CN113234740A

CN113234740A - Aquilaria sinensis terpene synthase

Info

Publication number: CN113234740A
Application number: CN202110489006.XA
Authority: CN
Inventors: 徐艳红; 魏建和; 唐小琳; 余翠翠
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-08-10
Anticipated expiration: 2041-04-30
Also published as: CN113234740B

Abstract

The invention relates to an aquilaria sinensis terpene synthase and provides a modified sesquiterpene synthase gene, which enables a common host microorganism to efficiently express by a genetic engineering technology: the synthetase for catalyzing and synthesizing the target product with high efficiency and good solubility of the sesquiterpene compound promotes the progress and development of the biological enzyme catalysis synthesis technology of the sesquiterpene compound.

Description

Aquilaria sinensis terpene synthase

Technical Field

The invention relates to an aquilaria sinensis terpene synthase, a catalytic product and application thereof, and belongs to the technical field of biology.

Background

The aquilaria is resin-containing wood induced by damaged aquilaria or aquilaria plants in the Thymelaeaceae, and is a valuable worldwide resource of rare or endangered south medicine. The Chinese eaglewood is mainly produced in places such as Guangdong, Hainan, Yunnan, Guangxi and the like in China, and the Aquilaria sinensis (Lour.) Gilg is the only genuine plant source for producing the eaglewood in China.

According to the record of Chinese pharmacopoeia (2010 edition), Chinese eaglewood has the efficacies of promoting qi circulation, relieving pain, warming middle energizer, arresting vomiting and receiving qi and relieving asthma, and is one of rare medicinal materials in China since ancient times. However, the yield of the agilawood is extremely low under natural conditions, and the wild aquilaria sinensis resource is seriously damaged and nearly endangered for a long time. The effective components of lignum Aquilariae Resinatum mainly comprise sesquiterpene compounds and 2- (2-phenylethyl) chromone. Dozens of sesquiterpenes have been found in agilawood essential oil. The sesquiterpene compound is the main source of fragrance of agilawood and is also the main substance for exerting the drug effect of the agilawood. Modern pharmacological research shows that the agilawood also has wider pharmacological effects such as pain easing, antibacterial, calming, anti-inflammatory, antioxidation, blood sugar reducing, anti-tumor and the like; lignum Aquilariae Resinatum has tranquilizing and hypnotic effects on central nerve, and has antidepressant effect. Therefore, people usually use agilawood incense to relax spirit and relieve stress.

The biosynthesis of sesquiterpenes belongs to the sesquiterpene branch of the isoprenoid metabolic pathway. In plants, they are synthesized by both Mevalonate (MAV) pathway located in the cytoplasm and erythrose phosphate (MEP) or deoxyxylitol phosphate (DXP) located in the plastid, through a series of enzymatic catalytic reactions to produce farnesyl pyrophosphate (FPP), a common substrate. Then, under the catalysis of terpene synthases (TPSs), various terpenes and intermediates thereof are formed by cyclization. It can be seen that TPS is a key enzyme gene for the synthesis of terpenoids. The cloning and identification of key enzyme genes of upstream pathways of terpene synthesis in aquilaria sinensis, including HMGR, HMGS, DXS, DXR, FPS and the like, are completed in the prophase of a subject group.

Sesquiterpene compounds are natural terpenoids containing 15 carbon atoms in the molecule, and most sesquiterpene compounds have pleasant volatile aromatic odor and are widely applied to high-grade perfumes. In addition, the sesquiterpenoids have various pharmacological actions and are used for clinical treatment, for example, the cyclic sesquiterpene artemisinin can be used for controlling and treating malaria symptoms.

Nerolidol is an organic compound with fragrance, and the fragrance is sweet, fresh, soft and beautiful with costus root; has good stability and durability at room temperature, and is commonly used for preparing essence such as rose type essence and lilac type essence. Recent studies have found that nerolidol inhibits fungal growth and has therapeutic effects on fungal keratitis under in vivo and in vitro conditions.

The agarol is also a sesquiterpene compound, is a main component of the agarol, can produce positive effect on the central nervous system, has good calming effect, can help people to eliminate tension and anxiety, has strong improving effect on sleep quality, can improve autoimmunity, and has good pain relieving effect. The linalool can be extracted and separated from the agilawood volatile oil, but the method is time-consuming and labor-consuming and has low yield.

