CN105647879A - Diterpene compound variediene synthesizing gene Au13192 and application thereof - Google Patents

Diterpene compound variediene synthesizing gene Au13192 and application thereof Download PDF

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CN105647879A
CN105647879A CN201610148945.7A CN201610148945A CN105647879A CN 105647879 A CN105647879 A CN 105647879A CN 201610148945 A CN201610148945 A CN 201610148945A CN 105647879 A CN105647879 A CN 105647879A
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gene
compound
variediene
diterpene compound
protein
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洪葵
殷如
柴沆镇
董治统
邓子新
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Wuhan University WHU
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Abstract

The invention discloses a diterpene compound variediene synthesizing gene Au13192 and application thereof, and belongs to the field of gene engineering. The diterpene compound synthesizing gene is derived from aspergillus 094102, a gene and a cDNA sequence of the diterpene compound are shown in SEQ ID NO.1 and 2, and a sequence of encoded Au 13192 protein is shown in SEQ ID NO.3. Au13192 protein has functions of catalyzing substrate chain length to extend and structure cyclizing, and the protein can catalyze DMAPP, GPP and FPP to react with IPP and generate variediene. The invention finds that an Au1392 gene has remarkable influence on yield of ophiobolin type compound in the aspergillus 094102, and the Au1392 gene can be used for preparing an Oph compound adjunctively. The invention provides a new resource for biosynthesis of the Oph compound, and provides possibility for improving biological yield of the compound.

Description

A kind of diterpene compound variediene synthetic gene Au13192 and application thereof
Technical field
The present invention relates to genetic engineering field, be specifically related to a kind of diterpene compound variediene synthetic gene Au13192 that clone obtains from aspergillus ustus 094102 (Aspergillusustus094102) and application thereof.
Background technology
Ophiobolin (Ophiobolin, hereinafter referred to as Oph) it is the class sesterterpene with 5-8-5 tricyclic structure, up to the present the ophiobolin compounds with 5-8-5 tri-ring mother nucleus structure delivered mainly has ophiobolin A, C, F, H, M, L (M.ORSENIGO.Estrazioneepurificazionedellacochliobolina, unatossinaprodottadaHelmintosporiumoryzae.Phytopath.Z.19 57, 29, 189 196), ophiobolin O, Q, R, S (CLTipton, TTovrea, GWhitehurst.TheeffectofophiobolinAonglucoseuptakebyanima lcells.Nutr.Rep.Int.23, 723-727 (1981)) etc. sesterterpene is made up of five isoprene units, is the infrequent types in terpenoid, only accounts for terpenoid sum less than 2%. there is presently no the medicine of sesterterpenoids, but some dimeric sesquiterpene compounds show good biological activity. the antibacterial activity (such as antifungal, antibacterium) of this compounds display wide spectrum and nematicide, antiviral isoreactivity, additionally various tumor cell strains is had notable cytotoxic activity (Au, T.K., Chick, W.S.H.&Leung, P.C.Thebiologyofophiobolins.LifeSci.67,733 742 (2000)).
OphA is as the first Oph compound being found, except there is multiple biological activity, or a kind of important pherylarsin oxide, it is possible to as probe (Leung, P.C., Taylor, W.A., Wang, J.H.&Tipton, C.L.Roleofcalmodulininhibitioninthemodeofactionofophiobo lina.PlantPhysiol.77,303-308 (1985); Peters, C.&Mayer, A.Ca2+/calmodulinsignalsthecompletionofdockingandtrigger salatestepofvacuolefusion.Nature.396,575-80 (1998)).Up-to-date research shows that OphA has potential application (Dasari in cerebral glioma and Parkinsonian treatment, R.etal.FungalmetaboliteophiobolinAasapromisinganti-gliom aagent:Invivoevaluation, structure-activityrelationshipanduniquepyrrolylationofpr imaryamines.BioorgMedChemLett.25,4544-4548 (2015); Xue, D.etal.3-anhydro-6-hydroxy-ophiobolinA, afungalsesterterpenefromBipolarisoryzaeinducedautophagya ndpromotedthedegradationof ��-synucleininPC12cells.BioorgMedChemLett.25,1464-1470 (2015)). Novel Medicine Research & Development Centre., North China Pharmacey Group once tested OphG and OphK to human colon cancer cell strain Hct-15/Hct-116; The inhibitory action of Breast cancer lines MDA-MB-231/MCF-7, human lung carcinoma cell line A549 and Leukemia K562 cell, it has been found that both compounds IC to above-mentioned cancerous cell50It is below 30 �� g/mL and in good dose-effect relationship (Zhu Jingtong, Fan Yuling, road Xinhua, Dong Yuesheng, Zheng Zhihui, Zhang Hua, He Jiangong. the experiment in vitro research of Dimeric sesquiterpene compound ophiobollin anti-tumor activity. " canceration distortion sudden change ", 2007,19 (5): 388-391). Studies have found that OphO can increase the sensitivity of the breast cancer cell of Adriamycin resistant, 0.1 ��M of OphO and adriamycin medication, the amycin IC50 that can make mdr cell is reduced to 6.67 �� 0.98 ��Ms from 74.00 �� 0.18 ��Ms, reduce about 11 times of (Sun, W.etal.Ophiobolin-Oreversesadriamycinresistanceviacellcy clearrestandapoptosissensitizationinadriamycin-resistant humanbreastcarcinoma (MCF-7/ADR) cells.MarDrugs.11,4570-84 (2013)).
