CN103454245B - A kind of method of qualitative and quantitative detection phytohormone activity gibberellin and special chip thereof - Google Patents
A kind of method of qualitative and quantitative detection phytohormone activity gibberellin and special chip thereof Download PDFInfo
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Abstract
The invention discloses a kind of method and special chip thereof of qualitative and quantitative detection phytohormone activity gibberellin.The method of active gibberellin whether is contained in a kind of auxiliary qualitative detection testing sample disclosed by the invention, surface plasma resonance technology is utilized to detect, comprise the steps: testing sample solution to flow through biologic sensor chip, after sample introduction, detect response A; Protein solution is flowed through biologic sensor chip, after sample introduction, detects contrast response; If response A is significantly greater than contrast response, then judge that testing sample candidate is as the sample containing active gibberellin.The present invention is from peptide level, construct a kind of can the SA chip of M37 coupling of stability and high efficiency of mass production, utilize surface plasma resonance technology, the qualitative and quantitative analysis to gibberellin can be realized, thus provide a kind of efficient, sensitive new method for the detection of gibberellin.
Description
Technical field
The present invention relates to a kind of method and special chip thereof of qualitative and quantitative detection phytohormone activity gibberellin.
Background technology
Gibberellin (Gibberellin, GA) is the class plant hormone extensively existed, and kind is very complicated, has identified 136 kinds at present.Gibberellin has active and nonactive dividing by function, and wherein tool activated GA number is less, has GA
1, GA
3, GA
4deng.
The chemical constitution of gibberellin is very similar, mostly is epimerism, and the gibberellin that textural difference is large also only exists the difference of double bond, hydroxy number or position, and therefore, the analysis of gibberellin detects has very large challenge.In the world the analysis and research of gibberellin are carried out already, at least can trace back to the sixties in 20th century even more early, but up to the present, the analyzing detecting method of really available gibberellin is also few.Early stage gibberellin determination is based on immune response, and by preparing haptens, immune animal carrys out Dispersal risk, thus achieves immune affinity chromatographic purification, radiommunoassay and the enzyme linked immunosorbent assay (ELISA) grown up afterwards.The advantage of immunoassays is that reaction conditions is gentle, can keep biologically active, be applicable to biological effect research, but shortcoming needs to prepare haptens and antibody, workload is large, and determination period is long, cost is high and be difficult to automation mechanized operation, and measure effect and reappearance also not satisfactory.After the eighties in 20th century, mass spectrum (MS) detect become gibberellin detect main force's instrument, by with tripping device, as gas chromatography (GC), high performance liquid chromatography (HPLC), the couplings such as Capillary Electrophoresis (CE) are analyzed, and these methods have higher automaticity, can realize qualitative and quantitative analysis simultaneously, but when detecting with MS, need the gibberellin standard model of cold labeling as internal reference, but some standard models are difficult to buy at home, and expensive.Therefore, the detection of gibberellin is still very difficult.
Surface plasma resonance (Surfaceplasmonresonance, SPR) technology is the optical bio detection technique developed rapidly in the world in recent years, the advantages such as it has that testing process is convenient and swift, sample is high without the need to mark, detection in real time, applied range, detection sensitivity, high throughput analysis, therefore can be widely used in the research of bio-molecular interaction.
Summary of the invention
An object of the present invention is to provide a kind of method whether containing active gibberellin in auxiliary qualitative detection testing sample, the method utilizes surface plasma resonance technology to detect, and comprises the steps:
Testing sample solution is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as response A; Protein solution is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as contrast response; If response A is significantly greater than contrast response, then judge that described testing sample candidate is as the sample containing active gibberellin.
Described testing sample solution is made up of albumen shown in testing sample, SEQIDNo.3 and damping fluid.
Described protein solution is made up of albumen shown in SEQIDNo.3 and damping fluid.
Described biologic sensor chip on the chip being suitable for using in surface plasma resonance technology, connects polypeptide shown in SEQIDNo.1 obtain.
Another object of the present invention is to provide a kind of auxiliary method comparing the active size of active gibberellin, and the method utilizes surface plasma resonance technology to detect, and comprises the steps:
Protein solution is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as contrast response; The solution of gibberellin I is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as response I; The solution of gibberellin II is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as response II; If response I and response II are all significantly greater than contrast response and response I is significantly greater than response II, then judge that the active candidate of gibberellin I is higher than gibberellin II.
Described protein solution is made up of albumen shown in SEQIDNo.3 and damping fluid.
