CN110305855A - Rhizoma Gastrodiae GeCPR gene and its application - Google Patents
Rhizoma Gastrodiae GeCPR gene and its application Download PDFInfo
- Publication number
- CN110305855A CN110305855A CN201910561632.8A CN201910561632A CN110305855A CN 110305855 A CN110305855 A CN 110305855A CN 201910561632 A CN201910561632 A CN 201910561632A CN 110305855 A CN110305855 A CN 110305855A
- Authority
- CN
- China
- Prior art keywords
- gecpr
- gene
- rhizoma gastrodiae
- halimasch
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 40
- IWDCLRJOBJJRNH-UHFFFAOYSA-N p-cresol Chemical class CC1=CC=C(O)C=C1 IWDCLRJOBJJRNH-UHFFFAOYSA-N 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 238000003786 synthesis reaction Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 235000012907 honey Nutrition 0.000 claims description 3
- 210000004885 white matter Anatomy 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- PUQSUZTXKPLAPR-KSSYENDESA-N 4-(beta-D-Glucopyranosyloxy) benzyl alcohol Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1ccc(CO)cc1 PUQSUZTXKPLAPR-KSSYENDESA-N 0.000 description 12
- PUQSUZTXKPLAPR-UJPOAAIJSA-N Gastrodin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(CO)C=C1 PUQSUZTXKPLAPR-UJPOAAIJSA-N 0.000 description 12
- 229930193974 gastrodin Natural products 0.000 description 12
- PUQSUZTXKPLAPR-NZEXEKPDSA-N helicidol Natural products O([C@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](CO)O1)c1ccc(CO)cc1 PUQSUZTXKPLAPR-NZEXEKPDSA-N 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 10
- 230000029087 digestion Effects 0.000 description 9
- 230000003115 biocidal effect Effects 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- BVJSUAQZOZWCKN-UHFFFAOYSA-N p-hydroxybenzyl alcohol Chemical compound OCC1=CC=C(O)C=C1 BVJSUAQZOZWCKN-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 101150053185 P450 gene Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- -1 cultivates 12h Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 2
- 108010045510 NADPH-Ferrihemoprotein Reductase Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- SCBJZDPIRGXHFV-JAJWTYFOSA-N 1-[(2S,3R,4S,5S,6R)-2,3,4,5-tetrahydroxy-6-(hydroxymethyl)oxan-2-yl]ethanone Chemical compound C(C)(=O)[C@]1(O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO SCBJZDPIRGXHFV-JAJWTYFOSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000019525 primary metabolic process Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 230000002557 soporific effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
- C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
- C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
- C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to Rhizoma Gastrodiae GeCPR gene and its applications, belong to technical field of bioengineering, the nucleotide sequence of the Rhizoma Gastrodiae GeCPR gene is as shown in SEQ ID N0:1；The protein amino acid sequence of the Rhizoma Gastrodiae GeCPR gene coding is as shown in SEQ ID N0:2；Using the gene constructed carrier for expression of eukaryon pH2-35S-GeCPR of GeCPR, halimasch is converted, positive colony transgenosis halimasch bacterial strain is obtained, 4- cresols can be converted to 4- salicylic alcohol.
Description
Technical field
The invention belongs to technical field of bioengineering, specifically, being related to Rhizoma Gastrodiae GeCPR gene and its application.
Background technique
Rhizoma Gastrodiae is a kind of mycotrophy type orchid, is a kind of rare Chinese medicine, can tranquilizing soporific, anti-blooming faint,
Improve memory, immunity can also be improved, important component Gastrodin and p-Hydroxybenzylalcohol in Rhizoma Gastrodiae, Gastrodin can controlled
The cardiovascular and cerebrovascular diseases such as similar myocardial hypertrophy, Alzheimer's disease are treated, the diseases aspect such as senile dementia, hyperglycemia has preferable treatment
Effect.
Known gastrodin synthesizing method has chemical synthesis and biosynthesis, and substrate needed for chemical synthesis has five acetyl-β-
D-Glucose, p-methyl phenol, p-Hydroxybenzylalcohol etc., these substances pass through a series of biochemical reactions, final synthetic gastrodin.
