CN108918638B - Test method for influence of long-term application of high-low nitrogen fertilizer on albumin of corn grains - Google Patents
Test method for influence of long-term application of high-low nitrogen fertilizer on albumin of corn grains Download PDFInfo
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Abstract
The invention discloses a test method for the influence of long-term application of high and low nitrogen fertilizers on corn kernel albumin, which researches the influence of long-term application of high and low nitrogen fertilizers on corn kernel albumin of Luyu 16 in summer by a proteomics method. The results showed that the albumin content in the control group was higher than in the two treatment groups and a distinct band was seen in the quantitative one-way SDS-PAGE. The two-dimensional electrophoresis result shows that the long-term low-nitrogen treatment has 7 different protein points, 1 up-regulation and 6 down-regulation compared with the control group; the long-term high nitrogen treatment had 6 differential protein spots, 1 up-regulation and 5 down-regulation compared to the control group. Through mass spectrum identification and functional classification, the protein and amino acid metabolism related protein, the energy metabolism related protein, the expression regulation and defense stress resistance related protein, the cell metabolism related protein and the function unknown protein are respectively identified.
Description
Technical Field
The invention belongs to the technical field of agriculture, and particularly relates to a test method for influence of long-term application of high and low nitrogen fertilizers on albumin of corn kernels.
Background
With the continuous improvement of the living standard of people, the requirements on the quality of crop products are higher and higher, the quality of agricultural products has a direct relation with external environmental factors, such as temperature, illumination, nutrients, moisture, soil and the like, and fertilizers are main nutrient supply sources in modern agricultural production, directly participate in coordinating nutrient metabolism and circulation, and inevitably greatly restrict the yield and the quality of crops. In recent years, nitrogen fertilizers have been researched more on corn kernel protein, most of the researches are the results of kernel protein content change and relatively short-term experiments, and no further researches are carried out, and the researches have no continuity on soil fertility, fertility and the like. The corn seed albumin is one of 4 main protein components of corn, and the corn material with different genetic backgrounds shows different protein bands through electrophoretic analysis, the band difference reflects the genotype difference to a certain extent, and the albumin bands of different seed grains of the same hybrid or inbred line are analyzed, so that whether genetic divergence exists among individuals can be seen, and the purity is checked. The research of utilizing protein electrophoresis technology to identify the purity of corn seeds has been reported. However, the research on the proteomics of the summer corn kernel albumin under the condition of long-term positioning fertilization is still blank.
Disclosure of Invention
In order to make up for the blank in the prior art, the invention provides a test method for the influence of long-term application of high and low nitrogen fertilizers on the albumin in corn kernels, which is based on a long-term positioning fertilization test of 33 years on non-calcareous moist soil, researches the influence of long-term application of high and low nitrogen fertilizers on the albumin in corn kernels, and provides scientific basis for scientific application of nitrogen fertilizers and genetic improvement of quality.
The technical scheme is as follows:
a test method for the influence of long-term application of high and low nitrogen fertilizers on the albumin of corn kernels comprises the following steps:
step 1, experimental design
The soil to be tested is non-calcareous moist soil, develops in alluvial matrix, is light in surface soil texture, has the organic matter content of 4.10g/kg, the total nitrogen content of 0.50g/kg, the total phosphorus content of 0.46g/kg, the available phosphorus content of 15mg/kg, the quick-acting potassium content of 38mg/kg and the cation replacement amount of 11.80 cmol/kg. The experiment was set up with 3 treatments: control, low nitrogen, high nitrogen, 3 replicates per treatment; the winter wheat-summer corn crop rotation is carried out in a test field, the crop is cultivated twice a year, the summer corn variety is Luyu 16, and the annual application amount of the high-nitrogen fertilizer is 276kg/hm2The annual application amount of the low-nitrogen fertilizer is 138kg/hm2。
2.1 extraction and determination of Kernel Albumin
Adding 2ml of water into 0.5g of sample, grinding to homogenate, adding 3ml of water, transferring to a centrifuge tube, washing a mortar with 3ml of water, oscillating for 5min, centrifuging at 4500r/min for 8min, pouring out supernatant, adding 8ml of water, stirring, oscillating for 5min, centrifuging, combining supernatants, adding water for 4 times, and fixing the volume to 50 ml. Respectively sucking 1ml of each sample solution, adding 5ml of Coomassie brilliant blue, mixing well, standing for 2min, and performing color comparison at 595 nm.
