CN108977426A - A kind of oil nanmu sesquiterpene synthase and its encoding gene and application - Google Patents
A kind of oil nanmu sesquiterpene synthase and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of oily nanmu sesquiterpene synthase and its encoding gene and applications.The present invention provides a kind of new sesquiterpene synthase --- oily nanmu sesquiterpene synthase SgSTPS1 and its encoding genes, oily nanmu sesquiterpene synthase SgSTPS1 albumen produces active sesquiterpene synthase after prokaryotic expression, catalysis substrate FPP generates β-carypohyllene, isocaryophyllene and α-carypohyllene, can be used for its mass production.
Description
Technical field
The invention belongs to field of plant genetic project technology, and in particular to it is a kind of oil nanmu sesquiterpene synthase and its coding base
Cause and application.
Background technique
Oily nanmu (Sindora glabra Merr.ex de Wit) is under the jurisdiction of Caesalpiniaceae oil Phoebe, primary to be distributed in China
Hainan Province is national second level Top-rated protected wild plants for the torrid zone and the high megaphanerophyte of south subtropics.The oily most important feature of nanmu is
Resinon can be secreted out of after its trunk xylem is impaired, it is flammable similar to diesel oil, therefore also known as " diesel oil tree ", in oil use, medicine
With, ornamental and material with etc. there is broader exploitation prospect.
Oily nanmu resin oil component mainly includes sesquiterpenoids, accounts for about 70% or more and Diterpenes, about 14% or more,
Middle α-copaene, β-carypohyllene and δ-cadinene three's content account for 2/3 of sesquiterpenoids or more.Oily nanmu oil also contains active constituent α-
Amorphene, cubebene, α-humulene, ring sativene etc., further, oily nanmu oil also contain the big Mang ox of volatile component
Alkene, α-aromadendrene, δ-cadinol, α-santalol and α-Java Cananga Oil alkene etc. represents the main component of fragrance, in terms of developing essential oil
Present huge potentiality to be exploited.Sesquiterpenoids is that more active one of field is studied in Natural Medicine Chemistry, tool
There is antitumor, antibacterial isoreactivity.However, the biosynthesis gene about terpene synzyme in oily nanmu is not isolated and identified also.
β-carypohyllene is widely used in the products such as food flavor essence, but its production mainly by be directly extracted from plants into
Row, and have no the report produced using biological engineering method.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of new sesquiterpene synthases and its encoding gene
And the application of sesquiterpene synthase.
The first purpose of the invention is to provide a kind of oily nanmu sesquiterpene synthase SgSTPS1, amino acid sequence such as SEQ
Shown in ID NO.2.
A second object of the present invention is to provide the encoding genes of the oily nanmu sesquiterpene synthase SgSTPS1.
It is preferred that the encoding gene, nucleotide sequence is as shown in SEQ ID NO.3.
It is preferred that the encoding gene, nucleotide sequence is as shown in SEQ ID NO.1.
Sequence with the oily nanmu sesquiterpene synthase SgSTPS1 amino acid with 50% or more similitude, or and its
Conservative variation's polypeptides, active fragment or reactive derivative with the same function, for example, by amino acid sequence by one or
Multiple replace, miss or add and the amino acid sequence formed also belongs to protection scope of the present invention.With the oily nanmu sesquialter
There is the nucleotide sequence of the encoding gene of terpene synzyme SgSTPS1 the sequence of 50% or more similitude to also belong to guarantor of the invention
Protect range.
The present invention also provides a kind of recombinant expressions of encoding gene containing the oily nanmu sesquiterpene synthase SgSTPS1
Carrier.
The expression vector, preferably expression vector pET-30a.
The present invention also provides a kind of genetic engineerings of encoding gene containing the oily nanmu sesquiterpene synthase SgSTPS1
Bacterium.
The genetic engineering bacterium, preferably Escherichia coli Top10 or e. coli bl21 (DE3).
Third object of the present invention is to provide the oily nanmu sesquiterpene synthase SgSTPS1 preparation β-carypohyllene,
Application in isocaryophyllene and/or α-carypohyllene.
