CN102269731A - Method for rapidly detecting deletion of soybean lipoxygenase - Google Patents
Method for rapidly detecting deletion of soybean lipoxygenase Download PDFInfo
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Abstract
The invention provides a method for rapidly detecting a deletion of soybean lipoxygenase, which is the improvement on the traditional SDS-PAGE (Sodium dDodecyl Sulfate Polyacrylamide Gel Electrophoresis) technology. The method provided by the invention is composed of five parts as follows: extraction of soybean Lox lipoxygenase, preparation of polyacrylamide gel, SDS-PAGE electrophoresis, coloration of glue and decoloration of glue, wherein the extraction of soybean Lox lipoxygenase and the preparation of polyacrylamide gel are essential parts of the invention; and the coloration and decoloration of the SDS-PAGE lipoxygenase glue are routinely common technologies. The method provided by the invention overcomes the disadvantage that the traditional technology cannot directly distinguish single deletion or double deletions in Lox-1, Lox-2 and Lox-3; and the invention provides the method which can be used for identifying deletion of Lox-1 and Lox-2 and has the advantages of simpleness and fastness in operation and direct and accurate identification result.
Description
Technical field
The present invention relates to a kind of detection method of protein, particularly relate to a kind of method that detects soybean lipoxygenase (lipoxygenase) disappearance by improved SDS-PAGE method.
Background technology
Soybean is one of picked-up source of most important vegetable oil and good protein in people of other countries' daily life, also is the primary raw material or the adjuvant of numerous food (as margarine, sausage), industrial product (as lubricating oil, printing ink).But because lipoxidase in the soya seeds (lipoxygenase, abbreviation Lox is called for short lipoxygenase, and it has three isodynamic enzymes, Lox-1, Lox-2, existence Lox-3).Linoleic acid in its catalysis seed etc. has suitable, suitable-1, the polybasic unsaturated fatty acid of 4-pentadiene structure, generate the derivant of hydrogen peroxide, finally be broken down into small-molecule substances such as volatile aldehyde, alcohol, ketone, these small-molecule substances are to cause the broken oxygen of meeting of soya seeds to produce stench root of distinguishing the flavor of.Because the existence of the stench flavor of soybean, influenced the value of exploiting and utilizing of bean product, the processing enterprise that with the soybean is raw material is in order to remove the stench flavor in the soya seeds, have to go ways such as raw meat and embedding to remove it by heating, but nonetheless also fail to remove fully stench flavor, increased heating and removed the raw meat cost, and because heating, also cause the sex change of soybean protein composition easily, reduced the nutritive value of soybean.Studies show that, in the soybean germplasm resources bank, have Lox-1, the soybean resource of single disappearance or two disappearances in-2 ,-3 by the artificial hybridization technology, can fundamentally be removed the stench flavor of soybean.Problem is how to screen the offspring of some or two disappearances that identify 3 isodynamic enzymes among the Lox from numerous filial generations.
Method at present commonly used mainly contains 3 kinds: polyacrylamide gel electrophoresis detection method (SDS-PAGE), isoelectric point electrophoresis (IEF-PAGE) and the beta carotene method of fading.Wherein, the most close with the present invention technology is the SDS-PAGE method.Judging that Lox is under the situation about whether lacking fully, the three can accurately judge, wherein the SDS-PAGE method is the most simple and easy to do method, and when will judging Lox-1, when whether certain lacks in-2 ,-3, the IEF-PAGE method is the most accurate, but detect the also the most complicated difficult grasp of step, time-consuming, it is also the highest to detect cost.Method is compared more accurately with the SDS-PAGE method but is also existed drawbacks such as detecting the cost height though beta carotene fades, though existing SDS-PAGE method detection method is simple and easy to do, but in the method that adopts aspect the crude protein extraction is the conventional soybean holoprotein institute method in common of extracting, promptly cut the powder 5-10mg of the cotyledon that the soya seeds hilum tosses about, put into the 1.5ml centrifuge tube, add protein extraction damping fluid (Tris-HCl, pH7.2), oscillator concussion about 10 seconds, leave standstill 15-30min, can utilize SDS-PAGE to go up sample and run glue, used separation gel also is a glue of analyzing 7.5% general concentration of large molecular weight protein, is not suitable for and the kinds of protein that three molecular weight are all very approaching big concerning molecular weight, and is some or two when whether lacking in judging Lox, almost can't accurately judge, exist the high shortcoming of erroneous judgement.