2.2 SDS-PAGE of Kernel Albumin
The electrophoresis device is a DYZC-24B type electrophoresis apparatus, and the thickness of the plate glue is 1 mm. The concentration of the separation gel is 12 percent, and the concentration of the concentrated gel is 5 percent. A200. mu.g sample was added to the wells. The voltage is 100V at the beginning, and after the bromophenol blue completely enters the concentrated gel interface, the electrophoresis is changed to 120V constant voltage electrophoresis till the end. After electrophoresis, stained with Coomassie brilliant blue, destained and scanned for imaging.
2.3 purification and quantification of Kernel Albumin
Precipitating the albumin extractive solution with acetone, centrifuging at 12000r/min for 1 hr, and dissolving with lysis solution. After purifying the protein with the 2-DClean-Up Kit, the protein was dissolved in an aqueous solution. Quantification was performed by the Bradford method using bovine serum albumin as a standard protein.
2.4 2-DE of Kernel Albumin
Taking the protein extract containing 200 mug protein, supplementing the volume to 450 mug protein by sample hydration buffer solution, covering a solid phase pH gradient linear adhesive tape with the length of 24cm and the pH value of 4-7 on the protein solution, passively hydrating for 17h, then performing isoelectric focusing, wherein the temperature is 20 ℃, the temperature is 100V, the time is 1h, the time is 300V, the time is 1h, the time is 500V, the time is 1h, the time is 1000V, the time is 2h, the time is 8000V, and the time is 10h, after the focusing is finished, respectively balancing the adhesive tape by adhesive tape balance buffer solution containing 1% DTT and 2.5% iodoacetamide for 15min, after the balancing is finished, closely attaching the adhesive tape on 12% polyacrylamide gel, performing second electrophoresis, dyeing the gel by adopting an improved silver staining method after the electrophoresis is finished, then scanning the image by a UMAX Powerlook2100X L scanner, analyzing the obtained image by a Mastera 6 analysis software, repeating the mass spectrometry, selecting the representative differential protein with the mass spectrum, and sending the protein to a Shenhua services Daji scientific and Zhenzhui.
Step 3, data processing
The data were statistically analyzed using DPS 9.50 and Microsoft Word 2003 tabulated.
Further, in the step 2.3, the lysis solution is 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 40 mmol/L DTT, 0.2% (W/V) IPG-Bufffer and has a pH value of 4-7.
Further, in the step 2.3, the hydration solution is 8 mol/L urea, 2% CHAPS, 20 mmol/L DTT, 0.5% IPG-Bufffer and has the pH value of 4-7.
Further, in step 2.4, the loading hydration buffer solution is hydration solution +0.002% bromophenol blue +0.012% Destreak Reagent.
Further, in step 2.4, the gel strip equilibration buffer of 2.5% iodoacetamide was 6 mol/L urea, 2% SDS, 0.075 mol/L Tris-HCl pH 8.8, 30% glycerol, 0.002% bromophenol blue.
Further, in step 2.4, in the second-direction electrophoresis, the time for each adhesive tape to run for 3W is 24-27 h.
The invention has the beneficial effects that:
the test results of the invention show that the albumin content of the control group is higher than that of the two treatment groups, and obvious difference bands can be seen in quantitative one-way SDS-PAGE. The two-dimensional electrophoresis result shows that the long-term low-nitrogen treatment has 7 different protein points, 1 up-regulation and 6 down-regulation compared with the control group; the long-term high nitrogen treatment had 6 differential protein spots, 1 up-regulation and 5 down-regulation compared to the control group. Through mass spectrum identification and functional classification, the protein and amino acid metabolism related protein, the energy metabolism related protein, the expression regulation and defense stress resistance related protein, the cell metabolism related protein and the function unknown protein are respectively identified.
Drawings
FIG. 1 is a one-way SDS-PAGE profile of different processed corn kernel albumins;
fig. 2 is a graph of the effect of long-term localized fertilization on summer corn kernel albumin, wherein fig. 2A: a two-dimensional electrophoresis chart of albumin of a control group for long-term positioning fertilization; FIG. 2B: positioning a two-dimensional electrophoresis chart of the albumin in the low-nitrogen fertilization for a long time; FIG. 2C: and (3) positioning a two-dimensional electrophoresis chart of the high-nitrogen fertilization albumin for a long time.
Detailed Description
The technical solutions of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.
