MnTPS12 and application thereof

Abstract

The application provides a machilus thunbergii TPS synthetase gene MnTPS12 The application is a key gene for synthesis of gamma-eudesmol and linalool in plant genetic engineering, and a new terpene synthase gene is cloned from machilus thunbergii for the first time MnTPS12 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. MnTPS12 The terpene synthase gene studied in the application can be used for preparing gamma-eudesmol and linalool, and for further preparing essential oil, essence and medicine containing gamma-eudesmol and linalool. MnTPS12 The gene fragment of the terpene synthase gene is constructed on a plant expression vector, and can be transformed into other plant materials by exogenous transformation, so as to obtain transgenic materials containing the terpene aroma gene, and provide an effective method for cultivating aroma and medicinal plants.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a Michelia macclurei TPS synthetase gene MnTPS12 and application thereof. BACKGROUND

[0002] Michelia macclurei Machilus nanmu is an important arbor tree species of Lauraceae Michelia, which is rich in various active metabolites and is quite effective in skin inflammation, foot swelling, diarrhea and the like. The polysaccharide of Michelia macclurei leaves has biological activities such as antioxidant and antitumor. Michelia macclurei has a high application potential in the fields of industrial timber, medicine and health care and the like. γ-Eudesmol and Linalool, as volatile metabolite components of Michelia macclurei, can be improved by means of genetic engineering technology and the like, and a new variety with high yield of γ-Eudesmol and Linalool is bred, thereby providing a new raw material source for the production of γ-Eudesmol and Linalool. γ-Eudesmol is a natural sesquiterpene compound, which has the core functions of antibacterial and anti-inflammatory, antioxidant and potential antitumor biological activity, and is mainly used in the fields of daily chemical skin care, medicine research and development and the like. Linalool, as a compound playing a key role in many fields such as spices, medicine and industry, has various pharmacological activities such as anti-inflammatory, antibacterial, antiviral and anticancer. Linalool can also be used for preparing some high-performance materials such as polymers with special performance, thereby providing more choices and possibilities for industrial production.

[0003] Although some progress has been made in the synthesis of γ-Eudesmol and Linalool, there are still some problems to be solved. In terms of microbial synthesis, although engineering strains have been constructed through genetic engineering, the yield is still low, the production cost is high, and it is difficult to compete with traditional chemical synthesis methods. During fermentation, microorganisms may produce some by-products, affecting the purity and quality of γ-Eudesmol and Linalool. In addition, the fermentation conditions of microorganisms are harsh, with strict requirements for temperature, pH value, dissolved oxygen and other environmental factors, which increases the complexity and cost of the production process. In terms of plant genetic engineering, 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, unstable promoter activity, etc., resulting in low gene expression efficiency and unsatisfactory γ-Eudesmol and Linalool synthesis. Plant genetic transformation technology also has certain limitations, with low transformation efficiency and abnormal growth and development of transformed plants. In addition, the production of γ-Eudesmol and Linalool through plant genetic engineering also needs to consider the impact on the ecological environment, such as the safety of transgenic plants. Terpenoid synthase (Terpenoid Synthase, TPS) is a class of key enzymes that catalyze the biosynthesis of terpenoids, playing a core role in plant secondary metabolism. Research on Michelia maudiae TPS synthase genes is expected to open up new ways to solve these problems. SUMMARY

[0004] To better understand the synthesis and regulation network of plant γ-Eudesmol and Linalool synthase genes and to improve the synthesis yield and efficiency of γ-Eudesmol and Linalool, the present application provides a Michelia maudiae TPS synthase gene MnTPS12 and its application, providing an effective way to solve the above problems.

[0005] To achieve the above objectives, the technical solutions provided by the present application are as follows:

[0006] The present application provides a Michelia maudiae TPS synthase gene MnTPS12 , the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0007] The present application also provides an amplification primer for the above-mentioned Michelia maudiae TPS synthase gene MnTPS12 , the primer sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0008] Further, the application also provides a Brintupia bipinnata TPS synthetase gene MnTPS12 Construct a prokaryotic expression vector.

