CN104630355B - A kind of rapid screening produces the method for gentiopicrin restructuring yeast strains - Google Patents
A kind of rapid screening produces the method for gentiopicrin restructuring yeast strains Download PDFInfo
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- DUAGQYUORDTXOR-GPQRQXLASA-N Gentiopicrin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](C=C)C2=CCOC(=O)C2=CO1 DUAGQYUORDTXOR-GPQRQXLASA-N 0.000 title claims abstract description 31
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention provide a kind of rapid screening produce gentiopicrin restructuring yeast strains method: 1) extract candidate strain genomic DNA, with this genomic DNA as template, carry out three-wheel PCR amplification respectively, obtain containing the restructuring yeast strains of the fragment of MECT gene, MECPS gene and GGPPS gene according to the screening of electrophoretic analysis result simultaneously；2) in YPD fluid medium, carry out fermentation culture, obtain producing gentiopicrin restructuring yeast strains by fermentation culture gained fermentation liquid carries out HPLC Analysis and Screening.Present invention greatly simplifies the screening process producing gentiopicrin restructuring yeast strains, method is simple, quick, efficient, cheap, highly versatile, is a kind of high-throughput screening method having and extensively using prospect.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of PCR-based amplification technique rapid screening and produce Radix Gentianae
The method of bitter glycosides restructuring yeast strains.
Background technology
Gentiopicrin (Gentiopicroside) is secoiridoid glycosides compound, is present in medicinal in a large number
In plant Radix Gentianae Macrophyllae (Gentianamacrophylla, G.macrophylla), it is the main of evaluation Gentiana medical material
Index, have antioxidation, antiinflammatory, protect the liver, the effect such as function of gallbladder promoting and antitumor.But the increasing along with clinical application amount
Add, wild resource transition is excavated, and causes the serious deficient of Radix Gentianae Macrophyllae resource.Therefore, in the urgent need to seeking and expanding
Big gentiopicrin novel medicine source approach meets its growing demand.
In recent years, finding and expanding gentiopicrin medicine source is a research field the most active, also achieves one
Fixed progress, mainly includes following two aspect: the artificial culture of (1) Radix Gentianae Macrophyllae；(2) biotechnology is utilized to improve
Gentiopicrin in Radix Gentianae Macrophyllae, including the cultivation of Radix Gentianae Macrophyllae tissue culture, suspension cell culture and hairy root.But,
Artificial culture exists that cycle length, survival rate be low, pesticide residues exceed standard and the drawback such as active ingredient is relatively low；Utilize group
Knit cultivation and suspension cell culture Radix Gentianae Macrophyllae cell produce gentiopicrin and all there is gentiopicrin yield wild effect,
Effective ingredient output increased in hairy root cultivating system is the most notable simultaneously.Therefore, along with the wild medicine of Radix Gentianae Macrophyllae
Increasingly reduce with plant resources, and utilize traditional plant culture technique to produce various the asking of gentiopicrin existence
Topic, promotes to utilize modern genetic engineering technology exploration to obtain novel medicine source approach and asks to solving gentiopicrin source
Topic has become a novel strategy having a high potential.
Low energy ion beam implantation technology is by Chinese Academy of Sciences's Hefei material science academy's plasma in the eighties in 20th century
Body and academy of agricultural sciences of the Anhui Province scientific research personnel emerging cross discipline ion beam bioengineering founding and grow up
Learning, in recent years, ion beam bioengineering technology is widely used in plant and the quality-improving of microorganism and plants matter
Innovation, and achieve great achievement.Mao Peihong etc. pass through Ar respectively+And N+Inject the blue Herba Ephedrae genome of mediation
The DNA random conversion in Hansenula yeast, it is thus achieved that inheritance stability Yeast engineering bacteria, its culture fluid epheday intermedia
The maximum output of alkali and pseudoephedrine is respectively 18.85mg/L and 4.11mg/L.And utilize ion implanting mediation sweet
Grass genomic DNA converts Hansenula yeast, it is thus achieved that the Yeast engineering bacteria of inheritance stability, its glycyrrhizic acid the highest
Yield reaches 114.49mg/L.Although low energy ion beam implantation technology has certain on the structure of recombination engineering
Directivity and purposiveness, but it also has bigger randomness, and after i.e. injecting, candidate's recombinant bacterial strain quantity is bigger,
So it is particularly important to set up a kind of high-throughout rapid screening method.At present, based on low energy ion beam implantation mediation
Synthetic biology method builds recombinant bacterial strain and produces natural product, and its screening technique used is usually according to producing
The chemical property design feature chemical reaction of thing is analyzed screening, utilizes low energy ion beam implantation such as Mao Peihong etc.
