CN103257173A - Chinese cabbage variety identification and seed purity testing method - Google Patents
Chinese cabbage variety identification and seed purity testing method Download PDFInfo
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- CN103257173A CN103257173A CN201310062681XA CN201310062681A CN103257173A CN 103257173 A CN103257173 A CN 103257173A CN 201310062681X A CN201310062681X A CN 201310062681XA CN 201310062681 A CN201310062681 A CN 201310062681A CN 103257173 A CN103257173 A CN 103257173A
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Abstract
The present invention discloses a Chinese cabbage variety identification and seed purity testing method performed by adopting seed protein ultrathin-layer isoelectric focusing electrophoresis. According to the method, Chinese cabbage varieties 87-114 and parent seeds thereof are adopted as materials, seed protein ultrathin-layer isoelectric focusing electrophoresis technology analysis is adopted to successfully obtain two female parent specific protein bands and 1 male parent specific protein band, variety identification and seed purity testing of 87-114 hybrid species can be performed according to the specific protein bands, and identifications of 200 87-114 random groups are performed to obtain the same identification result as the field identification result.
Description
Technical field
The invention belongs to plant variety and identify and seed purity inspection technology field, relate in particular to a kind of ultra-thin isoelectric focusing electrophoresis technology of seed-protein of utilizing and carry out Chinese cabbage cultivar evaluation and the seed purity method of inspection.
Background technology
It is indispensable important step in the seed production work that authenticity and variety are identified, is that the good heredity of assurance breeding is given full play to, and promotes agricultural production to continue the effective measures of stable and high yields; Be to prevent that breeding from mixing degeneration, improve the necessary means of seed quality and product quality; Be to overcome blindly to introduce a fine variety, transfer kind, avoid kind to obscure important step with mistake; It is the main foundation of correctly evaluating the seed grade, implementing excellent kind of favorable rates policy.The variety check has played huge promotion and guarantee effect to seed work and agricultural production in a word.
In production practices, owing to ignore the evaluation of seed authenticity and variety, often bring irretrievable loss to agricultural production.In producing as hybrid paddy rice, wrong sterile line when the cenospecies sowing, cause that No kernels or seeds are gathered, as in a year of scarcity; Yuexi, Anhui mistake is worked as the Shanyou 63 introduction of plant to the Hua Lian for No. 2 and is caused several hectares of good farmlands total crop failures.
Because reducing, variet complexity, variety can obviously reduce crop yield and quality.Early-maturing variety mixes late-maturing variety, will can not fully ripely influence crop yield because of late-maturing variety; Mix the not seed of disease-resistant variety in the disease-resistant variety, can reduce sowing quality and field plant resistance against diseases; High protein variety is mixed with the low protein variety seed, will reduce its main quality.The seed that variety is not high can cause crop growth inconsistent after broadcasting the field on the other hand, and the plant height differs, and maturation is inequality sooner or later, and field management and mechanical work are brought very big difficulty.Purity reduces more many, and the amplitude that influences output is also more big.Will descend 6%～7% according to surveying hybrid paddy rice breeding field parent (PO) every decline 1% seed farm of purity (F1) purity, land for growing field crops output (commodity food) then descends about 10%.No. 2, cross-bred rape Qin oil causes sterile strain to increase because the easy temperature influence of sterile line Ac often produces self-fertility, and hybrid purity descends, and causes the land for growing field crops to produce the underproduction significantly.
This shows, plant as transferring in each link of seed production, introduce a fine variety, check, seed purchase and business process all should pay attention to variety and identify, and set up the evaluation system of necessity, guarantee the variety and quality of agricultural seed.
Summary of the invention
At the problems referred to above, the invention provides a kind of ultra-thin isoelectric focusing electrophoresis of seed-protein that utilizes and carry out Chinese cabbage cultivar evaluation and the seed purity method of inspection.
