CN109824768A - The extracting method and electrophoretic detection of Rice Glutelin - Google Patents
The extracting method and electrophoretic detection of Rice Glutelin Download PDFInfo
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- CN109824768A CN109824768A CN201910071939.XA CN201910071939A CN109824768A CN 109824768 A CN109824768 A CN 109824768A CN 201910071939 A CN201910071939 A CN 201910071939A CN 109824768 A CN109824768 A CN 109824768A
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 58
- 235000009566 rice Nutrition 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 41
- 108010068370 Glutens Proteins 0.000 title claims abstract description 40
- 238000001514 detection method Methods 0.000 title claims abstract description 14
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 57
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 27
- 239000006228 supernatant Substances 0.000 claims abstract description 27
- 238000001962 electrophoresis Methods 0.000 claims abstract description 25
- 230000001376 precipitating effect Effects 0.000 claims abstract description 15
- 235000012054 meals Nutrition 0.000 claims abstract description 11
- 238000003756 stirring Methods 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
- 238000007605 air drying Methods 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 9
- 239000003292 glue Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 238000004043 dyeing Methods 0.000 claims description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 3
- 239000000975 dye Substances 0.000 claims description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 3
- 230000005611 electricity Effects 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 230000009182 swimming Effects 0.000 claims 1
- 238000001228 spectrum Methods 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 235000021329 brown rice Nutrition 0.000 description 3
- 230000031787 nutrient reservoir activity Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The extracting method and electrophoretic detection of present invention offer Rice Glutelin.Rice is ground into rice meal after natural air drying, soaks in water into rice meal, is centrifuged after stirring, removes supernatant, 0.03mol/L NaOH solution is added into precipitating, is centrifuged after stirring, takes supernatant, adjusts supernatant pH to 4.8, centrifugation, removes supernatant, and precipitating is washed, dried to get Rice Glutelin.When carrying out electrophoresis using the glutelin that the method for the present invention is extracted, obtained map bands of a spectrum are clear, and miscellaneous band is less.Rice Glutelin extracting method of the invention, is suitable for the identification of the rice varieties of separate sources, to instructing rice breeding etc. to be of great significance.
Description
Technical field
The present invention relates to field of biotechnology, specifically, being related to extracting method and the electrophoresis detection side of Rice Glutelin
Method.
Background technique
Rice is one of main cereal crops of the mankind, especially with Asian countries (such as China, Japan, South Asia country)
Based on.Protein in rice is generally acknowledged optimal vegetable protein, is divided by function, and storage protein and structure egg can be divided into
It is white.Most of protein is storage protein in rice paddy seed, is primarily present in endosperm, by dissolubility can be divided into globulin,
Albumin, alcohol soluble protein and glutelin.The ratio that alcohol soluble protein, globulin, albumin account for be respectively 1%-5%, 2%-10%,
2%-5%.Glutelin is storage protein important in rice, and content accounts for 80% of seed protein or so, it is in rice
It is only second to the main endosperm ingredient that starch is easy to be digested.Therefore, further investigation is carried out with important to glutelin
Meaning.
With the development of science and technology the research to rice protein is more deep.It is existing much to be reported about the research of glutelin,
But extracted according to the glutelin extracting method provided in document, when obtained glutelin carries out electrophoresis detection, often occur
The phenomenon that electrophorogram is unintelligible or miscellaneous band is more.Therefore, it needs to improve existing glutelin extracting method.
Summary of the invention
Bands of a spectrum are unintelligible when the present invention is for electrophoresis generally existing in current Rice Glutelin extracting method or foreign protein
The excessive problem of bands of a spectrum provides a kind of extracting method of Rice Glutelin that significant effect is promoted.
In order to achieve the object of the present invention, in a first aspect, the present invention provides the extracting method of Rice Glutelin, rice is through nature
After air-drying, it is ground into rice meal, is soaked in water into rice meal, is centrifuged after stirring, supernatant is removed, 0.03mol/ is added into precipitating
L NaOH solution is centrifuged after stirring, takes supernatant, adjusts supernatant pH to 4.8, and supernatant is removed in centrifugation, and precipitating is washed, dried to get water
Rice glutelin.
Further, rice meal 40g is taken, 40mL water is added, impregnates 1h；Moderate-speed mixer 15min, then 5000r/min from
Heart 10min；Supernatant is removed, 0.03mol/L NaOH solution (aq) 40mL, moderate-speed mixer 18min is added into precipitating；Then
5000r/min is centrifuged 10min；Supernatant, tune supernatant pH value to 4.8 are taken, then 10min is centrifuged with 5000r/min；Remove supernatant
Liquid, precipitating are washed (3 times), dry (natural drying), i.e. acquisition Rice Glutelin.- 20 DEG C of preservations.
In the present invention, the moderate-speed mixer refers to 140-170 times per minute hand operated mixing clockwise or counterclockwise.
Second aspect, the present invention provides the electrophoretic detection of Rice Glutelin, and the Rice Glutelin of said extracted is molten
In protein extract, after standing a period of time, centrifugation takes supernatant to carry out SDS-PAGE electrophoresis detection.