Since yield is lower in biosynthesis for Gastrodin, and synthetic method is mostly complicated or there are certain risks, improves Gastrodin
Yield becomes research hotspot.Cytochrome P450 gene can participate in being catalyzed a variety of primary cometabolism reactions in plant,
Main medicinal ingredient 4- salicylic alcohol and Gastrodin in Rhizoma Gastrodiae, both States Pharmacopoeia specifications total content cannot be below 0.25% and contain
Amount, wherein 4- salicylic alcohol is Gastrodin precursor substance, can be catalyzed and synthesized by Cytochrome P450.Cytochrome P450
Reductase (CPR) is the important component in Cytochrome P450 oxidative system (CYPs), it can transmit electronics extremely
The activated centre of CYP450 controls the speed of CYP450 redox reaction, participates in the primary and secondary metabolism of CYPs catalysis, mesh
It is preceding some researches show that in Escherichia coli heterogenous expression CYP450 the biosynthesis of Gastrodin may be implemented.
Summary of the invention
In order to solve the problems, such as background technique, the present invention provides GeCPR gene and its application, screening obtains day
The related gene GeCPR of numb CPR synthesis transfects honey by constructing GeCPR gene plant expression vector, then through Agrobacterium pMP90
Ring bacterium obtains the GeCPR transgenosis halimasch bacterial strain for having the function of synthesizing 4- salicylic alcohol.
To achieve the above object, the present invention is achieved through the following technical solutions:
Rhizoma Gastrodiae GeCPR gene, nucleotide sequence is as shown in SEQ ID N0:1.
The protein amino acid sequence of the Rhizoma Gastrodiae GeCPR gene coding is as shown in SEQ ID N0:2.
Application of the Rhizoma Gastrodiae GeCPR gene in 4- salicylic alcohol synthesis process specifically utilizes GeCPR
Gene constructed carrier for expression of eukaryon pH2-35S-GeCPR converts halimasch, obtains positive colony transgenosis halimasch bacterial strain, energy
4- salicylic alcohol is converted by 4- cresols.
Beneficial effects of the present invention: the present invention screens the related gene GeCPR for obtaining Rhizoma Gastrodiae CPR synthesis, passes through building
GeCPR gene plant expression vector, then halimasch is transfected through Agrobacterium pMP90, obtain the function with synthesis 4- salicylic alcohol
The GeCPR transgenosis halimasch bacterial strain of energy.
Detailed description of the invention
Fig. 1 is the PCR amplification of GeCPR full-length gene；
Fig. 2 is that carrier for expression of eukaryon pH2-35S-GeCPR digestion and PCR are detected, in figure, A: and digestion detection, M:
DL2000DNAmaker；1~2pH2-35S-GeCPR plasmid double digestion；3pENTR2B-GeCPR plasmid double digestion；B:PCR inspection
It surveys；
Fig. 3 is that transgenosis halimasch PCR is detected, in figure, M:DL5000DNAmaker, and 1~4: transgenosis honey bacterium, 5: non-turn
Gene halimasch, 6: blank control；
Fig. 4 is transgenosis halimasch transformation efficiency figure.
Specific embodiment
The acquisition of embodiment 1:GeCPR full-length gene
P450 gene is filtered out from transcript profile data with 5 ' end leading portion gene orders, passes through nested PCR amplification gene
Sequence:
The nest-type PRC first step, designs two upstream primers and universal primer, primer are synthesized by the Shanghai section of holding up.
First stage: upstream primer GeCPRF-1 (5 '-AGCAACAAAGGAATATGTGC-3 ') downstream primer UN36 (5 '-
GACTCGAGTCGACATC GATTTTTTTTTTTTTTTTTT-3 ') carry out gene magnification；
Second stage: it uses the first stage obtains after 10 times of product dilution, upstream primer GeCPRF-2 (5 '-
GGAGAGTCTGGTGAAGAAC-3 ') downstream primer UN36 (5 '-GACTCGAGTCGACATC GATTTTTTTTTTTTTTTTTT-
3 ') obtained target gene is detected and is spliced, online software detection http is carried out to the P450 gene that splicing obtains: //
Linuxl.soflberry.com/berry.phtml predicts the possible initiation codon of P450 and terminator codon, design primer
It is expanded, upstream primer: GeCPR-F (ggatccATGCAATCAGAGGCGATG) downstream primer: GeCPR-R
(ctcgaaTCACCAGACATCTCTCAG), product is recycled, carries out TA clone, picking positive colony is sequenced, is obtained
To correct full-length gene.
