CN115404228B - Lignum aquilariae resinatum sesquiterpene synthase protein TPS1, and coding gene and application thereof - Google Patents

Abstract

The invention discloses a lignum aquilariae resinatum sesquiterpene synthase protein TPS1, and a coding gene and application thereof. One technical scheme to be protected by the invention is protein shown as a sequence 2 or a sequence 4 in a sequence table. Experiments prove that the aquilaria sinensis TPS1 protein has the function of catalyzing FPP to synthesize the jacobscura blue alkene. The invention fills the research blank of the biological synthesis of the agilawood sesquiterpene, provides a basis for realizing the biological synthesis of the agilawood elegance threshold blue alkene type sesquiterpene, provides a feasible scheme for protecting wild agilawood resources, and has good research potential and wide application prospect.

Description

Lignum aquilariae resinatum sesquiterpene synthase protein TPS1, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to aquilaria sinensis sesquiterpene synthase protein TPS1, and a coding gene and application thereof.

Background

The lignum Aquilariae Resinatum is root, stem and thick branch of Eugenia caryophyllata (Syringa pinnatifolia hemsl.) of Syringa of Oleaceae after peeling and drying. The aquilaria sinensis has the curative effects of suppressing heryi, clearing heat, relieving pain and the like, and modern pharmacological researches also show that the medicinal material has a plurality of effects of protecting cardiac muscle, relieving pain, resisting tumor, resisting bacteria, resisting inflammation, resisting oxidation and the like. In Mongolian medicine treatment, shan Cheng Xiang is mainly used for treating heart and lung diseases such as coronary heart disease and angina pectoris. Chemical research of the eaglewood shows that lignans and terpenoids are main medicinal effect components of the eaglewood medicinal material. The volatile oil component mainly containing sesquiterpene is specific medicinal substances of lignum Aquilariae Resinatum, such as xanthone, calamine, humulone, bergamotene, and juniper, but related researches on biosynthesis of sesquiterpene of lignum Aquilariae Resinatum have not been reported.

7 kinds of sesquiterpenes separated from lignum Aquilariae Resinatum, including innafolone A, pinnatifide B, alashanoid K, alashanoid L, alashanoid M, 3-hydroxy-eremophilane-3, 11-diene-2, 9-dione, have been reported. However, the research on the biosynthesis of the aquilaria sinensis and the saphenous vein-like sesquiterpenes has not been reported yet.

Disclosure of Invention

The invention aims to solve the technical problem of how to biosynthesize the eaglewood and the Yabail blue alkene type sesquiterpene.

In order to solve the technical problems, the invention firstly provides a protein. The protein may be a protein of the following A1), A2), A3) or A4):

a1 Amino acid sequence is protein of sequence 2 in the sequence table,

a2 Amino acid sequence is protein of sequence 4 in the sequence table,

a3 Fusion proteins obtained by fusing protein tags at the carboxyl end or/and the amino end of the protein shown in A1) or A2),

a4 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues on the amino acid sequence shown in the sequence 2 in the sequence table, is derived from A1) or A2) and has the same function or has more than 80 percent of identity with the protein shown in A1) or A2).

The protein is derived from fresh stem segments of Syringa amurensis and consists of 555 amino acid residues, and the predicted protein molecular weight is about 65kDa.

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

Among the above proteins, the 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 80% or more identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In order to solve the technical problems, the invention also provides application of the protein in preparing the aquilaria sinensis sesquiterpene delphine synthase or preparing the aquilaria sinensis sesquiterpene. The protein may be a protein as described above.

Biological materials related to the proteins described above are also within the scope of the present invention.

The biomaterial may be any one of the following D1) to D6):

d1 A nucleic acid molecule encoding a protein as described above;

d2 An expression cassette comprising D1) said nucleic acid molecule;

d3 A recombinant vector comprising D1) said nucleic acid molecule, or a recombinant vector comprising D2) said expression cassette;

d4 A recombinant microorganism comprising D1) said nucleic acid molecule, or a recombinant microorganism comprising D2) said expression cassette, or a recombinant microorganism comprising D3) said recombinant vector;

d5 A nucleic acid molecule that promotes or enhances expression of a protein as described above;

d6 An expression cassette, a recombinant vector or a recombinant microorganism comprising the nucleic acid molecule of D5).

In the biological material described above, D1) the nucleic acid molecule may be a gene encoding the protein as shown in D1) D2) or D3) below:

d1 A cDNA molecule or a DNA molecule of which the coding sequence is a sequence 1 in a sequence table;

d2 Nucleotide is cDNA molecule or DNA molecule of sequence 3 in sequence table,

d3 A cDNA molecule or a DNA molecule which hybridizes with the cDNA or DNA molecule defined in d 2) and which codes for a protein having the same function.

In the above biological material, the expression cassette containing a nucleic acid molecule as described in D2) refers to a DNA capable of expressing the above protein in a host cell. The expression cassette may also include single or double stranded nucleic acid molecules of all regulatory sequences necessary for expression of the nucleic acid molecules of any of the proteins described above. The regulatory sequences are capable of directing the expression of any of the above proteins in a suitable host cell under conditions compatible with the regulatory sequences. Such regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences include promoters and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites of the vector in order to ligate the regulatory sequences with the coding region of the nucleic acid sequence encoding the protein, a ligated regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e.a nucleic acid sequence which is recognized by the host cell in which the nucleic acid sequence is expressed. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein. The promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The control sequence may also be a suitable transcription termination sequence, a sequence that is recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequences may also be suitable leader sequences, i.e., untranslated regions of mRNA which are important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the host cell of choice may be used in the present invention. The regulatory sequence may also be a signal peptide coding region which codes for an amino acid sequence attached to the amino terminus of the protein and which directs the encoded protein into the cell's secretory pathway. Signal peptide coding regions that direct the expressed protein into the secretory pathway of host cells used may be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein according to the growth of the host cell. Examples of regulatory systems are those that are capable of opening or closing gene expression in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those which enable the amplification of a gene. In these examples, the nucleic acid sequence encoding the protein should be operably linked to regulatory sequences.

