CN107058419B - Tripterygium wilfordii TwKS and applications of the TwCPS3 in Kaurane diterpine compound is prepared - Google Patents

Abstract

The present invention relates to the application of tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3 and tripterygium wilfordii kaurene synthase TwKS in Kaurane diterpine is prepared.By being recombinantly expressed to tripterygium wilfordii Ke Baji pyrophosphate synthase gene TwCPS3 and tripterygium wilfordii kaurene synthase gene TwKS, under the catalysis of recombinant protein, the precursor-derived thing Kaurane diterpine compound of gibberellin has successfully been synthesized using GGPP as substrate.The synthesis regulation of the diterpene compounds such as biosynthesis and triptolide of the present invention to gibberellin has great importance.

Description

Tripterygium wilfordii TwKS and applications of the TwCPS3 in Kaurane diterpine compound is prepared

Technical field

The present invention is urged to become reconciled yeast-leavened by building TwCPS3 and TwKS eucaryons and prokaryotic expression carrier by vitro enzyme Method successfully synthesizes 16 α-kaurene, it is related to the biosynthesis of gibberellin.Be related to tripterygium wilfordii growth and development regulation and control and The synthesis of active ingredient, belongs to Gene Engineering of Medicinal Plants field.

Background technology

Medicinal plant tripterygium wilfordii (Tripterygium wilfordii.Hook.f.) is Chinese herbal medicine simply, is widely used in The treatment of rheumatoid arthritis and inflammation.Ter penoids are the main active of tripterygium wilfordii, including triptolide (triptolide), triptophenolide (triptophenolide) and Celastrol (celastrol) etc..Now some researches show that Kaurane diterpine is the chief component in terpene natural products family, this quasi-molecule typically exhibits antibacterial, resists and swell A variety of important biomolecule activity such as knurl, antimalarial, antiviral.It is a kind of very promising mode from active ingredient of Chinese herbs developing new drug, Yet with the slow-growing of plant, along with content of these active ingredients in plant is few, thus greatly limit Its development.By seeking and explaining ter penoids, biosynthesis of the Kaurane diterpine in tripterygium wilfordii is particularly sought Approach and its regulatory mechanism, can be to improve target component content using biotechnology or directly produce active ingredient or intermediate Bring wide application space.

Gibberellin is a kind of Fourth Ring double terpene compound, has the thing of extremely important effect in terms of the growth regulating of plant Matter.Now some researches show that gibberellin plant seed sprouting, the growth of stem, bloom has important adjustment effect with result. Kaurane diterpine is a kind of double terpene compound, its biosynthesis of specific species and content for downstream gibberellin has weight Influence.Parsing from Kaurane diterpine compound precursor to gibberellin biological route of synthesis helps to regulate and control in tripterygium wilfordii The biosynthesis of triptolide, Celastrol isoreactivity component.

The content of the invention

The present invention is by tripterygium wilfordii Ke Baji pyrophosphate synthase gene Twcps3 and tripterygium wilfordii kaurene synthase gene Twks carries out the structure of expression vector, successfully synthesizes 16 α of precursor-kaurene of gibberellin, this is to triptolide etc. The biosynthetic controlling of active ingredient has great importance.

Specifically the present invention will clone the obtained cDNA clone of Twcps3 and Twks genes to prokaryotic expression carrier PMAL-c2X, recombinant expression carrier of the structure with Twcps3 and Twks genes；E.coli expressive host bacterium are transferred to, pass through induction Afterwards, expression product is added to using GGPP as in the external enzymatic reaction system of substrate, n-hexane extraction reacts after preliminary purification Product, the generation of Kaurane diterpine compound is detected using GC-MS, by showing the analysis result of GC-MS collection of illustrative plates, TwCPS3 and TwKS, which is used in combination, can be converted into GGPP Kaurane diterpine class compound, it is surprising that described In Kaurane diterpine class compound in addition to the gibberellin precursor ent-kaurene being currently known, additionally it is possible to produce Ent-16 α kaurenes, in addition it has also been found that a certain amount of manool in product.

By the way that the ORF of Twcps3 and Twks genes is cloned into carrier for expression of eukaryon pESC-Trp, structure carries Twcps3 And/or Twks genes recombinant expression carrier and the methods of pass through rite-directed mutagenesis, pass through yeast BY-T20 fermentation generation products, profit With Gas chromatographyMass spectrometry, the function of analysis Twcps3 and Twks genes.Result of study shows, eukaryotic expression GGPP can be converted into Kaurane diterpine, and the yield higher of ent-16 α kaurenes by TwCPS3 and TwKS.This hair The biosynthetic controlling of bright growth and development and active ingredient to tripterygium wilfordii is of great significance.

The present invention provides a kind of tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3 and tripterygium wilfordii kaurene synthase TwKS and exists Prepare the application in Kaurane diterpine, it is characterised in that using Mang ox base pyrophosphoric acid GGPP as substrate, catalysis produces shell China fir alkane type diterpenoid.

Wherein, the sequence of the tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3 is SEQ ID NO：1, tripterygium wilfordii shell The sequence of China fir alkene synthase TwKS is SEQ ID NO：3.

The present invention also provides tripterygium wilfordii Ke Baji pyrophosphate synthase gene Twcps3 and tripterygium wilfordii kaurene synthase gene Applications of the Twks in Kaurane diterpine is prepared, it is characterised in that recombination expression Twcps3 and Twks genes obtain TwCPS3 Kaurane diterpine is used to prepare with TwKS, the wherein sequence of tripterygium wilfordii Ke Baji pyrophosphate synthases gene Twcps3 is SEQ ID NO：The sequence of 2, tripterygium wilfordii kaurene synthase gene Twks are SEQ ID NO：4.

Tripterygium wilfordii Ke Baji pyrophosphate synthase gene Twcps3 and tripterygium wilfordii kaurene synthase gene of the present invention The wherein described Twcps3 and Twks genes of applications of the Twks in Kaurane diterpine is prepared pass through prokaryotic expression or eucaryon table Up to TwCPS3 and TwKS is obtained, the gene is preferably subjected to eukaryotic expression.

Tripterygium wilfordii Ke Baji pyrophosphate synthase gene Twcps3 and tripterygium wilfordii kaurene synthase gene of the present invention Applications of the Twks in Kaurane diterpine is prepared, built respectively with Twcps3 using prokaryotic expression carrier pMAL-c2X or The recombinant prokaryotic expression vector of Twks genes；Built respectively with Twcps3 or Twks bases using carrier for expression of eukaryon pESC-Trp The recombinant expression carrier of cause.

Tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3/ or its gene Twcps3 and tripterygium wilfordii kaurene of the present invention The application of synthase TwKS/ or its gene Twks in Kaurane diterpine is prepared, wherein the Kaurane diterpine includes Ent- kaurenes and ent-16 α kaurenes.

It is prepared by tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3 of the present invention and tripterygium wilfordii kaurene synthase TwKS Application in Kaurane diterpine, it is characterised in that manool is further included in product.

Tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3/ or its gene Twcps3 and tripterygium wilfordii kaurene of the present invention The application of synthase TwKS/ or its gene Twks in Kaurane diterpine is prepared, it is characterised in that tripterygium wilfordii Ke's Buckie Jiao's phosphorus Acid synthase TwCPS3/ or its gene Twcps3, uses or first at the same time with tripterygium wilfordii kaurene synthase TwKS/ or its gene Twks After use.

The present invention also provides a kind of method that enzymatic prepares Kaurane diterpine, it includes：(1) it is burnt with Mang ox base Phosphoric acid GGPP adds tripterygium wilfordii Ke Baji pyrophosphate synthase TwCPS3 for substrate, and n-hexane extraction 3 times, abandons organic phase, water intaking phase, Carry out dephosphorization after nitrogen drying, product that (2) are obtained into step (1) after dephosphorization add tripterygium wilfordii kaurene synthase TwKS into Row reaction, n-hexane extraction 3 times, takes n-hexane mutually to be dried up with nitrogen, and the product that (3) obtain step (2) carries out GC-MS points Analysis.

The present invention also provides a kind of method that enzymatic prepares Kaurane diterpine, it includes：(1) by Twcps3 genes Expression cassette and Twks expression casettes are building up on same carrier for expression of eukaryon Pesc-Trp, and structure obtains recombinant plasmid PESC- (Twcps3+Twks), transformed yeast screening obtain recombinant yeast；(2) by the positive recombinant yeast of PCR identifications into Row Fiber differentiation, collects bacterium solution and adds isometric n-hexane, and carrying out ultrasonic bacteria breaking, extraction, sodium sulphate drying, concentrated by rotary evaporation are done near, Saved backup for 4 DEG C after filtering；(3) GC-MS analyses are carried out to the product that step (2) obtains.

