CN103149263B - Method for identification of barley varieties by two-dimensional electrophoresis technology - Google Patents
Method for identification of barley varieties by two-dimensional electrophoresis technology Download PDFInfo
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Abstract
The invention provides a method for identification of barley varieties. The method comprises: peeling and crushing pure barley, then adding a buffer solution to perform extraction, then carrying out purification through a series of steps so as to obtain water-soluble protein of barley, subjecting different varieties of barley protein to electrophoresis by a two-dimensional electrophoresis technology so as to obtain clear and visualized protein maps, and carrying out contrastive analysis to search differential protein of different varieties, thus effectively identifying barley varieties.
Description
Technical field
The present invention is specifically related to a kind of method of identifying barley variety.
Background technology
Barley is the important source material of beer brewing, and barley variety is different can produce material impact to Fructus Hordei Germinatus manufacture and beer quality.Cause the basic reason of barley variety difference to be gene, so the barley of different cultivars also exists general difference at protein level.To the barley of different cultivars, there is very big-difference in wheat technique processed and finished beer quality.So very large on wheat processed and brewing impact for the barley variety purity of Beer Brewage, therefore Fructus Hordei Germinatus and Brewage enterprise very urgently find one method accurately and fast, and barley variety is detected and identified.
Identified that barley variety was except relying on outward appearance experience, main method is A-PAGE electrophoresis and the SSR mark based on DNA technique in the past.A-PAGE electrophoresis is to utilize the electrophoretic band of hordein as cultivar identification foundation, has easy and simple to handle, economic, feature efficiently.But because band is than comparatively dense, in the situation that otherness is little, easily cause the difficulty of certain qualification.SSR mark utilizes DNA technique to identify, although result is accurate, stricter to sample DNA extraction operative technique and equipment requirement, appraisal cost is higher, so there is certain difficulty in enterprise's application.
Along with the development of proteomics and analysis of protein technology, the Germplasm Identification of using the bidirectional electrophoresis technique of aqueous soluble protein to carry out becomes possibility.This research and utilization Two dimensional Electrophoresis of Proteins is analyzed the electrophoresis pattern of purebred barley aqueous soluble protein in conjunction with application software or magnify tool, and the kind of aqueous soluble protein can detect hundreds and thousands of kinds.By the comparison of interracial dielectrophoresis protein graphical spectrum, can find the otherness albumen of different cultivars barley, barley variety is carried out to precise Identification.It is accurate, quick, economical that this method has advantages of, and operation is relatively simple, is easily accepted by Fructus Hordei Germinatus manufacture and beer enterprise.
Summary of the invention
The invention provides a kind of method of identifying barley variety, after the peeling of purebred barley is pulverized with damping fluid lixiviate, purify by series of steps again, obtain the aqueous soluble protein of barley, large different cultivars aleuronat is carried out to electrophoresis with bidirectional electrophoresis technique, obtain clear and intuitive protein graphical spectrum, the otherness albumen of the different cultivars searching out by comparative analysis, can effectively identify the kind of barley.
Concrete steps are as follows:
The first step, protein extraction: barley is manually removed the peel rear liquid nitrogen grinding, getting 0.3g sample adds 0.9mL extract room temperature to extract 2h, and whirlpool shakes 3 ~ 5 times, the centrifugal 10min of 10000g, gets supernatant and adds equal-volume pH8.8Tris balance phenol, and room temperature is extracted 30min, the centrifugal 10min of 10000g, phenol is added to 4 times of volume cleaning fluid A, and-80 DEG C are extracted 3h, the centrifugal 10min of 10000g; The centrifugal albumen obtaining respectively with cleaning fluid A and cleaning fluid B respectively wash, after centrifugal 3 times, freeze drying; To obtain holoprotein sample dry powder and add 100 μ L lysates, room temperature vortex oscillation cracking 1h, 4 DEG C of cracking are spent the night, 13000ppm4 DEG C of centrifugal 10min, get 5 μ L supernatants and survey protein content with Coomassie brilliant blue method (by the cow's serum typical curve of accompanying drawing 1), by applied sample amount packing, prepare electrophoresis.In room temperature vortex oscillation cracking process, ultrasonic 4 times, each 10-20s, ultrasonic power 22W
Second step, dielectrophoresis: get albumen supernatant and add appropriate sample buffer, be splined on self-control adhesive tape, applied sample amount is 110 μ g; Self-control adhesive tape blending process can not produce bubble, increases every 10min200V etc. voltage linear in point focusing process.
