CN116478973A

CN116478973A - Plum sesquiterpene synthase CbTPS6 and related biological materials and application thereof

Info

Publication number: CN116478973A
Application number: CN202210039986.8A
Authority: CN
Inventors: 黄璐琦; 崔光红; 唐金富; 郭娟; 赵曼茜
Original assignee: Sichuan Honghe Biotechnology Co ltd
Current assignee: Sichuan Honghe Biotechnology Co ltd
Priority date: 2022-01-14
Filing date: 2022-01-14
Publication date: 2023-07-25

Abstract

The invention discloses a plum sesquiterpene synthase CbTPS6 and related biological materials and application thereof. The invention firstly discloses any one of the following proteins: a1 A protein consisting of the amino acid sequence shown in SEQ ID No. 2; a2 Fusion proteins obtained by connecting protein tags at the N end or/and the C end of the protein shown in the sequence 2; a3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2, has more than 90% of identity with the protein shown in the A1) and has the same function. The invention further discloses the protein related biological material and application thereof. The invention obtains the key enzyme gene synthesized by the monoterpene component from the plum slice tree for the first time, and proves that the protein CbTPS6 can catalyze FPP to produce (-) -beta-elemene, and has important theoretical and practical significance for regulating and producing plant terpenoid compounds and cultivating high-quality plum slice tree.

Description

Plum sesquiterpene synthase CbTPS6 and related biological materials and application thereof

Technical Field

The invention relates to the field of medicinal plant genetic engineering, in particular to a plum terpenoid synthase CbTPS6 and a related biological material and application thereof.

Background

The plum slice tree (physiolgical type of Cinnamomum burmannii) is evergreen arbor of Cinnamomum genus of Lauraceae family, is rich in dextroborneol ((+) -born), is a rare medicinal material, is also a high-grade spice, daily chemical cosmetic raw material and chemical product, and is widely applied to multiple industries such as medical treatment, beauty treatment, spice and the like. A new natural dextro borneol resource with production and utilization values is found in Guangdong in the south China vegetable garden of China academy of sciences, and the blank of producing natural dextro borneol in China is filled. The volatile oil contains a large amount of sesquiterpene components besides the monoterpene components such as dextrorotatory and the like. Wherein part of the sesquiterpenoids have remarkable pharmacological activity, for example, the volatile components of the plum contains trace amounts of beta-elemene. Beta-elemene is sesquiterpene with elemene skeleton, and the beta-elemene separated from volatile oil of Curcuma zedoaria is the main component of the antitumor medicine elemene emulsion developed by our country.

Terpene synthases (TPS) are a family of genes involved in the synthesis of an important class of Terpene secondary metabolites in the genome of the prune tree. CbTPS6 is a member of the family of the Meisson terpene synthase genes. At present, no related research on obtaining a key enzyme gene with beta-elemene synthesis capability from a plum tree has been found.

Disclosure of Invention

The technical problem to be solved by the invention is to obtain a novel meretrix terpene synthase which participates in the synthesis of sesquiterpene compounds so as to synthesize or prepare beta-elemene.

To solve the above problems, the present invention provides a protein, which is CbTPS6, derived from a Japanese apricot tree (physiolgical type of Cinnamomum burmannii), named Japanese apricot terpene synthase CbTPS6, which is a) or b) or c) as follows:

a) The amino acid sequence is a protein shown in the sequence 2;

b) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein represented by the sequence 2;

c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2.

Wherein sequence 2 consists of 561 amino acid residues.

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

Among the above proteins, a protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Biological materials related to CbTPS6 are also within the scope of the invention.

The related biological material of CbTPS6 provided by the invention is any one of the following A1) to A12):

a1 A nucleic acid molecule encoding CbTPS 6;

a2 An expression cassette comprising A1) said nucleic acid molecule;

a3 A) a recombinant vector comprising the nucleic acid molecule of A1);

a4 A recombinant vector comprising the expression cassette of A2);

a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);

a6 A) a recombinant microorganism comprising the expression cassette of A2);

a7 A) a recombinant microorganism comprising the recombinant vector of A3);

a8 A) a recombinant microorganism comprising the recombinant vector of A4);

a9 A transgenic plant cell line comprising the nucleic acid molecule of A1);

a10 A transgenic plant cell line comprising the expression cassette of A2);

a11 A transgenic plant cell line comprising the recombinant vector of A3);

a12 A) a transgenic plant cell line comprising the recombinant vector of A4).

