CN108315343A - A kind of method for synthesizing gene of production cyanidenon yeast strain and bacterial strain and application - Google Patents

Abstract

The present invention discloses method for synthesizing gene and bacterial strain and the application of a kind of production cyanidenon yeast strain, obtains fleabane flower genome cDNA and clones to obtain II gene of PAL, C4H, 4CL, CHS, CHI, FS through primer；Saccharomyces cerevisiae genome clones to obtain ADH2, ALD6, ACS, ACCI gene；With Golden gate clone technologies by the promoter and terminator plasmid construction T1 of said gene and primer amplification to T5 recombinant vectors；The two-way gene amplification fragment of recombinant vector is integrated into saccharomyces cerevisiae genome routine integration site and obtains recombinant bacterial strain by conventional method；Recombinant bacterial strain must be integrated by recombinating and being integrated into recombinant bacterial strain chromosome by homology arm with shuttle plasmid after F3 ' H, CPRI Gene Fusions；Integrate the yeast strain that recombinant bacterial strain screens to produce cyanidenon through auxotroph culture medium C M Trp.The present invention has the characteristics that cyanidenon yield height, synthetic method is environmentally protective.

Description

A kind of method for synthesizing gene of production cyanidenon yeast strain and bacterial strain and application

Technical field

The invention belongs to biotechnologies, and in particular to a kind of cyanidenon yield is high, at low cost, synthetic method is green The method for synthesizing gene and bacterial strain of the production cyanidenon yeast strain of environmental protection being transformed by gene chemical synthesis and application.

Background technology

Saccharomyces cerevisiae（Saccharomyces cerevisiae）It is first completion gene order-checking in eucaryote Model organism.Saccharomyces cerevisiae due to understanding with genetic background, higher homologous recombination efficiency, can preferably express P450 The advantages such as enzyme, good biological safety, genetic stability during the fermentation, are the platform bacterial strains being commonly used at present.

Cyanidenon（Luteolin）It is a kind of flavone compound of the generations such as plant response environment stress, because being initially From reseda（ResedaodorataL.）The tissues such as leaf, stem, branch in isolate and gain the name.It is distributed more widely in nature It is general, it is primarily present in numerous crude drugs such as tealeaves, honeysuckle, chrysanthemum, ginkgo, Perilla, Scutellaria.Cyanidenon has Valuable pharmacological effect and multiple biological activities, be the good natural drug for treating angiocardiopathy, the diseases such as hepatitis, have become For the prescription medicine of antiseptic, antioxidant, antitumor agent etc., it is existing that tight market demand has been presented in its yield in recent years Shape.First, using Resedaceae as the herbaceous plant of representative it is 1 year or perennial, growth cycle is long, and traditional planting scale is small. Secondly, chemical synthetic drug is to the pollution of the mankind and very harmful, and outlet exportation abroad especially European Union member countries are to Chinese products Solvent requirement is made that stringent limitation.Therefore, excellent short, at low cost, environmentally protective with its period of biosynthesis cyanidenon Gesture has obtained more and more extensive uses.

The present Research of cyanidenon synthesis：

Cyanidenon chemical formula：C₁₅H₁₀O₆；Chemical name：3', 4', 5,7- Luteolin（3',4',5,7- Tetrahydroxyflavone）.Cyanidenon（luteolin）It is a kind of representative natural fiavanoids compound, has good Good physiological activity, because plant extraction process has the shortcomings that content is low, and extraction yield is extremely low, cannot meet the market demand. Currently, cyanidenon is constantly explored by researcher in decades, fully synthetic and semisynthesis is generally used, to Meet market needs.

A. fully synthetic approach：Early in 1980, Lu Yuhua etc. was protected first using phloroglucin as raw material in chloromethyl methyl ether Group is protected to the lower first synthesizing chalcone precursor of the phenolic hydroxyl group protection of corresponding aldehyde, ketone, the compounds such as final obtained polyhydroxy flavones.It should Method time loss is mainly preparing chalcone reaction, which needs to undergo 6 day time, and time-consuming longer, total recovery but only has 3 ~ 4%, yield is relatively low.Fully synthetic route was transformed equal to 2003 through the dagger-axe summer, total recovery reaches 22%, and the time is shortened.

B. semi-synthetic approach：1998, Sun Zhizhong etc. was using dried orange peel extracts aurantiamarin as raw material, through using hydrolysis, taking off Methyl, dehydrogenation three-step reaction obtain the final product cyanidenon of tool physiological activity, total yield of products about 45%.And Peng Bin etc. It uses within 2009 and carries out microwave synthesis by raw material of rutin, it is 95% that purity, which is made, in the 1.5h that flows back under 300W microwave powers Cyanidenon, yield are up to 83.9%, and it is 99.4% product that purity is obtained after recrystallization.However, this research also be not scale, The example of complete approach production cyanidenon, therefore how to realize that the industrialized production of cyanidenon is increasingly paid close attention to as people Hot spot.

Invention content

The first object of the present invention is to provide that cyanidenon yield is high, at low cost, synthetic method is environmentally protective to be passed through The method for synthesizing gene of the production cyanidenon yeast strain of gene chemical synthesis transformation；The second object of the present invention is to provide a kind of the The bacterial strain that one mesh generates；Third of the present invention is designed to provide a kind of application of the bacterial strain of second mesh.

What the first object of the present invention was realized in：Including the extraction of fleabane flower tissue DNA, S. cervisiae gene gram Grand, construction of recombinant vector, genetic recombination, soluble fusion protein integration, screening step, are as follows：

A, fleabane flower tissue DNA extracts：Fleabane flower genome cDNA is obtained according to a conventional method, by fleabane flower genome cDNA through drawing Object design clone obtain phenylalanine lyase, cinnamic acid 4- hydroxylases, p-Coumaric Acid-coA ligases, chalcone synthase, The encoding gene of enzyme, namely chalcone isomerase and flavonoids synthetase II；

B, S. cervisiae gene cloning：Clone obtains alcohol dehydrogenase, acetaldehyde from saccharomyces cerevisiae genome according to a conventional method The encoding gene of dehydrogenase, acetyl-coenzyme A synthase and acetyl-CoA carboxylase 1；

C, construction of recombinant vector：Using Golden gate clone technologies, the gene for respectively obtaining step A and step B and by setting The promoter plasmid and terminator plasmid that the primer amplification counted obtains, common structure containing gene P1 (TDH3-ADH1), The T1 recombinant vectors of ALD6, ACS and T1 (tTPI1-tPGI), gene P2 (PGK1-TEF2), ADH2, ACCI and T2 (tADH1- TCYC1 the T3 recombinations of T2 recombinant vectors), gene P1 (TDH3-ADH1), PAL, 4CL and T3 (tFBA1-tPDC1) carry, gene The T4 recombinant vectors of P2 (PGK1-TEF2), C4H, CHI and T4 (tRPS2-tTDH1), gene P1 (TDH3-ADH1), CHS, FS II With the T5 recombinant vectors of T5 (tCCW12-tTRPL9A)；

D, genetic recombination：Utilize the two-way gene for five recombinant vectors that conventional yeasts genome integration processes obtain step C Amplified fragments are integrated into inside the routine integration site of saccharomyces cerevisiae genome W303, obtain the recombinant bacterial strain of production apiolin；

E, soluble fusion protein is integrated：By flavonoids -3'- hydroxylases, cytochrome reductase gene is fused rear and shuttles Plasmid YCplac22 recombinations, the recombinant bacterial strain chromosome that D steps obtain is integrated by the shuttle plasmid after recombination by homology arm It is interior, it obtains integrating recombinant bacterial strain；

F, it screens：The integration recombinant bacterial strain that E steps are obtained obtains production sweet-scented osmanthus by the M-Trp screenings of auxotroph culture medium C The yeast strain of careless element.

What the second object of the present invention was realized in：It is a kind of production cyanidenon yeast strain method for synthesizing gene obtain Production cyanidenon yeast strain.

What the third object of the present invention was realized in：A kind of production cyanidenon yeast strain is in preparing cyanidenon Using.