Eucalyptol is also a sesquiterpene compound widely found in the oil of many eucalyptus trees, and is a mixture of a-eucalyptol and β -eucalyptol.

Aquilaria sinensis terpene synthase belongs to sesquiterpene synthase.

At present, sesquiterpene compounds are mainly extracted from plants or chemically synthesized, but impurities are more, but the biosynthesis process of the sesquiterpene compounds does not meet the requirement of industrial production yet and remains in the basic research stage of a laboratory. With the rapid development of the bio-enzyme catalytic synthesis technology, people hope to utilize common host microorganisms such as escherichia coli and saccharomyces cerevisiae to efficiently express some synthetases for catalyzing and synthesizing sesquiterpene compounds with high efficiency and good solubility expression of target products, so as to promote the progress and development of the bio-enzyme catalytic synthesis technology of sesquiterpene compounds.

Disclosure of Invention

In order to solve the problems, the invention provides an aquilaria sinensis terpene synthase, a catalytic product and application thereof.

In one embodiment, the present invention provides a sesquiterpene synthase gene, the nucleic acid sequence of which is shown in SEQ ID No.1, and the protein sequence of which is shown in SEQ ID No. 2.

Secondly, the invention also provides a modified sesquiterpene synthase gene, and the nucleic acid sequence of the gene is shown in SEQ ID No. 3. One of the objectives of the modification is to make the amount of the hemiditerpene synthase-catalyzed substrate to produce a specific target product as high as possible (e.g., a major product or a minor major product); another purpose is to make the gene be expressed in Escherichia coli in a soluble manner, and to avoid the formation of inclusion bodies as much as possible.

In one embodiment, the present invention provides a method for catalytically synthesizing a terpenoid, comprising the steps of: the sesquiterpene synthase gene shown in SEQ ID No.3 is expressed in a recombination mode, the sesquiterpene synthase is purified, the sesquiterpene synthase is contacted with farnesyl pyrophosphate (FPP) (substrate) under the condition of enzymatic reaction, and terpenoids in the product are collected.

In one embodiment, the present invention provides a method for the bio-enzymatic synthesis of nerolidol, comprising the steps of: the sesquiterpene synthase gene shown in SEQ ID No.3 is expressed in a recombination mode, the sesquiterpene synthase is purified, the sesquiterpene synthase is contacted with FPP (substrate) under the enzymatic reaction condition, and nerolidol in the product is collected.

In one embodiment, the present invention further provides a method for the bio-enzymatic synthesis of linalool, comprising the following steps: and (3) recombining and expressing the sesquiterpene synthase gene shown in SEQ ID No.3, purifying the sesquiterpene synthase, contacting the sesquiterpene synthase with FPP (substrate) under an enzymatic reaction condition, and collecting the linalool in the product.

In one embodiment, the present invention also provides a method for the bio-enzymatic synthesis of eucalyptol, comprising the steps of: recombinantly expressing a sesquiterpene synthase gene shown in SEQ ID No.3, purifying the sesquiterpene synthase, contacting the sesquiterpene synthase gene with FPP (substrate) under an enzymatic reaction condition, and collecting cineole in a product.

In one embodiment, the present invention provides a method for the enzymatic simultaneous production of nerolidol, linalool and/or eucalyptol, comprising the steps of: the sesquiterpene synthase gene shown in SEQ ID No.3 is expressed in a recombination mode, the sesquiterpene synthase is purified and is in contact with FPP (substrate) under the enzymatic reaction condition, and nerolidol, linalool and/or eucalyptol in the products are collected respectively.

The host for recombinant expression is a prokaryote, e.g., a bacterium, specifically, e.g., E.coli.

The host for the recombinant expression is a eukaryote, for example a fungus, a plant cell or an animal cell, in particular a yeast.

A nucleic acid, the sequence of which is shown as SEQ ID No. 1.

A protein has a sequence shown in SEQ ID No. 2.

A nucleic acid, the sequence of which is shown as SEQ ID No. 3.

The biological material containing the nucleic acid is any one of the following A1) to A4):

A1) an expression cassette comprising the nucleic acid of claim 1;

A2) a recombinant vector comprising the nucleic acid of claim 1;

A3) a recombinant microorganism comprising the nucleic acid of claim 1;

A4) a transgenic plant cell line comprising the nucleic acid of claim 1.