The invention also relates to recombinant expression vectors comprising a nucleic acid molecule encoding any of the above proteins, a promoter and transcriptional and translational stop signals of the invention. In preparing the expression vector, a nucleic acid molecule encoding any of the above proteins may be located in the vector so as to be operably linked to appropriate expression control sequences. The recombinant expression vector may be any vector (e.g., a plasmid or virus) that facilitates recombinant DNA procedures and expresses a nucleic acid sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which it is to be introduced. The vector may be a linear or closed loop plasmid. The vector may be an autonomously replicating vector (i.e., a complete structure which exists extrachromosomal, the replication of which is independent of the chromosome), e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may comprise any mechanism that ensures self-replication. Alternatively, the vector is one that, when introduced into a host cell, will integrate into the genome and replicate with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids generally comprising the entire DNA to be introduced into the genome of the host cell, or a transposon may be used. The vector contains 1 or more selectable markers that facilitate selection of transformed cells. A selectable marker is a gene the product of which confers resistance to a biocide or virus, resistance to heavy metals, or confers prototrophy to an auxotroph, and the like. Examples of bacterial selectable markers are the dal genes from bacillus subtilis or bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline. The vector comprises elements that enable stable integration of the vector into the host cell genome or ensure autonomous replication of the vector in the cell independent of the cell genome. In the case of autonomous replication, the vector may also comprise an origin of replication, enabling the vector to replicate autonomously in the host cell in question. The origin of replication may carry mutations which render it temperature-sensitive in the host cell (see, e.g., fEhrlich,1978, proc. Natl. Acad. Sci. USA 75:1433). More than 1 copy of a nucleic acid molecule of the invention encoding any of the above proteins may be inserted into a host cell to increase the yield of the gene product. The copy number of the nucleic acid molecule may be increased by inserting at least 1 additional copy of the nucleic acid molecule into the host cell genome or inserting an amplifiable selectable marker with the nucleic acid molecule, and selecting cells containing amplified copies of the selectable marker gene and thus additional copies of the nucleic acid molecule by culturing the cells in the presence of a suitable selection reagent. The procedures used to construct the recombinant expression vectors of the present invention by ligating the elements described above are well known to those skilled in the art (see, e.g., sambrook et al, molecular cloning, A laboratory Manual, second edition, cold spring harbor laboratory Press, cold spring harbor, new York, 1989).

The term "operably linked" is defined herein as a conformation in which a regulatory sequence is located at a position relative to the coding sequence of a DNA sequence such that the regulatory sequence directs the expression of a protein.

Any of the following applications of the proteins described above and/or the biological materials described above are also within the scope of the present invention:

f1, the protein and/or the biological material are/is used for producing the agalloch eaglewood sesquiterpene;

f2, the protein and/or the biological material are/is used for preparing and producing the agalloch eaglewood sesquiterpene product.

In order to solve the technical problems, the invention also provides a method for preparing the agalloch eaglewood sesquiterpene jacara-blue alkene synthase. The method comprises the following steps: the gene encoding the protein is expressed in a prokaryotic microorganism to obtain the aquilaria sinensis sesquiterpene delbrueck blume synthase.

In the method, the expression of the encoding gene of the protein in the prokaryotic microorganism comprises the steps of introducing the encoding gene of the protein into a receptor microorganism to obtain a recombinant microorganism for expressing the agalloch eaglewood sesquiterpene jacara blue alkene synthase, culturing the recombinant microorganism, and expressing to obtain the agalloch eaglewood sesquiterpene jacara blue alkene synthase.

The expression described above may be induced expression. In the above method, the prokaryotic microorganism may be E.coli.

In order to solve the technical problems, the invention also provides a method for preparing the aquilaria sinensis sesquiterpene. The method comprises the following steps: catalyzing a substrate farnesyl ammonium salt FPP by using the protein as the agalloch eaglewood sesquiterpene delbrueckene synthase to obtain the agalloch eaglewood sesquiterpene.

Any of the following products containing the proteins described above and/or the biological materials described above also fall within the scope of the invention:

p1, producing a product of agalloch eaglewood sesquiterpene;

p2, preparing a product for producing the agalloch eaglewood sesquiterpene;

p3, producing the product of the agalloch eaglewood sesquiterpene jacarallon synthase.

TPS1 protein is found in the eaglewood of the feather leaf, and the TPS1 protein is proved to be the eaglewood sesquiterpene and the bergamot blue alkene synthase (Eremophilene synthase), and the substrate farnesyl ammonium pyrophosphate FPP can be catalyzed to obtain the eaglewood and the bergamot blue alkene sesquiterpenes. The invention fills the research blank of the biosynthesis of the linaloe sesquiterpene of the Mongolian medicine, provides a basis for producing the linaloe sesquiterpene in vitro, and has better research and development potential and application prospect.

Drawings

FIG. 1 is an RNA electrophoresis chart of eaglewood, wherein M represents a DNA marker, and 1-9 are eaglewood RNA.

FIG. 2 is a PCR amplification electrophoresis chart of a lignum Aquilariae Resinatum sesquiterpene synthase gene TPS1, wherein M represents a DNA marker, and 1-2 is a PCR amplification electrophoresis chart of TPS1.

FIG. 3 shows detection of the expression product induced by the agalloch eaglewood recombinant expression plasmid pET32a-TPS1. The numerical designations in the figures represent the different expression conditions, 1 and 8: inducing empty whole bacteria liquid, 2 and 9: empty supernatants were induced, 3 and 10: no-load precipitation was induced, 4 and 11: non-induced pET32a-TPS1 whole bacterial fluid, 5 and 12: inducing pET32a-TPS1 whole bacterial liquid, 6 and 13: induction of pET32a-TPS1 supernatant, 7 and 14: and (3) inducing pET32a-TPS1 to precipitate, wherein 1-7 is cultivated at 16 ℃, and 8-14 is cultivated at 30 ℃. Protein markers indicate the molecular weight of the protein, and the arrow indicates the TPS1 protein band.

