CN109706138A - A kind of Celastrus angulatus sesquiterpene synthase CaTPS2 and its gene order - Google Patents
A kind of Celastrus angulatus sesquiterpene synthase CaTPS2 and its gene order Download PDFInfo
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- CN109706138A CN109706138A CN201910091028.3A CN201910091028A CN109706138A CN 109706138 A CN109706138 A CN 109706138A CN 201910091028 A CN201910091028 A CN 201910091028A CN 109706138 A CN109706138 A CN 109706138A
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Abstract
The invention discloses a kind of Celastrus angulatus sesquiterpene synthase CaTPS2 and its gene orders, full length gene 1665bp, encode 554 amino acid, zymoprotein molecular size range is 64.13KDa, using pRS426-URA Yeast protein expression plasmid as carrier, it is host with Saccharomyces Cerevisiae in S accharomyces cerevisiae WQ011, realize the heterogenous expression of Celastrus angulatus sesquiterpene synthase CaTPS2, recombinant bacterium WQ011/pRS426-URA-CaTPS2 can generate α-eucalyptol (α-Eudesmol) and Selina-3,7 two kinds of (11)-diene sesquiterpenoids simultaneously.50mL shaking flask yield is γ-eucalyptol 0.15mg/L and sagittol 0.06mg/L.The acquisition of the gene has far-reaching significance the research of Celastrus angulatus sesquiterpenoid.
Description
Technical field
The invention belongs to enzyme gene engineering and enzyme engineering fields, and in particular to one kind derives from Celastrus angulatus (Celastrus
Angulatus sesquiterpene synthase CaTPS2 and gene order CaTPS-24009).
Background technique
Terpenoid is chemical structure and conformation natural products the most complicated, and presently found terpenoid has
80000 kinds or so, the one third of natural products is occupied, is distributed widely in plant and microorganism.The biology of terpenoid
The most basic structural unit that synthesis needs is dimethylallylpyrophosphate Dimethylallyl diphosphate, DMAPP
With isopentenyl pyrophosphate Isopentenyl diphosphate, IPP from mevalonate pathway and deoxy-D-xylulose sugar phosphoric acid
Ester approach.DMAPP and IPP the head and the tail polymerization of different number form monoterpene (C using the catalysis of special diterpene synthase10) sequiterpene
(C15) and diterpene (C20) etc. natural products.Sesquiterpenoids is one kind of structure and most species in terpenoid,
It is widely used in bio-pharmaceuticals, natural green agricultural insecticide and fungicide, fuel substitute and food grade spice and seasoning
The exploitation of the industries such as agent uses.
The sesquiterpenoids such as α-eucalyptol (α-Eudesmol) and Selina-3,7 (11)-diene have significant
Bioactivity.If α-eucalyptol can block calcium channel, inhibit the nerve of overacfivity, after brain being helped to be wound
Adjuvant treatment;Selina-3,7 (11)-diene has effects that clearing heat and detoxicating, smooth throat to stop cough.
Celastrus angulatus (Celastrus angulatus) belongs to Celastraceae (Celastraceae) Celastrus (Celastrus)
Perennial liana is distributed widely in the hills and mountain area of China the Yellow River, the Yangtze river basin.Insecticidal activity (the food refusal of Celastrus angulatus
Activity, narcotic activity, cytotoxicity) ingredient is mainly distributed in root skin.Research has shown that active insecticidal components master contained therein
If with β-dihydroagarofuran polyol ester sesquiterpenoids and its alkaloid, the difference of substituent group decides compound
Activity it is different.At present research be concentrated mainly on to the effective component Celastrus angulatus in Celastrus angulatus carry out separation with Structural Identification and
Action mechanism research, not to the pertinent literature of sesquiterpene synthase in Celastrus angulatus biosynthesis.Therefore bitter skin is studied
The function and its sequence signature of rattan sesquiterpene synthase are of great significance to the distribution of terpenoid.
Summary of the invention
It is an object of the invention to make up the deficiencies in the prior art, provide a kind of Celastrus angulatus sesquiterpene synthase CaTPS2 and
Its gene order additionally provides the plasmid comprising gene of the present invention and the host cell comprising the expression plasmid, and utilizes host
Cell (recombinant bacterium) inducing expression sesquiterpene synthase CaTPS2 generates sesquiterpenoids.