1 materials and methods
1.1 design of the experiment
The test was set up at the leiyang test station of Qingdao agricultural university, beginning in 1978. The tested soil is non-calcareous moist soil, develops in alluvial mother soil, has light surface soil texture, 4.10g/kg of organic matter content, 0.50g/kg of total nitrogen content and total phosphorus (P)2O5) The amount of the fertilizer is 0.46g/kg, and the soil available phosphorus (P)2O5) 15mg/kg, quick-acting potassium (K) in soil2O) was 38mg/kg, and the soil cation exchange amount was 11.80 cmol/kg. The experiment was set up with 3 treatments (control, low nitrogen, high nitrogen) each set up with 3 replicates. The crop rotation of winter wheat and summer corn is carried out in a test field, the crop is cultivated twice every year, and the variety of the summer corn is Luyu 16. The annual application amount of the high-quantity nitrogen fertilizer is 276kg/hm2The annual application amount of the low-nitrogen fertilizer is 138kg/hm2。
1.2 items and methods of measurement
1.2.1 extraction and determination of Kernel Albumin
Adding 2ml of water into 0.5g of sample, grinding to homogenate, adding 3ml of water, transferring to a centrifuge tube, washing a mortar with 3ml of water, oscillating for 5min, centrifuging at 4500r/min for 8min, pouring out supernatant, adding 8ml of water, stirring, oscillating for 5min, centrifuging, combining supernatants, adding water for 4 times, and fixing the volume to 50 ml. Respectively sucking 1ml of each sample solution, adding 5ml of Coomassie brilliant blue, mixing well, standing for 2min, and performing color comparison at 595 nm.
1.2.2 SDS-PAGE of Kernel Albumin
The electrophoresis apparatus is a DYZC-24B electrophoresis apparatus (six instruments factories in Beijing), and the thickness of the plate gel is 1 mm. The concentration of the separation gel is 12 percent, and the concentration of the concentrated gel is 5 percent. A200. mu.g sample was added to the wells. The voltage is 100V at the beginning, and after the bromophenol blue completely enters the concentrated gel interface, the electrophoresis is changed to 120V constant voltage electrophoresis till the end. After electrophoresis, stained with Coomassie brilliant blue, destained and scanned for imaging.
1.2.3 purification and quantification of Kernel Albumin
And (2) precipitating the albumin extracting solution by using acetone, centrifuging for 1h at 12000r/min, dissolving by using a lysis solution (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 40 mmol/L DTT, 0.2% (W/V) IPG-Bufffer, pH 4-7), purifying the protein by using a 2-DClean-Up Kit protein purification Kit, dissolving the protein by using an aqueous solution (8 mol/L urea, 2% CHAPS, 20 mmol/L DTT, 0.5% IPG-Bufffer, pH 4-7), and quantifying by using a Bradford method, wherein bovine serum albumin is used as a standard protein.
1.2.4 2-DE of Kernel Albumin
After the volume of the protein extract containing 200 mug protein is supplemented to 450 mug by sample hydration buffer (hydration solution +0.002% bromophenol blue +0.012% Destreak Reagent), a solid phase pH gradient linear gel strip of 24cm and pH 4-7 is covered above the protein solution, the protein solution is passively hydrated for 17h, then the protein solution is subjected to equal electrofocusing (20 ℃, 100V, 1 h; 300V, 1 h; 500V, 1 h; 1000V, 2 h; 8000V, 3 h; 8000, 10h), after the completion of the polyfocusing, the gel strip is respectively balanced by gel strip balance buffer (6 mol/L urea, 2% SDS, 0.075 mol/L Tris-HCl pH 8.8, 30% glycerol, 0.002% bromophenol blue) containing 1% DTT and 2.5% iodoacetamide, the mass spectrum is closely attached to the upper part of 12% polyacrylamide gel, the gel is subjected to second electrophoresis (3W, 24% bromophenol blue, 24-27. um) and then the gel is subjected to analytical scanning by a Powerk scanner, a limited electrophoresis scanner, a silver analyzer is used for analyzing the images, and the images are repeatedly scanned by a Powerk analyzer, and then analyzed by a Powerk analyzer.
1.3 data processing
The data were statistically analyzed using DPS 9.50 and Microsoft Word 2003 tabulated.
2 results and analysis
2.1 Effect of Long-term application of high-low nitrogen fertilizers on the content of albumin in corn kernels
TABLE 1 Effect of Long-term application of high-and low-nitrogen fertilizers on the Albumin content of corn kernels
Note: the same column of different letter representation reaches the significance level of difference
As can be seen from Table 1, the albumin content of corn kernels has difference after long-term high-low nitrogen fertilization treatment. There was no significant difference between the control and high nitrogen treatments, and there was a significant difference between the low nitrogen treatment and the control, high nitrogen treatment.