The method that enzymatic of the present invention prepares Kaurane diterpine, it is characterised in that in the step of GC-MS is analyzed 12.99th point of peak is manool, and the 13.03rd point of peak is ent- kaurenes, and the 13.96th point of peak is ent-16 α-shell China fir alkane.

Brief description of the drawings

Fig. 1 methyl jasmonates MJ induces the relative expression quantity of tripterygium wilfordii diterpene synthase gene different time

Fig. 2 host strain E.coli TransB (DE3) induced expression product SDS-PAGE protein electrophoresises analyze (C：It is negative right According to i.e. empty expression vector pMAL-c2X expression products；M：Protein Marker, band from top to bottom be respectively 200,116, 97.2、66.4、29.0、20.1、14.3、6.5KDa；Arrow represents purpose recombinant protein；1：The table of recombinant plasmid pMALTwCPS1 Reach；2：The expression product of recombinant plasmid pMALTwCPS3；3：The expression product of recombinant plasmid pMALTwKS；4：Recombinant plasmid The expression product of pMALTwCPS2；5：Recombinant plasmid pMALTwGES2 expression products；6：Recombinant plasmid pMALTwGES1 expression productions Thing)

Fig. 3 rite-directed mutagenesis primers design

Two diterpene synthase construction of eukaryotic expression vector of Fig. 4 tripterygium wilfordiis

Fig. 5 CPS catalysis substrate GGPP forms product extraction chromatography of ions figure, and the 13.83rd point of peak is CPP, mass spectrum lotus matter This is 275m/z.From top to bottom it is followed successively by TwCPS1, TwCPS2, TwCPS3, SmCPS1, AtCPS, empty carrier.

Fig. 6 (CPS+KS) catalysis GGPP forms product extraction chromatography of ions figure, and the 13.06th point of peak is miltirone two Alkene, the 13.03rd point of peak are that the peak of the 13.83rd point of ent-kaurene is CPP, and the 13.96th point of peak is ent-16 α-shell China fir alkane, the 13.46th point of peak is GGOH；Mass spectrum lotus matter this be 272m/z.

Fig. 7 TwCPS2C311D, TwKST318D catalysis GGPP form product extraction chromatography of ions figure, the 13.46th point of peak For GGOH；Mass spectrum lotus matter this be 275m/z, 272m/z.

Fig. 8 BY-T20/pESC-Trp-TwCPS3+TwKS, BY-T20/pESC-Trp-TwCPS3+TwKSA608M, BY- T20/pESC-Trp tunnings extract chromatography of ions figure, and the 13.03rd point of peak is ent-kaurene；Mass spectrum lotus matter this be 272m/z。

Fig. 9 BY-T20/pESC-Trp, BY-T20/pESC-Trp-TwCPS1, BY-T20/pESC-Trp-TwCPS3+ The peak of TwKS, BY-T20/pESC-Trp-TwCPS3+TwKSA608M tunning the 12.99th point of chromatography of ions figure of extraction is tear China fir alcohol, the 13.03rd point of peak is ent-kaurene, and the 13.83rd point of peak is CPP, and the 13.96th point of peak is ent-16 α-shellfish Shell China fir alkane；Mass spectrum lotus matter this be 272m/z.

Embodiment

Below in conjunction with specific embodiment, the present invention is further explained.These embodiments are merely to illustrate the present invention and do not have to In limiting the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to normal condition or presses According to the condition proposed by manufacturer.Unless otherwise defined, all professional and scientific terms used in text and this area are ripe It is identical to practice meaning known to personnel.In addition, any method similar or impartial to described content and material all can be applied to In the method for the present invention.The preferred methods and materials described herein are for illustrative purposes only.

Experimental method used in following embodiments is conventional method unless otherwise specified.Material used, reagent Deng being commercially available unless otherwise specified.

Tripterygium wilfordii (Tripterygium wilfordii Hook.F.) suspension cell is in document " tripterygium wilfordii 4- (5 '-two phosphorus Sour cytidine) -2-C- methyl Ds-erythrite kinase gene full-length clone and expression analysis CHINA JOURNAL OF CHINESE MATERIA MEDICAs, 2015,40 (21)：Mistake disclosed in 4165-4170 ", the public can obtain from Capital University of Medical Sciences's molecule crude drug with natural resources of Chinese medicinal materials laboratory.

SMARTerTM RACE cDNA Amplification Kit, PrimeSTAR GXL DNA Polymerase are purchased From Takara companies；PEASY-Blunt Simple Cloning Kit are purchased from Beijing Quanshijin Biotechnology Co., Ltd, GGPP is purchased from Sigma companies, catalog number G6025.

Embodiment one, tripterygium wilfordii Twcps3 and Twks Prokaryotic expression vector construction (Fig. 1)

Respectively using the carrier pMD-19-TPS plasmids containing tripterygium wilfordii Twcps3 and Twks full length gene cDNA as template, use Primer containing restriction enzyme site, carries out PCR amplification gene coding region (primer sequence is shown in Table 1), is inserted into prokaryotic expression carrier.DNA gathers Synthase uses high-fidelity DNA polymerase (PrimeSTAR HS DNA Polymerase).PCR parameters are 98 DEG C of 3min, and 1 follows Ring；98 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 2min30s, 30 circulations；72℃ 7min；4 DEG C of maintenances.Amplified production is through Gene JET Gel Extraction Kit glue reclaims (method is as follows).

Gene JET Gel Extraction Kit glue reclaim steps：

(1) take PCR product to be pre-mixed with 6 × loading buffer, on 1.5% Ago-Gel with low-voltage (about 5Vcm-1) electrophoresis 30-60min；

(2) gel containing DNA fragmentation with scalpel or slasher cutting, cuts close to DNA fragmentation, to subtract as far as possible Film, is placed on the 1.5mL centrifuge tubes weighed in advance and weighed by the content of small gel.The weight of recording film.Pay attention to avoiding growing Time exposes in the UV lamp, damages DNA and influences subsequent experimental；

(3) the Binding Buffer of 1: 1 amount are added (to be measured by weight, such as every 100 milligrams of Ago-Gels add into film 100 microlitres of Binding Buffer)；

(4) gel mixture 10min is incubated under conditions of 50-60 DEG C, during which overturns mixing 2-3 times, promotes glue to melt, Ensure that glue all dissolves, gel mixture is quickly vortexed mixing once before upper prop；

(5) most 800 μ L gels lysates are shifted to centrifuge 1min to gene recovery purifying column, 13000g, discard efflux, Then column is put back to identical collecting pipe；

(6) 700 μ LWash Buffer (being diluted with ethanol) are added and arrives Gene JET purification columns.13000g centrifuges 1min, Efflux is discarded, then column is put back to identical collecting pipe；

(7) sky GeneJET purification columns 13000g centrifugation 1min are centrifuged, thoroughly remove remaining Wash Buffer；

(8) Gene JET purification columns are transferred to a clean 1.5mL centrifuge tube, add 30-50 μ L ddH2O (can be 60 DEG C Preheating) in purification column film, 13000g centrifugations 1min；

(9) lose Gene JET purification columns and store the DNA of purifying at -20 DEG C.

PCR product after purified carries out double digestion with restriction enzyme, is oriented using NEB company's T 4DNA ligases Be connected into (method is as follows) in the expression vector pMAL-c2X through identical double digestion, connection product is inverted, blue white bacterium colony screening and The preliminary screening of positive colony, and sample presentation sequencing identification, obtain through be sequenced nucleotide sequence without mutation recombinant plasmid pMALTPS, And the recombinant plasmid pMALTPS of warp is converted to E. coli TransB (DE3) and expresses competent cell.It is same with this When, by the AtCPS (catalysis GGPP forms ent-CPP) of definite functions in arabidopsis, (catalysis ent-CPP forms mapping shellfish to AtKS Shell China fir alkene) and Radix Salviae Miltiorrhizae in the SmCPS1 (catalysis GGPP formed nor-CPP) of definite functions, SmKSL1 (catalysis nor-CPP is formed time Tanshinone diene) (the recombinant plasmid that SmCPS1, SmKSL1 are preserved using laboratory is building up on pMAL-c2X prokaryotic expression carriers PET32SmCPS1 and pET32SmKSL1), tripterygium wilfordii TwCPS3, TwKS are carried out with CPS, the KS in other plant source respectively pair This.

