CN101201334B - Method for identifying wheat variety using wheat water-solubility protein by high-efficiency capillary electrophoresis - Google Patents
Method for identifying wheat variety using wheat water-solubility protein by high-efficiency capillary electrophoresis Download PDFInfo
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- CN101201334B CN101201334B CN2006101652949A CN200610165294A CN101201334B CN 101201334 B CN101201334 B CN 101201334B CN 2006101652949 A CN2006101652949 A CN 2006101652949A CN 200610165294 A CN200610165294 A CN 200610165294A CN 101201334 B CN101201334 B CN 101201334B
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Abstract
The invention relates to a method to quickly verify the varieties of wheat, which comprises sampling; sample preparation; capillary electrophoresis; data processing analysis; and establishing standard high efficiency capillary electropherograms of wheat protoproteose to verify the types or idioplasm of wheat. The invention is characterized in that the end of a single seed without an embryo weighing about 15 to 20mg is taken, and is added to the ethanol of 20 percent in the way that 10 microlitres are added to every 1mg of the ethanol; the mixture is shaken for fifteen minutes in the room temperature, and is centrifugated at the speed of 10000rpm for ten minutes. The supernatant is the capillary electrophoresis specimen; the electrophoretic buffer during high efficiency capillary electrophoresis is phosphoric acid and glycine buffer solution of 0.1mol and the PH value is 2.5. Acetonitrile of 20 percent and hydroxyprolyl-methyl cellulose of 0.05 percent are added in the buffer solution. In addition, concrete electrophoretic conditions and parameters and flushing procedures are given. Compared with the prior method to verify the varieties of wheat, the method has the characteristics of quickness, accurateness, low cost, and high repeatability.
Description
Technical field
The present invention relates to a kind of rapid identification method of wheat breed, particularly separate wheat water-solubility protein fast and make up standard diagram and then realize authentication method on this basis wheat breed by the high performance capillary electrophoresis means.
Background technology
Wheat is one of most important cereal crops in the world, also is important protein matter source in the human grain consumption.China is world wheat production and consumption big country always, and sown area and gross output all rank first in the world.Water-solubility protein as wheat seed albumen important component part has been subjected to increasing attention, in wheat seed, water-solubility protein comprises albumin (albumin) and globulin (globulin), it mainly is the enzyme in some metabolic processes, inhibiting factor etc., as AMS, protease inhibitor, the regulation and control enzyme, the synzyme of special pathway, metabolic enzyme etc., main effect is the various metabolic processes of regulation and control wheat, to the function of food, the accumulation of starch, the rank of wheat gluten, the color of final use and particle etc. all has important role and influence.Albumin and globulin major sedimentary are in embryo, aleurone, and small part is present in endosperm, account for about 9% and 5% of seed albumen respectively.Also there is correlative study to prove that some high molecular albumins and globulin belong to wheat storage protein part.Studies show that water-solubility protein can be used as the biochemical marker of wheat quality and seed development research and cultivar identification.
In recent years along with the China market constant development of economy, at wheat seed production and operation, breeding of new variety and fields urgent need wheat breed authentication methods rapidly and efficiently such as protection of kind rights and interests and circulation and process.At present, identify that wheat breed mainly adopts methods such as identification of morphology, isodynamic enzyme evaluation, storage protein sds gel electrophoresis (SDS-PAGE) evaluation and acid polyacrylamide gel electrophoresis (A-PAGE) evaluation, reversed-phase high-performance liquid chromatography (RP-HPLC) evaluation, molecular markers for identification.Yet all there is limitation in various degree in more above-mentioned method, is subject to such environmental effects as morphological feature, poor reliability; Isodynamic enzyme, protein electrophorese disengaging time are long, time-consuming, in most cases needed 1-2 days could obtain analysis result, and resolution is low, often can't distinguish the kind that some sibships are near, its resolution and repeatability are also relatively poor, are difficult to carry out the comparison between quantification analysis and different experiments chamber gained result.The PAGE method also must have been used neurovirulent acrylamide and other contamination reagent in addition; Though RP-HPLC automaticity height, the instrument and equipment costliness, the analysis cost height, resolution also is difficult to satisfy the demand; The dna molecular marker technology, as PCR, RAPD, AFLP etc., Recent study is more, and accuracy is higher, but technical sophistication is time-consuming, and the appraisal cost height is difficult to widespread use.Therefore, all there are some shortcomings in existing wheat breed authentication method.
