Machilus rubescens TPS synthetase gene MnTPS12 and application thereof

Abstract

The Machilus rubescens TPS synthetase gene MnTPS12 provided by the invention is a key gene for synthesis of gamma-Eudesmol and Linalool in plant genetic engineering, a novel terpene synthetase gene MnTPS12 is obtained by cloning in Machilus rubescens for the first time, and the gene can catalyze FPP and GPP to generate gamma-Eudesmol and Linalool, and plays an important role in improving the content and resistance of plant terpene components. The terpene synthase gene MnTPS12 researched by the invention can be used for preparing gamma-Eudesmol and Linalool, and can be further used for preparing essential oil, essence and medicines containing the components of the gamma-Eudesmol and the Linalool. The gene segment of the terpene synthase gene MnTPS12 is constructed on a plant expression vector, other plant materials can be transformed through an external source, so that a transgenic material containing the terpene aroma gene is obtained, and an effective method is provided for cultivating aroma and medicinal plants.

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*. MnTPS12 And its applications. Background Technology

[0002] Runnan ( Machilus nanmu *Machilus*, an important arborescent tree species in the Lauraceae family, is rich in various active metabolites and is effective in treating skin inflammation, foot edema, and diarrhea. The polysaccharides in *Machilus* leaves possess antioxidant and antitumor bioactivities. It has high application potential in industrial timber, pharmaceuticals, and health care. γ-Eudesmol and Linalool, as volatile metabolites of *Machilus*, can be improved through genetic engineering to cultivate new varieties that produce high yields of γ-Eudesmol and Linalool, providing new raw material sources for their production. γ-Eudesmol is a natural sesquiterpene compound with core functions of antibacterial, anti-inflammatory, and antioxidant activity, and also possesses potential antitumor bioactivities. It is mainly used in daily chemical skincare and pharmaceutical research. Linalool, a compound playing a key role in fragrances, pharmaceuticals, and industry, possesses various pharmacological activities such as anti-inflammatory, antibacterial, antiviral, and anticancer effects. It can also be used to prepare high-performance materials, such as polymers with special properties, providing more choices and possibilities for industrial production.

[0003] Despite some progress in the synthesis of γ-Eudesmol and Linalool, several challenges remain. In microbial synthesis, while engineered strains have been constructed through genetic engineering, yields are still low and production costs are high, making it difficult to compete with traditional chemical synthesis methods. Microbial fermentation may also generate byproducts that affect the purity and quality of γ-Eudesmol and Linalool. Furthermore, microbial fermentation conditions are demanding, requiring strict control over environmental factors such as temperature, pH, and dissolved oxygen, increasing the complexity and cost of the production process. In plant genetic engineering, the difficulty of gene expression regulation is a major problem. The introduced γ-Eudesmol and Linalool synthase genes may be affected by various factors in plants, such as gene silencing and unstable promoter activity, leading to low gene expression efficiency and unsatisfactory synthesis of γ-Eudesmol and Linalool. Plant genetic transformation technology also has limitations, with low transformation efficiency and the possibility of abnormal growth and development in transformed plants. Furthermore, the production of γ-Eudesmol and Linalool through plant genetic engineering requires consideration of the ecological and environmental impacts, such as the safety of transgenic plants. Terpenoid synthases (TPS) are key enzymes catalyzing the biosynthesis of terpenoid compounds and play a central role in plant secondary metabolism. Research on the TPS synthase gene of *Machilus chinensis* holds promise for opening new avenues for addressing these issues. Summary of the Invention

[0004] To gain a deeper understanding of the synthetic regulatory network of plant γ-Eudesmol and Linalool synthase genes, and to improve the synthesis yield and efficiency of γ-Eudesmol and Linalool, this invention provides a *Machilus chinensis* TPS synthase gene. MnTPS12 Its application provides an effective way to solve the above problems.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows: This invention provides a Runan TPS synthase gene MnTPS12 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.

[0006] This invention also provides the above-mentioned *Lysimachia nummularia* TPS synthase gene. MnTPS12 The amplification primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0007] Furthermore, the present invention also provides a *Nasturtium nitidum* TPS synthase gene. MnTPS12 Construct prokaryotic expression vectors.

[0008] Furthermore, the present invention also provides the above-mentioned *Napier TPS synthase* gene. MnTPS12 Method for preparing the expressed protein MnTPS12.

[0009] Furthermore, the present invention also provides the above-mentioned *Lysimachia nummularia* TPS synthase gene. MnTPS12 The application of the expressed protein in the preparation of γ-Eudesmol and Linalool.

[0010] The present invention has the following beneficial effects: The TPS synthase gene provided by this invention MnTPS12 This gene is a key gene for the synthesis of γ-Eudesmol and Linalool in plant genetic engineering, effectively regulating their synthesis. Its expressed protein is used in the high-yield synthesis of γ-Eudesmol and Linalool, possessing significant application value. This invention is the first to clone a novel terpene synthase gene from *Machilus chinensis*. MnTPS12 This gene catalyzes the production of γ-Eudesmol and Linalool from FPP and GPP, playing a crucial role in increasing terpene content and plant resistance. This invention studies the terpene synthase gene. MnTPS12 It can be used to prepare γ-Eudesmol and Linalool, and further to prepare essential oils, fragrances, and pharmaceuticals containing γ-Eudesmol and Linalool. Terpenoid synthase gene. MnTPS12 The gene fragments are constructed on plant expression vectors, which can be used to exogenously transform other plant materials to obtain transgenic materials containing terpene aroma genes, providing an effective method for cultivating aromatic and medicinal plants. Attached Figure Description

[0011] Figure 1 PCR cloning MnTPS12 Gene electrophoresis diagram.

