CN104630355A - Method for quickly screening recombinant yeast strains producing gentiopicroside - Google Patents
Method for quickly screening recombinant yeast strains producing gentiopicroside Download PDFInfo
- Publication number
- CN104630355A CN104630355A CN201510036732.0A CN201510036732A CN104630355A CN 104630355 A CN104630355 A CN 104630355A CN 201510036732 A CN201510036732 A CN 201510036732A CN 104630355 A CN104630355 A CN 104630355A
- Authority
- CN
- China
- Prior art keywords
- gene
- yeast strains
- gentiopicrin
- screening
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- DUAGQYUORDTXOR-WULZUDSJSA-N Gentiopicrin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1[C@@H](C=C)C=2C(C(=O)OCC=2)=CO1 DUAGQYUORDTXOR-WULZUDSJSA-N 0.000 title claims abstract description 37
- 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 37
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000012216 screening Methods 0.000 title claims abstract description 33
- 239000012634 fragment Substances 0.000 claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 101710184086 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase Proteins 0.000 claims abstract description 15
- 101710201507 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, apicoplast Proteins 0.000 claims abstract description 15
- 101710161384 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, chloroplastic Proteins 0.000 claims abstract description 15
- 101100397240 Arabidopsis thaliana ISPD gene Proteins 0.000 claims abstract description 15
- 101000895629 Synechococcus sp. (strain ATCC 27264 / PCC 7002 / PR-6) Geranylgeranyl pyrophosphate synthase Proteins 0.000 claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 0.000 claims abstract description 12
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 12
- 238000001962 electrophoresis Methods 0.000 claims abstract description 6
- 238000004458 analytical method Methods 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 29
- 238000012408 PCR amplification Methods 0.000 claims description 21
- 238000010884 ion-beam technique Methods 0.000 claims description 15
- 238000002513 implantation Methods 0.000 claims description 14
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 238000011144 upstream manufacturing Methods 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 4
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000013537 high throughput screening Methods 0.000 abstract description 3
- 238000003752 polymerase chain reaction Methods 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 23
- 238000011160 research Methods 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 7
- KWGRBVOPPLSCSI-WPRPVWTQSA-N (-)-ephedrine Chemical compound CN[C@@H](C)[C@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WPRPVWTQSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 239000001177 diphosphate Substances 0.000 description 3
- 235000011180 diphosphates Nutrition 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229960002179 ephedrine Drugs 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000501743 Gentiana macrophylla Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- HGVJFBSSLICXEM-CRCLSJGQSA-N 2-methylerythritol Chemical compound OC[C@](O)(C)[C@@H](O)CO HGVJFBSSLICXEM-CRCLSJGQSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000235036 Debaryomyces hansenii Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000218671 Ephedra Species 0.000 description 1
- 241001465251 Ephedra sinica Species 0.000 description 1
- 206010056474 Erythrosis Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000007317 Farnesyltranstransferase Human genes 0.000 description 1
- 108010007508 Farnesyltranstransferase Proteins 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- 241001071795 Gentiana Species 0.000 description 1
- 240000008917 Glycyrrhiza uralensis Species 0.000 description 1
- 235000000554 Glycyrrhiza uralensis Nutrition 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- HGVJFBSSLICXEM-UHFFFAOYSA-N L-2-methyl-erythritol Natural products OCC(O)(C)C(O)CO HGVJFBSSLICXEM-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000336025 Ogataea parapolymorpha DL-1 Species 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 1
- 230000002279 cholagogic effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- -1 glycosides compound Chemical class 0.000 description 1
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000005468 ion implantation Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- KWGRBVOPPLSCSI-WCBMZHEXSA-N pseudoephedrine Chemical compound CN[C@@H](C)[C@@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WCBMZHEXSA-N 0.000 description 1
- 229960003908 pseudoephedrine Drugs 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for quickly screening recombinant yeast strains producing gentiopicroside. The method comprises the following steps: extracting genomic DNA of candidate strains, performing three rounds of PCR (polymerase chain reaction) amplification by taking the genomic DNA as a template, and screening the electrophoretic analysis results to obtain the recombinant yeast strains containing fragments of MECT gene, MECPS gene and GGPPS gene; and 2) performing fermentation culture in YPD liquid medium, performing HPLC (high performance liquid chromatography) analysis on the obtained fermentation liquid from the fermentation culture and screening the results to obtain the recombinant yeast strains producing gentiopicroside. By virtue of the method provided in the invention, the process for screening the recombinant yeast strains producing gentiopicroside is greatly simplified. The method is simple, quick, efficient, low in cost and strong in universality, and is a high-throughput screening method with a wide application prospect.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method that PCR-based amplification technique rapid screening produces gentiopicrin restructuring yeast strains.