It is preferred that the application, is with farnesyl pyrophosphate (FPP) for substrate, through oily nanmu sesquiterpene synthase SgSTPS1
Catalysis generates β-carypohyllene, isocaryophyllene and α-carypohyllene.
The present invention provides a kind of new sesquiterpene synthases --- oily nanmu sesquiterpene synthase SgSTPS1 and its coding base
Cause, the albumen produce active sesquiterpene synthase after prokaryotic expression, and catalysis substrate FPP generates β-carypohyllene, different China pink
Alkene and α-carypohyllene, can be used for its mass production.
Detailed description of the invention
Fig. 1 is target gene PCR fragment.
Fig. 2 is target gene bacterium colony PCR electrophoretogram.
Fig. 3 is the SDS-PAGE detection of destination protein SgSTPS1.
Fig. 4 is SDS-PAGE analysis destination protein SgSTPS1 supernatant purification result.
Fig. 5 is SDS-PAGE and the Western-blot detection of destination protein SgSTPS1.
Fig. 6 is GC-MS test map.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.Do not have in following instance
The dated experimental method of body, can conventionally carry out, or according to the operation instruction of production manufacturer used；It is used
Material, reagent etc., unless otherwise specified, can be obtained by commercial sources.
Embodiment 1
1. cloning oily nanmu sesquiterpene synthase gene SgSTPS1, cloning vector and conversion prokaryotic cell are constructed
The RNA of extract oil nanmu stem tissue, using reverse transcriptase M-MLV, reverse transcription reaction synthesizes cDNA.It is with the cDNA
Template, forward primer are as follows: 5'-ATGGGGCGACCCACGGCAA-3', reverse primer are as follows: 5'-
CTATTTGTTGACATCAATGGC-3' carries out PCR amplification using TakaRa company Ex Taq archaeal dna polymerase；PCR condition
Are as follows: 94 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 1min, 72 DEG C of 1min30s, 35 circulations；72 DEG C of extension 10min.PCR product is with 1%
Agarose gel electrophoresis detection, as a result as shown in Figure 1, the M of Fig. 1 be DNA marker, arrow be purpose gene SgSTPS1 piece
Section.
Target gene fragment is recycled using the method for agarose gel electrophoresis plastic recovery kit, target fragment is subjected to TA
Clone, is connected in carrier Pmd-18TA cloning vector, is then transformed into Escherichia coli Top10 clone strain, converts
Condition are as follows: 5 μ L connection products are added in 200 μ L competent cells, mix gently rear ice bath 30min；It is put into 42 DEG C of water rapidly
Heat shock 90s in bath is immediately placed on 2min on ice；800 μ L LB culture mediums are added, 37 DEG C are shaken culture 1h slowly；By bacterium solution 6000rpm
It is centrifuged 2min, abandons 600 μ L supernatants, suspension thalline is coated on the LB plate containing antibiotic (Amp), 37 DEG C of inversion dark cultures
12~16h.Positive colony screening, screening technique are carried out using bacterium colony PCR are as follows: the random picking single colonie from conversion plate is set
It is cultivated in fluid nutrient medium in 1.5mL centrifuge tube.Every pipe is numbered, each pipe takes 1 μ L to be used as template and carries out PCR inspection
It surveys, remaining culture is stored in 4 DEG C, and the bacterium colony of test positive is saved backup in plate or glycerol tube.PCR reaction system are as follows: 1
μ L bacterium solution, forward primer 5'-CGCCAGGGTTTTCCCAGTCACGAC-3', reverse primer 5'-
AGCGGATAACAATTTCACACAGGA-3', 1x Buffer, 0.25mM dNTP mixture, 0.5 μ L ExTaq DNA polymerization
Enzyme carries out PCR extension after mixing.PCR program are as follows: 94 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 1min, 72 DEG C of 1min30s, 35 are followed
Ring；72 DEG C of extension 10min.For bacterium colony PCR result as shown in Fig. 2, the M of Fig. 2 is DNAmarker, 1,2 and 7 be positive bacteria.Select sun
Property monoclonal colonies, extract plasmid after deliver sequencing.Through sequencing analysis, the sesquiterpene synthase gene SgSTPS1 cloned,
Its nucleotide sequence contains 1650 bases as shown in SEQ ID NO.1, and the albumen of coding is named as sesquiterpene synthase
SgSTPS1, totally 549 amino acid, specific amino acid sequence is as shown in SEQID NO.2.And it thus obtains sesquiterpene synthase
Gene SgSTPS1 is inserted into the recombinant plasmid successful conversion of Pmd-18TA cloning vector to the positive bacteria of Escherichia coli Top10, life
Entitled Top10-SgSTPS1.
