Machilus rubescens TPS synthetase gene MnTPS8 and application thereof

Abstract

According to the machilus rubescens TPS synthetase gene MnTPS8 and the application thereof, the gene is obtained from machilus rubescens through cloning for the first time and has beta-Eudesmol and Geraniol synthesis functions, and a new gene resource is provided for functional diversity and evolution analysis of a terpene synthetase gene family. Recombinant protein coded by the MnTPS8 gene has a unique dual-catalysis function, GPP can be catalyzed to generate Geraniol, FPP can be catalyzed to generate beta-Eudesmol, and compared with a single-function gene, the synchronous production process of two high-value terpenoids is greatly simplified, and the production cost is reduced. When the gene is used for heterologous expression or transgenic plant cultivation, synchronous large-scale production of beta-Eudesmol and Geraniol can be realized, the yield limitation of natural extraction is broken through, and the gene has remarkable economic value and industrial application prospect.

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*. MnTPS8 And its applications. Background Technology

[0002] β-Eudesmol and Geraniol are both terpenoid compounds with important biological activities and economic value. β-Eudesmol belongs to the sesquiterpenoid class and possesses various pharmacological activities such as antibacterial, anti-inflammatory, antitumor, and sedative effects, making it widely used in the pharmaceutical, health product, cosmetic, and fragrance industries. Geraniol belongs to the monoterpenoid class and is not only an important natural fragrance component with a refreshing rose aroma, but also possesses antibacterial, insecticidal, and antioxidant biological activities, resulting in strong demand in the food, daily chemical, and pharmaceutical fields.

[0003] Currently, the acquisition of β-Eudesmol and Geraniol mainly relies on extraction from natural plants. For example, β-Eudesmol can be extracted from plants such as eucalyptus, camphor, and nanmu, while Geraniol is mainly derived from plants such as rose, geranium, and lemongrass. However, the content of both in natural plants is generally low, and the yield is affected by factors such as growth environment, harvesting cycle, and extraction process, resulting in unstable yield, difficulty in controlling purity, and high production costs, which cannot meet the large-scale market demand. Although chemical synthesis can achieve the preparation of both, it has drawbacks such as complex reaction steps, numerous by-products, serious environmental pollution, and low optical purity of the products, which limit its industrial application.

[0004] The development of genetic engineering technology has provided new pathways for the large-scale preparation of terpenoids. Terpenoid synthases (TPSs) are key enzymes in the biosynthetic pathway of terpenoids. Monoterpenoid synthases catalyze the synthesis of monoterpenoids (such as Geraniol) from geranyyl pyrophosphate (GPP), while sesquiterpenoid synthases catalyze the synthesis of sesquiterpenoids (such as β-Eudesmol) from farnesyl pyrophosphate (FPP). In recent years, some plant-derived terpenoid synthase genes have been cloned and reported, but most genes only have the function of synthesizing a single product. If a bifunctional terpenoid synthase gene that can simultaneously catalyze the synthesis of β-Eudesmol and Geraniol could be obtained, the production difficulty and cost of these two high-value terpenoids would be significantly reduced, and it has important application prospects.

[0005] Machilus chinensis is an important economic tree species in southern my country, rich in various terpenoid compounds in its wood, branches, and leaves, suggesting the presence of multifunctional terpenoid synthase genes. However, to date, there are no reports on the cloning, sequence analysis, and functional verification of bifunctional terpenoid synthase genes from Machilus chinensis that synthesize both β-Eudesmol and Geraniol, limiting the progress of simultaneously large-scale production of these two high-value terpenoid compounds using genetic engineering techniques. Therefore, cloning a bifunctional terpenoid synthase gene from Machilus chinensis is necessary. MnTPS8 Clarifying its catalytic function is of great theoretical and practical significance for breaking through existing production technology bottlenecks and expanding gene resources for the biosynthesis of terpenoids. Summary of the Invention

[0006] To gain a deeper understanding of the synthetic regulatory network of β-Eudesmol and Geraniol synthase genes in plants, and to improve the synthesis yield and efficiency of β-Eudesmol and Geraniol, this invention provides a *Machilus chinensis* TPS synthase gene. MnTPS8 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. MnTPS8 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. MnTPS8 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. MnTPS8 Construct prokaryotic expression vectors.

[0010] Furthermore, the present invention also provides the above-mentioned *Napier TPS synthase* gene. MnTPS8 Methods for preparing expressed proteins.

[0011] Furthermore, the present invention also provides the above-mentioned *Lysimachia nummularia* TPS synthase gene. MnTPS8 The application of the expressed protein in the preparation of β-Eudesmol and Geraniol.