Fig. 4 is a post-peak ion flow diagram of TPS1 protein catalyzed FPP product extraction. Red arrow indicates the target product, a represents a control of induced empty supernatant obtained at 16 ℃; b represents a control of the induced empty supernatant obtained at 30 ℃; c represents a catalytic product ion flow diagram of induced pET32a-TPS1 supernatant obtained at 16 ℃; d represents the ion-flow diagram of the catalytic product obtained at 30℃for the induction of pET32a-TPS1 supernatant.

Fig. 5 is a mass spectrum and a standard quality spectrum of the catalyst product of aquilaria sinensis TPS1, namely, delbrueck sinensis blue alkene. A: mass spectrum of TPS1 crude enzyme catalytic product; b: standard jacobian mass spectrum. The abscissa is the mass-to-charge ratio (m/z) value of the ion and the ordinate is the intensity of the ion stream.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 full Length cloning of the lignum Aquilariae Resinatum sesquiterpene synthase Gene TPS1

1. Extraction of lignum Aquilariae Resinatum RNA

1mL of Trizol reagent was added to a 2mL centrifuge tube, then fresh stem samples of Syringa pubescens were ground to a fine powder under liquid nitrogen and rapidly added to the Trizol reagent-added centrifuge tube and vortexed. Centrifuge at 12000rpm for 10min, and take the supernatant into another centrifuge tube. Chloroform was added to the centrifuge tube in an amount of about 1/5 of the volume of the supernatant, vigorously shaken for 15s, left at room temperature for 2 to 3min, and then centrifuged at 12000rpm for 15min. The upper aqueous phase was aspirated into another centrifuge tube, into which was added isopropanol half the volume of the aqueous phase, mixed upside down and left at room temperature for 10min, after which it was centrifuged at 12000rpm for 10min. After centrifugation, the supernatant was discarded, and a small amount of white precipitate was observed at the bottom of the tube. The tube caps, tube walls and pellet in the centrifuge tube were washed with 75% ethanol and centrifuged at 12000rpm for 5min, the supernatant was discarded, and then the 75% ethanol in the previous step was changed to absolute ethanol and repeated. Finally, dry ethanol was blown dry to prevent the next reaction from being affected, and the RNA integrity was checked by agarose gel electrophoresis at 1% concentration (FIG. 1).

2. cDNA reverse transcription of lignum Aquilariae Resinatum

mu.L of the extracted RNA was aspirated using a pipette, and 2. Mu.L of Oligo (dT) 18 was added thereto, followed by centrifugation and mixing, followed by reaction at 70℃for 10min, and then ice bath for 2min. Then, 4. Mu.L of 5X Reverse Transcriptase M-MLV Buffer, 1. Mu.L of Recombinant RNase Inhibitor, 2. Mu.L of dNTP, 1. Mu.L of Reverse Transcriptase M-MLV (RNase H) were added to the solution obtained in the above step, reacted at 42℃for 60min, and kept at 70℃for 15min, and finally the obtained cDNA was stored in a refrigerator at-20 ℃.

3. Cloning of full-length sequence of lignum Aquilariae Resinatum TPS1

(1) The primers used for the TPS1 full length clone were:

an upstream primer: 5'-CCGCGAATTGGCCCTTAGCA-3'

A downstream primer: 5'-GAATTTGGTCGGAAACATTCTTCATTGATATCCTT-3'

(2) The reaction system used for TPS1 full length cloning is:

(3) The reaction procedure used for TPS1 full length cloning was:

(4) Agarose gel electrophoresis

Agarose gel powder was diluted to 100ml with 1X concentration of TAE buffer and 1.5% concentration and dissolved by heating in a microwave oven. Adding a drop of Ethidium Bromide (EB) into a fume hood, shaking, pouring into a glue-making plate into which a comb is inserted in the fume hood, and cooling and solidifying for later use. And (3) absorbing a proper amount of CDS sequence of the PCR product obtained in the step (3), namely TPS1 gene, if the PCR product is not pre-dyed, adding a proper concentration loading buffer, carrying out electrophoresis by using a TAE buffer solution with 1X concentration, setting the voltage to 200V, setting the current to 400mA, stopping electrophoresis when the strip runs to about three quarters, and observing and photographing by using a gel electrophoresis imager.

(5) PCR product gel recovery

Glue recovery was performed using Thermo Scientific GeneJET Gel Extraction Kit. The method comprises the following steps: a) Cutting a target PCR product strip (about 1600-1700 bp), adding into a centrifuge tube, and weighing; b) Adding Binding Buffer (100 mu L of Binding Buffer is added to each 100mg of glue) into a centrifuge tube (containing glue); c) Heating at 50-60 ℃ for about 10min, and after the glue is completely dissolved (if the obtained liquid is yellow, the next step can be directly carried out, and if orange yellow is developed, the pH value is required to be regulated by sodium acetate, so that the color of the liquid turns yellow, the next step is carried out); d) Transferring the solution obtained in the last step to a GeneJET purifying column, centrifuging at 12000rpm for 1min, and discarding the waste liquid; e) If the direct sequencing is needed, adding 100 mu L of Binding Buffer, and repeating the previous step; f) 700. Mu.L of Wash Buffer is added to a GeneJET purification column, the mixture is centrifuged at 12000rpm for 1min, and the waste liquid is discarded; g) Continuing to centrifuge for 1min to ensure that the liquid is completely removed; h) Transferring the GeneJET purification column to a clean 1.5ml centrifuge tube, adding 20-50 mu L of the absorption Buffer into the column, and centrifuging at 12000rpm for 1min to obtain a purified product, namely the full length sequence (CDS) (sequence 1 in a sequence table) of the purified TPS1 gene, wherein the translated protein sequence is (sequence 2 in the sequence table).

(6) LB Medium configuration

Weighing and preparing LB culture medium, wherein the concentration of Tryptone (Tryptone) is 10g/L, the concentration of Yeast Extract (Yeast Extract) is 5g/L, the concentration of sodium chloride is 10g/L, the concentration of agar powder is 8g/L, adding ultrapure water to the specified concentration, sealing by a sealing film, sterilizing at 121 ℃ for 20min, adding ampicillin solution to the final concentration of 100mg/L in an ultra-clean bench when the agar powder is not scalded after the agar powder is taken out, shaking uniformly, and pouring the agar powder into a flat plate. The liquid LB medium was prepared as above, but without adding agar powder, and was classified into an LB liquid medium without adding ampicillin and an LB liquid medium with adding ampicillin (the concentration of ampicillin was the same as that of the solid medium) as required.