[0012] Figure 2 : Pfam domain of the TPS gene family.

[0013] Figure 3 : MnTPS12 Gene expression in the roots, stems and leaves of *Machilus chinensis*.

[0014] Figure 4SDS-PAGE electrophoresis image of the purified product of pET-32a-MnTPS12 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.

[0015] Figure 5 GC-MS detection of in vitro catalytic reaction product spectra of MnTPS12 protein; where Figure A shows the catalytic products of the reaction between MnTPS12 and FPP; and Figure B shows the catalytic products of the reaction between MnTPS12 and GPP.

[0016] Figure 6 Mass spectrometry comparison diagrams; where, diagram A is the γ-Eudesmol mass spectrometry comparison diagram; diagram B is the Linalool mass spectrometry comparison diagram. Detailed Implementation

[0017] Example 1 MnTPS12 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.

[0018] 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'-ATGGCTCTTGTTTTGGGCTCT-3' (as shown in SEQ ID NO: 3), and downstream primer R1: 5'-CTACATAGGAACGGGTTCCACC-3' (as shown in SEQ ID NO: 4), and synthesized by Beijing Qingke Biotechnology Co., Ltd. Using the above cDNA as a template, [further details are needed]. PCR amplification was performed using Max DNA Polymerase polymerase, following the instructions in the manufacturer's manual.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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... MnTPS12 At the same time, its protein sequence was obtained.

[0023] MnTPS12 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... MnTPS12The coding sequence (CDS) of the gene is shown in SEQ ID: 1, totaling 1692 bp and encoding 563 amino acids. The amino acid sequence is shown in SEQ ID: 2. The protein molecular weight is 65.4 kDa, and the isoelectric point is 5.34. HMMER analysis of the conserved domains of MnTPS12 showed that it contains two conserved N-terminal and C-terminal domains, PF01397 and PF03936. Figure 2 Therefore, MnTPS12 is considered to be a TPS terpene synthase.

[0024] Example 2 MnTPS12 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'-CTCGTGAGGGGAAAGCCTAC-3' (as shown in SEQ ID NO: 5), R: 5'-GCAACATCTCCCATGCCAAC-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℃ for 3 min, denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, 39 cycles of denaturation and annealing, extension at 65℃ for 5 s, and final extension at 65℃ for 5 s. The reaction was performed using a BIO-RAD CFX ConnectReal-Time System (Bio-Rad). Each sample had three biological replicates and three technical replicates. Data were processed using the 2-ΔΔCt method to calculate the results. MnTPS12 The expression in different organizations. The results are as follows: Figure 3 As shown, MnTPS12 It is expressed at the highest level in the roots and at a lower level in the stems and leaves.

[0025] Example 3: Prokaryotic Expression Analysis of MnTPS12 1. Carrier Construction according to MnTPS12 The coding region of the gene was amplified by PCR using homologous recombination primers containing BamHI and HindIII restriction sites: F: 5'-GCCATGGCTGATATCGGATCCATGGCTCTTGTTTTGGGCTCTG-3' (as shown in SEQ ID NO: 9) and R: 5'- CTCGAGTGCGGCCGCAAGCTTCATAGGAACGGGTTCCACCAAC-3' (as shown in SEQ ID NO: 10), with the cDNA obtained in Example 1 as a template.

[0026] 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 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.

[0027] 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.

[0028] 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.

[0029] Example 4: In vitro enzyme catalytic function analysis of MnTPS12 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.

[0030] 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.

[0031] MnTPS12 The results of gene expression in prokaryotes are as follows Figure 4 As shown, the size of the pET-32a-MnTPS12 recombinant protein is approximately 84.6 kDa. SDS-PAGE gel electrophoresis results show that the size of the pET-32a-MnTPS12 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 catalyzed by pET-32a-MnTPS12 is the sesquiterpene γ-Eudesmol. Figure 5 When geraniol (GPP) is used as a substrate, the main product generated by catalysis is the monoterpene Linalool. Figure 5 B, 6B), indicating that MnTPS12 is a bifunctional enzyme that produces γ-Eudesmol and Linalool.

[0032] In summary, this invention is the first to clone a novel terpene synthase gene from *Machilus yunnanensis*. MnTPS12 This gene catalyzes the production of γ-Eudesmol and Linalool from FPP and GPP, playing a crucial role in increasing terpene content and plant resistance. This invention studies the terpene synthase gene. MnTPS12It can be used to prepare γ-Eudesmol and Linalool, and further to prepare essential oils, fragrances, and pharmaceuticals containing γ-Eudesmol and Linalool. Terpenoid synthase gene. MnTPS12 The gene fragments are constructed on plant expression vectors, which can be used to exogenously transform other plant materials to obtain transgenic materials containing terpene aroma genes, providing an effective method for cultivating aromatic and medicinal plants.

[0033] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A TPS synthase gene from *Agropyron cristatum* MnTPS12 Its features are: 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.

2. The Runnan TPS synthase gene as described in claim 1 MnTPS12 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 MnTPS12 The constructed prokaryotic expression vector.

4. A *Lysimachia christinae* TPS synthase gene as described in claim 1 MnTPS12 The application of the expressed protein in the preparation of γ-Eudesmol and Linalool.

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