In the above biological material, the nucleic acid molecule of A1) is as shown in any one of the following B1) to B5):

b1 A DNA molecule shown in a sequence 1 in a sequence table;

b2 A coding sequence is a DNA molecule shown as a sequence 1 in a sequence table;

b3 A DNA molecule shown in a sequence 4 in a sequence table;

b4 A coding sequence is a DNA molecule shown as a sequence 4 in a sequence table;

b5 Under stringent conditions with a DNA molecule defined by B1) or B2) or B3) or B4) and encoding CbTPS6.

Wherein, the sequence 1 in the sequence table consists of 1692 nucleotides, and codes the protein shown in the sequence 2.

The stringent conditions are hybridization and washing of the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing of the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA, and the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

In the above biological material, the expression cassette (CbTPS 6 gene expression cassette) described in A2) containing a nucleic acid molecule encoding CbTPS6 refers to DNA capable of expressing CbTPS6 in a host cell, and this DNA may include not only a promoter that initiates transcription of CbTPS6 but also a terminator that terminates transcription of CbTPS6. Further, the expression cassette may also include an enhancer sequence.

In the above biological material, the vector may be a plasmid, cosmid, phage or viral vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as agrobacterium.

In the above biological materials, none of the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs include propagation material.

The invention further provides application of the protein or the related biological material thereof.

The application is specifically as follows:

1) The application of the protein as terpene synthase;

2) The application of the related biological material in preparing terpene synthases;

3) The application of the protein or related biological material in preparing or synthesizing terpenoid;

4) The application of the protein or the related biological material in catalyzing farnesyl pyrophosphoric acid to form (-) -beta-elemene.

In the application, the terpenoid is (-) -beta-elemene.

The invention also provides a method for preparing CbTPS6.

The method for preparing the CbTPS6 comprises the steps of introducing a coding gene of the CbTPS6 into a receptor microorganism to obtain a recombinant microorganism expressing the CbTPS6, culturing the recombinant microorganism, and expressing the recombinant microorganism to obtain the CbTPS6.

In the above method, the recipient microorganism is a prokaryotic microorganism. Specifically, the prokaryotic microorganism is escherichia coli. More specifically, the E.coli is the E.coli expression strain Transetta (DE 3).

In the method, the coding gene of the CbTPS6 can be introduced into an escherichia coli expression strain Transetta (DE 3) through a recombinant plasmid pET32 a; the recombinant plasmid pET32a is characterized in that CbTPS6 is a recombinant expression vector which is constructed to a BamHI enzyme cutting site of a pET32a (+) vector by using a CbTPS6 gene shown in a sequence 1 and keeps other sequences of the pET32a (+) vector unchanged.

The invention further provides a process for preparing (-) - β -elemene comprising the step of catalyzing farnesyl pyrophosphate (FPP) with CbTPS6.

In the above method, an enzymatic buffer solution is added in the catalysis process, wherein the enzymatic buffer solution is composed of HEPES and MgCl ₂ DTT composition;

the concentration of HEPES in the enzymatic buffer is 50mM;

the MgCl ₂ The concentration in the enzymatic buffer was 10mM;

the concentration of the DTT in the enzymatic buffer is 5mM;

the pH value in the enzymatic buffer is 7.2.

The CbTPS6 gene is cloned from the cDNA of the plum tree, and experiments prove that: the CbTPS6 protein can catalyze FPP to form (-) -beta-elemene, has an important function on biosynthesis of monoterpene compounds such as (-) -beta-elemene in plum slice trees, provides an important basis for improving the content of active ingredient (-) -beta-elemene in plum slice trees or directly producing (-) -beta-elemene by utilizing a genetic engineering technology, and further has important theoretical and practical significance on regulating and producing plant terpenoid compounds and cultivating high-quality plum slice trees.

Drawings

FIG. 1 is a diagram of agarose gel electrophoresis of a clone of the plum CbTPS6 gene; m represents a Trans2K DNA Marker (the molecular weight standard of nucleic acid, the bands are 2000, 1000 and 750bp from top to bottom respectively), and CbTPS6 represents a CbTPS6 gene.