[0009] Further, the application also provides a preparation method of the expression protein MnTPS12 of the Brintupia bipinnata TPS synthetase gene MnTPS12 .

[0010] Further, the application also provides an application of the expression protein of the Brintupia bipinnata TPS synthetase gene MnTPS12 in preparation of γ-Eudesmol and Linalool.

[0011] The application has the following beneficial effects:

[0012] The Brintupia bipinnata TPS synthetase gene MnTPS12 provided by the application is a key gene for synthesis of γ-Eudesmol and Linalool in plant genetic engineering, and can effectively regulate synthesis of γ-Eudesmol and Linalool; the expression protein of the gene is applied to synthesis and preparation of γ-Eudesmol and Linalool, has high yield, and has important application value. MnTPS12 The application first clones a new terpene synthase gene MnTPS12 in Brintupia bipinnata, and the gene can catalyze FPP and GPP to generate γ-Eudesmol and Linalool, and plays an important role in improving plant terpene component content and resistance. MnTPS12 The terpene synthase gene studied in the application can be used for preparation of γ-Eudesmol and Linalool, and further preparation of essential oil, essence and medicine containing γ-Eudesmol and Linalool. The gene fragment of the terpene synthase gene

[0013] is constructed on a plant expression vector, and can be transformed into other plant materials by exogenous transformation, so as to obtain transgenic materials containing the terpene aroma gene, and provides an effective method for cultivating aroma and medicinal plants. MnTPS12 BRIEF DESCRIPTION OF DRAWINGS Figure 1

[0014] MnTPS12 : TPS gene family Pfam domain.

[0015] Figure 2 : Figure 3 Expression of the gene in rhizome leaves of Brintupia bipinnata.

[0016] MnTPS12: SDS-PAGE electrophoretogram of purified product of pET-32a-MnTPS12 recombinant protein; wherein, lane 1 is the supernatant after ultrasonic crushing; lane 2 is the supernatant after ultrasonic crushing; lanes 3-9 are 500 mM imidazole eluent.

[0017] Figure 4 : GC-MS detection spectrum of in vitro catalytic reaction product of MnTPS12 protein; wherein, A is the catalytic product of MnTPS12 and FPP reaction; B is the catalytic product of MnTPS12 and GPP reaction.

[0018] Figure 5 : Mass spectrum comparison chart; wherein, A is the mass spectrum comparison chart of γ-Eudesmol; B is the mass spectrum comparison chart of Linalool. DETAILED DESCRIPTION

[0019] Example One Figure 6 Gene Cloning

[0020] 1. Extraction of Total RNA from Machilus chinensis Leaves

[0021] Freshly collected Machilus chinensis leaves were used as the material, and total RNA was extracted using a kit Universal Plant Total RNA Kit (ER302-01, TRAN). The gun head and mortar used for RNA extraction were sterilized and sealed in a bag, and then subjected to high-pressure sterilization at 121°C for 20 min, followed by drying in a 60°C oven. After cooling, they were ready for use. The integrity of the RNA was detected by 1% agarose gel electrophoresis, and the concentration and purity were determined by a microspectrophotometer. 2. RCR Amplification and Recovery Purification

[0022] Using total RNA from Machilus chinensis leaves as the template, a single-stranded cDNA was synthesized using a TIANScript II RT Kit. According to the Machilus chinensis second-generation transcriptome sequencing sequence, primers were designed, with an upstream primer F1: 5'-ATGGCTCTTGTTTTGGGCTCT-3' (as shown in SEQ ID NO: 3) and a downstream primer R1: 5'-CTACATAGGAACGGGTTCCACC-3' (as shown in SEQ ID NO: 4), which were synthesized by Beijing Chengke Biological Company. The above cDNA was used as the template, and PCR amplification was performed using Max DNA Polymerase, according to the instructions.