Due to Oph compound good prospect in drug development, preparing compound in a large number is an important premise. Started to be related to ophiobolin compounds and the report of parent nucleus chemosynthesis thereof successively from 1992, but the research that the labyrinth of this compounds makes its chemosynthesis is extremely challenging, it is only completed complete synthesis (the KazuhiroTsuna of ophiobolin A up till now, NaoyoshiNoguchi, MasahisaNakada.Enantioselectivetotalsynthesisof (+)-ophiobolinA.Chemistry-AEuropeanJournal.2013,19,5,476 5486; KeLi, ChengWang, GangYin, etal.ConstructionofthebasicskeletonofophiobolinAandvarie colin.Organic&BiomolecularChemistry.2013,11,7550 7558), and synthesis step is loaded down with trivial details not easily operates, and preparing Oph hence with biological synthesis process is the important channel obtaining a large amount of compounds.
Summary of the invention
The primary and foremost purpose of the present invention is in that to provide a kind of diterpene compound variediene synthetic gene, and the structure of diterpene compound variediene is as shown in Figure 1. Another object of the present invention is to provide a kind of diterpene compound variediene synzyme. Another object of the present invention is in that the application of described gene or enzyme.
The purpose of the present invention is achieved through the following technical solutions:
In the biosynthetic process of research aspergillus ustus 094102 (CCTCCNO:M208153) metabolite Oph compound, the present invention is by knocking out the gene cluster relevant to terpenoid synthesis, the change of production of detection Oph compound, find POC13192 gene cluster (Fig. 2) knock out make Oph compound production and wild type under same culture conditions compared with significantly reduce (Fig. 3), it was shown that POC13192 gene cluster affects the synthesis of Oph compound.By to gene cluster gene function analysis, learning that Au13192 gene is relevant to terpenoid synthesis in POC13192 gene cluster, illustrates that Au13192 gene take part in the biosynthesis of Oph compound in POC13192 gene cluster. Carry out external enzymatic reaction by heterogenous expression Au13192 gene acquisition Au13192 albumen further and demonstrate the synthetic gene that Au13192 gene is a kind of diterpene compound variediene.
A kind of diterpene compound variediene synthetic gene, for cloning the Au13192 gene obtained from aspergillus ustus 094102, its gene order is such as shown in SEQIDNO.1. Described Au13192 gene contains 8 introns, and its cDNA is sized to 2127bp, and sequence is such as shown in SEQIDNO.2.
A kind of diterpene compound variediene synzyme, is the albumen of Au13192 gene code, and called after Au13192 albumen, its aminoacid sequence is such as shown in SEQIDNO.3. Au13192 albumen has catalytic substrate chain length and extends the function with structure cyclisation, and Au13192 albumen energy catalysis DMAPP (dimethylallylpyrophosphate), GPP (geranyl pyrophosphate) or FPP (farnesyl pyrophosphate) and IPP are synthesized variediene.
Au13192 gene can be carried out heterogenous expression by escherichia coli prokaryotic expression system and obtain by Au13192 albumen of the present invention, can be obtained the Au13192 albumen of purification by nickel ion Metal chelate affinity chromatography simultaneously.
A kind of carrier expressing above-mentioned Au13192 albumen, eucaryon containing above-mentioned Au13192 gene or prokaryotic expression carrier.
A kind of host cell expressing above-mentioned Au13192 albumen, containing above-mentioned carrier.
Diterpene compound variediene can be synthesized based on Au13192 albumen, Oph compound parent nucleus can be assisted to synthesize, and above-mentioned Au13192 gene or Au13192 albumen can be used for synthesizing diterpene compound variediene, synthesis terpenoid, auxiliary synthesis Oph compound (ophiobolin compounds).