FIG. 2 shows the analysis of the CbTPS6 protein expressed in E.coli by polyacrylamide gel electrophoresis (SDS-PAGE). Wherein M is Premixed Protein Marker (the molecular weight standard of the protein, the bands are 170, 130, 95, 72, 55 and 43kDa from top to bottom respectively), vector is the electrophoresis result of the supernatant of the control strain, and CbTPS6 is the electrophoresis result of the supernatant of recombinant plasmid pET32 a; the arrow indicates the recombinant plasmid pET32a, the target protein expressed by CbTPS6 (i.e. recombinant protein CbTPS 6).

FIG. 3 is a GC-MS analysis of the CbTPS6 enzymatic reaction product. Wherein A is an extraction ion flow diagram of a standard product (-) -beta-elemene, and B is an extraction ion flow diagram of a target compound of a supernatant of CbTPS6 recombinant bacterium; c is a mass spectrum of a standard product (-) -beta-elemene; d is a mass spectrum of a target compound of pET32a which is the supernatant of CbTPS6 recombinant bacteria.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

In the following examplesThe High-Fidelity DNA Polymerase BamHI restriction enzyme is New England Biolabs;

the rapid universal plant RNA extraction kit is a product of Beijing Hua Vietnam biotechnology Co., ltd;

TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, trans2K DNA Marker, pEASY-Uni Seamless Cloning and Assembly Kit, E.coli competent cell Transetta (DE 3) are products of Beijing full gold Biotechnology Co., ltd;

premixed Protein Marker (Low) is a product of Takara corporation;

PageRuler ^TM prestained Protein Ladder is a ThermoFisher Scientific company product;

the pET32a (+) vector is a product of Novagen company;

Ni-NTA Agarose is a product of Qiagen (catalog number: 30210);

protein purification and SDA-PAGENa ₂ HPO ₄ NaCl, DTT, PMSF, imidazole acrylamide/methylene bisacrylamide (30% solution) is a product of the division of bioengineering (Shanghai);

protein ultrafiltration tubes (Amicon-Ultra-15) are products from Millipore corporation (catalog number: UFC 903024);

the pESC-Leu vector is a product of Agilent company;

SD-Ura and SD-Ura-Leu are products of Beijing Pankeno technologies Co., ltd;

ZYMO RESEARCH Frozen-EZ Yeast Transformation II kit Zymo Research company;

BY4741 yeast strain (genotype: MATA his 3. Delta.1 leu 2. Delta.0 met 15. Delta.0 ura 3. Delta.0) is a product of Beijing Wash Vietnam Biotech Co., ltd;

farnesyl pyrophosphate (FPP) is a product of Sigma company with catalog number G6772 and CAS number 763-10-0;

(-) -beta-elemene is a control for Chinese inspection department, batch number: 100268-201903.

EXAMPLE 1 cloning of full-length cDNA sequence of the Mei-pian Tree CbTPS6 Gene

1. Extraction of Total RNA

The method is operated according to the instruction of a rapid universal plant RNA extraction kit of Beijing Hua Vietnam biotechnology Co Ltd, and total RNA of leaves of the plum tree is extracted.

2. Synthesis of first strand cDNA

The cDNA was obtained by reverse transcription, after the operation according to the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix instruction of Beijing full gold Biotechnology Co.Ltd.

The reverse transcription reaction system is shown in Table 1.

TABLE 1

The reverse transcription steps are as follows:

(1) To obtain higher synthesis efficiency, total RNA, anchore Oligo (dT) was used ₁₈ Primer and RNase-free Water are mixed in a PCR tube uniformly at 65 ℃ for 5min;

(2) 10.0. Mu.L of 2 XTS Reaction Mix and 1.0. Mu.L of the PCR Reaction Mix were added to the above-mentioned PCR tubeRT/RI Enzyme Mix, 1.0. Mu.L gDNA reverse, gently Mix;

(3) Carrying out reverse transcription reaction at 42 ℃ for 30min and 85 ℃ for 5s to obtain first strand cDNA;

(4) The first strand cDNA was stored at-20 ℃.