2, oily nanmu sesquiterpene synthase gene SgSTPS1 codon optimization and full genome synthesis, construction of expression vector and turn
Change prokaryotic cell
It is optimized using codon of the codon optimization software to expression SgSTPS1 albumen, the encoding gene after optimization
Base sequence as shown in SEQ ID NO.3.Sesquiterpene synthase after codon optimization is synthesized using full genome synthetic method
Gene SgSTPS1 full length sequence.By restriction enzyme site Nde I and Hind III to the SgSTPS1 gene and matter of optimization
Grain pET30a carries out double digestion, digestion system respectively are as follows: each 1 μ L of Nde I and Hind III, mrna concentration are 0.3 μ g, and plasmid is dense
Degree is 1 μ g, the distilled water of sterilizing is added to 30 μ L, the digestion time is 1h.It is purified after digestion using nucleic acid purification QIAquick Gel Extraction Kit
Recycling obtains the SgSTPS1 genetic fragment and pET30a carrier by the optimization of double digestion.By the SgSTPS1 of the optimization after digestion
Genetic fragment is connected in the expression vector pET30a after digestion, connect reaction condition are as follows: the SgSTPS1 gene of optimization and
PET30a plasmid (molar ratio 3:1), 1x connection Buffer, T4DNA ligase, after mixing, it is anti-that 15 DEG C of placement 16h complete connection
It answers, obtains recombinant plasmid (the SgSTPS1 gene of optimization is inserted into the recombinant plasmid after expression vector pET30a).Then by its turn
Change into Escherichia coli Top10 and e. coli bl21 (DE3) clone strain.Picking antibiotic (kan) screens the obtained positive
Bacterium colony extracts plasmid, and the accuracy of final expression vector is confirmed by enzyme cutting method and sequencing, thus obtains the sequiterpene that will optimize
Synthase gene SgSTPS1 is inserted into the recombinant plasmid successful conversion of pET30a expression vector to the positive bacteria of prokaryotic cell, life
Entitled Top10-SgSTPS1 (2) and BL21-SgSTPS1.The BL21-SgSTPS1 bacterium built is subjected to mass propgation, first
Picking monoclonal BL21-SgSTPS1 is inoculated into the LB culture medium of 4mL (kanamycin sulfate containing 50 μ g/mL), 37 DEG C,
200rpm, wait cultivate to OD600 be 0.5-0.8, into Tube propagation liquid be added final concentration 0.1mM IPTG, be respectively placed in later
15 DEG C and 37 DEG C of inducing expressions, obtain the bacterium solution of great expression sesquiterpene synthase SgSTPS1.
3, prokaryotic expression, protein purification and the albumen quality inspection of oily nanmu sesquiterpene synthase SgSTPS1
The bacterium solution of great expression sesquiterpene synthase SgSTPS1 after inducing is taken, 12000rpm is centrifuged 5min, removes supernatant
Liquid, be added PBS liquid be resuspended precipitating, be eventually adding SDS-PAGE sample-loading buffer and heat sample 10min at 100 DEG C, then from
The heart takes supernatant electrophoresis.Before electrophoresis when 10min, 100V pressure stabilizing electrophoresis, the 200V pressure stabilizing electricity after bromophenol blue indicator enters separation gel
Swimming to bromophenol blue band is migrated to from gel bottom 1cm, is taken out gel and is dyed with Coomassie brilliant blue dyeing liquor, then continues at destainer
In, decoloration to clear background.As a result see that Fig. 3, the M of Fig. 3 are albumen marker, Lane 0 is control, and Lane 1 is 15 DEG C of inductions
16h, Lane 2 is 37 DEG C of induction 16h, and arrow is purpose Protein S gSTPS1.