[0023]

[0024] ​​After the PCR reaction, the product was detected by 1% agarose gel electrophoresis, and the gel block containing the target fragment band was cut out under the ultraviolet lamp with a scalpel, and the gel was recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit. The recovery method was performed according to the instructions. Then the recovered product was detected by 1% agarose gel electrophoresis to ensure successful recovery.

[0025] 3. Cloning vector ligation

[0026] Using the Lethal Based Simple Fast Cloning Kit from Tiangen, the appropriate amount of recovered product obtained in the above steps was connected with the cloning vector pLB according to the size and concentration of the target fragment. The specific method was performed according to the instructions. After ligation, the DH5α E. coli competent cells (Novizen) were taken out of the -80°C ultra-low temperature refrigerator and placed on ice for low-temperature thawing; 50 μL of E. coli competent cells were added to 4 μL of ligation liquid, and incubated on ice for 30 min; 42°C water bath for 90 s, and placed on ice for 2 min; add 800 μL of LB liquid medium without antibiotics, 37°C, 225 rpm, cultivate for 1 h; centrifuge at 12000 rpm for 30 s, collect the bacterial cells, discard the supernatant, and spread on LB solid medium containing 100 μg / ml ampicillin; seal the plate with sealing film, and place it in a 37°C inverted culture for 12-16 h.

[0027] The recombinant plasmid was screened by blue-white spot screening, and the white single colony selected was used as the template. The primers used were pLB-F and pLB-R, and the method was performed according to the instructions. Finally, the product was detected by 1% agarose gel electrophoresis. The colony containing the target fragment size was selected as the candidate, and was picked into LB liquid medium containing 100 μg / ml ampicillin using a gun head, and was sent to Beijing Qikang Biological Company for DNA sequence sequencing.

[0028] The sequence obtained by sequencing was compared with the original sequence of the transcriptome, and the domain prediction was performed in HMMER to determine that the gene sequence obtained by the application was the full-length sequence of the TPS family, and the gene was named MnTPS12 , and the protein sequence was also obtained.

[0029] MnTPS12 The results of 1% agarose gel electrophoresis of the gene are shown in MnTPS12 , showing that a single band of the expected size was cloned. The sequence obtained after sequencing and comparison is Figure 1The coding region (CDS) sequence of the gene is shown in SEQ ID: 1, which is 1692 bp in total, encodes 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 MnTPS12 conservative domain shows that it contains two N-terminal and C-terminal conservative domains of PF01397 and PF03936 MnTPS12 Therefore, it is considered that MnTPS12 is a TPS terpene synthase.

[0030] Example Two Figure 2 Expression analysis of the gene

[0031] The roots, stems and leaves of Phoebe sheareri were selected, total RNA was extracted using the TIANGEN kit RNA Easy Fast Plant Tissue Kit, and the specific method was performed according to the instructions. Reverse transcription was performed using the Aikuer kit Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2, and the specific method was performed according to the instructions. qRT-PCR was performed using the Baoguang Bio SYBRPRIME qPCR kit (Fast HS). The qRT-PCR primers F: 5'-CTCGTGAGGGGAAAGCCTAC-3' (as shown in SEQ ID NO: 5) and R: 5'-GCAACATCTCCCATGCCAAC-3' (as shown in SEQ ID NO: 6) were designed using NCBI. The reference gene was Camphor ACTIN (ACT, KM086738.1), F: 5'-CCTCGACACACAGGCGTTAT-3' (as shown in SEQ ID NO: 7), and R: 5'-CCATGCTCGATGGGATATTTCA-3' (as shown in SEQ ID NO: 8). The reaction system was cDNA 1 μL, F primer 0.5 μL (10 μM), R primer 0.5 μL (10 μM), ddH2O 3 μL, SYBRPRIME qPCR kit (Fast HS) 5 μL. The detection program was 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 in a BIO-RAD CFX Connect Real-Time System (Bio-Rad), each sample was set with three biological replicates and three technical replicates, the data was processed using the 2-ΔΔCt method, and the expression of Phoebe sheareri MnTPS12 in different tissues was calculated. MnTPS12 As shown in Figure 3 the expression amount in the roots was the highest, and the expression amounts in the stems and leaves were less.