There is advantages that the Au13192 gene that present invention finds synthesis diterpene compound variediene, the Au13192 albumen of its coding can assist Oph compound parent nucleus to synthesize. The biosynthesis that the present invention is Oph compound provides new resource, and the biological yield for improving this compounds provides one to select. In the biosynthesis of Oph compound, the yield of Oph compound can be improved by coexpression Au13192 gene.
Accompanying drawing explanation
Fig. 1 is the structure chart of diterpene compound variediene.
Fig. 2 is the POC13192 gene cluster figure of aspergillus ustus 094102.
Fig. 3 is the comparison diagram of aspergillus ustus 094102POC13192 gene cluster knockout mutant strain �� POC13192 and wild type Oph compound production.
Fig. 4 is the protein A u13192 amino acid alignment result figure with EvVS albumen of aspergillus ustus 094102Au13192 gene code.
Fig. 5 is Au13192 gene cDNA amplification policy map.
Fig. 6 is the expression of results figure of Au13192 gene heterogenous expression albumen. M: albumen marker; 1: the BL21 (DE3) containing the pET28a carrier whole-cell protein figure before IPTG induces; 2: the BL21 (DE3) containing the pET28a carrier whole-cell protein figure after IPTG induces; 3: the BL21 (DE3) containing the pET28a-Au13192 carrier whole-cell protein figure before IPTG induces; 4: the BL21 (DE3) containing the pET28a-Au13192 carrier whole-cell protein figure after IPTG induces.
Fig. 7 is the purification result figure of Au13192 gene heterogenous expression albumen.
Fig. 8 is the GC-MS figure of the outer product of Au13192 albuminous body. A: mass-to-charge ratio is 272 times vitro reactions product GC chromatograms (I: with FPP and the IPP product GC chromatogram being substrate extracted; II: with GPP and the IPP product GC chromatogram being substrate; III: with DMAPP and the IPP product GC chromatogram being substrate; IV: with the IPP product GC chromatogram being substrate); B: vitro reactions product mass spectra figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention being done further detailed description, following embodiment is only used for the present invention is described, does not limit the scope of the invention.
Diterpene compound variediene synthetic gene provided by the present invention, is clone to obtain from aspergillus ustus 094102 (Aspergillusustus094102), and called after Au13192 gene, its gene order is such as shown in SEQIDNO.1. Au13192 gene contains 8 introns, and its cDNA is sized to 2127bp, and sequence is such as shown in SEQIDNO.2. The albumen of Au13192 gene code, called after Au13192 albumen, its aminoacid sequence is such as shown in SEQIDNO.3.
The homologous sequence searching for aspergillus ustus 094102Au13192 albumen from public database is compared, result display target protein Au13192 and EvVS (variediene the synzyme) (BinQin from Emericellavariecolor, YudaiMatsuda, TakahiroMori, etal.AnUnusualChimericDiterpeneSynthasefromEmericellavar iecolorandItsFunctionalConversionintoaSesterterpeneSynth asebyDomainSwapping.Angew.Chem.Int.Ed.54, 15 (2015)) homology higher (78%). Au13192 albumen belongs to mosaic type terpenoid synzyme, and its N end and C end are each responsible for terpenoid cyclisation and isopentene group forwarding function. Au13192 albumen contains two conserved domains, and wherein terpenoid cyclase domain contains two for identifying Mg2+Also two characteristics conserved motifs DDIED and DDYKN (Fig. 4) with identity function are contained with characteristic conserved motifs DNVVE and NDYFSFDIE, the E-IPPS domain of substrate.
Knocking out of embodiment 1POC13192 gene cluster
The method utilizing homologous recombination, replaces to blasticidin resistance gene by the POC13192 gene cluster in aspergillus ustus 094102 thalline, the situation of change of Oph content in detection mutant. Such as table 1 of culture medium prescription used in the present embodiment.
Table 1: culture medium prescription used in the present embodiment
1, the structure of POC13192 gene cluster knockout carrier
(1) by round pcr, the bleomycin gene order (Shble) containing Ptef1 and Pem7 promoter is obtained with pGAPZ �� A carrier for template amplification.
The primer sequence that amplification is used: Shble-F:CCCACACACCATAGCTTCAAAAT; Shble-R:AGCTTGCAAATTAAAGCCTTCG.
(2) fragment amplification obtained is connected with pMD18-T carrier by TA clone, builds pMD18-T-Shble carrier, and there are two multiple clone site these carrier S hble both sides, is respectively used to add the upper and lower homologous sequence of POC13192 gene cluster.
