CN103740670A - Kit for screening glyphosate N-acetyltransferase antiserum - Google Patents
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Abstract
The invention discloses a kit for screening glyphosate N-acetyltransferase antiserum. The invention provides an application of glyphosate N-acetyltransferase in preparation of the kit for preparing and screening glyphosate N-acetyltransferase antiserum. The amino acid sequence of the glyphosate N-acetyltransferase is shown in the table 1), namely amino acid residue of the 114-260th locus from the N-end in the sequence 2 in the sequence table, or table 2), namely the sequence 2 in the sequence table. Experiment shows that by utilizing the method in which the glyphosate N-acetyltransferase antiserum is prepared from prokaryotic expression protein for the first time, the antiserum of GAT (Glyphosate N-acetyltransferase) is successfully obtained and the antiserum can be also applied to specific detection on target protein of transgenic plant.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the sero-fast test kit of a kind of screening glyphosate N-acetyltransferase, also relate to the sero-fast preparation method of a kind of glyphosate N-acetyltransferase and application thereof.
Background technology
Glyphosate (glyphosate) is go out natural disposition, the outstanding weedicide of inner sucting conduction type of a kind of wide spectrum, because it has the features such as low cost, high-level efficiency, easily degraded and environmental influence are little, in agriculture production, is widely used.
The target enzyme of glyphosate is 5-enol pyruvoyl-shikimic acid-3-phosphate synthase (the 5-enolpyruvylshikimate-3-phosphate synthase in shikimic acid pathway (shikimate pathway); EPSPS) (Amrhein et al, 1980; Steinrucken and Amrhein, 1980).Glyphosate is the analogue of 5-phosphoenolpyruvic acid in shikimic acid pathway (phosphoenol pyruvate, PEP), and the two molecular structure is very similar.In shikimic acid pathway, glyphosate is competed EPSPS with PEP.The competitive power of glyphosate cause by force PEP enol group can not with shikimic acid triphosphoric acid (Shikimat-3-phosphate, S3P) combination, can not form 5-enol pyruvoyl-shikimic acid-3-phosphoric acid (5-enolpyruvylshikimate-3-phosphate synthase, EPSP); Finally suppressed the conversion of shikimic acid to phenylalanine (phenylalanine, Phe), tyrosine (tyrosine, Tyr) and tryptophane (trypotophan, Trp).Amino acid is must indispensable material in plant materials, in elementary, the secondary metabolism of protein synthesis and plant, plays an important role.Once amino acid whose synthetic obstruction, will affect the metabolism of plant, can cause plant death when serious.And shikimic acid pathway is the unique channel of synthetic styrene-acrylic propylhomoserin, tyrosine and tryptophane, after this approach is cut off by glyphosate, very fast death (Dill et al., 2010) of plant.
Glyphosate N-acetyltransferase (glyphosate N-acetyhransferase, GAT) can generate acetyl glyphosate by glyphosate acetylization, thereby makes glyphosate lose herbicidal activity.Utilize glyphosate acetyl to dissolve poison and can avoid the accumulation of glyphosate in plant materials, glyphosate all can be used at the whole growth cycle of crop, and be not subject to the restriction of growth and development stage, also can tolerate higher glyphosate concentration simultaneously.First Castle etc. are cloned into the gene of weak glyphosate N-acetyltransferase activity from Bacillus licheniformis (Bacillus licheniformis), by the method optimization of DNA shuffling, obtain gene (the Castle et al of strong degradation of glyphosate ability, 2004,2010).In China, Baoqing etc., to be subject to the long-term extreme total DNA of contaminated soil of glyphosate as material, obtain the gene of a high-resistance glyphosate, GAT(Baoqing etc. of pausing, 2006).Jin etc. have studied the nucleotide mutant site relevant to glyphosate resistance in this gene, and the method for reorganizing by DNA has obtained very highly active GAT(Jin et al, 2006), this is the gene that has China's independent intellectual property right.
Summary of the invention
An object of the present invention is to provide the application of glyphosate N-acetyltransferase.