The solution of described gibberellin I is made up of albumen shown in gibberellin I, SEQIDNo.3 and damping fluid.
The solution of described gibberellin II is made up of albumen shown in gibberellin II, SEQIDNo.3 and damping fluid.
Described biologic sensor chip the chip that uses in surface plasma resonance technology connects polypeptide shown in SEQIDNo.1 obtain.
Another object of the present invention is to provide a kind of method of quantitative detection of active gibberellin, and the method utilizes surface plasma resonance technology to detect, and comprises the steps:
Respectively the solution of the active gibberellin standard items of variable concentrations is flowed through biologic sensor chip, after sample introduction, detect response, record the solution sample introduction of each variable concentrations complete time corresponding response, with each response for ordinate, with the natural logarithm of each concentration for horizontal ordinate, production standard curve.
Gibberellins solution to be measured is flowed through biologic sensor chip, when sample introduction is complete, detects response, this response is substituted into typical curve, namely draws the concentration of gibberellin in testing sample solution.
The solution of described gibberellin standard items is made up of albumen shown in gibberellin standard items, SEQIDNo.3 and damping fluid.
Described Gibberellins solution to be measured is made up of albumen shown in gibberellin to be measured, SEQIDNo.3 and damping fluid.
In the solution of the active gibberellin standard items of described variable concentrations, the concentration of active gibberellin standard items is 100 μMs, 10 μMs, 1 μM, 0.1 μM, 0 μM.
Described biologic sensor chip the chip that uses in surface plasma resonance technology connects polypeptide shown in SEQIDNo.1 obtain.
Described gibberellin to be measured and gibberellin standard items are same gibberellin.
In above-mentioned arbitrary described method, described damping fluid is the PBS damping fluid of the pH7.4 of the Tween20 containing volumn concentration 0.005%.
In above-mentioned arbitrary described method, the sample size of described testing sample is 50 μ L, and sample introduction flow velocity is 30 μ L/min.
In above-mentioned arbitrary described method, shown in described SEQIDNo.1, polypeptide is connected with chip with the Streptavidin on chip by biotin.
In above-mentioned arbitrary described method, described active gibberellin is GA
3or GA
4.
Another object of the present invention is to provide and a kind ofly utilizes the biologic sensor chip used in surface plasma resonance technology detection of active gibberellin, and this chip the chip that uses in surface plasma resonance technology connects polypeptide shown in SEQIDNo.1 obtain.
In above-mentioned biologic sensor chip, shown in described SEQIDNo.1, polypeptide is connected with chip with the Streptavidin on chip by biotin.
Another object of the present invention is to provide a peptide species, shown in following (1) or (2):
(1) polypeptide shown in SEQIDNo.1;
(2) amino acid sequence shown in SEQIDNo.1 had a polypeptide of identical function through the replacement of one or several amino acid residue and/or disappearance and/or interpolation.
The application of aforementioned polypeptides in auxiliary qualitative and/or quantitative detection sample in active gibberellin also belongs to protection scope of the present invention.
The present invention is from peptide level, the polypeptide M37 of DELLA domain is comprised by synthesis, the mechanism of active GA concentration is depended on based on receptor protein GID1a and DELLA protein-interacting, construct a kind of can the SA chip of M37 coupling of stability and high efficiency of mass production, utilize surface plasma resonance technology, the qualitative and quantitative analysis to gibberellin can be realized, thus provide a kind of efficient, sensitive new method for the detection of gibberellin.
Accompanying drawing explanation
Fig. 1 is the qualification of GID1a-GST albumen.
Fig. 2 is the specificity analyses of M37 polypeptide chip.
Fig. 3 is the specificity analyses of M32 polypeptide chip.
Fig. 4 is the gibberellin activity analysis of M37 polypeptide chip.
Fig. 5 is the gibberellin activity analysis of M32 polypeptide chip.
Fig. 6 is active gibberellin concentration analysis.
Fig. 7 is active gibberellin content standard curve.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The experiment material used in following embodiment and source as follows:
Active gibberellin GA
3available from Sigma, article No. is G7645.
Active gibberellin GA
4available from Sigma, article No. is G7276.
IPTG(isopropyl-beta D-thio galactopyranoside) purchased from Merck, catalog number is CB420322.GSTrapFF affinity column is purchased from GE company.
Superdex20010/300GL purification column is purchased from GE company.
GST monoclonal antibody is purchased from Novagen company, and catalog number is 71097.