(3) connect product to be transformed in escherichia coli TOP10, screen positive transformant by ampicillin. Liquid culture positive transformant, extracts plasmid PCR checking, obtains pMD18-T-Shble plasmid.
(4) with aspergillus ustus 094102 genome for template, go out the sequence of 1226bp at POC13192 gene cluster upstream amplification, add suitable restriction enzyme site.
The primer sequence that amplification is used: POC13192L-F:GAATTCTAACTTCAAATCAGCGAGGA (EcoRI); POC13192L-R:TCTAGAATACCCACGGACCCAAC (XbaI).
(5) the POC13192 gene cluster fragment upstream that amplification obtains is connected with pMD18-T-Shble carrier, builds pMD18-T-13192L-Shble carrier.
(6) connect product to be transformed in escherichia coli TOP10, screen positive transformant by bleomycin. Liquid culture positive transformant, extracts plasmid PCR checking, obtains pMD18-T-13192L-Shble plasmid.
(7) with aspergillus ustus 094102 genome for template, go out the sequence of 1077bp at POC13192 gene cluster downstream amplification, add suitable restriction enzyme site.
The primer sequence that amplification is used: POC13192R-F:CCTGCAGGAGCAGCATCTTCGATTGG (SbfI); POC13192R-R:AAGCTTATTCTGGGACAGGAACTAG (HindIII).
(8) the POC13192 gene cluster segments downstream that amplification obtains is connected with pMD18-T-13192L-Shble carrier, builds pMD18-T-13192L-Shble-13192R.
(9) connect product to be transformed in escherichia coli TOP10, screen positive transformant by bleomycin. Liquid culture positive transformant, extracts plasmid PCR checking, obtains pMD18-T-13192L-Shble-13192R carrier. This carrier i.e. knocking out for POC13192 gene cluster.
2, the conversion of protoplast
(1) aspergillus ustus 094102 bacterial strain is applied to GMM flat board, cultivates 3-4d for 28 DEG C.
(2) collect spore in 10mL0.1%Tween-80 (it is generally required to collecting the spore of 4 flat boards), count with blood counting chamber.
(3) inoculation about 109Individual spore in 500mLLMM, 28 DEG C, 280rpm cultivate about 9h.
(4) with miracloth filter-cloth filtering LMM culture, 4 DEG C, the centrifugal 15min of 8000rpm collect the spore sprouted, wash once with 50mLMyceliumwashsolution.
(5) in the Osmoticmedium that spore precipitation is moved to containing 1%Driselase, 28 DEG C, 80rpm cultivate 4h, observe protoplast formation situation every 1/2h microscope.
(6) with miracloth filter-cloth filtering protoplast, the STCbuffer of 2 times of volumes is added, 4 DEG C, the centrifugal 8min collection protoplast of 6000rpm.
(7) protoplast is resuspended in STCbuffer so that it is final concentration reaches 108-109Individual protoplast/mL.
(8) preserve protoplast on ice, take out part dilution 10 times and carry out protoplast counting, other the protoplast transformation experiment carrying out PEG4000 mediation as early as possible.
(9) plasmid (pMD18-T-13192L-Shble-13192R) is knocked out with STCbuffer dilution is to be transformed so that it is final concentration reaches 10 �� g/100 �� L.
(10) 100 �� L are contained the STCbuffer knocking out plasmid and join in 100 �� L protoplasts, fully ice bath 50min after mixing.
(11) adding 1.25mLPEGsolution, after soft mixing, room temperature places 20min.
(12) add 5mLSTCbuffer, mix latter 4 DEG C, the centrifugal 10min of 3750rpm, abandon supernatant.
(13) softly add STCbuffer and be settled to 600 �� L.
(14) take the mixture after 200 �� L convert and coat SMM (Zeo+) on flat board, cultivate 3-5 days for 28 DEG C.
(15) transformant grown being carried out PCR checking, positive transformant is POC13192 deletion mutation strain �� POC13192.
3, the detection of Oph
(1) inoculation wild type aspergillus ustus 094102 or �� POC13192 spore are in No. 2 culture medium of fungus, cultivate 3d for 28 DEG C.
(2) inoculation bacterium solution is in solid fermentation culture medium, cultivates 18d for 20 DEG C.
(3) take 20g tunning, after adding 30mL80% acetone, use crusher in crushing.
(4), after ultrasonication 20min, 8000rpm is centrifuged 10min, takes supernatant.
(5) with the extraction into ethyl acetate 1 time of 2 times of volumes, dissolve with 15mL methanol (chromatographic grade) after being spin-dried for Rotary Evaporators.
