Identification method of pea seeds
The variety identification of pea seeds was performed by esterase isoenzyme electrophoresis, and the characteristic bands were used for rapid identification, which solved the problems of high cost and cumbersome process in the existing technology, and achieved rapid and accurate identification of different varieties of pea.
Patent Information
- Application Number
- CN202411897735.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-03-25
AI Technical Summary
The existing pea seed variety identification methods have high cost, cumbersome process, difficult operation, time-consuming and insufficient stability, making it difficult to meet the needs of rapid identification of varieties in seed trade.
Esterase isoenzyme electrophoresis was used to construct an electrophoresis map of the esterase isoenzyme of the pea seeds to be tested, and the characteristic bands of known pea seeds were identified quickly and accurately.
The rapid and accurate variety identification of dialogue peas, green peas, mahjong peas, Canada No. 1 and mahjong peas G2682 has been achieved, reducing costs and operational complexity, and improving identification efficiency and stability.
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Figure CN119688809A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of seed germplasm identification, and specifically to a method for identifying pea seeds. Background Art
[0002] Variety authenticity identification is an important part of seed inspection. Practice has shown that in seed production and management, factors such as biological mixing, mechanical mixing, genetic drift, and labeling errors all change the genetic authenticity and integrity of varieties to varying degrees. As seed production, processing, and circulation enter modernization and commercialization, the importance of pea variety authenticity identification has become more obvious. Therefore, it is necessary to strengthen the variety identification of pea seeds.
[0003] There are two main methods for variety authenticity identification: field planting identification and indoor rapid identification. Field plot planting identification is a phenotypic identification with a long cycle and high cost, which cannot meet the requirements for rapid variety identification in seed trade. Indoor rapid identification usually uses DNA molecular markers and seed storage protein electrophoresis identification. Pea seed variety identification using DNA molecular marker technology is not affected by environmental and time factors, and can be identified at the seed or seedling stage. It also has a large amount of information and can distinguish subtle differences that are difficult to identify with morphological markers. It is accurate and fast, and is an ideal technology for seed variety identification. Although DNA molecular marker identification has many advantages, the generally high cost, cumbersome process, difficult to master operation, relatively long time consumption and insufficient stability are obstacles to the practical application of DNA molecular markers. Seed storage protein electrophoresis identification uses genetic differences between varieties to form protein polymorphisms, and obtains polymorphic protein fingerprints through electrophoresis technology, which is used to identify the authenticity and purity of different varieties, namely protein fingerprint identification technology. The seed storage protein electrophoresis identification method is fast and reliable, and is not restricted by the external environment. However, the expression of germinating seeds, young sprouts or seedlings, and different stages of maturity is unstable, which affects the stability of the electrophoresis pattern. In addition, for genetically similar varieties, it is difficult to find specific proteins because the marker polymorphism of storage proteins is not rich, so it has not been promoted and applied in practice. Summary of the invention
[0004] Based on this, it is necessary for the present application to provide a method for variety identification of pea seeds, which is based on the characteristic bands in the electrophoresis patterns of various pea esterase isozymes and can quickly and accurately identify pea seeds.
[0005] The technical solution includes:
[0006] A method for identifying pea seeds, the method comprising the following steps:
[0007] Constructing the electrophoretic pattern of the esterase isozymes of the pea seeds to be tested;
[0008] Using the characteristic bands in the electrophoresis spectrum of the esterase isozymes of the known pea seeds, the variety of the pea seeds to be tested is determined;
[0009] The known pea seeds are one or more of white peas, green peas, hemp peas, Canada No. 1 and hemp peas G2682;
[0010] In the electrophoresis spectrum, the white pea has characteristic bands of P1, P2, P4 and P5, the green pea has characteristic bands of P1, P2, P3 and P4, the hemp pea has characteristic bands of P2 and P4, the Canada No. 1 has characteristic bands of P1, P2, P3 and P4, and the hemp pea G2682 has characteristic bands of P1, P2 and P4; the P3 characteristic band of Canada No. 1 has a higher intensity than the P3 characteristic band of the green pea;
[0011] The mobility Rf value of the P1 characteristic band is < the mobility Rf value of the P2 characteristic band < the mobility Rf value of the P3 characteristic band < the mobility Rf value of the P4 characteristic band < the mobility Rf value of the P5 characteristic band.
