Machilus rubescens TPS synthetase gene MnTPS6 and application thereof

Abstract

The invention belongs to but is not limited to the technical field of plant genetic engineering, and discloses a Nerolidol / Linalool synthetase gene MnTPS6 of machilus rubescens and an application of the Nerolidol / Linalool synthetase gene MnTPS6. The machilus nerolidol / Linalool synthetase gene MnTPS6 is a key gene for synthesis of the nerolidol / Linalool in plant genetic engineering, synthesis of the nerolidol / Linalool can be effectively regulated and controlled, expression protein of the machilus nerolidol / Linalool synthetase gene MnTPS6 is applied to synthesis and preparation of the nerolidol / Linalool, the yield is high, and the machilus nerolidol / Linalool synthetase gene MnTPS6 has important application value. Substrates of farnesyl pyrophosphate (FPP) and geranyl pyrophosphate (GPP) are added to the expression protein of the induced machilus nigrum Nerolidol / Linalool synthetase gene MnTPS6, the catalytic activity of the recombinant protein is detected, and a GC-MS (Gas Chromatography-Mass Spectrometer) detection component shows that the result shows that the MnTPS6 is a bifunctional enzyme for synthesizing Nerolidol / Linalool.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of plant genetic engineering technology, and particularly relates to a TPS synthase gene of *Machilus yunnanensis*. MnTPS6 And its applications. Background Technology

[0002] Nerolidol and Linalool are both important terpenoid compounds, widely used in fragrances, cosmetics, food additives, pharmaceutical intermediates, and biopesticides. Nerolidol has a soft floral and fruity aroma and is a key fragrance ingredient in high-end perfumes and skincare products; Linalool has a fresh lavender scent and is one of the most widely used terpenoid fragrance compounds globally. Both also possess antibacterial, anti-inflammatory, and insecticidal biological activities, showing significant application potential in the pharmaceutical and agricultural fields.

[0003] Currently, the acquisition of Nerolidol and Linalool mainly relies on natural plant extraction and chemical synthesis. Natural extraction methods are limited by factors such as plant growth cycles, environmental conditions, and harvesting seasons, resulting in problems such as low extraction efficiency, high costs, and unstable yields, making it difficult to meet the market's demand for large-scale production. While chemical synthesis methods can achieve large-scale production, the reaction conditions are harsh (high temperature, high pressure, strong acids and alkalis), easily producing toxic and harmful byproducts that pollute the environment. Furthermore, the purity and safety of the products are difficult to guarantee, which is inconsistent with the trend of green chemical development.

[0004] Terpene synthases (TPSs) are key rate-limiting enzymes in the biosynthetic pathway of terpenoids. Their function is to catalyze the polymerization of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to produce precursors such as geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP), converting them into specific terpenoid compounds. Among these, bifunctional TPSs, capable of simultaneously catalyzing the synthesis of two or more terpenoid products from GPP and FPP, possess greater application value due to their ability to achieve the simultaneous synthesis of multiple high-value terpenoids.

[0005] *Machilus chinensis*, an evergreen tree belonging to the genus *Machilus* in the family Lauraceae, is rich in terpenoids, suggesting the presence of highly efficient Nerolidol and Linalool synthase genes. However, to date, there are no reports on the cloning, identification, or application of Nerolidol and Linalool synthase genes from *Machilus chinensis*, limiting the development and utilization of this plant resource in linalool biosynthesis.

[0006] Therefore, cloning and identifying the Nerolidol and Linalool synthase genes from *Lysimachia christinae*, clarifying their functional characteristics, and then constructing an efficient biosynthetic system has become a key technical breakthrough that urgently needs to be achieved. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a Runan TPS synthase gene. MnTPS6 And its applications.

[0008] This invention provides a Nerolidol / Linalool synthase gene. MnTPS6 The Nerolidol / Linalool synthase gene of Runnan MnTPS6 The nucleotide sequence is shown in SEQ ID NO.1.