With respect to " capillary electrophoresis method of Rapid identification wheat breed " (CN200410037256.6) patented technology, the invention provides the high performance capillary electrophoresis method that another is identified wheat breed, the separation that is the water-solubility protein by wheat realizes with differentiating, rather than high efficiency separation by alcohol soluble protein in the wheat seed storage protein realizes with differentiating.
High performance capillary electrophoresis (High Performance Capillary Electrophoresis, HPCE) be a kind of new separation technology, have characteristics such as separation efficiency height, analysis speed be fast, showing great superiority aspect the compartment analysis of biomacromolecules such as protein, amino acid, nucleic acid.Because the seed water-solubility protein is formed complicated, heterogeneous high, isolation technics is the key of quick isolation identification efficiently.The apparatus of Capillary Electrophoresis mainly comprises five parts: high-voltage power supply, sampling system, capillary column, detecting device and register system.Make material capillaceous---the isoelectric point (pI) of fusion silica gel is about 1.5, and in alkalescence and subacidity (PH＞2.5) solution, the silicon hydroxyl (Si-OH) of molten silicon face is ionized into SiO
-, make its inner surface belt negative electricity.Negative charged surface is gathered the ion of opposite charges in solution, form electrostatic double layer between tube wall and solution.Under high effect of electric field, the fluid integral body that the hydrated cation in the electrostatic double layer causes will move towards the negative pole direction, and this phenomenon is called as electric osmose (Electroosmosis).Particle is subjected to electric field action on the one hand and directed moving, i.e. electrophoresis takes place in buffer solution in kapillary; Be subjected to the influence of electric osmose on the other hand.The vector that the final migration velocity of particle equals electrophoresis stream and electroosmotic flow with.Positive ion electrophoresis direction in the sample is consistent with the electroosmotic flow direction, so flow out at first; Neutral particle only flows to negative pole under the effect of electroosmotic flow, speed is slow than positive ion; Negative ion direction of motion is opposite with electric osmose,, will flow out after neutral particle during greater than electrophoretic velocity in electric osmose speed, if negative ion swimming flow velocity degree then can't flow out greater than percolation flow velocity.In general, electroosmotic flow speed is greater than electrophoretic velocity, so all components will move towards a direction in the sample, various like this particles have been realized separation because of the migration velocity difference in capillary medium.
The factor that influences the capillary electrophoresis separation effect is more, mainly comprises the modification of the determining of the selection of the type of operating voltage, temperature, buffer solution and concentration, pH value, adjuvant and concentration, capillary column etc.In actual applications, need at concrete detection thing these factors are optimized, to reach the optimal separation effect.
Summary of the invention
The high performance capillary electrophoresis method that the purpose of this invention is to provide a kind of quick separation wheat seed water-solubility protein, provide the running program and the condition of optimization, and then the HPCE collection of illustrative plates that passes through the wheat seed water-solubility protein is realized the evaluation to wheat breed, with respect to existing authentication method, this technology has fast, accurately, the characteristics of low, the favorable repeatability of cost.