The nucleic acid or the biological material is applied to preparing sesquiterpene compounds.

Preferably, the sesquiterpene compound is nerolidol, linalool and/or eucalyptol.

A method for preparing sesquiterpene compounds, comprising culturing a host containing said nucleic acid under conditions effective to produce a sesquiterpene synthase, reacting the sesquiterpene synthase with a substrate under enzymatic conditions, and isolating the sesquiterpene compounds.

Preferably, the host is modified with a recombinant expression vector comprising the nucleic acid, and is selected from the group consisting of a prokaryote, a fungus, a plant cell, and an animal cell.

A method for preparing nerolidol, linalool and/or eudesmol is characterized in that a host containing nucleic acid is cultured under the condition of producing sesquiterpene synthase to obtain sesquiterpene synthase, the sesquiterpene synthase and a substrate FPP are subjected to a reaction under an enzymatic condition, and then the nerolidol, the linalool and/or the eudesmol are separated.

A method for catalytically synthesizing terpenoids is characterized in that sesquiterpene synthase genes shown in SEQ ID No.3 are constructed into pET21 a-sesquiterpene synthase fusion expression vectors, the vectors are transformed into Escherichia coli E.coli Transetta (DE3), and positive clones are obtained through kanamycin screening; after the positive clone was cultured in liquid, 0.5mM IPTG was added to induce protein expression for 20 hours in a shaker at 25 ℃ (110 rpm/min).

The invention is superior to the prior art in that:

the invention provides a novel aquilaria sinensis terpene synthase and an aquilaria sinensis terpene synthase sequence with excellent soluble expression in prokaryotic cells.

Secondly, the invention provides a high-efficiency nerolidol biosynthesis method, and nerolidol can be obtained with high yield in the presence of a substrate FPP by adopting the aquilaria sinensis terpene synthase, wherein the yield of the nerolidol accounts for 47.12% of the total yield.

Thirdly, the invention provides a high-efficiency biosynthesis method of the linalool, and the linalool can be obtained with high yield in the presence of a substrate FPP by adopting the aquilaria sinensis terpene synthase, wherein the linalool accounts for 33.28% of the total yield.

Fourthly, the invention provides a high-efficiency biosynthesis method of eucalyptol, and eucalyptol can be obtained in high yield in the presence of a substrate FPP by adopting the aquilaria sinensis terpene synthase, wherein the yield of eucalyptol accounts for 19.05 percent of the total yield.

The invention has the advantages of obviously considering both the prokaryotic soluble expression of the aquilaria sinensis terpene synthase and the specific and high-efficiency catalytic activity to the target product, and obviously exceeding the level of the prior art. Meanwhile, for the biosynthesis methods of nerolidol, linalool and eucalyptol, the corresponding yield obtained by the method is higher, so that the method has wide industrial practical prospect.

Drawings

FIG. 1 Induction of fusion protein expression SDS-PAGE. Wherein:

m: protein marker (kd);

1,2 is PET-21a no-load transformation colibacillus;

3,4,5 and 6 are Escherichia coli of PET-21 a-AsTPS 13 recombinant plasmid.

1,3,5 with 1mM IPTG and 2,4,6 without IPTG (induction conditions of 37 ℃ for 6 hours at 200 rpm).

FIG. 2 MS spectrum of nerolidol as sesquiterpene product.

FIG. 3 MS spectrum of agarol as sesquiterpene product.

FIG. 4 MS spectra of eudesmol as sesquiterpene product.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 recombinant expression of sesquiterpene synthases

Step one, synthesizing the modified sesquiterpene synthase gene shown in SEQ ID No.1 or SEQ ID No.3 in a whole gene synthesis mode, connecting the sesquiterpene synthase gene to a pET-28a vector, and identifying the sesquiterpene synthase gene as a correct clone extraction plasmid for later use through sequencing and double enzyme digestion.

And step two, transforming the recombinant plasmid identified as positive into an Escherichia coli Transetta (DE3) competent cell, and simultaneously transforming the PET-21a no-load plasmid into the Escherichia coli Transetta (DE3) competent cell as a control.