[0009] Another object of the present invention is to provide a Nerolidol / Linalool synthase gene. MnTPS6 The expressed protein is MnTPS6, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0010] Another object of the present invention is the Nerolidol / Linalool synthase gene of *Linalool*. MnTPS6 The method for preparing the expressed protein MnTPS6 includes: based on the Nerolidol / Linalool synthase gene of Runnan. MnTPS6 A prokaryotic expression vector was constructed, and the resulting expression vector was transformed into a host bacterium. The resulting host bacterium was then cultured to express the MnTPS6 protein.

[0011] Furthermore, based on the Nerolidol / Linalool synthase gene of Runnan... MnTPS6 The primer pairs designed during the construction of the prokaryotic expression vector are shown in SEQ ID NO.9 and SEQ ID NO.10, and the plasmid vector used is pET-32a.

[0012] SEQ ID NO.9: GCCATGGCTGATATCGGATCCGCAAATTACCACCCAAGCATTTGG SEQ ID NO.10: CTCGAGTGCGGCCGCAAGCTTAGGCCACTGACTTGGAACG Another object of the present invention is the Nerolidol / Linalool synthase gene of *Linalool*. MnTPS6 The application of the expressed protein in the preparation of Nerolidol / Linalool.

[0013] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: First, the Nerolidol / Linalool synthase gene provided by this invention MnTPS6 This gene is a key gene in plant genetic engineering for the synthesis of Nerolidol / Linalool, effectively regulating its synthesis. Its expressed protein is used in the high-yield synthesis of Nerolidol / Linalool, possessing significant application value. [The text then abruptly shifts to a different topic:] The induced Nerolidol / Linalool synthase gene of *Machilus chinensis*... MnTPS6 The expressed protein was supplemented with substrates farnesyl pyrophosphate (FPP) and geranyyl pyrophosphate (GPP), and the catalytic activity of the recombinant protein was detected. Gas chromatography-mass spectrometry (GC-MS) analysis showed that the catalytic product with FPP was Nerolidol, and the catalytic product with GPP was Linalool, indicating that MnTPS6 is a bifunctional enzyme for the synthesis of Nerolidol / Linalool.

[0014] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: The core technical solution of this invention revolves around a bifunctional terpene synthase gene. MnTPS6 The cloning, expression, and application of this technology can lead to the formation of industrialized technologies covering multiple fields such as microbial fermentation production and plant breeding, with significant expected returns and commercial value. In the fragrance and cosmetics industry, Nerolidol and Linalool are high-end fragrance raw materials with huge global demand. This technology can achieve the simultaneous and efficient synthesis of both products through microbial fermentation (prokaryotic / eukaryotic engineered bacteria). In the pharmaceutical and agricultural fields… MnTPS6 The gene-encoded protein products possess natural antibacterial, anti-inflammatory, and insecticidal activities, and can be used as intermediates for biopesticides or raw materials for pharmaceuticals, aligning with the development trends of green agriculture and natural medicines. The engineered microbial fermentation system built based on this technology can rapidly achieve large-scale production of high-value-added intermediates, expanding the industrial chain. In the field of plant breeding, [this technology will be applied to]... MnTPS6 Gene transfer into economic crops such as tobacco, lavender, and roses can cultivate new aromatic plant varieties rich in both Nerolidol and Linalool. These new varieties significantly improve the yield and quality of essential oils, driving the upgrading of the aromatic plant cultivation industry and possessing substantial social and industrial benefits.

[0015] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally: This invention is the first to obtain from Runnan ( Machilus nanmu A terpene synthase gene with dual catalytic function was cloned from [the gene]. MnTPS6As a high-quality aromatic plant of the Lauraceae family, the gene resources related to terpenoid synthesis of *Machilus chinensis* have not yet been systematically explored. There are no reports on the cloning, identification, and functional verification of Nerolidol and Linalool bifunctional synthase genes from *Machilus chinensis*, either domestically or internationally. This technology fills the gap in the development and utilization of *Machilus chinensis* gene resources and enriches the gene library of plant bifunctional terpenoid synthases. Attached Figure Description

[0016] Figure 1 PCR cloning MnTPS6 Gene electrophoresis diagram.