Double digestion reaction is carried out using TaKaRa QuickCut enzymes, reaction system is following (50 μ L)：

After the completion of 1 digestion of restriction endonuclease, add 1 μ L restriction endonucleases 2 and continue digestion.Pay attention to：The relatively low inscribe of most suitable digestion temperature Enzyme should first carry out digestion, if most suitable digestion temperature is identical, can add at the same time while digestion.

DNA fragmentation coupled reaction is carried out using NEB T4DNA rapid ligation kits, coupled reaction system is as follows：

Coupled reaction (20 μ L systems)

* DNA and carrier mole this be about 3：1-10：1

25 DEG C of connection 5min (Connection Time can be appropriately extended).

Table 1 builds prokaryotic expression carrier primer sequence

Embodiment two, recombinant protein induced expression

Induced expression

100mmol·L-1IPTG：The IPTG for weighing 238.3mg is dissolved with the ddH2O of 10mL, -20 DEG C of guarantors of filtering packing Deposit；LB culture mediums：Trytone 1.0%, Yeast Extract0.5%, NaCl 1.0%, Agar1.5%, pH 7.0.

Operating procedure：

(1) recombinant plasmid correct and through sequence verification containing target gene is identified into digestion, takes 1 μ L to convert to 50 μ l In TransB (DE3) competent cell, coated plate LB+Amp (ampicillin sodium) solid plate, 37 DEG C are inverted culture 12-16h；

(2) picking monoclonal bacterium colony goes to the 2mL LB fluid nutrient mediums containing 100 μ gmL-1Amp after digestion is identified In, 37 DEG C of shaken cultivations to OD600 to 0.6-1.0；

(3) 4 DEG C of 1mL bacterium solutions 5000g centrifugation 5min are taken to collect thalline, with fresh LB+Amp fluid nutrient mediums suspension thalline, It is transferred in 100mL LB+Amp fluid nutrient mediums；

When (4) 37 DEG C of culture to host's strain densities (OD600) reach 0.6-1.0, appropriate IPTG derivants (final concentration is added About 0.4mM), (16 DEG C) Fiber differentiation 8h at low temperature；

(5) 4 DEG C of 3000g centrifugation 20min harvest thalline, 5mL HEPES buffer solutions (the 50mM HEPES, 100mM of precooling KCl, 7.5mM MgCl2,5mM DTT, 1mM PMSF, 5% glycerine, pH 7.2) it is resuspended；

(6) put carrying out ultrasonic bacteria breaking in ice bath (30% power, ultrasonic 5s, is spaced 5s, continues 3min)；Escherichia coli crush liquid and exist 4 DEG C, 15000g centrifugation 30min, take supernatant；

(7) supernatant is through albumen super filter tube concentration (4 DEG C of 5000g 40min) to 1.5mL.

Supernatant is taken to carry out sodium dodecyl sulfate-polypropylene acid amides (SDS-PAGE) detected through gel electrophoresis.

Carry out being expressed as compareing with empty plasmid pMAL-c2X conversion TransB (DE3) expression bacterium.

Embodiment three, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 2)

Preparation of reagents

30% acrylamide storage liquid (neurotoxicity, during operation with wear masks and gloves)：In fume hood, propylene is weighed Acid amides 29.2g, methylene diacrylamide 0.8g, after adding ddH2O to dissolve, constant volume to 100mL.Syringe needle filter filters postposition brown bottle In, 4 DEG C of preservations；

PH 8.8Tris-HCl separation gel buffer solutions：1.5M Tris-HCl are prepared, and adjust pH to 8.8,4 DEG C of preservations；

PH 6.8Tris-HCl concentrate glue buffer solution：1M Tris-HCl are prepared, and adjust pH to 6.8,4 DEG C of preservations；

10%SDS：Weigh SDS 1.0g, distilled water 10mL dissolvings, 4 DEG C of preservations；

10% ammonium persulfate (APS)：Take APS 1.0g, distilled water 10mL dissolvings, 4 DEG C of preservations；

TEMED (tetraethylethylenediamine) stoste；

5 × sample buffer (10mL)：The Tris-HCl (pH 6.8) of 0.6mL 1molL-1,5mL50% glycerine, 2mL 10% SDS, 0.5mL mercaptoethanol, 1% bromophenol blues of 1mL, 0.9mL distilled water.Several weeks can be preserved at 4 DEG C, or in -20 DEG C of guarantors Deposit the several months；

10 × electrophoretic buffer：Tris 30.38g are weighed, glycine 144g, SDS 10.8g, adds distilled water about 900mL, After adjusting pH 8.3,1000mL is settled to distilled water, puts 4 DEG C of preservations, dilute 10 times before use.

Example IV, sample preparation

Protein example is mixed with 5 × sample buffer in an Eppendorf pipe, is put into 100 DEG C of heating 5- 10min, room temperature 12000g centrifuge 3min, take supernatant point sample.

Embodiment five, electrophoresis

Operating procedure

(1) glass plate, sample comb are cleaned with detergent, is rinsed for several times, dried with ddH2O；

(2) glass plate is installed；

(3) press lower volume such as and prepare 10% separation gel 15mL (preparing two pieces of glue), mix；

(4) to separation gel is recorded between glass plate, 1mL ddH2O are added immediately and flatten glue surface, glue can gather after about 20min Close；

(5) press lower volume such as and prepare 5% concentration glue 5mL (preparing two pieces of glue), mix；

(6) upper strata ddH2O is inclined, filter paper blots, and records concentration glue, is inserted into sample comb；

(7) electrophoresis system is installed, adds electrophoretic buffer, loading 5-10 μ L；

(8) constant current 40mA, after bromophenol blue runs out of separation gel 30min, stops electrophoresis；

(9) offset plate is unloaded, peels off glue, dyeing, according to glue.

Embodiment six, coomassie brilliant blue staining

Preparation of reagents

Coomassie brilliant blue R250 dyeing liquor：0.25% coomassie brilliant blue R250 (W/V), 45% methanol (V/V), 10% ice second Acid；

Destainer (1000mL)：100mL glacial acetic acids, 250mL ethanol, distilled water complement to 1000mL.

Staining procedure

Room temperature dyeing 45min-60min (or micro-wave oven contaminates soon, high fire 20s, twice)；Cleaned 3-5 times with distilled water；Add Destainer, is placed on 100rpm shaking tables and decolourizes, and per 20min, destainer of replacement is transparent to glue, after the completion of decoloration, uses UMAX Powerlook 2100XL scanners carry out shining glue.

Embodiment seven, enzymatic reaction

Enzymatic reaction using GGPP as substrate

The concentration recombinant C PS albumen supernatant that Example two, step (7) obtain carries out enzymatic reaction.And with pMAL-c2X Empty carrier, pMAL-c2X-AtCPS, pET32a (+)-SmCPS1 conversion Escherichia coli TransB (DE3) expression competent cells, Crush the recombinant protein supernatant being concentrated to give and carry out enzymatic reaction as control.

Enzymatic reaction system is as follows：

Concentrate 182 μ L of recombinant protein supernatant

18 μ L of substrate GGPP (200 μM)

Reaction product is sufficiently mixed (pipette tips piping and druming), reacts 2h under room temperature (25 DEG C), dark environment；After reaction Extracted 3 times with n-hexane, add 0.5mL every time, abandon organic phase, stay water phase (to stay one layer of n-hexane covering water phase, prevent product oxygen Change)；Then the n-hexane in water phase is thoroughly dried up with N2, in order to avoid influence the dephosphorisation reaction of next step.

Dephosphorisation reaction system is as follows：

Reaction product is sufficiently mixed (pipette tips piping and druming), reacts 4h at 37 DEG C；Product after dephosphorization extracts 3 with n-hexane again It is secondary, 0.5mL is added every time, and extracting gained organic phase is merged together；Extracting solution is dried up with N2, and adds 60 μ L n-hexanes Dissolving, is analyzed for GC-MS.

