A zizyphus oleifera monoterpene synthase aotps1 gene, and the encoded protein and application thereof

Abstract

The application discloses a gene of eudesmolsynthase AoTPS1 and an application of the gene and a coded protein, and provides a gene AoTPS1 for regulating biosynthesis of a monoterpenoid compound eudesmol, wherein a nucleotide sequence is shown as SEQ ID NO. 1, the total length of the sequence is 1785 bp, 594 amino acids are coded, the molecular weight is 69.03 kDa, and a coded amino acid sequence is shown as SEQ ID NO. 2. The eudesmolsynthase AoTPS1 protein has the function of catalyzing synthesis of eudesmol in vitro, meanwhile, overexpression of the eudesmolsynthase AoTPS1 gene by using a genetic transformation technology can significantly increase the content of eudesmol in monoterpenoid volatile oil compounds. Therefore, the AoTPS1 gene is a key gene for regulating synthesis of volatile oil of Eucommia ulmoides Oliv., and can significantly promote biosynthesis of eudesmol in monoterpenoid volatile oil and improve the medicinal value of volatile oil of Eucommia ulmoides Oliv.

Description

Technical fields:

[0001] This invention belongs to the field of medicinal plant genetic engineering technology, specifically relating to the application of an Alpinia oxyphylla monoterpene synthase AoTPS1 gene and its encoded protein in regulating the biosynthesis of eucalyptol. Background technology:

[0002] Alpinia oxyphylla, the dried, mature fruit of a perennial plant belonging to the genus Alpinia of the ginger family, is known for its effects of warming the kidneys, consolidating essence, reducing urination, warming the spleen, stopping diarrhea, and reducing salivation. It is one of the "Four Great Southern Medicines" along with Areca catechu, Amomum villosum, and Morinda officinalis, and is mainly distributed in Hainan and southern Guangdong. The quality control of the traditional Chinese medicine Alpinia oxyphylla is mainly based on the content of volatile oils. It is rich in compounds such as 1,8-cineole, β-pinene, α-pinene, β-myrcene, camphor, and γ-terpinene (Van HT, Thang TD, Luu TN, Doan VD. An overview of the chemical composition and biological activities of essential oils from Alpinia genus (Zingiberaceae). RSCAdv.2021,11:37767-37783), with 1,8-cineole having the highest content. Eucalyptol, also known as eucalyptol or 1,8-cineole, is a colorless liquid with a pungent and cold taste, and a camphor-like odor. Studies have shown that eucalyptol has antibacterial, anti-inflammatory, anti-asthmatic, antioxidant, analgesic, and antispasmodic effects against influenza, colds, other respiratory infections, rhinitis, and sinusitis (Wang Y, Zhen D, Fu D, Fu Y, Zhang X, Gong G, Wei C. 1,8-cineole attenuates cardiac hypertrophy in heart failure by inhibiting the miR-206-3p / SERP1 pathway. Phytomedicine. 2021, 91:153672). Therefore, the discovery and identification of key enzyme genes encoding eucalyptol biosynthesis has significant medicinal and economic value.

[0003] Eucalyptol is a bicyclic monoterpene compound whose biosynthetic pathway in plants is relatively conservative, mainly catalyzed in chloroplasts / plasts. First, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) condense under the catalysis of geranyldiphosphate synthase (GPPS) to form geranylpyrophosphate (GPP). GPP is then cyclized by 1,8-cineole synthase (CINS) to form eucalyptol. (Karuppiah V, Ranaghan KE, Leferink NGH, Johannissen LO, Shanmugam M, NíCheallaigh A, Bennett NJ, Kearsey LJ, Takano E, Gardiner JM, van der Kamp MW, Hay S, Mulholland AJ, Leys D, Scrutton NS. Structural basis of catalysis in the bacterial monoterpene synthases linalool synthase and) 1,8-cineole synthase. ACS Catal. 2017, 7(9): 6268-6282; Ali M, Alshehri D, Alkhaibari AM, Elhalem NA, Darwish DBE. Cloning and characterization of 1,8-cineole synthase (SgCINS) gene from the leaves of Salvia guaranitica plant. Front Plant Sci. 2022, 13: 869432). Therefore, eucalyptol synthase is a key enzyme in the biosynthesis of eucalyptol and belongs to the monoterpene synthase family. Currently, the eucalyptol synthase genes that have been discovered and identified are mainly concentrated in bacteria, fungi, and Arabidopsis thaliana plants. However, there are few studies on plants of the genera Eucalyptus, Laurel, and Alpinia, which have high eucalyptol content. Therefore, mining monoterpene synthases or eucalyptol synthases from the traditional Chinese medicine Alpinia oxyphylla can not only expand the gene resources of eucalyptol synthases, but also provide an efficient protease source for building microbial engineered bacteria that produce high yields of eucalyptol volatile oil. Summary of the Invention:

[0004] The purpose of this invention is to provide a gene and encoded protein of monoterpene synthase AoTPS1, which can promote the biosynthesis of monoterpene volatile oil - eucalyptol.