2.2 one-way SDS-PAGE analysis of maize Kernel Albumin
Quantitative one-way electrophoresis images of different processed grain albumins are carried out, as shown in figure 1. It can be seen that the high-low nitrogen treatment clearly has protein bands with expression difference compared with the control group, so the detection of the dielectrophoresis is further carried out to detect the difference protein spots.
2.3 proteomics analysis of corn kernel albumin
As shown in fig. 2, the long-term low nitrogen-treated corn kernel albumin had 7 differential protein spots compared to the control group, wherein 1 spot expression was up-regulated and 6 spot expression was down-regulated; compared with a control group, the grain albumin treated by the long-term high nitrogen has 6 differential protein points, wherein the expression quantity of 1 point is up-regulated, and the expression quantity of 5 points is down-regulated. 3 of the differential protein spots of the high nitrogen treatment and the low nitrogen treatment were the same spot, so there were 10 differential protein spots in total.
The 10 differential protein spots 72, 102, 110, 115, 123, 133, 177, 213, 218 and 232 are detected by mass spectrometry, the detection results of the 10 spots are all positive, and the detection results are respectively 60S acidic ribosomal protein P3, general stress 39-chain dehydro protein/reductase SDR family protein, pectin esterase inhibitor domain protein precursor, rab28 protein, cysteine synthase subtype 3, glutathione S-transferase (GST), oxygenase2, glyoxalase I, embryonic development late-stage abundant protein D-34 and a hypothetical protein. The detailed information is shown in table 2.
2.4 functional Classification of Difference proteins
Through functional classification of the identified proteins, protein changes of grains under the treatment of long-term positioning of different nitrogen fertilizers are discussed. The 10 protein spots identified can be divided into five major classes according to their function: proteins involved in protein and amino acid metabolism 3: 213. 218, 72; 1 associated with energy metabolism: 102, and (b); 3 proteins related to expression regulation and defense stress resistance: 115. 132, 133; proteins associated with cellular metabolism: 110; 2 proteins with unknown functions: 232. 177.
Table 2 Mass spectrum identification results of long-term positioning fertilization of corn kernel albumin difference points
Discussion of 3
3.1 Long-term fixed-position application of high-low nitrogen fertilizers on the influence of the albumin of corn kernels
Nitrate nitrogen is an important component of mineral nitrogen in soil, and is converted into nitrate nitrogen under certain conditions, namely organic nitrogen or ammonium nitrogen. The nitrate nitrogen in the soil with long-term positioning application of high and low nitrogen fertilizers is obviously higher than that in the control group, and is mainly caused by unreasonable fertilization, low crop yield and low nutrient utilization rate. It can be seen from table 1 that long-term application of high and low nitrogen fertilizers results in a decrease in the albumin content of corn kernels.
3.2 functional analysis of differentially expressed proteins
3.2.1 proteins and proteins associated with amino acid metabolism
The ribosome of eukaryote is 80S, which includes two subunits of 60S and 40S size, and contains 4 RNA molecules and more than 80 different proteins. Many ribosomal proteins have been thought to have structural functions, but are also essential factors in translation. The 60S acidic ribosomal protein can participate in the biosynthesis of protein and has the function of regulating and controlling the development of plants. The 60S acidic ribosomal protein P3 identified in this experiment (point 213) was reduced in expression under long-term low nitrogen treatment conditions, whereas the control and high nitrogen treatment were not different, consistent with differences in albumin content. The nitrate ammonia in the soil with long-term positioning application of high and low nitrogen fertilizers is obviously higher than that in the control group, and is mainly caused by unreasonable fertilization, low crop yield and low nutrient utilization rate. This suggests that it may be that nitrogen over-expression of this protein is inhibited.
Pectinesterase, or pectin methylesterase, is an important pectinase, and is commonly present in different tissues and organs of higher plants, such as roots, stems, leaves, fruits and the like. It has important effects on the composition and degradation of cell walls, seed germination, root tip extension, seed cracking, fruit softening and ripening, disease resistance and the like. The pectin esterase inhibitor is a protein with an inhibiting effect on pectin methylesterase, and has a little regulating effect on the activity of the pectin methylesterase. The pectin esterase inhibitor domain protein precursor (point 218) constitutes the pectin esterase inhibitor and plays a certain role in regulation in the process of forming grains. Under the high-low nitrogen treatment, the expression of the protein is effectively inhibited, and the cell wall is possibly thickened.