Background technology
Gentiopicrin (Gentiopicroside) is secoiridoid glycosides compound, be present in medicinal plant bark of ash (Gentianamacrophylla in a large number, G.macrophylla) in, be the leading indicator evaluating Gentiana medicinal material, there is anti-oxidant, anti-inflammatory, protect the liver, cholagogic and the effect such as antitumor.But along with the increase of clinical application amount, wild resource transition are excavated, cause the serious scarcity of bark of ash resource.Therefore, in the urgent need to seeking and expanding gentiopicrin novel medicine source approach to meet its growing demand.
In recent years, searching and expansion gentiopicrin medicine source are very active research fields, also achieve certain progress, mainly comprise following two aspects: the artificial culture of (1) bark of ash; (2) utilize the gentiopicrin in biotechnology raising bark of ash, comprise the cultivation of bark of ash tissue culture, suspension cell culture and hairly root.But artificial culture exists that the cycle is long, surviving rate is low, pesticide residue exceed standard and the drawback such as effective ingredient is lower; Utilize tissue culture and suspension cell culture bark of ash cells produce gentiopicrin all to there is gentiopicrin output wild effect, the effective constituent output increased simultaneously in hairly root culture system is not remarkable.Therefore, along with bark of ash Wild Medicinal Plants Resources reduces increasingly, and the various problems utilizing traditional plant culture technique to produce gentiopicrin to exist, impel utilizing modern genetic engineering technology exploration to obtain novel medicine source approach to carry out source problem become a novel strategy had a high potential to solving gentiopicrin.
Low energy ion beam implantation technology be the eighties in 20th century by Hefei material science research institute of Chinese Academy of Sciences plasma body and academy of agricultural sciences of Anhui Province scientific research personnel found and the emerging cross discipline ion beam bioengineering grown up, in recent years, ion beam bioengineering technology is widely used in quality-improving and the germplasm innovation of plant and microorganism, and achieves great achievement.Mao Peihong etc. are respectively by Ar
+and N
+inject the blue Ephedra genome DNA of mediation in the random conversion of debaryomyces hansenii, obtain inheritance stability Yeast engineering bacteria, the production peak of its nutrient solution Ephedrine and pseudoephedrine is respectively 18.85mg/L and 4.11mg/L.And utilize ion implantation Mediated Glycyrrhiza uralensis genomic dna transformed into H polymorpha, obtain the Yeast engineering bacteria of inheritance stability, the production peak of its Potenlini reaches 114.49mg/L.Although low energy ion beam implantation technology has certain directivity and purpose on the structure of recombinant bacterial strain, it also has larger randomness, and after namely injecting, candidate's recombinant bacterial strain number ratio is comparatively large, so it is particularly important to set up a kind of high-throughout rapid screening method.At present, synthetic biology method based on low energy ion beam implantation mediation builds recombinant bacterial strain and produces natural product, its screening method adopted is generally that the chemical property DESIGNED FEATURE chemical reaction of foundation product carries out Analysis and Screening, as Mao Peihong etc. utilizes low energy ion beam implantation method to mediate Chinese ephedra genome transformed yeast, its screening method used is screen according to the characteristic color reaction of object product ephedrine, outcome research personnel obtain 9 strain ephedrine produced thereby yeast recombinant strain strains from the screening of the recombinant bacterial strain of strain more than 3000, but it is large to there is workload in this screening method, screening efficiency is low, add that color reaction needs the object product of certain output, this seriously constrains the extensive utilization of the synthetic biology of low energy ion beam implantation mediation.
Summary of the invention
A kind of rapid screening is the object of the present invention is to provide to produce the method for gentiopicrin restructuring yeast strains.