Deposition condition: pour electrophoretic buffer into, switch on power, 200V constant voltage electrophoresis 30min under room temperature, 500V constant voltage electrophoresis 30min, 1350V constant voltage electrophoresis 4h, carries out isoelectric focusing.
After completing etc. point focusing, adhesive tape is evenly extruded from glass tube, balance 30min in level pad, is transferred on 12% separation gel 20mA constant current vertical electrophoresis.Vertical electrophoresis does not use concentrated glue.
The 3rd step, dyeing and analysis: after electrophoresis finishes, gel is placed in to immobile liquid and fixes 1 ~ 2h, Coomassie brilliant blue method dyes, utilize gel imaging system to obtain image and complete the analysis of image by PDQuest software.
The above formula in, it is pure that extract agents useful for same is top grade, all the other reagent be analyze pure, institute's water is distilled water or ultrapure water, wherein:
Extract: 0.1M/L Tris-HCl pH8.8,50mM/L DTT, 20mM/L ethylenediamine tetraacetic acid, 1mM/L phenylmethylsulfonyl fluoride, 1 μ M/L pepsin inhibitor, and 10mM/L leupeptin, surplus is water;
The methanol solution of cleaning fluid A:100mM/L ammonium acetate, 10mM/L DTT;
90% ethanolic solution of cleaning fluid B:10mM/L DTT;
Lysate: 7M/L urea, 2M/L thiocarbamide, 4% (w/v) 3-[3-(courage acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt, 1% (w/v) DTT, 0.5% (v/v) carrier ampholyte pH310,2% (v/v) carrier ampholyte pH4 ~ 6,10mM/L phenylmethylsulfonyl fluoride, surplus is water;
Sample buffer: 7M/L urea, 2M/L thiocarbamide, 4% (w/v) 3-[3-(courage acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt, 1% (w/v) DTT, 0.5% (v/v) carrier ampholyte pH3 ~ 10,2% (v/v) carrier ampholyte pH4 ~ 6,10mM/L phenylmethylsulfonyl fluoride, 10mg/L bromophenol blue, surplus is water;
Self-control adhesive tape formula: 0.6g urea, 540 μ L distilled waters, 30% acrylamide solution 200 μ L, pH4 ~ 6 carrier ampholyte 24 μ L, pH310 carrier ampholyte solution 4.8 μ L, after light and slow mixing, add 5 μ L10% Ammonium Persulfate 98.5s and 4 μ L tetramethylethylenediamines, mix gently.Draw fast above-mentioned mixed liquor, slowly evenly pour into about 7cm, polymerized at room temperature 1h from one end of diameter 1mm, long 11cm glass tube;
In electrophoretic buffer, ability cathode electrophoresis damping fluid: 20mM/L sodium hydroxide solution 150mL, anodic electrophoresis damping fluid: 10mM/L phosphoric acid solution 200mL;
Level pad: 0.05M/L Tris-HCl (pH8.8), 30% (W/V) glycerine, 2.3% (W/V) SDS, 6mM/L urea, surplus is water;
Separation gel: 3.3mL distilled water, 4.0mL30% acrylamide, 2.5mL1.5M/L Tris-HCl, 0.1mL10%SDS, 0.1mL10% ammonium persulfate, 0.004mL tetramethylethylenediamine, surplus is water;
Immobile liquid: 10% acetic acid, 40% ethanol, surplus is water;
Dyeing liquor: 0.12% Coomassie brilliant blue G-250,10% ammonium sulfate, 10% phosphoric acid, 20% methyl alcohol, surplus is water.