Summary of the invention
At above-mentioned situation, technical matters to be solved by this invention provides a kind of fast and convenient, can with the naked eye just can distinguish simply exactly and differentiate Lox-1 and the single disappearance of Lox-2 or two disappearance whether new methods.
Method provided by the invention is the improvement to original SDS-PAGE method, the crude protein extracting method and the glue technology that are primarily aimed at prior art are improved, and the method after the improvement can be used for the evaluation of Lox-1 and Lox-2 disappearance, and simple to operate, quick, qualification result is intuitively accurate.
Concrete technical matters to be solved by this invention realizes by the following method:
Composition of the present invention: the present invention is by extraction, the preparation of polyacrylamide gel, the SDS-PAGE electrophoresis of soybean Lox lipoxygenase albumen, the dyeing of glue and five parts of decolouring constitute, wherein the preparation of the extraction of Lox lipoxygenase, polyacrylamide gel is core of the present invention place, and the dyeing of SDS-PAGE running gel and decolouring are conventional total technology.
The method of a kind of fast detecting soybean lipoxygenase disappearance provided by the invention is characterized in that may further comprise the steps:
(1) extraction of soybean Lox lipoxygenase albumen: cut seed fine powder 6～8mg, put into the centrifuge tube of 1.5mL, add and extract damping fluid, stir with oscillator, ultrasonic extraction 6～8min places 20～30min under the room temperature, after stirring once again, and 5000rmin
-1Centrifugal 5min gets supernatant 10 μ L-12 μ L and is used for the parsing of SDS-PAGE electrophoresis;
(2) preparation of polyacrylamide gel:
The preparation of gelling solution:
A liquid: be mixed with SDS by 4.0g/L, the mixed solution that 3M Tris-HCl forms, the pH value transfers to 8.8;
B liquid: be mixed with 0.48M HCl solution, adjust PH to 7.0 with Tris;
C liquid: be mixed with acrylamide, the mixed solution that the bisacrylamide of 8g/L is formed by 300g/L;
P liquid: the ammonium persulfate solution that is mixed with 15g/L;
E liquid: the riboflavin solution that is mixed with 0.04g/L;
The preparation of separation gel: preparation quality percent by volume is 6.75% polyacrylamide gel liquid, be that the glass plate of 16cm * 14cm is produced 2 pieces in the thick glue of 1mm (needing separation gel solution 36mL) and is example with the specification, formulated by solution: A liquid 9.0mL with lower volume, C liquid 8.1mL, P liquid 1.8mL, distilled water 17.1mL, N, N, N ', N '-tetramethylethylenediamine (TEMED) 0.024 μ L;
Concentrate the glue preparation: with the glue of preparing above-mentioned specification is example, and two boards need concentrate sol solution 12mL, formulated by the solution with lower volume: B liquid 3.2mL, C liquid 2.0mL, E liquid 2.6mL, P liquid 0.4mL, distilled water 3.8mL, N, N, N ', N '-tetramethylethylenediamine (TEMED) 0.012 μ L;
(3) SDS-PAGE electrophoresis:
The preparation of electrophoretic buffer: be mixed with glycocoll 14.4g/L, Tris 3.0g/L, the mixed solution of SDS 1.25g/L, standby;
(4) dyeing of glue and decolouring;
Wherein, the leaching process of soybean Lox lipoxygenase albumen at the opposition side of its plumular axis, cuts the seed fine powder for getting 1 in seed;
Wherein used extraction damping fluid is 0.05molL
-1Tris-HCl, pH 7.2, include volume parts and be 2% mercaptoethanol, and the bromophenol blue indicator is an amount of, and addition is 1mL.
The present invention does not limit the interpolation of described bromophenol blue indicator, should be understood that, an amount of interpolation should belong in those skilled in the art's the general knowledge scope for the bromophenol blue indicator.
Method provided by the present invention has the following advantages compared to prior art:
1, aspect Lox lipoxygenase protein extraction, the difference of new improved technology and conventional art is after original homogeneous vibration, holoprotein leaves standstill before the extracting, has carried out the ultrasonic Treatment about 8min, then, at room temperature leave standstill extracting 20～30min, stir once again, and in 5000rmin
-1Centrifugal 5min gets supernatant 10 μ L-12 μ L and is used for the parsing of SDS-PAGE electrophoresis.By ultrasonic Treatment, can increase the solubleness of protein, thereby be not easy to produce precipitation, and during the conventional art electrophoresis because albumen is deposited on easily and is difficult in the colloid move, cause Lox-1, the separation of Lox-2 and Lox-3 isodynamic enzyme and differentiate difficulty.