Enzymatic reaction using CPP as substrate

To TwKS carry out functional analysis enzymatic reaction be using through product (CPP) caused by CPS enzymatics GGPP as Reaction substrate, analyzes the chemical constitution of TwKS enzymatic products therefroms, so as to identify TwKS functions.Specific experiment process is as follows：

(1) enzymatic reaction system is formulated as follows, when reaction 2 is small under room temperature (25 DEG C) dark condition, GGPP is fully turned It is melted into CPP；

182 μ L of concentration restructuring KS albumen supernatant

18 μ L of substrate GGPP (200 μM)

(2) isometric TwKS enzymes are added into above-mentioned reaction mixture, and supplement MgCl2 to 10mM, in room temperature (25 DEG C) under dark condition, react overnight (12-16h)；

(3) after reaction, system extracts 3 times (each 0.5mL) with n-hexane, and gained n-hexane is mutually dried up with N2, so After add 60 μ L n-hexane dissolutions carry out GC-MS analyses.

Embodiment eight, reaction product GC-MS detections

GC-MS analysis conditions are：Take the sample introduction of 1 μ L, under the pattern of no shunting, 50 DEG C of holding 2min, 20 DEG C of min-1 liters To 300 DEG C, 20min is kept；250 DEG C of injector temperature, 250 DEG C, electron energy 70ev of ion source temperature, 20- is carried out to sample 650m/z range scans.GC-MS instruments are Thermo Scientific company's T hermo TRACE 1310/TSQ 8000gas Chromatograph, chromatographic column are DB-5ms (30m × 0.25mm).(Fig. 5,6)

Embodiment nine, rite-directed mutagenesis

Rite-directed mutagenesis operation is carried out to TwCPS2, TwKS using full formula gold reagent box Fast Mutagenesis System.

Rite-directed mutagenesis primer designs

In addition to mutational site, two primer lengths about 25-30bp, 5 ' end overlay regions include 15-20bp, 3 ' end extension areas Include at least 10bp；Mutational site is on two primers, respectively positioned at forward mutation assay primer overlay region downstream, close to overlapping Area, inverse transition primer 5 ' are held (see Fig. 3).

Rite-directed mutagenesis primer is designed according to above principle, it is at ' DXDD ' functional domain that TwCPS2DTDC (311) is prominent Become DTDD (311), TwCPS2C311D i.e., by TwKS DADT (318) is mutated into DADD (318), i.e. TwKST318D； At the 608th amino acids of TwKS, amino acid A is mutated into M, i.e. TwKSA608M, rite-directed mutagenesis primer sequence is shown in Table 2.

2 rite-directed mutagenesis primer sequence of table

Rite-directed mutagenesis reacts

Rite-directed mutagenesis reaction system：

(1) PCR reaction conditions：

(2) 10 μ LPCR products, the detection of 1.0% agarose gel electrophoresis are taken.Even if it was observed that a plurality of amplified band, if Purpose band size is correct, can continue with DMT digestion and conversion reaction；

(3) plus 1 μ LDMT enzymes are in PCR product, mix, 37 DEG C of incubation 1h；

(4) 2-5 μ LDMT enzymic digestions products are added in 50 μ LDMT competent cells, are gently mixed, ice bath 30min；

(5) 42 DEG C of accurate heat shock 45s, are immediately placed on 2min on ice；

(6) 250 μ L are added to balance to the LB culture mediums of room temperature, 37 DEG C of 225rpm cultures 1h；

(7) 200 μ L bacterium solution coated plates, 37 DEG C of inversion overnight incubations are taken.

(8) preliminary screening of blue white bacterium colony screening and positive colony, the same 2.2.3.1.4 of method, and sample presentation sequence verification energy Whether mutation succeeds.

Embodiment ten, yeast fermentation

Two diterpene synthase construction of eukaryotic expression vector of tripterygium wilfordii

Using double digestion method respectively by diterpene synthase gene TwCPS1, TwGES2 and combination TwCPS3+TwKS, TwCPS3 + TwKSA608M is building up on carrier for expression of eukaryon pESC-Trp, the same 3.2.1 of method.First by TwCPS1, TwGES2, TwKS, TwKSA608M PCR products (purifying) and vector plasmid pESC-Trp carry out double digestion with restriction enzyme, using NEB companies The orientation connection of T4DNA ligases, connection product is inverted, blue white bacterium colony is screened and the preliminary screening of positive colony, and sample presentation is surveyed Sequence identify, obtain through be sequenced nucleotide sequence without mutation recombinant plasmid pESCTwCPS1, pESCTwGES2, pESCTwKS and PESCTwKSA608M, then by TwCPS3PCR products (purifying) and recombinant plasmid pESCTwKS, pESCTwKSA608M with restricted Restriction endonuclease carries out double digestion, is oriented and connected using 4 DNA ligase of NEB company's Ts, and connection product is inverted, blue white bacterium colony screening And the preliminary screening of positive colony, and sample presentation sequencing identification, obtain through nucleotide sequence is sequenced without mutation recombinant plasmid pESC- (TwCPS3+TwKS)、pESC-(TwCPS3+TwKSA608M).See Fig. 4, primer sequence is shown in Table 3.

Table 3 builds carrier for expression of eukaryon primer sequence

It is prepared by competent yeast

SD-His solid plates：SD-His+2% glucose+4%Agar；Being not added with Agar then becomes respective liquid culture medium； SD-His-Trp solid plates：SD-His-Trp+2% glucose+4%Agar；Being not added with Agar then becomes respective liquid culture medium.

BY-T20 saccharomycete is in defective yeast bacterial strain BY4741 (genotype：MATa his3Δ1 leu2Δ0 0 ura3 Δs 0 of met15 Δs) on the basis of transformed, cause it to efficiently synthesize GGPP, and His can be covered, therefore by BY-T20 yeast Bacterium is coated with SD-His (scarce His) solid plate, and 30 DEG C are inverted culture 72h.

Competent yeast is done using ZYMO RESEARCH Frozen-EZ Yeast Transformation II kits Cell：

(1) from the single bacterium colony that picking newly activates on SD-His tablets, it is inoculated in 10mL SD-His fluid nutrient mediums, 30 Shaken cultivation is to OD600=0.8-1.0 or so at DEG C；

(2) room temperature, 500g centrifugation 4min, removes supernatant；

(3) 1 suspension thallines of 10mL Frozen-EZ Solution are added, room temperature, 500g centrifugation 4min, remove supernatant；

(4) 2 suspension thallines of 1mL Frozen-EZ Solution are added, packing is often managed into the 1.5mL EP pipes to sterilize 50μL；

(5) forbid using liquid nitrogen flash freezer competent cell, answer slow cooling to -70 DEG C (4 DEG C, 1h；- 20 DEG C, 1h；- 40 DEG C, 1h；- 70 DEG C of preservations).

Conversion

(1) 0.2-1 μ g recombinant plasmids (being less than 5 μ L) are taken to be mixed with 50 μ L competent cells；

(2) 500 μ L Frozen-EZ Solution 3 are added, are acutely mixed；

(3) 30 DEG C of incubation 45min, during which mix 2-3 times herein；

(4) bacterium solution for taking 50-150 μ L to be incubated, coating associated disadvantages type SD tablets (SD-His-Trp), after drying, is placed in 30 DEG C are inverted culture 48-96h.

Fermentation

Yeast plasmid extracts and identification

The single bacterium colony to grow on picking SD-His-Trp solid plates, is placed in 10mLSD-His-Trp fluid nutrient mediums In, 30 DEG C of 250rpm 48h；Plasmid is extracted, BY-T20 saccharomycete whether is transferred to specific primer PCR verification recombinant plasmids.

Yeast plasmid extracts (the small extraction reagent kit of Tiangeng company yeast plasmid)：

(1) column equilibration：Into adsorption column CP2, (adsorption column is put into collecting pipe) adds the equilibrium liquid BL of 500 μ L, 12000rpm centrifuges 1min, outwells the waste liquid in collecting pipe, and adsorption column places back in collecting pipe to (angel is worked as in same day processing With)；

(2) 1-5mL yeast cultures are taken, 12000rpm centrifugation 1min, absorb supernatant as far as possible (can pass through when bacterium solution is more Repeatedly centrifuge microorganism collection into a centrifuge tube)；

(3) 250 μ L solution YP1 (having added RNaseA) are added into thalline precipitation is resuspended, thorough suspension thalline, adds straight Footpath be 0.45-0.55mm pickling glass pearl, vortex oscillation 10min；

(4) 250 μ L solution YP2 are added into pipe, leniently spins upside down 6-8 times, thalline is fully mixed, room temperature is placed 5-10min (avoid acutely shaking, in order to avoid contaminating genomic dna)；

(5) 350 μ L solution YP3 are added into pipe, are leniently spun upside down 6-8 times immediately, fully mixes, occurs at this time White flock precipitate, 12000rpm centrifugations 20min；Supernatant fraction is taken, 12000rpm centrifugation 20min are centrifuged again, to obtain nothing The supernatant of minute white precipitation；

(6) carefully supernatant is added in adsorption column CP2 (adsorption column is put into collecting pipe), 12000rpm centrifugation 1min, Waste liquid is outwelled, adsorption column CP2 is put into collecting pipe；

(7) 500 μ L buffer solutions PD, 12000rpm centrifugation 1min are added into adsorption column CP2, outwell waste liquid；

(8) 600 μ L rinsing liquids (having added absolute ethyl alcohol) are added into adsorption column CP2,12000rpm centrifugation 1min, are outwelled Waste liquid, adsorption column CP2 is put into collecting pipe；

(9) repeat step 8；

(10) adsorption column CP2 is put into collecting pipe and is placed in 12000rpm centrifugation 2min, remove the rinsing liquid of remnants；

(11) adsorption column CP2 is placed in a clean 1.5mLEP pipe, 50-100 is added dropwise to the middle part of adsorbed film μ LddH2O, room temperature place 2min, and plasmid solution is collected into EP pipes by 12000rpm centrifugations 2min, preserves -20 DEG C.