The embodiment of the invention is achieved in that a kind of ultra-thin isoelectric focusing electrophoresis technology of seed-protein of utilizing carries out that Chinese cabbage cultivar is identified and the seed purity method of inspection, and described Chinese cabbage cultivar evaluation and the seed purity method of inspection may further comprise the steps:
Protein Extraction: get single seed, pulverize by grain, place centrifuge tube, extract protein;
The preparation gel;
Carry out electrophoretic analysis;
Electrophoresis film after fixing dyes in Coomassie brilliant blue solution;
Gel after the dyeing is placed in the destainer and decolours;
Film after decolouring is dried draws electrophoresis pattern through scanner scanning;
Carry out accuracy relatively.
Further, protein extracting method is: get single seed, pulverize by grain, place centrifuge tube; Every seed of Chinese cabbage adds 60 μ L deionized waters, extracts 1h under the room temperature; After extracting 1h, centrifuge tube is placed on the about 1min of mixing on the vortex mixer, puts into the centrifugal 10min of refrigerated centrifuge 10000rpm then;
Further, the preparation gel method is: (1) takes by weighing 0.08g taurine, 0.8g urea in small beaker; (2) add 5ml gel storage liquid piping and druming dissolving; (3) adding 220 μ L pH scopes is the ampholyte of 5-8; (4) add 30 μ L20% ammonium persulfate solutions; (5) 4 μ L TEMED; (6) mix after with all solution, be added on the film, cover glass cover; A slice 124 * 258mm Gel-Fix mylar (Serva) is launched to be placed on the glass plate, fit tightly for making film and glass plate, can on glass plate, drip some clear water earlier, spread film and drive bubble away with cylinder; Then the about 6.5ml of the gel solution for preparing is poured on film central authorities.To be put on the mylar through the loam cake glass plate that silylating reagent (Repel-Silane ES) handled, slowly put down then, gel is distributed in the space between the lower glass plate.The electrician's adhesive plaster that all posts thickness on rectangular two the long limits of loam cake glass plate and be 0.12mm is with as the interval between the last lower glass plate, thereby the polyacrylamide gel thickness of making is 0.12mm.After placing about 45min under the room temperature, gel polymerisation is finished.
Further, electrophoresis analytical method is: (1) has been put electrode strip and has been put belt transect; (2) with pipettor attract proteins extract 20 μ L, put in the aperture of a belt transect; (3) put sample after, put electrode at electrode filter paper, and push down with a glass plate, contact closely with the electrode paper slip to guarantee electrode.During focusing, temperature is set at 10 ℃, and voltage rises to 600V by 120V in 90min, and behind the 90min, focusing is finished; (4) fixing, the fixing 20min in 12% (W/V) trichloroacetic acid of the film after the focusing.
Further, colouring method is: the 50min that dyes in Coomassie brilliant blue solution of the electrophoresis film after fixing.
Further, discoloration method is: the gel after the dyeing is placed on the 20min that decolours in the destainer; Gel after the decolouring at room temperature dries and spends the night, but seals long preservation with transparent adhesive tape.
Further, the scanning of image method is: the film after decolouring is dried draws electrophoresis pattern through scanner scanning.
Further, the accuracy comparative approach is: random number is got 200 clean seeds from cenospecies 87-114 seed, extracts protein, utilizes the ultra-thin isoelectric focusing electrophoresis technical Analysis of protein seed purity.Random number is got 200 clean seeds and is carried out field planting and identify in addition, detects seed purity.The last ultra-thin isoelectric focusing electrophoresis qualification result of comparison protein and field planting result's consistance.
The invention provides a kind of ultra-thin isoelectric focusing electrophoresis of seed-protein that utilizes and carry out Chinese cabbage cultivar evaluation and the seed purity method of inspection, this method is material with Chinese cabbage cultivar 87-114 and parent's thereof seed, by the ultra-thin isoelectric focusing electrophoresis technical Analysis of seed-protein, successfully obtain 2 maternal specific protein bands and 1 male parent specific protein band, can carry out cultivar identification and the seed purity check of 87-114 cenospecies according to these specific protein bands.By the evaluation of 200 87-114 random populations, qualification result is identified consistent with the field.
Description of drawings
Fig. 1 is that the UTLIEF that the embodiment of the invention provides focuses on synoptic diagram;
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explaining the present invention, and be not used in restriction the present invention.