The present invention because saccharomyces cerevisiae is similar to many metabolic pathways of plant cell, then give birth to by safety and easy culture The good host of production cyanidenon is chosen to be yeast.Using the gene in fleabane flower source as background, design primer clones to obtain benzene Propane downstream pathway enzyme coding gene, including phenylalanine lyase（PAL）, cinnamic acid 4- hydroxylases（C4H）, to tonka-bean Acid-coA ligases（4CL）, chalcone synthase（CHS）, enzyme, namely chalcone isomerase（CHI）, flavonoids synthetase II（FSⅡ）Volume Code gene.Secondly, the intrinsic malcryscoa approach of S. cervisiae is introduced, clone obtains alcohol dehydrogenase from Yeast genome Enzyme（ADH2）, acetaldehyde dehydrogenase（ALD6）, acetyl coenzyme A synthase（ACS）, acetyl-CoA carboxylase 1（ACC1）Gene. Contain gene P1 (TDH3-ADH1), ALD6, ACS, T1 (tTPI1-tPGI) using Golden gate clone's constructing technology structures T1 recombinant vectors, P2 (PGK1-TEF2), the T2 recombinant vectors of ADH2, ACCI, T2 (tADH1-tCYC1), P1 (TDH3- ADH1), the T3 recombinant vectors of PAL, 4CL, T3 (tFBA1-tPDC1), P2 (PGK1-TEF2), C4H, CHI, T4 (tRPS2- TTDH1 the T5 recombinant vectors of T4 recombinant vectors, P1 (TDH3-ADH1), CHS, FS II, T5 (tCCW12-tTRPL9A)), it is above Recombinant vector is the cloned plasmids with kalamycin resistance.It is host with saccharomyces cerevisiae W303, utilizes conventional yeasts gene The two-way gene amplification fragment of above-mentioned five carriers is integrated into the routine integration site of Yeast genome W303 by group integration method Inside obtains the recombinant bacterial strain of production apiolin.

Flavonoids -3'- the '-hydroxylase genes that the present invention for yeast host obtain after codon optimization（F3’H）Table It is insoluble to reveal cell, to make F3 ' H enzymes that there is physiological activity, by F3 ' H and CPRI Gene Fusion to shuttle expression plasmid Y22, Active soluble fusion protein is produced, apiolin precursor synthesis final product cyanidenon can be catalyzed, lacked through overnutrition The M-Trp screenings of swaged culture medium C, obtain the yeast strain of production cyanidenon.The present invention is directed to Microbe synthesis flavonoid Object is limited by chassis cell itself heredity and biochemical characteristic, and especially malonyl coenzyme A synthesizes less in microbial body, And can not external source addition and become rate-limiting step.Related enzyme coding gene in cyanidenon de novo formation path is integrated into wine brewing In yeast chromosome body, lay a good foundation to improve cyanidenon yield by metabolic engineering from now on.

The present invention tunning of the yeast strain of optimized efficient liquid-phase chromatography method detection production cyanidenon, divides From the final product cyanidenon consistent with cyanidenon standard items retention time is identified, further liquid-matter is applied to join spectrometry （LC-MS）Further identify the final product cyanidenon that fermentation generates, 286.23 Da of molecular weight and configuration and natural products Cyanidenon is consistent.Cyanidenon easy interference flavonoid substances similar with other molecular structures even isomer can be bright Aobvious separation, no hangover, and the used time is shorter.The complete biosynthesis pathway of cyanidenon since glucose is low for its realization The high industrialization micro-organisms of cost, yield provide theory and technology support.

Therefore, the present invention will be assembled simultaneously by genetic engineering and metabolic engineering method from the gene of different plant tissues It is artificial constructed to become a gene cluster and synthesize active natural products, cyanidenon is incorporated in S. cervisiae from the beginning All genes of synthesis path become gene cluster, it is intended to shorten the production cycle, improve biosynthesis efficiency, are produced into reduce This, optimizes metabolic pathway so that fermenting and producing cyanidenon has low cost, the high development prospect of yield, gene of the invention It is environmentally protective to synthesize remodeling method.

Description of the drawings

Fig. 1 is Golden gate flow diagrams of the present invention；

Fig. 2 is that present invention production apiolin recombinates chassis strain construction flow chart；

Fig. 3 is plasmid construction DNA electrophoretograms of the present invention；

Fig. 4 is the connection diagram of target fragment of the present invention and plasmid vector；

Fig. 5 is the figure that the present invention describes Y22 plasmids；

Fig. 6 is sequencer map after F3 ' H and the CPRI Gene Fusions of the embodiment of the present invention；

Fig. 7 is gene sequencing figure after the recombinant plasmid transformed of the embodiment of the present invention.

Specific implementation mode

The present invention is further illustrated with reference to the accompanying drawings and examples, but is not subject in any way to the present invention Limitation, be based on present invention teach that made by any changes and modifications, all belong to the scope of protection of the present invention.

The method for synthesizing gene of the production cyanidenon yeast strain of the present invention includes the extraction of fleabane flower tissue DNA, wine brewing ferment Female bacterium gene cloning, construction of recombinant vector, genetic recombination, soluble fusion protein integration, screening step, are as follows：

A, fleabane flower tissue DNA extracts：Fleabane flower genome cDNA is obtained according to a conventional method, by fleabane flower genome cDNA through drawing Object design clone obtain phenylalanine lyase, cinnamic acid 4- hydroxylases, p-Coumaric Acid-coA ligases, chalcone synthase, The encoding gene of enzyme, namely chalcone isomerase and flavonoids synthetase II；

B, S. cervisiae gene cloning：Clone obtains alcohol dehydrogenase, acetaldehyde from saccharomyces cerevisiae genome according to a conventional method The encoding gene of dehydrogenase, acetyl-coenzyme A synthase and acetyl-CoA carboxylase 1；

C, construction of recombinant vector：Using Golden gate clone technologies, the gene for respectively obtaining step A and step B and by setting The promoter plasmid and terminator plasmid that the primer amplification counted obtains, common structure containing gene P1 (TDH3-ADH1), The T1 recombinant vectors of ALD6, ACS and T1 (tTPI1-tPGI), gene P2 (PGK1-TEF2), ADH2, ACCI and T2 (tADH1- TCYC1 the T3 recombinations of T2 recombinant vectors), gene P1 (TDH3-ADH1), PAL, 4CL and T3 (tFBA1-tPDC1) carry, gene The T4 recombinant vectors of P2 (PGK1-TEF2), C4H, CHI and T4 (tRPS2-tTDH1), gene P1 (TDH3-ADH1), CHS, FS II With the T5 recombinant vectors of T5 (tCCW12-tTRPL9A)；

D, genetic recombination：Utilize the two-way gene for five recombinant vectors that conventional yeasts genome integration processes obtain step C Amplified fragments are integrated into inside the routine integration site of saccharomyces cerevisiae genome W303, obtain the recombinant bacterial strain of production apiolin；

E, soluble fusion protein is integrated：By flavonoids -3'- hydroxylases, cytochrome reductase gene is fused rear and shuttles Plasmid YCplac22 recombinations, the recombinant bacterial strain chromosome that D steps obtain is integrated by the shuttle plasmid after recombination by homology arm It is interior, it obtains integrating recombinant bacterial strain；

F, it screens：The integration recombinant bacterial strain that E steps are obtained obtains production sweet-scented osmanthus by the M-Trp screenings of auxotroph culture medium C The yeast strain of careless element.

Phenylalanine lyase in the step A is PAL, and the cinnamic acid 4- hydroxylases are C4H, described to tonka-bean Acid-coA ligases are 4CL, and the chalcone synthase is CHS, and the enzyme, namely chalcone isomerase is CHI, the flavonoids synzyme II is FS II；Alcohol dehydrogenase in the step B is ADH2, and the acetaldehyde dehydrogenase is ALD6, and the acetyl coenzyme A closes Enzyme is ACS, and the acetyl-CoA carboxylase 1 is ACCI；Flavonoids -3'- hydroxylases in the E steps are F3 ' H, described thin Born of the same parents' pigment reductase is CPRI.

The step A include it is following step by step：

A1, into ground powdered fleabane flower tissue sample be added 10 times of volumes RLT and 1 times of volume PLANTaid, Stir into rapidly at room temperature homogenate after be packed into centrifuge tube acutely rock 13~17s of oscillation, then through 13000rpm centrifugation 5~ 10min obtains supernatant；

A2, it takes above-mentioned supernatant and blows and beats mixing immediately after the absolute ethyl alcohol of supernatant volume half is added, by the mixing of mixing Object addition genome is removed column and is placed in collecting pipe, through noresidue on centrifugation to centrifugation liquid in pipe whole filtration and film, abandons Fall filtrate；

A3, the genome removing column after above-mentioned centrifugation is put into centrifuge tube, is removed in column in genome and lysate is added RLTPlus, then 13000rpm centrifugations 30s, obtains collecting filtrate, and the absolute ethyl alcohol of 0.5 times of volume is then added and blows immediately Mixing is beaten, adsorption column RA, which is added, in the mixture of mixing is placed in collecting pipe, is all filtered through centrifugation to centrifugation liquid in pipe And noresidue on film, discard filtrate；

A4, protein liquid removal RW1 is added in the adsorption column RA after above-mentioned centrifugation, is placed at room temperature for 1min, is then centrifuged through 13000rpm 30s discards waste liquid, adds rinsing liquid RW, centrifuges 30s through 13000rpm, discards waste liquid；

A5, the adsorption column RA that A4 is obtained is put back in sky collecting pipe, removal rinsing liquid is centrifuged through 13000rpm, then by adsorption column RA is put into RNAase free centrifuge tubes, and RNAase free water are added at the intermediate position of adsorbed film and are placed at room temperature for 1min most centrifuges 1min through 12000rpm afterwards；