Cysteine synthase is a key enzyme in the cysteine-to-glutathione synthesis pathway, which may alter intracellular glutathione pools to resist oxidative stress under stress. Studies have shown that the cysteine synthase gene appears to encode an isoform of cysteine synthase and is under coordinated control by sulfur, nitrogen and light conditions. Cysteine synthase subtype 3 (point 72) constitutes cysteine synthase. Experiments show that the long-term positioning low-nitrogen treatment is not different from a control group, and the expression level of the high-nitrogen treatment is reduced, possibly that the expression of cysteine synthase is inhibited by the overhigh nitrogen. 3.2.2 proteins associated with energy metabolism
Short-chain dehydrogenases/reductases (SDRs) are a very large family of proteins. The SDR protein family gene now exceeds 10,000 sequences. The SDR protein family includes a number of different oxidoreductases, isomerases, lyases, and the like, which catalyze a variety of substrates, including steroids, vitamin a, prostaglandins, carbohydrates, alcohols, and many other small molecule compounds. Point 102 is the 39-chain dehydroprotein of general stress, which may be caused by the imbalance of nitrogen, phosphorus and potassium contents in soil due to long-term application of nitrogen fertilizer, and the reduction of stress protein content due to over-high nitrogen.
3.2.3 expression regulation and defense stress-resistance related protein
The rab28 protein (point 115) is a member of the rab protein family and is more studied in botany. It is expressed in the meristematic tissue of seed embryo and mature plant, and is located on the nucleus to regulate the expression of abscisic acid. The change of the abscisic acid content in the grains is consistent with the change of the grain filling rate, and the abscisic acid content and the grain filling rate are in extremely obvious positive correlation. The long-term positioning fertilization ensures that the nitrogen content in the soil is very high, the abscisic acid content is reduced, and the rab28 content is reduced.
Point 132 is glutathione S-transferase (GST): participating in the release of toxic substances. The high-nitrogen treatment is positioned for a long time, so that the reduction speed of the glutathione S-transferase is obviously lower than that of the low-nitrogen treatment, and experiments show that the content of the glutathione S-transferase is reduced under the condition of the low-nitrogen treatment.
The L EA protein can be induced by exogenous abscisic acid, dry early, osmotic and low-temperature stresses, and the content of abscisic acid in cells is reduced by long-term nitrogen fertilizer treatment, so that the expression quantity is reduced.
3.2.4 proteins associated with cellular metabolism
Glyoxalase I (point 110), an intracellular enzyme, is ubiquitous in a variety of cells, particularly mitochondria, and its activity extends throughout life, metabolism of methylglyoxalase in vivo is mainly achieved by condensing Methylglyoxal (MG) and Glutathione (GSH) into S-D-lactate glutathione under the action of glyoxalase I, reducing the physiological active concentration of MG, and then hydrolyzing S-D-lactate glutathione into non-toxic GSH and D-lactate under the action of G L OII.
3.2.5 other proteins
1, 2-dihydroxy-3-keto-5-methylenedioxygenase 2 (point 177) is an oxygenase whose function is unknown.
It is assumed that proteins (point 232) are a class of proteins that have been demonstrated to exist in vivo, but whose structure and function are unknown. Maize is rich in hypothetical proteins and it is currently unknown whether it is associated with long-term, localized application of nitrogen fertilizer.
In a word, proteins such as protein and amino acid metabolism related protein, energy metabolism related protein, expression regulation and defense stress-resistance related protein, cell metabolism related protein and the like coordinate and act together in the corn grain filling stage, and the influence of long-term application of nitrogen fertilizer on corn grain albumin is fully reflected. The experiment is believed to provide new ideas and suggestions for researching the protein level on the corn fertilization mechanism and the genetic improvement of the quality.
