CN102120762A - Arabidopsis anthocyanin pap1 purified protein and preparation method and applications thereof - Google Patents
Arabidopsis anthocyanin pap1 purified protein and preparation method and applications thereof Download PDFInfo
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Abstract
The invention belongs to the gene engineering field and particularly relates to the cloning, prokaryotic expression and protein purification of an Arabidopsis anthocyanin regulatory protein gene, an Arabidopsis anthocyanin pap1 purified protein and a preparation method and applications thereof. The preparation method comprises the following steps: utilizing the reverse transcription-polymerase chain reaction (RT-PCR) technology to clone the full-length cDNA of pap1 from the purple-red leaves of Arabidopsis, then utilizing a prokaryotic expression system to express the gene in Escherichia coli BL21, and adopting the nickel column method to purify and obtain the purified protein of Arabidopsis pap1. The invention also relates to the applications of the Arabidopsis anthocyanin pap1 purified protein in the preparation of Myb antibody and the detection of Myb protein expression.
Description
Technical field
The invention belongs to the genetically engineered field, be specifically related to a kind of clone, prokaryotic expression and protein purification of Arabidopis thaliana pigment regulatory protein gene, Arabidopis thaliana anthocyanidin pap1 purifying protein and its production and application.
Background technology
Anthocyanogen is the secondary metabolite that is distributed widely in the plant, and it is a class flavonoid that derives from phenylalanine, and it is water miscible, and is synthetic in tenuigenin, finally navigates to vacuole; It provides from orange/redness to purple/blue series of color.Anthocyanogen may cause corticocerebral disorder that therapeutic action is arranged for oxidative stress in addition, can also improve the learning and memory ability that causes the mouse of estrogen deficiency owing to oophorectomize.Manyly in recent years studies show that anthocyanogen has and comprise anti-oxidant, anti-inflammatory, antibiotic, anticancer and reduce a series of physiologically active of evidence of coronary heart diseases.Part myb class transcription factor is in the biosynthesizing of transcriptional level adjusted anthocyanogen, and Arabidopis thaliana pap gene just belongs to regulates the biosynthetic myb genoid of anthocyanogen.Up to the present, the anthocyanogen approach of all higher plants all is to be regulated and control by a cover transcription factor that comprises Myb, bHLH and WD40; Different with other plant, can act on whole phenylpropionic acid class route of synthesis at Arabidopsis thaliana PAP 1 (Myb)/bHLH/WD40 mixture; But the Myb class regulatory factor of Arabidopis thaliana is not also identified clear fully, and the anthocyanogen approach that TTG1-dependent form transcription complex is regulated is also known little about it.Pap1 is in the biosynthesizing of transcriptional level adjusted anthocyanogen, therefore many scholars have carried out big quantity research to pap1, the clone, the pap1 that comprise gene change Arabidopis thaliana, tomato, tobacco healing tissue etc., but prior art is not still for the proteic report of AtPAP1.