Method mediation Herba Ephedrae genome transformed yeast, its screening technique used is the spy according to purpose product ephedrine
Levying property color reaction screens, and outcome research personnel obtain 9 strains from the group bacterial strain screening of plant weight more than 3000 and produce fiber crops
Yellow alkali yeast recombinant strain strain, but there is workload greatly in this screening technique, and screening efficiency is low, adds that color reaction needs
Wanting the purpose product of certain yield, this extensive of synthetic biology seriously constraining low energy ion beam implantation mediation is transported
With.
Summary of the invention
It is an object of the invention to provide a kind of method that rapid screening produces gentiopicrin restructuring yeast strains.
For reaching above-mentioned purpose, present invention employs techniques below scheme:
1) recombinant bacterial strain primary dcreening operation: Radix Gentianae Macrophyllae total genomic dna conversion processing will be mediated through low energy ion beam implantation
After the yeast strain that sets out be seeded on YPD solid medium to carry out cultivation and obtain a large amount of transformant and (i.e. grow
Single bacterium colony on YPD solid medium), extract transformant genomic DNA, with transformant genomic DNA
For template, the specific fragment for MECT gene, MECPS gene and GGPPS gene enters respectively
Row three-wheel PCR expands, and respectively amplified production is carried out nucleic acid electrophoresis analysis, according to electrophoretic analysis result from
In transformant, screening obtains containing MECT genetic fragment, MECPS genetic fragment and GGPPS base simultaneously
Restructuring yeast strains because of fragment；
2) recombinant bacterial strain sieves again: by step 1) screen the restructuring yeast strains obtained in YPD fluid medium
In carry out fermentation culture, by fermentation culture gained fermentation liquid carries out HPLC Analysis and Screening, to obtain producing Radix Gentianae bitter
Glycosides restructuring yeast strains.
Use forward primer F1 and downstream primer R1 that the specific fragment of described MECT gene is carried out PCR
Amplification, the nucleotide sequence of forward primer F1 as shown in SEQ.ID.NO.1, the nucleotide of downstream primer R1
Sequence is as shown in SEQ.ID.NO.2.
Use forward primer F2 and downstream primer R2 that the specific fragment of described MECPS gene is carried out PCR
Amplification, the nucleotide sequence of forward primer F2 as shown in SEQ.ID.NO.3, the nucleotide of downstream primer R2
Sequence is as shown in SEQ.ID.NO.4.
Use forward primer F3 and downstream primer R3 that the specific fragment of described GGPPS gene is carried out PCR
Amplification, the nucleotide sequence of forward primer F3 as shown in SEQ.ID.NO.5, the nucleotide of downstream primer R3
Sequence is as shown in SEQ.ID.NO.6.
Beneficial effects of the present invention is embodied in:
Compared with existing screening technique, the present invention is by according to purpose product gentiopicrin biosynthetic metabolism approach
Multiple enzyme genes, as molecular marker, based on many wheel PCR amplification techniques, mediate based on low energy ion beam implantation
Restructuring yeast strains constructed by Radix Gentianae Macrophyllae total genomic dna transformed yeast technology carries out high flux screening, greatly
Simplify the screening process producing gentiopicrin restructuring yeast strains, the method is simple, quick, efficient, cheap,
Highly versatile, is a kind of high-throughput screening method having and extensively using prospect.