[0005] To achieve the above-mentioned objective, the present invention provides a monoterpene synthase AoTPS1 that regulates the biosynthesis of monoterpene volatile oil-eucalyptol, wherein AoTPS1 has the nucleotide sequence shown in SEQ ID NO.1.

[0006] The present invention provides a protein encoded by the above-mentioned Alpinia oxyphylla monoterpene synthase AoTPS1, the protein having an amino acid sequence as shown in SEQ ID NO.2.

[0007] The present invention also provides a recombinant vector containing the above-mentioned AoTPS1, wherein the recombinant vector is pET28-AoTPS1 and pCAMBIA1301-AoTPS1.

[0008] The present invention also provides an engineered bacterium containing the recombinant vector, wherein the engineered bacterium is Escherichia coli BL21 containing the pET28-AoTPS1 recombinant vector and Agrobacterium GV3101 (pSoup-p19) containing pCAMBIA1301-AoTPS1.

[0009] Another object of the present invention is to provide the application of the above-mentioned Alpinia oxyphylla monoterpene synthase AoTPS1 gene in promoting the in vitro production of the monoterpene compound eucalyptol through an Escherichia coli expression system.

[0010] Another object of the present invention is to provide the application of the above-mentioned monoterpene synthase AoTPS1 in promoting the accumulation of oleol in Eucalyptus oxyphylla leaves.

[0011] Another object of the present invention is to provide the application of the above-mentioned Alpinia oxyphylla monoterpene synthase AoTPS1 in Alpinia oxyphylla improvement breeding, wherein the application is preferably the application of Alpinia oxyphylla monoterpene synthase AoTPS1 in genetic engineering breeding for improving the quality of Alpinia oxyphylla volatile oil.

[0012] This invention provides an Alpinia oxyphylla monoterpene synthase AoTPS1 gene and its encoded protein, belonging to the field of medicinal plant genetic engineering technology. The nucleotide sequence of the Alpinia oxyphylla monoterpene synthase AoTPS1 gene is shown in SEQ ID NO.1. The full-length sequence is 1785 bp, encoding 594 amino acids, with a molecular weight of 69.03 kDa. The encoded amino acid sequence is shown in SEQ ID NO.2. Alpinia oxyphylla monoterpene synthase AoTPS1 possesses conserved functional domains DDXXD, RRX8W, and NSE / DTE, belonging to the TPS-b subfamily, which is a subfamily specific to monoterpene synthases in angiosperms. The AoTPS1 gene is expressed in the roots, leaves, and fruits of Alpinia oxyphylla, with the highest expression level, especially in the mid-fruit stage. Detection at different origins showed a significant positive correlation between the expression level of the AoTPS1 gene and the content of eucalyptol in Alpinia oxyphylla fruits. In this invention, the AoTPS1 protein of Alpinia oxyphylla has the function of catalyzing the synthesis of eucalyptol in vitro. Furthermore, overexpression of the AoTPS1 gene using genetic transformation technology can significantly increase the content of eucalyptol in monoterpene volatile oil compounds. This indicates that the AoTPS1 gene is a key gene regulating the synthesis of Alpinia oxyphylla volatile oil, and can significantly promote the biosynthesis of eucalyptol in monoterpene volatile oil, thereby enhancing the medicinal value of Alpinia oxyphylla volatile oil. Attached image description:

[0013] Figure 1 This is an electrophoresis image of the AoTPS1 gene amplification. The left side represents the AoTPS1 gene; the right side shows the nucleic acid molecular weight standards, with the bands from top to bottom being 2000, 1500, 1000, 750, 500, 250, and 100 bp.

[0014] Figure 2 This is the secondary structure of the aotropin monoterpene synthase AoTPS1 protein. Blue indicates α-helix, red indicates extended backbone, green indicates β-turn, and purple indicates random coil.

[0015] Figure 3 The tertiary structure of the Alginate monoterpene synthase AoTPS1 protein.

[0016] Figure 4 Phylogenetic analysis was performed on the AoTPS1 protein and its relationship to previously reported members of the terpene synthase family. The AoTPS1 protein clustered in the TPS-b subfamily and was most closely related to the Dendrobium officinale geranium synthase DoGES1 protein.

[0017] Figure 5The expression levels of the AoTPS1 gene in different plant parts and at different developmental stages of fruit were represented. Plant parts included roots, leaves, and fruit; fruit developmental stages included early, middle, and late stages. Each data point is represented by bars, which indicate ± standard error (n ≥ 10). Different lowercase letters indicate significant differences at p < 0.01. Figure 6 The expression levels of the AoTPS1 gene in different production areas (Guangdong, Hunan, and Guangxi) are represented by bar for each data set. The bar represents the mean ± standard error (n≥10), and different lowercase letters indicate significant differences between different production areas at p<0.01.