For achieving the above object, present invention employs following technical scheme:
1) recombinant bacterial strain primary dcreening operation: the yeast strain that sets out after low energy ion beam implantation mediation bark of ash total genomic dna conversion processing is seeded to and YPD solid medium carries out cultivation obtains a large amount of transformant (namely growing the single bacterium colony on YPD solid medium), extract transformant genomic dna, with transformant genomic dna for template, for MECT gene, the specific fragment of MECPS gene and GGPPS gene carries out three-wheel pcr amplification respectively, and respectively nucleic acid electrophoresis analysis is carried out to amplified production, screening from transformant according to electrophoretic analysis result obtains simultaneously containing MECT gene fragment, the restructuring yeast strains of MECPS gene fragment and GGPPS gene fragment,
2) recombinant bacterial strain sieves again: by step 1) screen the restructuring yeast strains that obtains carry out fermentation culture in YPD liquid nutrient medium, obtain producing gentiopicrin restructuring yeast strains by carrying out HPLC Analysis and Screening to fermentation culture gained fermented liquid.
Adopt the specific fragment of upstream primer F1 and downstream primer R1 to described MECT gene to carry out pcr amplification, the nucleotide sequence of upstream primer F1 is as shown in SEQ.ID.NO.1, and the nucleotide sequence of downstream primer R1 is as shown in SEQ.ID.NO.2.
Adopt the specific fragment of upstream primer F2 and downstream primer R2 to described MECPS gene to carry out pcr amplification, the nucleotide sequence of upstream primer F2 is as shown in SEQ.ID.NO.3, and the nucleotide sequence of downstream primer R2 is as shown in SEQ.ID.NO.4.
Adopt the specific fragment of upstream primer F3 and downstream primer R3 to described GGPPS gene to carry out pcr amplification, the nucleotide sequence of upstream primer F3 is as shown in SEQ.ID.NO.5, and the nucleotide sequence of downstream primer R3 is as shown in SEQ.ID.NO.6.
Beneficial effect of the present invention is embodied in:
Compared with existing screening method, the present invention passes through according to the multiple enzyme gene of object product gentiopicrin biosynthetic metabolism approach as molecule marker, based on many wheel pcr amplification technology, high flux screening is carried out to based on the restructuring yeast strains constructed by low energy ion beam implantation mediation bark of ash total genomic dna transformed yeast technology, greatly simplifie the screening process of producing gentiopicrin restructuring yeast strains, this method is simple, quick, efficient, cheap, highly versatile, is a kind of high-throughput screening method with extensive utilization prospect.
Accompanying drawing explanation
Fig. 1 is bark of ash total genomic dna electrophorogram in embodiment.
Fig. 2 is the (2-C-methyl D-tetrahydroxybutane-4-phosphoric acid cystyl transferring enzyme of MECT in embodiment, 2-C-Methyl--D-erythritol4 – phosphatecytidyltransferase) gene PCR amplification electrophorogram, wherein, M is Maker, 1,3 is starting strain, 2 is bark of ash STb gene, and 4 is recombinant bacterial strain.
Fig. 3 is (the 2-methyl erythrose-2 of MECPS in embodiment, 4-ring diphosphate synthase (2-C-methylerythritol 2,4-cyclo – Diphosphate synthetase) gene PCR amplification electrophorogram, wherein, M is Maker, 1 is bark of ash STb gene, and 2 is recombinant bacterial strain, and 3 is starting strain.
Fig. 4 is GGPPS in embodiment (ox base diphosphate synthase Geranylgeranyl-diphosphate synthase) gene PCR amplification electrophorogram, and wherein, M is Maker, and 1 is bark of ash STb gene, and 2,3,4 is starting strain, and 5 is recombinant bacterial strain.
Fig. 5 produces gentiopicrin recombinant bacterial strain HPLC collection of illustrative plates in embodiment.
Embodiment
Below in conjunction with drawings and Examples, the present invention is elaborated.
In order to the synthetic biology that mediates based on low energy ion beam implantation is built recombinant bacterial strain provide a kind of fast, the high-throughput screening method of efficient, highly versatile.The present invention with by low energy ion beam implantation technology by gentiopicrin route of synthesis portion gene or all genes involved import to recombinant bacterial strain in pastoris genomic dna for screening object, produce gentiopicrin restructuring yeast strains by repeatedly pcr amplification preliminary screening, and then utilize HPLC to analyze further it to determine.