According to the above method, the expression bacterium of amplification culture 3L, when growing to OD600=0.8, adds final concentration 0.1mM IPTG,
Thallus is collected after 15 DEG C of induction 16h.Full bacterium uses 50mM Tris (pH8.0), and 300mM NaCl, 50mM Imidazole contains 1%
The cracking of Triton X-100,1mM DTT, 1mM PMSF ultrasound, while with 50mM Tris (pH8.0), 300mM NaCl, 50mM
Imidazole buffer balances Ni-IDA affinity column, elutes target egg with the equilibration buffer of various concentration imidazoles later
It is white, and collect each elution fraction and carry out SDS-PAGE analysis detection.Analysis result is shown in that Fig. 4, the M of Fig. 4 are albumen marker,
Lane 1 is that full bacterium breaks supernatant after bacterium centrifugation, and Lane 2 is efflux after supernatant is incubated for Ni-IDA, and Lane 3-4 is 100mM
The elution fraction of Imidazole, Lane 5-11 are the elution fraction of 300mM Imidazole, and arrow is purpose albumen.
Through Ni-IDA affinitive layer purification, the preferable Lane 9-10 of purity levels is collected, and is carried out dialysis 1 × PBS,
10%Glycerol, pH7.4 are filtered after dialysing with 0.22 μm of film, and dispense jelly in -80 DEG C.Standard items are made with BSA, are used
Bradford method measures protein concentration.The albumen Western-blot test experience of oily nanmu sesquiterpene synthase SgSTPS1 operates
Process writes with reference to " protein electrophorese experimental technique " Guo Yaojun, as a result sees Fig. 5, Lane M1For SDS-PAGE albumen Marker,
Lane 1 is BSA albumen, and Lane 2 is SgSTPS1 albumen, Lane M2For Western-blot Marker, antibody Anti-
His。
4, β-carypohyllene biosynthesis
With farnesyl pyrophosphate (FPP) for substrate, enzymatic reaction system are as follows: bis- sulphur of 25mM Tris-HCI (pH7.4), 5mM
Threitol (DTT), 100mM potassium chloride, 5mM magnesium chloride, 10% glycerol, substrate farnesyl pyrophosphate (FPP) concentration are 50 μM, are added
Enter the oily nanmu sesquiterpene synthase SgSTPS1 albumen of 50 μ g after purification, is placed in 37 DEG C of reaction 1h.After reaction, micro- with solid phase
Extract 100 μm of headspace extraction volatile substance 30min of SPME fiber PDMS, 250 DEG C of desorption 3min sample introductions.Using GC-MS combined instrument
(Agilent GC-MS 7890B-5977A) detects catalysate.Gas chromatographic column be HP-5MS (30m ×
0.25mm), the high-purity He flow velocity of carrier gas is 1.0mL/min, temperature program are as follows: 50 DEG C of holding 1min are warming up to 5 DEG C/min speed
80 DEG C, 1min is kept, then be warming up to 220 DEG C with 10 DEG C/min speed, keep 10min, injector temperature is 250 DEG C, ion source
EI 70eV temperature is 230 DEG C, and interface temperature is 250 DEG C, and acquisition quality range is 30-200amu, and data are through 14 mass spectrum of NIST
Library searching, and compareed with standard spectrogram, identify each group swarming.Sample total ion current figure is shown in Fig. 6, retention time be 15.77,
Occur peak 1,2 and 3 at 15.97 and 16.42 respectively, compares through 14 data retrieval library searching of NIST, respectively correspond as the result is shown
Compound be isocaryophyllene (Isocaryophillene), β-carypohyllene (β-Caryophyllene) and α-carypohyllene
(Humulene), Similarity matching degree is respectively 98,99 and 98, and peak 1 as shown in the figure is isocaryophyllene
(Isocaryophillene), peak 2 is β-carypohyllene (β-Caryophyllene), and peak 3 is α-carypohyllene (Humulene).By
This shows that oily nanmu sesquiterpene synthase SgSTPS1 can be catalyzed FPP synthesis β-carypohyllene and a small amount of isocaryophyllene and α-China pink
Alkene.