Glyphosate N-acetyltransferase provided by the invention is in the application of preparing in resistance glyphosate N-acetyl-transferase serum;
Described glyphosate N-acetyltransferase is following 1) or 2):
The aminoacid sequence of described glyphosate N-acetyltransferase is following 1) or 2):
1) sequence 2 in sequence table is held 114-260 amino acids residue from N; For glyphosate N-acetyltransferase;
2) sequence 2 in sequence table, leads the glyphosate N-acetyltransferase of peptide for N holds fusion His label and chloroplast(id).
In an embodiment of the present invention, the glyphosate N-acetyltransferase of employing is the glyphosate N-acetyltransferase that N end fusion His label and chloroplast(id) are led peptide.
In above-mentioned application, the albumen that described glyphosate N-acetyltransferase obtains by prokaryotic expression.
Second object of the present invention is to provide a kind of method of preparing resistance glyphosate N-acetyl-transferase serum.
Method provided by the invention, comprises the steps:
1) glyphosate N-acetyltransferase encoding gene is imported to Host Strains and carry out prokaryotic expression, obtain glyphosate N-acetyltransferase;
2) by described glyphosate N-acetyltransferase immunize rabbit, collect serum, obtain resistance glyphosate N-acetyl-transferase serum.
In aforesaid method, in step 1), described glyphosate N-acetyltransferase encoding gene imports Host Strains by recombinant vectors and carries out prokaryotic expression.
In aforesaid method, described recombinant vectors is that described glyphosate N-acetyltransferase encoding gene is inserted in expression vector, obtains expressing the recombinant vectors of glyphosate N-acetyltransferase.
In aforesaid method, described expression vector is pET28a.
In aforesaid method, the nucleotides sequence of described glyphosate N-acetyltransferase encoding gene is classified sequence 1 in sequence table as from 5 ' end 109-780 position Nucleotide.
In an embodiment of the present invention, described recombinant vectors is BamH I and the Xho I restriction enzyme site that the sequence in sequence table 1 is inserted to the pET28a that contains His label from 5 ' end 109-780 position Nucleotide (coding N end fusion chloroplast(id) is led the glyphosate N-acetyltransferase of peptide), obtains expressing the recombinant vectors of glyphosate N-acetyltransferase;
The glyphosate N-acetyltransferase that above-mentioned recombinant vectors is expressed is the glyphosate N-acetyltransferase that N end fusion His label and chloroplast(id) are led peptide.
In aforesaid method, described Host Strains is intestinal bacteria, and described intestinal bacteria are specially BL121; Described rabbit is white rabbit.
The resistance glyphosate N-acetyl-transferase serum of being prepared by above-mentioned method is also the scope of protection of the invention.
Above-mentioned resistance glyphosate N-acetyl-transferase serum is also the scope of protection of the invention detecting the application whether containing in glyphosate N-acetyltransferase in testing sample.
In an embodiment of the present invention, testing sample is for turning GAT genetic tobacco.
Of the present invention experimental results show that, the glyphosate N-acetyltransferase that the present invention leads peptide by fusion chloroplast(id) is gene constructed to prokaryotic expression carrier, prokaryotic expression protein, and immune white rabbit prepares glyphosate N-acetyltransferase antiserum(antisera), and then apply this antiserum(antisera) and detect the expression that turns GAT in GAT genetic tobacco.The present invention utilizes prokaryotic expression protein to prepare the sero-fast method of glyphosate N-acetyltransferase first, has successfully obtained the antiserum(antisera) of GAT, and can be for the specific detection of transgenic plant target protein.By genetically engineered, obtained and had the ripe gradually of antiweed resistance farm crop technology, promoted developing rapidly and applying of antiweed farming, glyphosate N-acetyltransferase gene, as an efficient antiweed candidate gene, has a extensive future.The GAT antiserum(antisera) that the present invention obtains can rapidly and efficiently detect the expression of glyphosate N-acetyltransferase albumen, in the research of genetically modified crops and application, plays very important effect.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of GAT gene prokaryotic plasmid pET28a-GAT
Fig. 2 is GAT gene prokaryotic plasmid enzyme restriction evaluation figure
Fig. 3 demarcates protein concentration with SDS-PAGE in conjunction with the quantitative method of BSA
Fig. 4 is that the His label that GAT albumen is carried carries out Western blot detection
Fig. 5 is the Western blot detection that antiserum(antisera) specific detection turns GAT genetic tobacco
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
In experiment, required primer is synthetic by the Shanghai biological company limited of raw work, and plasmid extraction and DNA glue reclaim test kit purchased from Axygen company; Various restriction enzymes are purchased from NEB company.
