Artemisia argyi eudesma-5,7,15-triene synthase gene altps37, the product encoded by the gene and application thereof

Abstract

The present application relates to the technical field of genetic engineering, in particular to atractylodes lancea cymene synthase gene AlTPS37 and the product and application thereof, the gene AlTPS37 has the nucleotide sequence shown in (1) or (2): (1) the nucleotide sequence shown in SEQ ID NO.1; (2) the nucleotide sequence shown in SEQ ID NO.1 is substituted, deleted or added one or several nucleotides and the nucleotide sequence of the same functional protein expressed. The AlTPS37 gene is cloned and functionally identified from atractylodes lancea for the first time, it is confirmed that the proteinase coded by the gene can directly catalyze the substrate to generate gamma-cymene, beta-cymene and alpha-cymene. The biosynthesis molecular basis of the main active ingredient cymene in atractylodes lancea is fundamentally revealed, and the key technical blank in the field is filled.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the Atractylodes lancea leaf eucalyptus alcohol synthase gene AlTPS37 and its encoded products and applications. Background Technology

[0002] Atractylodes lancea (Thunb.) DC. is a perennial herbaceous plant of the Asteraceae family and one of the source plants of the traditional Chinese medicine Atractylodes lancea. Its dried rhizome is used medicinally and is a major traditional Chinese medicinal material with effects such as drying dampness and strengthening the spleen, dispelling wind and cold, and improving eyesight. It is widely used in clinical prescriptions and the production of traditional Chinese medicine preparations. Modern pharmacological studies have shown that eudesmol, including its isomers γ-eudesmol, β-eudesmol, and α-eudesmol, is one of the main active pharmaceutical ingredients in the volatile oil of Atractylodes lancea, exhibiting significant anti-inflammatory, anti-tumor, neuroprotective, and gastrointestinal regulatory effects. Therefore, the eudesmol content is often used as an important indicator for evaluating the quality of Atractylodes lancea.

[0003] The biosynthesis of terpenoids in plants is mainly accomplished through two classical pathways: the mevalonate (MVA) pathway in the cytoplasm and the methylerythritol phosphate (MEP) pathway in the plastids. These two pathways produce the universal five-carbon units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are then catalyzed by a series of terpene synthases (TPSs) to form diverse monoterpenoid, sesquiterpene, and diterpene skeletons. TPSs play a crucial role in plant growth and development; however, the functional genes of the major compounds in *Atractylodes lancea* have not been reported in detail, and the function of *TPS* genes in *Atractylodes lancea* remains to be elucidated.

[0004] Farnesyl pyrophosphate (FPP) is a common precursor to sesquiterpenes, while terpene synthase (TPS) is a key enzyme catalyzing the formation of specific carbon skeletons from FPP, determining the type of end product. Although terpenes (including eucalyptol) have significant medicinal value, their production heavily relies on direct extraction from plants such as *Atractylodes lancea*. This traditional approach has inherent limitations: little is known about *Atractylodes lancea* terpene synthases, particularly the key synthase genes responsible for eucalyptol biosynthesis. Most reported plant TPS-catalyzed products are complex, making it difficult to efficiently and specifically generate the target product. Currently, there is a lack of a clearly identified and functionally validated highly efficient eucalyptol synthase capable of producing eucalyptol (especially γ-eucalyptol) as the major product.

[0005] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0006] The purpose of this invention is to address the lack of a clearly identified and functionally validated highly efficient eucalyptol synthase in the prior art that can produce eucalyptol (especially γ-eucalyptol) as the main product. This invention provides the Atractylodes lancea eucalyptol synthase gene AlTPS37 and its encoded products and applications.

[0007] To achieve the above objectives, this invention discloses the Atractylodes lancea eucalyptus leaf alcohol synthase gene AlTPS37, wherein the gene AlTPS37 has the nucleotide sequence shown in (1) or (2):

[0008] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0009] (2) A nucleotide sequence of SEQ ID NO.1 that has been substituted, deleted or added with one or more nucleotides and expresses the same function of a protein.

[0010] SEQ ID NO.1:

[0011]

[0012] This invention also discloses the product encoded by the Atractylodes lancea eucalyptus leaf alcohol synthase gene AlTPS37, wherein the product encoded by the gene AlTPS37 has the amino acid sequence shown in (1) or (2):

[0013] (1) The amino acid sequence shown in SEQ ID NO.2;

[0014] (2) Without affecting its activity, the amino acid sequence shown in SEQ ID NO.2 is replaced, deleted or added with one or more amino acids to obtain the amino acid sequence.