Technical scheme of the present invention comprises sampling and specimen preparation, Capillary Electrophoresis and Data Management Analysis, by setting up the standard high performance capillary electrophoresis collection of illustrative plates of wheat water-solubility protein, and then realize wheat breed or germplasm are identified, it is characterized in that getting single seed and do not have embryo end (about 15-20mg), the ratio that adds 10 μ l in 1mg adds 20% ethanol, concussion was extracted 15 minutes under the room temperature, centrifugal 10 minutes of 10000rpm, and supernatant is the Capillary Electrophoresis sample; Electrophoretic buffer during high performance capillary electrophoresis (Buffer) is 0.1M phosphoric acid-glycine buffer, and this pH of buffer is 2.5, is added with 20% nitrile and 0.05% hydroxyprolyl-methylcellulose, and concrete deposition condition parameter is:
Capillary pipe length: 31.5cm
Capillary inner diameter: 50 μ m
Working voltage: 11.0kV
Capillary temperature: 35 ℃
Sample cell temperature: 15 ℃
Sample introduction: 10kV, 6-8 second
In technique scheme, for can the continuous detecting sample and guarantee the accuracy of testing result, when carrying out high performance capillary electrophoresis, kapillary be washed and must operate by cleaning procedure, according to the difference of flushing purpose, cleaning procedure specifically is divided into following four kinds of situations:
(1) will carry out the pre-service flushing before use for new kapillary, cleaning procedure is as follows:
H
2O 5 minutes
0.1MNaOH 10 minutes
H
2O 5 minutes
1MH
3PO
410 minutes
Electrophoretic buffer 60 minutes
(2) before the sample introduction kapillary is washed, cleaning procedure is as follows:
H
2O 5 minutes
0.1MNaOH 10 minutes
H
2O 5 minutes
1MH
3PO
410 minutes
Electrophoretic buffer 30 minutes
(3) when once identifying a plurality of sample of mensuration, will wash between twice sample detection, cleaning procedure is as follows:
1MH
3PO
43 minutes
Electrophoretic buffer 3 minutes
(4) detect the end back kapillary is washed, cleaning procedure is as follows:
1MH
3PO
445 minutes
H
2O 5 minutes
0.1MNaOH 2 minutes
H
2O 15 minutes
Advantage of the present invention is:
(1) amount of samples is few: only need the about 15-20mg of half granule seed repeatedly to analyze.Utilize this method can finish the separation of a sample in 10 minutes, efficient is apparently higher than the PAGE method of routine.
(2) resolution height: apparently higher than the conventional electrophoretic method, and collection of illustrative plates is stable.
(3) repeatability is high: the relative standard deviation of peak transit time and peak height (RSD) is respectively less than 1% and 3%.
(4) easy operating: analysis automated degree height from the specimen preparation to the Separation of Proteins, method is simple, easy operating.Can separate 30 samples continuously, by software control at every turn.
(5) analysis cost is low, pollution-free: only need cheap kapillary and some common agents, consumption seldom, nonhazardous, environmentally safe.
Description of drawings
Fig. 1. common wheat kind " Jimai 20 " water-solubility protein high performance capillary electrophoresis standard diagram;
Fig. 2. high performance capillary electrophoresis such as 7 Wheat Cultivars water-solubility proteins such as Zhengzhou 9023 grades is collection of illustrative plates relatively;
Fig. 3. the high performance capillary electrophoresis evaluation of fine quality (8331-1 is made among B-preferred No. 9 and the C-in A-Gaocheng 8901) and kind inferior (D-Huaihe River wheat 16, former winter of E-No. 6 and F-Shandong wheat 21) water-solubility protein.
Embodiment
Embodiment 1: the structure of wheat breed water-solubility protein standard HPCE finger-print
(1) sampling and specimen preparation
With wheat breed " Jimai 20 " is example.Get single seed, remove the embryo end with blade, accurately weighing (about 15-20mg) back is pricked with template and adz-eye hammer and is broken into fine powder, put into the 1ml centrifuge tube, the ratio that adds 10 μ l in 1mg adds 20% ethanol, concussion was extracted 15 minutes under the room temperature, centrifugal 10 minutes of 10000rpm, and supernatant is used for Capillary Electrophoresis.
(2) Capillary Electrophoresis
1. instrument and equipment adopts the BioFocus 3000 type efficient capillary electrophoresis apparatus systems (be furnished with software and be used for system operation and data processing) of Bio-Rad company production to carry out the CE analysis.
2. kapillary fused-silica capillary column, internal diameter are that 50 μ m, length are 31.5cm (detecting length 26.5cm), and skin scribbles the polyimide protective seam, and inwall is coating not all.
3. electrophoretic buffer 100mm phosphoric acid-glycine buffer (pH2.50 is added with 20% nitrile and 0.05% hydroxyprolyl-methylcellulose HPMC).The preparation method of 500ml phosphoric acid-glycine buffer: 85% phosphatase 11 .6ml, glycocoll 2.0g, acetonitrile 100ml, HPMC 0.25g adds distilled water and is settled to 500ml, and regulating pH is 2.50, through 0.45 μ m membrane filtration, 4 ℃ of preservations.