A6, if it is expected that 30 μ g of RNA yield ＞, add the RNase free water of 30~50 μ l simultaneously to the intermediate position of adsorbed film A5 step by step is repeated, merges washing lotion or the eluent using first time twice and is added back to adsorption column repetition A4 mono- time step by step, Obtain fleabane flower total serum IgE；

A7, take above-mentioned fleabane flower total serum IgE as the sides Kit template reference RevertAidTM Fist Stand cDNA Synthesis Method reverse transcription synthesizes the first chain cDNA, and end reaction keeps 5min in 70 DEG C of metal baths, finally according to DNA purification kit operation streams Cheng Jinhang is purified, and obtains fleabane flower genome cDNA

The A7 is using fleabane flower total serum IgE as template, with reference to RevertAidTM Fist Stand cDNA step by step Synthesis Kit methods sequentially add the Oligo of total serum IgE no more than 1 μ g, 1 μ l in the PCR reaction tubes of no RNAase (dT) 18Primer complements to 12 μ l with DEPC water, and 5 × Reaction Buffer of 4 μ l, 1 μ l is then added RiboLock RNase Inhibitor, the 10mMdNTPMix of 2 μ l, RevertAid M-MuLV RT of 1 μ l, 20 μ l Total and mixing most centrifuge 1min through 13000rpm afterwards, discard waste liquid, then through 42 DEG C of metal bath 60min, end reaction is at 70 DEG C Metal bath keeps 5min, is finally purified according to DNA purification kit operating processes, obtains fleabane flower genome cDNA.

Double-promoter sequence P1 (TDH3-ADH1), P2 (PGK1-TEF2) in the step C and double terminator sequence T1 (tTPI1-tPGI)、T2(tADH1-tCYC1)、T3(tFBA1-tPDC1)、T4(tRPS2-tTDH1)、T5(tCCW12- TTRPL9A it) is obtained respectively by designed primer amplification.

The Golden gate clone technologies of the step C include it is following step by step：

C1, promoter is called by PCR from promoter and termination word bank and terminates subcomponent, PCR amplification promoter and termination Subsequence；

The design of primers of C2, target gene, including initiation codon end design of primers and terminator codon end design of primers；

C3、Golden Gate Assembling：Gene that step A and step B are obtained and by each segment of designed primer By equimolar number hybrid reaction, digests each gene primer with BsaI restriction enzyme sites respectively with BsaI restriction enzymes and expanded As a result the product of increasing and terminator plasmid vector with BsaI restriction enzyme sites generate the target fragment with cohesive end and contain The linear plasmid of terminator, then connected the target fragment for being respectively provided with cohesive end and linear plasmid with T4 Ligase ligases Connect recombination；

C4, selected integration site and marker gene Marker select corresponding carrier by common digestion connection to integrating position Point is replaced, then by entire integration site 1+Marker+ homology arms 1 by way of PCR（site1-marker-L1）Or Integration site 2+ homology arms 3 (site2-L3) expand；

C5, by segment that C4 is expanded step by step, Saccharomyces cerevisiae transformant culture to transformant is grown with treated, obtains five The two-way gene amplification fragment of a recombinant vector.

Middle initiation codon end design of primers is the C2 step by step：CC+GGTCTC+A+ ATCG+ complementary portion sequences or CC+GGTCTC+A+AGGT+ complementary portion sequences；

Terminator codon end design of primers is：CC+GGTCTC+A+GAAT+ complementary portion sequences or CC+GGTCTC+A+ GATG+ complementary portion sequences, if a wherein initiation codon end interface is ATCG, terminator codon during cohesive end interface selects End interface is GAAT, then it is AGGT, terminator codon that another gene interface in combination, which is selected as initiation codon end interface, End interface is GATG.

Auxotroph culture medium in the F-step saves histidine, with 1M sodium hydroxide solutions and 1M hydrochloric acid Solution tune pH value is to fluid nutrient medium 5.6 and solid medium 6.5.

The present invention produces the production cyanidenon yeast strain that the method for synthesizing gene of cyanidenon yeast strain obtains.

The present invention produces application of the cyanidenon yeast strain in preparing cyanidenon.

Embodiment

1 fleabane flower tissue DNA extracts

1.1 material

Table 1 carries drawn material

1.2 method

The centrifuge tube for being respectively provided with three parallel samples of Liquid nitrogen storage respectively organized comprising fleabane flower is taken out, each centrifuge tube takes 2g（Following unit, which is least unit, to be equipped with by multiple, similarly hereinafter）Tissue sample obtains a 6g（Following unit is minimum Unit can be equipped with by multiple, similarly hereinafter）Tissue mixing sample, for RNA extract.Quickly weigh 100mg（Following unit is Least unit can be equipped with by multiple, similarly hereinafter）, it is put into mortar, liquid nitrogen is added and is ground into powder rapidly.Total serum IgE is using north The RNRN12-PLANTeasy Reagent Kit kits that Jing Aidelai biotech companies provide are extracted according to flow. Extraction process is as follows：

1.2.1 step

The first step：10 times of volumes are added in the ground powdered fleabane flower tissue sample put into container（1ml）RLT With 1 times of volume（100µl）PLANTaid, rapid stirring at room temperature makes tissue and lysate RLT be come into full contact at once to inhibit RNA enzyme activity, is sufficiently stirred into homogenate.PLANTaid helps to extract secondary metabolites and the pigments such as plant polyose polyphenol The high complex sample of content.Then homogenate is transferred in 1.5ml centrifuge tubes acutely rock oscillation 15s, then through 13000rpm from 5~10min of the heart precipitates the fragment that cannot be cracked and the PLANTaid for being combined with polysaccharide polyphenol, obtains supernatant.

Second step：Take the 480 above-mentioned supernatants of μ l（To improve yield, it can remove in column limit of power and add on more in DNA Clear liquid）It goes in addition new centrifuge tube, the absolute ethyl alcohol of supernatant volume half is added（0.5 volume）, it is heavy to be likely to occur at this time It forms sediment, but does not influence extraction process, mixing is blown and beaten immediately, by the mixture of mixing（It is less than 720 μ l every time, it mostly can be at twice It is added）A genome is added to remove in column（Column is removed to be put into collecting pipe）, 2min is centrifuged through 13000rpm, discards waste liquid. Centrifugal force and centrifugation time, which can be increased, ensures that centrifuging liquid in pipe all filters, noresidue on film.

Third walks：Genome after above-mentioned centrifugation is removed pillar to be placed in 2ml centrifuge tubes, removes in column and adds in genome Then 500 μ l lysate RLTPlus centrifuge 30s through 13000rpm, collect filtrate（RNS is in filtrate）, with micropipettor compared with Accurate estimation filtered solution volume（Usually 450~500 μ l or so, loss volume should subtract when filtration）, 0.5 times of volume is added Absolute ethyl alcohol, be likely to occur precipitation at this time, but do not influence extraction process, blow and beat mixing immediately.At once by the mixing of mixing Object（Each small 720 μ l, can mostly be added in two portions）It is added in adsorption column RA（Adsorption column is put into collecting pipe）, through 13000rpm 2min is centrifuged, filtrate is discarded.Liquid all go by filtration after ensuring centrifugation, is not remained on film, if it is necessary, centrifugation can be increased Power and centrifugation time.

4th step：700 μ l protein liquid removal RW1 will be added in adsorption column RA after above-mentioned centrifugation, be placed at room temperature for 1min, then 30s is centrifuged through 13000rpm, discards waste liquid.It adds and 500 μ l rinsing liquids RW is added（It first checks whether and absolute ethyl alcohol has been added）, 30s is centrifuged through 13000rpm, discards waste liquid.500 μ l rinsing liquid RW are added, come again.

5th step：The adsorption column RA that 4th step obtains is put back in sky collecting pipe, 2min is centrifuged through 13000rpm, it is attached as possible Rinsing liquid is removed, in case residual ethanol inhibits downstream reaction in rinsing liquid.It then takes out adsorption column RA and is put into RNAase free centrifugations The RNAase free water of 30~50 μ l are added at the intermediate position of adsorbed film according to expected RNA yield in Guan Zhong（Exist in advance Yield can be improved in heating in 70~90 DEG C of water-baths）, it is placed at room temperature for 1min, most centrifuges 1min through 12000rpm afterwards.

6th step：If it is expected that RNA yield>30 μ g add the RNase free water of 30~50 μ l to repeat the 5th step, close And washing lotion or the eluent using first time are added back to the 4th step of adsorption column repetition one time twice, obtain fleabane flower total serum IgE.

Fleabane flower total serum IgE purity and extraction quality testing：

The total serum IgE Eon microplate spectrophotometers that extraction is obtained（Microplate reader）Its purity is tested, the test of microplate reader is opened Nucleic acid pattern measures g/ulOD260/280=1.932 a concentration of 1.489 μ of RNA.Meet>The sample requirement of 500 ng/ μ L is dense Degree, total amount>50 μ g, OD260/280 values illustrate that RNA purity is very high in 1.8 ~ 2.2 ranges.Matter can further be extracted The verification of amount.Total serum IgE integrality is verified using gel electrophoresis analysis result, as a result requires 28S:18S≥2.Deposition condition： 1 × tbe buffer liquid, agar gel compound concentration 0.8%, 110V voltages, electrophoresis 10min.Range estimation blue bands run out of sample holes 2 ~ When 3 cm are about at the 2/3 of gel, take out.The test strip under gel imager.Band is clear, not disperse, the first band It is about 2 with second strip brightness ratio:1, show that extracted total serum IgE integrality is fine, can be used as template and continue in next step, reversion Record cDNA.