The present invention is starting strain with multiple-shaped nuohan inferior yeast DL-1 (H.polymorpha DL-1 originates as ATCC No.26012), utilizes low energy ion beam implantation to mediate bark of ash total genomic dna and transforms starting strain at random.
(1) extraction of bark of ash total genomic dna:
The fresh tender leaf of bark of ash gathers in bark of ash planting base, Taibai County, Shaanxi, cleans freezen protective at being placed on-20 DEG C.What the extraction of bark of ash total genomic dna adopted is classical CTAB method: bark of ash tender leaf shreds, liquid nitrogen grinding is to finely powdered, add the 2%CTAB damping fluid (2%CTAB of preheating, 100mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 0.5% beta-mercaptoethanol), after abundant mixing, 65 DEG C of incubation 1h (shaking up once every 10min), the centrifugal 10min of 4000rpm, get supernatant liquor, equal-volume adds chloroform-isoamyl alcohol (24:1), shake to milk sap, leave standstill 10min, the centrifugal 20min of 4000rpm, get upper strata aqueous phase liquid, add the Virahol of equal-volume-20 DEG C of precoolings, adularescent floss occurs,-20 DEG C of standing 30min, the centrifugal 10min of 12000rpm, abandoning supernatant, 70% washing with alcohol twice, dry in 65 DEG C of water-baths, TE dissolves preservation, get 20 μ L volumes and carry out detected through gel electrophoresis.As a result, bark of ash total genomic dna electrophoretic band very clear neat (see Fig. 1), shows that the bark of ash total genomic dna purity that CTAB method used is extracted is high, quality good, is very suitable for the pcr amplification of next step.
(2) foundation of system is screened
The determination of key enzyme and searching of gene order thereof in gentiopicrin anabolism, and design of primers is carried out to it, carry out three-wheel pcr amplification with bark of ash total genomic dna for template, optimize and determine amplification condition, set up the preliminary screening protocol of recombinant bacterial strain;
Design of primers: according to gentiopicrin biosynthesizing feature, determine that the key enzyme in gentiopicrin synthesis is respectively MECT, MECPS, GGPPS, then in NCBI gene database, its homogenic protein sequence is searched for respectively, according to five protein sequences that the comparison of blast protein sequence finds its homology higher respectively, according to codehop website (
http:// blocks.fhcrc.org/codeh op.html) carry out specific primer design respectively.The concrete specific primer sequence of each gene is in table 1, and primer is synthesized by Shanghai Sheng Gong biotechnology limited-liability company.
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), ddH
2it is 20 μ L that O adjusts final volume.Taq PCR MasterMix is that TIANGEN Biotech (Beijing) Co., Ltd. produces.
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 DEG C of 7min.Amplified production uses 1% agarose gel electrophoresis to detect, and result obtains the object fragment that institute's amplified production is about 450bp in MECT 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 DEG C of 7min.Amplified production uses 1% agarose gel electrophoresis to detect, and result obtains the object fragment that institute's amplified production is 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 of 100s; 72 DEG C of 7min.Amplified production uses 1% agarose gel electrophoresis to detect, and result obtains the object fragment that institute's amplified production is about 1300bp in GGPPS gene.
(3) low energy ion beam implantation mediation bark of ash total genomic dna transforming Hansenula starting strain
The preparation of bacteria suspension: by the polymorpha strain (starting strain) 37 DEG C of freezen protective activation 3 ~ 4h, then in 37 DEG C, YPD liquid nutrient medium (massfraction 1% yeast extract paste, massfraction 2% peptone, massfraction 2% glucose) in carry out seed liquor cultivate 8 ~ 10h, obtain seed liquor.Adopt blood counting chamber counting process determination extension rate, with protection liquid (massfraction 0.5% D/W and massfraction 0.5% aqueous sucrose solution, volume ratio is 1:1), seed liquor is diluted, make cell concentration be 1.0 × 10
7cFU/mL, obtains bacteria suspension.
Prepared by mycoderm: 0.1mL bacteria suspension is spread evenly across the sterilized petri dishes central authorities that diameter is 90mm, dries up make sterilized petri dishes forms mycoderm with sterile wind.