4. capillary column purging method
A. new kapillary preprocessor:
H
2O 5 minutes
0.1MNaOH 10 minutes
H
2O 5 minutes
1M H
3PO
410 minutes
Electrophoretic buffer 60 minutes
B. flushing before the sample introduction:
H
2O 5 minutes
0.1M NaOH 10 minutes
H
2O 5 minutes
1M H
3PO
410 minutes
Electrophoretic buffer 30 minutes
C. sample room cleaning procedure:
1M H
3PO
43 minutes
Electrophoretic buffer 3 minutes
D. finish the experiment flushing:
1M H
3PO
45 minutes
H
2O 5 minutes
0.1MNaOH 2 minutes
H
2O 15 minutes
5. move the electrophoresis parameter
Working voltage: 15kV
Capillary temperature: 35 ℃
Sample cell temperature: 15 ℃
Sample introduction: 10kV, 6 seconds
Electrode: by+to-
6. data processing utilizes software to carry out data processing, simultaneously electrophoresis result is converted to the ASC sign indicating number, carries out the Excel Treatment Analysis.
(3) fingerprint map construction makes up standard finger-print according to parameters such as the transit time of each protein component, peak height, peak areas.The standard diagram of wheat breed " Jimai 20 " as shown in Figure 1.
The separation sign and the cultivar identification of embodiment 2 Wheat Cultivars water-solubility proteins
(1) experimental implementation: sampling and specimen preparation, Capillary Electrophoresis, standard diagram make up and are equal to embodiment 1.
(2) HPCE atlas analysis:, analyze its electrophoresis composition characteristic according to the isoparametric difference of transit time, peak height, peak area of different cultivars water-solubility protein component.Each kind repeats electrophoresis 3-5 time; calculate the relative standard deviation (RSD) of peak transit time, peak height, peak area; obtain the mean value and the relative standard deviation of each water-solubility protein component, as the standard electrophoresis pattern of variety authentication and purity evaluation or the protection of kind power.
(3) kind or Germplasm Identification: when carrying out variety authentication and kind power protection evaluation, 10 seeds are analyzed in each kind grab sample, and each sample repeated isolation 3 times is carried out kind or Germplasm Identification according to the difference of electrophoresis pattern.Variety is identified and is got 50-100 grain seed at random, and repeated isolation 3 times compares with standard diagram then, calculates variety.
Collection of illustrative plates such as HPCE such as 6 Wheat Cultivars water-solubility proteins such as Zhengzhou 9023 grades more as shown in Figure 2.
Screening of embodiment 3 wheat high-quality resources and quality-improving marker assisted selection
(1) experimental implementation: sampling and specimen preparation, Capillary Electrophoresis, atlas analysis are equal to embodiment 1 and with embodiment 2.
(2) the high-quality protein subunit is identified and the high-quality resource screening: by the comparative analysis to different cultivars or germ plasm resource material water dissolubility protein electrophoresis collection of illustrative plates, identify the differential protein subunit that only in fine quality or germplasm, occurs, as the mark of high-quality protein Screening and Identification.For example, we have found and the closely-related protein labeling of high-quality that by the analysis to a large amount of kinds this protein protomer (Fig. 3 arrow indication) and gluten strength height correlation can carry out Rapid identification to high-quality and kind inferior by this mark.6 different high-qualitys such as Gaocheng 8901 grades and wheat breed water-solubility protein Capillary Electrophoresis collection of illustrative plates inferior are more as shown in Figure 3.
(3) marker assisted selection: selected the parent by what identify among the embodiment 2 with the closely-related water-solubility protein mark of quality, preparing hybrid combination, (general F in early days from generation to generation
2-F
4Generation) carries out the quality-improving assisted Selection by this mark, can improve quality breeding efficient greatly.
