Using farnesyl pyrophosphate (FPP) and geraniyl pyrophosphate (GPP) as substrates, via the ginger lotus terpene synthase gene... CaTPS2 Or, the ginger terpene synthase CaTPS2 catalyzes the production of nerolidol and / or linalool.

4. A recombinant vector, characterized in that, Contains the ginger lotus terpene synthase gene as described in claim 1 CaTPS2 .

5. A recombinant bacterium comprising the recombinant vector of claim 4.

6. A method for preparing nerolidol and / or linalool, characterized in that, Using farnesyl pyrophosphate (FPP) and geraniyl pyrophosphate (GPP) as substrates, nerolidol and / or linalool are produced by the catalysis of the ginger terpene synthase CaTPS2 described in claim 2.

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