[0015] SEQ ID NO.2:

[0016] MSTVQENVVRATASFPLDIWGDQFLVCDQQEEQDGVEQVVEDLKEEVRKE

[0017] ILAALNVPAEHTNLLKLVDAIQRLGIAYYFEEEINEVLKHIYVSNGDNWT

[0018] GGCPSLWFRLLRQQGFFVSCDIFNNYKDKDGSFKECLANDVQGLLDLYEA

[0019] AYMRVQGEDILDDALVFTRTRLDDISKDPLRGTNTDSTQIQEALKQPLLK

[0020] RLPRLEALRYIPFYQQQASHNKYLLKLAKLGFNQVQSLHKKELSQLSKWW

[0021] KGYDVTNFPYARNRLVECYFWAQGVYFEPKYSQSRIFLAKNLATASILD

[0022] DTYDAYGTYEELKIFTEAIQRWSITCLDMLPEYMKPLYQMVIDVYKEMEE

[0023] IMADEEKAYYLNNAIESMKEFIGSYMTEAKWGNEGYIPTTEEHISVALIS

[0024] SGTKHLVTTSFVGMHDIITEESFKWVSTNPPLIKAAAAVGRFLDDIVSHK

[0025] EEQERKHAASVVECYMEQFDVTEDHVYDLVNKKIDQAWKEIVRESLMCKD

[0026] VPMALIMRAINFARGMEVMYKGQDNYTHMGDEMINHIKSLFVDSIST.

[0027] The present invention also discloses a recombinant expression vector comprising the Atractylodes macrocephala eucalyptus leaf alcohol synthase gene AlTPS37 as described in claim 1.

[0028] The recombinant expression vector is plasmid p416GAL1.

[0029] When constructing the AlTPS37 gene of this invention into the expression vector, any strong or inducible promoter can be added before its transcription initiation nucleotide, provided that it is within the same reading frame as the coding sequence to ensure translation of the entire sequence. To facilitate the identification and screening of transgenic plant cells or plants, the vector can be modified or altered during construction, for example, by adding selectable markers. Commonly used markers include genes for antibiotic resistance enzymes and biosafety markers, as well as genes for color-changing enzymes or luminescent compounds such as GUS or GFP.

[0030] This invention also discloses a specific primer pair for the AlTPS37 gene of Atractylodes lancea leaf ethanol synthase, wherein the specific primer pair includes an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4. The specific primer pair can be used to amplify the nucleotide sequence of the AlTPS37 gene by polymeric chain reaction (PCR).

[0031] SEQ ID NO.3:

[0032] AGAACTAGTGGATCCCAAAAAAATGTCTACTGTACAAGAAAACGTTGT.

[0033] SEQ ID NO.4:

[0034] TAACTAATTACATGACTCGAGGTCGATCATGTACTGATAGAGTCAACG.

[0035] This invention also discloses the application of the Atractylodes lancea eucalyptus leaf alcohol synthase gene AlTPS37 in the preparation of sesquiterpenoid compounds.

[0036] Compared with existing technologies, the advantages of this invention are as follows: This invention successfully cloned a gene encoding an atractylodes terpene synthase (AlTPS) from the rhizome of Atractylodes lancea. This enzyme can be applied to pathways prepared using farnesyl pyrophosphate (FPP) as a substrate. The gene provided by this invention can be used to increase the content of atractylodes terpenes through genetic engineering techniques. Attached Figure Description

[0037] Figure 1 Agarose gel electrophoresis image of Atractylodes lancea terpenoid synthase AmTPS37;

[0038] Figure 2 Predictive analysis of the structure and functional domains of AlTPS37, a terpene synthase of Atractylodes lancea;

[0039] Figure 3 The tertiary structure of the AlTPS37 protein, a gene for terpenoid synthase in Atractylodes lancea;

[0040] Figure 4 SPME-GC-MS analysis of the eukaryotic expression product of AlTPS37 recombinant protein is shown. In the figure, A is the chromatogram of the empty vector, B is the chromatogram of α-Eudesmol, C is the chromatogram of β-Eudesmol, D is the chromatogram of γ-Eudesmol, and E is the chromatogram of the recombinant plasmid. Detailed Implementation