Accompanying drawing explanation
Fig. 1 is Radix Gentianae Macrophyllae total genomic dna electrophoretogram in embodiment.
Fig. 2 be MECT in embodiment (2-C-methyl D-erithritol-4-phosphoric acid cystyl transferring enzyme,
2-C-Methyl--D-erythritol4 phosphatecytidyltransferase) gene PCR amplification electrophoretogram, its
In, M is Maker, and 1,3 is starting strain, and 2 is Radix Gentianae Macrophyllae STb gene, and 4 is recombinant bacterial strain.
Fig. 3 is (the 2-methyl erythrose-2,4-ring diphosphate synthase of MECPS in embodiment
(2-C-methylerythritol 2,4-cyclo Diphosphate synthetase) gene PCR amplification electrophoretogram, its
In, M is Maker, and 1 is Radix Gentianae Macrophyllae STb gene, and 2 is recombinant bacterial strain, and 3 is starting strain.
Fig. 4 is that (cattle base diphosphonic acid closes GGPPS in embodiment
Enzyme Geranylgeranyl-diphosphate synthase) gene PCR amplification electrophoretogram, wherein, M is Maker,
1 is Radix Gentianae Macrophyllae STb gene, and 2,3,4 is starting strain, and 5 is recombinant bacterial strain.
Fig. 5 is product gentiopicrin recombinant bacterial strain HPLC collection of illustrative plates in embodiment.
Detailed description of the invention
With embodiment, the present invention is elaborated below in conjunction with the accompanying drawings.
There is provided a kind of quick, high to the synthetic biology mediated based on low energy ion beam implantation be built recombinant bacterial strain
Effect, the high-throughput screening method of highly versatile.The present invention is to close gentiopicrin by low energy ion beam implantation technology
The recombinant bacterial strain that one-tenth approach portion gene or all related gene import in pastoris genomic dna is right for screening
As, produce gentiopicrin restructuring yeast strains by repeatedly PCR amplification Preliminary screening, and then utilize HPLC
Its further analysis is determined.
The present invention is with multiple-shaped nuohan inferior yeast DL-1 (H.polymorpha DL-1 originates as ATCC No.26012)
For starting strain, low energy ion beam implantation mediation Radix Gentianae Macrophyllae total genomic dna is utilized to convert starting strain at random.
(1) extraction of Radix Gentianae Macrophyllae total genomic dna:
The fresh tender leaf of Radix Gentianae Macrophyllae gathers in Radix Gentianae Macrophyllae planting base, Taibai County, Shaanxi, cleans freezing at being placed on-20 DEG C guarantor
Deposit.The extraction of Radix Gentianae Macrophyllae total genomic dna uses the CTAB method of classics: Radix Gentianae Macrophyllae tender leaf shreds, and liquid nitrogen grinds
Be milled to fine-powdered, add preheating 2%CTAB buffer (2%CTAB, 100mmol/L Tris-HCl, 20
Mmol/L EDTA, 1.4mol/L NaCl, 0.5% beta-mercaptoethanol), fully after mixing, 65 DEG C of incubation 1h
(shaking up once every 10min), 4000rpm is centrifuged 10min, takes supernatant, and equal-volume adds chloroform-isoamyl
Alcohol (24:1), shakes to emulsion, stands 10min, 4000rpm and is centrifuged 20min, takes upper strata aqueous phase liquid,
Adding the isopropanol of-20 DEG C of pre-coolings of equal-volume, have White Flocculus to occur ,-20 DEG C stand 30min, 12000rpm
Centrifugal 10min, abandoning supernatant, 70% washing with alcohol twice, to dry in 65 DEG C of water-baths, TE dissolves preservation,
Take 20 μ L volumes and carry out detected through gel electrophoresis.As a result, Radix Gentianae Macrophyllae total genomic dna electrophoretic band is the most whole
Together (see Fig. 1), show that the Radix Gentianae Macrophyllae total genomic dna purity that CTAB method used is extracted is high, quality is good, non-
It is very suitable for the PCR amplification of next step.