(6) take 1mL methanol solution, be placed in chromatogram bottle through 0.22 ��m of membrane filtration, be HPLC sample.
(7) sample being carried out HPLC-UV detection: sample introduction 10 �� L, flow velocity 1mL/min, full wavelength detecting, �� max=230-240nm, mobile phase is methanol: water=85:15 (v/v), rinses 30min, time=8-30min.
(8) oph yield is calculated according to standard curve: y=3E+07x-76040 [R2=0.995] (Waters); Y=65482x+17.89 [R2=0.998] (Agilent).
By wild type aspergillus ustus 094102 and POC13192 deletion mutation strain �� POC13192oph Yield compari@, it was shown that the disappearance of POC13192 reduces Oph yield (Fig. 3).
For verifying the explanation POC13192 impact on oph yield further, below the core gene Au13192 of POC13192 gene cluster is carried out heterogenous expression, this gene function of Validation in vitro.
Embodiment 2Au13192 gene clone
1, the DNA extraction of aspergillus ustus 094102 bacterial strain
(1) aspergillus ustus 094102 bacterium solution is coated on GMM flat board, cultivate about 3d for 28 DEG C.
(2) from the spore that picking GMM culture plate is appropriate, it is suspended in 1mLddH2O is to muddy, and the concussion of vortex instrument makes mixing. 12000rpm is centrifuged 2min, collects spore, outwells supernatant.
(3) Buffer10.4mL is added, mixing. Buffer1:50mMTris-Hcl, 10mMEDTA, 100 �� gRNaseA, pH8.0.
(4) add Buffer20.4mL, mix latter 65 DEG C and hatch 20min, be cooled to room temperature. Buffer2:200mMNaoH, 1%SDS.
(5) Buffer30.6mL is added, the centrifugal 10min of 12000rpm after mixing, the 1.5mL centrifuge tube that transfer supernatant is extremely new. Buffer3:3M potassium acetate, pH5.5.
(6) adding the 3MNaAc (pH5.2) of 1/8 times of volume and the isopropanol of 0.6��0.8 times of volume, 30min placed by 4 DEG C of refrigerators.
(7) the centrifugal 20min of 12000rpm, outwells supernatant.
(8) washing twice with 70% ethanol 0.6mL, 12000rpm is centrifuged 2min, outwells supernatant.
(9) being dissolved in sterilized water 10 �� L after drying ,-40 DEG C save backup.
2, the amplification of Au13192 gene cDNA
Au13192 gene has 9 exons (E1��E9), adopts the method for Over-lapPCR to get up to obtain Au13192 gene cDNA by 9 exon splicings. Design primer makes the overlay region having 18-22bp between each adjacent segment, and wherein E2, E7 fragment is less, will expand out (Fig. 5) in its sequential design to the primer of adjacent segment.
(1) with aspergillus ustus 094102 genome for template, the primer (table 2) of each fragment is utilized to expand 9 exon fragment E1, E3, E4, E5, E6, E8, E9 obtaining Au13192 gene respectively. Reaction system is: the Pfu high-fidelity DNA polymerase of 1 NewEnglandBiolabs company of the �� L U.S., 32 �� L sterilizing deionized waters, 5 �� NFbuffer that 10 �� LPfu high-fidelity DNA polymerases carry, the each 2.5 �� L (10 ��Ms) of forward, reverse primer, aspergillus ustus 094102 genomic templates 2 �� L.
Table 2 expands the primer of Au13192 gene extron
(2) taking 1 �� LE1, E3 fragment respectively is template, and with E1F, E3R for primer, under Pfu high-fidelity DNA polymerase effect, amplification obtains E1:E3.
(3) taking 1 �� LE4, E5 fragment respectively is template, and with E4F, E5R for primer, under Pfu high-fidelity DNA polymerase effect, amplification obtains E4:E5.
(4) not taking 1 �� LE6, E8, E9 fragment is template, and with E6F, E9R for primer, under Pfu high-fidelity DNA polymerase effect, amplification obtains E6:E9.
(5) not taking 1 �� LE1:E3, E4:E5, E6:E9 fragment is template, and with E1F, E9R for primer, under Pfu high-fidelity DNA polymerase effect, amplification obtains Au13192 gene cDNA.
Pcr amplification condition is: 95 DEG C of denaturation 3min; 95 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 35 circulations; 72 DEG C of abundant extension 10min.