Nonactive gibberellin GA
20, provided by Institute of Chemistry, Academia Sinica Chen Yi laboratory.
Recombinant bacterial strain Rosetta
tM(DE3) pLysS/pGEX-KG-GID1a document " Zhou Min, Zhao Zhuoya, Xu Wenzhong, Xiao's wave, Wang Ruozhong. the optimizing research of arabidopsis gibberellin acceptor AtGID1a prokaryotic expression.2011.20 (1), 103-107 " be disclosed in, the public can obtain from the thick laboratory of Institute of Botany, Chinese Academy of Sciences.
SA chip in surface plasma resonance technology is purchased from GE company, and catalog number is BR-1000-32.
PBS damping fluid (pH7.4): 137mMNaCl, 2.7mMKCl, 10mMNa
2hPO
4, 2mMKH
2pO
4.
The preparation of the SA chip of embodiment 1, polypeptide coupling
One, the sequence of polypeptide M37 and M32 and preparation
(1) artificial synthetic polypeptide M37, and biotin labeling is carried out to its N-end.
The sequence of the M37 of synthesis is: NEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEV(SEQIDNo.1);
The M37 sequence of N-terminal biotin mark is:
Biotin-NEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEV；
(2) artificial synthetic polypeptide M32, and biotin labeling is carried out to its N-end.
The sequence of the M32 of synthesis is: NEEDDGNGMVLGYKVRSSEMADVAQKLEQLEV(SEQIDNo.2);
The M32 sequence of N-terminal biotin mark is:
Biotin-NEEDDGNGMVLGYKVRSSEMADVAQKLEQLEV。
Two, polypeptide is connected on SA chip
SA chip is the chip used in surface plasma resonance biological sensor.Interaction of biomacromolecules analyser Biacore3000 proceeds as follows: with 50mMNaOH(containing 1MNaCl) rinse SA chip three times with the flow velocity of 10 μ L/min, each 1min; Polypeptide M37 and M32 to 50 μ g/mL is diluted respectively with the PBS damping fluid of pH7.4, polypeptide solution is flowed through SA chip with the flow velocity of 10 μ L/min respectively, polypeptide is combined with the Streptavidin of chip surface, until reach predetermined coupling level, be about 1000RU, obtain the SA chip of M37 coupling and the SA chip of M32 coupling.
The purifying of embodiment 2, receptor protein GID1a and qualification
One, recombinant bacterial strain Rosetta
tM(DE3) pLysS/pGEX-KG-GID1a is seeded in LB nutrient culture media and cultivates, and is 0.6-0.8 through second incubation in OD value, and adding IPTG to final concentration is 0.1mM induction, and at 16 DEG C, cultivate 12h under 200rpm, 5000g is centrifugal, collects thalline.
Two, broken with low temperature ultrasonic after PBS damping fluid (pH7.4) resuspended thalline.
Three, under 4 DEG C of conditions, the centrifugal 40min of 10000g, gets supernatant.
Four, by supernatant 0.45 μm of membrane filtration.
Five, protein purification.The GID1a protein band GST label of pGEX-KG-GID1a plasmid expression, therefore prioritizing selection affinity chromatography purifying during purifying.
(1) supernatant GSTrapFF affinity column is carried out initial purification.
(2) exist with Superdex20010/300GL purification column
on carry out secondarily purified to the GID1a-GST albumen after affinity purification.
Six, collect purification of samples and carry out 12%SDS-PAGE electrophoresis detection.
Electrophoresis result as shown in Figure 1A.
GID1a in Figure 1A is the GID1a with GST label.
The amino acid sequence of GID1a is as shown in SEQIDNo.3.
The molecular weight theoretical value of GID1a-GST is 66.6kD, and the sample strip size after purifying and destination protein GID1a-GST's is in the same size.
Seven, the Westernblot of purification of samples detects
Form the feature of fusion according to GID1a albumen and GST, utilize the monoclonal antibody of GST to carry out Westernblot detection to the protein sample after purifying.
Go to wet after SDS-PAGE is separated for the protein sample of purifying on pvdf membrane, and carry out immuning hybridization, the dilution ratio of the monoclonal antibody of GST is 1:4000.
Results of hybridization as shown in Figure 1B.
GID1a in Figure 1B is the GID1a with GST label.
Figure 1B shows that the monoclonal antibody of protein sample and GST has hybridization signal, and the size of hybridising band and destination protein GID1a-GST's is in the same size, proves that the protein sample after purifying is GID1a-GST albumen.