[0018] Figure 7 This is a schematic diagram of the pET28-AoTPS1 overexpression vector. The accession number for pET28 is KJ782405.

[0019] Figure 8 SDS-PAGE electrophoresis image of AoTPS1 protein and its in vitro catalytic formation of eucalyptol. The bar represents ± standard error (n≥6) for each data set. ** indicates a significant difference between the control and treatment groups as found in Student's t-test at p<0.01. Figure 9 This is a schematic diagram of the pCABIA1301-AoTPS1 overexpression vector. The accession number for pCABIA1301 is AF234297.

[0020] Figure 10 To investigate the effect of AoTPS1 gene overexpression on the increase of the monoterpene compound eucalyptol content. For each data point, bar represents ± standard error (n≥6), and ** indicates a significant difference (p<0.01) between the control group and the overexpression group as determined by Student's t-test.

[0021] Figure 11 This is the mass spectrum of the eucalyptol standard in GC-MS / MS analysis. Detailed Implementation

[0022] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0023] Unless otherwise specified in the following examples, all experimental methods can be performed using conventional methods, such as those described in J. Sambrook et al.'s *Molecular Cloning: A Laboratory Manual*, F. Osborne et al.'s *A Concise Laboratory Manual of Molecular Biology*, or the manufacturer's instructions for use of the products employed.

[0024] The materials used in this example: *Alpinia oxyphylla* was cultivated at the Medicinal Botanical Garden of Zhejiang University of Traditional Chinese Medicine (N30°5′, E119°53′; Hangzhou, China); the polysaccharide and polyphenol plant RNA extraction kit was purchased from Huayueyang Biotechnology Co., Ltd. (catalog number: 0416-50); the reverse transcriptase M-MLV kit was purchased from Promega (catalog number: M1701); *Escherichia coli* DH5α (catalog number: DL1001) and BL21 (catalog number: EC1002) were purchased from Shanghai Weidi Biotechnology Co., Ltd.; the HiPure Gel PureMicro Kit was purchased from Guangzhou Meiji Technology Co., Ltd. (catalog number: D2110-02); 2× Hieff PCR MasterMix was purchased from YEASEN (product number: 10136ES03); The HD Cloning Kit was purchased from Takara (catalog number: 639648); pMD18-TVector was purchased from Takara (catalog number: D101A); the SYBR Premix Ex Taq Kit was purchased from Takara (catalog number: DRR420A); and TaKaRa LATaq was purchased from Takara (catalog number: RR52A). LB, MS, and SD media were commonly used media in this field, and their formulations were based on J. Sambrook et al.'s *Molecular Cloning: A Laboratory Manual*. Unless otherwise specified, all materials and reagents used in the following examples were commercially available.

[0025] Example 1: Cloning and Bioinformatics Analysis of the Alpinia oxyphylla Monoterpene Synthase AoTPS1 Gene

[0026] (1) Extraction of total RNA from Alpinia oxyphylla fruit and synthesis of the first strand of cDNA

[0027] 100 mg of freshly harvested Alpinia oxyphylla fruit was ground into powder under liquid nitrogen. Total RNA was extracted from the fruit using a polysaccharide and polyphenol plant RNA extraction kit (Huayueyang Biotechnology Co., Ltd., catalog number: 0416-50). The RNA was analyzed using a 1.0% agarose gel electrophoresis system (Biorad, California, USA) and NanoDrop gel electrophoresis. TM The content and purity of total RNA from Alpinia oxyphylla fruit were determined using a 2000°C micro-volume spectrophotometer (Thermo Scientific, Wisconsin, USA). 1000 ng of purified total RNA from Alpinia oxyphylla fruit was used to synthesize the first strand of cDNA according to the instructions of the reverse transcriptase M-MLV kit (Promega, catalog number: M1701). The reaction product was diluted to the required concentration and stored at -80°C.

[0028] (2) Amplification and sequence analysis of the AoTPS1 gene, an enzyme in the production of Alpinia oxyphylla monoterpenes.