2, aspect the preparation of polyacrylamide gel: the concentration of polyacrylamide gel is dropped to 6.75% from original 7.5%, the grid space of gel is strengthened, be convenient to the Lox isodynamic enzyme and on separation gel, move.The employed glue of conventional art is 7.5% polyacrylamide gel, isodynamic enzyme Lox-1 and Lox-2 bands of a spectrum almost are in same horizontal level (Fig. 1), Fig. 1 is with reference to herd too (ダ イ ズ Xin Pin Seed " い Chi ひ め " breeds と そ characteristic, Jiu Zhou Red Okinawa Farming already studies セ Application タ one Reported announcement, 2002, No. 40, PP:79-86) from the plumage deer, when utilizing the method to carry out the discriminating of resource screening of Lox isodynamic enzyme disappearance and filial generation disappearance proterties, cause the erroneous judgement of isodynamic enzyme disappearance proterties easily.Fig. 2 is of the present invention 6.75% new polypropylene acrylamide gel electrophoresis pattern, whether can distinguish sample by this figure is deletant, as shown in Figure 2, the position of isodynamic enzyme Lox-1 and Lox-2 bands of a spectrum, than Fig. 1 evident difference is arranged, when the isodynamic enzyme disappearance character identification of screening that isodynamic enzyme is lacked resource and filial generation, be difficult for causing erroneous judgement, have higher using value.
Description of drawings
Fig. 1 is that conventional SDS-PAGE electrophoresis detects lipoxygenase disappearance collection of illustrative plates;
Fig. 2 is soybean lipoxygenase of the present invention (lipoxygenase) disappearance fast detecting electrophoresis pattern.
Embodiment
Further illustrate beneficial effect of the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit the scope of the invention.
Experiment material: maternal ♀: CS8000 (Lox-3null) (cv., China), male parent
FJ307 (Lox-1 ,-2 ,-3triple-null) (cv.Japan), 11 from CS8000 (♀) * FJ307
F
2The sample of colony.
The extraction of soybean Lox lipoxygenase albumen: respectively get 1 in seed respectively,, cut seed fine powder 6mg, put into the centrifuge tube of 1.5mL, add 1mL and extract damping fluid (0.05molL with the blade of cutting a sheet of paper at the opposition side of its plumular axis
-1Tris-HCl, pH 7.2, include 2% mercaptoethanol, the bromophenol blue indicator is an amount of), stirring with oscillator, ultrasonic extraction 6min places 20min under the room temperature, after stirring once again, 5000rmin
-1Centrifugal 5min gets supernatant 10 μ L and is used for the parsing of SDS-PAGE electrophoresis.
The preparation of polyacrylamide gel:
The preparation of gelling solution:
A liquid: SDS 2.0g, Tris 181.2g uses 1molL
-1HCl transfers to 8.8 with the pH value, and last adding distil water is settled to 500mL;
C liquid: Acrylamide 30g, add Bisacrylamide 0.8g, adding distil water is settled to 100mL.
P liquid: ammonium persulfate 1.5g, adding distil water is to 100mL;
B liquid: 1molL
-1Behind the abundant mixing of HCl 240mL adding distil water 200mL, adjust PH to 7.0 with Tris, last adding distil water is settled to 500mL;
E liquid: lactochrome 4mg, adding distil water 100mL mixing;
The preparation of electrophoretic buffer: glycocoll Glycine 14.4g, Tris 3.0g, SDS 1.25g, adding distil water 1000mL mixing is standby;
The preparation of dyeing liquor: Kao Masi light blue G-250 1.5g, add 440mL ethanol (analyzing alcohol), it is standby that 60mL acetate, distilled water transfer to the 1000mL mixing;
The preparation of destainer: graduated cylinder is measured 200mL methyl alcohol, 50mL acetate, and adding distil water is standby to the 1000mL mixing;
The assembling of glass plate: (the glass plate specification is 16cm * 14cm) with glass plate to prepare the thick common SDS-PAGE for 1mm of glue, with the inside surface wiped clean airing of 70% ethanol with glass plate, place leakproof glue sealed silicon adhesive tape (or silicone tube), 1 block of plate is with 4 large size macrolabias, each side 2, be fixed on the glass plate and (be as the criterion), vertically be positioned on the horizontal experiment table not leak glue;
The preparation of separation gel: the polyacrylamide gel liquid of preparation 6.75%: press A liquid 9.0mL, C liquid 8.1mL, P liquid 1.8mL, the sequencing of distilled water 17.1mL joins in the Erlenmeyer flask of 100mL, after fully mixing, add TEMED 24 μ L, once again behind the mixing, on average be poured in the slit of two cover glass plates, it is an amount of that the top of glue face splashes into distilled water gently, prevents glue face drying, after room temperature placement 20～30min glue solidifies, outwell top distilled water, it is stood upside down in desktop, it is standby to remove residual moisture;
Concentrate the glue preparation: add B liquid 3.2mL in the following order one by one, C liquid 2.0mL, E liquid 2.6mL, P liquid 0.4mL behind the abundant mixing of distilled water 3.8mL, adds TEMED 12 μ L mixings again, on average be poured into the upper strata of separation gel, insert comb, room temperature is placed 20～30min.The condensing gelling fixedly pincers of dropping back down admittedly rinses out gently with distilled water and to solidify incomplete cull, wraps with preservative film, and 4 ℃ to be inverted refrigeration standby.