Wherein, the protein extract are as follows: 0.125mol/L Tris-HCl pH6.8,4%SDS, 20% glycerol, 4mol/
L urea element, 5% beta -mercaptoethanol.
Electrophoretic detection above-mentioned the following steps are included:
1) 0.05g Rice Glutelin is taken to be dissolved in 1mL protein extract, oscillation mixes, 4 DEG C of standing 30min；Then
10000r/min is centrifuged 5min, and supernatant is taken to carry out SDS-PAGE electrophoresis；
2) electrophoresis terminates, and takes out gel, fixed in solution of trichloroacetic acid；
3) dyeing and decoloration: the gel after fixation is transferred in coomassie brilliant blue R_250 solution, shaking table dyeing, then
Decoloration.
Further, step 1) carry out the prepared gel of SDS-PAGE electrophoresis are as follows: 15% separation gel and 4% concentration
Glue；In separation gel, voltage 100V, until voltage is down to 80V, electrophoresis total duration 2.5h when concentration glue.
Gel is fixed 2h in 10% solution of trichloroacetic acid by step 2).
Shaking speed 70r/min in step 3) dyes 2h.
Preferably, the sample volume of SDS-PAGE electrophoresis is 7 holes μ L/.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) Rice Glutelin extracting method provided by the invention, has the advantages that step is simple and quick.With it is reported
Method is compared, and is shortened extraction time, while the reduction of NaOH (aq) concentration, is not only effectively slowed down the gelatinization situation of starch,
And drug dosage is saved.
(2) when carrying out electrophoresis using the glutelin that the method for the present invention is extracted, obtained map bands of a spectrum are clear, miscellaneous band
It is less.
(3) Rice Glutelin extracting method of the invention, is suitable for the identification of the rice varieties of separate sources, to guidance
Rice breeding etc. is of great significance.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis detection result in the embodiment of the present invention 1 under three kinds of Rice Glutelin extracting methods；Its
In, A corresponds to method one, and B corresponds to method two, and C corresponds to method three.
It is glutelin key band at 57kDa, 37-39kDa, 22-23kDa arrow meaning in figure.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
The percentage sign " % " being related in the present invention refers to mass percent if not specified；But the percentage of solution
Than unless otherwise specified, referring to the grams in 100mL solution containing solute.
The extraction and electrophoresis detection of 1 Rice Glutelin of embodiment
1, the preparation of rice sample: for rice after natural air drying, each Rice Samples weigh 100g.First it is ground into brown rice machine
Brown rice, then brown rice is ground into polished rice by rice polisher.Polished rice is milled into powder through flour mill, and -20 DEG C of refrigerators save backup.
2, it the measurement of rice protein content: is measured using kjeldahl apparatus.Selected sample gross protein value is shown in Table
1。
1 sample gross protein value of table
3, Rice Glutelin extracting method:
Method one: this method has been reported.Rice meal 40g is weighed in beaker, the NaOH of 400mL 0.05mol/L is added
(aq), stirring and leaching 2h at room temperature；Leaching liquor is centrifuged 10min under 3500r/min；It is heavy that supernatant tune pH to 4.8 carries out acid.
Then 3500r/min is centrifuged 10min, collects precipitating.By precipitating washing 3 times, 4h, -20 DEG C of preservations are spontaneously dried.
Method two: this method has been reported.Rice meal 40g is weighed in beaker, 40mL water is added, impregnates 1h, moderate-speed mixer
15min.The NaOH (aq), moderate-speed mixer 18min of 40mL 0.03mol/L is added.Then 5000r/min is centrifuged 10min.It takes
Clearly in beaker, pH to 4.8 is adjusted.Then 5000r/min is centrifuged 10min.Precipitating washing 3 times spontaneously dries 4h, -20 DEG C of preservations.
Method three: the method for the present invention.Rice meal 40g is weighed in beaker, 40mL water is added, impregnates 1h, moderate-speed mixer
15min.Centrifugation, 5000r/min, 10min.Supernatant is removed, precipitating is transferred in beaker, and the NaOH of 40mL 0.03mol/L is added
(aq), moderate-speed mixer 18min.Centrifugation, 5000r/min, 10min.It takes supernatant in beaker, adjusts pH to 4.8.Then 5000r/
Min is centrifuged 10min.Precipitating washing 3 times spontaneously dries 4h, -20 DEG C of preservations.
4, the preparation of gel: 15% separation gel and 4% concentration glue is selected to carry out SDS-PAGE electrophoresis tests respectively.
5, the preparation of protein extract: 0.125mol/L Tris-HCl pH6.8,4%SDS, 20% glycerol, 4mol/L urea
Element, 5% beta -mercaptoethanol.