Summary of the invention
The objective of the invention is to obtain purifying PAP1 albumen, be used to make the PAP1 polyclonal antibody.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Arabidopis thaliana anthocyanidin pap1 purifying protein is got by following method preparation:
(1) reported the entire reading frame frame of sequence (NM_104541.3) according to NCBI, design primer PAP1-T5 ' (5
CCATGGAGGGTTCGTCCAAAGGG3 ', line place is restriction enzyme site NcoI) and PAP1-T3 ' (5 '
CTCGAGCTAATCAAATTTCACAGTCTCTCCATC3 ', line place is restriction enzyme site XhoI) carry out the amplification of cDNA total length, for convenient follow-up prokaryotic expression research, introduce NcoI and XhoI site at 5 of PAP1-T5 ' and PAP1-T3 ' ' end respectively; Total RNA is a template with Arabidopis thaliana red-purple blade, with using PAP1-T5 ' and PAP1-T3 ' to increase behind the synthetic cDNA of M-MLV ThermoScript II;
(2) reclaim the pap1 full length fragment, and be connected on the pMD18-T carrier, adopt the alkaline lysis method of extracting plasmid, cut by enzyme and detect acquisition recombinant plasmid pMD18-pap1;
(3) make up prokaryotic expression carrier: use NcoI and XhoI that plasmid pMD18-pap1 and pET32a-xyk are carried out double digestion and obtain pap1 gene and pET32a carrier respectively, reclaim respectively after the leakage of electricity swimming, connect 16h at 16 ℃ then, obtain prokaryotic expression carrier pET32a-pap1;
(4) PAP1 albumen pronucleus expression: use the thermal stimulus method that pET32a-pap1 is changed in the e. coli bl21, under IPTG induces, grope the optimum expression condition, under optimum condition, carry out great expression;
(5) PAP1 protein purification: collect thalline, carry out ultrasonication, cross the nickel post and carry out purifying, the albumen behind the collection purifying, the albumen behind the purifying are used to make Myb antibody and the Myb protein expression detects.
The preparation method of Arabidopis thaliana anthocyanidin pap1 purifying protein comprises the steps:
(1) reported the entire reading frame frame of sequence (NM_104541.3) according to NCBI, design primer PAP1-T5 ' (5
CCATGGAGGGTTCGTCCAAAGGG3 ', line place is restriction enzyme site NcoI) and PAP1-T3 ' (5 '
CTCGAGCTAATCAAATTTCACAGTCTCTCCATC3 ', line place is restriction enzyme site XhoI) carry out the amplification of cDNA total length, for convenient follow-up prokaryotic expression research, introduce NcoI and XhoI site at 5 of PAP1-T5 ' and PAP1-T3 ' ' end respectively; Total RNA is a template with Arabidopis thaliana red-purple blade, with using PAP1-T5 ' and PAP1-T3 ' to increase behind the synthetic cDNA of M-MLV ThermoScript II;
(2) reclaim the pap1 full length fragment, and be connected on the pMD18-T carrier, adopt the alkaline lysis method of extracting plasmid, cut by enzyme and detect acquisition recombinant plasmid pMD18-pap1;
(3) make up prokaryotic expression carrier: use NcoI and XhoI that plasmid pMD18-pap1 and pET32a-xyk are carried out double digestion and obtain pap1 gene and pET32a carrier respectively, reclaim respectively after the leakage of electricity swimming, connect 16h at 16 ℃ then, obtain prokaryotic expression carrier pET32a-pap1;
(4) PAP1 albumen pronucleus expression: use the thermal stimulus method that pET32a-pap1 is changed in the e. coli bl21, under IPTG induces, grope the optimum expression condition, under optimum condition, carry out great expression;
(5) PAP1 protein purification: collect thalline, carry out ultrasonication, cross the nickel post and carry out purifying, the albumen behind the collection purifying.
The application of Arabidopis thaliana anthocyanidin pap1 purifying protein in preparation Myb antibody.
The application of Arabidopis thaliana anthocyanidin pap1 purifying protein in the Myb protein expression detects.
The present invention utilizes the RT-PCR technology to clone the pap1 full-length cDNA from Arabidopis thaliana red-purple blade, and then utilizes prokaryotic expression system that this gene is expressed in e. coli bl21, adopts nickel post method purifying then, obtains the purifying protein of Arabidopis thaliana PAP1.
The clone of pap1 gene of the present invention, prokaryotic expression and purifying:
Reported the entire reading frame frame of sequence (NM_104541.3) according to NCBI, design primer PAP1-T5 ' (5
CCATGGAGGGTTCGTCCAAAGGG3 ', line place is restriction enzyme site NcoI) and PAP1-T3 ' (5 '
CTCGAGCTAATCAAATTTCACAGTCTCTCCATC3 ', line place is restriction enzyme site XhoI) carry out the amplification of cDNA total length, for convenient follow-up prokaryotic expression research, introduce NcoI and XhoI site at 5 of PAP1-T5 ' and PAP1-T3 ' ' end respectively.Total RNA is a template with Arabidopis thaliana red-purple blade, with using PAP1-T5 ' and PAP1-T3 ' to increase behind the synthetic cDNA of M-MLV ThermoScript II (Promega company).