And step three, after the selected bacteria are activated, transferring a large number of shake bacteria to ensure that the initial OD value is lower than 0.1, and measuring the OD value by using an ultraviolet spectrophotometer. Shaking at 37 deg.C and 200rpm for 2h 40min, adding 0.5mM IPTG when OD value is increased to about 0.5, inducing at 16 deg.C and 150rpm for 16h, then centrifuging at 12000rpm for 15min, and collecting thallus.

And step four, resuspending the thalli by a1 XPBS buffer solution, repeatedly freezing and thawing for 4-5 times to fully crack the thalli, boiling in boiling water for 5min to denature protein, and detecting by 10% SDS-PAGE electrophoresis. It was found that the band of interest was induced (FIG. 1).

Example 2 purification of sesquiterpene synthases

Protein mass expression was performed according to defined induction conditions.

Step one, centrifuging at 5000rpm for 20min, collecting cells for next affinity purification.

Step two, resuspend cells with 40mL binding buffer (containing protease inhibitor) and sonicate. Centrifuging at 4 deg.C 8000 r/min for 30min, filtering the supernatant with 0.45 μm filter, and standing the precipitate on ice.

Step three, taking 1mL of Ni-NTA His Bind resin, adding the balanced Ni-NTA His Bind resin into the filtered supernatant, and mixing the filtered supernatant with 1mL of Ni²⁺The resin homogenate was mixed well and bound on ice for 4 h.

And step four, putting the mixture on a column, and allowing the solution to flow out.

Step five, washing the resin with washing buffer solution, 10mL each time, repeating for 3 times.

And step six, eluting the target protein by using the elution buffer solution, repeating for 3 times, wherein each time is 1mL, and collecting the eluent.

And seventhly, taking a small amount of samples from each tube to carry out SDS-PAGE, and detecting the protein purification condition.

Preferably, the six steps set three different elution concentrations: 200, 150 and 300 mmol/L.

The target protein is purified pET-21 a-sesquiterpene synthase His-tag fusion protein and is detected by SDS-PAGE.

Example 3 in vitro enzymatic reaction of sesquiterpene synthases

The reaction system contained 25mM Tris-HCl (pH7.0), 10% glycerol, 100mM Mg2SO4,5mM DTT, 46uM FPP or GPP (from sigma), purified protein 50uL, ddH2O to make up to 200 uL. Mixing the above solutions, and placing into 30 deg.C metal bath for 1 hr.

Example 4 product detection and isolation

Inserting the solid phase microextraction SPME fiber head aged by GC into an adsorption bottle, slowly pushing a handle to expose the extraction fiber with the adsorption coating in a sample to absorb volatile substances for 80min, and performing the extraction process in a water bath kettle at 80 ℃.

The catalytic products were assayed using an Agilent GC-MS (available from SynTECH, Agilent/Germany, model 7890B-5977A).

The GC program was: ionization mode E1, electron energy 70eV, carrier gas helium gas, flow rate of 1mL/min, injection inlet temperature of 250 deg.C, initial temperature of 80 deg.C, rising to 220 deg.C at 5 deg.C/min, storing for 10min, rising to 240 deg.C at 10 deg.C/min, and maintaining for 3 min. The scanning mass range is 50-500 amu.

Adsorbing volatile substances generated by protein catalysis by SPME, replacing sesquiterpene synthase protein by 250mmol/L NEB, keeping other components unchanged, and performing GC-MS detection on the catalytic product as a control.

The results show that:

the sesquiterpene synthases expressed in SEQ ID No.3 have three main catalytic products with FPP as substrate (see FIGS. 2-4), which are nerolidol (47.12%), linalool (33.28%) and eucalyptol (19.50%).

In conclusion, the invention provides a new aquilaria sinensis terpene synthase, optimizes the performance of soluble expression in pronucleus, and obviously improves the yield of nerolidol, linalool and eucalyptol in the biosynthesis method.

While the invention has been described in detail with respect to specific embodiments thereof, the foregoing description is not intended to limit the scope of the invention.