Extract plasmid be used for PCR identification recombinant plasmid whether be transferred in BY-T20 saccharomycete.

PCR reaction systems：

PCR reaction conditions：

(remarks：* the fragment ＞ 3kb of PCR product are represented, and often add 1kb then elongating temperature extension 1min)

1.0% agarose gel electrophoresis detects, if there is purpose fragment band, can prove that recombinant plasmid is successfully transferred to Thalline.

Galactolipin induction fermentation

Induction type SD-His-Trp fluid nutrient mediums：SD-His-Trp+2% galactolipins (filtration sterilization)

(1) the positive bacterium solution (the BY-T20 saccharomycete that i.e. recombinant plasmid is successfully transferred to) for identifying PCR, transfers into 50mL In SD-His-Trp fluid nutrient mediums, 30 DEG C of 250rpm cultivate 12-16h；

(2) thalline is collected by centrifugation in room temperature 5000g 5min, is transferred to 100mL induction type SD-His-Trp fluid nutrient mediums In, 30 DEG C of 250rpm induce 72h；

Tunning extracts

Target component is terpenoid, fat-soluble, is soluble in n-hexane, therefore it is solvent extraction target to choose n-hexane Terpenoid.Extraction step is as follows：

(1) collect fermentation and complete bacterium solution, add isometric n-hexane；

(2) carrying out ultrasonic bacteria breaking 1h, during which multiple oscillation, which mixes, shakes；

(3) separatory funnel is extracted twice；The extract of mixing twice, adds (the 120 DEG C of drying of suitable anhydrous sodium sulfate 30min), side edged shakes, the moisture for extract of going out；

(4) it is concentrated on Rotary Evaporators and closely does,

(5) concentrate is drawn, crosses 0.22 μm of PTFE syringe needle filter, filtered fluid is stored in liquid phase bottle, and sealed membrane is close Close, be stored in 4 DEG C of refrigerators.

Extract GC-MS is detected

GC-MS analysis conditions are：Take the sample introduction of 1 μ L, under the pattern of no shunting, 50 DEG C of holding 2min, 20 DEG C of min-1 liters To 300 DEG C, 20min is kept；250 DEG C of injector temperature, 250 DEG C, electron energy 70ev of ion source temperature, 20- is carried out to sample 650m/z range scans.GC-MS instruments are Thermo Scientific company's T hermo TRACE 1310/TSQ 8000gas Chromatograph, chromatographic column are DB-5ms (30m × 0.25mm).(Fig. 7,8,9)

Described above is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common Technical staff is in the essential scope of the present invention, the variations, modifications, additions or substitutions made, should also belong to the protection of the present invention Scope, protection scope of the present invention are subject to claims.

SEQUENCE LISTING

<110>The Capital University of Medical Sciences

<120>Tripterygium wilfordii TwKS and applications of the TwCPS3 in Kaurane diterpine compound is prepared