[0029] Using the obtained Alpinia oxyphylla fruit cDNA as a template, Primer ATGTCTACTCGTCAAGCAATCT and Primer CTAGATTTGGATGGGTTCGAGT were designed for the Alpinia oxyphylla monoterpene synthase AoTPS1 gene using Primer Premier 5.0 (Premier Biosoft, California, USA). The corresponding upstream primer ATGTCTACTCGTCAAGCAATCT and downstream primer CTAGATTTGGATGGGTTCGAGT were then applied using 2×Hieff The gene sequence of the Alpinia oxyphylla monoterpene synthase AoTPS1 was obtained by PCR amplification using PCR Master Mix (YEASEN, catalog number: 10136ES03), as shown in SEQ ID NO.1. PCR reaction system: 1.0 μL template, 2× Hieff PCR MasterMix 12.5 μL, upstream primer 1 μL, downstream primer 1 μL, and sterile ddH2O added to a total volume of 25 μL. PCR reaction program: 98℃ pre-denaturation for 3 min, followed by 35 cycles (98℃ 10 s, 60℃ 30 s, 72℃ 1 min), and final extension at 72℃ for 5 min. PCR products were detected using a 1.0% agarose gel electrophoresis system (Biorad, California, USA). The target fragment was recovered using the HiPureGel Pure Micro Kit (Guangzhou Meiji Technology Co., Ltd., catalog number: D2110-02) and ligated into a pMD18-T Vector (Takara, catalog number: D101A). The ligation product was transformed into *E. coli* DH5α (Shanghai Weidi Biotechnology Co., Ltd., catalog number: DL1001) using the heat shock method and plated on a plate containing 100 μg / mL of PCR MasterMix. -1 The ampicillin antibiotic was incubated overnight at 37°C on LB agar plates. Single colonies were picked as templates, and colony PCR was performed using the aforementioned upstream and downstream sequences as primers for verification. Positive clones were sent to Zhejiang Shangya Biotechnology Co., Ltd. for bacterial sequencing. The nucleotide sequence of AoTPS1 was obtained as shown in SEQ ID NO. 1.

[0030] The open reading frame of the nootropic monoterpene synthase AoTPS1 is 1785 bp. Figure 1 The protein, encoding 594 amino acids (amino acid sequence shown in SEQ ID NO.2), has a predicted molecular weight of 69.03 kDa and a molecular formula of C2. 3085 H 4800 N 842 O 913 S 23The theoretical isoelectric point (pI) is 5.41, the aliphatic index is 88.64, the instability index is 48.47, and the grand average of hydropathicity is -0.361, indicating that the Alpinia oxyphylla monoterpene synthase AoTPS1 protein is a hydrophilic protein. The Alpinia oxyphylla monoterpene synthase AoTPS1 protein contains a conserved Terpene_synth_N domain (PF01397; E-value 4×e) at positions 65-242. -48 Meanwhile, the conserved Terpene_synth_C functional domain (PF03936; E-value is 2.7×e) exists at positions 273-536. -97 According to the pLoc-mPlant subcellular localization prediction software, the Alpinia oxyphylla monoterpene synthase AoTPS1 protein is located in chloroplasts.

[0031] Analysis using the biological software SOPM showed that the secondary structure of the Alpinia monoterpene synthase AoTPS1 protein contains 66.84% alpha helix, 3.37% beta turn, 26.43% random coil, and 3.37% extended strand. Figure 2 The tertiary structure of the aoTPS1 protein, an enzyme in the alginic monoterpene synthase, was deduced using SWISS-MODEL software as follows: Figure 3 As shown, its GMQE value is 0.88, and the sequence homology reaches 83.5%.

[0032] Further phylogenetic analysis showed that the Alpinia oxyphylla monoterpene synthase AoTPS1 protein belongs to the TPS family and possesses the conserved functional domains DDXXD, RRX8W, and NSE / DTE of terpene synthase family members. AoTPS1 protein clusters in the TPS-b subfamily, a subfamily specific to angiosperm monoterpene synthases, and is closely related to the Dendrobium officinale geranium synthase DoGES1 protein and the grape α-terpineol synthase protein. Figure 4 ).

[0033] Example 2: Analysis of the expression pattern of the AoTPS1 gene for improving intelligence

[0034] Different tissues (roots, leaves, and fruits) of Alpinia oxyphylla were collected, and total RNA was extracted and reverse transcribed according to the method in Example 1-(1). Real-time quantitative PCR was performed using upstream primers (CGTCGACAGATGGGACTTAAC) and downstream primers (CATCACCCGGTAACCTTCTTC) designed based on the AoTPS1 gene. Amplification was performed using the SYBR Premix ExTaq Kit (Takara, catalog number: DRR420A). The reaction program was 95℃ denaturation for 20s, followed by 40 cycles (95℃ 20s, 60℃ 1min). Data were obtained after running a 480 Instrument real-time quantitative PCR test (Roche Diagnostics, Mannheim, Germany), and then analyzed using 2... -ΔΔCT The relative expression levels of each sample were calculated. The internal control was the EF-1α gene, with the upstream primer shown as GTCGTGTTGAGACTGGAATGT and the downstream primer shown as TGGTGCATCTCTACGGATTTG.

[0035] The results showed that the AoTPS1 gene was expressed in the roots, leaves, and fruits of Alpinia oxyphylla, with the highest expression level observed in the fruits. Figure 5 Furthermore, we analyzed the expression level of the AoTPS1 gene at different developmental stages (early, middle, and late) of the fruit. The results showed that as the fruit matured, the expression level of the AoTPS1 gene first increased and then decreased, reaching its highest level in the middle stage of fruit development. Figure 5 This is consistent with the variation trend of eucalyptol content in different developmental stages of Alpinia oxyphylla, indicating that the AoTPS1 gene may be involved in regulating the biosynthesis of eucalyptol in Alpinia oxyphylla.