[0041] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0042] The technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0043] The PrimeScript™ II 1st Strand cDNA Synthesis Kit was purchased from TakaraBio; the non-toxic 4S Green Plus nucleic acid dye was purchased from Shanghai Sangon Biotech Co., Ltd.; the EasyPure Quick Gel Extraction Kit and the pEASY-Blunt Zero Cloning Kit were purchased from Beijing TransGen Biotech Co., Ltd.; high-fidelity restriction endonucleases such as Phusion, BamHI, and SalI were purchased from NEB Biotechnology (Beijing) Co., Ltd.; the ZYMO RESEARCH Frozen-EZ Yeast Transformation II™ yeast competent cell preparation kit was purchased from ZYMO RESEARCH; SD / -Ura Broth, SC / -Ura Broth, and other yeast auxotrophic media were purchased from Beijing Huayueyang Biotechnology Co., Ltd.; D-(+)-galactose was purchased from Sangon Biotech Co., Ltd.; primers were synthesized by General Biotech (Anhui) Co., Ltd.; and other reagents were imported or domestically produced analytical grade reagents.

[0044] Example 1

[0045] Cloning of the AlTPS37 gene, a terpene synthase in Atractylodes lancea:

[0046] Cloning of AlTPS37 utilizes forward primers:

[0047] Upstream primer: P1: 5' ATGTCTACTGTACAAGAAAACGTTG 3'; Downstream primer: P2: 5'TCATGTACTGATAGAGTCAACGAAA 3'.

[0048] PCR amplification was performed using the full-length sequence encoding the AlTPS37 gene, a terpene synthase from Atractylodes lancea, as a template. The PCR reaction volume was (50 μL): 2 μL template cDNA, 2 μL each of primers primer-F and primer-R, 25 μL Phusion High-Fidelity, and the remaining reaction volume was made up with sterile double-distilled water. PCR reaction conditions: 95 ℃ pre-denaturation for 2 min, 95 ℃ denaturation for 20 s, 56 ℃ annealing for 20 s, 72 ℃ extension for 1 min, 40 cycles, followed by a final extension at 72 ℃ for 5 min, and storage at 4 ℃. Figure 1 As shown, the size of the PCR amplification product was determined by agarose gel electrophoresis to be between 1000bp and 2000bp, which is consistent with the length of the target gene. Thus, the clone sequence of the Atractylodes terpenoid synthase AlTPS37 gene was obtained.

[0049] Example 2

[0050] Bioinformatics analysis of the AlTPS37 gene:

[0051] The full-length cDNA of the Atractylodes terpenoid synthase gene obtained in Example 1, AlTPS37, has an open reading frame (ORF) length of 1644 bp, and its detailed sequence is shown in SEQ ID NO.1 in the sequence listing. The AlTPS37 gene sequence was subjected to nucleotide homology searches in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR databases using the BLAST program in the NCBI database. This gene showed high homology at the amino acid level with TPS genes from other species and possessed a typical TPS functional domain (…). Figure 2 The tertiary structure of the AlTPS37 protein was predicted using the online software Swiss Model, such as... Figure 3 As shown, the AlTPS37 protein model A0A5P8H4P8.1.A has a score of 0.93 and a protein sequence similarity of 77.76%.

[0052] Example 3

[0053] Construction of the eukaryotic expression vector for the AlTPS37 gene:

[0054] Using the cDNA of the AlTPS37 gene as a template, BamHI and SaIⅠ were selected as double restriction sites, and specific upstream and downstream primers were designed (as shown in Table 1) for PCR amplification. The underlined parts in the primers are the restriction sites.

[0055] Table 1. Specific upstream and downstream primers

[0056]

[0057] The PCR reaction system (50 μL) consisted of: 2 μL template cDNA, 2 μL each of primers primer-F and primer-R, 25 μL Phusion High-Fidelity, and the remaining reaction volume was made up with sterile double-distilled water. PCR conditions were: 95 °C pre-denaturation for 2 min, 95 °C denaturation for 20 s, 56 °C annealing for 20 s, 72 °C extension for 1 min, 40 cycles followed by a final extension at 72 °C for 5 min, and storage at 4 °C. The amplified product was detected by 1% agarose gel electrophoresis and then recovered from the gel. The expression vector p416GAL1 plasmid was digested with BamHI and SaIⅠ, and then recovered from the gel. The recovered target fragment was ligated to the expression vector p416GAL1 using a seamless splicing kit at 50 °C for 60 min. The ligation product was transformed into *E. coli* Trans1-T1 competent cells, and single clones were picked for colony PCR positive testing, sequencing, and plasmid extraction. The recombinant plasmid AlTPS37-P416-Ura with the correct target sequence was preserved for expression transformation.