<130>Nothing

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 816

<212> PRT

<213>Tripterygium wilfordii

<400> 1

Met Val Ile Met Ser Ser His Gln Ile Phe Ser Val Ser Ser Ser Ser

1 5 10 15

Tyr Leu His Pro Pro Leu Leu Phe Pro Gly Leu Ser Ser Tyr Thr Thr

20 25 30

Lys Asp Lys Arg Val Cys Tyr Phe Asp Ser Thr Arg Leu Ile Cys Arg

35 40 45

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

50 55 60

Gln Asn Gly Leu Pro Leu Ile Lys Trp Arg Glu Ile Val Glu Asp Asp

65 70 75 80

Ile Gln Glu Gln Glu Glu Pro Leu Lys Val Ser Leu Glu Asn Gln Ile

85 90 95

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

100 105 110

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

115 120 125

Glu Asn Ile His His Pro Thr Gly Ser Pro Gln Phe Pro Ser Thr Leu

130 135 140

Gln Trp Ile Ala Asn Asn Gln Leu Pro Asp Gly Ser Trp Gly Asp Pro

145 150 155 160

Tyr Val Phe Leu Ala His Asp Arg Leu Ile Asn Thr Leu Ala Cys Val

165 170 175

Ile Ala Leu Lys Lys Trp Asn Ile His Pro Asn Lys Tyr Lys Lys Gly

180 185 190

Leu Ser Phe Val Lys Glu Asn Ile Ser Lys Leu Glu Lys Glu Asn Glu

195 200 205

Glu His Met Pro Ile Gly Phe Glu Ile Ala Phe Pro Ser Leu Leu Glu

210 215 220

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

225 230 235 240

Gln Asp Ile Tyr Ala Lys Arg Asp Leu Lys Leu Thr Arg Ile Pro Lys

245 250 255

Asp Ile Met His Asn Val Pro Thr Thr Leu Leu His Ser Leu Glu Gly

260 265 270

Leu Pro Gly Leu Asp Trp Glu Lys Leu Val Arg Leu Gln Cys Lys Asp

275 280 285

Gly Ser Phe Leu Phe Ser Pro Ser Ser Thr Ala Cys Ala Leu Met His

290 295 300

Thr Lys Asp Gly Asn Cys Phe Ser Tyr Leu Asn Asn Met Val Gln Lys

305 310 315 320

Phe Asn Gly Gly Val Pro Asn Val Tyr Pro Val Asp Leu Phe Glu His

325 330 335

Ile Trp Ser Val Asp Arg Leu Leu Arg Leu Gly Ile Ser Arg Phe Phe

340 345 350

Gln Pro Glu Ile Lys Gln Cys Leu Asp His Val His Arg Tyr Trp Thr

355 360 365

Lys Asp Gly Ile Cys Trp Ala Arg Asn Ser His Val Gln Asp Ile Asp

370 375 380

Asp Thr Ser Met Gly Phe Arg Leu Leu Arg Leu His Gly Tyr Glu Val

385 390 395 400

Ser Pro Asp Val Phe Lys Gln Phe Arg Lys Gly Asp Glu Phe Val Cys

405 410 415

Phe Ala Gly Gln Ser Asn Gln Ala Ile Thr Gly Ile Tyr Asn Leu Tyr

420 425 430

Arg Ala Ser Gln Leu Met Phe Pro Glu Glu Pro Ile Leu Val Glu Ala

435 440 445

Lys Lys Phe Ala Arg Asn Phe Leu Arg Glu Lys Arg Ala Val Asn Glu

450 455 460

Leu Leu Asp Lys Trp Ile Ile Thr Lys Asp Leu Pro Gly Glu Val Gly

465 470 475 480

Phe Ala Leu Asp Val Pro Trp Tyr Ala Cys Leu Pro Arg Val Glu Thr

485 490 495

Arg Leu Tyr Ile Glu Gln Tyr Gly Gly Gln Asp Asp Val Trp Ile Gly

500 505 510

Lys Ser Leu Tyr Arg Met Pro Tyr Val Asn Asn Asn Ile Tyr Leu Glu

515 520 525

Leu Ala Lys Leu Asp Tyr Asn Asn Cys Gln Ser Leu His Arg Thr Glu

530 535 540

Trp Asp Asn Ile Gln Glu Trp Tyr Glu Glu Tyr Asn Val Gly Gly Phe

545 550 555 560

Gly Val Ser Lys Arg Ser Leu Leu Lys Thr Tyr Phe Val Ala Thr Ala

565 570 575

Ser Ile Phe Glu Pro Glu Arg Ser Val Glu Arg Leu Val Trp Thr Lys

580 585 590

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

595 600 605

Glu Glu Arg Ile Glu Phe Val Asn Glu Phe Gln Lys Phe Pro Lys Thr

610 615 620

Arg Gly Tyr Ile Lys Gly Arg Arg Leu Asp Gly Lys Met Ala Thr Lys

625 630 635 640

Glu Val Ile Glu Met Val Phe Ala Ala Leu Asn His Phe Ser Leu Asp

645 650 655

Ser Leu Val Val His Gly Gln Asp Ile Thr His His Leu Tyr Gln Ser

660 665 670

Trp Val Lys Trp Val Leu Thr Trp Gln Glu Glu Gly Asp Arg Arg Glu

675 680 685

Gly Glu Ala Glu Leu Leu Val Gln Thr Ile Asn Leu Met Ala Gly His

690 695 700

Thr Pro Ser Gln Glu Leu Leu Tyr Glu Arg Leu Phe Lys Leu Thr Asn

705 710 715 720

Lys Val Cys His Gln Leu Gly His Tyr His His Leu Asn Lys Asp Lys

725 730 735

Gln Pro Gln Gln Val Glu Asp Asn Gly Gly Tyr Asn Asn Ser Asn Pro

740 745 750

Glu Ser Ile Ser Lys Leu Gln Ile Glu Ser Asp Met Arg Glu Leu Val

755 760 765

Gln Leu Val Leu Asn Ser Ser Asp Gly Met Asp Ser Asn Ile Lys Gln

770 775 780

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

785 790 795 800

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

805 810 815

<210> 2

<211> 2748

<212> DNA

<213>Tripterygium wilfordii

<400> 2

acacagagtt agacaaagtg aatagttttg gtttaggatg atcccatgca cctattagcg 60

ggaatctcct gggaagtcgt ttctgtcttg ttgccctggc ccgccacctc ctccctctta 120

ttacttttga atccttttaa gccccctttt tgctctgtct ctctctctct ctctgtgtct 180

ctgcaaataa acattaacac aaatccaatc aatcatggtg atcatgtcct ctcatcaaat 240

cttctctgtt tcttcctcct cctaccttca ccctcctctg cttttcccag gattaagttc 300

ctacactact aaagacaaac gagtctgcta ctttgatagc actcgcttaa tctgccgtgc 360

aatatccaag cctgctcgca caactccaga atactcgggc gtgcttcaaa acggtctgcc 420

attgataaag tggcgtgaga ttgtggagga tgacatacaa gaacaagaag aacctcttaa 480

ggtttccttg gaaaaccaga ttagacaagg tgtcgacatt gttaagtcga tgttgggatc 540

aatggaggat ggcgagatta gcatttcagc ttatgacact gcatgggttg cacttgttga 600

aaacattcac cacccaactg gttctcctca attcccatcc actcttcaat ggattgccaa 660

caatcagctc cccgatggct cctggggtga tccctacgtg ttcttggcac acgatcgatt 720

aatcaatacg ttagcttgtg ttatcgctct caagaaatgg aatattcacc ccaacaaata 780

caaaaaagga ttgtcgtttg tgaaagagaa cataagcaag ctagaaaagg agaatgagga 840

gcacatgcca ataggatttg aaatcgcttt cccatcactt cttgagatgg ctaaaaaact 900

agggattgaa atacctgatg attgtccagc cttgcaagac atctatgcca agagagatct 960

caaactcaca aggataccaa aggacataat gcataatgtg cccacaacac tactccatag 1020

cttggaagga ttgccaggcc tggattggga aaagcttgta agattgcaat gcaaagatgg 1080

gtcattcttg ttctctccat cttccactgc ctgtgcactc atgcacacta aagatggcaa 1140

ttgcttctcc tacttgaaca acatggtaca aaagttcaat ggaggagtac cgaatgtcta 1200

cccggttgac ctgttcgaac atatttggtc tgttgatcgc ttgctacgcc taggaatctc 1260

cagatttttt cagccagaaa tcaaacaatg tcttgatcat gttcacagat attggaccaa 1320

agacggaatt tgttgggcaa gaaattcgca tgttcaagat attgatgaca catcaatggg 1380

attcaggttg cttagattgc atggatacga ggtttctcct gacgtgttta agcaatttag 1440

aaaaggcgac gagttcgttt gtttcgcggg acaatcaaat caggcgatta ctggaatcta 1500

taacctgtat agggcttctc aattgatgtt ccctgaagag cctattcttg tggaagccaa 1560

aaaatttgca agaaatttct tgagggaaaa aagagctgtt aatgaactac tagataaatg 1620

gatcataacc aaagatttac caggcgaggt ggggtttgcc ttggatgttc catggtacgc 1680

atgcttgcct agagttgaga cgaggcttta cattgaacaa tacggtggcc aagatgacgt 1740

atggattggc aagtctcttt acaggatgcc ctatgtcaac aacaatatat atctggagct 1800

agcaaagcta gactacaaca attgccaatc cttgcatcgc actgaatggg acaatatcca 1860

agagtggtac gaagaataca atgttggagg atttggggtc agcaaaagga gccttcttaa 1920

gacatatttt gtagctacag ccagtatttt tgaacccgaa agatcggtag agcgactcgt 1980

ttggacaaag actgcaatct tgttagaagc tattgcatct tattttaaaa attctagaga 2040

agaaaggatt gaatttgtaa atgaattcca aaaatttccc aagacaagag gttacattaa 2100

aggaaggaga ttagatggaa agatggcaac aaaagaagtt attgagatgg tgtttgcagc 2160

actaaatcat ttttcattgg attcattggt ggtgcatggt caagacatta ctcaccattt 2220

atatcaatcc tgggtaaagt gggtgctgac gtggcaagag gaaggagaca gaagagaagg 2280

agaagcagag ctattggtgc agacaataaa cttgatggct ggccacacac cctctcagga 2340

gttgttgtat gagcgactct tcaaactcac aaacaaggtt tgccatcagc ttggtcacta 2400

tcaccacctc aacaaagaca aacaacctca acaggtagaa gacaatggag gatacaataa 2460

ttcaaaccct gaaagcattt caaagctcca aatagagagt gacatgagag agctagtgca 2520

gctggtacta aattcttcag atggcatgga ctctaatatc aagcaaacat tcttaacagt 2580

cacaaagagc ttttactaca atgctttcac tcatcctggg actgtcaact accatattgc 2640

caaagtactc tttgaaagag tgtaagatta gttgacattt gaatattgaa aataatgttg 2700