In following embodiment, the construction process of part plasmid used is as follows:
The structure of plasmid p35S-2301-GUS: with Hind III and EcoR I double digestion pBI121(Beijing Baeyer enlightening Bioisystech Co., Ltd, article No.: MP-091), reclaim the endonuclease bamhi (with the fragment of p35S-GUS-Nos-ter) of about 3000bp, cut pCAMBIA2301(Beijing with the same enzyme of process and pushed up prosperous Bioisystech Co., Ltd, article No.: MCV037) carrier framework of about 11500bp connects, and forms intermediate carrier p35S-2301-GUS;
The structure of plasmid p2301-35S-GAT: the DNA molecular that sequence 1 is led to peptide-glyphosate N-acetyltransferase from the coding shown in the Nucleotide of 5 ' end 109-780 position replaces the carrier that the gus gene between Xba I and the Sac I restriction enzyme site of p35S-2301-GUS carrier obtains.
The preparation of embodiment 1, resistance glyphosate N-acetyl-transferase serum
One, label-the lead prokaryotic expression of peptide-glyphosate N-acetyltransferase
1, the amplification of glyphosate N-acetyltransferase (GAT) gene
According to GAT gene order, design primer upstream primer (GATF-B): 5 '-GGATCCTCTAGAATGGCGCAAGTTAG-3 ' (sequence 3); Downstream primer (GATR-X): 5 '-CTCGAGGAGCTCTTATGCGATCCTCTT-3 ' (sequence 4), upstream primer increases BamH I restriction enzyme site, and downstream primer increases Xho I restriction enzyme site.
Take plasmid p2301-35S-GAT as template, carry out pcr amplification with GATF-B and GATR-X, obtain the PCR product of 696bp.
Through order-checking, this PCR product has sequence 1 in sequence table and, from the Nucleotide shown in 5 ' end 109-780 position, for N holds fusion chloroplast(id), leads the glyphosate N-acetyltransferase encoding gene of peptide.
2, the structure of recombinant vectors
The PCR product obtaining with BamH I and Xho I double digestion above-mentioned 1, reclaims the enzyme of about 670bp and cuts product, and this enzyme is cut to product and the carrier pET28a(Novagen cutting through same enzyme, Cat.No.69864-3) connect, the connection product obtaining, transforms bacillus coli DH 5 alpha, screening recombinant vectors.
By BamH I and Xho I double digestion for recombinant vectors, result as shown in Figure 2, obtains about 700bp(arrow indication) the positive recombinant vectors of fragment.
Enzyme is cut and identified that positive recombinant vectors checks order, this recombinant vectors is sequence in sequence table 1 to be merged to chloroplast(id) from the N end shown in the Nucleotide of 5 ' end 109-780 position lead the recombinant vectors obtaining between the BamH I of glyphosate N-acetyltransferase encoding gene insertion vector pET28a of peptide and Xho I restriction enzyme site, and the His label on this DNA molecular and pET28a carrier forms N and holds fusion His label and chloroplast(id) to lead the encoding gene of the glyphosate N-acetyltransferase of peptide, the nucleotides sequence of this encoding gene is classified the sequence 1 in sequence table as, coding N end fusion His label and chloroplast(id) are led the glyphosate N-acetyltransferase of peptide, the aminoacid sequence of this fusion rotein is the sequence 2 in sequence table.This recombinant vectors called after pET28a-GAT(structural representation as shown in Figure 1).
In DNA molecular shown in sequence 1, in 5 ' end 1-108 position, be the sequence on pET28a carrier, 109-339 position Nucleotide is the encoding gene that chlorophyll signal is led peptide, and 340-780 position Nucleotide is GAT sequence.
Protein sequence shown in sequence 2, is that chlorophyll signal is led peptide in N-terminal 33-113 amino acids, and 114-260 amino acids is GAT, and 5-10 amino acids is His label; Express a fusion rotein that is about 30kDa.