The technical scheme is that
A kind of Celastrus angulatus sesquiterpene synthase is denoted as CaTPS2, and amino acid sequence is as shown in SEQ ID NO.1.
The gene for encoding above-mentioned Celastrus angulatus sesquiterpene synthase CaTPS2, is denoted as CaTPS-24009, nucleotide sequence is such as
Shown in SEQ ID NO.2.
A kind of recombinant plasmid, is denoted as pRS426-URA-CaTPS2, contains coding Celastrus angulatus sesquiterpene synthase CaTPS2's
Gene, galactose-inducible promoter PGal1,10, uracil auxotrophy screens label.
A kind of recombinant bacterium, is denoted as WQ011/pRS426-URA-CaTPS2, contains above-mentioned recombinant plasmid.
The beneficial effects of the present invention are: the invention discloses a kind of Celastrus angulatus sesquiterpene synthase CaTPS2 and its gene sequences
Column, full length gene 1665bp encode 554 amino acid, and zymoprotein molecular size range is 64.13KDa, with pRS426-URA ferment
Female protein expressing plasmid is carrier, is host with Saccharomyces Cerevisiae in S accharomyces cerevisiae WQ011, realizes hardship
The heterogenous expression of skin rattan sesquiterpene synthase CaTPS2, recombinant bacterium WQ011/pRS426-URA-CaTPS2 can generate α-simultaneously
Two kinds of sesquiterpenoids of eucalyptol (α-Eudesmol) and Selina-3,7 (11)-diene.50mL shaking flask yield is γ-eucalyptol
0.15mg/L and sagittol 0.06mg/L, production and utilization to sequiterpene are of great significance, while the acquisition of the gene
Forming Mechanism and biotic mechanism study to Celastrus angulatus sesquiterpenoid lay the foundation.
Detailed description of the invention
Fig. 1 is WQ011/pRS426-URA-CaTPS2 restructuring yeast strains validating DNA Ago-Gel map (purpose item
Band size is 1665bp);
M: stranded DNA molecule ruler;Swimming lane 1 is CaTPS-24009 gene DNA sample;
Fig. 2 is external standard method standard curve;
Fig. 3 is WQ011/pRS426-URA-CaTPS2 restructuring yeast strains product GC-MS map;
A is the GC map of tunning;B is that the mass spectrogram at the peak retention time 11.82min and NIST14 compose α-folium eucalypti in library
Alcohol mass spectrogram compares;C is that the mass spectrogram at the peak retention time 12.43min and NIST14 compose Selina-3,7 (11)-diene in library
Mass spectrogram compares.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.
Sesquiterpene synthase gene source of the present invention is collected in Shaanxi on May 11st, 2017 in Celastrus angulatus plant, plant
Save Yangling Area northwest agricultural design of university museum park.
It selects the Celastrus angulatus plant of fresh acquisition to carry out RNA extraction, and its cDNA sequence, structure is obtained by reverse transcription PCR
Build cDNA library.Be sequenced using PacBio ISO-Seq platform, by Interpro (http: //
Www.ebi.ac.uk/interpro BLAST is carried out in) compares acquisition with PF_03936Terpene synthase family
The target gene of structural domain.Using cDNA sequence as template, design primer expands CaTPS-24009 gene order, and passes through digestion
It is connected to acquisition pRS426-URA-CaTPS2 yeast host expression plasmid on WQ011/pRS426-URA-CaTPS2 plasmid.Using
Lithium acetate yeast chemical transformation passes through recombinant plasmid pRS426-URA-CaTPS2 conversion such as Saccharomyces cerevisiae host WQ011
URA deficiency screening flat board, screening obtain WQ011/pRS426-URA-CaTPS2 restructuring yeast strains, realize times of Celastrus angulatus
The high efficient expression of hemiterpene synzyme CaTPS2.
WQ011/pRS426-URA-CaTPS2 bacterial strain Celastrus angulatus sesquiterpene synthase gene C aTPS- described in embodiment 1.
24009 clone and expression
Celastrus angulatus total serum IgE is extracted using RNAprep pure Plant Kit.Using Clontech SMARTer PCR
CDNA Synthesis Kit the first chain of synthesis cDNA, the first chain cDNA synthesize the second chain cDNA by PCR amplification, then double-strand
DNA builds library after secondary PCR expands, for SMRTbell, and is sequenced using PacBio ISO-Seq platform.