(7) Vector ligation and transformation

Vector ligation was performed using pEASY-Blunt Zero Cloning Kit (Beijing full gold Biotechnology Co., ltd., CU 101). 4 mu L of CDS of the TPS1 gene purified in the step (5) is taken, 1 mu L of pEASY-Blunt Zero Cloning Vector is added, the mixture is gently mixed and then reacted at room temperature for 30min to obtain a connection product of TPS1 and a cloning vector, and then a centrifuge tube is placed on ice. E.coli Trans1-T1 competent cells (Beijing full gold biotechnology Co., ltd., CD 501) were thawed on ice in advance, the centrifuge tube used was also cooled on ice, 50. Mu.L of each tube was rapidly dispensed when the competent cells had just been thawed, and the connection product of TPS1 and the cloning vector of the previous step was added to the competent cells, and after mixing, the ice bath was carried out for 30min, followed by heat shock in a water bath at 42℃for 30s, and immediately placed on ice for 2min. Then, 450. Mu.L of ampicillin-free liquid LB medium was added to the resultant after transformation into cells, and the resultant was subjected to shaking culture at 200rpm and 37℃for 1 hour. After removal, the culture was centrifuged at 2000rpm for 4min, 300. Mu.L of the supernatant medium was aspirated, and the remainder was mixed and spread on LB solid medium containing ampicillin, followed by overnight culture at 37 ℃.

(8) Monoclonal culture and validation

The monoclonal colony grown on the culture medium cultivated overnight in the step (7) is picked and cultivated in 1ml LB culture medium containing ampicillin, 200rpm and 37 ℃ for 6 hours, 1 mu L of bacterial liquid is taken for PCR verification, and the system is as follows:

bacterial liquid PCR reaction procedure:

and determining the PCR product of the bacterial liquid as a target size band, and then carrying out Sanger first-generation sequencing to finally obtain a positive monoclonal colony containing a TPS1 gene vector, wherein the agarose gel electrophoresis diagram of the PCR product of the positive monoclonal colony is shown in fig. 2, and the sequencing result shows that the positive monoclonal colony contains the full length of the sequence 1 in the sequence table, namely the CDS sequence of the gene coding for the agalloch eaglewood sesquiterpene jacaralloch blue alkene synthase TPS1 (Eremophilene synthase), and the amino acid sequence of the corresponding agalloch eaglewood sesquiterpene jacaralloch blue alkene synthase TPS1 protein is shown as the sequence 2 in the sequence table. The TPS1 protein was predicted to be 65kDa in size.

EXAMPLE 2 prokaryotic expression of the lignum Aquilariae Resinatum sesquiterpene synthase Gene TPS1

Construction of pET32a-TPS1 expression vector

The CDS sequence (sequence 1 in the sequence table) of the TPS1 gene obtained in example 1 is used as a template, the digestion site which exists in the target expression vector but does not exist in the inserted gene is analyzed and selected by using NEBcUTter2.0 on-line software, a primer with the digestion recognition site is designed, and the joints are added at the two ends of the CDS sequence of the cDNA of the TPS1 gene through PCR amplification. TPS1 was designed with BamHI and SacI cleavage sites. The primer pET32a vector has two His tags, and for His tag expression in the subsequent prokaryotic expression, the stop codon needs to be removed in reverse primer design, and the primers used are as follows. The underlined sequence is the cleavage site recognition sequence.

The reaction system of TPS1 with linker gene is as follows:

the PCR procedure for TPS1 was as follows:

expression vector linearization pET-32a (preserved in this laboratory, related reference: jin B, et al functional diversification of kaurene synthase-like genes in Isodon rubescens [ J ]. Plant Physiology,2017,174:943-955. Public available from the applicant in duplicate copies of the invention) was double digested with restriction enzymes BamHI and SacI to give a linearized pET-32a vector. Meanwhile, double digestion is carried out on the CDS sequence of the TPS1 gene added with the digestion recognition site, and a target gene fragment after double digestion is obtained, wherein the digestion system is as follows. Wherein the endonucleases used by the related subsequent ligation vectors of the TPS1 gene are BamHI and SacI, and the digestion conditions are 37 ℃ for 3 hours.

The target gene fragment after double cleavage was ligated to the linearized pET32a vector using Uni Seameless Assembly Kit. Reacting at 50deg.C for 15min, and cooling on ice. Finally obtaining recombinant expression vector pET32a-TPS1. The recombinant expression vector pET32a-TPS1 is obtained by replacing a fragment (small fragment) between BamHI and SacI recognition sites of pET32a (+) with 1 st-1665 th nucleotides of a sequence 1 in a sequence table, and keeping other sequences of pET32a (+) unchanged. The pET32a-TPS1 recombinant vector contains a recombinant TPS1 gene TPS1-Trx-His (sequence 3 in a sequence table) with Trx and His tag sequences, and can express a recombinant TPS1 protein TPS1-Trx-His with the Trx and His tags shown in sequence 4 in the sequence table.

2. Prokaryotic expression TPS1 protein

The recombinant gene expression vector pET32a-TPS1 is subjected to expansion culture, then recombinant plasmid pET32a-TPS1 is extracted, the extracted recombinant plasmid pET32a-TPS1 is transformed into expression competent cells, and the cells used for transformation are competent cells of escherichia coli Rossetta strain Transetta (DE 3) (Beijing full gold biotechnology Co., ltd., CD 801-02) to obtain recombinant escherichia coli DE3/pET32a-TPS1. Meanwhile, transforming the pET32a (+) empty plasmid into a Transetta (DE 3) competent cell to obtain recombinant escherichia coli DE3/pET32a.