3, expression condition and the purifying of GAT fusion rotein in intestinal bacteria
Recombinant vectors pET28a-GAT is transformed to intestinal bacteria BL121 competent cell, obtain recombinant bacterium BL121/pET28a-GAT(extraction plasmid BamH I and Xho I enzyme and cut evaluation, obtain the positive of 696bp).
A recombinant bacterium BL121/pET28a-GAT37 ℃ shaking culture is spent the night (12h), by 1:100, be transferred in 1L LB liquid nutrient medium, shaking culture adds IPTG (final concentration 1mg/L) when OD600 is between 0.4-0.6, induce, 18 ℃ of temperature, time 8h, collects thalline.Concentration is ultrasonication (arrange-broken time: 2min of ultrasonic disruption instrument working conditions after 50mM, the pH value resuspended collection thalline of PBS that is 7.4; Power: 40%; Working hour: 2s; Time of having a rest: 2s), the centrifugal 10min of 1,2000rpm, gets respectively supernatant liquor and precipitation, carries out SDS-PAGE electrophoresis.
Result confirmation, exogenous protein expression is mainly present in lysate supernatant liquor, and relative molecular weight is about 30kDa, conforms to expection size, for N holds fusion His label and chloroplast(id), leads the glyphosate N-acetyltransferase of peptide.
By His affinity column (Beijing Xin Jingke Bioisystech Co., Ltd for supernatant liquor, pillar length is 10ml, diameter 15mm) carry out purifying, fill filler Ni-NTA His-Band Resin(QIAGEN company, article No.: N76045), crossing column flow rate is 1ml/min, contains respectively the elution buffer of 10mM, 20mM, 50mM, 100mM, 200mM, 300mM and 400mM imidazoles with PBS preparation, use respectively the above buffer solution elution of 5 column volumes, and collect respectively.Collect liquid and carry out SDS-PAGE electrophoresis, result shows that 50mM imidazole concentration is the suitableeest wash-out concentration.Collect the albumen of 50mM imidazoles wash-out, the N that obtains purifying holds fusion His label and chloroplast(id) to lead the glyphosate N-acetyltransferase of peptide.
The N end fusion His label of purifying and chloroplast(id) are led to the glyphosate N-acetyltransferase dialysis enrichment of peptide, first dialysis tubing is cut into the segment of suitable length (10-20cm); In 2% (W/V) sodium bicarbonate of large volume and 1mmol/l EDTA (pH8.0), dialysis tubing is boiled to 10min; Thoroughly clean dialysis tubing with distilled water; Be placed in 1mmol/l EDTA (pH8.0) it is boiled to 10min; After cooling, deposit in 4 ℃, must guarantee that dialysis tubing is immersed in solution all the time, from now, taking dialysis tubing is to wear gloves; With front, at the in-built full water of dialysis tubing, then discharge, it is cleaned up.By containing N end, merge His label and chloroplast(id) and lead the elutriant of the glyphosate N-acetyltransferase of peptide and pack in the dialysis tubing of processing, dialysis tubing length is 1cm/ml; Dialyse with the PBS of following concentration, 50mM PBS(is containing 6M imidazoles) 3h, 50mM PBS(is containing 5M imidazoles) 3h, and 50mM PBS(is containing 4M imidazoles) 3h, 50mM PBS(is containing 3M imidazoles) 3h, 50mM PBS(is containing 2M imidazoles) 3h, 50mM PBS(is containing 1M imidazoles) 3h, and 50mM PBS(is containing 0.5M imidazoles) 3h, 50mM PBS3h, physiological saline (0.9%NaCl) spends the night (12h), obtains concentrated N and holds fusion His label and chloroplast(id) to lead the glyphosate N-acetyltransferase of peptide.
Glyphosate N-acetyltransferase and a certain amount of BSA albumen of holding fusion His label and chloroplast(id) to lead peptide concentrated N run SDS-PAGE jointly, according to the brightness of BSA band, the concentration (Fig. 3) of estimation fusion rotein, the concentration that the fusion rotein N end fusion His label that guestimate is concentrated and chloroplast(id) are led the glyphosate N-acetyltransferase of peptide is about 3mg/ml.