Low energy ion beam implantation: ion source is N
+, Implantation Energy is 15 ~ 25KeV, and dosage is 1.5 × 10
16~ 2.5 × 10
16ions/cm
2, mycoderm is placed on the aseptic target platform of ion implanter vacuum target chamber, 1 × 10
-3with 10s pulse mode under Pa vacuum tightness, interval 10s injects N
+, then immediately bark of ash total genomic dna solution is laid on mycoderm and makes it fully contact.
Yeast incubation and wash-out: with the mycoderm 2min on sterile glass spatula repeatedly wash-out sterilized petri dishes after 37 DEG C of incubation 2h, obtain elutriant.
Slat chain conveyor: get elutriant 0.1mL, YPD solid medium (massfraction 1% yeast extract paste, massfraction 2% peptone, massfraction 2% glucose is spread evenly across under aseptic condition, massfraction 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 research object, extract its genomic dna respectively, using bark of ash STb gene (i.e. bark of ash total genomic dna) as positive control, starting strain DNA is as negative control, with MECT gene (AAZ80386.1) for target gene, carry out pcr amplification, screening is containing candidate's recombinant bacterial strain of MECT gene fragment.Fig. 2 is shown in electrophoretic analysis, and result shows, has 132 strain restructuring yeast strains can amplify MECT Gene Partial fragment band in 1072 strain transformants, aobvious positive findings, and gene stripe size is at about 450bp, consistent with scheduled target stripe size; The pcr amplification being template with starting strain DNA then occurs without any specific band.
2) using step 1) in 132 strain restructuring yeast strains as research object, extract its genomic dna respectively, using bark of ash STb gene as positive control, starting strain DNA is as negative control, with MECPS (AAY40863.1) for target gene, carry out second and take turns pcr amplification, screening is simultaneously containing candidate's restructuring yeast strains of MECPS gene fragment.Fig. 3 is shown in electrophoretic analysis, and result shows, has 22 strain recombinant bacterial strains can amplify MECPS Gene Partial fragment band in 132 strain restructuring yeast strains, aobvious positive findings, and gene stripe size is at about 750bp, consistent with scheduled target stripe size; The pcr amplification being template with starting strain DNA then occurs without any specific band.
3) using step 2) in 22 strain recombinant bacterial strains as research object, extract its genomic dna respectively, using bark of ash STb gene as positive control, starting strain DNA is as negative control, with GGPPS (AAQ72786.1) for target gene, carry out third round pcr amplification, screening is simultaneously containing candidate's restructuring yeast strains of GGPPS gene fragment.Fig. 4 is shown in electrophoretic analysis, and result shows, has 8 strain recombinant bacterial strains can amplify GGPPS Gene Partial fragment band in 22 strain recombinant bacterial strains, aobvious positive findings, and gene stripe size is at about 1300bp, consistent with scheduled target stripe size; The pcr amplification being template with starting strain DNA 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 filtered out in (4) for research object, carry out liquid fermenting, described fermentation process is: be inoculated in by recombinant bacterial strain in YPD liquid nutrient medium, in 37 DEG C, cultivate 48 ~ 72h under 150r/min speed conditions.Extract fermented liquid, utilize HPLC to carry out further identification and analysis, Fig. 5 is shown in by HPLC analyzing and testing collection of illustrative plates.Result has 3 strain recombinant bacterial strains to show positive findings, goes out peak at 13.8min place, and namely have gentiopicrin to produce in recombinant bacterial strain, output is about 2.216mg/L.
Through multiple batches of experiments, can determine that the positive findings Probabilistic Stability utilizing the recombinant bacterial strain that in (4), method is determined to determine after fermentation and HPLC remains on 25% ~ 40%.
(6) genetic stability of gentiopicrin restructuring yeast strains is produced
3 strain recombinant bacterial strains are carried out respectively seed liquor (with seed liquor preparation method in above-mentioned (3)) to cultivate, get 2mL seed liquor to be inoculated in 500mL YPD liquid nutrient medium and to carry out shake-flask culture, in 37 DEG C, under 125r/min condition, after Secondary Culture 8 generation, extract fermented liquid, restructuring fermenting performance genetic stability is measured.All can genetic stability in 3 strain recombinant bacterial strains, gentiopicrin output concentrates between 2.216 ~ 2.011mg/L.Show, screen the product gentiopicrin recombination microzyme obtained and there is good genetic stability.