3. Primer design

According to the transcriptome data of the leaves of the plum slice, an Open Reading Frame (ORF) sequence is obtained, and cloning primers CbTPS6-F1 and CbTPS6-R1 are designed based on the ORF, wherein the sequences of the primers are as follows:

CbTPS6-F1：5’-ATGGCTCTTGTTTCTGGTTCTGG-3’；

CbTPS6-R1：5’-TTAAATAGGAATGGGTTCCACTAAC-3’。

4. PCR amplification

Using the first strand cDNA obtained in the step 2 as a template, and adopting high-fidelity enzymePCR amplification is carried out on the High-Fidelity DNA Polymerase, cbTPS6-F1 and CbTPS6-R1 primers to obtain PCR amplification products, the result of the agarose gel electrophoresis of the gene clone is shown in FIG. 1, and sequencing is carried out on the PCR amplification products.

Wherein, the PCR amplification procedure is as follows:

pre-denaturation at 98℃for 3min;98℃for 20s,55℃for 20s,72℃for 1min,35 cycles; extending at 72℃for 5min.

Sequencing results showed that: the sequence of the PCR amplification product was shown in sequence 1, and the gene shown in sequence 1 was designated as CbTPS6, which encodes a protein consisting of 561 amino acid residues, designated as CbTPS6, and the amino acid sequence of the protein was shown as sequence 2.

Example 2 obtaining and functional analysis of the Mei-pian tree CbTPS6 protein

1. Obtaining of the CbTPS6 protein of the Mei-pian Tree

1. Construction of recombinant vectors

The CbTPS6 gene shown in the sequence 1 is constructed to the BamHI enzyme cutting site of a pET32a (+) vector (Novagen company) by adopting pEASY-Uni Seamless Cloning and Assembly Kit of Beijing full gold biotechnology limited company, and other sequences of the pET32a (+) vector are kept unchanged, so that a recombinant plasmid pET32a:: cbTPS6 (verified by sequencing) is obtained.

The method comprises the following specific steps:

1) The PCR amplification product obtained in example 1 was used as a template, and the primers CbTPS6-F2 and CbTPS6-R2 were used for PCR amplification, and the purified PCR product was obtained by recovering and purifying. Wherein the primer sequences are as follows (the sequences shown underlined are the vector homology regions):

the primer sequences are as follows (the sequences shown underlined are the vector homology regions):

CbTPS6-F2：

5’-CCATGGCTGATATCGGAATGCCTCTTGTTTTGGGCTCTGG-3’；

CbTPS6-R2：

5’-ACGGAGCTCGAATTCGGTTAAATAGGAATGGGTTCCACTA-3’。

2) The pET32a (+) vector (Novagen) was digested with the restriction enzyme BamHI, and the linearized vector backbone was recovered.

3) And (3) taking the purified PCR product obtained in the step (1), operating according to the specification of Peking full-scale gold biotechnology Co., ltd., pEASY-Uni Seamless Cloning and Assembly Kit, and cloning the PCR product to the linearized vector skeleton of the step (2) to obtain the recombinant plasmid pET32a: cbTPS6.

2. Recombinant bacterium acquisition

Converting the recombinant plasmid pET32a into an escherichia coli expression strain Transetta (DE 3) (purchased from Beijing full-scale gold biotechnology Co., ltd.) to obtain pET32a CbTPS6 recombinant bacterium; meanwhile, a pET32a (+) vector without a target gene is used for transforming an escherichia coli expression strain Transetta (DE 3) as a control bacterium.

3. Induction expression of recombinant protein CbTPS6

pET32a is selected, cbTPS6 recombinant bacteria and control bacteria are respectively inoculated in 2mL of LB liquid medium (100 mg/L ampicillin) and cultured overnight at 37 ℃ in a shaking way. The following day is 1:100 diluted and added into 200mL LB liquid medium, and shake cultured at 37 ℃ until OD ₆₀₀ 0.6-0.8 h transferred to 18℃and shaken for 1 hour, IPTG was added to a final concentration of 0.5mM, and the shaking culture was continued at 18℃for 24 hours to induce the expression of the target protein. Centrifuging the bacterial liquid with 8000g for 5min, discarding the supernatant, collecting pET32a (CbTPS 6 recombinant bacteria) and control bacteria, and storing in a refrigerator at-80deg.C for use.

4. Recombinant protein CbTPS6 extraction

Extracting the proteins in the recombinant strain CbTPS6 and the control strain.