According to the CaTPS-24009 gene order SEQ ID NO.2 that sequencing obtains, design primer amplifying target genes draw
Object sequence is as follows:
Primer CaTPS-24009-F:5'CGCGGATCCATGGCAGCAACCAACCAATC 3'(SEQ ID NO.3)
Primer CaTPS-24009-R:5'CCGCTCGAGTTAATTATCTTGGATTGGTATTTGT3'(SEQ ID NO.4)
The cDNA sequence obtained using reverse transcription is template, using above-mentioned primer, PCR amplification CaTPS-24009 gene order.
Reaction system is 50ul, including ddH2O 18ul, dNTP mixture 1ul, CaTPS-24009-F primer 1ul, CaTPS-
24009-R primer 1ul, DNA profiling 1ul, 2 × Phanta Max 25ul, Fidelity DNA polymerase 1ul.Amplification
Process are as follows: 95 DEG C of initial denaturation 3min;95 DEG C of denaturation 15s, 60 DEG C of annealing 30s, 72 DEG C of extension 1.5min, 35 recycle;72 DEG C of ends
Extend 5min.
PCR product through 1.5% Ago-Gel test strip size, with Universal DNA Purification
Kit recycles target fragment.
The DNA fragmentation and pRS426-URA plasmid purified with BamHI and XhoI digestion, the product after digestion pass through
Universal DNA Purification Kit recycles to obtain endonuclease bamhi, is connected by T4DNA ligase ligase
It connects, obtains recombinant plasmid.Digestion system are as follows: 1.5 μ L of BamHI1.5 μ L, XhoI, 42 μ L, cut smart of DNA fragmentation or plasmid
buffer 5μL.37 DEG C of water-bath 2h.Linked system are as follows: 7 μ L of purpose digestion genetic fragment and 1 μ L, T4 ligase of digested plasmid, 1 μ
L, 10 × T4ligase buffer1 μ L.16 DEG C of connections overnight.
Using lithium acetate transformation method, yeast conversion is carried out, operating procedure is as follows:
(1) Wine brewing yeast strain WQ011 scribing line YPD plate, 30 DEG C of cultures, until growing single colonie about 1-3mm.It is flat from YPD
One ring saccharomycete of plate picking is inoculated in containing in 5mL YPD fluid nutrient medium, 180r/min, and 30 DEG C, 14~16h of shaking table culture.
(2) according to 1% inoculum concentration, bacterium solution is accessed in 50mL YEPD fluid nutrient medium, 180r/min, 30 DEG C of cultures are extremely
OD600=0.5 (about 3-5h).
(3) 5000r/min, 4 DEG C of refrigerated centrifuge 6min collect thallus.
(4) 25mL aseptic deionized water suspension thalline, 5000r/min, 4 DEG C of refrigerated centrifuge 6min is added.
(5) it is primary to repeat previous action.
(6) thallus is resuspended with the LiAc buffer that 1mL concentration is 0.1mol/L, stands 5min.5000r/min, 4 DEG C of freezings
After being centrifuged 5min, discard supernatant.
(7) thallus is resuspended with the LiAc buffer of 500 μ L 0.1mol/L, every 100 μ L is dispensed into a centrifuge tube, is made
At competent yeast cells.
(8) ssDNA is cooling rapidly on ice after boiling 12min.
(9) in the competent yeast cells dispensed, the carrier or linear DNA of 100-500ng is added.Rotation adds later
Enter in following mixture.The ingredient of transformation mixture is as follows: 1) PEG-4000 50% (w/v), 620 μ l;2)LiAc1.0mol/
L, 90 μ l;3) SS-DNA (10mg/ml), 40 μ l.
30min is incubated in (10) 30 DEG C of incubators.
(12) 42 DEG C of water-bath heat shock 20min.
(15) 6000r/min is centrifuged 1min, discards part supernatant, and thallus is resuspended, takes 100 μ L coated plates, 30 DEG C of culture 2-3
It, grows transformant.
(16) a little single colonie of picking, the 20mM NaOH solution that 50 μ l degermings are added mix.