Reclaim the pap1 full length fragment, and be connected to the pMD18-T carrier (precious biotechnology company limited, Takara) on, adopt the alkaline lysis method of extracting plasmid, cut to detect by enzyme and obtain recombinant plasmid pMD18-pap1.
Make up prokaryotic expression carrier
Use NcoI and XhoI that plasmid pMD18-pap1 and pET32a-xyk are carried out double digestion and obtain pap1 gene and pET32a carrier respectively, reclaim respectively after the leakage of electricity swimming, connect 16h at 16 ℃ then, obtain prokaryotic expression carrier pET32a-pap1.
The PAP1 albumen pronucleus expression
Use the thermal stimulus method that pET32a-pap1 is changed in the e. coli bl21, under IPTG induces, grope the optimum expression condition, under optimum condition, carry out great expression.
The PAP1 protein purification
Collect thalline, carry out ultrasonication, cross the nickel post and carry out purifying, the albumen behind the collection purifying.
Albumen behind the purifying is used to make Myb antibody and the Myb protein expression detects.
Description of drawings:
Fig. 1 is total RNA;
Fig. 2 is the amplification of pap1 gene;
Fig. 3 is a large amount of amplifications of pap1;
Fig. 4 reclaims for pap1 glue;
Fig. 5 is that the PCR of pap1 bacterium liquid detects;
Fig. 6 is NcoI and XhoI double digestion;
Fig. 7 is the pET32a-pap1 plasmid;
Fig. 8 detects for 32a-pap1PCR;
Fig. 9 is that NcoI and XhoI double digestion detect;
M:protein marker-0431 among Figure 10; 1: contrast, IPTG is the inductive total protein not; 2,3,4,5:1mM IPTG induce 2,4,6 respectively, the total protein of 8h;
Figure 11 is the structure of prokaryotic expression carrier pET32a-pap1.
Embodiment
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiments of the invention, but do not limit the present invention with this.
Embodiment 1:
Reagent and instrument
Reagent is mainly molecular biology experiment reagent.Various restriction enzymes, Long-taq polysaccharase, Taq polysaccharase, RNA enzyme inhibitors, dNTP etc. are precious biotechnology company limited (Dalian, Takara) product, ThermoScript II is a Promaga company, plasmid extraction kit is available from vast Tyke Bioisystech Co., Ltd, and TRIzo1 Reagent RNA extracts test kit available from invitrogen company.All the other reagent are homemade analytical pure.
Instrument is molecular biology and genetically engineered laboratory common instrument equipment.
All primer sequences are synthetic, and all (Sangon) carry out in Shanghai Shanghai life worker with gene sequencing.
Method therefor is ordinary method if no special instructions among the present invention.
The clone of pap1 gene of the present invention, prokaryotic expression and purification process basic step: the entire reading frame frame according to NCBI has reported sequence (NM_104541.3), design primer PAP1-T5 ' (5
CCATGGAGGGTTCGTCCAAAGGG3 ', line place is restriction enzyme site NcoI) and PAP1-T3 ' (5 '
CTCGAGCTAATCAAATTTCACAGTCTCTCCATC3 ', line place is restriction enzyme site XhoI) carry out the amplification of cDNA total length, for convenient follow-up prokaryotic expression research, introduce NcoI and XhoI site at 5 of PAP1-T5 ' and PAP1-T3 ' ' end respectively.Total RNA is a template with Arabidopis thaliana red-purple blade, with using PAP1-T5 ' and PAP1-T3 ' to increase behind the synthetic cDNA of M-MLV ThermoScript II (Promega company).