The present invention's application bidirectional electrophoresis technique binding analysis software, by the Two-dimensional Gel Electrophoresis of comparison different cultivars, finds the otherness protein site of different cultivars barley, thereby quick and precisely identifies barley variety.The present invention simultaneously can provide reference for Process of Beer Brewing, evaluation barley quality.Compare other document discrimination methods and there is obvious Stability and veracity.
Brief description of the drawings
Fig. 1, bovine serum albumin(BSA) (BSA) typical curve.
Fig. 2, different cultivars barley Two-dimensional Gel Electrophoresis, wherein, a is that No. 4, sweet beer, b are No. 7, sweet beer, c No. 7, d are that draw No. 1 in west in order to cultivate wheat, e is single No. 2.
The differential protein of Fig. 3, different cultivars barley distributes, and wherein, a is that No. 4, sweet beer, b are No. 7, sweet beer, c No. 7, d are that draw No. 1 in west in order to cultivate wheat, and e is singly No. 2.
Fig. 4, No. 6 barley dielectrophoresis figure of sweet beer, wherein magnification region is differential protein spot region.
Fig. 5, No. 4 barley dielectrophoresis figure of sweet beer are wherein differential protein region in square frame.
Embodiment
The qualification of embodiment mono-, several barley varieties
The first step, protein extraction:
Get manually liquid nitrogen grinding after peeling of five kinds of purebred barleys (No. 4, sweet beer, No. 7, sweet beer, No. 7, west are drawn No. 1 to cultivate wheat, single No. 2), get 0.3g sample and add 0.9mL extract room temperature to extract 2h, the centrifugal 10min of 10000g.Get supernatant and add equal-volume pH8.8Tris balance phenol, room temperature is extracted 30min, the centrifugal 10min of 10000g.Phenol is added to 4 times of volume cleaning fluid A, and-80 DEG C are extracted 3h, the centrifugal 10min of 10000g.The centrifugal albumen obtaining is respectively with cleaning fluid A and cleaning fluid B washing, after centrifugal 3 times, and freeze drying.To obtain holoprotein sample dry powder and add 100 μ L lysates, room temperature vortex oscillation cracking 1h, 4 DEG C of cracking are spent the night.13000ppm4 DEG C of centrifugal 10min, gets 5 μ L supernatants and surveys protein content with Coomassie brilliant blue method, by applied sample amount packing, prepares electrophoresis.
Wherein, in bovine serum albumin(BSA) (BSA) typical curve, protein concentration and absorbance relation are as follows:
| Absorbance (595nm) | Protein concentration (μ g/ μ L) |
| 0.034 | 0.4 |
| 0.042 | 1 |
| 0.105 | 2 |
| 0.196 | 4 |
| 0.307 | 6 |
Show that bovine serum albumin(BSA) (BSA) typical curve is y=15.597x-0.0501, R
2=0.9942
Second step, dielectrophoresis:
Get albumen supernatant and add appropriate sample buffer, be splined on self-control adhesive tape;
Deposition condition: pour electrophoretic buffer (ability cathode electrophoresis damping fluid: 20mM/L NaOH 150mL, anodic electrophoresis damping fluid: 10mM/L phosphoric acid 200mL) into.Switch on power, 200V constant voltage electrophoresis 30min under room temperature, 500V constant voltage electrophoresis 30min, 1350V constant voltage electrophoresis 4h, carries out isoelectric focusing.
After completing etc. point focusing, adhesive tape is evenly extruded from glass tube, balance 30min in level pad, is transferred on 12% separation gel 20mA constant current electrophoresis.