The recombinant escherichia coli DE3/pET32a-TPS1 bacterial liquid is subjected to expansion culture by using LB culture liquid containing ampicillin in a ratio of 1:50, the bacterial liquid is cultured for about 3 hours at 37 ℃ and 200rpm, and OD value at 600nm is measured to be between 0.6 and 1.0, so that uninduced pET32a-TPS1 whole bacterial liquid and uninduced empty whole bacterial liquid are obtained. . Adding IPTG to the uninduced pET32a-TPS1 whole bacterial liquid and the uninduced empty whole bacterial liquid to a final concentration of 500 mu M, then culturing at the same temperature for 12 hours overnight, taking two temperatures of 16 ℃ and 30 ℃ and keeping the rotating speed at 200rpm for induced expression, collecting fermentation liquor, and naming fermentation liquor obtained by induced expression of recombinant escherichia coli DE3/pET32a-TPS1 as induced pET32a-TPS1 whole bacterial liquid, and naming fermentation liquor obtained by induced expression of recombinant escherichia coli DE3/pET32a as induced empty whole bacterial liquid.

Centrifuging the induced pET32a-TPS1 whole bacterial liquid and the induced empty whole bacterial liquid at 4 ℃ for 10min at a rotation speed of 5000rpm, discarding the supernatant, and then cleaning and centrifuging the supernatant again by using sterilized water to ensure that the culture medium is completely cleaned. Subsequently, 500. Mu.L of PBS buffer was added, and after mixing, the mixture was sonicated on ice for 3s at 30% energy and suspended for a total of 10min. After ultrasonic crushing, reserving a part of the mixture as whole bacterial liquid, centrifuging the rest of the mixture at 12000rpm for 20min at 4 ℃, and sucking the supernatant to obtain an induced pET32a-TPS1 supernatant and an induced empty supernatant respectively; the precipitated fraction was added to the same volume of PBS buffer as the supernatant and mixed to obtain induced pET32a-TPS1 precipitation and induced empty precipitation, respectively, and SDS-PAGE was performed as follows.

3. Polyacrylamide gel electrophoresis (SDS-PAGE)

Firstly, preparing SDS-PAGE separating gel according to the following formula, adding a proper amount of separating gel into a clamped glass plate, adding water to avoid drying and smoothening the gel surface; after the separation gel is solidified, water is removed, SDS-PAGE concentrated gel is prepared, the SDS-PAGE concentrated gel is added above the solidified separation gel, a comb is inserted, and the SDS-PAGE concentrated gel is solidified for standby.

SDS-PAGE separating gel formula

SDS-PAGE concentrated gel formula

And (3) respectively taking a part of the precipitation mixed solution of the recombinant bacterial solution and the empty bacterial solution obtained in the step (2), respectively adding SDS-PAGE Buffer, uniformly mixing, boiling in water for 5min, and then taking and loading the solution. The operation was first performed at 80V for 30min, and then 120V was performed for 1h. After removal of the gel, the gel was stained with coomassie brilliant blue R250 dye for 2h and decolorized for about 4h, and analysed by photographic preservation (fig. 3). Wherein the TPS1 protein has a predicted size of 65kDa and the recombinant protein TPS1-Trx-His has a predicted size of 85kDa, and the results in FIG. 3 show that recombinant protein TPS1-Trx-His is detected in the induced pET32a-TPS1 whole bacterial liquid, the induced pET32a-TPS1 supernatant and the induced pET32a-TPS1 precipitate (the band size is about 80kDa and is close to the predicted value); no recombinant protein TPS1-Trx-His band was detected in the induced empty whole bacterial liquid, the induced empty supernatant and the induced empty pellet.

Example 3 functional verification of the lignum Aquilariae Resinatum sesquiterpene synthase Gene TPS1

1. Enzymatic reactions

The enzymatic reaction system was 400. Mu.L in total, and contained 282. Mu.L of the induced pET32a-TPS1 supernatant (abbreviated as TPS1 crude enzyme) obtained in example 2, with ammonium farnesyl pyrophosphate (Farnesyl pyrophosphate ammonium salt, FPP) (Sigma-Aldrich Co., U.S.A., F6892-1 VL) as a substrate (final concentration: 50. Mu.M), HEPES (Sigma-Aldrich Co., U.S.A., H7006) (pH 7.2) to 50mM, the balance comprising magnesium chloride to 7.5mM, HEPES (pH 7.2) to a final concentration of 50mM, glycerol 5%, and DTT (dithiothreitol) (Promega Co., U.S.A., V3151) to 5mM, the balance being deionized water. After the above system is uniformly mixed, 500 mu L of n-hexane is added above the mixture to cover the mixture, so that the escape of volatile sesquiterpene products is prevented, and the mixture is reacted in a water bath at 30 ℃ for 1h to obtain an enzymatic reaction catalytic product (TPS 1 crude enzyme catalytic product). After the reaction, vortex and mix well, centrifuge at 10000rpm for 5min, take the upper layer normal hexane organic phase to carry on the ingredient measurement. The enzyme reaction system added in example 2 to induce empty supernatant to other substances and the same content is used as a control to obtain an enzyme reaction catalysis product.

2. Product detection

The enzymatic reaction catalytic product was measured using a GC-MS gas chromatograph-mass spectrometer after filtration using a 0.22 μm polytetrafluoroethylene organic filter membrane under conditions of an initial temperature of 100 ℃, holding for 3.5min, rising to 170 ℃ at a rate of 20 ℃/min, then slowly rising to 180 ℃ at a rate of 0.5 ℃/min, then rising to 250 ℃ at a rate of 25 ℃/min, and finally rising to 300 ℃ at a rate of 5 ℃/min, holding for 5min. The temperature of the sample inlet is 280 ℃, the ion source is EI, the electron energy is 70eV, the ion source temperature is 230 ℃, and the scanning range is 40-400 m/z. The ion flow diagram of the blank bacterial liquid and the recombinant bacterial liquid containing recombinant protein TPS1-Trx-His after the product after the FPP is catalyzed is shown in figure 4, and compared with an empty load when pET32a is taken as a carrier (shown as pET32a-empty in A and B in figure 4), the target product (shown as pET32a-TPS1 in C and D in figure 4) is successfully obtained by catalyzing TPS1 enzyme at 16 ℃ and 30 ℃, and the catalytic product has a single peak at 8.63min and is single. Further from the mass spectrum, it can be seen that the TPS1-Trx-His catalyzed sesquiterpene product (FIG. 5A) is compared with the mass spectrum of jacobian (FIG. 5B), so that the TPS 1-encoding enzyme functions to catalyze the production of jacobian, which can be named as jacobian 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> Chinese institute of traditional Chinese medicine