[0036] Example 3: Analysis of eucalyptol content in Alpinia oxyphylla fruits from different origins

[0037] The content of eucalyptol in Alpinia oxyphylla from different origins was determined according to the Alpinia oxyphylla section of the Pharmacopoeia of the People's Republic of China. 1.0 g of powdered Alpinia oxyphylla from different origins (passed through a No. 4 sieve) was accurately weighed and placed in a stoppered conical flask. 5 mL of anhydrous ethanol was accurately added, and the mixture was sonicated for 30 minutes. The filtrate was then used for gas chromatography detection. A capillary column with polyethylene glycol 20000 as the stationary phase was used. The initial temperature was 45℃, maintained for 10 minutes, increased to 100℃ at a rate of 2℃ / min, and then increased to 200℃ at a rate of 10℃ / min, maintained for 5 minutes. The injection port temperature was 220℃, the detector temperature was 250℃, and the split ratio was 1:100. 1 μL each of the test solution and the standard solution (eucalyptol, Sigma-Aldrich, catalog number: 00020590) were injected into the gas chromatograph, and the chromatograms were recorded. The eucalyptol content can be calculated using the peak area normalization method. Any chromatographic peak in the test solution chromatogram smaller than the main peak area of ​​the reference solution can be ignored (0.05%).

[0038] The results showed that the peak area RSD% of eucalyptol was 1.06%, and it achieved baseline separation from adjacent impurity peaks, meeting the testing requirements of the Pharmacopoeia of the People's Republic of China. The eucalyptol content in Alpinia oxyphylla fruit varied significantly from different producing areas (Guangdong Province, Hainan Province, and Guangxi Zhuang Autonomous Region), with the highest eucalyptol content observed in Guangdong Province. Figure 6 Furthermore, we analyzed the expression level of the AoTPS1 gene in Alpinia oxyphylla from Guangdong Province, Hainan Province, and Guangxi Zhuang Autonomous Region. The results showed that the expression level of the AoTPS1 gene was significantly positively correlated with the content of eucalyptol in Alpinia oxyphylla (Pearce correlation coefficient R). 2 =0.965, p<0.001). Therefore, the AoTPS1 gene regulates the accumulation of eucalyptol in Alpinia oxyphylla fruit.

[0039] Example 4: Obtaining the AoTPS1 protein and synthesizing eucalyptol in vitro

[0040] (1) Construction of a prokaryotic expression vector for monoterpene synthase AoTPS1

[0041] Based on the nucleotide sequence of the monoterpene synthase AoTPS1 (SEQ ID NO.1), upstream primers (AATGGGTCGCGGATCCATGTCTACTCGTCAAGCAATCTCC) and downstream primers (GTGCGGCCGCAAGCTTGATTTGGATGGGTTCGAGTATCAC) were designed at the BamHI and HindIII restriction sites, respectively, to amplify the AoTPS1 sequence with high fidelity. Simultaneously, the prokaryotic expression vector pET28 was digested with both BamHI and HindIII, and... The HD Cloning Kit (Takara, catalog number: 639648) was used to construct the recombinant expression vector pET28-AoTPS1 (…). Figure 7 (2) Expression and purification of pET28-AoTPS1 prokaryotic expression vector

[0042] The correctly sequenced recombinant plasmid pET28-AoTPS1 was transformed into E. coli competent cells BL21(DE3). Positive clones were screened and identified, and single clones were picked and added to 5 mL of culture medium, incubated at 37°C and 230 rpm for 12 h. The bacterial culture was then transferred 1:100 to 100 mL of culture medium and incubated at 37°C and 230 rpm for approximately 2.5 h. The OD value was then measured. 600 The pH value was between 0.4 and 0.6. IPTG (final concentration 1 mmol / L) was added for induction, and the cells were incubated overnight at 18°C ​​and 160 rpm. The induced bacterial culture was centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in 2 mL of pre-chilled Tris·HCl (25 mM, pH 7.4). The cells were collected. The cells were transferred to 50 mL centrifuge tubes and sonicated in an ice-water bath (coarse rotor, 5 s sonication, 5 s pause) for 40 min until the liquid was clear and non-viscous. After centrifugation at 4°C and 12000 rpm for 10 min, the supernatant was collected. 0.1 mL of Ni-NTA was added to the supernatant, and the mixture was incubated at 4°C and 200 rpm for approximately 1 h. The column was packed, and the flow through was collected. After removing impurities, 2 mL of wash buffer was added, and the process was repeated 4 times. 0.5 mL of elute buffer was added for elution, and the process was repeated 5–7 times, collecting the cells tube by tube. PD-10 was first activated with 25 mL of Tris·HCl, and then 2.5 mL of sample was added to PD-10 for desalting. 3.5 mL of Tris·HCl was added to collect the sample, and the nickel column was washed.