[0012] In one embodiment, the determination basis includes at least one of the following:
[0013] If there are characteristic bands of P1, P2, P4 and P5, the peas to be tested are white peas;
[0014] If there are characteristic bands of P2 and P4, the pea to be tested is pea oleifera;
[0015] If there are characteristic bands of P1, P2 and P4, the pea to be tested is G2682;
[0016] If there are characteristic bands of P1, P2, P3 and P4, the peas to be tested are green peas or Canada No. 1;
[0017] When the peas to be tested are green peas or Canada No. 1, when the intensity of the P3 characteristic band is weaker than or equal to the intensity of the P3 characteristic band in green peas, the peas to be tested are determined to be green peas, otherwise they are determined to be Canada No. 1.
[0018] In one embodiment, the mobility Rf value of the characteristic bands P1 to P5 is 0.4-0.7.
[0019] Optionally, the mobility Rf value of the P1 characteristic band is 0.45, the mobility Rf value of the P2 characteristic band is 0.56, the mobility Rf value of the P3 characteristic band is 0.61, the mobility Rf value of the P4 characteristic band is 0.65, and the mobility Rf value of the P5 characteristic band is 0.69.
[0020] In one embodiment, the construction of the electrophoretic pattern of esterase isozymes of pea seeds includes:
[0021] extracting the esterase isozyme of the pea seeds; and
[0022] The extracted esterase isozymes were subjected to polyacrylamide gel electrophoresis and staining.
[0023] Optionally, the steps of gel preparation, sample preparation and sample spotting are further included before polyacrylamide gel electrophoresis.
[0024] In one embodiment, the polyacrylamide gel comprises a separation gel and a stacking gel;
[0025] Optionally, the separation gel concentration is 10%-15%;
[0026] Optionally, the concentration of the concentrated gel is 3%-5%.
[0027] In one embodiment, the separation gel is prepared by comprising the following raw materials: gel storage solution, separation gel buffer, distilled water, coagulant and coagulant.
[0028] Optionally, the gel storage solution comprises 29.1% by mass of acrylamide and 0.9% by mass of bisacrylamide.
[0029] Optionally, the separation gel buffer comprises 1 mol / L-1.5 mol / L trishydroxyaminomethane-hydrochloric acid, and the pH value is 8.7-8.9.
[0030] Optionally, the volume ratio of the gel storage solution, separation gel buffer, distilled water, coagulant and solidifying agent is (660-1000):500:(470-810):10:1.
[0031] Optionally, the concentration of the separation gel is 12%, and the volume ratio of the gel storage solution, separation gel buffer, distilled water, coagulant and coagulant is 800:500:670:10:1.
[0032] In one embodiment, the concentrated gel is prepared from the following raw materials: gel storage solution, concentrated gel buffer, distilled water, coagulant and coagulant.
[0033] Optionally, the gel storage solution comprises 29.1% by mass of acrylamide and 0.9% by mass of bisacrylamide.
[0034] Optionally, the stacking gel buffer comprises 0.5 mol / L-0.8 mol / L tris(hydroxyaminomethane)-hydrochloric acid, and the pH value is 6.5-6.8.
[0035] Optionally, the concentration of the concentrated gel is 5%, and the volume ratio of the gel storage solution, concentrated gel buffer, distilled water, coagulant and coagulant is 160:250:584:5:1.
[0036] In one embodiment, the extraction of esterase isozymes from pea seeds comprises: mixing bean powder from pea seeds with an extract, grinding on ice, ultrasonically extracting, and collecting esterase isozymes from pea seeds.
[0037] Optionally, the ratio of the mass (g) of the soy powder to the volume (mL) of the extract is 0.5:(1.0-1.5).
[0038] Optionally, the extract comprises 1 mol / L-1.5 mol / L trishydroxyaminomethane-hydrochloric acid, and the pH value is 8.0-8.5.
[0039] In one embodiment, the method for preparing the dyeing solution used for dyeing comprises the steps of dissolving α-naphthyl acetate, β-naphthyl acetate, galactocyanate and acetone, and then diluting with a phosphoric acid solution.
[0040] Optionally, the mass ratio of the α-naphthyl acetate, the β-naphthyl acetate and the chloranthate blue is 1:1:(2-2.5).
[0041] Optionally, the volume ratio of the mass of the α-naphthyl acetate to the dyeing solution is 1:(3-3.5).
[0042] Optionally, in the dyeing solution, the volume fraction of acetone is 3%-4%.
[0043] Optionally, the concentration of the phosphoric acid solution is 0.1 moL / L-0.2 moL / L, and the pH value is 6.0-6.4.