<120> lignum Aquilariae Resinatum sesquiterpene synthase protein TPS1, and coding gene and application thereof

<130> GNCSQ211299

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1668

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> Artificial sequence

<400> 1

atggcgcagg ctcttgttgc aaatcctatc gataacaaaa aggagattgt tcgtccagta 60

gccaacttct ctcccagctt atggggcgaa cagtttatca atttttcgtt tgatactgag 120

ttagcagaga aatatgaaga ggagatcgaa cagttaaaat atgaagtgaa aagcatgcta 180

acagctcctg gaaaagacat ggtggagacg atgaatttga tcgaaacact tgagcgcttg 240

ggcatctcat ttcactttgc aaatgagatc gaagaactac tacaacgttt ctttaatctc 300

aactcaaact atgcagatca tgaagcctat gatttgtaca ctgttgcact tcattttcgt 360

ttgttcagac aacatggcta ccgaatgtct tgtgatatct ttaaaagatt cacagacgag 420

actgggaaat tcaaagagtc tatcaagagc gatgcaagtg gattgcttag cttatatgaa 480

gctgcatatt taagagtgca tggagaagat atactagaag acgccctcgt cttcacaacg 540

gagaacctac aatccatggc accaaaacta agctctaccc tggggaaaca agtagctcat 600

gccctcgtac aatgcatcca ttttgggaac ccaagaattg agtcacgtaa ctttatctcc 660

atctaccaag aagatgagtt caagaatgaa atgttgctga ggtttgccaa attagactat 720

aattcattgc aaatgttgca taaaaaggaa ctctatgaag tttcaaggtg gtggaaggat 780

ttggaccttg tctctaagct tccttatgct agagatagag ttgtagaatg ctttttctgg 840

gcaatgggag tttatcatga accacaatat tccgttgctc gaatcatgct caccaaaacc 900

attgcaatga cgtcgataat agatgataca tatgacgcct atggtgtagt tgaagaactt 960

gaaattttta cggaggccat tcaaaagtgg gatattagcg agattgatcg gctaccagag 1020

tatataaagc cattttacag tgctcttcta aatctatatg aggagtttga cgaagaacta 1080

gcaaaggaag gacggtctta tgcagtctac tatgcaaaag aagcactaaa agaacttgtg 1140

aggacgtacc atgtggaagc caagtggttc attgaaggat acttgccacc attttctgag 1200

tacatgagca atgccttaat aacatgtact tatgtttacc atacaactac atccttgttg 1260

ggaatcaaat ccgtcaccaa ggaagaattt gaatggctaa gcaataaacc taaaatgctt 1320

gttgcaagcc tcataatatg tcggctcgtt gatgacattg ctacctatga ggttgagaag 1380

gaaaggggcc aaattgcaac tggcattgaa tcatacatgc aagagaatgg tgtaactaaa 1440

gaagtggcta ttgataagtt ttttgaaatg gttacaaatg catggaagga tatcaatgaa 1500

gaatgtttcc gaccaaattc ctctccgaga gaaattctga tgcgaattct aaaccttgaa 1560

cgcattattg atgtaactta caaaggcaat gaagatggat acacgcaacc acagaaagtt 1620

ctaatgcctc acatcattgc tttgtttatt gatcccatcg gcatctaa 1668

<210> 2

<211> 555

<212> PRT

<213> Syringa pinnatifolia hemsl (Syzygium aromaticum)