The glyphosate N-acetyltransferase of holding fusion His label and chloroplast(id) to lead peptide the N of purifying carries out Western blot detection (two is anti-purchased from the H7403 of Sigma company), result as described in Figure 4, can find out, the band of arrow indication fusion rotein, illustrates that the glyphosate N-acetyltransferase that N holds fusion His label and chloroplast(id) to lead peptide is able to effective expression in intestinal bacteria.
Two, the preparation of resistance glyphosate N-acetyl-transferase serum
1, the preparation of resistance glyphosate N-acetyl-transferase serum
1) prepare Freund's Freund's incomplete adjuvant: in small beaker, put into 1.5g lanolin and 8ml paraffin oil, in warm water bath, melt, after mixing on magnetic stirring apparatus, after autoclaving, at 4 ℃, save backup;
2) concentrated N end is merged to His label and chloroplast(id) and lead glyphosate N-acetyltransferase and adjuvant 1:1 equal volume amounts mixing in small beaker of peptide, with syringe and syringe needle, repeatedly push away and inhale so that emulsification is complete, emulsification 6mg albumen.
3) rabbit with individual large (2-3kg), healthy, without the purebred white rabbit of immunity for well, select female rabbit of buck or not mating all can.
4) immunity: adopt intramuscular injection.
The plump place of choosing outside rabbit's foot or on the muscle of inner side, after cropping, with 70% ethanol disinfection, with No. 9 syringe needles injection albumen adjuvant emulsion liquid, the approximately dark 2cm of inserting needle.Specific design is as follows:
First week: (1) ear vein is got blood, prepares a small amount of normal serum;
(2). intramuscular injection emulsified protein 2/3 amount, subcutaneous 3 injection 1/3 amounts.
Second week: muscle, subcutaneous injection, consumption as above.
The 3rd week: muscle, subcutaneous injection, consumption as above.
Six～seven weeks: get blood, survey is tired, bloodletting when the highest of tiring, prepared antiserum(antisera).
5) blood sampling: the utensil used of taking a blood sample will be through strict sterilization, and before blood sampling, immunize rabbit will feed water, 12-24h stops eating.By ear vein, take a blood sample, step is as follows:
(1) immunize rabbit is packed in fixed case, head and ear are all exposed.Artificial fixing also can.
(2) hard of hearing outer rim vein place scrape hair, with sterilizing the alcohol edge innner and outer sides of will taking a blood sample in one's ear.
(3) embrocate with the absorbent cotton that is moistened with dimethylbenzene at ear middle part, makes the ear congestion of blood vessel, and blood flow accelerates, and is coated with one deck Vaseline on it.
(4), at ear edge vasodilation end, by razor sharpened, along vessel directions, longitudinally scratch the otch (while after this taking a blood sample, incision site moves to ear base portion from end again) of 1-2mm.During appropriateness, blood flows out immediately, and blood collecting, in sterilizing small beaker, is got blood 20-40ml at every turn.Do not allow blood alcohol exposure, to avoid haemolysis.
(5) dry absorbent cotton hemostasis by compression for cutting part, and clean residual bloodstain on the rabbit ear, prevents that rabbit scratching from causing and bleeds or infect.
(6) can continuous several times take a blood sample, every minor tick 3-4d.As the decline of tiring, can again inject appropriate antigen, to maintain longer blood sampling time, during blood sampling, strengthen the nutrition of animal.
7, serum separating out and preserving.
(1) after collection blood is put being solidified under room temperature, build and put 37 ℃ of incubator 1-2h.
(2) with scalper, along beaker edge, peel off gently sludged blood.
(3) build and put 4 ℃ of refrigerator overnight.
(4) serum is separated out in sucking-off, centrifugal 10min under 3,000rpm.
(5) supernatant liquor is resistance glyphosate N-acetyl-transferase serum, by 1/1000 amount, adds NaN3 anticorrosion.℃ preservation of aseptic subpackaged rear sealing-20.
The functional study of embodiment 2, resistance glyphosate N-acetyl-transferase serum
With whether containing target protein GAT in resistance glyphosate N-acetyl-transferase serum detection testing sample, adopt Western blot to carry out, specific as follows:
1, the preparation of testing sample
Turn GAT genetic tobacco for GAT gene (sequence 1 is from 5' end 340-780 position Nucleotide) is proceeded to by p2301-35S-GAT in wild-type tobacco (kind is the western cigarette Nicotiana of coral tabacum va.Xanthi nc), what obtain turns GAT genetic tobacco.