The building of embodiment 2:GeCPR carrier for expression of eukaryon
The GeCPR full-length gene target fragment of recycling is connected with 18T carrier, is transformed into bacillus coli DH 5 alpha, is obtained
Escherichia coli with PMD18T-GeCPR carrier；
The strain Escherichia coli activation of correct PMD18T-GeCPR carrier will be sequenced, picking single bacterium falls within Amp resistance LB
37 DEG C of culture 12h in fluid nutrient medium, while by PENTRTM- 2B is activated in the culture medium kind containing Kan antibiotic, is used
The plasmid extraction kit bought in the raw work in Shanghai extracts PMD18T-GeCPR and PENTRTM- 2B plasmid, while using I He of BamH
Xho I is respectively to plasmid pMD18T-GeCPR and pENTRTM- 2B carrier synchronizes double digestion, and the digestion system used is as follows: matter
Grain 6 μ L, BamH I 1 μ L, Xho I 1 μ L, 10 × Buffer 2 μ L, ddH210 μ L of O), purpose is recycled using plastic recovery kit
Target fragment after recycling is attached by genetic fragment, converts the Yu Hanyou Kan antibiotic into DH5 α competent cell
Plate on 37 DEG C of culture 12h, picking single colonie, which is transferred in Kan resistance LB culture medium, cultivates 12h, extract plasmid carry out double digestion
Detection, detects correct entry clones carrier pENTRTM2B-GeCPR carries out LR with purpose carrier pH2GW7.0 and reacts, and converts DH5
α, is transferred to 37 DEG C of culture 12h on the plate containing Spe (50 μ g/mL) antibiotic, the bacterium colony that the single bacterium of picking is formed be transferred to containing
It is cultivated in the LB liquid medium of identical antibiotic, plasmid extracts, and synchronizes double digestion detection to the plasmid of acquisition,
Obtain pH2-35S-GeCPR carrier.
It will be screened on all carriers built all culture mediums containing antibiotic, and extract plasmid, pass through confrontation
Grain synchronizes double digestion and PCR verifying, detects the correctness of recombinant plasmid, and carry out PCR and detect these the result shows that pH2-
35S-GeCPR vector construction success (as shown in Figure 2).
Embodiment 3: conversion halimasch
PH2-35S-GeCPR carrier is transferred in Agrobacterium competence pMP90 using electrotransformation, is containing Spe antibiosis
It is coated on the plate of element, is inverted 37 DEG C, 12h of culture, picking single colonie is carried out Liquid Culture, carried out using specific primer
Bacterium solution PCR spreads cultivation bacterium solution after detection is correct, transfects halimasch using the method that Agrobacterium infects halimasch.After transfection
Halimasch is applied in the PDA culture medium containing Hyg (100mg/L), and 26 DEG C of culture 26d pickings grow intact mycelia and train in liquid
It supports and is cultivated in base 2 weeks, the halimasch of successful conversion is not free of hygromycin gene, can not contain Hyg (100mg/L)
Normal growth on culture medium.
Using common wild type halimasch and transgenosis halimasch as template, drawn with the specificity of designed GeCPR gene
Object carries out PCR amplification, and whether verifying foreign gene is inserted into.As a result as shown in figure 3, the result of PCR: WT (wild type halimasch) is no
The foreign gene of 2094bp segment can be amplified, and transgenic strain can amplify target fragment, it was demonstrated that the success of GeCPR gene
It is transferred in halimasch.
Embodiment 4: the transformation efficiency of transgenosis halimasch 4- salicylic alcohol
By HPLC high performance liquid chromatography, the effective medicinal ingredient Gastrodin precursor substance tetrahydroxy benzene methanol of halimasch is detected
Transformation efficiency, 2,4,6,8,10, the tetrahydroxy benzene methanol concentration that generates afterwards for 24 hours are specifically detected, as a result as shown in figure 4,4-
The yield of 4- salicylic alcohol is basically unchanged between 10h, is increased again after catalysis for 24 hours.
The present invention obtains cytochrome P450 reductase (GeCPR) gene in Rhizoma Gastrodiae by clone, by constructing GeCPR base
Halimasch is transfected because of carrier for expression of eukaryon, then through mediated by agriculture bacillus, screening obtains the transgenosis halimasch bacterial strain of positive colony, energy
Success converts 4- cresols to the precursor substance 4- salicylic alcohol of Gastrodin, not only contributes to the yield for improving Gastrodin, also
Environmental pollution can be reduced.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention rather than limits, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from defined by claims of the present invention.