3, target DNA reclaims
After PCR primer electrophoresis, use the GelExtractionKit (100) (article No.: D2500-01) of OMEGA company of Germany, from agarose gel, reclaim target DNA fragment. Concrete operation step is as follows:
(1) use trishydroxymethylaminomethane-acetic acid (referred to as TAE) buffer to make the agarose gel of 1.0%, target DNA is carried out agarose gel electrophoresis.
(2) under uviol lamp, cut the gel containing target DNA fragment, exhaust the liquid (now should excise the gel without target DNA part as far as possible, reduce gel volume as far as possible, improve the DNA response rate) of gel surface with napkin.
(3) chopping blob of viscose (can accelerate the blob of viscose thawing time after blob of viscose chopping, improve the DNA response rate).
(4) weigh blob of viscose weight, calculate blob of viscose volume. Add Bindingbuffer, 50-60 DEG C in the ratio of every 100mg agarose gel 400 �� L and process about 10min (mixing once every 2min, until blob of viscose melts completely).
(5) being transferred to by the sol solution of thawing in the adsorption column of collecting pipe, room temperature 10000 �� g is centrifuged 1min, abandons filtrate.
(6) adding 500 �� LXP2 (Bindingbuffer) to adsorption column, room temperature 10000 �� g is centrifuged 1min, outwells the filtrate in collecting pipe, is put back in collecting pipe by adsorption column.
(7) adding 700 �� LSPW (Washbuffer) to adsorption column, room temperature 10000 �� g, from 1min, outwells the filtrate in collecting pipe, is put back in collecting pipe by adsorption column.
(8) adding 300 �� LSPW (Washbuffer) to adsorption column, room temperature 10000 �� g is centrifuged 1min, outwells the filtrate in collecting pipe, is put back in collecting pipe by adsorption column.
(9) room temperature 10000 �� g uncaps centrifugal 2min.
(10) being put into by adsorption column in another clean 1.5mL plastic centrifuge tube, the centre at adsorption column mould adds 30-50 �� LElutionbuffer or sterilized water, and after room temperature places 2-5min, room temperature 10000 �� g is centrifuged 1min.
(11) reclaim product to check with the agarose gel electrophoresis of 1%.
4, the clone of target DNA
The target DNA reclaimed is connected 2-4h in 16 DEG C with pEASY-Blunt carrier, connects product and converts escherichia coli TOP10 competent cell, adopts method Preliminary screening positive colony of quickly inspection after incubated overnight, and method is as follows:
(1) with sterile toothpick converting about 20 monoclonal of picking on flat board, at the flat lining out of LA containing ammonia benzyl resistance (100 �� g/mL), 8h is cultivated in 37 DEG C.
(2) in 1.5mL plastic centrifuge tube, add 30 �� LSTE solution (20mMTris-HCl, 25mMEDTA, 75mMNaCl), the monoclonal picking respectively that will be enlarged by after cultivating with toothpick processes about 2min to above-mentioned plastic centrifuge tube mesoscale eddies so that it is fully mix.
(3) adding equal-volume chloroform: phenol: isoamyl alcohol (v:v:v=24:25:1), after jog mixing, room temperature 10000 �� g is centrifuged 5min.
(4) supernatant detects with the agarose gel electrophoresis of 1%, primarily determines that whether the plasmid that clone contains inserts exogenous sequences.
With the plasmid Mini Kit of Mike Lai Bo bio tech ltd, Wuhan, the clone containing exogenous sequences being carried out plasmid extraction, concrete operations are as follows:
(1) monoclonal is accessed in the 5mLLB culture medium containing ammonia benzyl resistance (100 �� g/mL), under 37 DEG C of conditions, after 220r/min incubated overnight, transferase 12 mL bacterium solution is to 2mL plastic centrifuge tube, in the centrifugal 1min of room temperature 12000 �� g, abandons supernatant.
(2) add 250 �� LBuffer1, acutely shake in vortex oscillator, make thalline fully resuspended.
(3) adding 250 �� LBuffer2, gentle reverse centrifuge tube 5-6 time, until system becomes limpid.
(4) adding 350 �� LBuffer3, gentle reverse centrifuge tube 3-5 time is with abundant mixing.
(5) the centrifugal 10min of room temperature 12000 �� g, carefully proceeds in the adsorption column of collecting pipe by supernatant, and room temperature 12000 �� g is centrifuged 1min, outwells the liquid in collecting pipe, adsorption column is put back to collecting pipe.
(6) add 500 �� LBufferD, room temperature 12000 �� g and be centrifuged 1min, outwell the liquid in collecting pipe, adsorption column is put back to collecting pipe.
(7) add 600 �� LBufferW, room temperature 12000 �� g and be centrifuged 1min, outwell the liquid in collecting pipe, adsorption column is put back to collecting pipe.