aggtcattgt agataaccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2748

<210> 3

<211> 781

<212> PRT

<213>Tripterygium wilfordii

<400> 3

Met Met Ser Leu Ser His Pro Asn Cys Ile Arg His Ser Ser Leu Pro

1 5 10 15

Ile Ser Ala Thr Leu Pro Lys Ser Lys Ser Glu Val Leu Thr Glu Thr

20 25 30

Asp Ala Thr Ile Leu Tyr Phe Gln Glu Thr Lys Glu Arg Ile Lys Lys

35 40 45

Met Phe Asp Lys Ala Glu Leu Ser Val Ser Ala Tyr Asp Thr Ala Trp

50 55 60

Val Ala Met Val Ser Ser Pro Asn Ser His Gln Ala Pro Cys Phe Pro

65 70 75 80

Glu Cys Val Asn Trp Leu Leu Asp Asn Gln Leu Ser Asp Gly Ser Trp

85 90 95

Gly Leu Pro Pro His His Pro Ser Leu Val Lys Asp Ala Leu Ser Ser

100 105 110

Thr Leu Ala Cys Leu Leu Ala Leu Lys Arg Trp Gly Leu Gly Glu Gln

115 120 125

Gln Met Thr Lys Gly Leu Gln Phe Ile Glu Ser Asn Phe Thr Ser Ile

130 135 140

Asn Asp Glu Glu Gln His Thr Pro Ile Gly Phe Asn Ile Ile Phe Pro

145 150 155 160

Gly Met Ile Glu Thr Ala Ile Asp Met Asn Leu Asn Leu Pro Leu Arg

165 170 175

Ser Glu Asp Ile Asn Val Met Leu His Asn Arg Asp Leu Glu Leu Arg

180 185 190

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

195 200 205

Gly Met Gly Lys Leu Gln Asp Trp Glu Met Val Met Lys Tyr Gln Arg

210 215 220

Lys Asn Gly Ser Leu Phe Asn Ser Pro Ser Thr Thr Ala Ala Ala Leu

225 230 235 240

Ser Gln Leu Gly Asn Ala Gly Cys Phe His Tyr Ile Asn Ser Leu Ile

245 250 255

Ala Lys Phe Gly Asn Ala Val Pro Thr Val Tyr Pro Ser Asp Lys Tyr

260 265 270

Ala Leu Leu Cys Met Ile Glu Ser Leu Glu Arg Leu Gly Ile Asp Ser

275 280 285

His Phe Ser Gln Glu Ile Arg Asp Val Leu Glu Glu Thr Tyr Arg Cys

290 295 300

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

305 310 315 320

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

325 330 335

Pro Leu Thr Gln Tyr Ala Glu His His Phe Ser Arg Ser Phe Gly Gly

340 345 350

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

355 360 365

Phe Val Ile Phe Pro Glu Glu Ser Gly Leu Glu Lys Gln Met Ser Trp

370 375 380

Thr Asn Gln Phe Leu Lys Gln Glu Phe Ser Asn Gly Thr Thr Arg Ala

385 390 395 400

Asp Arg Phe Arg Lys Tyr Phe Ser Ile Glu Val His Asp Thr Leu Lys

405 410 415

Phe Pro Phe His Ala Asn Val Glu Arg Leu Ala His Arg Arg Asn Ile

420 425 430

Glu His His His Val Asp Asn Thr Arg Ile Leu Lys Thr Ser Tyr Cys

435 440 445

Phe Ser Asn Ile Ser Asn Ala Asp Phe Leu Gln Leu Ala Val Glu Asp

450 455 460

Phe Asn Arg Cys Gln Ser Ile His Arg Glu Glu Leu Lys His Leu Glu

465 470 475 480

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

485 490 495

Lys Met Ala Tyr Cys Tyr Phe Ser Ala Ala Gly Thr Cys Phe Ser Pro

500 505 510

Glu Leu Ser Asp Ala Arg Ile Ser Trp Ala Lys Asn Ser Val Leu Thr

515 520 525

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

530 535 540

Ala Asn Leu Val His Leu Leu Glu Asn Trp Asp Ala Asn Gly Ser Pro

545 550 555 560

His Tyr Cys Ser Glu Pro Val Glu Ile Ile Phe Ser Ala Leu Arg Ser

565 570 575

Thr Ile Cys Glu Ile Gly Asp Lys Ala Leu Ala Trp Gln Gly Arg Ser

580 585 590

Val Thr His His Val Ile Glu Met Trp Leu Asp Leu Leu Lys Ser Ala

595 600 605

Leu Arg Glu Ala Glu Trp Ala Arg Asn Lys Val Val Pro Thr Phe Asp

610 615 620

Glu Tyr Val Glu Asn Gly Tyr Val Ser Met Ala Leu Gly Pro Ile Val

625 630 635 640

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

645 650 655

Arg Ser Pro Glu Phe His Asn Leu Phe Lys Leu Met Ser Ile Cys Gly

660 665 670

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

675 680 685

Lys Leu Asn Ser Val Leu Leu His Met Ile His Ser Gly Ser Gly Thr

690 695 700

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

705 710 715 720

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

725 730 735

Pro Arg Ala Cys Lys Asp Leu Phe Trp Lys Met Val Gln Val Leu His

740 745 750

Leu Phe Tyr Met Asp Gly Asp Gly Phe Ser Ser Pro Asp Met Met Leu

755 760 765

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

770 775 780

<210> 4

<211> 2548

<212> DNA

<213>Tripterygium wilfordii

<400> 4

aagtctggtg catgatttga agaaaattat tatttgggca gagaggggac tctactgacc 60

actgagtaat attcttcata tatagcttgt ttgggattct ggtgccatga tgtctctatc 120

gcatcccaat tgtattaggc actcctcttt accaatatca gcaaccttac cgaagagtaa 180

atcagaagta ctgaccgaaa ccgatgccac cattctgtat tttcaagaga ctaaagaaag 240

aattaagaag atgtttgata aggccgaact ctctgtttcg gcatatgaca ccgcttgggt 300

agcaatggta tcatctccta attcccatca ggccccttgc ttccctgagt gcgtaaattg 360

gttgttggat aatcaactct ctgatggttc ttggggtctt cctcctcatc atccctcgtt 420

agttaaagat gctttgtcat ccactttagc atgtctcctt gctttgaagc gatggggtct 480

tggtgaacaa caaatgacaa aaggactcca atttattgaa tcaaatttca cttcaattaa 540

tgatgaggag caacacaccc caattggatt caatataatt tttcctggga tgattgagac 600

tgctatagat atgaatttga atcttcctct aagatcggaa gacataaatg tgatgcttca 660

caatagagac ttggagctta ggagaaataa attcgaggga agggaagcgt acttagccta 720

tgtttccgaa ggaatgggaa aattacagga ttgggaaatg gttatgaaat atcagagaaa 780

gaatgggtca ctgttcaatt caccgtctac tactgcagct gctcttagcc aacttggaaa 840

tgctggttgt tttcattaca tcaactcact tatagcaaag tttgggaatg cagttccgac 900

agtttatcct tctgataaat atgctcttct ttgtatgatt gagagtcttg agaggctggg 960

aattgatagt catttcagtc aggaaattag agatgtactg gaggaaacct acagatgctg 1020

gttgcaggga gatgaagaga tattttccga tgctgacaca tgtgccatgg catttcgaat 1080

attacgtgta catggatatg aagtctcttc agatccatta actcaatatg ctgaacatca 1140

tttctcccgt tcttttggag gccatttgaa ggattttagt actgccttgg agttattcaa 1200

agcttcacaa tttgtaatat tcccagagga atcaggtctc gaaaaacaga tgtcgtggac 1260

aaatcaattt ctaaagcagg aattctctaa tgggacaact cgtgcagata gattccgcaa 1320

atattttagc atagaagtac atgatactct taagtttccc ttccatgcaa atgtggaacg 1380

cttagcacac aggagaaata tagagcatca ccatgtagat aacacaagaa tattaaagac 1440

ttcatattgt ttttcaaata tcagcaacgc tgattttctg caactggcgg tggaagactt 1500

caataggtgc caatcaatac accgcgaaga actcaaacat cttgagaggt gggttgtgga 1560

gaccaaatta gacaggctaa agtttgctag acagaaaatg gcatactgtt acttttctgc 1620

tgctggaaca tgcttctctc ctgaactatc agatgctcgc atctcatggg ccaaaaacag 1680

tgtgcttaca actgttgccg acgacttctt tgacattgtg gggtcggaag aggaactggc 1740

aaaccttgta cacttgcttg agaattggga tgcaaatggt agccctcatt attgttctga 1800

gccagttgaa atcatatttt cagcacttcg tagcacaatt tgtgagatcg gagacaaagc 1860

attagcatgg caagggagaa gtgtgacaca ccatgttatt gagatgtggt tggatttgct 1920

aaagtctgcg ttaagggaag ccgagtgggc gagaaacaaa gtagtgccaa cattcgatga 1980

atacgtggaa aatggatatg tgtctatggc cttgggacca attgttcttc cagcagtcta 2040

tctcatcggg cctaaagttt cagaggaggt tgtccgaagc cctgaattcc ataacctctt 2100

caaacttatg agcatttgtg gacgtctaat caatgacacc cgcacattta agagggaatc 2160

cgaggctggg aaattgaatt ctgttttgtt gcacatgatc cacagtggta gtggaacaac 2220

ggaggaagaa gctgttgaaa agattagagg tttgattgcg gatggacaaa gagaactgct 2280

aaggctagtg ttgcaggaga aagacagcat agttcctaga gcttgcaagg acttgttctg 2340

gaaaatggtc caagtgttgc acctatttta catggatgga gatggattct cttcacctga 2400

catgatgctc aatgcagtaa acgctttgat acgtgaaccc atctctctat aattgcaggc 2460

tctacccttt acccattgaa aggattttac aataaattgg gatggattgt gtgttttcag 2520

ccaaaaaaaa aaaaaaaaaa aaaaaaaa 2548

<210> 5

<211> 33

<212> DNA

<213>Artificial sequence

<400> 5

cgcggatcca tggtgatcat gtcctctcat caa 33

<210> 6

<211> 36

<212> DNA

<213>Artificial sequence

<400> 6

gcactgcagt tacactcttt caaagagtac tttggc 36

<210> 7

<211> 35

<212> DNA

<213>Artificial sequence

<400> 7

cgcggatcca tgatgtctct atcgcatccc aattg 35

<210> 8