6, electrophoresis detection: taking 0.05g glutelin to be dissolved in 1mL protein extract, vibrates 1min, mixes.It is stood in 4 DEG C
30min.Then 10000r/min is centrifuged 5min.Supernatant is taken to carry out electrophoresis tests, sample volume is 7 holes μ L/.In separation gel, electricity
Pressure is 100V, until voltage is down to 80V when concentration glue.Electrophoresis time is 2.5h.
7, proteopexy: electrophoresis terminates, and takes out gel.It is placed in 10% solution of trichloroacetic acid and fixes 2h.
8, dyeing and decoloration: gel is placed in 40mL coomassie brilliant blue R_250 solution, and shaking table dyes 2h.Then it decolourizes.
Glutelin electrophorogram is as shown in Figure 1, as seen from Figure 1, method one extracts obtained glutelin and carries out electrophoresis
Experiment, map is fuzzy, completely invisible bands of a spectrum (A)；The map clarity that method two obtains is preferable, but miscellaneous band is more (B)；Side
The glutelin map that method three obtains is clear, and bands of a spectrum are clearly demarcated (C), and extraction effect basic phase of the method three in two rice varieties
Together, illustrate that this method has broad applicability, stability is good.
Extraction and electrophoresis detection that different operation personnel carry out Rice Glutelin according to the method described above are changed, as a result basic one
It causes.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. the extracting method of Rice Glutelin, which is characterized in that rice is ground into rice meal after natural air drying, to rice meal
In soak in water, be centrifuged after stirring, remove supernatant, into precipitating be added 0.03mol/L NaOH solution, be centrifuged, take after stirring
Clear liquid adjusts supernatant pH value to 4.8, and supernatant is removed in centrifugation, and precipitating is washed, dried to get Rice Glutelin.
2. 40mL water is added the method according to claim 1, wherein taking rice meal 40g, 1h is impregnated；Middling speed is stirred
15min is mixed, then 5000r/min is centrifuged 10min；Supernatant is removed, 0.03mol/L NaOH solution 40mL, middling speed are added into precipitating
Stir 18min；Then 5000r/min is centrifuged 10min；Supernatant, tune supernatant pH value to 4.8 are taken, then is centrifuged with 5000r/min
10min；Supernatant is removed, precipitating is washed, dried, i.e. acquisition Rice Glutelin.
3. the electrophoretic detection of Rice Glutelin, which is characterized in that the Rice Glutelin that claims 1 or 2 is extracted to be dissolved in
In protein extract, after standing a period of time, centrifugation takes supernatant to carry out SDS-PAGE electrophoresis detection；
Wherein, the protein extract are as follows: 0.125mol/L Tris-HCl pH6.8,4%SDS, 20% glycerol, 4mol/L urea
Element, 5% beta -mercaptoethanol.
4. according to the method described in claim 3, characterized by comprising the following steps:
1) 0.05g Rice Glutelin is taken to be dissolved in 1mL protein extract, oscillation mixes, 4 DEG C of standing 30min；Then
10000r/min is centrifuged 5min, and supernatant is taken to carry out SDS-PAGE electrophoresis；
2) electrophoresis terminates, and takes out gel, fixed in solution of trichloroacetic acid；
3) dyeing and decoloration: the gel after fixation is transferred in coomassie brilliant blue R_250 solution, then shaking table dyeing is decolourized.
5. according to the method described in claim 4, it is characterized in that, step 1) carries out the prepared gel of SDS-PAGE electrophoresis
Are as follows: 15% separation gel and 4% concentration glue；In separation gel, voltage 100V, until voltage is down to 80V when concentration glue, electricity
Swimming Association duration 2.5h.
6. according to the method described in claim 4, it is characterized in that, step 2) is fixed in 10% solution of trichloroacetic acid by gel
2h。
7. according to the method described in claim 4, it is characterized in that, shaking speed 70r/min in step 3), dyes 2h.
8. according to the described in any item methods of claim 4-7, which is characterized in that the sample volume of step 1) SDS-PAGE electrophoresis is
7 holes μ L/.
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| WO2012088295A1 (en) * | 2010-12-21 | 2012-06-28 | The Chinese University Of Hong Kong | A cost-effictive method for expression and purification of recombinant proteins in plants |
| CN104642715A (en) * | 2015-03-10 | 2015-05-27 | 安徽天利粮油集团股份有限公司 | Method for extracting rice germ protein |
| CN108634088A (en) * | 2018-05-16 | 2018-10-12 | 武汉轻工大学 | The extraction separation method of cadmium-binding protein in a kind of rice |
-
2019
- 2019-01-25 CN CN201910071939.XA patent/CN109824768A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012088295A1 (en) * | 2010-12-21 | 2012-06-28 | The Chinese University Of Hong Kong | A cost-effictive method for expression and purification of recombinant proteins in plants |
| CN104642715A (en) * | 2015-03-10 | 2015-05-27 | 安徽天利粮油集团股份有限公司 | Method for extracting rice germ protein |
| CN108634088A (en) * | 2018-05-16 | 2018-10-12 | 武汉轻工大学 | The extraction separation method of cadmium-binding protein in a kind of rice |
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Application publication date: 20190531 |