Sequence table
<110>Tropical Foresty Inst., Chinese Academy of Foresty Sciences
<120>a kind of oily nanmu sesquiterpene synthase and its encoding gene and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1650
<212> DNA
<213>oily nanmu (Sindora glabra Merr.ex de Wit)
<400> 1
atggggcgac ccacggcaaa cttcagccca agtgtttggg gaaatcagtt cctttccatt 60
gcatcaggtc ccttgctgaa gaacaaagaa gcggaaattc atcaacattt actgaatctg 120
aaagaacaag tgaggaaaca actaaaaaat ggtgttgaag agccttctga gaagctaaac 180
ttgatcgata ccattcaacg gcttggagtg tctcaccatt ttgaaagaga gattgaagaa 240
tcattgaaac atctacataa gaatcctcct tcttcctgga atgccgaaga tatcgatgca 300
catcttttaa gcatttctct ctggtttcgg ttgcttagac aacaaggcta ttatgtctca 360
tgtgatgtat ttgataaatt caaagatgac aaaggcatgt tcaagacaac attgattgat 420
gatgtggaag gaatgttggc cttgtatgaa gctacatatc tcggcattcg tggggaagaa 480
attcttgatc aagtgctaga atttacaatg tttcatctta aatcaagatt ggaaggcatg 540
acaccctacc ttcgagaaag agcagatcgt gctttgtact gtcctataaa caaaggttta 600
ccacgaattg agacgcgata tttcatttcg atgtactcaa aaaaggattc tcgaaatgat 660
ctgttgctag aatttgccat gctagacttc aatatcctgc agcaacaata tcagaaggaa 720
ttaagccacc tatcagagtg gtacaaaaaa ttagattttg taagcaaggt tccttacacc 780
agagacagaa ttgttgaagg ctacttttgg cctttgggag catactttga gaatcaatat 840
aacaaaggaa gaataattgt aagcaaactg atattggttc ttacagcttt ggatgatacc 900
tatgatgcct acggtactgt agatgagctc aaactcttca ctgaagcaat taagagatgg 960
gatattaata tggtggcttc tctccctgaa tgcatgaaag tggtattcca agctatttta 1020
gatttgttgg atgaaatgga gttgctaacg gaagcggatg gaatatcatg ctttgttgaa 1080
tatgttaaac cagcgcttca agatttagca aaatcatact tgcttgaagc tgagtggagg 1140
gacaaaggct atataccaac atatgaggag tatatagcaa atggagtttt ttcttgtggt 1200
tacccagctg ttgaaatggc gtccttactt ggtcttggaa aaattgcaac caaagaagtg 1260
tttgattgga tttctaatgt acctaaaatt gttagagctt catcaattat gtgtagattg 1320
acagatgatt tggcttctca taagtttgaa caaaacagag aacatgttgg atctgcaatt 1380
gagtgttaca tgaagcaata tggggtttca gaagaagaag cctataaaat gctgctaaag 1440
gagatagaga atggatggaa ggatcttaac gaagagtaca tgaagccaaa tggtgttcca 1500
aaggtggtac ttaagtgtgt attgaatttt tcacgagtga ttgagtttct gtacggtcac 1560
tttgttgata aatacacaaa tgcagagatg ctgaaggatc acattgcttc actttttgtg 1620
gatcccattg ccattgatgt caacaaatag 1650
<210> 2
<211> 549
<212> PRT
<213>oily nanmu (Sindora glabra Merr.ex de Wit)
<400> 2
Met Gly Arg Pro Thr Ala Asn Phe Ser Pro Ser Val Trp Gly Asn Gln
1 5 10 15
Phe Leu Ser Ile Ala Ser Gly Pro Leu Leu Lys Asn Lys Glu Ala Glu
20 25 30
Ile His Gln His Leu Leu Asn Leu Lys Glu Gln Val Arg Lys Gln Leu
35 40 45
Lys Asn Gly Val Glu Glu Pro Ser Glu Lys Leu Asn Leu Ile Asp Thr
50 55 60
Ile Gln Arg Leu Gly Val Ser His His Phe Glu Arg Glu Ile Glu Glu
65 70 75 80
Ser Leu Lys His Leu His Lys Asn Pro Pro Ser Ser Trp Asn Ala Glu
85 90 95
Asp Ile Asp Ala His Leu Leu Ser Ile Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln Gln Gly Tyr Tyr Val Ser Cys Asp Val Phe Asp Lys Phe Lys