[0044] In one embodiment, the electrode buffer in the polyacrylamide gel electrophoresis comprises tris(hydroxymethyl)aminomethane and glycine, wherein each 1000 mL of 10× electrode buffer comprises 0.60 g-1.0 g of tris(hydroxymethyl)aminomethane and 2.88 g-3 g of glycine; and / or,
[0045] The conditions of the polyacrylamide gel electrophoresis include a voltage of 80V-90V. After the sample to be tested enters the separation gel, the electrophoresis is continued at a voltage of 120V-125V for 2h-3h.
[0046] Compared with the traditional technology, this application has the following beneficial effects:
[0047] The present application uses esterase isozyme electrophoresis to identify white peas, green peas, hemp peas, Canada No. 1, and hemp peas G2682, and analyzes the characteristic bands of each pea seed in the electrophoresis pattern (including the number, position, and intensity of the bands) to quickly and accurately distinguish the five types of peas. BRIEF DESCRIPTION OF THE DRAWINGS
[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.
[0049] Figure 1 Schematic diagram of the electrophoresis patterns of five pea esterase isozymes. DETAILED DESCRIPTION
[0050] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.
[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.
[0052] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.
[0053] Isozymes have obvious species, tissue and developmental stage specificity, and can be used as physiological indicators as well as reliable genetic markers. The isozyme spectrum of plant seeds is the result of long-term natural or artificial hybridization and breeding, and is the product of gene expression. It records the blood relationship of species during development and illustrates the profound genetic differences between varieties, which are not easily affected by environmental conditions. Therefore, isozymes are important markers for identifying genetic differences between varieties, which is of great significance in seed variety identification. As a reliable molecular marker technology, esterase isozyme electrophoresis has the characteristics of being fast, intuitive, sensitive, simple, not easily affected by the external environment, and rich in spectrum types. It makes up for the shortcomings of traditional methods of classification based on species morphology, color and other characteristics. There are abundant esterase isozymes in pea seeds, and the electrophoresis bands have distinct characteristics and can be distinguished from each other. The esterase isozyme electrophoresis method can be used to identify pea seeds. It is crucial to maintain the activity of esterase isozymes during the operation of pea seed esterase isozyme electrophoresis technology.
[0054] An embodiment of the present application provides a method for identifying pea seeds, the method comprising the following steps ab:
[0055] Step a: constructing the electrophoresis pattern of the esterase isozymes of the pea seeds to be tested.
[0056] Step b: using the characteristic bands in the electrophoresis spectrum of the esterase isozymes of known pea seeds to determine the variety of the pea seeds to be tested.
[0057] In a specific example, the pea seeds are known to be one or more of white peas, green peas, hemp peas, Canada No. 1, and hemp peas G2682.
[0058] In a specific example, in the electrophoresis spectrum, white peas have characteristic bands of P1, P2, P4 and P5, green peas have characteristic bands of P1, P2, P3 and P4, hemp peas have characteristic bands of P2 and P4, Canada No. 1 has characteristic bands of P1, P2, P3 and P4, and hemp peas G2682 have characteristic bands of P1, P2 and P4; the intensity of the characteristic band P3 of Canada No. 1 is higher than that of the characteristic band P3 of the green peas.
[0059] In a specific example, the mobility Rf value of the P1 characteristic band < the mobility Rf value of the P2 characteristic band < the mobility Rf value of the P3 characteristic band < the mobility Rf value of the P4 characteristic band < the mobility Rf value of the P5 characteristic band.
[0060] In a specific example, the electrophoresis pattern of the esterase isozymes of known pea seeds is established in the same way as the electrophoresis pattern of the esterase isozymes of the pea seeds to be tested, and the electrophoresis patterns of the above-mentioned 5 known pea seeds are obtained. The electrophoresis bands of the above-mentioned 5 pea seeds are compared to find out the band characteristics of different samples, and make "fingerprints" of different tested samples to identify the tested pea seed varieties.
[0061] In a specific example, the judgment basis includes at least one of the following:
[0062] If there are characteristic bands of P1, P2, P4 and P5, the peas to be tested are white peas;
[0063] If there are characteristic bands of P2 and P4, the pea to be tested is pea oleifera;
[0064] If there are characteristic bands of P1, P2 and P4, the pea to be tested is G2682;
[0065] If there are characteristic bands of P1, P2, P3 and P4, the peas to be tested are green peas or Canada No. 1;
[0066] When the peas to be tested are green peas or Canada No. 1, when the intensity of the P3 characteristic band is lower than or equal to the intensity of the P3 characteristic band in green peas, the peas to be tested are determined to be green peas, otherwise they are determined to be Canada No. 1 (or when the intensity of the P3 characteristic band is higher than or equal to the intensity of the P3 characteristic band in Canada No. 1, the peas to be tested are determined to be Canada No. 1).