The 3rd step, dyeing and analysis:
After electrophoresis finishes, gel is put into immobile liquid and fix 1h, Blue silver Coomassie brilliant blue method dyes, and utilizes gel imaging system to obtain image and completes the analysis of image by PDQuest software (also can use other magnify tools).
The Two-dimensional Gel Electrophoresis of the each purebred barley being obtained by Fig. 2, can determine the otherness protein site (square frame demonstration) of each kind, and Fig. 3 is the enlarged drawing of otherness protein site, and contrast difference's protein site can make a distinction every kind of barley variety accurate and visually.
Embodiment bis-, No. 6 qualifications of sweet beer
The first step, protein extraction:
Get the sweet beer of purebred barley and manually remove the peel rear liquid nitrogen grinding No. 6, get 0.3g sample and add 0.9mL extract room temperature to extract 2h, the centrifugal 10min of 10000g.Get supernatant and add equal-volume pH8.8Tris balance phenol, room temperature is extracted 30min, the centrifugal 10min of 10000g.Phenol is added to 4 times of volume cleaning fluid A, and-80 DEG C are extracted 3h, the centrifugal 10min of 10000g.The centrifugal albumen obtaining originally Yong cleaning fluid A and cleaning fluid B washing, after centrifugal 3 times, and freeze drying.To obtain holoprotein sample dry powder and add 100 μ L lysates, room temperature vortex oscillation cracking 1h, 4 DEG C of cracking are spent the night.13000ppm4 DEG C of centrifugal 10min, the 5 μ L supernatant Bradford methods of getting are surveyed protein content, by applied sample amount packing, prepare electrophoresis.
Second step, dielectrophoresis:
Get albumen supernatant and add appropriate sample buffer, be splined on self-control adhesive tape;
Deposition condition: pour electrophoretic buffer (ability cathode electrophoresis damping fluid: 20mM/L NaOH 150mL, anodic electrophoresis damping fluid: 10mM/L phosphoric acid 200mL) into.Switch on power, 200V constant voltage electrophoresis 30min under room temperature, 500V constant voltage electrophoresis 30min, 1350V constant voltage electrophoresis 4h, carries out isoelectric focusing.
After completing etc. point focusing, adhesive tape is evenly extruded from glass tube, balance 30min in level pad, is transferred on 12% separation gel 20mA constant current electrophoresis.
The 3rd step, dyeing and analysis:
After electrophoresis finishes, gel is put into immobile liquid and fix 1h, Blue silver Coomassie brilliant blue method dyes, and utilizes gel imaging system to obtain image and completes the analysis of image by PDQuest software (also can use other magnify tools).
The otherness albumen of the dielectrophoresis Fig. 4 obtaining and Fig. 2 same area is compared, and result shows all not identical, and Fig. 4 region is shown as the differential protein region of this kind.This kind barley can be distinguished.
Embodiment tri-, No. 4 barley inspections of sweet beer
The first step, protein extraction:
Get the sweet beer of purebred barley and manually remove the peel rear liquid nitrogen grinding No. 4, get 0.3g sample and add 0.9mL extract room temperature to extract 2h, the centrifugal 10min of 10000g.Get supernatant and add equal-volume pH8.8Tris balance phenol, room temperature is extracted 30min, the centrifugal 10min of 10000g.Phenol is added to 4 times of volume cleaning fluid A, and-80 DEG C are extracted 3h, the centrifugal 10min of 10000g.The centrifugal albumen obtaining originally Yong cleaning fluid A and cleaning fluid B washing, after centrifugal 3 times, and freeze drying.To obtain holoprotein sample dry powder and add 100 μ L lysates, room temperature vortex oscillation cracking 1h, 4 DEG C of cracking are spent the night.13000ppm4 DEG C of centrifugal 10min, the 5 μ L supernatant Bradford methods of getting are surveyed protein content, by applied sample amount packing, prepare electrophoresis.