[0012] The present invention has the following beneficial effects: This invention is the first to clone a bifunctional terpene synthase gene from *Machilus yunnanensis* that possesses both β-Eudesmol and Geraniol synthesis functions. MnTPS8The nucleotide and amino acid sequences of the gene were determined, filling the gap in the study of bifunctional terpene synthase genes of *Lysimachia nummularia* and providing new gene resources for the functional diversity and evolutionary analysis of the terpene synthase gene family. MnTPS8 The recombinant protein encoded by this gene possesses a unique dual-catalytic function, simultaneously catalyzing the production of Geraniol from GPP and β-Eudesmol from FPP. Compared to single-function genes, this significantly simplifies the simultaneous production process of these two high-value terpenoids, reducing production costs. Using this gene for heterologous expression or transgenic plant cultivation enables the simultaneous large-scale production of β-Eudesmol and Geraniol, overcoming the yield limitations of natural extraction and demonstrating significant economic value and industrial application prospects. This technical approach is simple and efficient, and the expressed product is easy to purify, laying a solid technical foundation for subsequent industrial applications. Furthermore, this gene provides a core tool gene for metabolic engineering research in terpenoid synthesis. Attached Figure Description

[0013] Figure 1 PCR cloning MnTPS8 Gene electrophoresis diagram.

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

[0015] Figure 3 : MnTPS8 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-MnTPS8 recombinant protein; lane 1 is the precipitate suspension after ultrasonic disruption; lane 2 is the supernatant after ultrasonic disruption; lanes 3-8 are 500 mM imidazole elution buffer.

[0017] Figure 5 GC-MS detection of in vitro catalytic reaction product spectra of MnTPS8 protein; where Figure A shows the catalytic products of the reaction between MnTPS8 and FPP; and Figure B shows the catalytic products of the reaction between MnTPS8 and GPP.

[0018] Figure 6 Mass spectrometry comparison diagrams; where, diagram A is the β-Eudesmol mass spectrometry comparison diagram; diagram B is the Geraniol mass spectrometry comparison diagram. Detailed Implementation

[0019] Example 1 MnTPS8 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'-ATGGCTTCCCTAAGTTCCTCTCC-3' (as shown in SEQ ID NO: 3), and downstream primer R1: 5'-TCACAAAGGGATAGGTTCGACG-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... MnTPS8 At the same time, its protein sequence was obtained.

[0025] MnTPS8 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... MnTPS8 The coding sequence (CDS) of the gene is shown in SEQ ID: 1, totaling 1674 bp and encoding 557 amino acids. The amino acid sequence is shown in SEQ ID: 2. The protein molecular weight is 63.9 kDa, and the isoelectric point is 5.52. HMMER analysis of the conserved domains of MnTPS8 showed that it contains two conserved N-terminal and C-terminal domains, PF01397 and PF03936. Figure 2 Therefore, it is believed that MnTPS8 It is a TPS terpene synthase.

[0026] Example 2 MnTPS8 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 Biotech. qRT-PCR primers were designed using NCBI: F: 5'-TGGTATGGGAGACGAGGTGA-3' (as shown in SEQ ID NO: 5), R: 5'-CGCGATACACCATGCTCCTT-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. MnTPS8 The expression in different organizations. The results are as follows: Figure 3 As shown, MnTPS8 It is expressed at the highest level in the roots and at a lower level in the stems and leaves.

[0027] Example 3: MnTPS8 Prokaryotic Expression Analysis 1. Carrier Construction according to MnTPS8The coding region of the gene was amplified by PCR using homologous recombination primers containing BamHI and HindIII restriction sites: F: 5'-GCCATGGCTGATATCGGATCCATGGCTTCCCTAAGTTCCTCTCC-3' (as shown in SEQ ID NO: 9) and R: 5'-CTCGAGTGCGGCCGCAAGCTTCAAAGGGATAGGTTCGACGAACATTG-3' (as shown in SEQ ID NO: 10), with the cDNA obtained in Example 1 as a template.

[0028] Linearization of the pET-32a prokaryotic expression vector involved digestion with BamHI and HindIII restriction enzymes, followed by incubation at 37°C for 30 min, then at 80°C for 10 min, and finally induced with Novizan. The II One Step Cloning Kit was used to perform homologous recombination between the linearized vector and the amplified target fragment, following the 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 MnTPS8 Add 50 μg of protein, 10 mM MgCl2, 10 mM DTT, 5 μg 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] MnTPS8 The results of gene expression in prokaryotes are as follows Figure 4 As shown, the recombinant pET-32a-MnTPS8 protein is approximately 83.1 kDa. SDS-PAGE gel electrophoresis results show that the size of the recombinant pET-32a-MnTPS8 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-MnTPS8 is the sesquiterpene β-Eudesmol. 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. An oreocallis taylori TPS synthase gene MnTPS8 characterized in that: The gene MnTPS8 The nucleotide sequence is shown as SEQ ID NO. 1 and the amino acid sequence is shown as SEQ ID NO.

2.

2. The amplification primer of the TPS synthase gene of the genus Ficus as claimed in claim 1, characterized by: MnTPS8 The sequence of the amplification primer is shown as SEQ ID NO. 3 and SEQ ID NO.

4. ​ 3. A TPS synthase gene of Brintupia bipunctata as claimed in claim 1 MnTPS8 prokaryotic expression vector constructed.

4. Use of the expressed protein of the TPS gene of Guaiacum officinale of claim 1 in the preparation of β-Eudesmol and Geraniol. MnTPS8 of claim 1 in the preparation of β-Eudesmol and Geraniol.

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