[0032] Example 3: Prokaryotic Expression Analysis of MnTPS12

[0033] 1. Carrier Construction

[0034] 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.

[0035] 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.

[0036] 2. Recombinant protein expression

[0037] 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.

[0038] 3. Recombinant protein purification

[0039] The supernatant was slowly passed through the Ni column, and the impurity proteins were removed with 3 column volumes of PBS buffer containing 20 mM imidazole, and then the target protein was eluted with 14 ml of PBS buffer containing 500 mM imidazole, and one tube was taken every 2 ml. The precipitate, supernatant and eluate were sampled and detected using SDS-PAGE. The eluate containing the target protein was desalted, and then the protein concentration was determined.

[0040] Example Four In Vitro Enzymatic Function Analysis of MnTPS12

[0041] Take 50 μg of protein, 10 mM of MgCl2, 10 mM of DTT, 5 μg of FPP / GPP / FPP+GPP, and 25 mM of HEPES (pH 7.4) to make up to 100 μL. Incubate at 30°C for 3 h. After the reaction is completed, 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. Take the organic phase and filter it into a sample bottle using a 0.22 μm filter head, and then perform GC-MS detection.

[0042] Use a TRACE 1300 gas chromatograph-ISQ 7000 mass spectrometer detector (GC-MS) and an HP-5MS column (30 m x 250 μm x 0.25 μm film thickness) with helium as the carrier, and the carrier speed is 1.5 mL / min. Set the initial column temperature to 40°C, and equilibrate for 5 min, then increase the temperature to 300°C at a rate of 8°C / min for 30 min. The mass spectrometry conditions are as follows: ion transmission line temperature 290°C, solvent delay 3 min, ion scan range 40 m / z to 600 m / z, and the rest of the parameters are according to the system default. Use an EI source as the ion source of the mass spectrometer. The collected mass spectrum is analyzed using the NIST mass spectrum library.

[0043] MnTPS12 The results of the prokaryotic expression of the gene are shown in MnTPS12 The size of the pET-32a-MnTPS12 recombinant protein is about 84.6 Kda, and the SDS-PAGE gel electrophoresis result shows that the size of the pET-32a-MnTPS12 recombinant protein is close to the expected size. The in vitro enzyme activity identification is shown in Figure 4 When farnesyl pyrophosphate (FPP) is used as the substrate, the main product catalyzed by pET-32a-MnTPS12 is the sesquiterpenoid γ-Eudesmol Figure 5 A, 6A), and when geranyl pyrophosphate (GPP) is used as the substrate, the main product catalyzed is the monoterpene Linalool Figure 5 B, 6B), indicating that MnTPS12 is a bifunctional enzyme that generates γ-Eudesmol and Linalool.

[0044] In summary, the present application first clones a new terpenoid synthase gene from Machilus breviflora Figure 5 , which can catalyze FPP and GPP to generate γ-Eudesmol and Linalool, and plays an important role in improving the content of plant terpenoid components and resistance. The terpenoid synthase gene MnTPS12 studied in the present application can be used for preparing γ-Eudesmol and Linalool, and for further preparing essential oil, essence and medicine containing γ-Eudesmol and Linalool. The gene fragment of the terpenoid synthase gene MnTPS12 MnTPS12 is constructed on a plant expression vector, and can be transformed into other plant materials by exogenous transformation, thereby obtaining transgenic materials containing the terpenoid aroma gene, and providing an effective method for cultivating aroma and medicinal plants.

[0045] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and all shall be included in the protection scope of the present application.

Claims

1. A TPS synthase gene from *Agropyron cristatum* MnTPS12 Its features are: The gene MnTPS12 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. MnTPS12 The encoded 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 sequences of the amplification primers 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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