Second step, dielectrophoresis:
Get albumen supernatant and add appropriate sample buffer, be splined on self-control adhesive tape;
Deposition condition: pour electrophoretic buffer (ability cathode electrophoresis damping fluid: 20mM/L NaOH 150mL, anodic electrophoresis damping fluid: 10mM/L phosphoric acid 200mL) into.Switch on power, 200V constant voltage electrophoresis 30min under room temperature, 500V constant voltage electrophoresis 30min, 1350V constant voltage electrophoresis 4h, carries out isoelectric focusing.
After completing etc. point focusing, adhesive tape is evenly extruded from glass tube, balance 30min in level pad, is transferred on 12% separation gel 20mA constant current electrophoresis.
The 3rd step, dyeing and analysis:
After electrophoresis finishes, gel is put into immobile liquid and fix 1h, Blue silver Coomassie brilliant blue method dyes, and utilizes gel imaging system to obtain image and completes the analysis of image by PDQuest software (also can use other magnify tools).
The dielectrophoresis figure (Fig. 5) obtaining is compared with Fig. 2, with wherein No. 4 otherness albumen regions of sweet beer are identical, prove that the method repeatability is fine, can meet and put into practice needs.
In sum, the present invention has advantages of aspect barley variety accurate, quick, economical in qualification, along with the acquisition of the Two-dimensional Gel Electrophoresis Profiles of the each kind of barley, can set up the dielectrophoresis database of each barley variety, for protein comparison and the germ plasm resource analysis of domestic and international different barley varieties provide strong reference.
Claims (4)
1. a method of utilizing dielectrophoresis qualification barley variety, concrete operation step is as follows:
The first step, protein extraction: barley is manually removed the peel rear liquid nitrogen grinding, getting 0.3g sample adds 0.9mL extract room temperature to extract 2h, and whirlpool shakes 3～5 times, the centrifugal 10min of 10000g, gets supernatant and adds equal-volume pH8.8Tris balance phenol, and room temperature is extracted 30min, the centrifugal 10min of 10000g, phenol is added to 4 times of volume cleaning fluid A, and-80 DEG C are extracted 3h, the centrifugal 10min of 10000g; The centrifugal albumen obtaining successively with cleaning fluid A and cleaning fluid B respectively wash, after centrifugal 3 times, freeze drying; To obtain holoprotein sample dry powder and add 100 μ L lysates, room temperature vortex oscillation cracking 1h, 4 DEG C of cracking are spent the night, and 13000rpm4 DEG C of centrifugal 10min gets 5 μ L supernatants and surveys protein content with Coomassie brilliant blue method, by applied sample amount packing, prepares electrophoresis;
Second step, dielectrophoresis:
Get albumen supernatant and add appropriate sample buffer, be splined on self-control adhesive tape, applied sample amount is 110 μ g;
Deposition condition: pour electrophoretic buffer into, switch on power, 200V constant voltage electrophoresis 30min under room temperature, 500V constant voltage electrophoresis 30min, 1350V constant voltage electrophoresis 4h, carries out isoelectric focusing.