7th step：The synthesis of first chain cDNA of fleabane flower total serum IgE reverse transcription：

First, the first chain cDNA is synthesized by template reverse transcription of total serum IgE.Method is with reference to RevertAidTM Fist Stand cDNA Synthesis Kit（Thermo Fischer Scient Inc. of the U.S.）Operation manual, in the PCR reaction tubes of no RNAase according to Secondary addition template total serum IgE is no more than 1 μ g, draws 1 μ l of Oligo (dT) 18Primer, and 12 μ l are complemented to DEPC water, 5 × 4 μ l, RiboLock RNase Inhibitor of Reaction Buffer, 12 μ l, RevertAid M- of μ l, 10mMdNTPMix 1 20 μ l of μ l, Total of MuLV RT.Whole operation need to be completed on ice.By solution mixing, 13000rpm centrifuges 1min.42℃ Metal bath 60min, end reaction inactivate reverse transcriptase in 70 DEG C of holding 5min of metal bath.Reaction system such as table 2：

2 fleabane flower total serum IgE the first chain of reverse transcription cDNA of table reflects system

Secondly, the first chain cDNA products are purified：According to DNA purification kits（Tiangeng biochemical technology company, Beijing）Operating process It is purified, obtains fleabane flower genome cDNA.The nucleic acid pattern test purification quality and dense of purified product Eon microplate reader Degree.

2 design of primers and gene chemical synthesis

The primer of corresponding gene is designed using primer-design software Primer premier 6.0.Primer sequence such as table 3.

3 gene primer sequence of table

The 2 μ l of product of the first chain cDNA of fleabane flower total serum IgE reverse transcription synthesis after purification are taken to be packed into RNase andDNase In the 200 μ l PCR reaction tubes of free, 5 × Phusion buffer, the 16 μ l of institute's amplification gene 10 μ l and 40 μ l are sequentially added 2.5mM dNTP, 2 μ l Phusion Enzyme, Mg²⁺5 μ l are added, with ddH₂O complements to 200 μ l.Whole operation need to be It carries out on ice.Pcr amplification reaction system such as table 4：

4 pcr amplification reaction system of table

Dosage is analogized by unit in table 4.

Used gene：

PAL, phenylalanine ammonia lyase（EC：4.3.1.24）；

4-coumarate: CoA ligase（EC：6.2.1.12 44）；

C4H, cinnamate 4-hydroxylase（EC：1.14.13.11 1）；

CHI, chalcone isomerase（EC：5.5.1.6 8）；

CHS, chalcone synthase（EC：2.3.1.74）；

FS Ⅱ , flavone synthase Ⅱ（EC：1.14.13.87）；

F3‘H, flavonoid 3’ hydroxylase（EC：1.14.13.21）；

ALD6, ACS, ADH2, ACCI, CPRI.

The above gene, Ethanol Dehydrogenase 2 (ADH2), Acetaldehyde Dehydrogenase 6 (ALD6), Acetyl CoA Synthase (ACS), Acetyl CoA Carboxylase I (ACCI) are because in yeast genes It can clone to obtain in group, gene order can be by Yeast GenBank SGD（Saccharomyces Genome Database, http://www.yeastgenome.org/）It downloads website.In addition, Cytochrome ReductaseI （CPRI）For the cytochrome reductase I from arabidopsis, gene order is downloaded by the websites NCBI（Gene ID: 828554）, And it clones to obtain by arabidopsis gene group.Flavonoids -3'- hydroxylases（F3'H）By Suzhou gold after yeast host codon optimization Only intelligence synthesizes.Remaining 6 gene, by fleabane flower genome cDNA (Accession number in NCBI SRA database:SRA111764 it) clones to obtain through design of primers.Promoter plasmid PMD-P1 (TDH3-ADH1), PMD-P2 (PGK1-TEF2) the double-promoter sequence P1 (TDH3-ADH1), P2 (PGK1-TEF2) in and terminator plasmid p-T1 (tTPI1-tPGI)、p-T2 (tADH1-tCYC1)、p-T3(tFBA1-tPDC1)、p-T4 (tRPS2-tTDH1)、p-T5 (tCCW12-tTRPL9A) double terminator sequence T1 (tTPI1-tPGI) in, T2 (tADH1-tCYC1), T3 (tFBA1- TPDC1), T4 (tRPS2-tTDH1), T5 (tCCW12-tTRPL9A) are obtained by designed primer amplification respectively.In addition, piece It is advance by this laboratory that section integrates homology arm sequence, flag sequence and plasmid with selected marker that Lian Chengzhong is used It prepares, packing is spare.

3 ethanol precipitations

The sodium acetate of 1/10 volume is added（3mol/l, PH=5.2）It is mixed well in DNA solution, it is 0.3 to make its ultimate density 3mol/l.2 times of ice-cold ethyl alcohol of volume are added, is mixed well again after mixing, is placed in 15~30min in -20 DEG C. 12000 rpm centrifuge 10 min, are carefully removed from supernatant, suck drop all on tube wall.1/2 volume of centrifuge tube is added 70% ethyl alcohol, 12000 rpm centrifuge 2 min, are carefully removed from supernatant, suck drop all on tube wall.It will uncap at room temperature EP pipes be placed on laboratory table that so that residual liquid is evaporated into dry.Add suitable ddH₂O dissolving DNAs precipitate.Measured concentration.

The conversion of 4 E. coli competents

（1）Liquid Culture：It is fallen with transfer needle picking single bacterium in the EP pipes equipped with 1ml LB liquid mediums, 37 DEG C of constant temperature oscillations Culture, 220rpm cultivate 45min.

（2）Spare resistant panel is taken out from -4 DEG C of refrigerators, it is spare.2 μ l are taken to be connected with target fragment under aseptic condition Plasmid culture solution -80 DEG C of refrigerators of entrance in spare 50 μ l DH5 α competence bacterium solutions, mixing, ice bath 30min, 42 DEG C of heat Swash 45s, takes out, rapid 2~3min of ice bath.

（3）The 450 sterile LB liquid mediums of μ l are added in each centrifuge tube（Without antibiotic）, mixing is placed on 37 DEG C and shakes Bed, 150 rpm vibrate 45min, recovery thalline.（LB culture mediums cannot be in sudden and violent free air）.

（4）It takes out, centrifuges 30s through 13000rpm, draw 300 μ l supernatants, discard.

（5）By remaining supernatant in EP pipes and precipitation mixing.Aseptically be applied to taken out it is spare containing corresponding anti- In the LB resistant panels of raw element, even spread is opened, and tablet is placed in 37 DEG C of constant incubators, is inverted 12~16h of culture.

（6）Picking grow up to after bacterium colony, the 15ml equipped with 5ml liquid LBK/ LBA that transfers sterilized in glass tube.37 DEG C, 220 12~16h of rpm incubator overnight cultures.5min is centrifuged through 13000 rpm, discards supernatant.According to plasmid extraction kit Operational manual extract plasmid.

（7）Each gene is verified with EasyTaq archaeal dna polymerases and corresponding primer, PCR is prepared and verifies system.PCR reactants System such as table 5.

5 PCR reaction systems of table

The preparation and conversion of 5 competent yeast cells

A. the preparation of competent yeast cells：

（1）From one fresh saccharomyces cerevisiae monoclonal of picking on YPD tablets to the YPD fluid nutrient mediums of 10ml, through 30 DEG C, 250 rpm overnight incubations；

（2）Between the OD600 values for measuring overnight culture are 3.0~5.0；

（3）It is 0.2~0.4 that the YPD overnight cultures of 10 ml, which are diluted to OD600 values,；

（4）Continue 3~6h of culture in 28~30 DEG C of shaking tables, its OD600 value is made to reach 0.6~1.0；

（5）5 min are centrifuged in room temperature 1500rpm and collect yeast cells, abandon supernatant；

（6）Yeast cells is washed with 10ml washing lotions, then centrifuging 5 min in room temperature 1500rpm is collected by centrifugation cell, abandons supernatant；

（7）Yeast cells is resuspended with the TE/LiAc of 1 ml, is dispensed with 50 μ 1 of every pipe.

B. the conversion of yeast cells

（1）50 μ l competent cells are taken, adds and waits for each 2 μ 1 of Pignus pignoris grain, mixing；

（2）500 μ l conversion solution are added（PEG/LiAc, ssDNA）, attack tube wall mixing；

（3）30 DEG C of water-bath 1h, every 15min attack tube wall mixings；

（4）The YPD culture solutions of 1ml are added, through 30 DEG C of shaking table culture 1h；

（5）3500rpm centrifuges 5min, stays precipitation, abandons supernatant；

（6）Precipitation is resuspended with the TE of 150 μ l, is coated with corresponding auxotroph SD tablets, tablet is then inverted in 30 DEG C of cultures.