The invention provides a kind of ultra-thin isoelectric focusing electrophoresis of seed-protein that utilizes and carry out Chinese cabbage cultivar evaluation and the seed purity method of inspection, concrete steps are as follows:
One, instrument and equipment
Isoelectric focusing electrophoresis groove, low-temperature circulating water bath, high pressure bistable electrophoresis apparatus power supply, horizontal shaking table, whirlpool
Vortex mixer, pipettor, glass plate, Gel-Fix mylar
Two, medicine and reagent
Acrylamide, N; N '-methylene diacrylamide, ampholyte, ammonium persulfate, N; N; N ', N '-tetramethylethylenediamine, hydrophobic silane, taurine, urea, sodium chloride, ethylene chlorhydrin, arginine, lysine, ethylenediamine, aspartic acid, glutamic acid, 95% ethanol, acetic acid, trichloroacetic acid
Three, test method
(1) reagent preparation
1. extract: deionized water;
2. gel is store liquid: acrylamide 6.63g, and N, N '-methylene diacrylamide 0.17g, the distilled water dissolving is settled to 100ml.
3.20% ammonium persulfate solution: the dissolving of 0.2g ammonium persulfate adding distil water is settled to 1ml.
4. negative electrode damping fluid: the 0.472g arginine, 0.364%g lysine, the dissolving of 12ml ethylenediamine adding distil water is settled to 100ml.
5. anode electrode damping fluid: the 0.332g aspartic acid, the dissolving of 0.368g glutamic acid adding distil water is settled to 100ml.
6. gel stationary liquid: the dissolving of 12g trichloroacetic acid adding distil water is settled to 300ml.
7. dyeing liquor: contain 0.015% (W/V) coomassie brilliant blue R250,0.045% (W/V) Coomassie brilliant blue G250,11% (V/V) acetic acid, 18% ethanol (V/V) and 71% (V/V) distilled water.
8. destainer: contain 30% (V/V) ethanol, 5% (V/V) acetic acid and 65% (V/V) distilled water
(2) running program
1. Protein Extraction
Get single seed, pulverize by grain, place centrifuge tube.Every seed of Chinese cabbage adds 60 μ L deionized waters, extracts 1h under the room temperature; After extracting 1h, centrifuge tube is placed on the about 1min of mixing on the vortex mixer, puts into the centrifugal 10min of refrigerated centrifuge 10000rpm then.
2. preparing gel
(1) takes by weighing 0.08g taurine, 0.8g urea in small beaker;
(2) add 5ml gel storage liquid piping and druming dissolving.
(3) adding 220 μ L pH scopes is the ampholyte of 5-8;
(4) add 30 μ L20% ammonium persulfate solutions;
(5)4μL?TEMED
(6) mix after with all solution, be added on the film, cover glass cover.
A slice 267 * 193mm Gel-Fix mylar (Serva) is launched to be placed on the glass plate (to fit tightly for making film and glass plate, can on glass plate, drip some clear water earlier, spread film and drive bubble away with cylinder), then the gel solution (about 6.5ml) for preparing is poured on film central authorities.To be put on the mylar through the loam cake glass plate that silylating reagent (Repel-Silane ES) handled, slowly put down then, gel is distributed in the space between the lower glass plate.The electrician's adhesive plaster that all posts thickness on rectangular two the long limits of loam cake glass plate and be 0.12mm is with as the interval between the last lower glass plate, thereby the polyacrylamide gel thickness of making is 0.12mm.After placing about 45min under the room temperature, gel polymerisation is finished.
3. electrophoresis
(1) electrode strip and some belt transect puts
Electrophoresis adopts monofocal, namely respectively puts an electrode paper slip on the gel both sides, on one side the electrode paper slip with cathode buffer liquid soak into, another side electrode paper slip soaks into cathode buffer liquid.To put belt transect (52 hole) then and be placed on the position of anodal 1.5cm, press so that the some belt transect contacts (see figure 1) fully with gel with light finger.
(2) point sample
With pipettor attract proteins extract 20 μ L, put in the aperture of a belt transect.
(3) electrophoresis
After having put sample, put electrode at electrode filter paper, and push down with a glass plate, contact closely with the electrode paper slip to guarantee electrode.During focusing, temperature is set at 10 ℃, and voltage rises to 600V by 120V in 90min.Behind the 90min, focusing is finished.