The above results shows, the present invention can obtain the recombination microzyme producing gentiopicrin by high frequency zone.
(7) the shake flask fermentation experiment of gentiopicrin restructuring yeast strains is produced
Recombination microzyme seed liquor (with seed liquor preparation method in above-mentioned (the 3)) 50mL of inheritance stability is inoculated in 5L YPD liquid nutrient medium and carries out fermentation culture, 37 DEG C, cultivate 96h under 125r/min condition after, extract and concentrated broth, further HPLC analysis is carried out to fermented liquid, measure its gentiopicrin, interpretation of result obtains content and is about 2.008mg/L, and result shows that the recombinant bacterial strain obtained by this method screening may be used for further fermentation culture research.
Claims (4)
1. rapid screening produces a method for gentiopicrin restructuring yeast strains, it is characterized in that: comprise the following steps:
1) recombinant bacterial strain primary dcreening operation: the yeast strain that sets out after low energy ion beam implantation mediation bark of ash total genomic dna conversion processing is seeded to and YPD solid medium carries out cultivation obtains transformant, extract transformant genomic dna, with transformant genomic dna for template, for MECT gene, the specific fragment of MECPS gene and GGPPS gene carries out three-wheel pcr amplification respectively, and respectively nucleic acid electrophoresis analysis is carried out to amplified production, screening from transformant according to electrophoretic analysis result obtains simultaneously containing MECT gene fragment, the restructuring yeast strains of MECPS gene fragment and GGPPS gene fragment,
2) recombinant bacterial strain sieves again: by step 1) screen the restructuring yeast strains that obtains carry out fermentation culture in YPD liquid nutrient medium, obtain producing gentiopicrin restructuring yeast strains by carrying out HPLC Analysis and Screening to fermentation culture gained fermented liquid.
2. a kind of rapid screening produces the method for gentiopicrin restructuring yeast strains according to claim 1, it is characterized in that: adopt the specific fragment of upstream primer F1 and downstream primer R1 to described MECT gene to carry out pcr amplification, the nucleotide sequence of upstream primer F1 is as shown in SEQ.ID.NO.1, and the nucleotide sequence of downstream primer R1 is as shown in SEQ.ID.NO.2.
3. a kind of rapid screening produces the method for gentiopicrin restructuring yeast strains according to claim 1, it is characterized in that: adopt the specific fragment of upstream primer F2 and downstream primer R2 to described MECPS gene to carry out pcr amplification, the nucleotide sequence of upstream primer F2 is as shown in SEQ.ID.NO.3, and the nucleotide sequence of downstream primer R2 is as shown in SEQ.ID.NO.4.
4. a kind of rapid screening produces the method for gentiopicrin restructuring yeast strains according to claim 1, it is characterized in that: adopt the specific fragment of upstream primer F3 and downstream primer R3 to described GGPPS gene to carry out pcr amplification, the nucleotide sequence of upstream primer F3 is as shown in SEQ.ID.NO.5, and the nucleotide sequence of downstream primer R3 is as shown in SEQ.ID.NO.6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510036732.0A CN104630355B (en) | 2015-01-23 | 2015-01-23 | A kind of rapid screening produces the method for gentiopicrin restructuring yeast strains |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510036732.0A CN104630355B (en) | 2015-01-23 | 2015-01-23 | A kind of rapid screening produces the method for gentiopicrin restructuring yeast strains |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104630355A true CN104630355A (en) | 2015-05-20 |
| CN104630355B CN104630355B (en) | 2016-08-24 |
Family
ID=53209599
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510036732.0A Expired - Fee Related CN104630355B (en) | 2015-01-23 | 2015-01-23 | A kind of rapid screening produces the method for gentiopicrin restructuring yeast strains |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104630355B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079630A (en) * | 2019-05-10 | 2019-08-02 | 陕西科技大学 | A kind of method that quick screening produces rhodioside restructuring yeast strains |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923848A (en) * | 2014-04-28 | 2014-07-16 | 陕西科技大学 | hansenula polymorpha recombinant strain and application thereof in biosynthesis of paclitaxel |