6 Golden gate flows（Such as Fig. 1）

A. promoter is called by PCR from promoter and termination word bank and terminates subcomponent, PCR amplification promoter and terminator sequence.

ddH₂O 14.7 µl

10Xbuffer 2 µl

dNTP 1.6 µl

Primer F 0.5 µl

Primer R 0.5 µl

Top taq（Quan Shijin） 0.2 µl

template 0.5 µl(10ng)

total 20 µl

200 μ l are expanded to by ratios such as above-mentioned systems, often 50 μ l amplifications of pipe, reaction condition are as follows for point 4 pipes：

PCR product gel electrophoresis is verified：

（1）If without miscellaneous band, can direct purification use, after gel imager is taken pictures, preserve picture.

（2）If there is apparent miscellaneous band, recycled by gel-purified on probation.

Note：Wherein terminate subcomponent（Carrier framework）It need to be digested with DpnI, 37 DEG C of the DpnI of 2 μ l is added in purified product Reaction two hours（To ensure positive rate, the carrier framework used for the first time can do a positive control conversion, and knot is seen in directly conversion Fruit）；Obtained fragment concentrations cannot be too low, at least more than 50ng/ μ l.

B. the design of primers of target gene

（1）Initiation codon end design of primers can select：

CC (protection base)+GGTCTC (BsaI recognition sites)+A+ ATCG (cohesive end of restriction enzyme site)+complementary portion Sequence or CC (protection base)+GGTCTC (BsaI recognition sites)+A+AGGT (cohesive end of restriction enzyme site)+complementary portion Sub-sequence；

（2）Terminator codon end design of primers can select：

CC (protection base)+GGTCTC (BsaI recognition sites)+A+ GAAT (cohesive end of restriction enzyme site)+complementary portion Sequence or CC (protection base)+GGTCTC (BsaI recognition sites)+A+GATG (cohesive end of restriction enzyme site)+complementary portion Sub-sequence, if a wherein initiation codon end interface is ATCG during cohesive end interface selects, terminator codon end interface is GAAT, then it is AGGT that another gene interface in combination, which is selected as initiation codon end interface, and terminator codon end interface is GATG.(to sum up one sharing 4 kinds of combinations).

c. Golden Gate Assembling

Each segment presses equimolar number（50~100 fmol）Hybrid reaction：It is digested respectively with BsaI restriction enzymes and carries BsaI The product that each gene primer of restriction enzyme site is expanded（Including double-promoter sequence）With the terminator with BsaI restriction enzyme sites As a result plasmid vector generates the target fragment with cohesive end and the linear plasmid containing terminator.T4 Ligase connections are used again The target fragment for being respectively provided with cohesive end is connected recombination with linear plasmid by enzyme.Reaction system is as follows：

Carrier framework（Terminate subcomponent）（200ng）

Promoter element and the equimolar amount of carrier framework

Gene 1 and the equimolar amount of carrier framework

Gene 2 and the equimolar amount of carrier framework

10XNEB T4 buffer(NEB) 1.5µl

100XBSA*(NEB) 0.15µl

BsaI(NEB) 1µl

NEB T4 Ligase (NEB) 1µl

ddH₂O is supplied to 15 μ l

Total 15µl

Example：If selecting the PCR product of PMD-T1 as carrier framework, 200ng is added（81.44fmol）, start then P1 is added Subcomponent is 73.70ng（81.44fmol）, other to obtain in accordance with the law.

Reaction condition is as follows：

16℃ 4min（It is high to connect enzymatic activity）× 30~50cycles

50℃ 5min（If having in fragment reaction containing BsaI restriction enzyme sites, this step must be omitted）

50℃ 5min（Inactivation）

Obtained product directly converts DH5 α Escherichia coli, cultivates 12~16h, the verification of picking single bacterium colony.After verifying correctly Plasmid is the two-way genetic fragment of template PCR amplifications：

ddH₂O 14.7 µl

10Xbuffer 2 µl

dNTP 1.6 µl

PrimerF 0.5 µl

PrimerR 0.5 µl

Top taq（Quan Shijin） 0.2 µl

Template 0.5 µl

total 20 µl

200 μ l are expanded to by ratios such as above-mentioned systems, often 50 μ l amplifications of pipe, reaction condition are as follows for point 4 pipes：

PCR product gel electrophoresis is verified：

If I. without miscellaneous band, can direct purification it is on probation.

If II. there is apparent miscellaneous band, recycled by gel-purified on probation.

D. integration site 1+Marker+ homology arms 1（site1-marker-L1）And integration site 2+ homology arms 3 (site2-L3)：

The integration site and marker gene Marker of oneself are selected, selects corresponding carrier by common digestion connection to integrating Site is replaced, then by entire 1+Marker+ homology arms 1 of integration site by way of PCR（site1-marker- L1）Or integration site 2+ homology arms 3 (site2-L3) amplification.

PCR amplification systems：

ddH₂O 14.7 µl

10Xbuffer 2 µl

dNTP 1.6 µl

PKan-A-F 0.5 µl

Pkan-A-R 0.5 µl

Top taq（Quan Shijin） 0.2 µl

Template 0.5µl（10ng）

Total 20 µl

200 μ l are expanded to by ratios such as above-mentioned systems, often 50 μ l amplifications of pipe, reaction condition are as follows for point 4 pipes：

PCR product gel electrophoresis is verified：

（1）If without miscellaneous band, can direct purification it is on probation.

（2）If there is apparent miscellaneous band, recycled by gel-purified on probation.

E. multiple clips cotransformation yeast（Lithium acetate transformation method）

（1）Picking single bacterium colony shakes training 12h overnight in corresponding culture medium.

（2）With the value of the OD600 of the bacterium solution of spectrophotometric measurement culture.

（3）It is that 0.2OD is transferred in the fresh YPAD culture mediums of 50ml with first OD600 values（50 × 0.2=OD of calculation formula Value × extension rate × switching volume）.

Example：It is 0.25, i.e., 50 × 0.2=0.25 × 20 × switching volume to measure 20 times of dilution and shake cultivation liquid OD600 overnight, It is 2ml that can be accumulated in the hope of chovr body, so being the bacterium solution of the fresh YPAD culture mediums+2ml of 48ml.

（4）4~5h of activation allows yeast to rise in value two generation bacterium solution OD600 values 0.8~0.9.

（5）Boil ssDNA（If the ssDNA used is well-done, this step operation can be saved）.

（6）3000rpm centrifuges 5min and collects thalline, with the ddH of 25ml₂O is washed twice（DdH used₂O is preferably to go out on the same day Bacterium）.

（7）1ml water is resuspended in sterile 1.5ml centrifuge tubes.（It is used in super-clean bench）

（8）13000rpm centrifuges 30s and collects thalline.

（9）Packing is resuspended in 1ml water, and often for converting, the bacterium solution of packing is centrifuged 20s on palm centrifuge abandons 100 μ l of pipe On reset and add transformation system.

（The above operation must be completed in super-clean bench, ensure gnotobasis）.

Transformation system matches：

PEG3350（50%（W/V）Filtration sterilization） 240µl

LiAc 1.0M（Filtration sterilization） 36 µl

SSDNA（2.0 mg/ml） 50 µl

34 μ l of cotransformation DNA segments and water

Total 360µl

Segment needed for cotransformation is added 400ng by each segment and mixes mixing.

（10）Thalline is resuspended with the transformation system mixed, 20min is kept the temperature in 30 DEG C of incubators.

（11）42 DEG C of heat shock 40min coated plates, inversion are put in 2~3d of culture in 30 DEG C of incubators, and son to be transformed is longer, chooses Selected monoclonal proposes genome verification.

（It is control, the influence for being competence state with exclusion that an expression plasmid of yeast can be converted when conversion.）

The extraction of cyanidenon product in 7 zymotic fluids

The successful transformant of picking conversion, which enters, to be had been loaded in 5ml liquid CM-Trp culture medium 15ml sterile centrifugation tubes, in 30 DEG C, 220 rpm 4~5h of shaken cultivation measure bacterium solution OD values, when reaching 0.8~0.9OD, are transferred for 0.2OD with 600 value of initial OD To the CM-Trp fresh liquid culture mediums equipped with 50ml（Calculation formula：50 × 0.2=OD values × extension rate × switching volume）'s In 250ml triangular flasks, in 30 DEG C, 220 rpm shaken cultivations are fermented.

Every product in 4h, sample detection zymotic fluid after starting fermentation：900 μ l bacterium solutions are drawn to enter equipped with 900 μ l first In the sterile EP tube of alcohol, about 40 min of ultrasonic disruption cell wall in ultrasonic wave fluid cylinder is floated on, is taken out, is centrifuged through 1300 rpm 10 min draw supernatant, are filtered, are fitted into the sample injection bottle of high-efficient liquid phase analysis detection with the sterile filter in 0.22 μm of aperture, Wait for that HPLC is detected.