[0017] Figure 2 Schematic diagram of the Pfam domain of the TPS gene family.

[0018] Figure 3 : MnTPS6 A schematic diagram showing gene expression in the roots, stems, and leaves of *Machilus chinensis*.

[0019] Figure 4 SDS-PAGE electrophoresis image of the purified product of pET-32a-MnTPS6 recombinant protein; lane 1 is the precipitate suspension after ultrasonic disruption; lane 2 is the supernatant after ultrasonic disruption; lanes 3-9 are 500 mM imidazole elution buffer.

[0020] Figure 5 : Schematic diagram of the in vitro catalytic reaction products of MnTPS6 protein detected by GC-MS; where Figure A shows the catalytic products of the reaction between MnTPS6 and FPP; Figure B shows the catalytic products of the reaction between MnTPS6 and GPP.

[0021] Figure 6 Mass spectrometry comparison images; where image A is the Nerolidol mass spectrometry comparison image; image B is the Linalool mass spectrometry comparison image. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] This invention provides a Nerolidol / Linalool synthase gene. MnTPS6 The Nerolidol / Linalool synthase gene of Runnan MnTPS6 The nucleotide sequence is shown in SEQ ID NO.1.

[0024] SEQ ID NO.1: This invention provides a Nerolidol / Linalool synthase gene. MnTPS6 The expressed protein is MnTPS6, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0025] SEQ ID NO.2: MSLIIQSLPHWSRIPPRPPQLSQFQNSSRPKPVIQASQVQRNALQIARRSANYHPSIWDPQYIESLTSPYGDECFGARLEELKLEAKRLLEATVEPLSWLELVDTIQRLGVAYHFEDEIKEGLDGVYGVGAHAGDDLYTAALRFR LLRQHGYGVAPDIFNKFLENERTFKACTSLDAKGLLSLYEASHTMIHGEEVLEEAKEFSVKHLNYLMGNLQSNLREQVQHALEMPLHWRMPRLEAKNYIDVNGRSDERNMVLLELARLDFNFVQSKHQEELREVSRWWRDLGLAKK LEFSRDRLVENYLWAMGIAPEPKFSNCRKGLTKLISILTVIDDIYDVYGSLDELELFTEAVKRWDIEALETLPEYMKICYMALFNFVHEVSYDILKDYGWNILPFIREEWKRLCMSYLVEAKWFGNGNKPALDEYLRNGWISVGG PVAMVHAYFLQGRPIRKDSINFLDHGSELIYWSSVATRLNDDLGTSKAEMKRGDVPKAVECHMIQTGGSHEDAREHIKGLVRDCWKKMNEECLKCSLPYSYVETVLNMVRTAHCIYQHGDGIGTSTGVTQDRIISLICEPVPSQWP This invention provides the Nerolidol / Linalool synthase gene from *Linalool*. MnTPS6 The method for preparing the expressed protein MnTPS6 includes: based on the Nerolidol / Linalool synthase gene of Runnan. MnTPS6 A prokaryotic expression vector was constructed, and the resulting expression vector was transformed into a host bacterium. The resulting host bacterium was then cultured to express the MnTPS6 protein.

[0026] The Nerolidol / Linalool synthase gene based on Runnan MnTPS6The primer pairs designed during the construction of the prokaryotic expression vector are shown in SEQ ID NO.9 and SEQ ID NO.10, and the plasmid vector used is pET-32a.

[0027] SEQ ID NO.9: GCCATGGCTGATATCGGATCCGCAAATTACCACCCAAGCATTTGG SEQ ID NO.10: CTCGAGTGCGGCCGCAAGCTTAGGCCACTGACTTGGAACG This invention provides the Nerolidol / Linalool synthase gene from *Linalool*. MnTPS6 The application of the expressed protein in the preparation of Nerolidol / Linalool.