The SDS-PAGE electrophoresis: the electrophoretic buffer for preparing is an amount of, poured in the vertical electrophoresis groove (not being as the criterion) to have the colloid bottom surface, glass plate is installed on the vertical electrophoresis groove, notice that the bottom surface of glue does not enter bubble; Last groove is poured electrophoretic buffer into, makes liquid level not have the glass plate breach.Extract sample 10 μ L with micro syringe or multichannel micropipettor, slowly inject each swimming lane.Deposition condition: voltage 100V, about electrophoresis time 1h, treat that the bromophenol blue indicator is by after concentrating glue, voltage is risen to 150V, about electrophoresis time 4h, when the bromophenol blue indicator is about to by the colloid bottom, stop electrophoresis, take off colloid, put into dyeing liquor, jog dyeing 1h takes out and puts into destainer, more than the jog decolouring 4h, on common fluorescent glass lamp box, confirm the miss status of Lox isodynamic enzyme, subsequently can be with after the colloid drying, scanning is preserved.
The result identifies: as shown in Figure 2, wherein, the swimming lane of the left and right sides be respectively maternal ♀: CS8000 (Lox-3null) (cv., China), male parent
FJ307 (Lox-1 ,-2 ,-3triple-null) (cv.Japan), 1-11 is respectively CS8000 (♀) * FJ307
F
2The electrophoresis pattern of the sample segment of colony.Can distinguish sample No.1 by this figure, 5,6,8 is Lox-2 ,-3 deletants, and No.2,4,7 is Lox-1 ,-3 deletants, No.3,9,11 is the Lox-3 deletant, No.10 is Lox-1 ,-2 ,-3 complete deletants.As shown in Figure 2, there is evident difference the position of isodynamic enzyme Lox-1 and Lox-2 bands of a spectrum than Fig. 1, when the isodynamic enzyme disappearance character identification of screening that isodynamic enzyme is lacked resource and filial generation, is difficult for causing erroneous judgement, has higher using value.
The extraction of soybean Lox lipoxygenase albumen: respectively get 1 in seed respectively,, cut seed fine powder 8mg, put into the centrifuge tube of 1.5mL, add 1mL and extract damping fluid (0.05molL with the blade of cutting a sheet of paper at the opposition side of its plumular axis
-1Tris-HCl, pH 7.2, include 2% mercaptoethanol, the bromophenol blue indicator is an amount of), stirring with oscillator, ultrasonic extraction 8min places 30min under the room temperature, after stirring once again, 5000rmin
-1Centrifugal 5min gets supernatant 12 μ L and is used for the parsing of SDS-PAGE electrophoresis.
The preparation of polyacrylamide gel, the SDS-PAGE electrophoresis, dyeing and decolorization are carried out according to the method for embodiment 1.
The above only is the preferred embodiments of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and can carry out many changes to it in the spirit and scope that claim of the present invention limited, revise, even the equivalence change, but all will fall within the scope of protection of the present invention.