<211> 39

<212> DNA

<213>Artificial sequence

<400> 8

cgcgtcgact tatagagaga tgggttcacg tatcaaagc 39

<210> 9

<211> 39

<212> DNA

<213>Artificial sequence

<400> 9

cgcggatcca tgtctcttca gtatcatgtt ctaaactcc 39

<210> 10

<211> 37

<212> DNA

<213>Artificial sequence

<400> 10

gcactgcagc tagacttttt gaaacaagac tttggag 37

<210> 11

<211> 32

<212> DNA

<213>Artificial sequence

<400> 11

cgcggatcca tgtctatcaa ccttcgctcc tc 32

<210> 12

<211> 35

<212> DNA

<213>Artificial sequence

<400> 12

cgcgtcgact caagttaaag attcttcctg taagc 35

<210> 13

<211> 32

<212> DNA

<213>Artificial sequence

<400> 13

cgcagcttcg cgataccgat gataccgcca tg 32

<210> 14

<211> 36

<212> DNA

<213>Artificial sequence

<400> 14

tcatcggtat cgcgaagctg cgaatttctt gtccag 36

<210> 15

<211> 35

<212> DNA

<213>Artificial sequence

<400> 15

gagatatttt ccgatgctga cgattgtgcc atggc 35

<210> 16

<211> 37

<212> DNA

<213>Artificial sequence

<400> 16

atcgtcagca tcggaaaata tctcttcatc tccctgc 37

<210> 17

<211> 32

<212> DNA

<213>Artificial sequence

<400> 17

gttggatttg ctaaagtcta tgttaaggga ag 32

<210> 18

<211> 32

<212> DNA

<213>Artificial sequence

<400> 18

atagacttta gcaaatccaa ccacatctca at 32

<210> 19

<211> 35

<212> DNA

<213>Artificial sequence

<400> 19

cgcggatccg atgcatagtt tgttaatgaa aaagg 35

<210> 20

<211> 36

<212> DNA

<213>Artificial sequence

<400> 20

tcggggccca aatctactct ctcaaagagt actttg 36

<210> 21

<211> 37

<212> DNA

<213>Artificial sequence

<400> 21

cgcggatccg atggtgatca tgtcctctca tcaaatc 37

<210> 22

<211> 35

<212> DNA

<213>Artificial sequence

<400> 22

tcggggccca acactctttc aaagagtact ttggc 35

<210> 23

<211> 37

<212> DNA

<213>Artificial sequence

<400> 23

atttgcggcc gcaatgatgt ctctatcgca tcccaat 37

<210> 24

<211> 37

<212> DNA

<213>Artificial sequence

<400> 24

cccatcgata ctagagagat gggttcacgt atcaaag 37

<210> 25

<211> 37

<212> DNA

<213>Artificial sequence

<400> 25

atttgcggcc gcaatgatgt ctctatcgca tcccaat 37

<210> 26

<211> 37

<212> DNA

<213>Artificial sequence

<400> 26

cccatcgata ctagagagat gggttcacgt atcaaag 37

<210> 27

<211> 34

<212> DNA

<213>Artificial sequence

<400> 27

cgcggatccg atggattttt cagattcctc aatt 34

<210> 28

<211> 35

<212> DNA

<213>Artificial sequence

<400> 28

tcggggccca aaatgaaaca tggtgagaat tttgg 35

Claims (12)

1. tripterygium wilfordii Ke Baji pyrophosphate synthases T_wCPS3 and tripterygium wilfordii kaurene synthase TwKS are preparing Kaurane diterpine Application in ent-16 α kaurenes, it is characterised in that using Mang ox base pyrophosphoric acid GGPP as substrate, catalysis produces kaurene Type diterpene compound ent-16 α kaurenes；

Wherein, the sequence of the tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3 is SEQ ID NO：1, tripterygium wilfordii kaurene The sequence of synthase TwKS is SEQ ID NO：3.

2. tripterygium wilfordii Ke Baji pyrophosphate synthase gene Twcps3 and tripterygium wilfordii kaurene synthase gene Twks are preparing shell Application in China fir alkane type diterpene ent-16 α kaurenes, it is characterised in that recombination expression Twcps3 and Twks genes obtain TwCPS3 and TwKS is used to prepare Kaurane diterpine ent-16 α kaurenes, wherein tripterygium wilfordii Ke Baji pyrophosphate synthases The sequence of gene Twcps3 is SEQ ID NO：The sequence of 2, tripterygium wilfordii kaurene synthase gene Twks are SEQ ID NO：4.

3. tripterygium wilfordii Ke Baji pyrophosphate synthase gene Twcps3 described in claim 2 and tripterygium wilfordii kaurene synthase gene Applications of the Twks in Kaurane diterpine ent-16 α kaurenes are prepared, wherein Twcps3 the and Twks genes pass through Prokaryotic expression or eukaryotic expression obtain TwCPS3 and TwKS.

4. tripterygium wilfordii Ke Baji pyrophosphate synthase gene Twcps3 described in Claims 2 or 3 and tripterygium wilfordii kaurene synthase base Because of applications of the Twks in Kaurane diterpine ent-16 α kaurenes are prepared, divided using prokaryotic expression carrier pMAL-c2X The recombinant prokaryotic expression vector of Twcps3 and Twks genes Gou Jian not carried；Built respectively using carrier for expression of eukaryon pESC-Trp Recombinant eukaryon expression vector with Twcps3 and Twks genes.

5. application as claimed in claim 1, it is characterised in that manool is further included in product.

6. application as claimed in claim 2, it is characterised in that manool is further included in product.

7. application as claimed in claim 1, it is characterised in that tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3/ or its gene Twcps3, uses or successively uses at the same time with tripterygium wilfordii kaurene synthase TwKS/ or its gene Twks.

8. application as claimed in claim 2, it is characterised in that tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3/ or its gene Twcps3, uses or successively uses at the same time with tripterygium wilfordii kaurene synthase TwKS/ or its gene Twks.

9. a kind of method that enzymatic prepares Kaurane diterpine ent-16 α kaurenes, it comprises the following steps：

(1) tripterygium wilfordii Ke Baji pyrophosphate synthase TwCPS3, n-hexane extraction 3 are added by substrate of Mang ox base pyrophosphoric acid GGPP It is secondary, organic phase, water intaking phase are abandoned, dephosphorization is carried out after nitrogen drying；

(2) product obtained into step (1) after dephosphorization adds tripterygium wilfordii kaurene synthase TwKS and is reacted, n-hexane extraction Take 3 times, take n-hexane mutually to be dried up with nitrogen；

(3) GC-MS analyses are carried out to the product that step (2) obtains, the 13.96th point of peak is ent-16 α kaurenes；

Wherein, the sequence of the tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3 is SEQ ID NO：1, tripterygium wilfordii kaurene The sequence of synthase TwKS is SEQ ID NO：3.

10. a kind of method that enzymatic prepares Kaurane diterpine ent-16 α kaurenes, it comprises the following steps：

(1) Twcps3 expression casettes and Twks expression casettes are building up on same carrier for expression of eukaryon pESC-Trp, Structure obtains recombinant plasmid pESC- (Twcps3+Twks), and transformed yeast screening obtains recombinant yeast；

(2) the positive recombinant yeast of PCR identifications is subjected to Fiber differentiation, collects bacterium solution and add isometric n-hexane, ultrasound is broken Bacterium, extraction, sodium sulphate drying, concentrated by rotary evaporation are done near, are saved backup for 4 DEG C after filtering；

(3) GC-MS analyses are carried out to the product that step (2) obtains, the 13.96th point of peak is ent-16 α kaurenes；

Wherein, the sequence of the tripterygium wilfordii Ke Baji pyrophosphate synthases TwCPS3 is SEQ ID NO：1, tripterygium wilfordii kaurene The sequence of synthase TwKS is SEQ ID NO：3.

11. the method that enzymatic as claimed in claim 9 prepares Kaurane diterpine ent-16 α kaurenes, its feature exist The 12.99th point of peak is manool in the step of GC-MS is analyzed, and the 13.03rd point of peak is ent- kaurenes.

12. the method that enzymatic as claimed in claim 10 prepares Kaurane diterpine ent-16 α kaurenes, its feature exist The 12.99th point of peak is manool in the step of GC-MS is analyzed, and the 13.03rd point of peak is ent- kaurenes.