[0028] Example 1 MnTPS6 Gene cloning 1. Extraction of total RNA from *Machilus yunnanensis* leaves Freshly collected leaves of *Machilus yunnanensis* were used as material, and a reagent kit was used. Total RNA was extracted using the Universal PlantTotal RNA Kit (ER302-01, TRAN). The pipette tips and mortar used for RNA extraction were placed in sterile sealed bags, autoclaved at 121°C for 20 minutes, and then dried in a 60°C oven. After cooling, the RNA was used for processing. RNA integrity was assessed by 1% agarose gel electrophoresis, and concentration and purity were determined using a micro-spectrophotometer.

[0029] 2. RCR amplification and purification Single-stranded cDNA was synthesized using total RNA from *Machilus yunnanensis* leaves as a template and the TIANScript II RT Kit. Primers were designed based on the second-generation transcriptome sequencing sequence of *Machilus yunnanensis*: upstream primer F1: 5'-ATGAGTTTGATCATTCAATCTCTTCC-3' (as shown in SEQ ID NO: 3), and downstream primer R1: 5'-TCAAGGCCACTGACTTGGAACG-3' (as shown in SEQ ID NO: 4). The cDNA was synthesized by Beijing Qingke Biotechnology Co., Ltd. Using the above cDNA as a template, single-stranded cDNA was synthesized using… PCR amplification was performed using Max DNA Polymerase polymerase, following the instructions in the manufacturer's manual.

[0030] After the PCR reaction, the products were detected by 1% agarose gel electrophoresis. The gel containing the target fragment band was cut out under UV light using a scalpel and recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit, following the manufacturer's instructions. The recovered products were then detected by 1% agarose gel electrophoresis to ensure successful recovery.

[0031] 3. Cloning vector ligation Using the Tiangen Lethal Based Simple Fast Cloning Kit, according to the size and concentration of the target fragment, take an appropriate amount of the recovered product obtained in the above steps and ligate it with the cloning vector pLB, following the instructions. After ligation, remove DH5α E. coli competent cells (Novizan) from the -80℃ ultra-low temperature freezer and thaw them on ice; add 4μL of the ligation solution to 50μL of E. coli competent cells and incubate on ice for 30min; heat shock in a 42℃ water bath for 90s, then place on ice for 2min; add 800μL of antibiotic-free LB liquid medium, incubate at 37℃ and 225rpm for 1h; centrifuge at 12000rpm for 30s, collect the cells, discard the supernatant, and spread them on LB solid medium containing 100μg / ml ampicillin; seal the plates with sealing film and incubate upside down at 37℃ for 12-16h.

[0032] Recombinant plasmids were screened using a blue-white screening method, with the selected white single colonies used as templates. The universal primers for the pLB vector, pLB-F and pLB-R, were used, and the method was performed according to the manufacturer's instructions. Finally, the products were detected by 1% agarose gel electrophoresis. Colonies containing the target fragment size were selected as candidate bacteria, picked up with a pipette tip, and placed in LB liquid medium containing 100 μg / ml ampicillin. These samples were then sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing.

[0033] The sequence obtained from sequencing was compared with the original transcriptome sequence, and domain prediction was performed using HMMER. This confirmed that the gene sequence obtained in this invention is the full-length sequence of the TPS family, and the gene was named... MnTPS6 At the same time, its protein sequence was obtained.

[0034] MnTPS6 The results of 1% agarose gel electrophoresis of the gene are as follows: Figure 1 As shown, the clone yielded a single band that matched the estimated size. Sequencing alignment yielded... MnTPS6The coding sequence (CDS) of the gene is shown in SEQ ID: 1, totaling 1749 bp and encoding 582 amino acids. The amino acid sequence is shown in SEQ ID: 2. The protein molecular weight is 67.1 kDa, and the isoelectric point is 5.80. HMMER analysis of the conserved domains of MnTPS6 showed that it contains two conserved N-terminal and C-terminal domains, PF01397 and PF03936. Figure 2 Therefore, MnTPS6 is considered to be a TPS terpene synthase.