The surface plasma resonance (SPR) of embodiment 3, sample is analyzed
One, pass into PBS damping fluid (be the Tween20 of 0.005% containing volumn concentration), rinse chip surface.
Two, when RU value is steady, prepare testing sample with PBS damping fluid (be the Tween20 of 0.005% containing volumn concentration), sample detects with the flow velocity of 30 μ L/min, and sample size is 50 μ L.
Three, the response situation of (95s) at the end of com-parison and analysis sample introduction.
Four, pass into PBS damping fluid (be the Tween20 of 0.005% containing volumn concentration) 2min, analysis thing is dissociated from chip surface.
Five, the NaOH regeneration chip of 5 μ L10mM is passed into.
The specificity analyses of the SA chip of embodiment 4, polypeptide coupling
One, four negative control samples are set.BSA protein solution (7.5 μMs), GA
4(100 μMs) and BSA(7.5 μM) mixed solution, and in order to get rid of GST label in GID1a-GST fusion interference arrange GST protein solution (7.5 μMs), GA
4(100 μMs) and GST(7.5 μM) mixed solution.
Two, respectively SPR analysis is carried out to the SA chip of M37 coupling and the SA chip of M32 coupling.Concrete steps are with embodiment 3.
The SPR analysis result of the SA chip of M37 coupling as shown in Figure 2.
In Fig. 2: Aa: analysis thing is GA
4(100 μMs) and BSA(7.5 μM) mixed solution; Ab: analyzing thing is BSA protein solution (7.5 μMs); Ba: analysis thing is GA
4(100 μMs) and GST(7.5 μM) mixed solution; Bb: analyzing thing is GST protein solution (7.5 μMs).
Fig. 2 result shows, and before and after four kinds of protein sample sample introductions, response does not all change, and namely the polypeptide M37 of protein sample and chip surface does not interact.Result shows that the specificity of chip is good, may be used for the transactional analysis with receptor protein GID1a.
The SPR analysis result of the SA chip of M32 coupling as shown in Figure 3.
In Fig. 3: Aa: analysis thing is GA
4(100 μMs) and BSA(7.5 μM) mixed solution; Ab: analyzing thing is BSA protein solution (7.5 μMs); Ba: analysis thing is GA
4(100 μMs) and GST(7.5 μM) mixed solution; Bb: analyzing thing is GST protein solution (7.5 μMs).
Fig. 3 result shows, and before and after four kinds of protein sample sample introductions, response does not all change, and namely the polypeptide M32 of protein sample and chip surface does not interact.Result shows that the specificity of chip is good, may be used for the transactional analysis with receptor protein GID1a.
The SA chip of embodiment 5, polypeptide coupling is to the qualitative detection of gibberellin
One, the SA chip of M37 coupling is to the qualitative detection of gibberellin
(1) add the different GA samples of same concentrations in the receptor protein GID1a of same concentrations, be respectively the highest active GA
4, the GA that activity is taken second place
3, do not have activated GA
20, in addition, the contrast that does not add GA is set.
Four detection samples are:
GA
4(1 μM) and GID1a(7.5 μM) mixed solution, GA
3(1 μM) and GID1a(7.5 μM) mixed solution, GA
20(1 μM) and GID1a(7.5 μM) mixed solution, GID1a solution (7.5 μMs).
(2) SPR analysis is carried out to the SA chip of M37 coupling.Concrete steps are with embodiment 3.
The SPR analysis result of the SA chip of M37 coupling as shown in Figure 4.
In Fig. 4: the analysis thing that a-d is corresponding is followed successively by: GA
4(1 μM) and GID1a(7.5 μM) mixed solution, GA
3(1 μM) and GID1a(7.5 μM) mixed solution, GA
20(1 μM) and GID1a(7.5 μM) mixed solution, GID1a(7.5 μM) solution.
Fig. 4 result shows, and adds and do not have activated GA in receptor protein GID1a
20after, with the interactional response of polypeptide M37 and GID1a response when not adding GA equally large (56RU); GA is added in receptor protein GID1a
3and GA
4after, be significantly greater than with the interactional response of polypeptide M37 and do not add GA and add GA
20the response of GID1a sample, and add GA
4time response (97RU) significantly to be greater than and add GA
3time response (69RU).
In triplicate, result is all consistent with above-mentioned conclusion in experiment.Therefore, can by comparing the response detecting sample response value and whether be significantly greater than GID1a solution, judge that whether the sample that detects is the candidate sample containing active gibberellin, and when detecting sample response value and being significantly greater than the response of GID1a solution, can the active size of active gibberellin contained by judgement sample further.