3.3 essentially, the variety identification is the genotype identification. The protein is the primary product of the structural gene expression, can be regarded as a marker of the structural gene, and the analysis of the inter-species relationship is to analyze and identify the protein with various molecular forms, if the protein has enough polymorphism, the analysis method can detect the polymorphism, and then the polymorphism of the protein is identified, so that the genetic composition of the crop variety is known, and the variety is identified. The experimental result shows that no genetic difference exists in long-term positioning application of the high-low nitrogen fertilizer, and only the content of the protein of the Luyu 16 corn kernel is changed.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (3)
1. A test method for the influence of long-term application of high and low nitrogen fertilizers on the albumin of corn kernels is characterized by comprising the following steps:
step 1, experimental design
The soil to be tested is non-calcareous moist soil, develops in alluvial matrix, is light in surface soil texture, has the organic matter content of 4.10g/kg, the total nitrogen content of 0.50g/kg, the total phosphorus content of 0.46g/kg, the available phosphorus content of 15mg/kg, the quick-acting potassium content of 38mg/kg and the cation replacement amount of 11.80 cmol/kg; the experiment was set up with 3 treatments: control, low nitrogen, high nitrogen, 3 replicates per treatment; the winter wheat-summer corn crop rotation is carried out in a test field, the crop is cultivated twice a year, the summer corn variety is Luyu 16, and the annual application amount of the high-nitrogen fertilizer is 276kg/hm2The annual application amount of the low-nitrogen fertilizer is 138kg/hm2；
Step 2, measurement items and method
2.1 extraction and determination of Kernel Albumin
Adding 2m L water into 0.5g of sample, grinding to homogenate, adding 3m L water, transferring to a centrifuge tube, washing the mortar with 3m L water, oscillating for 5min, centrifuging at 4500r/min for 8min, pouring out supernatant, adding 8m L water, stirring, oscillating for 5min, centrifuging, pouring out supernatant for 4 times, mixing the poured-out supernatant, diluting with water to 50m L, sucking supernatant 1m L, adding Coomassie brilliant blue 5m L, mixing, standing for 2min, and carrying out color comparison at 595 nm;
2.2 SDS-PAGE of Kernel Albumin
The electrophoresis device is a DYZC-24B type electrophoresis apparatus, and the thickness of the plate gel is 1 mm; the concentration of the separation gel is 12 percent, and the concentration of the concentration gel is 5 percent; adding 200 microgram samples into the point sample holes; the voltage is 100V at the beginning, and after the bromophenol blue completely enters a concentrated gel interface, the electrophoresis is changed into 120V constant voltage electrophoresis till the end; after electrophoresis, staining with Coomassie brilliant blue, decoloring and scanning to form an image;
2.3 purification and quantification of Kernel Albumin
Precipitating the grain albumin extracting solution by using acetone, centrifuging for 1h at 12000r/min, dissolving by using a lysis solution, purifying protein by using a 2-DClean-Up Kit protein purification Kit, and dissolving the protein by using a hydration solution, wherein the Bradford method is adopted for quantification, bovine serum albumin is taken as a standard protein, the lysis solution comprises 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 40 mmol/L DTT, 0.2% IPG-buffer and has the pH of 4-7, and the hydration solution comprises 8 mol/L urea, 2% CHAPS, 20 mmol/L DTT, 0.5% IPG-buffer and has the pH of 4-7;
2.4 2-DE of Kernel Albumin
Taking the grain protein extract containing 200 micrograms of protein, supplementing the volume to 450 micrograms L by using a sample hydration buffer solution, covering a solid phase pH gradient linear adhesive tape with the volume of 24cm and the pH of 4-7 above the protein solution, passively hydrating for 17 hours, then carrying out isoelectric focusing at 20 ℃, 100V, 1h, 300V, 1h, 500V, 1h, 1000V, 2h, 8000V, 3h, 8000V, 10h, respectively balancing the adhesive tape by using an adhesive tape balancing buffer solution containing 1% of DTT and 2.5% of iodoacetamide after the completion of the focusing for 15min, closely attaching the adhesive tape above 12% polyacrylamide gel after the completion of the balancing, carrying out secondary electrophoresis, staining the gel by adopting an improved silver staining method after the completion of the electrophoresis, then scanning an image by using a UMAX Powerlok 2100X L scanner, analyzing the obtained image by using a camera plasma Immunity 6 analysis software, repeating the mass spectrum for 3 times, selecting a differential protein sample hydration buffer solution with the difference, wherein the difference is reageMateik +0.012% hydration buffer solution;
step 3, data processing
The data were statistically analyzed using DPS 9.50 and Microsoft Word 2003 tabulated.
2. The method for testing the effect of long-term application of high and low nitrogen fertilizers on the albumin in corn kernels as claimed in claim 1, wherein in step 2.4, the 2.5% iodoacetamide gel strip equilibration buffer is 6 mol/L urea, 2% SDS, 0.075 mol/L Tris-HCl pH 8.8, 30% glycerol, 0.002% bromophenol blue.
3. The method for testing the influence of long-term application of high and low nitrogen fertilizers on the albumin in corn kernels according to claim 1, wherein in the step 2.4, the second-direction electrophoresis is performed for 24-27 h at 3W per rubber strip.
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