SEQUENCE LISTING

<110> institute of medicinal plants of academy of Chinese medical science

<120> an aquilaria sinensis terpene synthase

<130> 2021.03.03

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1632

<212> DNA

<213> Artificial Synthesis

<400> 1

atggcggcag agacgaatcg tcctcttgcg catttccagg ccgacatctg ggaggaacgt 60

ttcgtatcat cccctcatct tcatctggaa acggagcaag catcggagca ccaaaagctg 120

aaggaacagg tgagggaact cctgttagcc ggactggaca agccttggga acaactggac 180

ttgatcaact tgatccagcg cttgggcata ggttaccatt tcgaggatga aatcgaaaac 240

ctcttacaag gaattcacaa gaatctcgat acttgccttg aagaaaatga taacctacac 300

ggcatttcac tactgtttcg cctgcttagg caatctggcc tgcccgtttc atgcgacgtg 360

tttaacaagt ttaaggatga aagtgggaag ttgaaggaat ctctaagtag agatgttata 420

gggctgttga gcttgcatga ggctgcatgt gtgaggctgc atggagaaga tatacttgag 480

caggtacttg atttcaccac gaagcacctc gaatccattg caaaggacac atcttcaagc 540

ccaaaactcg caacacaagc aagtcaggca ttgaagtacc ctgttcggaa gaacatccca 600

aggctggatg ctaaacacta tatctctgtg tatcctttgc tcaacactcc tgttgtattg 660

acttttgcta agttggactt caatatctta caaaagttgc atcgaaatga gctgagggag 720

ttagcaaggt ggtggaaaga tttggatatt ccaaagagac taccatatgc aagggataga 780

ataacggaat tattcttttg ggtactagcg gtgtattatg agccttgtta cgcactgggt 840

aggaagatat tgaccaaagt tatagctctt acttctcttc ttgacgacat gtacgatgct 900

tatgggacaa ttgaagaact agagctcctt acacaagcca ttcaaaggtg ggatagaaac 960

gcaatggatg ggttgaataa tgagtgcagc aaagagttgt atcagatatt gctggacgtg 1020

tatgatgaaa ttggggagga catggccaaa cttgagaaat cctatcgcct caactacgct 1080

gtagaaatga tgaagaagca agcaagaatt tacttgacag aagctagatg gtgcagccaa 1140

aactatacac ccatattgga ggagtacctt aagctgggaa tccacaccag cacttgccct 1200

ctactcatac tttcgtcttt gcttgggatg ggagacaata taagcagaga tgcttttgag 1260

tggatcttgt ccacccctaa gattcttgtt gcttcttcat taatcacaag actcttagat 1320

gacataatga ccgacgagtt tgagaaaaaa cgggggcacg tagactctgc catagattgt 1380

tatatgaaag agcacagggt gacgaaacag gaggcagtga aggagttgta caagcaaatt 1440

gagagtgcat ggaaggacat gaatgaagag cttttgcggc caacagaggt gtccaaggca 1500

gtgctcatgc gaatgctcaa cttcacccgt agtaaccagg tcgtgtatgc cgatgcagat 1560

tcctacacct ttcctgatta tctaaaggac tttgtggctg ctttgctcgt tcaccaactc 1620

cctcttgatt aa 1632

<210> 2

<211> 543

<212> PRT

<213> Artificial Synthesis

<400> 2

Met Ala Ala Glu Thr Asn Arg Pro Leu Ala His Phe Gln Ala Asp Ile

1 5 10 15

Trp Glu Glu Arg Phe Val Ser Ser Pro His Leu His Leu Glu Thr Glu

20 25 30

Gln Ala Ser Glu His Gln Lys Leu Lys Glu Gln Val Arg Glu Leu Leu

35 40 45

Leu Ala Gly Leu Asp Lys Pro Trp Glu Gln Leu Asp Leu Ile Asn Leu

50 55 60

Ile Gln Arg Leu Gly Ile Gly Tyr His Phe Glu Asp Glu Ile Glu Asn

65 70 75 80

Leu Leu Gln Gly Ile His Lys Asn Leu Asp Thr Cys Leu Glu Glu Asn

85 90 95

Asp Asn Leu His Gly Ile Ser Leu Leu Phe Arg Leu Leu Arg Gln Ser

100 105 110

Gly Leu Pro Val Ser Cys Asp Val Phe Asn Lys Phe Lys Asp Glu Ser

115 120 125

Gly Lys Leu Lys Glu Ser Leu Ser Arg Asp Val Ile Gly Leu Leu Ser

130 135 140