(8) again add 300 �� LBufferW, room temperature 12000 �� g and be centrifuged 1min.
(9) adsorption column is transferred in another clean 1.5mL plastic centrifuge tube, the unsettled plasmid adding the centrifugal 1min eluting clone of 50-100 �� LBufferE the rear chamber gentle and quiet 1min that puts, room temperature 12000 �� g of centre at adsorbed film.
By the plasmid high-fidelity restricted enzyme HindIII of NewEnglandBiolabs company of the U.S. obtained and NotI double digestion (2 �� LCutsmartbuffer, 0.5 �� LHindIII, 0.5 �� LNotI, 1 �� L plasmid DNA, 16 �� LddH2O, 37 DEG C process 2h) after, the agarose gel electrophoresis with 1% detects, it is determined that whether the exogenous dna fragment that clone is carried is target DNA fragment. Taking the 1mL clone bacterium solution containing target DNA fragment and deliver the order-checking of Beijing Qing Kexin industry Bioisystech Co., Ltd, sequencing result confirms that this clone contains target DNA fragment really, and sudden change does not occur in target DNA fragment. Recombiant plasmid called after by this positive colony-Blunt-Au13192��
The expression of embodiment 3Au13192 albumen and purification
1, containing the structure of Au13192 gene coli expression carrier
With restricted enzyme HindIII and NotI from the recombiant plasmid containing genes of interest-Blunt-Au13192 cuts purpose fragment, simultaneously with HindIII and NotI double digestion pET28a carrier, after agarose gel electrophoresis separates, reclaims test kit with OMEGA glue and be separately recovered genes of interest fragment and the large fragment of pET28a carrier. T4DNA ligase is utilized to be inserted in pET28a carrier by Au13192 gene forward, product will be connected and convert escherichia coli TOP10 competent cell, after clone is quickly checked and is identified with plasmid enzyme restriction checking, it is thus achieved that Recombinant protein expression carrier pET28a-Au13192.
The pET28a-Au13192 carrier built is proceeded to e. coli bl21 (DE3) through heat-shock transformed method, verifies the function of Au13192 gene further.
2, the expression of Au13192 gene
Recombiant plasmid pET28a-Au13192 and pET28a plasmid are proceeded in expressive host bacterium e. coli bl21 (DE3) by heat-shock transformed method respectively, with the plasmid Mini Kit of Mike Lai Bo bio tech ltd, Wuhan, transformant is carried out plasmid extraction, identify through restricted enzyme HindIII and NotI digestion verification, be successfully obtained the E. coli expression strains respectively containing recombiant plasmid pET28a-Au13192 and pET28a plasmid.
The single colony inoculation of the picking e. coli bl21 (DE3) containing recombiant plasmid pET28a-Au13192 and pET28a plasmid is to the 5mLLB culture medium containing kalamycin resistance (50 �� g/mL) respectively, in 37 DEG C, 220r/min incubated overnight. Fresh bacterium solution is seeded in the 5mLLB culture medium containing kalamycin resistance (50 �� g/mL) by the inoculum concentration by 1%, continues to cultivate 2-3h to OD600When reaching 0.4-0.6, adding IPTG to final concentration of 0.1mM, at 37 DEG C, inducing culture is about 3-5h (taking out 1mL bacterium solution before induction respectively as comparison). Take out the bacterium solution 200 �� L before induction and after induction respectively, add 5 �� SDS-PAGE sample buffer and boil 30min, room temperature 12000 �� g is centrifuged 10min, respectively taking 10 �� L of supernatant liquid and carry out SDS-PAGE detection, result (Fig. 6) shows destination protein successful expression in e. coli bl21 (DE3).
3, the purification of Au13192 albumen
The e. coli bl21 containing recombiant plasmid pET28a-Au13192 (DE3) bacterium solution taking 5mL incubated overnight is seeded in the 500mLLB culture medium containing kalamycin resistance (50 �� g/mL) by the inoculum concentration of 1%, and under 37 DEG C of conditions, 220r/min is cultured to OD600When reaching 0.4-0.6, adding IPTG to final concentration of 0.1mM, at 37 DEG C, inducing culture is about 3-5h. Then the coli somatic after the centrifugal 5min collection abduction delivering of room temperature 5000r/min, uses aseptic ddH2O suspension thalline, removes supernatant (repeating twice, thoroughly clean cell) after the centrifugal 5min of room temperature 5000r/min. Afterwards with HEPES buffer suspension thalline, after room temperature 5000r/min is centrifugal, remove supernatant. 10mLHEPES buffer is added in somatic cells, fully after mixing, cell suspension is put ultrasonic in ice-water bath (parameter: ultrasonic 2s, interval 5s, power 300W) and processes 30min. Then 4 DEG C, the centrifugal 60min of 13000rpm, take supernatant and carry out protein purification with after 0.45 ��m of membrane filtration. Purification process is as follows:
(1) take appropriate nickel bead substrate upper prop, make the dehydrated alcohol in substrate fully flow out under gravity.