Get and turn GAT genetic tobacco 0.2g blade liquid nitrogen grinding and become powder, add 150 μ l2 × SDS albumen sample-loading buffer and ddH
2o vibration mixes, and 100 ℃ of sex change 10min, put rapidly 5min on ice, and the centrifugal 10min of 12,000rpm transfers to supernatant liquor in new centrifuge tube, standby, obtains total protein, is testing sample.
2、Western?blot
1) electrophoresis
According to the form below 1, resolving gel concentration 12.5% and concentrated gum concentration 4.5% are prepared SDS-PAGE gel,
Table 1 is SDS-PAGE gel formula
The total protein solution preparing is got to appropriate loading, electrophoresis (electrophoresis apparatus is the Mini-Protean3Electrophoresis Module of Bio-Rad company), 80V voltage, after sample enters concentrated glue, the about 2h tetrabromophenol sulfonphthalein of 120V is run out of glue, and electrophoresis finishes;
2) transferring film
By the nitrocellulose filter cutting out (nitrocellulose filter membrane, NC film, Amersham Pharmacia company) and filter paper wetting in distilled water, balance 2min in transferring film damping fluid, by Bio-Rad transferring film system, wet and turn 200mA constant current, 1.5h (or Mini-Protean3Elecrophoresis transfer Cell electricity turn, 10V constant voltage, 30min);
3) sealing
The film taking a turn for the better is placed in confining liquid to 37 ℃ of 1h (or 4 ℃ spend the night) at least;
4) primary antibodie reaction
In confining liquid, add the resistance glyphosate N-acetyl-transferase serum of being prepared by embodiment 1,37 ℃ of reaction 2h (or 4 ℃ spend the night);
5) two anti-reactions: TBST damping fluid is washed film 3 times, each 5min, presses the goat anti-rabbit igg (purchased from SIGMA company) of the dilution proportion alkaline phosphate ester enzyme labelling of 1:5000 with the dilution of TBST damping fluid, and 37 ℃ are reacted 1h;
6) colour developing: TBST damping fluid is washed film, 3 times, each 3-5min, is placed in by film the alkaline phosphatase damping fluid that contains 330 μ g/ml NBT and 165 μ g/ml BCIP and develops the color clearly to band, uses distilled water termination reaction.
Take the total protein (preparation method is the same) of wild-type tobacco (N) blade as contrast.
Result as shown in Figure 5, what arrow was indicated is object band, detect turn GAT genetic tobacco (T) plant in the expression of target protein GAT can be detected specifically, there is not specific band in wild-type tobacco (N), illustrates that prepared antiserum(antisera) can be used for the GAT of the expression such as transgenic plant to carry out specific detection.
Claims (10)
1. glyphosate N-acetyltransferase is in the application of preparing in resistance glyphosate N-acetyl-transferase serum;
The aminoacid sequence of described glyphosate N-acetyltransferase is following 1) or 2):
1) sequence 2 in sequence table is held 114-260 amino acids residue from N;
2) sequence 2 in sequence table.
2. application according to claim 1, is characterized in that:
Described glyphosate N-acetyltransferase is the albumen obtaining by prokaryotic expression.
3. a method of preparing resistance glyphosate N-acetyl-transferase serum, comprises the steps:
1) glyphosate N-acetyltransferase encoding gene is imported to Host Strains and carry out prokaryotic expression, obtain glyphosate N-acetyltransferase;
2) by described glyphosate N-acetyltransferase immunize rabbit, collect serum, obtain resistance glyphosate N-acetyl-transferase serum.
4. method according to claim 3, is characterized in that:
In step 1), described glyphosate N-acetyltransferase encoding gene imports Host Strains by recombinant vectors and carries out prokaryotic expression.
5. method according to claim 4, is characterized in that: described recombinant vectors, for described glyphosate N-acetyltransferase encoding gene is inserted in expression vector, obtains expressing the recombinant vectors of glyphosate N-acetyltransferase.
6. method according to claim 5, is characterized in that: described expression vector is pET28a.
