Machilus rubescens TPS synthetase gene MnTPS10 and application thereof

Abstract

The invention provides a machilus TPS synthetase gene MnTPS10 and application thereof, the terpene synthetase gene MnTPS10 with a dual catalytic function is cloned from machilus, the gene can catalytically synthesize Hedycarylol and Geraniol at the same time, the research on the dual-function terpene synthetase gene in machilus plants is enriched, and the application of the gene in the machilus plants is developed. Important gene resources and theoretical basis are provided for deeply researching the diversity synthesis mechanism of terpenoids. The MnTPS10 gene disclosed by the invention has a unique dual catalytic function, two high-value terpenoids can be respectively synthesized by utilizing different terpenoid precursors (GPP and FPP), and compared with a synthetase gene with a single function, the MnTPS10 gene disclosed by the invention is wider in application range, the biosynthesis cost can be reduced, and the production efficiency can be improved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a TPS synthase gene of *Machilus yunnanensis*. MnTPS10 And its applications. Background Technology

[0002] Terpenes are the most diverse class of plant secondary metabolites, widely involved in physiological processes such as plant growth, development, and defense responses. They also have significant applications in medicine, fragrances, and agriculture. Hedycaryol, a bicyclic sesquiterpene compound, possesses potential pharmacological activities such as anti-inflammatory, antibacterial, and antitumor effects, and is an important component of natural fragrances. Geraniol, a monoterpene alcohol, has a refreshing rose aroma and is widely used in the fragrance and flavor industry. It also possesses insecticidal and antibacterial biological activities, and has significant application prospects in agriculture and medicine.

[0003] Currently, the acquisition of Hedycaryol and Geraniol mainly relies on natural plant extraction and chemical synthesis. Natural plant extraction suffers from problems such as limited raw material supply, low product content, complex extraction processes, and high costs, and over-exploitation can damage the ecological environment. Chemical synthesis, on the other hand, has drawbacks such as multiple reaction steps, numerous by-products, serious environmental pollution, and difficulty in guaranteeing product purity, making it difficult to meet the market demand for natural and safe products.

[0004] Biosynthesis technology, with its advantages of being green, efficient, and environmentally friendly, has become an important direction for the large-scale production of natural products. Terpenoid synthases are key rate-limiting enzymes in the biosynthetic pathway of terpenoid compounds, and the cloning and functional verification of their encoding genes are the core of realizing the biosynthesis of terpenoid compounds. Currently, terpenoid synthase genes that catalyze the synthesis of Hedycaryol or Geraniol have been cloned from some plants, but synthase genes that simultaneously catalyze the synthesis of these two different types of terpenoid compounds (sesquiterpenes and monoterpenes) have not yet been discovered.

[0005] *Machilus chinensis*, a timber species belonging to the genus *Machilus* in the Lauraceae family, is widely distributed in southern my country. Rich in various terpenoid compounds, it is an important resource for discovering novel terpenoid synthase genes. However, to date, there are no reports on the cloning, identification, and application of terpenoid synthase genes from *Machilus chinensis*. Therefore, isolating and cloning synthase genes from *Machilus chinensis* capable of simultaneously catalyzing the synthesis of Hedycaryol and Geraniol has significant theoretical and practical value for achieving the efficient biosynthesis of these two high-value terpenoid compounds. Summary of the Invention

[0006] To gain a deeper understanding of the synthetic regulatory network of Hedycaryol and Geraniol synthase genes in plants, and to improve the synthesis yield and efficiency of Hedycaryol and Geraniol, this invention provides a *Machilus chinensis* TPS synthase gene. MnTPS10 Its application provides an effective way to solve the above problems.

[0007] To achieve the above objectives, the technical solution provided by this invention is as follows: This invention provides a Runan TPS synthase gene MnTPS10 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0008] This invention also provides the above-mentioned *Lysimachia nummularia* TPS synthase gene. MnTPS10 The amplification primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0009] Furthermore, the present invention also provides a *Nasturtium nitidum* TPS synthase gene. MnTPS10 Construct prokaryotic expression vectors.

[0010] Furthermore, the present invention also provides the above-mentioned *Napier TPS synthase* gene. MnTPS10 Methods for preparing expressed proteins.

[0011] Furthermore, the present invention also provides the above-mentioned *Lysimachia nummularia* TPS synthase gene. MnTPS10 The application of the expressed protein in the preparation of Hedycaryol and Geraniol.

[0012] The present invention has the following beneficial effects: This invention clones a terpene synthase gene with dual catalytic function from *Phoebe zhennan*. MnTPS10 This gene can simultaneously catalyze the synthesis of Hedycaryol and Geraniol, enriching the research on dual-function terpene synthase genes in the genus *Machilus*, and providing important gene resources and theoretical basis for in-depth research on the diverse synthetic mechanisms of terpene compounds. This invention... MnTPS10This gene possesses a unique dual catalytic function, enabling the synthesis of two high-value terpenoid compounds from different terpenoid precursors (GPP and FPP). Compared to single-function synthase genes, its application range is wider, reducing biosynthetic costs and improving production efficiency. Using the gene of this invention to construct recombinant vectors and engineered bacteria, large-scale, green production of Hedycaryol and Geraniol can be achieved through microbial fermentation. Compared to traditional plant extraction and chemical synthesis methods, this method offers advantages such as low cost, high yield, environmental friendliness, high product purity, and freedom from natural condition limitations. It effectively solves the problem of obtaining these two compounds in existing technologies and has broad prospects for industrial application. The technical solution of this invention is simple and easy to implement, with high gene cloning efficiency, high success rate of recombinant vector construction, and mild induction expression conditions for engineered bacteria, facilitating widespread application. It also provides a reference paradigm for discovering terpenoid synthase genes with multiple catalytic functions from other plants. Attached Figure Description

[0013] Figure 1 PCR cloning MnTPS10 Gene electrophoresis diagram.