[0035] Example 2 MnTPS6 Gene expression analysis Total RNA was extracted from the roots, stems, and leaves of *Machilus chinensis* using the TIANGEN RNA Easy Fast PlantTissue Kit, following the manufacturer's instructions. Reverse transcription was performed using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2, following the manufacturer's instructions. qRT-PCR was performed using the SYBRPRIME qPCR kit (Fast HS) from Baoguang Biotechnology. qRT-PCR primers were designed using NCBI: F: 5'-ATCCAACGGCTTGGGGTG-3' (as shown in SEQ ID NO: 5), R: 5'-CGAAGAAGCCGGAACCGT-3' (as shown in SEQ ID NO: 6). The internal reference gene was *Cinnamomum camphora* ACTIN (ACT, KM086738.1), F: 5'-CCTCGACACACAGGCGTTAT-3' (as shown in SEQ ID NO: 7), R: 5'-CCATGCTCGATGGGATATTTCA-3' (as shown in SEQ ID NO: 8). The reaction mixture consisted of 1 μL cDNA, 0.5 μL (10 μM) F primer, 0.5 μL (10 μM) R primer, 3 μL ddH2O, and 5 μL SYBRPRIME qPCR kit (Fast HS). The assay program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, 39 cycles of denaturation and annealing, extension at 65°C for 5 s, and final extension at 95°C for 5 s. The reaction was performed using a BIO-RAD CFX Connect Real-Time System (Bio-Rad), with three biological replicates and three technical replicates per sample, employing 2... -ΔΔCt The method processes the data and calculates Runnan. MnTPS6 The expression in different organizations. The results are as follows: Figure 3 As shown, MnTPS6 It is expressed at the highest level in the roots and at a lower level in the stems and leaves.

[0036] Example 3: MnTPS6 Prokaryotic Expression Analysis 1. Carrier Construction According to TargetP-2.0 (https: / / services.healthtech.dtu.dk / services / TargetP-2.0 / ), MnTPS4 is predicted to contain a transport peptide at amino acid positions 49-50. Therefore, we will design homologous recombination primers based on the position of the truncated transport peptide. MnTPS6 The coding region of the gene was amplified by PCR using homologous recombination primers containing BamHI and HindIII restriction sites: F: 5'- GCCATGGCTGATATCGGATCCGCAAATTACCACCCAAGCATTTGG-3' (as shown in SEQ ID NO: 9) and R: 5'- CTCGAGTGCGGCCGCAAGCTTAGGCCACTGACTTGGAACG-3' (as shown in SEQ ID NO: 10).

[0037] Linearization of the pET-32a prokaryotic expression vector was performed using BamHI and HindIII restriction enzymes at 37°C for 30 min, followed by 80°C for 10 min, and then with novizan. The II One Step Cloning Kit performs homologous recombination between the linearized vector and the target fragment, following the manufacturer's instructions. The ligation product is transformed into DH5α E. coli competent cells (Novizan), and the recombinant prokaryotic expression vector is obtained by colony PCR identification and sequencing.

[0038] 2. Recombinant protein expression The identified recombinant plasmid DNA was transformed into BL21(DE3) competent cells (Novizan). Single colonies were picked and inoculated into 50 ml of LB liquid medium containing 100 μg / ml ampicillin, and cultured overnight at 37°C and 225 rpm. The culture was expanded at a 1:100 ratio; when OD600 ≈ 0.5, 0.1 mM IPTG was added, and the cells were induced at 16°C and 140 rpm for 20 h; the cells were collected by centrifugation at 4000 rpm for 20 min; the supernatant was discarded, and the cells were resuspended in 30 ml of PBS buffer; the cells were sonicated and centrifuged at 12000 rpm and 4°C for 20 min. The supernatant was transferred to a new centrifuge tube to obtain the precipitate and supernatant.