Leu His Glu Ala Ala Cys Val Arg Leu His Gly Glu Asp Ile Leu Glu

145 150 155 160

Gln Val Leu Asp Phe Thr Thr Lys His Leu Glu Ser Ile Ala Lys Asp

165 170 175

Thr Ser Ser Ser Pro Lys Leu Ala Thr Gln Ala Ser Gln Ala Leu Lys

180 185 190

Tyr Pro Val Arg Lys Asn Ile Pro Arg Leu Asp Ala Lys His Tyr Ile

195 200 205

Ser Val Tyr Pro Leu Leu Asn Thr Pro Val Val Leu Thr Phe Ala Lys

210 215 220

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

225 230 235 240

Leu Ala Arg Trp Trp Lys Asp Leu Asp Ile Pro Lys Arg Leu Pro Tyr

245 250 255

Ala Arg Asp Arg Ile Thr Glu Leu Phe Phe Trp Val Leu Ala Val Tyr

260 265 270

Tyr Glu Pro Cys Tyr Ala Leu Gly Arg Lys Ile Leu Thr Lys Val Ile

275 280 285

Ala Leu Thr Ser Leu Leu Asp Asp Met Tyr Asp Ala Tyr Gly Thr Ile

290 295 300

Glu Glu Leu Glu Leu Leu Thr Gln Ala Ile Gln Arg Trp Asp Arg Asn

305 310 315 320

Ala Met Asp Gly Leu Asn Asn Glu Cys Ser Lys Glu Leu Tyr Gln Ile

325 330 335

Leu Leu Asp Val Tyr Asp Glu Ile Gly Glu Asp Met Ala Lys Leu Glu

340 345 350

Lys Ser Tyr Arg Leu Asn Tyr Ala Val Glu Met Met Lys Lys Gln Ala

355 360 365

Arg Ile Tyr Leu Thr Glu Ala Arg Trp Cys Ser Gln Asn Tyr Thr Pro

370 375 380

Ile Leu Glu Glu Tyr Leu Lys Leu Gly Ile His Thr Ser Thr Cys Pro

385 390 395 400

Leu Leu Ile Leu Ser Ser Leu Leu Gly Met Gly Asp Asn Ile Ser Arg

405 410 415

Asp Ala Phe Glu Trp Ile Leu Ser Thr Pro Lys Ile Leu Val Ala Ser

420 425 430

Ser Leu Ile Thr Arg Leu Leu Asp Asp Ile Met Thr Asp Glu Phe Glu

435 440 445

Lys Lys Arg Gly His Val Asp Ser Ala Ile Asp Cys Tyr Met Lys Glu

450 455 460

His Arg Val Thr Lys Gln Glu Ala Val Lys Glu Leu Tyr Lys Gln Ile

465 470 475 480

Glu Ser Ala Trp Lys Asp Met Asn Glu Glu Leu Leu Arg Pro Thr Glu

485 490 495

Val Ser Lys Ala Val Leu Met Arg Met Leu Asn Phe Thr Arg Ser Asn

500 505 510

Gln Val Val Tyr Ala Asp Ala Asp Ser Tyr Thr Phe Pro Asp Tyr Leu

515 520 525

Lys Asp Phe Val Ala Ala Leu Leu Val His Gln Leu Pro Leu Asp

530 535 540

<210> 3

<211> 1632

<212> DNA

<213> Artificial Synthesis

<400> 3

atggcagcag aaaccaatcg cccgctggcc cattttcagg cagatatttg ggaagaacgt 60

tttgtgagca gtccgcatct gcatctggaa accgaacagg caagtgaaca tcagaaactg 120

aaagaacagg ttcgcgaact gctgctggca ggtctggata aaccgtggga acagctggat 180

ctgattaatc tgattcagcg tctgggtatt ggctatcatt ttgaagatga aattgagaat 240

ctgctgcagg gtattcataa aaatctggat acctgcctgg aagaaaatga taatctgcat 300

ggtattagtc tgctgtttcg tctgctgcgc cagagcggcc tgccggttag ctgcgatgtt 360

tttaataagt ttaaggatga gagcggtaaa ctgaaagaga gtctgagccg tgatgtgatt 420

ggtctgctga gtctgcatga agcagcctgc gtgcgcctgc atggtgaaga tattctggaa 480

caggtgctgg attttaccac caaacatctg gaaagtattg caaaagatac cagcagcagc 540

ccgaaactgg ccacccaggc aagccaggcc ctgaaatatc cggttcgtaa aaatattccg 600

cgtctggatg caaaacatta tattagtgtt tacccgctgc tgaatacccc ggtggttctg 660

acctttgcaa aactggattt taatattctg cagaaactgc atcgtaatga actgcgcgaa 720

ctggcacgtt ggtggaaaga tttggatatt ccgaaacgtc tgccgtatgc ccgtgatcgt 780