SEQ ID No.1
2041
DNA
Artificial sequence
1
1 ATGCAATCAG AGGCGATGAA GTCGTCTCCG CTGGATCTTC TCTCCGCTAT CCTCACCGGC
61 AGACGAGGCG GGGAGGGCGA TTCCATTCCC GGGAATCAAG AGCTTCTTGT TCTACTTGCG
121 ACTTCAATGG CGATGCTTGT TGGCTGCGTG CTATTGATAC TATGGCGCCG TTCGTCCAAT
181 AAGAAATCTG CTTTCAAAGC CGAGCCACCG CGGCCGGCGG CCTTGAGGGT GTCCCTGGAG
241 CCGGAGATTG ACGACGGGAA GAAGAAGGTC ATTGTACTCT TCGGAACGCA GACCGGTACT
301 GCTGAAGGGT TCGCGAAGAC GTTGGCGGAG GAAGCAAAGG CACGGTACGA TAAAGCCGCT
361 TTCAAAGTTG TTGATCTGGA TGATTACGCA GCTGATGATG ATGAGTATGA AGAGAAGATG
421 AAGAAGGAGA CTCTAGCCTT GTTCTTCATG GCAACGTATG GAGATGGAGA ACCGACTGAT
481 AATGCTGCGA GGTTCTACAA ATGGTTTTCG GAGGGCAAAG AGAGGGGCAT TTGGCTAGAG
541 AATCTCACAT ATGCGGTATT TGGACTGGGC AACAGACAGT ATGAACACTT CAATAAGGTC
601 GCAAAGGTTG TTGATGACAT CTTAGCTGAG CAAGGCGGAA AATGTCTTGT TCCTGTTGGC
661 CTTGGGGATG ATGATCAATG CATTGAGGAT GATTTCACTG CATGGAAAGA ACAGCTCTGG
721 ACCGAGCTGG ATCAGTTGCT TCGAGATGAA GATGATGTAG CAGGCGGAAC TTATACTGTT
781 GCAGTGCCTG AATATCGTGT TGTATTCATT GATTTGCCAG AGGCATCACA CACAGAAAAG
841 GGTTGGAATC TTGTGAATGG AAGTGCTGTT TGTGATGTTA ACCATCCTTG CAGGGCAAAT
901 GTAGCTGTAA GAAGGGAACT TCATTCTCCT GCTTCAGATC GTTCCTGCAT TCATCTGGAG
961 TTTGACATAC ATGGCACTGG TCTTATGTAT GAGGCAGGAG ATCATGTTGG TTTATATGCT
1021 GAAAATAGCT TGGAAACAGT GGAGGAAGCA GAAAAGTTAC TAGGCTATGC ACCAGATACA
1081 TTCTTTTCTA TTCATGCTGA CAAAGAAGAC GGGACACCAC TCAGCGGTGG TGCTCTTGCT
1141 TCTCCATTTC CATCTCCCTG CACATTGAGA ACTGCGTTGG CTCGATATGC TGACCTGTTA
1201 AGTTCTCCCA AAAAGGCTTC TTTAACCGCT TTGGCTGCTC ATGCGTCTGA TCCAATTGAA
1261 GCTGAACGGT TGAGATTTCT GGCTTCCCCT TCCGGAAAGG ATGAGTATTC TCAGTGGGTA
1321 ATAGCTAGCC AGAGGAGTCT TTTGGAAGTA ATGGCCGAGT TCCCTTCAGC CAAGCCACCT
1381 CTAGGTGTTT TCTTTGCAGC AATTGCTCCA AGATTACAGC ATCGCTTTTA TTCTATATCA
1441 TCTTCTTCAA GGATGTACCC AACTAGAATT CATGTGACAT GTGCTCTAGT GTATGGACCA
1501 ACACTGACCG GAAGGATTCA TAAGGGAGTA TGCTCGACTT GGATGAAGAA TGCAATTCCA
1561 TTGGAGGGAA GCGAAAATTG CAGCTGGGCT CCTATATTTG TAAGGCAATC AAATTTTAAG