(17) it is placed in PCR instrument, according to program: 99 DEG C, 5min;4 DEG C, 1min is recycled 3 times.
(18) bacterium solution is taken out, 8000r/min is centrifuged 5 minutes, takes supernatant as pcr template.
(19) reaction system is 10ul, including CaTPS-24009-F primer 1ul, CaTPS-24009-R primer 1ul, DNA
Template 3ul, 2 × Taq Master Mix 5ul.Amplification procedure are as follows: 95 DEG C of initial denaturation 3min;95 DEG C of denaturation 15s, 60 DEG C of annealing
30s, 72 DEG C of extension 1.5min, 35 circulations;72 DEG C extend 5min eventually.
(20) whether PCR product is correct through 1.5% Ago-Gel test strip size.
The result shows that (as shown in Figure 1): the transformant selected has single band between 1200bp and 2000bp, it was demonstrated that turns
Beggar is positive clone molecule.
2. induction fermentation of embodiment
(1) transformant WQ011/pRS426-URA-CaTPS2 is forwarded to 50mL synthetic media (without URA)
In, 180r/min, 30 DEG C, shaking table culture 30h.
(2) final concentration 10g/L galactolipin inducing expression is added in 30h to 90-120h, is added in 36h and 72h dense eventually
10g/L ethyl alcohol is spent as supplementary carbon source.
(3) fermentation liquid carries out GC-MS detection using 3mL n-hexane extraction tunning 15min.
Embodiment 3.GC-MS detects tunning
(1) GC-MS detection method
Chromatographic column: TG-5MS;Ion source;EI, 70eV;Sample volume: 1uL;Injector temperature: 200 DEG C;Detector temperature: 280
℃;Column temperature: 240 DEG C;Temperature programming: 70 DEG C of initial temperature, 10 DEG C/min is warming up to 280 DEG C, maintains 5min.
(2) yield is calculated using external standard method.It is accurate respectively to prepare 0.02,0.04,0.06,0.08 and 1.0mg/L standard items,
Using peak area as ordinate, concentration is that abscissa draws standard curve (as shown in Figure 2).
The result shows that: the target peak in GC-MS spectrogram, by being sent out compared with the reference mass spectrometric data in NIST14 database
Existing, chromatographic peak of the WQ011/pRS426-URA-CaTPS2 bacterial strain fermentation liquor N-hexane extract in retention time 11.82min is
α-eucalyptol (α-Eudesmol), 50mL shaking flask yield are 0.15mg/L (as shown in Fig. 3 A, 3B).In retention time 12.43min
Chromatographic peak be Selina-3,7 (11)-diene, 50mL shaking flask yield be 0.06mg/L (as shown in Fig. 3 A, 3C).
It is all using made by description of the invention and accompanying drawing content the foregoing is merely the preferred embodiment of the present invention
Several improvement or deformation, being applied directly or indirectly in other relevant technical fields, also should be regarded as protects in patent of the invention
It protects in range.
<110>University Of Tianjin
<120>a kind of Celastrus angulatus sesquiterpene synthase CaTPS2 and its gene order
<130>
<160> 4
<170>
<210> 1
<211> 554
<212> PRT
<213>Celastrus angulatus
<400> 1
Met Ala Ala Thr Asn Gln Ser Ala Glu Val Ser Arg Arg Leu Ala Asn
5 10 15
Phe Ala Pro Ala Ile Trp Gly His Asp Asp Phe Ala Ser Phe Thr Ser
20 25 30
Asp Gln Asp Ser Val Tyr Gly Leu Tyr Thr Lys Arg Val Glu Glu Leu