[0043] The purified AoTPS1 protein was detected by SDS-PAGE electrophoresis. The SDS-PAGE gel was prepared as follows: 12% separating gel (4 mL sterile water, 3.3 mL 30% acrylamide mixture, 2.5 mL 1.5 mol / L Tris (pH 8.8), 100 μL 10% SDS, 100 μL 10% ammonium persulfate, 4 μL TEMED), stacking gel (1.4 mL sterile water, 330 μL 30% acrylamide mixture, 250 μL 1 mol / L Tris (pH 6.8), 20 μL 10% SDS, 20 μL 10% ammonium persulfate, 2 μL TEMED). After adding the sample to the separating gel, mix gently and pipette into the glass plate interlayer. Add the gel to 1 cm from the top and seal with 1 mL of ethanol. After 30 min, discard the ethanol and add the stacking gel. Add 16 μL of protein sample to 4 μL of loading buffer, mix well, and heat to 100°C for 5 min. Load 20 μL of sample into each well. Electrophoresis is initially performed at 60 V for 30 min, then adjusted to 120 V until the bands separate to the bottom of the gel. After electrophoresis, stain and destain with Coomassie Brilliant Blue rapid staining solution until the bands are clearly visible. Figure 8 ).

[0044] (3) In vitro enzyme activity detection of AoTPS1 protein for Alpha-6

[0045] Take 100 μg of purified AoTPS1 recombinant protein, add 1 mL of prepared MOPSO buffer (pH 7.0, 5 mM dithiothreitol, 10 mM MgCl2, 10 mM GPP, total reaction volume 200 μL), mix the solution and incubate at 30 °C for 1 h, vortex for 5 min after reaction, centrifuge at 12000 rpm to separate the organic phase, and take 2 μL of product for GC-MS detection.

[0046] The product was added to an EP tube and concentrated to 500 μL using a nitrogen blower. After being transferred to an inner liner tube, it was detected by GC-MS (QP2010SE; Shimadzu Corporation, Tokyo, Japan). The conditions were: column type: HP-5 (30m × 0.25mm × 0.25μm), injection volume: 1 μL, injection port temperature: 240℃, carrier gas flow rate: 1 mL / min, GC column oven temperature program: 60℃ for 3 min, then increased to 240℃ at a rate of 4℃ / min, and held at 240℃ for 20 min. Mass spectrometry was performed in Scan mode with a mass-to-nucleus ratio range of 20-200. GC-MS results showed that, with the participation of the substrate GPP, the AoTPS1 recombinant protein produced a specific product, while the control group did not produce any product. Comparison with standards and mass spectral library searches revealed the production of eucalyptol, indicating that the AoTPS1 protein has the ability to catalyze the synthesis of eucalyptol in vitro. Figure 8 ).

[0047] Example 5: Overexpression of the AoTPS1 gene promotes the biosynthesis of eucalyptol.

[0048] (1) Construction of a vector for overexpressing monoterpene synthase AoTPS1

[0049] Based on the nucleotide sequence of the monoterpene synthase AoTPS1 (SEQ ID NO.1), upstream primers (CCATGATTACGAATTCATGTCTACTCGTCAAGCAATCTCC) and downstream primers (ATGCCTGCAGGTCGACGATTTGGATGGGTTCGAGTATCAC) were designed at the BamHI and HindIII restriction sites, respectively. The AoTPS1 sequence was obtained by PCR amplification. Simultaneously, the plant binary expression vector pCAMBIA1301 was digested with both BamHI and HindIII. pCAMBIA1301-AoTPS1 was constructed using the HD Cloning Kit (Takara, catalog number: 639648). Figure 9 (2) The recombinant plasmid pCAMBIA1301-AoTPS1 was transformed into Agrobacterium GV3101 (pSoup-p19).

[0050] The pCAMBIA1301-AoTPS1 recombinant plasmid was transformed into Agrobacterium GV3101 (pSoup-p19) using a heat shock method. The specific steps were as follows: 1 μg of recombinant plasmid pCAMBIA1301-AoTPS1 was mixed with 100 μL of Agrobacterium GV3101 (pSoup-p19) competent cells, incubated on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, and then rapidly transferred to a 37°C water bath for 5 min, followed by incubation on ice for 5 min. 700 μL of antibiotic-free LB medium was added, and the mixture was incubated on a shaker at 28°C and 100 rpm for 2–3 h. The culture was then plated onto 25 mL LB agar plates (containing 50 mg / mL of the medium). -1 Kanamycin). Invert the plate and incubate it in a 28°C incubator until colonies grow (about 48 hours). Select positive clones for subsequent experiments.