The method comprises the following specific steps: pET32 a:. CbTPS6 recombinant and control cells were pre-chilled with 5mL HEPES buffer (25mM HEPES,5M MgCl) ₂ 5M DTT, pH 7.0) was resuspended; placing the mixture into ice bath for ultrasonic bacteria breaking (30% power, ultrasonic for 5s, interval for 5s, lasting for 5min, repeating for 1 time), centrifuging 12000g at 4 ℃ for 30min to respectively obtain pET32a: cbTPS6 recombinant bacteria supernatant and control bacteria supernatant, namely protein solution.

SDS-PAGE of pET32a: cbTPS6 recombinant supernatant and control supernatant was performed and the results are shown in FIG. 2. As can be seen from the figure, the recombinant strain supernatant of pET32 a:CbTPS 6 has recombinant plasmid pET32 a:CbTPS 6 expressed recombinant protein CbTPS6, and the recombinant protein CbTPS6 has the size of about 88kDa and is consistent with the expected size. The control bacterial supernatant was free of the corresponding protein.

2. Analysis of the enzymatic Activity of recombinant protein CbTPS6

1. Enzymatic Activity

(1) And (3) taking pET32a, namely carrying out enzymatic reaction on the supernatant of the CbTPS6 recombinant bacterium to obtain an enzymatic reaction product. Wherein, the specific steps of the enzymatic reaction are as follows:

the total enzymatic reaction system was 0.2mL, and 190. Mu.L of pET32a:: cbTPS6 recombinant strain supernatant was taken (the pET32a:: cbTPS6 recombinant strain supernatant contained an enzymatic buffer, i.e., HEPES buffer (25mM HEPES,5MMgCl) ₂ 5M DTT, pH 7.0)), 10. Mu.L of farnesyl pyrophosphate (FPP) as a substrate was added, mixed well, and the total enzymatic reaction system was covered with 200. Mu.L of n-hexane and sealed with a liquid, and left at 30℃for 2 hoursAfter that time, pET32a is obtained as a supernatant enzymatic reaction product of CbTPS6 recombinant bacteria for GC-MS analysis.

3. GC-MS analysis

Detecting the target compound of the supernatant of the recombinant strain pET32a (CbTPS 6): the GC-MS analysis system is Thermo TRACE 1310/TSQ 8000gas chromatograph, the sample injection amount is 1 mu L, the split mode is adopted, and the gas chromatographic column is Agilent J&W Cyclodex-B chiral column (30 m×0.25mm×0.25 μm), helium flow rate 1.0mL/min, sample inlet temperature 220 ℃, ion source temperature 200 ℃, temperature program 50 ℃ for 2min, temperature program 3 ℃ min ^-1 To 150℃and maintained for 5min,10℃min ^-1 The sample was scanned over the 50-500m/z range to 220℃with electron energy of 70 eV.

The GC-MS analysis results are shown in FIG. 3: the CbTPS6 recombinant protein can catalyze FPP to form (-) -beta-elemene ((+) -elemene), and the recombinant protein CbTPS6 is sesquiterpene synthase.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> Sichuan Hongchong Biotech Co., ltd

<120> Mei pian tree sesquiterpene synthase CbTPS6 and related biological materials and applications thereof

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atgcctcttg ttttgggctc tggccattct gatgtcccaa actcaaacca atcacaggga 60