35 40 45
Lys Val Gln Val Arg Asp Leu Leu Leu Ser Thr Asn Asp Ile Val Glu
50 55 60
Lys Val Glu Leu Ile Asp Leu Leu Gly Arg Leu Gly Ile Ser Tyr His
65 70 75 80
Phe Glu Ser Glu Ile Glu Asn Gln Leu Met Gln Asn Leu Asn Ile Ile
85 90 95
Lys Thr Lys Leu Ile Asp Asp Asn Asp Tyr Ser Leu Tyr Ala Val Ala
100 105 110
Leu Leu Phe Arg Val Phe Arg Gln Tyr Gly Tyr Lys Ile Ser Cys Asp
115 120 125
Val Phe Glu Lys Phe Glu Gly Asp Asp Gly Lys Phe Lys Met Ser Leu
130 135 140
Ala Ser Asp Val Glu Gly Met Leu Ser Leu Tyr Glu Ala Ser His Leu
145 150 155 160
Ser Met His Gly Glu Asp Val Leu Asp Glu Ala Leu Val Phe Ser Lys
165 170 175
Thr Ser Leu His Ser Met Val Thr Gln Leu Asn Pro His Phe Ala Asn
180 185 190
Gln Val Thr His Ala Leu Gln Gln Pro Tyr His Lys Gly Ile Pro Arg
195 200 205
Ile Glu Ser Arg Lys Tyr Ile Asn Phe Tyr Glu Glu Asp Glu Ser Arg
210 215 220
Asn Glu Ile Met Leu Lys Leu Ala Lys Ile Asp Phe Asn Arg Val Gln
225 230 235 240
Leu Leu His Gln Gln Glu Leu Ser His Val Ser Arg Trp Tyr Lys Asp
245 250 255
Leu Gln Ile Ala Ser Lys Phe Pro Tyr Ala Arg Asp Arg Ile Ala Glu
260 265 270
Ile Tyr Met Trp Thr Val Gly Ser Asn Phe Glu Pro His Tyr Gly Arg
275 280 285
Thr Arg Ile Phe Leu Thr Lys Ser Val Thr Met Ile Ser Ile Leu Asp
290 295 300
Asp Thr Tyr Asp Ala Tyr Gly Thr Ile Glu Glu Leu Arg Leu Leu Thr
305 310 315 320
Asp Ala Val Glu Arg Trp Asp Val Gly Ala Ile Asp Glu Leu Pro Asp
325 330 335
Tyr Met Lys Val Leu Tyr Lys Met Ile Ile Asn Leu Tyr Asp Glu Phe
340 345 350
Glu Asn Glu Leu Lys Asn Glu Gly Arg Ser Ser Cys Val Ala Tyr Ala
355 360 365
Arg Asp Ala Leu Arg Glu Met Val Lys Ala Tyr His Val Glu Ala Glu
370 375 380
Trp Cys Asn Lys Gly Tyr Ile Pro Thr Phe Asp Glu Tyr Met Glu Asn
385 390 395 400
Ala Leu Ile Thr Ser Cys Tyr His Ala Ile Pro Ala Ala Cys Phe Leu
405 410 415
Gly Met Gly Glu Ile Ala Gly Thr Lys Glu Phe Glu Trp Leu Lys Ser
420 425 430
Ile Pro Lys Ile Ile Arg Ala Ser Glu Met Ile Gly Arg Leu Met Asp
435 440 445
Asp Ile Met Ser His Lys Glu Glu Gln Glu Arg Gly His Val Ala Ser
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Ser Ile Glu Cys Phe Met Lys Gln Tyr Gly Val Ser Ser Glu Glu Glu
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Val Val Gln Asp Phe Gln Ile Arg Ile Ala Asn Ala Trp Lys Asp Ile
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Asn Glu Glu Cys Val Arg Pro Thr Ala Val Ser Leu His Leu Leu Met
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Pro Ile Leu Asn Leu Thr Arg Ile Ile Asp Val Val Tyr Lys Asn Glu
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Asp Gly Tyr Ser Asn Pro Val Asn Leu Lys Glu His Val Lys Ala Leu
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Phe Ile Gln Gln Ile Pro Ile Gln Asp Asn
545 550 554
<210> 2
<211> 1665
<212> DNA
<213>Celastrus angulatus
<400> 2
atggcagcaa ccaaccaatc cgcggaggtt tcacggcggc tggccaactt cgcccccgcc 60
atttggggtc acgatgactt tgcttctttt acttctgatc aagactcggt gtatggattg 120
tatacaaaga gagtggagga gctgaaagta caagtgagag atttgttgct gtctacaaat 180
gacattgtgg agaaagtcga gttgatcgac ttgttgggtc gtcttggtat ttcatatcac 240
tttgaaagtg agattgaaaa ccagcttatg cagaatctca acataatcaa aactaaactc 300
atcgatgata atgactacag tttatacgct gtagcacttc tattccgcgt tttcagacaa 360