<400> 2

Met Ala Gln Ala Leu Val Ala Asn Pro Ile Asp Asn Lys Lys Glu Ile

1 5 10 15

Val Arg Pro Val Ala Asn Phe Ser Pro Ser Leu Trp Gly Glu Gln Phe

20 25 30

Ile Asn Phe Ser Phe Asp Thr Glu Leu Ala Glu Lys Tyr Glu Glu Glu

35 40 45

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

50 55 60

Lys Asp Met Val Glu Thr Met Asn Leu Ile Glu Thr Leu Glu Arg Leu

65 70 75 80

Gly Ile Ser Phe His Phe Ala Asn Glu Ile Glu Glu Leu Leu Gln Arg

85 90 95

Phe Phe Asn Leu Asn Ser Asn Tyr Ala Asp His Glu Ala Tyr Asp Leu

100 105 110

Tyr Thr Val Ala Leu His Phe Arg Leu Phe Arg Gln His Gly Tyr Arg

115 120 125

Met Ser Cys Asp Ile Phe Lys Arg Phe Thr Asp Glu Thr Gly Lys Phe

130 135 140

Lys Glu Ser Ile Lys Ser Asp Ala Ser Gly Leu Leu Ser Leu Tyr Glu

145 150 155 160

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

165 170 175

Val Phe Thr Thr Glu Asn Leu Gln Ser Met Ala Pro Lys Leu Ser Ser

180 185 190

Thr Leu Gly Lys Gln Val Ala His Ala Leu Val Gln Cys Ile His Phe

195 200 205

Gly Asn Pro Arg Ile Glu Ser Arg Asn Phe Ile Ser Ile Tyr Gln Glu

210 215 220

Asp Glu Phe Lys Asn Glu Met Leu Leu Arg Phe Ala Lys Leu Asp Tyr

225 230 235 240

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

245 250 255

Trp Trp Lys Asp Leu Asp Leu Val Ser Lys Leu Pro Tyr Ala Arg Asp

260 265 270

Arg Val Val Glu Cys Phe Phe Trp Ala Met Gly Val Tyr His Glu Pro

275 280 285

Gln Tyr Ser Val Ala Arg Ile Met Leu Thr Lys Thr Ile Ala Met Thr

290 295 300

Ser Ile Ile Asp Asp Thr Tyr Asp Ala Tyr Gly Val Val Glu Glu Leu

305 310 315 320

Glu Ile Phe Thr Glu Ala Ile Gln Lys Trp Asp Ile Ser Glu Ile Asp

325 330 335

Arg Leu Pro Glu Tyr Ile Lys Pro Phe Tyr Ser Ala Leu Leu Asn Leu

340 345 350

Tyr Glu Glu Phe Asp Glu Glu Leu Ala Lys Glu Gly Arg Ser Tyr Ala

355 360 365

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

370 375 380

Val Glu Ala Lys Trp Phe Ile Glu Gly Tyr Leu Pro Pro Phe Ser Glu

385 390 395 400

Tyr Met Ser Asn Ala Leu Ile Thr Cys Thr Tyr Val Tyr His Thr Thr

405 410 415

Thr Ser Leu Leu Gly Ile Lys Ser Val Thr Lys Glu Glu Phe Glu Trp

420 425 430

Leu Ser Asn Lys Pro Lys Met Leu Val Ala Ser Leu Ile Ile Cys Arg

435 440 445

Leu Val Asp Asp Ile Ala Thr Tyr Glu Val Glu Lys Glu Arg Gly Gln

450 455 460

Ile Ala Thr Gly Ile Glu Ser Tyr Met Gln Glu Asn Gly Val Thr Lys

465 470 475 480

Glu Val Ala Ile Asp Lys Phe Phe Glu Met Val Thr Asn Ala Trp Lys

485 490 495

Asp Ile Asn Glu Glu Cys Phe Arg Pro Asn Ser Ser Pro Arg Glu Ile

500 505 510

Leu Met Arg Ile Leu Asn Leu Glu Arg Ile Ile Asp Val Thr Tyr Lys

515 520 525

Gly Asn Glu Asp Gly Tyr Thr Gln Pro Gln Lys Val Leu Met Pro His

530 535 540

Ile Ile Ala Leu Phe Ile Asp Pro Ile Gly Ile

545 550 555

<210> 3

<211> 2229

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> Artificial sequence

<400> 3

atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120

ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatccatggc gcaggctctt gttgcaaatc ctatcgataa caaaaaggag 540

attgttcgtc cagtagccaa cttctctccc agcttatggg gcgaacagtt tatcaatttt 600

tcgtttgata ctgagttagc agagaaatat gaagaggaga tcgaacagtt aaaatatgaa 660

gtgaaaagca tgctaacagc tcctggaaaa gacatggtgg agacgatgaa tttgatcgaa 720

acacttgagc gcttgggcat ctcatttcac tttgcaaatg agatcgaaga actactacaa 780

cgtttcttta atctcaactc aaactatgca gatcatgaag cctatgattt gtacactgtt 840

gcacttcatt ttcgtttgtt cagacaacat ggctaccgaa tgtcttgtga tatctttaaa 900

agattcacag acgagactgg gaaattcaaa gagtctatca agagcgatgc aagtggattg 960

cttagcttat atgaagctgc atatttaaga gtgcatggag aagatatact agaagacgcc 1020

ctcgtcttca caacggagaa cctacaatcc atggcaccaa aactaagctc taccctgggg 1080

aaacaagtag ctcatgccct cgtacaatgc atccattttg ggaacccaag aattgagtca 1140

cgtaacttta tctccatcta ccaagaagat gagttcaaga atgaaatgtt gctgaggttt 1200

gccaaattag actataattc attgcaaatg ttgcataaaa aggaactcta tgaagtttca 1260

aggtggtgga aggatttgga ccttgtctct aagcttcctt atgctagaga tagagttgta 1320

gaatgctttt tctgggcaat gggagtttat catgaaccac aatattccgt tgctcgaatc 1380

atgctcacca aaaccattgc aatgacgtcg ataatagatg atacatatga cgcctatggt 1440

gtagttgaag aacttgaaat ttttacggag gccattcaaa agtgggatat tagcgagatt 1500

gatcggctac cagagtatat aaagccattt tacagtgctc ttctaaatct atatgaggag 1560

tttgacgaag aactagcaaa ggaaggacgg tcttatgcag tctactatgc aaaagaagca 1620

ctaaaagaac ttgtgaggac gtaccatgtg gaagccaagt ggttcattga aggatacttg 1680

ccaccatttt ctgagtacat gagcaatgcc ttaataacat gtacttatgt ttaccataca 1740

actacatcct tgttgggaat caaatccgtc accaaggaag aatttgaatg gctaagcaat 1800

aaacctaaaa tgcttgttgc aagcctcata atatgtcggc tcgttgatga cattgctacc 1860

tatgaggttg agaaggaaag gggccaaatt gcaactggca ttgaatcata catgcaagag 1920

aatggtgtaa ctaaagaagt ggctattgat aagttttttg aaatggttac aaatgcatgg 1980

aaggatatca atgaagaatg tttccgacca aattcctctc cgagagaaat tctgatgcga 2040

attctaaacc ttgaacgcat tattgatgta acttacaaag gcaatgaaga tggatacacg 2100

caaccacaga aagttctaat gcctcacatc attgctttgt ttattgatcc catcggcatc 2160

gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc 2220

ggctgctaa 2229

<210> 4

<211> 742

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<220>

<223> Artificial sequence

<400> 4

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Met Ala Gln Ala Leu Val Ala Asn Pro Ile Asp