115 120 125
Asp Asp Lys Gly Met Phe Lys Thr Thr Leu Ile Asp Asp Val Glu Gly
130 135 140
Met Leu Ala Leu Tyr Glu Ala Thr Tyr Leu Gly Ile Arg Gly Glu Glu
145 150 155 160
Ile Leu Asp Gln Val Leu Glu Phe Thr Met Phe His Leu Lys Ser Arg
165 170 175
Leu Glu Gly Met Thr Pro Tyr Leu Arg Glu Arg Ala Asp Arg Ala Leu
180 185 190
Tyr Cys Pro Ile Asn Lys Gly Leu Pro Arg Ile Glu Thr Arg Tyr Phe
195 200 205
Ile Ser Met Tyr Ser Lys Lys Asp Ser Arg Asn Asp Leu Leu Leu Glu
210 215 220
Phe Ala Met Leu Asp Phe Asn Ile Leu Gln Gln Gln Tyr Gln Lys Glu
225 230 235 240
Leu Ser His Leu Ser Glu Trp Tyr Lys Lys Leu Asp Phe Val Ser Lys
245 250 255
Val Pro Tyr Thr Arg Asp Arg Ile Val Glu Gly Tyr Phe Trp Pro Leu
260 265 270
Gly Ala Tyr Phe Glu Asn Gln Tyr Asn Lys Gly Arg Ile Ile Val Ser
275 280 285
Lys Leu Ile Leu Val Leu Thr Ala Leu Asp Asp Thr Tyr Asp Ala Tyr
290 295 300
Gly Thr Val Asp Glu Leu Lys Leu Phe Thr Glu Ala Ile Lys Arg Trp
305 310 315 320
Asp Ile Asn Met Val Ala Ser Leu Pro Glu Cys Met Lys Val Val Phe
325 330 335
Gln Ala Ile Leu Asp Leu Leu Asp Glu Met Glu Leu Leu Thr Glu Ala
340 345 350
Asp Gly Ile Ser Cys Phe Val Glu Tyr Val Lys Pro Ala Leu Gln Asp
355 360 365
Leu Ala Lys Ser Tyr Leu Leu Glu Ala Glu Trp Arg Asp Lys Gly Tyr
370 375 380
Ile Pro Thr Tyr Glu Glu Tyr Ile Ala Asn Gly Val Phe Ser Cys Gly
385 390 395 400
Tyr Pro Ala Val Glu Met Ala Ser Leu Leu Gly Leu Gly Lys Ile Ala
405 410 415
Thr Lys Glu Val Phe Asp Trp Ile Ser Asn Val Pro Lys Ile Val Arg
420 425 430
Ala Ser Ser Ile Met Cys Arg Leu Thr Asp Asp Leu Ala Ser His Lys
435 440 445
Phe Glu Gln Asn Arg Glu His Val Gly Ser Ala Ile Glu Cys Tyr Met
450 455 460
Lys Gln Tyr Gly Val Ser Glu Glu Glu Ala Tyr Lys Met Leu Leu Lys
465 470 475 480
Glu Ile Glu Asn Gly Trp Lys Asp Leu Asn Glu Glu Tyr Met Lys Pro
485 490 495
Asn Gly Val Pro Lys Val Val Leu Lys Cys Val Leu Asn Phe Ser Arg
500 505 510
Val Ile Glu Phe Leu Tyr Gly His Phe Val Asp Lys Tyr Thr Asn Ala
515 520 525
Glu Met Leu Lys Asp His Ile Ala Ser Leu Phe Val Asp Pro Ile Ala
530 535 540
Ile Asp Val Asn Lys
545
<210> 3
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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atgggtcgtc cgaccgcaaa ttttagtccg agcgtttggg gcaaccagtt tctgtccatt 60
gcaagcggtc cgctgctgaa aaacaaagaa gcggaaattc accagcatct gctgaatctg 120
aaagagcagg tccgcaaaca gctgaaaaac ggcgttgaag aaccgagcga aaaactgaac 180
ctgatcgata ccattcaacg tctgggcgtt agccaccatt tcgaacgcga aatcgaagag 240
agcctgaaac acctgcacaa aaatccgccg agtagctgga acgcagaaga tattgacgcg 300
catctgctgt ccatcagtct gtggtttcgt ctgctgcgtc aacagggtta ttacgttagc 360
tgcgacgtct tcgacaaatt caaagacgac aaaggcatgt tcaaaaccac cctgatcgac 420
gacgtcgaag gtatgttagc gctgtacgaa gcaacctatc tgggtattcg cggcgaagaa 480
atcctggatc aggttctgga gttcaccatg ttccacctga aaagccgtct ggaaggtatg 540