(2) with the aseptic ddH of 20mL2O rinses pillar.
(3) with 20mLHEPES buffer balance pillar.
(4) supernatant containing target protein is added in pillar, collect stream and wear liquid, carry out SDS-PAGE detection.
(5) respectively with containing 25mM, 50mM, 75mM, 100mM and 250mM imidazoles eluent (imidazole solution with the dilution of HEPES buffer) carry out eluting, collect effluent, carry out SDS-PAGE detection.
SDS-PAGE testing result shows when carrying out eluting with the eluent containing 75mM imidazoles, it is thus achieved that purer albumen (Fig. 7). Concentrating this purer protein solution to after 2.5mL, the protein solution after concentration being carried out desalting processing with the PD-10 desalting column (article No. is 17-0851-01) of GEHealthcare with the pipe that is concentrated by ultrafiltration of 10kDa, concrete operations are as follows:
(1) with 10mL equilibration buffer pillar, abandon waste liquid, repeat 4 times.
(2) add 2.5mL protein liquid to being completely immersed in pillar, abandon waste liquid.
(3) add 3.5mL eluent and to collect-80 DEG C of preservations of the effluent protein solution of desalting processing (this effluent be) standby.
The functional analysis of embodiment 4Au13192 albumen
In order to determine the function of Au13192 albumen, with the DMAPP (dimethylallylpyrophosphate) of Sigma-Aldrich, GPP (geranyl pyrophosphate), FPP (farnesyl pyrophosphate) for substrate, add IPP (isopentenylpyrophosphate) respectively and design vitro reactions.Reaction system (200 �� L): 50 ��Ms of DMAPP (or GPP, FPP), 50 ��Ms of IPP, 20mMHEPES (pH7.4), 2mMDTT (dithiothreitol, DTT), 5mMMgCl2With 100 �� gAu13192 albumen, 30 DEG C of reaction 3h. After reaction terminates, with equal-volume n-hexane extraction 3 times, with Nitrogen evaporator, organic solvent is dried up, after 50 �� L n-hexane dissolutions, carry out GC-MS (gas chromatography-mass spectrum) detection. Heating schedule: 60 DEG C of constant temperature 2min, then it is warmed up to 150 DEG C with the speed of 30 DEG C/min, then it is warmed up to 180 DEG C with the speed of 10 DEG C/min, then it is warmed up to 210 DEG C with the speed of 2 DEG C/min, keep 210 DEG C of 5min.
With DMAPP, GPP and FPP for substrate, add IPP respectively, all detect the material that molecular weight is 272 when retention time is 12.7min, this material is diterpene compound variediene (m/z=55,67,91,105,119,133,147,203,230,257,272 etc.) (Fig. 8) by mass spectrogram analysis.
Result above shows that Au13192 albumen can be substrate with DMAPP, GPP and FPP, adds synthesis diterpene compound variediene during IPP respectively. It can thus be appreciated that Au13192 albumen has catalytic substrate chain length extends the function with structure cyclisation simultaneously, it is possible to DMAPP de novo synthesis variediene.

Claims (7)

1. a diterpene compound variediene synzyme, it is characterised in that: its aminoacid sequence is such as shown in SEQIDNO.3; The structure of described diterpene compound variediene is as follows:
2. the gene of diterpene compound variediene synzyme described in coding claim 1, it is characterised in that: its gene order is such as shown in SEQIDNO.1.
3. gene according to claim 2, it is characterised in that: its cDNA sequence is such as shown in SEQIDNO.2.
4. the carrier of the diterpene compound variediene synzyme that a kind is expressed described in claim 1, it is characterised in that: for the eucaryon containing gene described in Claims 2 or 3 or prokaryotic expression carrier.
5. the host cell of the diterpene compound variediene synzyme that a kind is expressed described in claim 1, it is characterised in that: containing the carrier described in claim 4.
6. the synzyme described in claim 1 or the application in synthesis variediene of the gene described in Claims 2 or 3.
7. the synzyme described in claim 1 or the gene described in the Claims 2 or 3 application in preparing terpenoid.
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