[0067] In a specific example, the mobility Rf value of the characteristic bands P1 to P5 is 0.4-0.7.
[0068] In a specific example, the mobility Rf value of the P1 characteristic band is 0.45, the mobility Rf value of the P2 characteristic band is 0.56, the mobility Rf value of the P3 characteristic band is 0.61, the mobility Rf value of the P4 characteristic band is 0.65, and the mobility Rf value of the P5 characteristic band is 0.69.
[0069] In a specific example, the construction of the electrophoresis pattern of esterase isozymes of pea seeds includes the following steps S10-S20:
[0070] Step S10: extracting the esterase isozymes of the pea seeds.
[0071] In a specific example, the step of extracting esterase isozymes from pea seeds includes: mixing bean powder from pea seeds with an extracting solution, grinding on ice, ultrasonic extraction, and collecting esterase isozymes from pea seeds.
[0072] In a specific example, the ratio of bean powder mass (g) to the volume (mL) of the extract is 0.5:(1.0-1.5); preferably, the ratio of bean powder mass (g) to the volume (mL) of the extract is 0.5:1.5. The electrophoresis result of the esterase isozyme extracted at this ratio is the best. Too much or too little bean powder and extract will affect the extraction of esterase isozymes.
[0073] In a specific example, the extracting solution includes 1 mol / L-1.5 mol / L tris(hydroxyaminomethane)-hydrochloric acid solution, and the pH value is 8.0-8.5. Preferably, the extracting solution includes 1 mol / L tris(hydroxyaminomethane)-hydrochloric acid, and the pH value is 8.0.
[0074] Specifically, 0.5 g of soybean powder from different samples were taken, 1.5 mL of 1 mol / L trishydroxyaminomethane (Tris)-hydrochloric acid (HCl) extract with a pH value of 8.0 was added respectively, the samples were fully ground in an ice-water bath, ultrasonically extracted for 30 min, centrifuged at 10,000 r / min and 4°C for 15 min, the supernatant was taken to obtain the esterase isozyme solution, and stored in a refrigerator at 0°C to 4°C.
[0075] Step S20: performing polyacrylamide gel electrophoresis and staining on the obtained esterase isozymes.
[0076] In a specific example, the supporting medium of polyacrylamide gel electrophoresis is composed of a separation gel and a concentrated gel. Optionally, the concentration of the separation gel is 10%-15%, preferably, the concentration of the separation gel is 12%. Preferably, the concentration of the concentrated gel is 3%-5%, and the concentration of the concentrated gel is 5%. When the concentration of the separation gel is around 12%, the separation effect is better; when the concentration of the separation gel is less than 10%, the enzyme band is dragged and the stained enzyme band is less; when the concentration of the separation gel is greater than 15%, the gel is relatively brittle, and the gel is easily broken during the separation, staining and photosensitive process of the gel from the glass plate.
[0077] For example, the concentration of the separation gel is 12%. Usually, 12% refers to the percentage of the total mass of acrylamide and methylenebisacrylamide to the total mass of the gel solution.
[0078] In a specific example, the separation gel is prepared by including the following raw materials: gel storage solution, separation gel buffer, distilled water, coagulant and coagulant.
[0079] In a specific example, the gel stock solution includes 29.1% by mass of acrylamide and 0.9% by mass of bisacrylamide.
[0080] In a specific example, the separation gel buffer comprises 1 mol / L-1.5 mol / L tris(hydroxyaminomethane)-hydrochloric acid, and the pH value is 8.7-8.9. Preferably, the separation gel buffer comprises 1.5 mol / L tris(hydroxyaminomethane)-hydrochloric acid, and the pH value is 8.9.
[0081] In a specific example, the volume ratio of the gel storage solution, separation gel buffer, distilled water, coagulant and coagulant is (660-1000): 500: (470-810): 10: 1. Preferably, the concentration of the separation gel is 12%, and the volume ratio of the gel storage solution, separation gel buffer, distilled water, coagulant and coagulant is 800: 500: 670: 10: 1.
[0082] In a specific example, the concentrated gel is prepared from the following raw materials: gel storage solution, concentrated gel buffer, distilled water, coagulant and coagulant. In a specific example, the gel storage solution includes 29.1% by mass of acrylamide and 0.9% by mass of bisacrylamide.
[0083] In a specific example, the concentration of the stacking gel is 5%, and the volume ratio of the gel storage solution, the stacking gel buffer, the distilled water, the coagulant and the coagulant is 160:250:584:5:1.