1621 CTGCCTGCAG ATACCTCAGT GCCCATTATC ATGATTGGAC CTGGAACTGG CTTGGCCCCC
1681 TTCCGTGGCT TCCTACAGGA GAGGCTGGTG TTAAAAGAGT CCGGTGCTGA ACTCGGCCCT
1741 GCTATGCTCT TCTTTGGATG CAGGAATCGG AAAATGGATT TCATTTATGA AGACGAGCTT
1801 AAGAATTTTG CTGAGGCTGG AGTGGTTTCT GAGCTCATTG TGACTTTCTC TCGGGAGGGA
1861 GCAACAAAGG AATATGTGCA GCATAAAATG GTCGAAAAGG CATCTGATAT TTGGGATGTT
1921 ATATCTAAAG GCGGTTACAT TTATGTATGT GGTGATGCTA AAGGCATGGC CAAAGATGTT
1981 CACCGCACTC TGCATACTAT TGTTCAGGAA CAGGGCTGTC TAGATAGCTC GAAGACGGAG
2041 AGTCTGGTGA AGAACCTGCA AATGCAAGGA AGGTATCTGA GAGATGTCTG GTGA
SEQ ID No.2
661
Amino acid sequence
2
1 MQSEAMKSSP LDLLSAILTG RRGGEGDSIP GNQELLVLLA TSMAMLVGCV LLILWRRSSN
61 KKSAFKAEPP RPAALRVSLE PEIDDGKKKV IVLFGTQTGT AEGFAKTLAE EAKARYDKAA
121 FKVVDLDDYA ADDDEYEEKM KKETLALFFM ATYGDGEPTD NAARFYKWFS EGKERGIWLE
181 NLTYAVFGLG NRQYEHFNKV AKVVDDILAE QGGKCLVPVG LGDDDQCIED DFTAWKEQLW
241 TELDQLLRDE DDVAGGTYTV AVPEYRVVFI DLPEASHTEK GWNLVNGSAV CDVNHPCRAN
301 VAVRRELHSP ASDRSCIHLE FDIHGTGLMY EAGDHVGLYA ENSLETVEEA EKLLGYAPDT
361 FFSIHADKED GTPLSGGALA SPFPSPCTLR TALARYADLL SSPKKASLTA LAAHASDPIE
421 AERLRFLASP SGKDEYSQWV IASQRSLLEV MAEFPSAKPP LGVFFAAIAP RLQHRFYSIS
481 SSSRMYPTRI HVTCALVYGP TLTGRIHKGV CSTWMKNAIP LEGSENCSWA PIFVRQSNFK
541 LPADTSVPII MIGPGTGLAP FRGFLQERLV LKESGAELGP AMLFFGCRNR KMDFIYEDEL
601 KNFAEAGVVS ELIVTFSREG ATKEYVQHKM VEKASDIWDV ISKGGYIYVC GDAKGMAKDV
661 HRTLHTIVQE QGCLDSSKTE SLVKNLQMQG RYLRDVW*
Claims (4)
1. Rhizoma Gastrodiae GeCPR gene, it is characterised in that: the nucleotide sequence of the Rhizoma Gastrodiae GeCPR gene such as SEQ ID N0:1 institute
Show.
2. Rhizoma Gastrodiae GeCPR gene according to claim 1, it is characterised in that: the egg of the Rhizoma Gastrodiae GeCPR gene coding
White matter amino acid sequence is as shown in SEQ ID N0:2.
3. application of the Rhizoma Gastrodiae GeCPR gene according to claim 1 in 4- salicylic alcohol synthesis process.