accccgtatt tacgcgaacg cgcagatcgc gcactgtatt gtccgatcaa caaaggcctg 600
ccgcgtattg aaacccgcta tttcatcagc atgtacagca aaaaagacag ccgcaacgac 660
ctgctgttag aattcgcgat gctggacttc aacatcctgc agcagcagta ccagaaagaa 720
ctgagccacc tgagcgagtg gtacaaaaaa ctggacttcg tcagcaaagt tccgtatacc 780
cgcgatcgca tcgttgaagg ttatttctgg ccgctgggcg catattttga aaaccagtac 840
aacaaaggcc gcatcatcgt cagcaaactg attctggttc tgaccgcgtt agacgatacc 900
tatgacgcat acggtaccgt tgacgaactg aaactgttca ccgaagcgat caaacgctgg 960
gacattaaca tggttgcctc tctgccggaa tgcatgaaag tcgttttcca ggcgatcctg 1020
gacttactgg acgaaatgga actgctgacc gaagctgacg gtattagctg cttcgtcgaa 1080
tatgttaaac cggcactgca ggatctggct aaaagctacc tgttagaagc ggagtggcgc 1140
gataaaggct atatcccgac ctacgaagaa tatatcgcga acggcgtttt tagctgcggt 1200
tatccggccg ttgaaatggc aagcttactg ggtctgggca aaattgcgac caaagaagtc 1260
ttcgactgga tcagcaacgt cccgaaaatc gttcgcgctt cctctattat gtgtcgtctg 1320
accgacgatc tggcgagcca taaattcgaa cagaatcgcg aacacgttgg ttccgctatt 1380
gagtgctaca tgaaacagta cggcgtcagc gaagaagaag cgtacaaaat gctgctgaaa 1440
gagatcgaga acggctggaa agacctgaac gaagaatata tgaaaccgaa cggggttccg 1500
aaagtcgttc tgaaatgcgt cctgaacttc agccgcgtta ttgaattcct gtacggccat 1560
ttcgtcgaca aatacaccaa cgcggagatg ctgaaagatc acatcgcgag cctgttcgtt 1620
gatccgattg ccatcgacgt caacaaataa 1650
Claims (10)
1. a kind of oil nanmu sesquiterpene synthase SgSTPS1, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.2.
2. the encoding gene of oil nanmu sesquiterpene synthase SgSTPS1 described in claim 1.
3. encoding gene according to claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.3.
4. encoding gene according to claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.1.
5. a kind of recombinant expression carrier of the encoding gene containing oily nanmu sesquiterpene synthase SgSTPS1 as claimed in claim 2.
6. expression vector according to claim 5, which is characterized in that be expression vector pET-30a.
7. a kind of genetic engineering bacterium of the encoding gene containing oily nanmu sesquiterpene synthase SgSTPS1 as claimed in claim 2.
8. genetic engineering bacterium according to claim 7, which is characterized in that be Escherichia coli Top10 or e. coli bl21
(DE3)。
9. oil nanmu sesquiterpene synthase SgSTPS1 described in claim 1 is in preparation β-carypohyllene, isocaryophyllene and/or α-stone
Application in bamboo alkene.
10. application according to claim 9, which is characterized in that be using farnesyl pyrophosphate as substrate, through oily nanmu sequiterpene
Synzyme SgSTPS1 catalysis generates β-carypohyllene, isocaryophyllene and α-carypohyllene.
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| CN109852600A (en) * | 2018-12-25 | 2019-06-07 | 天津大学 | A kind of calyculus tongue fur sesquiterpene synthase MTb and its gene order |
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