[0051] (3) Agrobacterium-mediated transient transformation of the AoTPS1 gene in Alpinia oxyphylla leaves

[0052] Positive clones containing pCAMBIA1301-AoTPS1 were picked and placed in 100 mL of LB liquid medium (containing 50 mg / mL). -1 Kanamycin), incubate at 28°C with shaking at 180 rpm for 16 h, until OD... 600=0.6~0.8. Collect the bacterial culture by centrifugation at 5000×g for 5 min at room temperature. Resuspend the precipitated Agrobacterium in 100 mL of osmotic buffer (0.2 mM acetylsalicylic acid, 10 mM MgCl2, 10 mM MES, pH 5.7), centrifuge at 5000×g for 5 min at room temperature, discard the supernatant, and adjust the OD with osmotic buffer. 600 ≈0.6, activated at 25℃ in the dark for 2 hours. Healthy leaves of *Alpinia oxyphylla* were selected, pre-cultured in the dark for 2 days, and then infected with activated bacterial solution containing the AoTPS1 gene for 10 minutes. After 24 hours of dark culture in a tissue culture room at 23±2℃, the leaves were cultured under light for 2 days. Positive plants were screened using semi-quantitative PCR for subsequent experiments. Uninfected leaves served as a control. Results showed that, compared with the control group, the content of eucalyptol was significantly increased in *Alpinia oxyphylla* leaves overexpressing AoTPS1 (≈0.6, 25℃ room temperature, dark, static). Figure 10 This indicates that the monoterpene synthase AoTPS1 has the activity of eucalyptol synthase in plants and can catalyze the synthesis of eucalyptol.

[0053] The specific sequence list information is as follows:

[0054] >AoTPS1 nucleotide sequence (SEQ ID NO.1)