[0084] In a specific example, the stacking gel buffer comprises 0.5 mol / L-0.8 mol / L tris(hydroxyaminomethane)-hydrochloric acid, and the pH value is 6.5-6.8. Preferably, the stacking gel buffer comprises 0.5 mol / L tris(hydroxyaminomethane)-hydrochloric acid, and the pH value is 6.8.
[0085] In one specific example, the coagulant includes 10% ammonium persulfate and the coagulant includes tetramethylethylenediamine.
[0086] In a specific example, the polyacrylamide gel electrophoresis further includes gel preparation, sample preparation and sample spotting steps.
[0087] In a specific example, the gel preparation includes the step of injecting separation gel and concentration gel into an empty gel plate. In a specific example, the volume ratio of separation gel to concentration gel is 2:1.
[0088] Specifically, 4 mL of separation gel was injected, followed by distilled water. After the separation gel solidified, the distilled water was discarded, the distilled water between the glass plates was absorbed with a filter paper strip, and then 2 mL of concentrated gel was injected, and the sample comb was inserted.
[0089] In a specific example, the sample preparation includes mixing the esterase isoenzyme solution and the indicator (such as bromophenol blue) in a volume ratio of 4:1 to prepare a loading buffer.
[0090] In one specific example, the bromophenol blue indicator includes 0.25% bromophenol blue and 40% sucrose.
[0091] In a specific example, spotting includes removing the sample comb after the concentrated gel solidifies, adding electrode buffer to the electrophoresis tank, placing the electrophoresis tank at 0°C-4°C, and spotting 15μL-20μL (preferably 20μL) of loading buffer into each well.
[0092] In a specific example, the electrode buffer comprises tris and glycine, wherein each 1000 mL of 10× electrode buffer comprises 0.60 g-1.0 g of tris and 2.88 g-3 g of glycine. Preferably, each 1000 mL of 10× electrode buffer comprises 0.60 g of tris and 2.88 g of glycine.
[0093] In a specific example, electrophoresis is performed at a voltage of 80 V to 90 V, and after the indicator (e.g., bromophenol blue) enters the separation gel, electrophoresis is continued at a voltage of 120 V to 125 V. Preferably, electrophoresis is performed at a voltage of 80 V, and after the indicator (e.g., bromophenol blue) enters the separation gel, electrophoresis is continued at a voltage of 120 V for 2 h to 3 h.
[0094] In a specific example, the staining step includes ending the electrophoresis when the indicator reaches about 1 cm from the bottom of the gel plate, removing the gel plate, placing it in a staining solution, staining at a constant temperature of 36°C-38°C (e.g., 37°C) until the bands are clear, rinsing with water, and obtaining the sample electrophoresis bands.
[0095] In a specific example, the method for preparing the dyeing solution includes the steps of dissolving α-naphthyl acetate, β-naphthyl acetate, galactocyanate and acetone, and then diluting with a phosphoric acid solution.
[0096] In a specific example, the mass ratio of α-naphthyl acetate, β-naphthyl acetate and Caesalpinia blue is 1:1:(2-2.5). Preferably, the mass ratio of α-naphthyl acetate, β-naphthyl acetate and Caesalpinia blue is 1:1:2.
[0097] In a specific example, the mass ratio of α-naphthyl acetate to the volume ratio of the dyeing liquid is 1:(3-3.5). Preferably, the mass ratio of α-naphthyl acetate to the volume ratio of the dyeing liquid is 1:3.
[0098] In a specific example, in the dyeing solution, the volume fraction of acetone is 3%-4%.
[0099] Specifically, the preparation method of the dyeing solution used for dyeing includes: dissolving 50 mg of α-naphthyl acetate, 50 mg of β-naphthyl acetate and 100 mg of Janus blue with about 5 mL of acetone, and then diluting to 150 mL with phosphoric acid solution.
[0100] In a specific example, the concentration of the phosphoric acid solution is 0.1 mol / L-0.2 mol / L, and the pH value is 6.0-6.4. Preferably, the concentration of the phosphoric acid solution is 0.1 mol / L, and the pH value is 6.4.
[0101] In a specific example, the Cinnamon Blue is Cinnamon Blue RR or Cinnamon Blue B.
[0102] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.
[0103] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.