(2) foundation of system is screened
The determination of key enzyme and the lookup of gene order thereof in gentiopicrin anabolism, and it is carried out primer
Design, carries out three-wheel PCR amplification with Radix Gentianae Macrophyllae total genomic dna for template, optimizes and determine amplification condition, building
The preliminary screening protocol of vertical recombinant bacterial strain；
Design of primers: according to gentiopicrin biosynthesis feature, determine the key enzyme in gentiopicrin synthesis respectively
For MECT, MECPS, GGPPS, in NCBI gene database, then search for its homologous genes respectively
Protein sequence, find, according to blast protein sequence comparison, five protein sequences that its homology is higher respectively,
According to codehop website (http://blocks.fhcrc.org/codeh op.html) carry out specific primer respectively
Design.The concrete specific primer sequence of each gene is shown in Table 1, and primer is had by the raw work biological engineering share in Shanghai
Limit company synthesizes.
Table 1 MECT, MECPS, GGPPS specific primer sequence
Reaction system 20 μ L:2 × Taq PCR MasterMix 10 μ L, primer (concentration is 10 μMs) each 1 μ L,
Template 2 μ L (concentration is 2 μ g/mL), ddH2It is 20 μ L that O adjusts final volume.Taq PCR MasterMix
Produce for TIANGEN Biotech (Beijing) Co., Ltd..
MECT Gene response program: 94 DEG C of 5min；Circulate 35 times: 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s；
72℃7min.Amplified production uses 1% agarose gel electrophoresis detection, and it is MECT that result obtains institute's amplified production
The purpose fragment of about 450bp in gene.
MECPS Gene response program: 94 DEG C of 5min；Circulate 35 times: 94 DEG C of 30s, 61 DEG C of 30s, 72 DEG C of 45s；
72℃7min.Amplified production uses 1% agarose gel electrophoresis detection, and result obtains institute's amplified production and is
The purpose fragment of about 750bp in MECPS gene.
GGPPS Gene response program: 94 DEG C of 5min；Circulate 35 times: 94 DEG C of 30s, 58.5 DEG C of 30s, 72 DEG C
100s；72℃7min.Amplified production uses 1% agarose gel electrophoresis detection, and result obtains institute's amplified production
For the purpose fragment of about 1300bp in GGPPS gene.
(3) low energy ion beam implantation mediation Radix Gentianae Macrophyllae total genomic dna converts multiple-shaped nuohan inferior yeast starting strain
The preparation of bacteria suspension: the polymorpha strain (starting strain) 37 DEG C of freezen protective is activated 3～4h,
Then in 37 DEG C, YPD fluid medium (mass fraction 1% yeast extract, mass fraction 2% peptone,
Mass fraction 2% glucose) in carry out seed liquor cultivate 8～10h, obtain seed liquor.Use blood counting chamber meter
Number method determines extension rate, with protection liquid (mass fraction 0.5% D/W and mass fraction 0.5% sugarcane
Sugar aqueous solution, volume ratio is 1:1) seed liquor is diluted, making cell concentration is 1.0 × 107CFU/mL,
Obtain bacteria suspension.
Prepared by Mycoderma: 0.1mL bacteria suspension is spread evenly across the sterilized petri dishes central authorities of a diameter of 90mm, uses
Sterile wind dries up and makes to be formed on sterilized petri dishes Mycoderma.
Low energy ion beam implantation: ion source is N+, Implantation Energy is 15～25KeV, and dosage is
1.5×1016～2.5 × 1016ions/cm2, Mycoderma is placed on the aseptic target platform of ion implantation apparatus vacuum target chamber,
1×10-3With 10s pulse mode under Pa vacuum, interval 10s injects N+, immediately after by total for Radix Gentianae Macrophyllae genome
DNA solution is laid on Mycoderma and makes it be fully contacted.