The extracting method for producing apiolin strain fermentation product is identical as the extracting method of cyanidenon product in zymotic fluid.

8 fermentation production HPLC detection methods

Cyanidenon molecular weight 286.23 has maximum absorption band at 253nm and 350nm.Most common acetonitrile-acetic acid system System, methanol-phosphate aqueous solution system, fail the absorption peak for preferably isolating cyanidenon, chromatographic peak separating effect is less Ideal has trailing phenomenon, and the used time is long compared with this law.The reason is that cyanidenon is there are isomer, while flavonoids molecule knot Structure is similar, causes appearance time close, is not readily separated.Classical HPLC methods are optimized, it is preferable to have obtained separating degree High-efficiency liquid chromatography method for detecting.Using Phenomen companies of U.S. Kinetex 5 μm of LC column, 250 × 4.6 mm, C18 columns.Sample size 20 μ l, 1.0 ml/min of flow velocity, 30 DEG C of column temperature.Under the chromatographic condition, test sample is with reference substance in phase There is absorption peak at the retention time answered, and reaches baseline separation with other components.The mobile phase used is A phases-methanol, B - 0.1% formic acid of phase, both gradient changes ratio are clearly separated until ingredient to be measured and other interference components.Method is as follows：

0 min methanol 20%（A）0.1% formic acid 80%（B）

3 min methanol 20%（A）0.1% formic acid 80%（B）

5 min methanol 40%（A）0.1% formic acid 60%（B）

23 min methanol 60%（A）0.1% formic acid 40%（B）

35 min methanol 20%（A）0.1% formic acid 80%（B）

40 min methanol 20%（A）0.1% formic acid 80%（B）

Realize that the structure of production apiolin chassis bacterial strain needs flow as shown in Figure 2：

The structure for producing the recombination chassis bacterial strain of apiolin, in order to be integrated into apiolin synthesis way in saccharomyces cerevisiae genome Diameter relative enzyme gene, by way of Golden gate homologous recombination double crossing overs, by the catalyzing enzyme base from glucose to apiolin Because being integrated into inside the routine integration site of yeast W303 genomes, the bacterial strain of production apiolin is as a result obtained.Integrator gene process In constructed cloned plasmids the plasmid that cultivation liquid is extracted shaken with 12~16h of single bacterium colony carry out PCR verifications, just by result True plasmid is delivered Jin Weizhi biotechnologies company and is sequenced, and correctly each gene order is and long without mutational site for sequencing result Degree meets each gene, and it is positive correct clone to illustrate obtained cloned plasmids.

9 gene magnifications

9.1 gene magnification

200 μ l PCR reaction systems of each gene are prepared respectively, these genes include：ALD6、ACS、ADH2、ACCI、PAL、 10 genes of 4CL, C4H, CHI, CHS, FS and double-promoter sequence P1 (TDH3-ADH1), P2 (PGK1-TEF2) and double terminations Subsequence T1 (tTPI1-tPGI), T2 (tADH1-tCYC1), T3 (tFBA1-tPDC1), T4 (tRPS2-tTDH1), T5 (tCCW12-tTRPL9A)。

By each system mixing, 1 min is centrifuged through 13000 rpm.It is divided into 4 pipes, often 50 μ l of pipe, are put into PCR instrument and are expanded Reaction.

Recycling, the purifying of 9.2 target gene

5 μ l amplified productions and 6 × Loading buffer of 1 μ l are taken to mix, loading separately adds Marker（1kb）2 μ l are compared, fine jade Sepharose（1%）Electrophoretic analysis, 110 V, 40 min.DNA bands are estimated with gel imager.Purpose band is correct, it is clear, There are the non-specific miscellaneous bands of 100bp or more, preservation of taking pictures gel extraction to be carried out, according to gel reclaims kit（The full formula gold in Beijing） Specification operated.Band is clear and without non-specific miscellaneous band, shows that DNA cloning result is correct, is directly purified, pressed According to DNA purification kits（The full formula gold in Beijing）Specification operated.The concentration and purity of the target gene of recycling, purifying make It is tested with the nucleic acid pattern of Eon microplate reader, concentration and purity meet the requirements（Table 6）.

6 mrna concentration of table and purity

The connection of 9.3 target fragments and plasmid vector

Two target gene, double-promoter sequence, the linear plasmid comprising double terminators are expanded according to Goldengate methods Purified product prepares digestion, coupled reaction system in proportion.Recombinant plasmid（It is kalamycin resistance）Composition such as table 7.

7 recombinant plasmid of table forms

After reaction, connection product is placed on ice rapidly.Often 15 μ l systems of pipe add 37 DEG C of the DpnI enzymes of 1 μ l, digest 2h.It takes Go out, carries out next step conversion.

9.4 recombinant plasmid transformed E. coli competents

A. it is equipped in the EP pipes of the 1.5ml of 50 μ l competent cells and 7.5 μ l connection products is added.30min is placed on ice, is taken out, Heat shock 45s in 42 DEG C of metal baths, takes out place 3min on ice immediately.The sterilized liquid LB of 450 μ l are added into reaction system Solution, through 37 DEG C, 220rpm recoveries 45min.Palm centrifuge, 20s draw 350 μ l supernatants, discard, stay about 100 μ l supernatants Liquid, which is slowly inhaled to beat in EP bottom of the tube, with pipettor, is resuspended thalline；Resuspended bacterium solution is sucked on LBK solid plates, with the glass of sterilizing Glass spreading rod smoothens bacterium solution；Tablet is inverted in 12~16h in 37 DEG C of constant incubators.

B. the bacterium colony after picking grows up to, the 15ml equipped with 5ml liquid LBK that transfers have sterilized in glass tube.Through 37 DEG C, 220 12~16h of rpm incubator overnight cultures.It is added in the 2ml cryopreservation tubes for 50% glycerine crossed through high pressure sterilization equipped with 500 μ l 1ml crosses the bacteria suspension of liquid shaking table culture, is slowly inhaled with liquid-transfering gun after beating mixing, sets and preserves bacterial strain in -80 DEG C of ultra low temperature freezers.It will Remaining 4ml bacteria suspensions centrifuge 5min through 13000 rpm, discard supernatant, are carried according to the operational manual of plasmid extraction kit Plasmid is taken to precipitate.The plasmid concentration and purity tested with the nucleic acid pattern of EON microplate reader meet the requirements.

9.5 PCR verify recombinant plasmid

After reaction, 5 μ l products is taken to add 6 × Loading buffer of 1 μ l, with 1% agarose 1 × TAE 110V, 40min, Electrophoresis detection, another plus 2 μ l 1kb Marker are compared.Stripe size should be：P1（1452bp）、P2（1350bp）、ADH2 （1048bp）、ALD6（1503bp）、ACS（1959bp）、

ACCI（6702bp）、PAL（2136bp）、C4H（1518bp）、4CL（1624bp）、CHS（1199bp）、CHI（718bp）、 FSⅡ（1562bp）、T1（3766bp）、T2（2826bp）、T3（3585bp）、T4（3590bp）、T5（3514bp）.Electrophoresis result Show the band of institute's amplification gene in correct position（Fig. 3）.

To ensure that recombinant plasmid correctly connects, digestion verification is further taken, i.e., respectively with two single enzymes of recombinant plasmid tool The restriction enzyme of enzyme site prepares endonuclease reaction system, digests recombinant plasmid, and reaction condition is 37 DEG C, 1.5h.To digestion Product is detected with 1% agarose gel electrophoresis, and electrophoresis result shows that the band of endonuclease bamhi appears in correct position, can Send sequencing.

9.6 sequencings and sequence analysis

By the correct positive colony plasmid sample presentation sequencing of recombination to construct, existed by the gold return sequence that only intelligence sequencing company is sequenced Blast is carried out with target sequence in fleabane flower genome compare analysis online on the websites NCBI, as a result without mutation, sequence is consistent, Show that this Success in Experiment obtains recombinant plasmid.

9.7 production apiolin chassis strain constructions and tunning detection

By each slice unit（Such as Fig. 4）It is thin that cotransformation system transformed yeast competence is prepared in proportion according to Goldengate methods Born of the same parents, selection markers are CM-His histidine auxotrophs.The single bacterium colony libation at an ancient wedding ceremony grown up to sterile toothpick picking enters equipped with 5 ml liquid In the glass tube of sterilizing of body CM-His culture mediums, mixing makes thalline be uniformly distributed, and through 30 DEG C, 220 rpm, 12~16h are overnight Shaking table culture.

600 values of OD of shaking table culture bacterium solution are stayed overnight in detection, at UV2600 UV spectrophotometer measuring 600nm wavelength Light absorption value, in the triangular flask equipped with 50 ml liquid CM-His of transferring at 0.5~0.8 OD, through 30 DEG C, 200 rpm shaking tables Culture.It is sampled every 4h, prepares zymotic fluid celery extract, HPLC detections.Tunning detects to obtain celery through HPLC after 48h Dish element product peak.