[0104] Example 1
[0105] This embodiment provides a method for identifying the variety of pea seeds using esterase isozyme electrophoresis, comprising the following steps:
[0106] 1) Extraction of sample esterase isozymes: dry seeds of white pea, green pea, hemp pea, Canada No. 1 and hemp pea G2682 (white pea, green pea, hemp pea and Canada No. 1 were purchased from the retail market, and hemp pea G2682 was provided by the Seed Resource Bank of the Chinese Academy of Agricultural Sciences. Canada No. 1 is a kind of hemp pea. Peas with brown spots on the surface of peas are collectively referred to as hemp peas. There is no clear distinction between species at present. Ordinary hemp peas and hemp peas with the name Canada No. 1 were purchased from the retail market. Hemp pea G2682 has no clear species, but is provided by the Seed Resource Bank of the Chinese Academy of Agricultural Sciences, with the code G2682) were selected, and the pea seeds were crushed into powder by a grinder, and passed through a 60-mesh sieve to obtain bean powder of different varieties of peas. Take 0.5 g of each sample of soybean powder, add 1.5 mL of 1 mol / L trishydroxyaminomethane (Tris)-hydrochloric acid (HCl) extract at pH 8.0, grind thoroughly in an ice-water bath, extract with ultrasound (frequency 40 kHz, temperature 4°C) for 30 min, centrifuge at 10000 r / min, 4°C for 15 min, take the supernatant, obtain the esterase isozyme solution, and store it in a refrigerator at 0°C-4°C.
[0107] 2) Assemble the electrophoresis tank, check for leaks, and inject glue: Use the plastic sealing strips that match the DYY-6C electrophoresis device to seal the vertical plate electrophoresis tank glass plates; fill the space between the glass plates with distilled water, let it stand for a few minutes, and observe whether the liquid level drops. If the liquid level does not change, the electrophoresis tank is successfully assembled. Discard the distilled water, use filter paper strips to absorb the distilled water between the glass plates, and inject 4mL of 12% separation gel (note that there should be no bubbles during the injection process). After the separation gel is injected, slowly fill the space between the glass plates with distilled water. After the separation gel solidifies, discard the distilled water. At this time, the separation gel interface is a flat straight line. Next, use filter paper strips to absorb the remaining water between the glass plates (note that the separation gel should not be touched during the water absorption process). Finally, inject 2mL of 5% concentrated gel (note that there should be no bubbles during the injection process). After the concentrated gel is injected, insert the sample comb immediately.
[0108] Among them, 12% separation gel is made by mixing 2.0mL gel storage solution, 1.25mL separation gel buffer, 1.675mL water, 25μL 10% ammonium persulfate, and 2.5μL tetramethylethylenediamine. The volume ratio of each component is gel storage solution: separation gel buffer: distilled water: 10% ammonium persulfate: tetramethylethylenediamine = 800:500:670:10:1; the gel storage solution is 29.1% acrylamide (Acr) + 0.9% bisacrylamide (Bis); the separation gel buffer is 1.5 mol / L trihydroxyaminomethane (Tris)-hydrochloric acid (HCl) with a pH of 8.9.
[0109] Among them, 5% stacking gel is made by mixing 0.4mL gel storage solution, 0.625mL stacking gel buffer, 1.46mL water, 12.5μL 10% ammonium persulfate, and 2.5μL tetramethylethylenediamine. The volume ratio of each component is gel storage solution: stacking gel buffer: distilled water: 10% ammonium persulfate: tetramethylethylenediamine = 160:250:584:5:1; the gel storage solution is 29.1% acrylamide (Acr) + 0.9% bisacrylamide (Bis), and the stacking gel buffer is 0.5mol / L trihydroxyaminomethane (Tris)-hydrochloric acid (HCl) with a pH of 6.8.
[0110] 3) Sample preparation: Esterase isozyme solution and bromophenol blue indicator (0.25% bromophenol blue + 40% sucrose) are mixed in a volume ratio of 4:1 to prepare a loading buffer. When spotting, 20 μL of loading buffer is required, that is, 16 μL of esterase isozyme preparation solution and 4 μL of bromophenol blue indicator are mixed to prepare the loading buffer.
[0111] 4) Sample loading: After the concentrated gel solidifies (about 30 min), remove the sample comb, pour in the electrode buffer (0.60 g of tris(hydroxymethyl)aminomethane + 2.88 g of glycine, dissolved in distilled water and fixed to 1000 mL, pH 8.3), place the electrophoresis tank at 4°C, and load 20 μL of sample buffer into each well.
[0112] 5) Electrophoresis: After spotting, turn on the power supply and adjust the voltage to 80V. After the indicator (bromophenol blue) enters the separation gel, run electrophoresis at a constant voltage of 120 V for about 2.5 hours.