Claims (2)
1. high performance capillary electrophoresis method of identifying wheat breed by wheat water-solubility protein, comprise sampling and specimen preparation, Capillary Electrophoresis and Data Management Analysis, by setting up the standard high performance capillary electrophoresis collection of illustrative plates of wheat water-solubility protein, and then realize wheat breed or germplasm are identified, it is characterized in that getting single seed and do not have embryo end 15-20mg, the ratio that adds 10 μ l in 1mg adds 20% ethanol, concussion was extracted 15 minutes under the room temperature, centrifugal 10 minutes of 10000rpm, supernatant is the Capillary Electrophoresis sample; Electrophoretic buffer during high performance capillary electrophoresis is 0.1M phosphoric acid-glycine buffer, and this buffer solution ph is 2.5, and being added with volume ratio is the hydroxyprolyl-methylcellulose that 20% acetonitrile and quality account for damping fluid volume 0.05%, and concrete deposition condition parameter is:
Capillary pipe length: 31.5cm
Capillary inner diameter: 50 μ m
Working voltage: 11.0kv
Capillary temperature: 35 ℃
Sample cell temperature: 15 ℃
Sample introduction: 10kv, 6-8 second.
2. high performance capillary electrophoresis method according to claim 1, it is characterized in that for can the continuous detecting sample and guarantee the accuracy of testing result, when carrying out high performance capillary electrophoresis, kapillary is washed and must operate by cleaning procedure, according to the difference of flushing purpose, cleaning procedure specifically is divided into following four kinds of situations:
(1) will carry out the pre-service flushing before use for new kapillary, cleaning procedure is as follows:
(2) before the sample introduction kapillary is washed, cleaning procedure is as follows:
(3) when once identifying a plurality of sample of mensuration, will wash between twice sample detection, cleaning procedure is as follows:
1M H
3PO
43 minutes
Electrophoretic buffer 3 minutes
(4) detect the end back kapillary is washed, cleaning procedure is as follows:
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| CN101793865A (en) * | 2010-03-29 | 2010-08-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting content of human seralbumin in human milk |
| CN101949884A (en) * | 2010-08-02 | 2011-01-19 | 浙江银象生物工程有限公司 | Method for detecting content of epsilon-polylysine in rice by capillary electrophoresis |
| CN103575793A (en) * | 2013-10-26 | 2014-02-12 | 盐城工学院 | Method for constructing capillary electrophoresis fingerprints of chrysanthemum buds and standard fingerprint |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1046979A (en) * | 1990-02-02 | 1990-11-14 | 河北省农产商品质量鉴督检验站 | Method for rapidly identifying purity of crop seeds |
| SU1658927A1 (en) * | 1989-02-06 | 1991-06-30 | Белорусский Научно-Исследовательский Институт Земледелия | Method for selection of winter rye and wheat early forms |
| CN1430058A (en) * | 2003-01-29 | 2003-07-16 | 天津师范大学 | Method of detecting crop seed purity |
| CN1570622A (en) * | 2004-04-30 | 2005-01-26 | 首都师范大学 | Capillary electrophoresis method for fast identification of wheat varieties |
| CN1570623A (en) * | 2004-04-30 | 2005-01-26 | 首都师范大学 | Acidic capillary electrophoresis identification method for high molecular weight glutelin subunit of wheat |
| US6936703B2 (en) * | 2000-06-22 | 2005-08-30 | K.U. Leuven Research And Development | Biocatalyst inhibitors |
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2006
- 2006-12-15 CN CN2006101652949A patent/CN101201334B/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1658927A1 (en) * | 1989-02-06 | 1991-06-30 | Белорусский Научно-Исследовательский Институт Земледелия | Method for selection of winter rye and wheat early forms |
| CN1046979A (en) * | 1990-02-02 | 1990-11-14 | 河北省农产商品质量鉴督检验站 | Method for rapidly identifying purity of crop seeds |
| US6936703B2 (en) * | 2000-06-22 | 2005-08-30 | K.U. Leuven Research And Development | Biocatalyst inhibitors |
| CN1430058A (en) * | 2003-01-29 | 2003-07-16 | 天津师范大学 | Method of detecting crop seed purity |
| CN1570622A (en) * | 2004-04-30 | 2005-01-26 | 首都师范大学 | Capillary electrophoresis method for fast identification of wheat varieties |
| CN1570623A (en) * | 2004-04-30 | 2005-01-26 | 首都师范大学 | Acidic capillary electrophoresis identification method for high molecular weight glutelin subunit of wheat |
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