Chromatographic condition：1260 type high performance liquid chromatograph of Agilent, using Kinetex H15-168747 type analysis columns, stream Dynamic phase：A phases are methanol, B phases are 0.1% formic acid, flow velocity：1mL/min, column temperature：30℃.

Detection method setting is identical with the setting of cyanidenon HPLC detection methods.The work produced with Beijing Suo Laibao companies Industry grade apiolin is standard items, and applied sample amount is 20 μ l.Tunning occurs inhaling in the vicinity corresponding retention time 19min Peak is received, is consistent with the HPLC chromatogram result of apiolin standard items.

Further, tunning is through the connection spectrometer detection of LC-MS liquid matter, Mass Spectrometry Conditions：Mass spectrograph Bruker- MicrOTOF-II, ESI ion source, positive ion mode.Ionic conditions after optimization are：Ion spray voltage is 4500V；Capillary Tube temperature degree is set as 400 DEG C, and nitrogen is reaction system dry gas, and flow velocity is set as 1mL/min, and Detection wavelength is set as 350nm, drying temperature are set as 180 DEG C.Molecular weight of the tunning near 19 min is 270.24 Da（m/z, 271.0638,[M+H]+）, the mass spectral results of this and apiolin standard items are consistent.

10. the structure and product detection of F3 ' H expression vectors

The fusion of 10.1 target genes

Distinguish PCR amplification F3 ' two genes of H and CPRI with pre-designed primer, obtains F3 ' H genes and CPRI gene pieces Section.Due to flavonoids -3'- hydroxylases（F3’H）It is shown as in intracellular inactive, to obtain final product cyanidenon, uses and melt The mode of gene is closed by two Gene Fusions of F3 ' H genes and CPRI genes, gives expression to active F3 ' H.Using TMHMM Server predicts the secondary structure of sequence at the website (version 2.0), inputs F3 ' H gene sequences, the results show that its 24 amino acid long-chains of N-terminal are accredited as film calmodulin binding domain CaM；CPRI gene orders are inputted, the results show that 46 amino acid of its N-terminal Sequence is accredited as film calmodulin binding domain CaM.Therefore, F3 ' H sense primers tF3'HFre is since the 73rd nucleotide, including one SalI restriction enzyme list enzyme sites, downstream primer tF3'HRre include Gly-Ser-ThrLinker regional sequence and The nucleotide of the upstreams CPRI 18, in order to limit the formation for the secondary structure that interference F3 ' two zymoproteins of H and CPRI interact, Add the regions GST Linker.CPRI sense primers tCPRIFre is since the nucleotide of the upstreams CPRI 139, including tF3 ' H 22, downstream nucleotide and Gly-Ser-Thr Linker regional sequences have one in CPRI downstream primer tCPRIRre sequences A XbaI restriction enzymes list enzyme site and terminator codon.Pass through PCR amplification with primer pair tF3'HFre and tCPRIRre Two segment compositions of equimolar F3 ' H and CPRI are become tool by reaction, and there are one setting up password and terminator codons The fusion segment tF3 ' H-tCPRI of one entry.

PCR system needs to do the pre-reaction of homology arm combination, reaction process before adding primer pair tF3'HFre and tCPRIRre It is as follows：98 DEG C, 30S, one cycle；98 DEG C, 10S, 72 DEG C, 2min, 72 DEG C, 1min 15S, 5 cycles.It stands after circulation terminates I.e. plus respective primer pair, pcr amplification reaction is carried out.TF3 ' the H-tCPR fragment products of integration are through 1% gel electrophoresis analysis, knot Fruit（Fig. 6）Show to merge segment structure correctly.

Purified with the requirement of DNA purification kits to specifications, recycle PCR product, the nucleic acid pattern through EON microplate reader Detection conforms to quality requirements.Further disappeared with SalI restriction enzymes and XbaI restriction enzymes after fusion segment is purified Change, 37 DEG C, after 1.5h, is placed on 10min in 80 DEG C of metal baths rapidly, inactivates restriction enzyme, be stored in -4 DEG C of refrigerators It is interior, it is spare.

The structure of 10.2 shuttle plasmids

Due to needing the screening after escherichia coli cloning and yeast cell to express after fusion segment and plasmid recombination of setting out Secondary screening afterwards uses Ycplac22 shuttle plasmids（Have both ammonia benzyl chloramphenicol resistance and Tryptophan auxotrophic mutant selection markers）Into Row plasmid recombinates.Ycplac22 original plasmids are digested altogether with SalI restriction enzymes and XbaI restriction enzymes, generation Linear plasmid segment is analyzed through 1% agarose gel electrophoresis, the nucleic acid pattern of the concentration and purity EON microplate reader of linear plasmid The result of detection conforms to quality requirements.

According to linear plasmid carrier segments：It is 1 to merge segment molar ratio：3 ratio prepares coupled reaction system.After mixing 20s is centrifuged in being placed on palm centrifuge, makes system each component mixing.Coupled reaction system is positioned over 22 DEG C of metal baths, is protected 20min is held, is taken out.Liquid-transfering gun draws the DH5 α competent escherichia coli cells that 10 μ l connection reaction products convert 50 μ l.Reaction System is as follows（Fig. 5）：

Y22 2.7 µl

Overlap 6.8 µl

5×Ligase buffer 4 µl

T4Ligase 1 µl

ddH₂O 5.5 µl

The conversion of 10.3 recombinant plasmids

After the transformant grown is shaken bacterium recovery in liquid LBA culture mediums, plasmid is extracted, with SalI and XbaI restriction enzymes 37 DEG C of enzyme digests 1.5h altogether.1% agarose gel electrophoresis analyze digestion products the result shows that, successfully construct recombinant plasmid.It is pure Change recombinant plasmid, EON its concentration of microplate reader nucleic acid mode detection and purity conform to quality requirements, -4 DEG C of preservations, spare.Simultaneously Song Jinwei intelligence company is sequenced, the results showed that sequence is without mutation, i.e., successful construction recombination plasmid.It is converted with prepared transformation system Competent yeast cells, the tablet with CM-Trp selection markers be used to screen transformant.The single bacterium colony conversion that picking has been grown Son extracts Yeast genome and verifies each segment of fusion after recovery.The analysis result of 1% agarose gel electrophoresis shows fusion Segment is successfully integrated into Yeast genome.Song Jinwei intelligence company is sequenced, as a result（Fig. 7）Show sequence without mutation, i.e. recombinant plasmid Successfully it is building up in Yeast genome.

10.4 yeast transformant tunnings detect

Glass tube equipped with 5ml sterilized liquid CM-Trp culture mediums is used for the yeast transformant of recovery picking, in 30 DEG C, 220 rpm stay overnight 12~16h of shaking table culture.Detection yeast transformant shakes 600 values of OD of cultivation liquid, ultraviolet point of UV2600 overnight Light photometer detects light absorption value at 600nm wavelength, transfers into the triangular flask equipped with 50ml liquid CM-His at 0.5~0.8 OD In, 30 DEG C, 200rpm shaking table cultures.Every 4h, sampling prepares zymotic fluid reseda extract, HPLC detections.HPLC after 60h Detection obtains the chromatographic peak of cyanidenon from tunning.

Chromatographic condition：The prompt 1260 type high performance liquid chromatographs of peace, analytical column：Kinetex H15-168747, A in mobile phase It is mutually methanol, 0.1% formic acid of B phases, 350 nm of Detection wavelength, flow velocity is set as 1mL/min, column temperature：30℃.With Beijing Suo Laibao The technical grade cyanidenon of company's production is standard items, and applied sample amount is 20 μ l.

There is absorption peak in 6 vicinity min of corresponding retention time in tunning, with cyanidenon standard items HPLC chromatogram result is consistent.

Further, tunning is through the connection spectrometer detection of LC-MS liquid matter, Mass Spectrometry Conditions：Mass spectrograph Bruker- MicrOTOF-II, ESI ion source, positive ion mode.Ionic conditions after optimization are：Ion spray voltage is 4500V；Capillary Tube temperature degree is set as 400 DEG C, and nitrogen is reaction system dry gas, and 1mL/min is arranged in flow velocity, and Detection wavelength is set as 350nm, Drying temperature is set as 180 DEG C.Molecular weight of the tunning near 6min is 286.23 Da（m/z,287.0545,[M+H] +）, this is consistent with the mass spectral results of cyanidenon standard items.The molecular weight of apiolin is 270.24Da（m/z,271.0606,[M+ H]+）, this is consistent with the mass spectral results of apiolin standard items.