aatggaaaat ccaatgtaga aaggaaatct gcaaattatc accctgacat ctggggtgat 120

cgtttcatta aagcctccat tgatgacttg aaacctgatg aattaactca agaacgggct 180

aatgagttga aggaagaagt gaatagaatg tttatcaatg tcaatgatca tttgcaagaa 240

ctgaatttga tcgatgccat ccaacgcctc ggagtggcgt atcactttga aaccgagatt 300

gctgaggccc tgctcaggat atacaacacc tatgataagg aggatgatag tgatgatttg 360

cacactgtcg ctcttcgttt tcgactgcta aggcaagagg ggtacaatgc ctcacccaac 420

gtgttcaaca agtttataga tggagaaagc aagttcaaga aaccgttggc tactgacatt 480

cgcgggattc ttagtttata tgaagcagca tatatgggaa tacctggaga agatatattg 540

gatgaagcca ttgcttttac taaagaacat cttaacttgg cacttcctca tcttgaatca 600

cctctctcaa ccctagttac gcttgcccta gagttacctt tgcgcaaaca tattgagagg 660

ctacagtcaa ggtattacat ctcaatctat caacaagaga aggcaaagaa caacatctta 720

ttagagtttg caaagttgga tttcaatata ttgcagttat tgcatcgaaa ggagctaaag 780

gaaatttcaa tgtggtggaa gaagtgggat tttggtgtga agctaccatt cattagagaa 840

agagtggtgg agtgctactt ttgggtaatg gcagtatatt ttgaaccaca atattcccaa 900

gctagaatca taacaactaa aattttattg ttagcatcag tgatggatga cacctatgat 960

gtatatgcta caacagatga attggaacca tacacagatg caatccaaag gtgggatcga 1020

agcgccatag atcagctgcc tgattacatg aagttacatt tttgtgcact cttagacact 1080

gtctatgaat ttgaagagga actttctcat gaggggaaag cctatcgtat ttcatacttg 1140

aaagaagttt ataaggaact ctccaaaaac tattatattg aacaacagtg gacacattcg 1200

ggatatgtgc caacattaga agagtatatg aaagtagctt taatcactgg tgcataccat 1260

atgctgacac tagtttcata tgttggcatg agagatgttg caactaaaga agcctttgaa 1320

tgggtgaaaa atatgccaaa actggtccaa gctgcttcta tagtttgtcg attcaaggat 1380

gatatccaat caaaccagct tgaacaaatg agaggacatg tggcatcatg tattcaaatc 1440

gacatgaagg agaatggaag cacatatgaa gaggcttgcg aaaagtttaa aagtatggct 1500

gcaaatgcat ggaaagacat caataaggaa tgtctgaatc ctacagtggt tcctatgcct 1560

ctcctcatgc ggactgtaaa tctcgcacgt gttattgaag tcctatacca acacagagat 1620

ggctacacta attccacaca tgagaccaaa gatcaaatct ctttagtgtt agtggaaccc 1680

attcctattt aa 1692

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Met Pro Leu Val Leu Gly Ser Gly His Ser Asp Val Pro Asn Ser Asn