[0113] 6) Staining and result observation: End the electrophoresis when the indicator moves to about 1 cm from the bottom of the gel plate. Remove the gel plate and place it in esterase isozyme staining solution (50 mg of α-naphthyl acetate + 50 mg of β-naphthyl acetate + 100 mg of Janus Blue RR or Janus Blue B, first dissolved in about 5 mL of acetone, then diluted to 150 mL with pH 6.4, 0.1 mol / L phosphoric acid solution). Stain at 37°C until the bands are clear. After washing with water, obtain the sample electrophoresis bands.
[0114] 7) Identification of the tested pea seed varieties: The electrophoresis bands of the tested samples are obtained by the above method, and the electrophoresis bands of the tested samples are compared to find out the differences in the bands of different tested samples, which are regarded as the "fingerprints" of different tested samples and used to identify the tested pea seed varieties.
[0115] The esterase isozyme electrophoresis method was used to identify the varieties of white peas, green peas, hemp peas, Canada No. 1, and hemp peas G2682. There were significant differences in the electrophoresis bands of the five samples, among which the common bands P2 and P4 could be used as characteristic identification bands; white peas had four bands, among which band P5 was unique to white peas and could be used as an identification band; hemp peas had two bands, which could be distinguished from other varieties; hemp peas G2682 had three bands, which could be distinguished from other varieties; green peas and Canada No. 1 both had four bands, which could be distinguished from several other peas, green peas P3 was a weak band, and Canada No. 1 P3 was a second-strong band, and they could be distinguished from each other through the P3 band. The band patterns (number, position, intensity) of the pea esterase isozyme electrophoresis patterns were able to distinguish the five peas (Table 1, Figure 1 ).
[0116] Table 1 Rf values of electrophoresis bands of five pea esterase isozymes
[0117]
[0118] Note: Rf = the position ratio of each band in the lane to the front of bromophenol blue.
[0119] Example 2
[0120] The difference from Example 1 is that the separation gel concentration is 7.5%, and its formula is shown in Table 2.
[0121] Example 3
[0122] The difference from Example 1 is that the separation gel concentration is 10%, and its formula is shown in Table 2.
[0123] Example 4
[0124] The difference from Example 1 is that the separation gel concentration is 12%, and its formula is shown in Table 2.
[0125] Example 5
[0126] The difference from Example 1 is that the separation gel concentration is 15%, and its formula is shown in Table 2.
[0127] Table 2
[0128]
[0129] The effects of different separation gel concentrations on the electrophoresis results of pea esterase isozymes in Examples 2 to 5 are shown in Table 3. From the perspective of enzyme bands and staining effects, the electrophoresis results of 12% separation gel and 15% separation gel concentrations are better, the enzyme bands are not dragged and there are more dyed enzyme bands. However, the gel prepared by 15% separation gel concentration is relatively brittle, and the gel is easily broken during the separation, staining and photosensitive process of the gel from the glass plate. Therefore, when the separation gel concentration is 12%, the pea esterase isozyme enzyme bands are clearly discernible, and the separation effect is better.
[0130] Table 2 Effect of separation gel concentration on electrophoresis of pea esterase isozymes
[0131]
[0132] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0133] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.
Claims
1. A method for identifying pea seeds, characterized in that: The identification method comprises the following steps: Constructing the electrophoresis pattern of the esterase isozymes of the pea seeds to be tested; Using the characteristic bands in the electrophoresis spectrum of the esterase isozymes of the known pea seeds, the variety of the pea seeds to be tested is determined; The known pea seeds are one or more of white peas, green peas, hemp peas, Canada No. 1 and hemp peas G2682; In the electrophoresis spectrum, the white pea has characteristic bands of P1, P2, P4 and P5, the green pea has characteristic bands of P1, P2, P3 and P4, the hemp pea has characteristic bands of P2 and P4, the Canada No. 1 has characteristic bands of P1, P2, P3 and P4, and the hemp pea G2682 has characteristic bands of P1, P2 and P4; the P3 characteristic band of Canada No. 1 has a higher intensity than the P3 characteristic band of the green pea; The mobility Rf value of the P1 characteristic band is < the mobility Rf value of the P2 characteristic band < the mobility Rf value of the P3 characteristic band < the mobility Rf value of the P4 characteristic band < the mobility Rf value of the P5 characteristic band.
2. The identification method according to claim 1, characterized in that: The judgment basis includes at least one of the following: If there are characteristic bands of P1, P2, P4 and P5, the peas to be tested are white peas; If there are characteristic bands of P2 and P4, the pea to be tested is pea oleifera; If there are characteristic bands of P1, P2 and P4, the pea to be tested is G2682; If there are characteristic bands of P1, P2, P3 and P4, the peas to be tested are green peas or Canada No. 1; When the peas to be tested are green peas or Canada No. 1, when the P3 characteristic band intensity is lower than or equal to the P3 characteristic band intensity in green peas, the peas to be tested are determined to be green peas, otherwise they are determined to be Canada No.