Two, the SA chip of M32 coupling is to the qualitative detection of gibberellin
(1) two detection sample is:
GA
4(100 μMs) and GID1a(7.5 μM) mixed solution and GID1a solution (7.5 μMs).
(2) SPR analysis is carried out to the SA chip of M32 coupling.Concrete steps are with embodiment 3.
The SPR analysis result of the SA chip of M32 coupling as shown in Figure 5.
In Fig. 5: a:GA
4(100 μMs) and GID1a(7.5 μM) mixed solution, b:GID1a(7.5 μM) solution.
Fig. 5 shows, and passes into GID1a(7.5 μM) solution, response does not change, and namely GID1a does not interact with the M32 of chip surface.Pass into containing 100 μMs of GA
4receptor protein GID1a(7.5 μM) sample, same not interact with the M32 of chip surface.
Fig. 4 and Fig. 5 contrast can find, GID1a and M37 is not adding active GA
4with add GA
4shi Jun has obvious response to increase, and illustrates that the SA chip of M37 coupling may be used for the detection of gibberellin.And GID1a and M32 is not adding active GA
4with add GA
4time response all there is no significant change compared with the response before sample introduction, show that M32 can not identify with receptor protein GID1a, illustrate that the SA chip of M32 coupling can not be used for the detection of gibberellin.
The SA chip of embodiment 6, M37 coupling is to the quantitative detection of gibberellin
The GA of variable concentrations is added in the receptor protein GID1a of one, same concentrations
4sample, active GA
4the concentration of sample is respectively: 100 μMs, 10 μMs, 1 μM, 0.1 μM, 0 μM.
Five detection samples are:
GA
4(100 μMs) and GID1a(7.5 μM) mixed solution, GA
4(10 μMs) and GID1a(7.5 μM) mixed solution, GA
4(1 μM) and GID1a(7.5 μM) mixed solution, GA
4(0.1 μM) and GID1a(7.5 μM) mixed solution, GID1a solution (7.5 μMs).
Two, SPR analysis is carried out to the SA chip of M37 coupling.Concrete steps are with embodiment 3.
Result as shown in Figure 6.
In Fig. 6: the analysis thing that a-e is corresponding is followed successively by: GA
4(100 μMs) and GID1a(7.5 μM) mixed solution, GA
4(10 μMs) and GID1a(7.5 μM) mixed solution, GA
4(1 μM) and GID1a(7.5 μM) mixed solution, GA
4(0.1 μM) and GID1a(7.5 μM) mixed solution, GID1a solution (7.5 μMs).
Result shows, along with GA
4the reduction of concentration, GID1a and the interactional response of chip surface M37 polypeptide are reduction trend (being followed successively by: 159RU, 142RU, 109RU, 82RU), and response is all greater than and does not add GA
4time response (80RU).
Three, the making of active gibberellin content standard curve
(1) five detection sample is:
GA
4(100 μMs) and GID1a(7.5 μM) mixed solution, GA
4(10 μMs) and GID1a(7.5 μM) mixed solution, GA
4(1 μM) and GID1a(7.5 μM) mixed solution, GA
4(0.1 μM) and GID1a(7.5 μM) mixed solution, GID1a solution (7.5 μMs).
(2) SPR analysis is carried out.Concrete steps are with embodiment 3.
(3) will containing 0.1 μM of GA
4sample and M37 effect time response be set to 0RU, the corresponding conversion of response (being respectively 77RU, 60RU, 27RU) of other samples.With GA
4the natural logarithm value of concentration is horizontal ordinate, and the response after conversion is ordinate mapping.As shown in Figure 7, equation is result: y=11.46ln (x)+186.2, R
2=0.986.
In triplicate, result is all consistent with above-mentioned conclusion in experiment.Therefore, determine within the scope of 0.1 μM-100 μMs, can by the Analyzing on Size active gibberellin GA of response
4relative content, response is larger, and content is higher, and response is less, and content is lower.
Claims (8)
1. whether contain a method for active gibberellin in auxiliary qualitative detection testing sample, utilize surface plasma resonance technology to detect, comprise the steps:
Testing sample solution is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as response A; Protein solution is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as contrast response; If response A is significantly greater than contrast response, then judge that described testing sample candidate is as the sample containing active gibberellin;
Described testing sample solution is made up of albumen shown in testing sample, SEQIDNo.3 and damping fluid;
Described protein solution is made up of albumen shown in SEQIDNo.3 and damping fluid;
Described biologic sensor chip on the chip being suitable for using in surface plasma resonance technology, connects polypeptide shown in SEQIDNo.1 obtain;
Polypeptide shown in SEQIDNo.1 is connected with chip with the Streptavidin on chip by biotin.