1 5 10 15

Gln Ser Gln Gly Asn Gly Lys Ser Asn Val Glu Arg Lys Ser Ala Asn

20 25 30

Tyr His Pro Asp Ile Trp Gly Asp Arg Phe Ile Lys Ala Ser Ile Asp

35 40 45

Asp Leu Lys Pro Asp Glu Leu Thr Gln Glu Arg Ala Asn Glu Leu Lys

50 55 60

Glu Glu Val Asn Arg Met Phe Ile Asn Val Asn Asp His Leu Gln Glu

65 70 75 80

Leu Asn Leu Ile Asp Ala Ile Gln Arg Leu Gly Val Ala Tyr His Phe

85 90 95

Glu Thr Glu Ile Ala Glu Ala Leu Leu Arg Ile Tyr Asn Thr Tyr Asp

100 105 110

Lys Glu Asp Asp Ser Asp Asp Leu His Thr Val Ala Leu Arg Phe Arg

115 120 125

Leu Leu Arg Gln Glu Gly Tyr Asn Ala Ser Pro Asn Val Phe Asn Lys

130 135 140

Phe Ile Asp Gly Glu Ser Lys Phe Lys Lys Pro Leu Ala Thr Asp Ile

145 150 155 160

Arg Gly Ile Leu Ser Leu Tyr Glu Ala Ala Tyr Met Gly Ile Pro Gly

165 170 175

Glu Asp Ile Leu Asp Glu Ala Ile Ala Phe Thr Lys Glu His Leu Asn

180 185 190

Leu Ala Leu Pro His Leu Glu Ser Pro Leu Ser Thr Leu Val Thr Leu

195 200 205

Ala Leu Glu Leu Pro Leu Arg Lys His Ile Glu Arg Leu Gln Ser Arg

210 215 220

Tyr Tyr Ile Ser Ile Tyr Gln Gln Glu Lys Ala Lys Asn Asn Ile Leu

225 230 235 240

Leu Glu Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Leu Leu His Arg

245 250 255

Lys Glu Leu Lys Glu Ile Ser Met Trp Trp Lys Lys Trp Asp Phe Gly

260 265 270

Val Lys Leu Pro Phe Ile Arg Glu Arg Val Val Glu Cys Tyr Phe Trp

275 280 285

Val Met Ala Val Tyr Phe Glu Pro Gln Tyr Ser Gln Ala Arg Ile Ile

290 295 300

Thr Thr Lys Ile Leu Leu Leu Ala Ser Val Met Asp Asp Thr Tyr Asp

305 310 315 320

Val Tyr Ala Thr Thr Asp Glu Leu Glu Pro Tyr Thr Asp Ala Ile Gln

325 330 335

Arg Trp Asp Arg Ser Ala Ile Asp Gln Leu Pro Asp Tyr Met Lys Leu

340 345 350

His Phe Cys Ala Leu Leu Asp Thr Val Tyr Glu Phe Glu Glu Glu Leu

355 360 365

Ser His Glu Gly Lys Ala Tyr Arg Ile Ser Tyr Leu Lys Glu Val Tyr

370 375 380

Lys Glu Leu Ser Lys Asn Tyr Tyr Ile Glu Gln Gln Trp Thr His Ser

385 390 395 400

Gly Tyr Val Pro Thr Leu Glu Glu Tyr Met Lys Val Ala Leu Ile Thr

405 410 415

Gly Ala Tyr His Met Leu Thr Leu Val Ser Tyr Val Gly Met Arg Asp

420 425 430

Val Ala Thr Lys Glu Ala Phe Glu Trp Val Lys Asn Met Pro Lys Leu

435 440 445

Val Gln Ala Ala Ser Ile Val Cys Arg Phe Lys Asp Asp Ile Gln Ser

450 455 460

Asn Gln Leu Glu Gln Met Arg Gly His Val Ala Ser Cys Ile Gln Ile

465 470 475 480

Asp Met Lys Glu Asn Gly Ser Thr Tyr Glu Glu Ala Cys Glu Lys Phe

485 490 495

Lys Ser Met Ala Ala Asn Ala Trp Lys Asp Ile Asn Lys Glu Cys Leu

500 505 510

Asn Pro Thr Val Val Pro Met Pro Leu Leu Met Arg Thr Val Asn Leu

515 520 525

Ala Arg Val Ile Glu Val Leu Tyr Gln His Arg Asp Gly Tyr Thr Asn

530 535 540

Ser Thr His Glu Thr Lys Asp Gln Ile Ser Leu Val Leu Val Glu Pro

545 550 555 560

Ile Pro Ile

Claims

1. A protein which is a protein of the following a) or b) or c):

a) The amino acid sequence is a protein shown in the sequence 2;

2. The biological material related to the protein of claim 1, which is any one of the following:

a1 A nucleic acid molecule encoding the protein of claim 1;

a2 An expression cassette comprising A1) said nucleic acid molecule;

a3 A) a recombinant vector comprising the nucleic acid molecule of A1);

a4 A recombinant vector comprising the expression cassette of A2);

a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);

a6 A) a recombinant microorganism comprising the expression cassette of A2);

a7 A) a recombinant microorganism comprising the recombinant vector of A3);

a8 A) a recombinant microorganism comprising the recombinant vector of A4);

a9 A transgenic plant cell line comprising the nucleic acid molecule of A1);

a10 A transgenic plant cell line comprising the expression cassette of A2);

a11 A transgenic plant cell line comprising the recombinant vector of A3);

a12 A) a transgenic plant cell line comprising the recombinant vector of A4).

3. The related biological material according to claim 2, wherein A1) the nucleic acid molecule is any one of the following B1) to B5):

b1 A DNA molecule shown in a sequence 1 in a sequence table;

b3 A DNA molecule shown in a sequence 4 in a sequence table;

b5 A DNA molecule which hybridizes under stringent conditions to a DNA molecule as defined in B1) or B2) or B3) or B4) and which codes for a protein according to claim 1.

4. Use of the protein of claim 1 as a sesquiterpene synthase.

5. Use of the related biological material of claim 2 for the preparation of sesquiterpene synthases.

6. Use of the protein of claim 1 or the related biological material of claim 2 for the preparation or synthesis of monoterpene compounds.

7. Use of the protein of claim 1 or the related biomaterial of claim 2 to catalyze formation of (-) - β -elemene from farnesyl pyrophosphate.

8. A method for preparing the protein of claim 1, characterized in that: the method comprises introducing a gene encoding the protein of claim 1 into a recipient microorganism to obtain a recombinant microorganism expressing the protein of claim 1, culturing the recombinant microorganism, and expressing the protein of claim 1.

9. A method for preparing (-) -beta-elemene, which is characterized in that: the method comprising the step of catalyzing farnesyl pyrophosphate with the protein of claim 1.