Claims (10)

1. a kind of method for synthesizing gene of production cyanidenon yeast strain, it is characterised in that including the extraction of fleabane flower tissue DNA, make Brewer yeast bacterium gene cloning, construction of recombinant vector, genetic recombination, soluble fusion protein integration, screening step, specific steps are such as Under：

A, fleabane flower tissue DNA extracts：Fleabane flower genome cDNA is obtained according to a conventional method, by fleabane flower genome cDNA through drawing Object design clone obtain phenylalanine lyase, cinnamic acid 4- hydroxylases, p-Coumaric Acid-coA ligases, chalcone synthase, The encoding gene of enzyme, namely chalcone isomerase and flavonoids synthetase II；

B, S. cervisiae gene cloning：Clone obtains alcohol dehydrogenase, acetaldehyde from saccharomyces cerevisiae genome according to a conventional method The encoding gene of dehydrogenase, acetyl-coenzyme A synthase and acetyl-CoA carboxylase 1；

C, construction of recombinant vector：Using Golden gate clone technologies, the gene for respectively obtaining step A and step B and by setting The promoter plasmid and terminator plasmid that the primer amplification counted obtains, common structure contain gene P1, ALD6, ACS and T1 The T3 recombinations of T1 recombinant vectors, the T2 recombinant vectors of gene P2, ADH2, ACCI and T2, gene P1, PAL, 4CL and T3 carry, gene The T5 recombinant vectors of the T4 recombinant vectors of P2, C4H, CHI and T4, gene P1, CHS, FS II and T5；

D, genetic recombination：Utilize the two-way gene for five recombinant vectors that conventional yeasts genome integration processes obtain step C Amplified fragments are integrated into inside the routine integration site of saccharomyces cerevisiae genome W303, obtain the recombinant bacterial strain of production apiolin；

E, soluble fusion protein is integrated：By flavonoids -3'- hydroxylases, cytochrome reductase gene is fused rear and shuttles Plasmid YCplac22 recombinations, the recombinant bacterial strain chromosome that D steps obtain is integrated by the shuttle plasmid after recombination by homology arm It is interior, it obtains integrating recombinant bacterial strain；

F, it screens：The integration recombinant bacterial strain that E steps are obtained obtains production sweet-scented osmanthus by the M-Trp screenings of auxotroph culture medium C The yeast strain of careless element.

2. producing the method for synthesizing gene of cyanidenon yeast strain according to claim 1, it is characterised in that in the step A Phenylalanine lyase be PAL, the cinnamic acid 4- hydroxylases be C4H, the p-Coumaric Acid-coA ligases be 4CL, The chalcone synthase is CHS, and the enzyme, namely chalcone isomerase is CHI, and the flavonoids synthetase II is FS II；The step B In alcohol dehydrogenase be ADH2, the acetaldehyde dehydrogenase is ALD6, and the acetyl coenzyme A synthase is ACS, and the acetyl is auxiliary Enzyme A carboxylases 1 are ACCI；Flavonoids -3'- hydroxylases in the E steps are F3 ' H, and the cytochrome reductase is CPRI。

3. producing the method for synthesizing gene of cyanidenon yeast strain according to claim 2, it is characterised in that the step A packet Include it is following step by step：

A1, into ground powdered fleabane flower tissue sample be added 10 times of volumes RLT and 1 times of volume PLANTaid, Stir into rapidly at room temperature homogenate after be packed into centrifuge tube acutely rock 13~17s of oscillation, then through 13000rpm centrifugation 5~ 10min obtains supernatant；

A2, it takes above-mentioned supernatant and blows and beats mixing immediately after the absolute ethyl alcohol of supernatant volume half is added, by the mixing of mixing Object addition genome is removed column and is placed in collecting pipe, through noresidue on centrifugation to centrifugation liquid in pipe whole filtration and film, abandons Fall filtrate；

A3, the genome removing column after above-mentioned centrifugation is put into centrifuge tube, is removed in column in genome and lysate is added RLTPlus, then 13000rpm centrifugations 30s, obtains collecting filtrate, and the absolute ethyl alcohol of 0.5 times of volume is then added and blows immediately Mixing is beaten, adsorption column RA, which is added, in the mixture of mixing is placed in collecting pipe, is all filtered through centrifugation to centrifugation liquid in pipe And noresidue on film, discard filtrate；

A4, protein liquid removal RW1 is added in the adsorption column RA after above-mentioned centrifugation, is placed at room temperature for 1min, is then centrifuged through 13000rpm 30s discards waste liquid, adds rinsing liquid RW, centrifuges 30s through 13000rpm, discards waste liquid；

A5, the adsorption column RA that A4 is obtained is put back in sky collecting pipe, removal rinsing liquid is centrifuged through 13000rpm, then by adsorption column RA is put into RNAase free centrifuge tubes, and RNAase free water are added at the intermediate position of adsorbed film and are placed at room temperature for 1min most centrifuges 1min through 12000rpm afterwards；

A6, if it is expected that 30 μ g of RNA yield ＞, add the RNase free water of 30~50 μ l simultaneously to the intermediate position of adsorbed film A5 step by step is repeated, merges washing lotion or the eluent using first time twice and is added back to adsorption column repetition A4 mono- time step by step, Obtain fleabane flower total serum IgE；

A7, take above-mentioned fleabane flower total serum IgE as the sides Kit template reference RevertAidTM Fist Stand cDNA Synthesis Method reverse transcription synthesizes the first chain cDNA, and end reaction keeps 5min in 70 DEG C of metal baths, finally according to DNA purification kit operation streams Cheng Jinhang is purified, and obtains fleabane flower genome cDNA.

4. producing the method for synthesizing gene of cyanidenon yeast strain according to claim 3, it is characterised in that the A7 substeps Suddenly be using fleabane flower total serum IgE as template, with reference to RevertAidTM Fist Stand cDNA Synthesis Kit methods, Oligo (dT) 18Primer of total serum IgE no more than 1 μ g, 1 μ l are sequentially added in the PCR reaction tubes of no RNAase, are used DEPC water complements to 12 μ l, and 5 × Reaction Buffer of 4 μ l, the RiboLock RNase of 1 μ l is then added Inhibitor, the 10mMdNTPMix of 2 μ l, RevertAid M-MuLV RT of 1 μ l, the Total of 20 μ l and mixing, most pass through afterwards 13000rpm centrifuges 1min, discards waste liquid, then through 42 DEG C of metal bath 60min, end reaction keeps 5min in 70 DEG C of metal baths, finally It is purified according to DNA purification kit operating processes, obtains fleabane flower genome cDNA.

5. producing the method for synthesizing gene of cyanidenon yeast strain according to claim 2, it is characterised in that in the step C Double-promoter sequence P1, P2 and double terminator sequence T1, T2, T3, T4, T5 obtained respectively by designed primer amplification.

6. producing the method for synthesizing gene of cyanidenon yeast strain according to claim 5, it is characterised in that the step C Golden gate clone technologies include it is following step by step：It is

C1, promoter is called by PCR from promoter and termination word bank and terminates subcomponent, PCR amplification promoter and termination Subsequence；

The design of primers of C2, target gene, including initiation codon end design of primers and terminator codon end design of primers；

C3、Golden Gate Assembling：Gene that step A and step B are obtained and by each segment of designed primer By equimolar number hybrid reaction, digests each gene primer with BsaI restriction enzyme sites respectively with BsaI restriction enzymes and expanded As a result the product of increasing and terminator plasmid vector with BsaI restriction enzyme sites generate the target fragment with cohesive end and contain The linear plasmid of terminator, then connected the target fragment for being respectively provided with cohesive end and linear plasmid with T4 Ligase ligases Connect recombination；

C4, selected integration site and marker gene Marker select corresponding carrier by common digestion connection to integrating position Point is replaced, then by entire integration site 1+Marker+ homology arms 1 or integration site 2+ homology arms by way of PCR 3 amplifications；

C5, by segment that C4 is expanded step by step, Saccharomyces cerevisiae transformant culture to transformant is grown with treated, obtains five The two-way gene amplification fragment of a recombinant vector.

7. producing the method for synthesizing gene of cyanidenon yeast strain according to claim 6, it is characterised in that the C2 substeps Initiation codon end design of primers is in rapid：CC+GGTCTC+A+ ATCG+ complementary portion sequences or CC+GGTCTC+A+AGGT+ Complementary portion sequence；

Terminator codon end design of primers is：CC+GGTCTC+A+GAAT+ complementary portion sequences or CC+GGTCTC+A+ GATG+ complementary portion sequences, if a wherein initiation codon end interface is ATCG, terminator codon during cohesive end interface selects End interface is GAAT, then it is AGGT, terminator codon that another gene interface in combination, which is selected as initiation codon end interface, End interface is GATG.

8. producing the method for synthesizing gene of cyanidenon yeast strain according to claim 2, it is characterised in that in the F-step Auxotroph culture medium save histidine, with 1M sodium hydroxide solutions and 1M hydrochloric acid solution tune pH value to liquid training Support base 5.6 and solid medium 6.5.

9. the production wood that the method for synthesizing gene for producing cyanidenon yeast strain described in a kind of claim 1 to 8 any one obtains The plain yeast strain of rhinoceros grass.

10. producing application of the cyanidenon yeast strain in preparing cyanidenon described in a kind of claim 9.