[0014] Figure 2 : Pfam domain of the TPS gene family.

[0015] Figure 3 : MnTPS10 Gene expression in the roots, stems and leaves of *Machilus chinensis*.

[0016] Figure 4 SDS-PAGE electrophoresis image of the purified product of pET-32a-MnTPS10 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.

[0017] Figure 5 GC-MS detection of in vitro catalytic reaction product spectra of MnTPS10 protein; where Figure A shows the catalytic products of the reaction between MnTPS10 and FPP; and Figure B shows the catalytic products of the reaction between MnTPS10 and GPP.

[0018] Figure 6 Mass spectrometry comparison images; where image A is the mass spectrometry comparison image of Hedycaryol; image B is the mass spectrometry comparison image of Geraniol. Detailed Implementation

[0019] Example 1 MnTPS10 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.

[0020] 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'-ATGTCTCTTGTCCTAGGCTCAGGCC-3' (as shown in SEQ ID NO: 3), and downstream primer R1: 5'-TCAAATAGGAATAGGATTAACAA-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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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... MnTPS10 At the same time, its protein sequence was obtained.

[0025] MnTPS10 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... MnTPS10 The coding sequence (CDS) of the gene is shown in SEQ ID: 1, totaling 1701 bp and encoding 566 amino acids. The amino acid sequence is shown in SEQ ID: 2. The protein molecular weight is 65.5 kDa, and the isoelectric point is 5.15. HMMER analysis of the conserved domains of MnTPS10 showed that it contains two conserved N-terminal and C-terminal domains, PF01397 and PF03936. Figure 2 Therefore, it is believed that MnTPS10 It is a TPS terpene synthase.

[0026] Example 2 MnTPS10 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'-ATATGAAGGGGCATGGAGGC-3' (as shown in SEQ ID NO: 5), R: 5'-TGCGCTCTTTGGTCTCATGT-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 65°C for 5 s. The reaction was performed using a BIO-RAD CFX ConnectReal-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. MnTPS10 The expression in different organizations. The results are as follows: Figure 3 As shown, MnTPS10 It is expressed at the highest level in the roots and at a lower level in the stems and leaves.

[0027] Example 3: MnTPS10 Prokaryotic Expression Analysis 1. Carrier Construction according to MnTPS10The coding region of the gene was amplified by PCR using homologous recombination primers containing BamHI and HindIII restriction sites: F: 5'-GCCATGGCTGATATCGGATCCATGTCTCTTGTCCTAGGCTCAGG-3' (as shown in SEQ ID NO: 9) and R: 5'-CTCGAGTGCGGCCGCAAGCTTAATAGGAATAGGATTAACAAGCACTAATGAGATGC-3' (as shown in SEQ ID NO: 10), with the cDNA obtained in Example 1.

[0028] The pET-32a prokaryotic expression vector was linearized by digestion with BamHI and HindIII restriction enzymes at 37°C for 30 min, followed by 80°C for 10 min. Homologous recombination of the linearized vector with the target fragment was then performed using the Novizan ClonExpress® II One Step Cloning Kit, following the manufacturer's instructions. The ligation product was transformed into DH5α E. coli competent cells (Novizan), and the recombinant prokaryotic expression vector was obtained by colony PCR identification and sequencing.

[0029] 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.

[0030] 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.

[0031] Example 4: In vitro enzyme catalytic function analysis of MnTPS10 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.

[0032] 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.

[0033] MnTPS10 The results of gene expression in prokaryotes are as follows Figure 4 As shown, the recombinant pET-32a-MnTPS10 protein is approximately 84.8 kDa. SDS-PAGE gel electrophoresis results show that the size of the recombinant pET-32a-MnTPS10 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 catalyzed by pET-32a-MnTPS10 is the sesquiterpene Hedycaryol. Figure 5 When geraniol (GPP) is used as a substrate, the main product generated by catalysis is the monoterpene Geraniol. Figure 5 B, 6B).

Claims

1. A TPS synthase gene from *Agropyron cristatum* MnTPS10 Its features are: The gene MnTPS10 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The Runnan TPS synthase gene as described in claim 1 MnTPS10 The amplification primers are characterized by: The amplification primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.

4.

3. A *Lysimachia christinae* TPS synthase gene as described in claim 1 MnTPS10 The constructed prokaryotic expression vector.

4. A *Lysimachia christinae* TPS synthase gene as described in claim 1 MnTPS10 The application of the expressed protein in the preparation of Hedycaryol and Geraniol.

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