Yeast incubation and eluting: with on sterile glass spatula eluting sterilized petri dishes repeatedly after 37 DEG C of incubation 2h
Mycoderma 2min, it is thus achieved that eluent.
Flat board is cultivated: takes and is spread evenly across YPD solid medium (matter under eluent 0.1mL, aseptic condition
Amount mark 1% yeast extract, mass fraction 2% peptone, mass fraction 2% glucose, mass fraction 2%
Agar powder) upper 37 DEG C of cultivation 48h, obtain 1072 strain transformants.
(4) PCR method is utilized to carry out primary dcreening operation
1) using 1072 strain transformants as object of study, its genomic DNA is extracted respectively, with Radix Gentianae Macrophyllae STb gene
(i.e. Radix Gentianae Macrophyllae total genomic dna), as positive control, starting strain DNA is as negative control, with MECT
Gene (AAZ80386.1) is target gene, carries out PCR amplification, the screening time containing MECT genetic fragment
Select recombinant bacterial strain.Electrophoretic analysis is shown in that Fig. 2, result show, has 132 plant weight group ferment in 1072 strain transformants
Mother strains can amplify MECT Gene Partial fragment band, and aobvious positive findings, gene stripe size is 450
About bp, consistent with scheduled target stripe size；PCR with starting strain DNA as template expands then
Occur without any specific band.
2) using step 1) in 132 strain restructuring yeast strains as object of study, extract its genomic DNA respectively,
Using Radix Gentianae Macrophyllae STb gene as positive control, starting strain DNA is as negative control, with MECPS
(AAY40863.1) it is target gene, carries out second and take turns PCR amplification, the screening gene sheet Han MECPS simultaneously
Candidate's restructuring yeast strains of section.Electrophoretic analysis is shown in that Fig. 3, result show, in 132 strain restructuring yeast strains
There are 22 strain recombinant bacterial strains can amplify MECPS Gene Partial fragment band, aobvious positive findings, gene bar
Band size is at about 750bp, consistent with scheduled target stripe size；And with starting strain DNA as template
PCR amplification then occurs without any specific band.
3) using step 2) in 22 strain recombinant bacterial strains as object of study, extract its genomic DNA respectively,
Using Radix Gentianae Macrophyllae STb gene as positive control, starting strain DNA is as negative control, with GGPPS
(AAQ72786.1) it is target gene, carries out the amplification of third round PCR, the screening gene Han GGPPS simultaneously
Candidate's restructuring yeast strains of fragment.Electrophoretic analysis is shown in that Fig. 4, result show, has 8 in 22 strain recombinant bacterial strains
Strain recombinant bacterial strain can amplify GGPPS Gene Partial fragment band, aobvious positive findings, gene stripe size
At about 1300bp, consistent with scheduled target stripe size；And the PCR with starting strain DNA as template
Amplification then occurs without any specific band.
(5) HPLC (high performance liquid chromatography) is utilized to analyze primary dcreening operation recombinant bacterial strain
With the 8 strain recombinant bacterial strains that filter out in (4) as object of study, carry out liquid fermentation, described fermentation side
Method is: be inoculated in by recombinant bacterial strain in YPD fluid medium, in 37 DEG C, train under 150r/min speed conditions
Support 48～72h.Extracting fermentation liquid, utilize HPLC to carry out further identification and analysis, HPLC analyzes detection figure
Spectrum is shown in Fig. 5.Result has 3 strain recombinant bacterial strains to show positive findings, goes out in peak, i.e. recombinant bacterial strain at 13.8min
Having gentiopicrin to produce, yield is about 2.216mg/L.
Test through multiple batches, it may be determined that utilize method determines in (4) recombinant bacterial strain in fermentation and
The positive findings Probabilistic Stability determined after HPLC is maintained at 25%～40%.