[0058] Example 4

[0059] AlTPS37 eukaryotic expression and functional analysis:

[0060] (1) Yeast conversion:

[0061] Take 2 μL of p416GAL1-AlTPS37 recombinant plasmid and p416GAL1 respectively

[0062] Add a blank vector (as a negative control) to 50 μL of competent cells, then add 500 μL of Frozen-EZ YeastSolution 3 and mix thoroughly. Incubate at 30°C for 45 min, gently tapping the mixture 2-3 times with your finger during the incubation period to ensure even mixing. Spread 150 μL of the mixture onto an SD-Ura plate and incubate at 30°C until single colonies appear. Pick a single colony from the SD-Ura plate and transfer it to 4 mL of SD-Ura liquid medium. Incubate at 30°C and 200 rpm for 2-3 days. Then, transfer 1 mL of the bacterial culture to 10 mL of SD-Ura liquid medium and incubate at 30°C and 200 rpm for 2-3 days to expand the culture.

[0063] (2) Galactose-induced expression and incubation:

[0064] Centrifuge 10 mL of the culture obtained from the above steps at 5000 x g for 5 min to collect the bacteria. In a clean bench, discard the waste liquid, add 10 mL of SC-Ura liquid medium containing 2% galactose, mix well by pipetting, and induce at 30°C and 200 rpm for 2-3 days. Take 5 mL of the induced bacterial culture into a 20 mL headspace vial and induce at 30°C and 200 rpm for 2 days.

[0065] (3) Functional identification:

[0066] Volatile components in the induced bacterial culture were detected using HS-SPME-GC-MS. Extraction conditions: headspace extraction for 50 min at 60 ℃, followed by elution for 8 min, and then GC-MS separation and identification. Chromatographic conditions: Agilent DB-WAX UI column (30 m × 0.25 mm × 0.25 µm). Injection temperature 250 ℃, split ratio 20:1; initial temperature 40 ℃, hold for 3 min, increase to 200 ℃ at 5 ℃ / min, hold for 5 min, then increase to 240 ℃ at 5 ℃ / min, hold for 5 min. High-purity helium was used as the carrier gas at a flow rate of 1 mL / min. Mass spectrometry conditions: electron impact (EI) ionization source, ionization voltage 70 eV, ionization source temperature 230 ℃, scan range 33–600 amu. The products were qualitatively analyzed using comparisons with standards, such as… Figure 4 As shown, the results revealed that AlTPS37 in *Saccharomyces cerevisiae* catalyzes the production of γ-eudesmol (γ-Eudesmol, peak 1), α-eudesmol (α-Eudesmol, peak 2), and β-eudesmol (β-Eudesmol, peak 3) by FPP. γ-eudesmol was the major product (peak area 30.79%), while α-eudesmol (7.57%) and β-eudesmol (3.99%) were the minor major products. Therefore, the experimental results indicate that the products of the induced expression reaction contain three sesquiterpenoid components (α-eudesmol, β-eudesmol, and γ-eudesmol), demonstrating the catalytic activity of the AlTPS37 recombinant protein.

[0067] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. The AlTPS37 gene of *Atractylodes lancea* leaf ethanol synthase, characterized in that, The gene AlTPS37 has the nucleotide sequence shown in (1) or (2): (1) The nucleotide sequence shown in SEQ ID NO.1; (2) A nucleotide sequence of SEQ ID NO.1 that has been substituted, deleted or added with one or more nucleotides and expresses the same function of a protein.

2. The product encoded by the Atractylodes lancea leaf eucalyptus alcohol synthase gene AlTPS37 as described in claim 1, characterized in that, The product encoded by the gene AlTPS37 has the amino acid sequence shown in (1) or (2): (1) The amino acid sequence shown in SEQ ID NO.2; (2) Without affecting its activity, the amino acid sequence shown in SEQ ID NO.2 is replaced, deleted or added with one or more amino acids to obtain the amino acid sequence.

3. A recombinant expression vector, characterized in that, The recombinant expression vector includes the Atractylodes macrocephala eucalyptus leaf alcohol synthase gene AlTPS37 as described in claim 1.

4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector is plasmid P416-Ura.

5. The specific primer pair for the *Atractylodes lancea* leaf eucalyptus alcohol synthase gene AlTPS37 as described in claim 1, characterized in that, The specific primer pair includes an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.

4.

6. The application of the Atractylodes lancea leaf alcohol synthase gene AlTPS37 as described in claim 1 in the preparation of sesquiterpenoid compounds.

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