165 170 175

Asn Lys Lys Glu Ile Val Arg Pro Val Ala Asn Phe Ser Pro Ser Leu

180 185 190

Trp Gly Glu Gln Phe Ile Asn Phe Ser Phe Asp Thr Glu Leu Ala Glu

195 200 205

Lys Tyr Glu Glu Glu Ile Glu Gln Leu Lys Tyr Glu Val Lys Ser Met

210 215 220

Leu Thr Ala Pro Gly Lys Asp Met Val Glu Thr Met Asn Leu Ile Glu

225 230 235 240

Thr Leu Glu Arg Leu Gly Ile Ser Phe His Phe Ala Asn Glu Ile Glu

245 250 255

Glu Leu Leu Gln Arg Phe Phe Asn Leu Asn Ser Asn Tyr Ala Asp His

260 265 270

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

275 280 285

Gln His Gly Tyr Arg Met Ser Cys Asp Ile Phe Lys Arg Phe Thr Asp

290 295 300

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

305 310 315 320

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

325 330 335

Leu Glu Asp Ala Leu Val Phe Thr Thr Glu Asn Leu Gln Ser Met Ala

340 345 350

Pro Lys Leu Ser Ser Thr Leu Gly Lys Gln Val Ala His Ala Leu Val

355 360 365

Gln Cys Ile His Phe Gly Asn Pro Arg Ile Glu Ser Arg Asn Phe Ile

370 375 380

Ser Ile Tyr Gln Glu Asp Glu Phe Lys Asn Glu Met Leu Leu Arg Phe

385 390 395 400

Ala Lys Leu Asp Tyr Asn Ser Leu Gln Met Leu His Lys Lys Glu Leu

405 410 415

Tyr Glu Val Ser Arg Trp Trp Lys Asp Leu Asp Leu Val Ser Lys Leu

420 425 430

Pro Tyr Ala Arg Asp Arg Val Val Glu Cys Phe Phe Trp Ala Met Gly

435 440 445

Val Tyr His Glu Pro Gln Tyr Ser Val Ala Arg Ile Met Leu Thr Lys

450 455 460

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

465 470 475 480

Val Val Glu Glu Leu Glu Ile Phe Thr Glu Ala Ile Gln Lys Trp Asp

485 490 495

Ile Ser Glu Ile Asp Arg Leu Pro Glu Tyr Ile Lys Pro Phe Tyr Ser

500 505 510

Ala Leu Leu Asn Leu Tyr Glu Glu Phe Asp Glu Glu Leu Ala Lys Glu

515 520 525

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

530 535 540

Val Arg Thr Tyr His Val Glu Ala Lys Trp Phe Ile Glu Gly Tyr Leu

545 550 555 560

Pro Pro Phe Ser Glu Tyr Met Ser Asn Ala Leu Ile Thr Cys Thr Tyr

565 570 575

Val Tyr His Thr Thr Thr Ser Leu Leu Gly Ile Lys Ser Val Thr Lys

580 585 590

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

595 600 605

Leu Ile Ile Cys Arg Leu Val Asp Asp Ile Ala Thr Tyr Glu Val Glu

610 615 620

Lys Glu Arg Gly Gln Ile Ala Thr Gly Ile Glu Ser Tyr Met Gln Glu

625 630 635 640

Asn Gly Val Thr Lys Glu Val Ala Ile Asp Lys Phe Phe Glu Met Val

645 650 655

Thr Asn Ala Trp Lys Asp Ile Asn Glu Glu Cys Phe Arg Pro Asn Ser

660 665 670

Ser Pro Arg Glu Ile Leu Met Arg Ile Leu Asn Leu Glu Arg Ile Ile

675 680 685

Asp Val Thr Tyr Lys Gly Asn Glu Asp Gly Tyr Thr Gln Pro Gln Lys

690 695 700

Val Leu Met Pro His Ile Ile Ala Leu Phe Ile Asp Pro Ile Gly Ile

705 710 715 720

Glu Leu Arg Arg Gln Ala Cys Gly Arg Thr Arg Ala Pro Pro Pro Pro

725 730 735

Pro Leu Arg Ser Gly Cys

740

Claims (10)

1. A protein characterized in that: the protein is the protein of A1) or A2) as follows:

a1 Amino acid sequence is protein of sequence 2 in the sequence table,

a2 Amino acid sequence is protein of sequence 4 in the sequence table.

2. Use of a protein as or in the preparation of a agalloch eaglewood sesquiterpene, a meropenem synthase, said protein being as described in claim 1.

3. A biological material associated with the protein of claim 1, characterized in that: the biomaterial is any one of the following D1) to D4):

d1 A nucleic acid molecule encoding the protein of claim 1;

d2 An expression cassette comprising D1) said nucleic acid molecule;

d3 A recombinant vector comprising D1) said nucleic acid molecule, or a recombinant vector comprising D2) said expression cassette;

d4 A recombinant microorganism comprising D1) said nucleic acid molecule, or a recombinant microorganism comprising D2) said expression cassette, or a recombinant microorganism comprising D3) said recombinant vector.

4. A biomaterial according to claim 3, wherein: d1 The nucleic acid molecule is a gene encoding the protein as shown in d 1) or d 2) below:

d1 A DNA molecule with a coding sequence of a sequence 1 in a sequence table;

d2 Nucleotide is a DNA molecule of a sequence 3 in a sequence table.

5. Use of a protein as claimed in claim 1 and/or a biomaterial as claimed in claim 3 or 4 for the production of a shan agalloch sesquiterpene.

6. A method for preparing agalloch eaglewood sesquiterpene jacarallorene synthase, comprising the following steps: expressing the gene encoding the protein of claim 1 in a prokaryotic microorganism to obtain the agalloch eaglewood sesquiterpene jacara-blue alkene synthase.

7. The method according to claim 6, wherein: expressing the gene encoding the protein of claim 1 in a prokaryotic microorganism comprises introducing the gene encoding the protein of claim 1 into a recipient microorganism to obtain a recombinant microorganism expressing the agalloch eaglewood sesquiterpene jacara blue alkene synthase, culturing the recombinant microorganism, and expressing to obtain the agalloch eaglewood sesquiterpene jacara blue alkene synthase.

8. The method according to claim 6 or 7, characterized in that: the expression is induced expression.

9. A method for preparing aquilaria sinensis sesquiterpene, comprising the following steps: catalyzing a substrate farnesyl ammonium pyrophosphate with the protein of claim 1 as a agalloch eaglewood sesquiterpene delphine synthase to obtain the agalloch eaglewood sesquiterpene.

10. A product comprising the protein of claim 1 and/or any one of the following biological materials of claim 3 or 4:

p1, producing a product of agalloch eaglewood sesquiterpene;

p2 is used for producing the product of the agalloch eaglewood sesquiterpene and the delphine synthase.