(6) hereditary stability of gentiopicrin restructuring yeast strains is produced
3 strain recombinant bacterial strains carry out seed liquor (with seed liquor preparation method in above-mentioned (3)) respectively cultivate,
Take 2mL seed liquor to be inoculated in 500mL YPD fluid medium and carry out shake-flask culture, in 37 DEG C, 125r/min
Under the conditions of, after Secondary Culture 8 generation, extract fermentation liquid, restructuring fermenting performance hereditary stability is surveyed
Fixed.3 strain recombinant bacterial strains all can be stablized heredity, gentiopicrin yield concentrate on 2.216～2.011mg/L it
Between.Showing, the product gentiopicrin recombinant yeast that screening obtains has preferable hereditary stability.
The above results shows, the present invention can obtain the recombinant yeast producing gentiopicrin with high frequency zone.
(7) the shake flask fermentation experiment of gentiopicrin restructuring yeast strains is produced
By recombinant yeast seed liquor (with seed liquor preparation method in above-mentioned (the 3)) 50mL of inheritance stability
Be inoculated in 5L YPD fluid medium and carry out fermentation culture, 37 DEG C, cultivate 96h under the conditions of 125r/min
After, extract and concentrated broth, fermentation liquid is carried out further HPLC analysis, measures its gentiopicrin, knot
Fruit analyze content is about 2.008mg/L, result show by the method screening obtain recombinant bacterial strain can use
Study in further fermentation culture.
Claims (1)
1. the method that a rapid screening produces gentiopicrin restructuring yeast strains, it is characterised in that: include following
Step:
1) recombinant bacterial strain primary dcreening operation: Radix Gentianae Macrophyllae total genomic dna conversion processing will be mediated through low energy ion beam implantation
After the yeast strain that sets out be seeded on YPD solid medium to carry out cultivation and obtain transformant, extract transformant
Genomic DNA, with transformant genomic DNA as template, for MECT gene, MECPS gene with
And the specific fragment of GGPPS gene carries out three-wheel PCR amplification respectively, and respectively amplified production is carried out core
Acid electrophoretic analysis, screen from transformant according to electrophoretic analysis result obtain containing simultaneously MECT genetic fragment,
MECPS genetic fragment and the restructuring yeast strains of GGPPS genetic fragment；
2) recombinant bacterial strain sieves again: by step 1) screen the restructuring yeast strains obtained in YPD fluid medium
In carry out fermentation culture, by fermentation culture gained fermentation liquid carries out HPLC Analysis and Screening, to obtain producing Radix Gentianae bitter
Glycosides restructuring yeast strains；
Use forward primer F1 and downstream primer R1 that the specific fragment of described MECT gene is carried out PCR
Amplification, the nucleotide sequence of forward primer F1 as shown in SEQ.ID.NO.1, the nucleotide of downstream primer R1
Sequence is as shown in SEQ.ID.NO.2；
Use forward primer F2 and downstream primer R2 that the specific fragment of described MECPS gene is carried out PCR
Amplification, the nucleotide sequence of forward primer F2 as shown in SEQ.ID.NO.3, the nucleotide of downstream primer R2
Sequence is as shown in SEQ.ID.NO.4；
Use forward primer F3 and downstream primer R3 that the specific fragment of described GGPPS gene is carried out PCR
Amplification, the nucleotide sequence of forward primer F3 as shown in SEQ.ID.NO.5, the nucleotide of downstream primer R3
Sequence is as shown in SEQ.ID.NO.6.
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| CN103966112A (en) * | 2014-04-28 | 2014-08-06 | 陕西科技大学 | Hansenula polymorpha recombination strain and application thereof in biosynthesis of gentiopicroside |
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| CN103966112A (en) * | 2014-04-28 | 2014-08-06 | 陕西科技大学 | Hansenula polymorpha recombination strain and application thereof in biosynthesis of gentiopicroside |
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