4, fixing
Film after the focusing fixing 20min in 12% (W/V) trichloroacetic acid.
5, dyeing
Electrophoresis film after the fixing 50min that in Coomassie brilliant blue solution, dyes.
6, decolouring
Gel after the dyeing is placed on the 20min that decolours in the destainer.Gel after the decolouring at room temperature dries and spends the night, but seals long preservation with transparent adhesive tape.
7, scanning of image
Film after decolouring is dried draws electrophoresis pattern through scanner scanning.
8, accuracy relatively
Random number is got 200 clean seeds (by crop seeds inspection procedure GB/T3543-1995 requirement) from cenospecies 87-114 seed, extracts protein, utilizes the ultra-thin isoelectric focusing electrophoresis technical Analysis of protein seed purity.Random number is got 200 clean seeds and is carried out field planting and identify in addition, detects seed purity.The last ultra-thin isoelectric focusing electrophoresis qualification result of comparison protein and field planting result's consistance.
Four, interpretation of result
(1) the ultra-thin isoelectric focusing electrophoresis qualification result of seed-protein
Utilize electrophoretic techniques to judge F
1Whether seed is real hybrid seed, mainly be to see whether it has the characteristic protein matter band of male parent, female parent simultaneously, if any, then be real hybrid seed; Not having the male parent characteristic strip if only have maternal characteristic strip, then is " pseudostationary "; If not only do not have the male parent characteristic strip but also do not have maternal characteristic strip, it then is variet complexity.
2 maternal specific protein bands (FMB1 and FMB2) and 1 male parent specific protein band (MMB1) are found in this research in this combination.By maternal specific protein band and male parent specific protein band, just can carry out cultivar identification and purity check to cabbage hybrid 87-114.
Utilize the ultra-thin isoelectric focusing electrophoresis of seed-protein to identify the purity of 200 87-114 Chinese cabbage seeds, the result is 99%.
(2) field planting qualification result
Proterties such as main cotyledon shape, color and true leaf shape, color, the fine hair according to seedling stage of field planting evaluation has or not, the closed degree of growth evolution degree, the centre of sphere of the leaf-head in heading stage, leaf-head shape, blade obvolvent mode, ball top form, centre of sphere color type are checked the purity of seed.2 results show that the purity of 87-114 Chinese cabbage seeds is 98.5%.
Chinese cabbage
Five, conclusion
The ultra-thin isoelectric focusing electrophoresis technology of this research and utilization seed-protein is carried out Chinese cabbage cultivar evaluation and seed purity check, found that, land for growing field crops qualification result and indoor electrophoresis assay are identical, and illustrate that the ultra-thin isoelectric focusing electrophoresis technical system evaluation Chinese cabbage cultivar authenticity of the protein that utilizes this research to set up and purity result are accurate.
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. one kind is utilized the ultra-thin isoelectric focusing electrophoresis technology of seed-protein to carry out Chinese cabbage cultivar evaluation and the seed purity method of inspection, it is characterized in that described Chinese cabbage cultivar is identified and the seed purity method of inspection may further comprise the steps:
Protein Extraction: get single seed, pulverize by grain, place centrifuge tube, extract protein;
The preparation gel;
Carry out electrophoretic analysis;
Electrophoresis film after fixing dyes in Coomassie brilliant blue solution;
Gel after the dyeing is placed in the destainer and decolours;
Film after decolouring is dried draws electrophoresis pattern through scanner scanning;
Carry out accuracy relatively.
2. Chinese cabbage cultivar as claimed in claim 1 is identified and the seed purity method of inspection, it is characterized in that protein extracting method is: get single seed, pulverize by grain, place centrifuge tube; Every seed of Chinese cabbage adds 60 μ L deionized waters, extracts 1h under the room temperature; After extracting 1h, centrifuge tube is placed on the about 1min of mixing on the vortex mixer, puts into the centrifugal 10min of refrigerated centrifuge 10000rpm then.