4. application of the Rhizoma Gastrodiae GeCPR gene according to claim 3 in 4- salicylic alcohol synthesis process, feature exist
In: the gene constructed carrier for expression of eukaryon pH2-35S-GeCPR of GeCPR is utilized, halimasch is converted, obtains positive colony transgenosis honey
Ring bacteria strain can convert 4- cresols to 4- salicylic alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910561632.8A CN110305855B (en) | 2019-06-26 | 2019-06-26 | Gastrodia elata GeCPR gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910561632.8A CN110305855B (en) | 2019-06-26 | 2019-06-26 | Gastrodia elata GeCPR gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110305855A true CN110305855A (en) | 2019-10-08 |
| CN110305855B CN110305855B (en) | 2022-03-22 |
Family
ID=68076293
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910561632.8A Active CN110305855B (en) | 2019-06-26 | 2019-06-26 | Gastrodia elata GeCPR gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110305855B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763663A (en) * | 2020-07-09 | 2020-10-13 | 昆明理工大学 | Gastrodia elata glucosyltransferase gene and application thereof |
| CN113528551A (en) * | 2021-08-03 | 2021-10-22 | 昆明理工大学 | Gastrodia elata superoxide dismutase gene and application thereof |
Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999023226A1 (en) * | 1997-10-30 | 1999-05-14 | The Regents Of The University Of Michigan | Methods and compositions for use of benzylalcohol acetyl transferase |
| US20030070188A1 (en) * | 1997-07-15 | 2003-04-10 | Daphna Havkin-Frenkel | Vanillin biosynthetic pathway enzyme from Vanilla planifolia |
| US20070207209A1 (en) * | 2004-08-27 | 2007-09-06 | Murphy Christopher J | Trophic factor combinations for nervous system treatment |
| CN101094827A (en) * | 2004-10-27 | 2007-12-26 | 第一三共株式会社 | Benzene compound having two or more substituents |
| CN101586116A (en) * | 2008-10-14 | 2009-11-25 | 昆明理工大学 | Plant expression vector of arabidopsis thaliana cytosolic malate dehydrogenase gene and application thereof |
| US7723498B2 (en) * | 2004-06-04 | 2010-05-25 | University Of Connecticut | Directed evolution of recombinant monooxygenase nucleic acids and related polypeptides and methods of use |
| CN101928720A (en) * | 2010-05-14 | 2010-12-29 | 昆明理工大学 | Prokaryotic expression vector of dependent ADH (Alcohol Dehydrogenase) of arabidopsis glutathione as well as construction method and application thereof |
| CN102686585A (en) * | 2009-10-07 | 2012-09-19 | 住友化学株式会社 | Heterocyclic compound and its use for control of an arthropod pest |
| WO2014052961A1 (en) * | 2012-09-28 | 2014-04-03 | Industrial Cooperation Foundation Chonbuk National University | Pvax copolymer and pvax microparticles comprising the same |
| EP2749644A1 (en) * | 2012-12-27 | 2014-07-02 | Eviagenics S.A. | Recombinant host cell for biosynthetic production of vanillin |
| CN105039274A (en) * | 2015-07-13 | 2015-11-11 | 中国农业科学院蔬菜花卉研究所 | Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster |
| US20150322465A1 (en) * | 2012-12-27 | 2015-11-12 | Rhodia Operations | Recombinant host cell for biosynthetic production |
| CN105121647A (en) * | 2012-11-01 | 2015-12-02 | 不列颠哥伦比亚大学 | Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof |
| CN106701696A (en) * | 2016-12-16 | 2017-05-24 | 江苏省中国科学院植物研究所 | Cytochrome P450 reductase 1 of lycoris aurea as amaryllidaceae plant as well as coding gene and application thereof |
| CN106834246A (en) * | 2016-12-30 | 2017-06-13 | 江苏省中国科学院植物研究所 | Amrallid Lycoris aurea cytochrome P450 reductase 2 and its encoding gene and application |
| CN107988247A (en) * | 2017-10-27 | 2018-05-04 | 昆明理工大学 | Halimasch selection markers and the method for building transgenic strain |
| CN108337892A (en) * | 2015-01-30 | 2018-07-27 | 埃沃尔瓦公司 | Steviol glycoside is produced in the recombination host |
| CN109477120A (en) * | 2016-07-20 | 2019-03-15 | 弗门尼舍有限公司 | Vetiver |
| CN109468351A (en) * | 2018-11-27 | 2019-03-15 | 湖南美可达生物资源股份有限公司 | The method of efficient Enzyme catalyzed synthesis sanguinarine and Chelerythrine |
| CN112522220A (en) * | 2019-08-27 | 2021-03-19 | 中国医学科学院药用植物研究所 | Gene cloning primer, function and application of salvia miltiorrhiza CYP71BE37 participating in tanshinone biosynthesis |