[0055] ATGTCTACTCGTCAAGCAATCTCCATTTGCGCTCCTCCCATGATTTCCGTCCTCTCTCGTC

[0056] GGCCAATGATCGTTGTTGCCGTGGAGCACTGTGGCCAACGGACGTTCCGGCGAACTCTG

[0057] CAAGTCCGATCATGCAGTGCCACTAGTAATGTAGCTACCTTGCGGCGGTCTGGGAATTAT

[0058] CCGCAGAACATATGGACGGACGAGAGCGTGCAATCCCTCACGAGCACCTCCACGGAGC

[0059] AACGGGAAGAGAAACGCGAGAGAATAAACGTACTGAAGGAGCAGACGAGGAATCTGA

[0060] TACTCGAGCAGCAGCAAGTAGCAGAGCAGCTTCGACTGATCGACCACCTGCAACAGCT

[0061] CGGCGTGGCTTACCACTTCAAAGACGAGATCGCAGACGTTTTGAGTCGTCTTCATGCGT

[0062] CTTTAGACGGCGTGAGCTTGCAGCTGGAGGACGATCTCCATGCCACAGCGCTGCTCTTC

[0063] AGGCTCCTCAGAGCCAATGGCTTCTCTGTTTCACAAGATTTGTTCAAGACATTTAGAGAC

[0064] GAGAAGGGAAACTTCGAAGTTCGCTGTGAGGACCAGATCAGAGGGCTTCTGAGCCTCT

[0065] ACGAAGCTTCCTACCTCGAGAAGGAAGGAGAGATTTTGCTGAAGGAAGCCATGGATTTT

[0066] GCGACCGAGAAGCTGAAAGGATTCATGGAGGAGGGATCTGGCAGTCTCGGTCTCAGAG

[0067] AGCAGGTGGCCCATGCGCTGCAGCTTCCACTGAATTGGCGGCTGGAGAGAGTGCAGCA

[0068] CAGGTGGTTCATTGAAGCATGCCATGGTGACGGCACCATCAACCCTCTCCTGCTGGAAT

[0069] TTGCTAAGCTCGACTTCAATATTGTTCAGGACATATACAAGAGTGAACTCAGAGAGCTCT

[0070] CCAGATGGTGGTCAGACCTTGGGCTTTCAGAGGAGCTGCCATTTTTCAGAGACAGATTA

[0071] ACTGAGAACTATCTGTGGGCAGTCGGTTTCACCTATGAACCAGATAGCTGGAGATGCAG

[0072] AATGATCGAAACAAAGGCAATCTGTTTTATTACACTCATAGATGATATTTATGATGTCTAT

[0073] GGGACCTTAGACGAACTCCAGCTCTTCACTGATGCCGTCGACAGATGGGACTTAACTGC

[0074] AGTCGACGAGCTTCCAGAGTACATGAAGCTATGCTTCTTTGCACTATTCAACATGGTGCA

[0075] CGAAGAAGGTTACCGGGTGATGAAGGAGAAGGGCTTGAACATAGTGCCGAACTTAAAG

[0076] AGAACATGGGGAAAACAATGCAAAGCCTACTTTCAGGAAGCAAAATGGTTCCACCATG

[0077] GCCAAACCCCGAAGCTTAAGGAGTACTTGGAGAATGGATGGGTATCAGTCTCCGGTCCG

[0078] AACATTCTATTTAGTGCTTACTGTGCTTTCAAAGACTTAGATAGAGAGGCCTTGAAAAGT

[0079] TTCCCTAGTTACCATGTGATCACACGCTCCTCTAGCATGCTCCTCCGTCTTTACGATGATA

[0080] TGGGCACTTCGACGGATGAGATAGAAAGAGGCGATGTGGCGAAATTTATTCAATGCAGC

[0081] ATGCACGAAAAAGGTGTTTCAGAGGAAGTAGCACGGAGGGAGATAAGAGAATTGGTGA

[0082] AGGAACATTGGAGAGTAGTGAATGAAGCTCTTAGTTGGGATTCTCCATTTGAGGAGTAC

[0083] TTCAAGAATGTAGCAGTTAATCTTGCTCGGACGTCGCAGTTCTTTTATCAGAATGGGGAT

[0084] GGGTATGCCAAGGCGGATCGAGAAACTAAGTCTCAAATTATTTCTGTGATACTCGAACCC

[0085] ATCCAAATCTAG

[0086] >AoTPS1 Amino Acid Sequence (SEQ ID NO.2)

[0087] MSTRQAISICAPPMISVLSRRPMIVVAVEHCGQRTFRRTLQVRSCSATSNVATLRRSGNYPQN

[0088] IWTDESVQSLTSTSTEQREEKRERINVLKEQTRNLILEQQQVAEQLRLIDHLQQLGVAYHFK

[0089] DEIADVLSRLHASLDGVSLQLEDDLHATALLFRLLRANGFSVSQDLFKTFRDEKGNFEVRC

[0090] EDQIRGLLSLYEASYLEKEGEILLKEAMDFATEKLKGFMEEGSGSLGLREQVAHALQLPLN

[0091] WRLERVQHRWFIEACHGDGTINPLLLEFAKLDFNIVQDIYKSELRELSRWWSDLGLSEELPF

[0092] FRDRLTENYLWAVGFTYEPDSWRCRMIETKAICFITLIDDIYDVYGTLDELQLFTDAVDRWD

[0093] LTAVDELPEYMKLCFFALFNMVHEEGYRVMKEKGLNIVPNLKRTWGKQCKAYFQEAKWF

[0094] HHGQTPKLKEYLENGWVSVSGPNILFSAYCAFKDLDREALKSFPSYHVITRSSSMLLRLYD

[0095] DMGTSTDEIERGDVAKFIQCSMHEKGVSEEVARREIRELVKEHWRVVNEALSWDSPFEEYF

[0096] KNVAVNLARTSQFFYQNGDGYAKADRETKSQIISVILEPIQIAoTPS1 Nucleotide sequence amplification primers

[0097] upstream primer

[0098] ATGTCTACTCGTCAAGCAATCT

[0099] Downstream primer

[0100] The real-time quantitative PCR (RT-qPCR) primers used in CTAGATTTGGATGGGTTCGAGTAoTPS1

[0101] upstream primer

[0102] CGTCGACAGATGGGACTTAAC

[0103] Downstream primer:

[0104] CATCACCCGGTAACCTTCTTC

[0105] The upstream primer of the real-time quantitative PCR (RT-qPCR) primers used for the internal reference gene EF-1α

[0106] GTCGTGTTGAGACTGGAATGT

[0107] Downstream primer:

[0108] TGGTGCATCTCTACGGATTTG

[0109] Primers used for constructing recombinant plasmid pET28-AoTPS1

[0110] Upstream primer:

[0111] Downstream primer: AATGGGTCGCGGATCCATGTCTACTCGTCAAGCAATCTCC

[0112] Primers used for constructing the recombinant plasmid pCAMBIA1301-AoTPS1

[0113] Upstream primer:

[0114] CCATGATTACGAATTCATGTCTACTCGTCAAGCAATCTCC downstream primer:

[0115] ATGCCTGCAGGTCGACGATTTGGATGGGTTCGAGTATCAC.

Claims

1. A gene for an alginate monoterpene synthase, AoTPS1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the monoterpene synthase AoTPS1 gene as described in claim 1.

3. A recombinant vector containing the monoterpene synthase AoTPS1 gene as described in claim 1.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is pET28-AoTPS1 or pCAMBIA1301-AoTPS1.

5. An engineered bacterium containing the recombinant vector as described in claim 3 or 4.

6. The engineered bacteria according to claim 5, characterized in that, The engineered bacteria are Escherichia coli BL21 containing the pET28-AoTPS1 recombinant vector or Agrobacterium GV3101 (pSoup-p19) containing pCAMBIA1301-AoTPS1.

7. The application of the monoterpene synthase AoTPS1 according to claim 2 in promoting the biosynthesis of eucalyptol in Alpinia oxyphylla.

8. The application of the monoterpene synthase AoTPS1 gene as described in claim 1 in the breeding of Alpinia oxyphylla for improvement, wherein the application is the application of the monoterpene synthase AoTPS1 gene in genetic engineering breeding to promote the biosynthesis of eucalyptol in Alpinia oxyphylla.

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