attaccgaac tgtttttctg ggttctggcc gtttattatg aaccgtgcta tgccctgggt 840

cgtaaaattc tgaccaaagt gattgccctg accagtctgc tggatgatat gtatgatgca 900

tacggtacca ttgaagaact ggaactgctg acccaggcca ttcagcgttg ggatcgcaat 960

gccatggatg gtctgaataa tgaatgtagt aaagaactgt accagattct gctggatgtt 1020

tatgatgaaa ttggtgaaga tatggccaaa ctggaaaaaa gctatcgcct gaattatgca 1080

gttgaaatga tgaaaaagca ggcccgtatc tatctgaccg aagcacgctg gtgtagtcag 1140

aattataccc cgattctgga agaatatctg aaactgggca ttcataccag cacctgcccg 1200

ctgctgattc tgagcagcct gctgggcatg ggtgacaata ttagccgtga tgcctttgaa 1260

tggattctga gcaccccgaa aattctggtt gccagtagcc tgattacccg cctgctggat 1320

gacattatga ccgatgaatt tgaaaagaaa cgcggtcatg tggatagtgc cattgattgt 1380

tatatgaaag aacatcgcgt taccaaacag gaagcagtta aagaactgta taaacagatt 1440

gaaagcgcct ggaaagatat gaatgaagaa ctgctgcgtc cgaccgaagt tagcaaagca 1500

gtgctgatgc gcatgctgaa ttttacccgt agtaatcagg tggtgtatgc cgatgcagat 1560

agctatacct ttccggatta tctgaaagat tttgtggccg ccctgctggt gcatcagctg 1620

ccgctggatt aa 1632

Claims

1. A nucleic acid, the sequence of which is shown as SEQ ID No.1 or SEQ ID No. 3.

2. The biomaterial containing the nucleic acid according to claim 1, which is any one of the following A1) to A4):

A1) an expression cassette comprising the nucleic acid of claim 1;

A2) a recombinant vector comprising the nucleic acid of claim 1;

A3) a recombinant microorganism comprising the nucleic acid of claim 1;

A4) a transgenic plant cell line comprising the nucleic acid of claim 1.

3. Use of the nucleic acid of claim 1 or the biomaterial of claim 2 in the preparation of a sesquiterpene compound.

4. Use according to claim 3, wherein the sesquiterpene compound is nerolidol, linalool and/or eucalyptol.

5. A method of producing a sesquiterpene comprising culturing a host comprising a nucleic acid of claim 1 under conditions effective to produce a sesquiterpene synthase, reacting the sesquiterpene synthase with a substrate under enzymatic conditions, and isolating the sesquiterpene.

6. The method of claim 5, wherein the host is modified with a recombinant expression vector comprising the nucleic acid, and wherein the host is selected from the group consisting of a prokaryote, a fungus, a plant cell, and an animal cell.

7. The method of claim 5 or 6, wherein the sesquiterpene compound is nerolidol, linalool and/or eucalyptol.

8. A method for preparing nerolidol, linalool and/or eudesmol, comprising culturing a host containing the nucleic acid of claim 1 under conditions capable of producing a sesquiterpene synthase, reacting the sesquiterpene synthase with a substrate FPP under enzymatic conditions, and separating nerolidol, linalool and/or eudesmol.

9. The method of claim 8, wherein the host is modified with a recombinant expression vector comprising the nucleic acid, and wherein the host is selected from the group consisting of a prokaryote, a fungus, a plant cell, and an animal cell.

10. A protein has a sequence shown in SEQ ID No. 2.