tatggttata aaatatcttg tgacgtgttt gagaagtttg agggagatga tggaaagttt 420
aaaatgagtc tagctagtga tgtggagggg atgctaagct tgtatgaagc ttctcacttg 480
agcatgcatg gagaggatgt tttggatgaa gcactagtgt tttcaaaaac ttctcttcat 540
tccatggtga cccaattgaa cccacacttt gcaaaccaag ttactcatgc actgcaacag 600
ccttatcaca agggcatacc aagaatagag tcgagaaaat acatcaattt ctacgaagaa 660
gatgagtctc gaaatgaaat tatgctcaaa ttagcgaaaa ttgattttaa tcgagtacaa 720
ttgttgcacc aacaagagct aagtcatgtt tcaaggtggt acaaagactt gcagattgct 780
tcaaaatttc cttatgcaag agatagaatt gctgaaatct acatgtggac tgttgggtct 840
aattttgaac cacattatgg acgtacccgc atttttctta ctaaaagtgt gacaatgata 900
tcaattttag atgacacata tgatgcatac ggtacaattg aagaactccg actcttgacc 960
gatgcagttg aaaggtggga cgttggtgcc attgatgaac taccagatta catgaaagtt 1020
ctttacaaga tgattataaa tctctacgat gaattcgaga acgaattgaa aaatgaagga 1080
agatcttctt gtgtcgctta tgctagagac gcgctaagag aaatggtgaa agcctaccac 1140
gttgaagcag agtggtgcaa caaaggttac ataccaacat ttgatgagta catggagaat 1200
gcattgatca caagctgtta tcatgcaatt ccagctgcat gttttctagg catgggagaa 1260
attgcaggaa caaaagaatt tgaatggctc aaaagcatcc caaaaattat tagagcttct 1320
gagatgatcg gtcgtcttat ggacgacata atgtcacata aggaggagca agagaggggg 1380
catgtcgcgt caagtattga gtgctttatg aagcaatatg gtgtgtcgag tgaggaagag 1440
gtggttcaag attttcaaat taggattgcg aatgcttgga aggatattaa tgaagaatgt 1500
gtgagaccaa ctgctgtgtc tcttcatctt ctgatgccaa ttttgaacct aacacgcatc 1560
atagatgttg tctacaagaa tgaagatggg tattcaaatc cagtgaattt gaaggagcat 1620
gtcaaggctt tgttcattca acaaatacca atccaagata attaa 1665
<210> 3
<211> 29
<212> DNA
<213>artificial sequence
<400> 3
cgcggatcca tggcagcaac caaccaatc 29
<210> 4
<211> 34
<212> DNA
<213>artificial sequence
<400> 4
ccgctcgagt taattatctt ggattggtat ttgt 34
Claims (4)
1. a kind of Celastrus angulatus sesquiterpene synthase, is denoted as CaTPS2, which is characterized in that its amino acid sequence such as SEQ ID NO.1
It is shown.
2. encoding the gene of Celastrus angulatus sesquiterpene synthase CaTPS2 described in claim 1, it is denoted as CaTPS-24009, feature
It is, nucleotide sequence is as shown in SEQ ID NO.2.
3. a kind of recombinant plasmid, is denoted as pRS426-URA-CaTPS2, which is characterized in that containing gene described in claim 2, partly
Lactose inducible promoter PGal1,10, uracil auxotrophy screens label.
4. a kind of recombinant bacterium, is denoted as WQ011/pRS426-URA-CaTPS2, which is characterized in that contain recombination described in claim 3
Plasmid.
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LI, WG等: ""Characterization of trans-Nerolidol Synthase from Celastrus angulatus Maxim and Production of trans-Nerolidol in Engineered Saccharomyces cerevisiae"", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
李瑞红等: ""白姜花倍半萜合成酶基因的克隆及表达"", 《园艺学报》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109679931A (en) * | 2019-01-30 | 2019-04-26 | 天津大学 | A kind of Celastrus angulatus acyltransferase 35019 and its gene order |
CN109679943A (en) * | 2019-01-30 | 2019-04-26 | 天津大学 | A kind of Celastrus angulatus sesquiterpene synthase CaTPS3 and its gene order |
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