After completing etc. point focusing, adhesive tape is evenly extruded from glass tube, balance 30min in level pad, is transferred on 12% separation gel 20mA constant current vertical electrophoresis;
The 3rd step, dyeing and analysis:
After electrophoresis finishes, gel is placed in to immobile liquid and fixes 1～2h, Coomassie brilliant blue method dyes, and utilizes gel imaging system to obtain image and completes the analysis of image by PDQuest software;
In the above step, it is pure that extract agents useful for same is top grade, all the other reagent be analyze pure, institute's water is distilled water or ultrapure water, wherein:
Extract: 0.1M/L Tris-HCl pH8.8,50mM/L DTT, 20mM/L ethylenediamine tetraacetic acid, 1mM/L phenylmethylsulfonyl fluoride, 1 μ M/L pepsin inhibitor, and 10mM/L leupeptin, surplus is water;
The methanol solution of cleaning fluid A:100mM/L ammonium acetate, 10mM/L DTT;
90% ethanolic solution of cleaning fluid B:10mM/L DTT;
Lysate: 7M/L urea, 2M/L thiocarbamide, 4% (w/v) 3-[3-(courage acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt, 1% (w/v) DTT, 0.5% (v/v) carrier ampholyte pH3～10,2% (v/v) carrier ampholyte pH4～6,10mM/L phenylmethylsulfonyl fluoride, surplus is water;
Sample buffer: 7M/L urea, 2M/L thiocarbamide, 4% (w/v) 3-[3-(courage acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt, 1% (w/v) DTT, 0.5% (v/v) carrier ampholyte pH3～10,2% (v/v) carrier ampholyte pH4～6,10mM/L phenylmethylsulfonyl fluoride, 10mg/L bromophenol blue, surplus is water;
Self-control adhesive tape formula: 0.6g urea, 540 μ L distilled waters, 30% acrylamide solution 200 μ L, pH4～6 carrier ampholyte 24 μ L, pH3～10 carrier ampholyte solution 4.8 μ L, after light and slow mixing, add 5 μ L10% Ammonium Persulfate 98.5s and 4 μ L tetramethylethylenediamines, mix gently.Draw fast above-mentioned mixed liquor, slowly evenly pour into about 7cm, polymerized at room temperature 1h from one end of diameter 1mm, long 11cm glass tube;
In electrophoretic buffer, ability cathode electrophoresis damping fluid: 20mM/L sodium hydroxide solution 150mL, anodic electrophoresis damping fluid: 10mM/L phosphoric acid solution 200mL;
Level pad: 0.05M/L Tris-HCl pH8.8,30% (W/V) glycerine, 2.3% (W/V) SDS, 6mM/L urea, surplus is water;
Separation gel: 3.3mL distilled water, 4.0mL30% acrylamide, 2.5mL1.5M/L Tris-HCl, 0.1mL10%SDS, 0.1mL10% ammonium persulfate, 0.004mL tetramethylethylenediamine, surplus is water;
Immobile liquid: 10% acetic acid, 40% ethanol, surplus is water;
Dyeing liquor: 0.12% Coomassie brilliant blue G-250,10% ammonium sulfate, 10% phosphoric acid, 20% methyl alcohol, surplus is water.
2. a kind of method of utilizing dielectrophoresis qualification barley variety according to claim 1, is characterized in that in the first step in room temperature vortex oscillation cracking process, ultrasonic 4 times, and each 10-20s, ultrasonic power 22W.
3. a kind of method of utilizing dielectrophoresis qualification barley variety according to claim 1, is characterized in that second step self-control adhesive tape blending process can not produce bubble, increases every 10min200V etc. voltage linear in point focusing process.
4. a kind of method of utilizing dielectrophoresis qualification barley variety according to claim 1, is characterized in that second step vertical electrophoresis only uses 12% separation gel, does not use concentrated glue.
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| CN107344961A (en) * | 2017-07-03 | 2017-11-14 | 中国农业科学院烟草研究所 | A kind of method for extracting No. 0/1 microspecies differential protein peptide fragment of tobacco black shank bacterium and the otherness peptide fragment of identification extraction |
| CN108007997A (en) * | 2017-12-06 | 2018-05-08 | 上海市农业科学院 | A kind of shiitake mushroom hypha total protein extraction and separation method for dielectrophoresis |
| CN109916988B (en) * | 2019-03-21 | 2021-09-21 | 上海交通大学 | Meat quality identification method based on isoelectric focusing electrophoresis technology |
| CN111505094A (en) * | 2020-04-28 | 2020-08-07 | 上海交通大学 | Food feed species tracing identification method based on isoelectric focusing electrophoresis |
| CN113189182B (en) * | 2021-04-25 | 2023-05-23 | 湖南省作物研究所 | Method for identifying purity of cabbage type rape hybrid seeds by using water-soluble protein |
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