7. according to arbitrary described method in claim 3-6, it is characterized in that: the nucleotides sequence of described glyphosate N-acetyltransferase encoding gene is classified sequence 1 in sequence table as from 5 ' end 109-780 position Nucleotide.
8. according to arbitrary described method in claim 3-7, it is characterized in that: described Host Strains is intestinal bacteria, and described intestinal bacteria are specially BL121; Described rabbit is white rabbit.
9. by resistance glyphosate N-acetyl-transferase serum that in claim 3-8 prepared by arbitrary described method.
10. whether resistance glyphosate N-acetyl-transferase serum claimed in claim 9 contains the application in glyphosate N-acetyltransferase in detection testing sample.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981199A (en) * | 2014-05-15 | 2014-08-13 | 中国农业科学院生物技术研究所 | Glyphosate resistance gene-containing expression vector and application thereof |
| CN108395477A (en) * | 2018-02-06 | 2018-08-14 | 中国农业科学院生物技术研究所 | Purposes of the monoclonal antibody FB9b in detecting GAT transgenic crops |
| CN108414768A (en) * | 2018-02-06 | 2018-08-17 | 中国农业科学院生物技术研究所 | A kind of gold mark detection test paper item of resistance glyphosate GAT transgenic crops |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1531594A (en) * | 2000-10-30 | 2004-09-22 | �����ɷ� | Novel glyphosate N-acetyltransferase (GAT) genes |
| CN103454424A (en) * | 2013-08-20 | 2013-12-18 | 山东农业大学 | Quantitative enzyme-linked immuno sorbent assay (ELISA) detection method of neomycin phosphotransferase (NPT II) double-antibody sandwich in transgenic crops |
-
2014
- 2014-01-13 CN CN201410014620.0A patent/CN103740670B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1531594A (en) * | 2000-10-30 | 2004-09-22 | �����ɷ� | Novel glyphosate N-acetyltransferase (GAT) genes |
| CN103454424A (en) * | 2013-08-20 | 2013-12-18 | 山东农业大学 | Quantitative enzyme-linked immuno sorbent assay (ELISA) detection method of neomycin phosphotransferase (NPT II) double-antibody sandwich in transgenic crops |
Non-Patent Citations (4)
| Title |
|---|
| CASTLE,L.A ET AL: "glyphosate N-acetyltransferase [synthetic construct]", 《GENBANK: AAU04689.1》, 1 May 2005 (2005-05-01) * |
| HARRY J. KLEE ET AL: "Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase:sequence analysis and manipulation to obtain glyphosate-tolerant plants", 《MOL GEN GENET》, vol. 210, 31 December 1987 (1987-12-31) * |
| KLEE,H.J ET AL: "EPSP [Arabidopsis thaliana]", 《GENBANK: CAA29828.1》, 14 November 2006 (2006-11-14) * |
| SIEHL D.L: "Chain A,Glyphosate N-acetyltransferase Bound to Oxidized Coa and Sulfate", 《PDB:2JDC_A》, 17 April 2013 (2013-04-17) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981199A (en) * | 2014-05-15 | 2014-08-13 | 中国农业科学院生物技术研究所 | Glyphosate resistance gene-containing expression vector and application thereof |
| CN103981199B (en) * | 2014-05-15 | 2017-01-18 | 中国农业科学院生物技术研究所 | Glyphosate resistance gene-containing expression vector and application thereof |
| CN108395477A (en) * | 2018-02-06 | 2018-08-14 | 中国农业科学院生物技术研究所 | Purposes of the monoclonal antibody FB9b in detecting GAT transgenic crops |
| CN108414768A (en) * | 2018-02-06 | 2018-08-17 | 中国农业科学院生物技术研究所 | A kind of gold mark detection test paper item of resistance glyphosate GAT transgenic crops |
| CN108395477B (en) * | 2018-02-06 | 2020-07-03 | 中国农业科学院生物技术研究所 | Application of monoclonal antibody FB9b in detection of GAT transgenic crops |
| CN108414768B (en) * | 2018-02-06 | 2020-10-30 | 中国农业科学院生物技术研究所 | Gold-labeled detection test strip for glyphosate-resistant GAT transgenic crops |
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