-
2019
- 2019-06-26 CN CN201910561632.8A patent/CN110305855B/en active Active
Patent Citations (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030070188A1 (en) * | 1997-07-15 | 2003-04-10 | Daphna Havkin-Frenkel | Vanillin biosynthetic pathway enzyme from Vanilla planifolia |
| WO1999023226A1 (en) * | 1997-10-30 | 1999-05-14 | The Regents Of The University Of Michigan | Methods and compositions for use of benzylalcohol acetyl transferase |
| US7723498B2 (en) * | 2004-06-04 | 2010-05-25 | University Of Connecticut | Directed evolution of recombinant monooxygenase nucleic acids and related polypeptides and methods of use |
| US20070207209A1 (en) * | 2004-08-27 | 2007-09-06 | Murphy Christopher J | Trophic factor combinations for nervous system treatment |
| CN101094827A (en) * | 2004-10-27 | 2007-12-26 | 第一三共株式会社 | Benzene compound having two or more substituents |
| CN101586116A (en) * | 2008-10-14 | 2009-11-25 | 昆明理工大学 | Plant expression vector of arabidopsis thaliana cytosolic malate dehydrogenase gene and application thereof |
| CN102686585A (en) * | 2009-10-07 | 2012-09-19 | 住友化学株式会社 | Heterocyclic compound and its use for control of an arthropod pest |
| CN101928720A (en) * | 2010-05-14 | 2010-12-29 | 昆明理工大学 | Prokaryotic expression vector of dependent ADH (Alcohol Dehydrogenase) of arabidopsis glutathione as well as construction method and application thereof |
| US20150290239A1 (en) * | 2012-09-28 | 2015-10-15 | Industrial Cooperation Foundation Chonbuk National University | Pvax copolymer and pvax microparticles comprising the same |
| WO2014052961A1 (en) * | 2012-09-28 | 2014-04-03 | Industrial Cooperation Foundation Chonbuk National University | Pvax copolymer and pvax microparticles comprising the same |
| CN105121647A (en) * | 2012-11-01 | 2015-12-02 | 不列颠哥伦比亚大学 | Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof |
| CN105283547A (en) * | 2012-12-27 | 2016-01-27 | 罗地亚经营管理公司 | Recombinant host cell for biosynthetic production |
| EP2749644A1 (en) * | 2012-12-27 | 2014-07-02 | Eviagenics S.A. | Recombinant host cell for biosynthetic production of vanillin |
| US20150322465A1 (en) * | 2012-12-27 | 2015-11-12 | Rhodia Operations | Recombinant host cell for biosynthetic production |
| CN108337892A (en) * | 2015-01-30 | 2018-07-27 | 埃沃尔瓦公司 | Steviol glycoside is produced in the recombination host |
| CN105039274A (en) * | 2015-07-13 | 2015-11-11 | 中国农业科学院蔬菜花卉研究所 | Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster |
| CN109477120A (en) * | 2016-07-20 | 2019-03-15 | 弗门尼舍有限公司 | Vetiver |
| CN106701696A (en) * | 2016-12-16 | 2017-05-24 | 江苏省中国科学院植物研究所 | Cytochrome P450 reductase 1 of lycoris aurea as amaryllidaceae plant as well as coding gene and application thereof |
| CN106834246A (en) * | 2016-12-30 | 2017-06-13 | 江苏省中国科学院植物研究所 | Amrallid Lycoris aurea cytochrome P450 reductase 2 and its encoding gene and application |
| CN107988247A (en) * | 2017-10-27 | 2018-05-04 | 昆明理工大学 | Halimasch selection markers and the method for building transgenic strain |
| CN109468351A (en) * | 2018-11-27 | 2019-03-15 | 湖南美可达生物资源股份有限公司 | The method of efficient Enzyme catalyzed synthesis sanguinarine and Chelerythrine |
| CN112522220A (en) * | 2019-08-27 | 2021-03-19 | 中国医学科学院药用植物研究所 | Gene cloning primer, function and application of salvia miltiorrhiza CYP71BE37 participating in tanshinone biosynthesis |
Non-Patent Citations (8)
| Title |
|---|
| CHANG-QING YANG等: ""Characterization of two NADPH: Cytochrome P450 reductases from cotton (Gossypium hirsutum)"", 《PHYTOCHEMISTRY》 * |
| H. UMESHA SHETTY等: ""Identification and regional distribution in rat brain of radiometabolites of the dopamine transporter PET radioligand [11C]PE2I"", 《EUR J NUCL MED MOL IMAGING》 * |
| NCBI: ""NADPH--cytochrome P450 reductase-like [Phalaenopsis equestris]"", 《GENBANK DATABASE》 * |
| NCBI: ""PREDICTED: Phalaenopsis equestris NADPH--cytochrome P450 reductase-like(LOC110021752), mRNA"", 《GENBANK DATABSE》 * |
| 徐德宏等: ""天麻素生物合成的研究进展"", 《中草药》 * |
| 程婕等: ""细胞色素P450氧化还原酶的研究进展"", 《中国药理学通报》 * |