1.
3. The identification method according to claim 1 or 2, characterized in that: The mobility Rf values of the characteristic bands from P1 to P5 are 0.4-0.7; Optionally, the mobility Rf value of the P1 characteristic band is 0.45, the mobility Rf value of the P2 characteristic band is 0.56, the mobility Rf value of the P3 characteristic band is 0.61, the mobility Rf value of the P4 characteristic band is 0.65, and the mobility Rf value of the P5 characteristic band is 0.
69.
4. The identification method according to claim 1 or 2, characterized in that: The construction of the electrophoretic pattern of esterase isozymes of pea seeds includes: extracting the esterase isozyme of the pea seeds; and The extracted esterase isozymes were subjected to polyacrylamide gel electrophoresis and staining; Optionally, the steps of gel preparation, sample preparation and sample spotting are further included before polyacrylamide gel electrophoresis.
5. The identification method according to claim 4, characterized in that: Polyacrylamide gels include separation gels and stacking gels; Optionally, the concentration of the separation gel is 10%-15%; Optionally, the concentration of the concentrated gel is 3%-5%.
6. The identification method according to claim 5, characterized in that: The separation gel is prepared from the following raw materials: gel storage solution, separation gel buffer, distilled water, coagulant and coagulant; Optionally, the gel storage solution comprises 29.1% by mass of acrylamide and 0.9% by mass of bisacrylamide; Optionally, the separation gel buffer comprises 1 mol / L-1.5 mol / L tris(hydroxyaminomethane)-hydrochloric acid, with a pH value of 8.7-8.9; Optionally, the volume ratio of the gel storage solution, separation gel buffer, distilled water, coagulant and coagulant is (660-1000):500:(470-810):10:1; Optionally, the concentration of the separation gel is 12%, and the volume ratio of the gel storage solution, separation gel buffer, distilled water, coagulant and coagulant is 800:500:670:10:
1.
7. The identification method according to claim 5, characterized in that: The concentrated gel is prepared from the following raw materials: gel storage solution, concentrated gel buffer, distilled water, coagulant and coagulant; Optionally, the gel storage solution comprises 29.1% by mass of acrylamide and 0.9% by mass of bisacrylamide; Optionally, the stacking gel buffer comprises 0.5 mol / L-0.8 mol / L tris(hydroxyaminomethane)-hydrochloric acid, with a pH value of 6.5-6.8; Optionally, the concentration of the concentrated gel is 5%, and the volume ratio of the gel storage solution, concentrated gel buffer, distilled water, coagulant and coagulant is 160:250:584:5:
1.
8. The identification method according to claim 4, characterized in that: The extraction of esterase isozymes from pea seeds comprises: mixing bean powder from pea seeds with an extracting solution, grinding on ice, ultrasonically extracting, and collecting esterase isozymes from pea seeds; Optionally, the ratio of the mass (g) of the soy powder to the volume (mL) of the extract is 0.5:(1.0-1.5); Optionally, the extract comprises 1 mol / L-1.5 mol / L trishydroxyaminomethane-hydrochloric acid, and the pH value is 8.0-8.
5.
9. The identification method according to claim 4, characterized in that: The preparation method of the dyeing solution used in the dyeing comprises the steps of: dissolving α-naphthyl acetate, β-naphthyl acetate, janodine blue and acetone, and then diluting with a phosphoric acid solution; Optionally, the mass ratio of the α-naphthyl acetate, the β-naphthyl acetate and the caesium blue is 1:1:(2-2.5); Optionally, the mass ratio of the α-naphthyl acetate to the volume ratio of the dyeing solution is 1:(3-3.5); Optionally, in the dyeing solution, the volume fraction of acetone is 3%-4%; Optionally, the concentration of the phosphoric acid solution is 0.1 moL / L-0.2 moL / L, and the pH value is 6.0-6.
4.
10. The identification method according to claim 4, characterized in that: The electrode buffer in the polyacrylamide gel electrophoresis comprises tris(hydroxymethyl)aminomethane and glycine, wherein each 1000 mL of 10× electrode buffer comprises 0.60 g-1.0 g of tris(hydroxymethyl)aminomethane and 2.88 g-3 g of glycine; and / or, The conditions of the polyacrylamide gel electrophoresis include a voltage of 80V-90V. After the sample to be tested enters the separation gel, the electrophoresis is continued at a voltage of 120V-125V for 2h-3h.