| 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 |
| CN103981130A (en) * | 2014-05-09 | 2014-08-13 | 虞龙 | Glutamine transaminase high-yielding strain and application thereof |
-
2015
- 2015-01-23 CN CN201510036732.0A patent/CN104630355B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923848A (en) * | 2014-04-28 | 2014-07-16 | 陕西科技大学 | hansenula polymorpha recombinant strain and application thereof in biosynthesis of paclitaxel |
| 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 |
| CN103981130A (en) * | 2014-05-09 | 2014-08-13 | 虞龙 | Glutamine transaminase high-yielding strain and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| SANG-MIN KIM等: "Cloning and functional characterization of 2-C-methyl-D-erythritol4-phosphate cytidyltransferase (GbMECT) gene from Ginkgo biloba", 《PHYTOCHEMISTRY》, vol. 67, no. 14, 10 July 2006 (2006-07-10), pages 1435 - 1441 * |
| 付云芳: "低能N+注入介导秦艽基因组DNA转化酵母的研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》, no. 12, 15 December 2013 (2013-12-15), pages 006 - 15 * |
| 钱卫东等: "一步重组法构建产龙胆苦苷酵母重组菌的研究", 《陕西科技大学学报》, vol. 32, no. 5, 25 October 2014 (2014-10-25), pages 123 - 128 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079630A (en) * | 2019-05-10 | 2019-08-02 | 陕西科技大学 | A kind of method that quick screening produces rhodioside restructuring yeast strains |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104630355B (en) | 2016-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103416223B (en) | Method for improving cordycepin output in cordyceps militaris fermentation broth | |
| CN101831481B (en) | New method for preparing Iturin A and homolugues thereof | |
| CN103820337B (en) | A kind of S. cervisiae for fermentative production Yeast Nucleic Acid and application thereof | |
| CN111690690B (en) | Saccharomyces cerevisiae for producing farnesene | |
| CN103409485A (en) | Method for improving adenosine fermentation output through feeding organic nitrogen source | |
| CN105087455A (en) | Genetic engineering strain for producing phenazine-1-carboxylic acid and application of genetic engineering strain | |
| CN105420154A (en) | Double knockout recombinant rhodococcus as well as construction method and application thereof | |
| CN104726505A (en) | Method for producing three-carbon compounds by using gene engineering cyanobacteria | |
| CN102342218B (en) | Method for producing cordyceps sinensis hyphae by virtue of solid-state fermentation | |
| CN108410753A (en) | A kind of method of composite bacteria agent of degrading straw and preparation method thereof, degrading straw | |
| CN103882080A (en) | Effective method for preparing avermectin | |
| CN104911169A (en) | Method for culturing cordyceps militaris mycelium and plasmin | |
| CN102839130B (en) | Bacterial strain for producing gliotoxin and method for producing gliotoxin by adopting bacterial strain | |
| CN107365715A (en) | A kind of preparation method and application of the Candida glycerolgenesis engineering bacteria of the benzyl carbinol of high yield 2 | |
| CN104046586A (en) | Genetically engineered bacteria and application of genetically engineered bacteria to production of (2R, 3R)-2,3-butanediol | |
| CN104419657A (en) | D-lactic acid producing strain with high growth rate and acid producing velocity and application thereof | |
| CN103865814B (en) | A kind of ganoderan high-yielding engineering bacterial strain Rmust and construction process thereof | |
| CN104152483A (en) | Application of argJ gene in fermentation production of L-citrulline | |
| CN104630355A (en) | Method for quickly screening recombinant yeast strains producing gentiopicroside | |
| CN105886638A (en) | Method for detecting salidroside synthetase gene expression activity by using dual reference genes | |
| CN103409486A (en) | Method for improving adenosine fermentation output through feeding hypoxanthine | |
| CN106631440A (en) | Compound microbial organic fertilizer for taxus chinensis as well as preparation method and application thereof | |
| CN101805704A (en) | Candidatropicalis for producing ribonucleic acid with high yield and application thereof | |
| CN104388418A (en) | Method for breeding gibberellic acid high-producing strains by performing low-energy ion induced mutation on gibberella | |
| CN114958613A (en) | Trichoderma spirillum RS05 and application thereof in preventing and treating brown rot of dendrocalamus latiflorus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160824 |