Reclaim the pap1 full length fragment, and be connected to the pMD18-T carrier (precious biotechnology company limited, Takara) on, adopt the alkaline lysis method of extracting plasmid, cut to detect by enzyme and obtain recombinant plasmid pMD18-pap1.
Make up prokaryotic expression carrier
Use NcoI and XhoI that plasmid pMD18-pap1 and pET32a-xyk are carried out double digestion and obtain pap1 gene and pET32a carrier respectively, reclaim respectively after the leakage of electricity swimming, connect 16h at 16 ℃ then, obtain prokaryotic expression carrier pET32a-pap1.
The PAP1 albumen pronucleus expression
Use the thermal stimulus method that pET32a-pap1 is changed in the e. coli bl21, under IPTG induces, grope the optimum expression condition, under optimum condition, carry out great expression.
The PAP1 protein purification
Collect thalline, carry out ultrasonication, cross the nickel post and carry out purifying, the albumen behind the collection purifying.
Albumen behind the purifying is used to make Myb antibody and the Myb protein expression detects.
Particularly,
The extraction of RNA
Test materials is the red-purple blade of florescence Arabidopis thaliana, and guanidine isothiocyanate method is adopted in the extraction of the total RNA of blade, and the RNA of extraction is standby in-20 ℃ of preservations.
The clone of Arabidopis thaliana pap1 gene
Entire reading frame according to existing sequence (NM_104541.3) sets up meter primer PAP1-T5 ' (5
CCATGGAGGGTTCGTCCAAAGGG3 ', line place is a restriction enzyme site, down together) and PAP1-T3 ' (5 '
CTCGAGCTAATCAAATTTCACAGTCTCTCCATC3 ') carries out the amplification of cDNA total length,, introduce NcoI and XhoI site at 5 of PAP1-T5 ' and PAP1-T3 ' ' end respectively for convenient follow-up prokaryotic expression research.Total RNA is a template with Arabidopis thaliana red-purple blade, with using PAP1-T5 ' and PAP1-T3 ' to increase behind the synthetic cDNA of M-MLV ThermoScript II (Promega company).Amplified production carries out electrophoresis, observations in gel imaging system on 1.2% sepharose.The PCR product reclaims the back and is connected (purpose fragment DNA0.1-0.2pmol with the pMD18-T carrier; T-carrier 50 μ g/ml, 0.5 μ l; Ligation Solution I 3 μ l, 16 ℃ connect 8-12h), Transformed E .coliDH5 α obtains recon pMD-pap1 and (competence intestinal bacteria E.coli DH5 α 100 μ l is added among the plasmid system 6 μ l; Mixed solution ice bath 20min, 42 ℃ of thermal stimulus 45s, ice bath 2min; Add 900 μ l SOC, 37 ℃ of shaking table 200rpm 60min; After cultivating end, the centrifugal 1min of 10000rpm; Inhale on super clean bench and remove supernatant, when remain about 0.1ml, use the rifle mixing, access has on the LB flat board of resistance, is coated with even with aseptic triangle glass stick; 37 ℃ of incubated overnight; The picking positive bacteria is dropped into the row checking) and deliver to Shanghai and give birth to the order-checking of worker bio-engineering corporation.Carry out sequence homology analysis among order-checking back and the NCBI.