Claims (3)
1. the method for fast detecting soybean lipoxygenase disappearance is characterized in that may further comprise the steps:
(1) extraction of soybean Lox lipoxygenase albumen: cut seed fine powder 6～8mg, put into the centrifuge tube of 1.5mL, add and extract damping fluid, stir with oscillator, ultrasonic extraction 6～8min places 20～30min under the room temperature, after stirring once again, and 5000rmin
-1Centrifugal 5min gets supernatant 10 μ L-12 μ L and is used for the parsing of SDS-PAGE electrophoresis;
(2) preparation of polyacrylamide gel:
The preparation of gelling solution:
A liquid: be mixed with SDS by 4.0g/L, the mixed solution that 3M Tris-HCl forms, the pH value transfers to 8.8;
B liquid: be mixed with 0.48M HCl solution, adjust PH to 7.0 with Tris;
C liquid: be mixed with acrylamide, the mixed solution that the bisacrylamide of 8g/L is formed by 300g/L;
P liquid: the ammonium persulfate solution that is mixed with 15g/L;
E liquid: the riboflavin solution that is mixed with 0.04g/L;
The preparation of separation gel: preparation quality percent by volume is 6.75% polyacrylamide gel liquid, be that the glass plate of 16cm * 14cm is produced the thick glue of 1mm and is example for 2 pieces with the specification, need separation gel solution 36mL, by solution composition: A liquid 9.0mL, C liquid 8.1mL, P liquid 1.8mL, distilled water 17.1mL, N with lower volume, N, N ', N '-tetramethylethylenediamine 24 μ L;
Concentrate the glue preparation: with the glue of preparing above-mentioned specification is example, and two boards need concentrate sol solution 12mL, by the solution composition with lower volume: B liquid 3.2mL, C liquid 2.0mL, E liquid 2.6mL, P liquid 0.4mL, distilled water 3.8mL, N, N, N ', N '-tetramethylethylenediamine 12 μ L;
(3) SDS-PAGE electrophoresis:
The preparation of electrophoretic buffer: be mixed with glycocoll 14.4g/L, Tris 3.0g/L, the mixed solution of SDS 1.25g/L, standby;
(4) dyeing of glue and decolouring.
2. the method for a kind of fast detecting soybean lipoxygenase disappearance as claimed in claim 1 is characterized in that getting 1 in seed, at the opposition side of its plumular axis, cuts the seed fine powder.
3. the method for a kind of fast detecting soybean lipoxygenase disappearance as claimed in claim 1 is characterized in that described extraction damping fluid is 0.05molL
-1Tris-HCl, pH 7.2, include volume fraction and be 2% mercaptoethanol, and the bromophenol blue indicator is an amount of, and addition is 1mL.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103160579A (en) * | 2013-02-21 | 2013-06-19 | 安徽农业大学 | Multiple polymerase chain reaction (PCR) primer used for identifying soybean deletants of Lox1 and Lox3 and method used for identifying soybean deletants of Lox1 and Lox3 |
| CN108333245A (en) * | 2018-01-16 | 2018-07-27 | 东北农业大学 | A kind of method for building up of lipoxygenase and the new soybean germplasms of the double missings of 7S globulin |
| CN110763750A (en) * | 2019-09-29 | 2020-02-07 | 东北农业大学 | Method for identifying lipoxygenase deficiency of soybean variety |
| CN112665950A (en) * | 2021-01-28 | 2021-04-16 | 安徽佳创生物科技有限公司 | Method for detecting high-density lipoprotein in serum |
| CN113848202A (en) * | 2021-09-03 | 2021-12-28 | 东北农业大学 | Screening and identifying method for soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid progeny |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160579A (en) * | 2013-02-21 | 2013-06-19 | 安徽农业大学 | Multiple polymerase chain reaction (PCR) primer used for identifying soybean deletants of Lox1 and Lox3 and method used for identifying soybean deletants of Lox1 and Lox3 |
| CN103160579B (en) * | 2013-02-21 | 2014-11-12 | 安徽农业大学 | Multiple polymerase chain reaction (PCR) primer used for identifying soybean deletants of Lox1 and Lox3 and method used for identifying soybean deletants of Lox1 and Lox3 |
| CN108333245A (en) * | 2018-01-16 | 2018-07-27 | 东北农业大学 | A kind of method for building up of lipoxygenase and the new soybean germplasms of the double missings of 7S globulin |
| CN108333245B (en) * | 2018-01-16 | 2020-02-28 | 东北农业大学 | Method for establishing lipoxygenase and 7S globulin double-deletion soybean new germplasm |
| CN110763750A (en) * | 2019-09-29 | 2020-02-07 | 东北农业大学 | Method for identifying lipoxygenase deficiency of soybean variety |
| CN112665950A (en) * | 2021-01-28 | 2021-04-16 | 安徽佳创生物科技有限公司 | Method for detecting high-density lipoprotein in serum |
| CN113848202A (en) * | 2021-09-03 | 2021-12-28 | 东北农业大学 | Screening and identifying method for soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid progeny |
| CN113848202B (en) * | 2021-09-03 | 2024-03-01 | 东北农业大学 | Soybean lipoxygenase and screening and identifying method of 7S and 11S globulin subunit deletion hybrid offspring |
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