2. the auxiliary method comparing the active size of active gibberellin, utilizes surface plasma resonance technology to detect, comprises the steps:
Protein solution is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as contrast response; The solution of gibberellin I is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as response I; The solution of gibberellin II is flowed through biologic sensor chip, after sample introduction, detects response, be denoted as response II; If response I and response II are all significantly greater than contrast response and response I is significantly greater than response II, then judge that the active candidate of gibberellin I is higher than gibberellin II;
Described protein solution is made up of albumen shown in SEQIDNo.3 and damping fluid;
The solution of described gibberellin I is made up of albumen shown in gibberellin I, SEQIDNo.3 and damping fluid;
The solution of described gibberellin II is made up of albumen shown in gibberellin II, SEQIDNo.3 and damping fluid;
Described biologic sensor chip the chip that uses in surface plasma resonance technology connects polypeptide shown in SEQIDNo.1 obtain;
Polypeptide shown in SEQIDNo.1 is connected with chip with the Streptavidin on chip by biotin.
3. a method for quantitative detection of active gibberellin, utilizes surface plasma resonance technology to detect, comprises the steps:
Respectively the solution of the active gibberellin standard items of variable concentrations is flowed through biologic sensor chip, after sample introduction, detect response, record the solution sample introduction of each variable concentrations complete time corresponding response, with each response for ordinate, with the natural logarithm of each concentration for horizontal ordinate, production standard curve;
Gibberellins solution to be measured is flowed through biologic sensor chip, when sample introduction is complete, detects response, this response is substituted into typical curve, namely draws the concentration of gibberellin in testing sample solution;
The solution of described gibberellin standard items is made up of albumen shown in gibberellin standard items, SEQIDNo.3 and damping fluid;
Described Gibberellins solution to be measured is made up of albumen shown in gibberellin to be measured, SEQIDNo.3 and damping fluid;
In the solution of the active gibberellin standard items of described variable concentrations, the concentration of active gibberellin standard items is 100 μMs, 10 μMs, 1 μM, 0.1 μM, 0 μM;
Described biologic sensor chip the chip that uses in surface plasma resonance technology connects polypeptide shown in SEQIDNo.1 obtain;
Polypeptide shown in SEQIDNo.1 is connected with chip with the Streptavidin on chip by biotin.
4. according to the arbitrary described method of claim 1-3, it is characterized in that: described damping fluid is the PBS damping fluid of the pH7.4 of the Tween20 containing volumn concentration 0.005%.
5., according to the arbitrary described method of claim 1-3, it is characterized in that: sample size is 50 μ L, sample introduction flow velocity is 30 μ L/min.
6., according to the arbitrary described method of claim 1-3, it is characterized in that: described active gibberellin is GA
3or GA
4.
7. utilizing the biologic sensor chip used in surface plasma resonance technology detection of active gibberellin, is the chip that uses in surface plasma resonance technology connects polypeptide shown in SEQIDNo.1 obtain;
Shown in described SEQIDNo.1, polypeptide is connected with chip with the Streptavidin on chip by biotin.
8. the application of a peptide species in auxiliary qualitative and/or quantitative detection sample in active gibberellin;
The amino acid sequence of described polypeptide is as shown in SEQIDNo.1.
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102156193A (en) * | 2011-03-31 | 2011-08-17 | 中国科学院植物研究所 | Method for detecting target protein in plants and special SPR (selective posterior rhizotomy) biosensor for method |
| CN103197081A (en) * | 2013-04-17 | 2013-07-10 | 无锡优创生物科技有限公司 | Protein chip and preparation and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1,and DELLA Proteins;Yuko Yamamoto 等;《The Plant Cell》;20101130;第22卷;3589－3602 * |
| Gibberellin Signaling: A Theme and Variations on DELLA Repression;Amber L. Hauvermale 等;《Plant Physiology》;20120930;第160卷;83－92 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018020442A1 (en) * | 2016-07-26 | 2018-02-01 | University Of Johannesburg | Ligand binding assay for detecting gibberellins |
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| Publication number | Publication date |
|---|---|
| CN103454245A (en) | 2013-12-18 |
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