3. Chinese cabbage cultivar signing as claimed in claim 1 and the seed purity method of inspection is characterized in that, the preparation gel method is: (1) takes by weighing 0.08g taurine, 0.8g urea in small beaker; (2) add 5ml gel storage liquid piping and druming dissolving; (3) adding 220 μ L pH scopes is the ampholyte of 5-8; (4) add 30 μ L20% ammonium persulfate solutions; (5) 4 μ L TEMED; (6) mix after with all solution, be added on the film, cover glass cover; A slice 124 * 258mm Gel-Fix mylar (Serva) is launched to be placed on the glass plate, fit tightly for making film and glass plate, can on glass plate, drip some clear water earlier, spread film and drive bubble away with cylinder; Then the about 6.5ml of the gel solution for preparing is poured on film central authorities; To be put on the mylar through the loam cake glass plate that silylating reagent (Repel-Silane ES) handled, slowly put down then, gel is distributed in the space between the lower glass plate; The electrician's adhesive plaster that all posts thickness on rectangular two the long limits of loam cake glass plate and be 0.12mm is with as the interval between the last lower glass plate, thereby the polyacrylamide gel thickness of making is 0.12mm; After placing about 45min under the room temperature, gel polymerisation is finished.
4. Chinese cabbage cultivar as claimed in claim 1 is identified and the seed purity method of inspection, it is characterized in that electrophoresis analytical method is: (1) has put electrode strip and some belt transect; (2) with pipettor attract proteins extract 20 μ L, put in the aperture of a belt transect; (3) put sample after, put electrode at electrode filter paper, and push down with a glass plate, contact closely with the electrode paper slip to guarantee electrode; During focusing, temperature is set at 10 ℃, and voltage rises to 600V by 120V in 90min, and behind the 90min, focusing is finished; (4) fixing, the fixing 20min in 12% (W/V) trichloroacetic acid of the film after the focusing.
5. Chinese cabbage cultivar as claimed in claim 1 is identified and the seed purity method of inspection, it is characterized in that colouring method is: the 50min that dyes in Coomassie brilliant blue solution of the electrophoresis film after fixing.
6. Chinese cabbage cultivar as claimed in claim 1 is identified and the seed purity method of inspection, and it is characterized in that discoloration method is: the gel after the dyeing is placed on the 20min that decolours in the destainer; Gel after the decolouring at room temperature dries and spends the night, but seals long preservation with transparent adhesive tape.
7. Chinese cabbage cultivar as claimed in claim 1 is identified and the seed purity method of inspection, it is characterized in that the scanning of image method is: the film after decolouring is dried draws electrophoresis pattern through scanner scanning.
8. Chinese cabbage cultivar as claimed in claim 1 is identified and the seed purity method of inspection, it is characterized in that, the accuracy comparative approach is: random number is got 200 clean seeds from cenospecies 87-114 seed, extracts protein, utilizes the ultra-thin isoelectric focusing electrophoresis technical Analysis of protein seed purity; Random number is got 200 clean seeds and is carried out field planting and identify in addition, detects seed purity; The last ultra-thin isoelectric focusing electrophoresis qualification result of comparison protein and field planting result's consistance.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107860814A (en) * | 2017-12-08 | 2018-03-30 | 丽珠集团丽珠制药厂 | A kind of protein isoelectric focusing electrophoresis method and its with adhesive dispenser |
| CN110554081A (en) * | 2019-10-17 | 2019-12-10 | 东莞太力生物工程有限公司 | Isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein |
-
2013
- 2013-02-28 CN CN201310062681.XA patent/CN103257173B/en not_active Expired - Fee Related
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| 王晓峰等: "超薄等电聚焦电泳技术及其在种子纯度鉴定中的运用", 《种子》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107860814A (en) * | 2017-12-08 | 2018-03-30 | 丽珠集团丽珠制药厂 | A kind of protein isoelectric focusing electrophoresis method and its with adhesive dispenser |
| CN107860814B (en) * | 2017-12-08 | 2023-08-01 | 丽珠集团丽珠制药厂 | Protein isoelectric focusing electrophoresis method and glue distributing device thereof |
| CN110554081A (en) * | 2019-10-17 | 2019-12-10 | 东莞太力生物工程有限公司 | Isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein |
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