Construction of prokaryotic expression vector and abduction delivering
Extract behind the plasmid pMD-pap1 with NcoI and XhoI double digestion and reclaim the pap1 fragment.This fragment is connected to the plasmid pET-32a (+) that cuts through same enzyme goes up formation recombinant vectors pET-pap1, be converted into expressive host bacterium BL21, the positive bacterium colony of picking carries out abduction delivering after the Screening and Identification.To contain pET-pap1 recombinant bacterial strain 37 ℃ of shaking culture in the LB liquid nutrient medium and spend the night, and be inoculated on the identical LB substratum in 1: 100 ratio and be cultured to OD
600Be 0.6-0.8, add IPTG to final concentration be 1mM, cultivate 0,2,4,6 respectively, collect bacterium liquid behind the 8h and be used for analyzing total albumen.4 ℃, the centrifugal 1min of 12000rpm abandon supernatant liquor, the precipitation with 100 μ L sds gel sample loading buffers (Tri s-HCl 50mM, pH 6.8; SDS 2%; DTT 100mM; Tetrabromophenol sulfonphthalein 0.1%; Glycerine 10%) resuspended, boil 5min, the centrifugal 1min of 12000rpm gets 20 μ L supernatant liquors and carries out the SDS-PAGE detection.With Xylene Brilliant Cyanine G R-250 dyeing (0.1% Xylene Brilliant Cyanine G R-250,40% methyl alcohol, 10% glacial acetic acid), decolouring (25% methyl alcohol, 6% glacial acetic acid) back record result.
The proteic purifying of soluble analysis PAP1 of recombinant protein
Thalline behind a large amount of abduction deliverings is through the ultrasonic treatment thalline, and 4 ℃ of centrifugal 20min of 12000rpm keep respectively and go up cleer and peaceful precipitation.Supernatant partly uses the Ni post to carry out purifying, low salt buffer balance columns material in the elder generation, supernatant liquor upper prop then, resuspended post material, ice bath 30min, discard effluent liquid, 15 times of column volume low salt buffer wash-outs, 10 times of column volume high-salt buffer wash-outs, 5 times of column volume low salt buffer wash-outs, 2 times of column volume elutriant wash-outs are collected sample liquid.SDS-PAGE detects contrast supernatant, precipitation and purification result.
Claims (4)
1. Arabidopis thaliana anthocyanidin pap1 purifying protein, by following method preparation and get:
(1) reported the entire reading frame frame of sequence (NM_104541.3) according to NCBI, design primer PAP1-T5 ' (5
CCATGGAGGGTTCGTCCAAAGGG3 ', line place is restriction enzyme site NcoI) and PAP1-T3 ' (5 '
CTCGAGCTAATCAAATTTCACAGTCTCTCCATC3 ', line place is restriction enzyme site XhoI) carry out the amplification of cDNA total length, for convenient follow-up prokaryotic expression research, introduce NcoI and XhoI site at 5 of PAP1-T5 ' and PAP1-T3 ' ' end respectively; Total RNA is a template with Arabidopis thaliana red-purple blade, with using PAP1-T5 ' and PAP1-T3 ' to increase behind the synthetic cDNA of M-MLV ThermoScript II;
(2) reclaim the pap1 full length fragment, and be connected on the pMD18-T carrier, adopt the alkaline lysis method of extracting plasmid, cut by enzyme and detect acquisition recombinant plasmid pMD18-pap1;
(3) make up prokaryotic expression carrier: use NcoI and XhoI that plasmid pMD18-pap1 and pET32a-xyk are carried out double digestion and obtain pap1 gene and pET32a carrier respectively, reclaim respectively after the leakage of electricity swimming, connect 16h at 16 ℃ then, obtain prokaryotic expression carrier pET32a-pap1;
(4) PAP1 albumen pronucleus expression: use the thermal stimulus method that pET32a-pap1 is changed in the e. coli bl21, under IPTG induces, grope the optimum expression condition, under optimum condition, carry out great expression;
(5) PAP1 protein purification: collect thalline, carry out ultrasonication, cross the nickel post and carry out purifying, the albumen behind the collection purifying, the albumen behind the purifying are used to make Myb antibody and the Myb protein expression detects.