[0039] 3. Recombinant protein purification Equilibrate the Ni column with 3 column volumes of PBS buffer. Slowly pass the supernatant through the Ni column. Remove contaminating proteins with 3 column volumes of PBS buffer containing 20 mM imidazole. Elute the target protein with 14 ml of PBS buffer containing 500 mM imidazole, using 2 ml tubes per elution. Prepare samples from the precipitate, supernatant, and eluent, and analyze using SDS-PAGE. Desalt the eluent containing the target protein, and then determine the protein concentration.

[0040] Example 4: In vitro enzyme catalytic function analysis of MnTPS6 Add 50 μg of protein, 10 mM MgCl2, 10 mM DTT, 5 μg FPP / GPP / FPP+GPP, and 25 mM HEPES (pH 7.4) to a final volume of 100 μL. Incubate at 30 °C for 3 h. After the reaction is complete, add 150 μL of n-hexane, shake vigorously for 5 min, and centrifuge at 12000 rpm for 10 min to separate the oil and water phases. Pipette the organic phase and filter it through a 0.22 μm filter into a sample vial for GC-MS analysis.

[0041] A TRACE 1300 gas chromatography-ISQ 7000 mass spectrometry (GC-MS) detector and an HP-5MS column (30 m × 250 μm × 0.25 μm film thickness) were used with helium as the carrier gas at a flow rate of 1.5 mL / min. The initial column temperature was set at 40 °C, equilibrated for 5 min, and then increased to 300 °C at a rate of 8 °C / min for 30 min. Mass spectrometry conditions were: ion transfer line temperature 290 °C, solvent delay 3 min, ion scan range 40 m / z to 600 m / z, with other parameters set to system defaults. An EI source was used as the ion source for the mass spectrometer. The acquired mass spectra were analyzed using the NIST mass library.

[0042] MnTPS6 The results of gene expression in prokaryotes are as follows Figure 4 As shown, the recombinant MnTPS6 protein pET-32a-MnTPS6 is approximately 80.6 kDa. SDS-PAGE gel electrophoresis results show that the size of the pET-32a-MnTPS6 recombinant protein is close to the expected size. In vitro enzyme activity identification is as follows... Figure 5 As shown, when farnesyl pyrophosphate (FPP) is used as a substrate, the main product generated by pET-32a-MnTPS6 catalysis is the sesquiterpene Nerolidol (…). Figure 5 When geraniol (GPP) is used as a substrate, the main product generated by catalysis is the monoterpene Linalool. Figure 5 B, 6B).

[0043] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A Nerolidol / Linalool synthase gene from Linalool. MnTPS6 Its characteristics are, The Nerolidol / Linalool synthase gene of Runnan MnTPS6 The nucleotide sequence is shown in SEQ ID NO.

1.

2. A Nerolidol / Linalool synthase gene from Linalool. MnTPS6 The expressed protein MnTPS6 is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. The Nerolidol / Linalool synthase gene as described in claim 2 MnTPS6 A method for preparing the expressed protein MnTPS6, characterized in that, include: Based on the Nerolidol / Linalool synthase gene of Runnan MnTPS6 A prokaryotic expression vector was constructed, and the resulting expression vector was transformed into a host bacterium. The resulting host bacterium was then cultured to express the MnTPS6 protein.

4. The preparation method according to claim 3, characterized in that, The Nerolidol / Linalool synthase gene based on Runnan MnTPS6 The primer pairs designed during the construction of the prokaryotic expression vector are shown in SEQ ID NO.9 and SEQ ID NO.10, and the plasmid vector used is pET-32a.

5. A Nerolidol / Linalool synthase gene as described in claim 1 MnTPS6 The application of the expressed protein in the preparation of Nerolidol / Linalool.

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