| 肖舒卉: ""天麻GeCPR与共生蜜环菌Lac基因克隆及其功能鉴定"", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 钟贝芬等: ""基于细胞色素P450 单加氧酶介导的4-甲酚氧化降解途径的天麻素生物合成"", 《湖南师范大学自然科学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763663A (en) * | 2020-07-09 | 2020-10-13 | 昆明理工大学 | Gastrodia elata glucosyltransferase gene and application thereof |
| CN111763663B (en) * | 2020-07-09 | 2022-04-15 | 昆明理工大学 | Gastrodia elata glucosyltransferase gene and application thereof |
| CN113528551A (en) * | 2021-08-03 | 2021-10-22 | 昆明理工大学 | Gastrodia elata superoxide dismutase gene and application thereof |
| CN113528551B (en) * | 2021-08-03 | 2023-03-24 | 昆明理工大学 | Gastrodia elata superoxide dismutase gene and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110305855B (en) | 2022-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100569938C (en) | Can produce the Cladosporium endogenetic fungus of trans-resveratrol | |
| CN109988722A (en) | A kind of method of recombinant Saccharomyces cerevisiae bacterial strain and its application and production tyrosol and/or rhodioside | |
| CN112899177B (en) | Recombinant yarrowia lipolytica expressing myrosinase TGG4 and application thereof | |
| CN104357418A (en) | Applications of glycosyltransferase and mutants thereof to synthesis of ginsenoside Rh2 | |
| CN113755354A (en) | Recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application thereof | |
| CN110343709A (en) | A kind of arctic nocardia lasso trick peptide gene cluster and its cloning and expression method | |
| CN107460203A (en) | A kind of recombinant bacterium and construction method and purposes for producing rhodioside and the like | |
| CN110305855A (en) | Rhizoma Gastrodiae GeCPR gene and its application | |
| CN114058525A (en) | High-yield squalene genetic engineering bacterium and construction method and application thereof | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| CN116987603A (en) | Recombinant saccharomyces cerevisiae strain for high yield of cannabigerolic acid as well as construction method and application thereof | |
| CN110117582B (en) | Fusion protein, encoding gene thereof and application thereof in biosynthesis | |
| CN112080452B (en) | High-yield phenyllactic acid bacillus licheniformis genetically engineered bacterium, method for producing phenyllactic acid and application | |
| CN111154665B (en) | Recombinant yarrowia lipolytica and construction method and application thereof | |
| CN116716270A (en) | Glycosyltransferase and application thereof in biocatalysis synthesis of salidroside | |
| CN107460152A (en) | Produce recombinant bacterium, construction method and the purposes of rhodioside and the like | |
| CN113817757A (en) | Recombinant yeast engineering strain for producing cherry glycoside and application | |
| CN110004099A (en) | A kind of fermentation method for producing of rhodioside | |
| CN113969288A (en) | High-yield farnesol gene engineering bacterium and construction method and application thereof | |
| CN107810269A (en) | Novel promoter and application thereof | |
| CN106010999A (en) | Gene engineering strain, culturing method and application of gene engineering strain | |
| CN108424859B (en) | Construction and application of gene engineering bacteria for producing citicoline | |
| CN105586281B (en) | Proline is for enhancing purposes, the purposes of gene, expression vector of bacterial strain tolerance and application thereof, bacterial strain and application thereof | |
| CN109266591A (en) | A kind of genetic engineering bacterium and construction method using phenol production protocatechuic acid (3,4- dihydroxy-benzoic acid) | |
| CN114921465B (en) | Regulatory element CsiZ and application thereof in improvement of synthetic yield of terpenoid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240202 Address after: East Floor 1, Building C4, Biological Innovation Park, No. 666, Gaoxin Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province 430000 Patentee after: Hubei Chang'e Biological Co.,Ltd. Country or region after: China Address before: 650093 No. 253, Xuefu Road, Wuhua District, Yunnan, Kunming Patentee before: Kunming University of Science and Technology Country or region before: China |
|
| TR01 | Transfer of patent right |