2. the preparation method of Arabidopis thaliana anthocyanidin pap1 purifying protein comprises the steps:
(1) reported the entire reading frame frame of sequence (NM_104541.3) according to NCBI, design primer PAP1-T5 ' (5
CCATGGAGGGTTCGTCCAAAGGG3 ', line place is restriction enzyme site NcoI) and PAP1-T3 ' (5 '
CTCGAGCTAATCAAATTTCACAGTCTCTCCATC3 ', line place is restriction enzyme site XhoI) carry out the amplification of cDNA total length, for convenient follow-up prokaryotic expression research, introduce NcoI and XhoI site at 5 of PAP1-T5 ' and PAP1-T3 ' ' end respectively; Total RNA is a template with Arabidopis thaliana red-purple blade, with using PAP1-T5 ' and PAP1-T3 ' to increase behind the synthetic cDNA of M-MLV ThermoScript II;
(2) reclaim the pap1 full length fragment, and be connected on the pMD18-T carrier, adopt the alkaline lysis method of extracting plasmid, cut by enzyme and detect acquisition recombinant plasmid pMD18-pap1;
(3) make up prokaryotic expression carrier: use NcoI and XhoI that plasmid pMD18-pap1 and pET32a-xyk are carried out double digestion and obtain pap1 gene and pET32a carrier respectively, reclaim respectively after the leakage of electricity swimming, connect 16h at 16 ℃ then, obtain prokaryotic expression carrier pET32a-pap1;
(4) PAP1 albumen pronucleus expression: use the thermal stimulus method that pET32a-pap1 is changed in the e. coli bl21, under IPTG induces, grope the optimum expression condition, under optimum condition, carry out great expression;
(5) PAP1 protein purification: collect thalline, carry out ultrasonication, cross the nickel post and carry out purifying, the albumen behind the collection purifying.
3. the application of the described Arabidopis thaliana anthocyanidin of claim 1 pap1 purifying protein in preparation Myb antibody.
4. the application of the described Arabidopis thaliana anthocyanidin of claim 1 pap1 purifying protein in the Myb protein expression detects.
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| CN102660553A (en) * | 2012-01-13 | 2012-09-12 | 昆明理工大学 | Yunnan red pear [delta]PybHLH gene and prokaryotic expression vector and application thereof |
| CN103333902A (en) * | 2013-07-11 | 2013-10-02 | 中国热带农业科学院橡胶研究所 | Method for increasing content of cyanidin in kochia scoparia |
| CN108018290A (en) * | 2017-12-05 | 2018-05-11 | 西北农林科技大学 | Anthocyanin synthesis control gene and its application |
| CN111896734A (en) * | 2020-05-29 | 2020-11-06 | 湖北新纵科病毒疾病工程技术有限公司 | 2019-nCoV double-target antibody detection microsphere complex combination, preparation method, kit and kit using method |
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| CN101475960A (en) * | 2009-01-06 | 2009-07-08 | 华南农业大学 | Use of gene AtPAP15 for improving soybean plant strain organophosphorus absorption |
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| CN101475960A (en) * | 2009-01-06 | 2009-07-08 | 华南农业大学 | Use of gene AtPAP15 for improving soybean plant strain organophosphorus absorption |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660553A (en) * | 2012-01-13 | 2012-09-12 | 昆明理工大学 | Yunnan red pear [delta]PybHLH gene and prokaryotic expression vector and application thereof |
| CN103333902A (en) * | 2013-07-11 | 2013-10-02 | 中国热带农业科学院橡胶研究所 | Method for increasing content of cyanidin in kochia scoparia |
| CN108018290A (en) * | 2017-12-05 | 2018-05-11 | 西北农林科技大学 | Anthocyanin synthesis control gene and its application |
| CN108018290B (en) * | 2017-12-05 | 2020-11-10 | 西北农林科技大学 | Plant anthocyanin synthesis control gene and application thereof |
| CN111896734A (en) * | 2020-05-29 | 2020-11-06 | 湖北新纵科病毒疾病工程技术有限公司 | 2019-nCoV double-target antibody detection microsphere complex combination, preparation method, kit and kit using method |
| CN111896734B (en) * | 2020-05-29 | 2024-04-19 | 湖北新纵科病毒疾病工程技术有限公司 | 2019-NCoV double-target antibody detection microsphere complex combination, preparation method, kit and kit using method |
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Application publication date: 20110713 |