CN103333234A - Method for extracting apoplast antifreeze proteins from winter rape roots - Google Patents
Method for extracting apoplast antifreeze proteins from winter rape roots Download PDFInfo
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Abstract
The invention relates to a method for extracting antifreeze proteins, in particular to a method for extracting apoplast antifreeze proteins from winter rape roots. According to the method, apoplast antifreeze proteins are successfully extracted from cold-induced winter rape roots. The method for extracting apoplast antifreeze proteins from winter rape roots is characterized by comprising the following steps: (1) sampling; (2) extracting a buffer solution for preparing; (3) slitting; (4) immersing; (5) vacuumizing; and (6) centrifuging. According to the method, apoplast antifreeze proteins are successfully and rapidly extracted from cold-induced winter rape roots, so that technical support is provided for further research of the cold resistance mechanism of winter rape.
Description
Technical field
The present invention relates to the antifreeze protein extracting method, relate in particular to winter rape root apoplast antifreeze protein extracting method, that utilizes this method success has extracted the apoplast antifreeze protein from cold winter rape root of inducing.
Background technology
Low temperature is one of main environment factor that influences the growth of northern China plant normal growth.When plant runs into low temperature stress, a series of Physiology and biochemistries can take place in the body change to adapt to low temperature stress, also can synthesize and secrete multiple proteins-cold temperature induced protein simultaneously.Antifreeze protein (AFPs) is the class in the cold temperature induced protein, can produce in Different Organs such as the root of plant, stem, leaf, also may reside in the callus under the conditions of tissue culture, in suspended culture cell and the suspending nutrient solution.They are very complicated in the location of cell, can be present in the various kinds of cell devices such as intercellular space, cell walls, nucleus, cytolemma, tenuigenin, find at present that the antifreeze protein content in non-plastid that separates is higher in freezingtolerant plant and have ice crystal shape effect, heat stagnation effect (Thermal Hysteresis, TH), recrystallization retarding effect (Recrystallization Inhibition).Three characteristics of AFPs show that it is directly related with the formation of ice crystal at low temperatures, and therefore the protection to plant has substantivity, is the essential substance basis of plant cold resistance.
Antifreeze proteins in 1964 are after (AFPs) be found for the first time in bloom first larva, and through semicentennial research, today, people all found the existence of antifreeze protein (AFPs) in multiple biologies such as fish, insect, plant, bacterium and fungi.Up to now, the AFPs of fish, insect research very deeply and system, and in the plant discovery of AFPs than later.1992, the first time such as Griffith from obtain winter rye (Secalecereale) the blade apoplast that the extracellular freezes through can standing of cold acclimation and partial purification AFPs, the research of having opened plant antifreeze protein.People have also found AFPs in many plants such as Stem and leaf of Mongolian Ammopiptanthus (Ammopiptanthus monglicus), alpine plant Tang Gute Root of Kirilow Rhodiola, Radix Dauci Sativae (Daucus carota), serpentgrass (Polygonumviviparum) and winter wheat afterwards.
Winter rape is that northern very important farm crop, particularly northern winter rape will just can turn green through cold very long winter, bolting, bloom and set seeds.Root is winter rape unique organ of surviving the winter in winter.Therefore studying the root antifreeze protein is one of effective way of northern winter rape winter resistance evaluation.The research of rape antifreeze protein does not still have maturation method.The invention provides winter rape root apoplast antifreeze protein extracting method.Utilize this method to extract apoplast albumen from the rape root fast and effectively, for the cold-resistant mechanism of research winter rape provides technical support.
Summary of the invention
The objective of the invention is to avoid the deficiencies in the prior art, a kind of extracting method of winter rape root apoplast antifreeze protein is provided.By success of the present invention extracted the apoplast antifreeze protein from cold winter rape root of inducing, for the further cold-resistant mechanism of research winter rape provides technical support.
For achieving the above object, the technical scheme that the present invention takes is: a kind of extracting method of winter rape root apoplast antifreeze protein, and its principal feature is to include following steps:
(1) sampling: in 10 to December or in January, second, dig out the rape root in the field, in 3-5 minute, put into ice chest, take back the laboratory, be stored in 3-5 ℃ of refrigerator;
(2) extract damping fluid 1000ml and press the preparation of column weight amount volume:
0.05M/L Tris-Hcl6.057g transfers ph to 8.0;
0.01M/L the EDTA-Na2.2H2O(disodium ethylene diamine tetraacetate) 3.72g;
0.02M/L the 3.52g VC(vitamin c);
Be settled to 1000ml with ddH2O, the damping fluid for preparing is preserved in 3-5 ℃ of environment;
(3) slitting: remove soil that the rape root carries, remove the root part that thirst, flowing water flushing 10-12min, foreign material are used ddH till cleaning
2O flushing 2 times, blot surface-moisture with filter paper after, cut after root cleaned that to be cut into 1-2cm rectangular open;
(4) embathe: embathe the root of cutting into inch strips with above-mentioned damping fluid;
(5) vacuumize: the rape root of cutting into inch strips is put into the container (Erlenmeyer flask is best) of band plug, adds the above-mentioned damping fluid of 80-100ml, vacuumize, be stabilized to 0.07-0.08Mpa after, continue to vacuumize 7-10min, the time is difficult for long;
(6) centrifugal: outwell damping fluid, with filter paper inhale remove the root surface solution after, put it in the syringe, syringe is put into the 50ml centrifuge tube, be 6100-6200rpm at rotating speed, 3-5 ℃, centrifugal 15min collects the centrifuge tube bottom liquid, is non-plastid extract.
The extracting method of described winter rape root apoplast antifreeze protein also includes following steps:
(7) mercaptoethanol solution preparation formula
0.5mol/L Tris-HCL(PH=6.8)12.5％
Glycerine 10%
Beta-mercaptoethanol 5%
ddH2O72.5％
(8) purify:
A. the cold acetone that step (6) extract is added 5 times of volumes ,-20 ℃ of precoolings behind-20 ℃ of precipitation 10-20h, are 10000rpm at rotating speed, and 4 ℃, 30min is centrifugal, and the acetone that inclines is deposited in-20 ℃ of volatilization acetone, obtains the lyophilized protein sample;
B. use the lyophilized protein sample of mercaptoethanol mixed solution dissolving step (8) A, fully dissolving is 10000rpm at rotating speed again, and 4 ℃, 15min is centrifugal, gets supernatant liquor, and is standby-20 ℃ of preservations.
Beneficial effect of the present invention: present method proposes and can not or be difficult in the viewpoint that the rape root extracts albumen at the rape root at some scholar, through constantly attempting and groping, extract the apoplast antifreeze protein by the quick of success of the present invention from cold winter rape root of inducing, provide technical support for further studying the cold-resistant mechanism of winter rape.
Description of drawings
Fig. 1. No. 6 blades of Gansu Province oil and root apoplast protein concentration detect;
Fig. 2. No. 6 blades of different times Gansu Province oil and root apoplast protein electrophoresis collection of illustrative plates;
Fig. 3. mass spectrum is identified discrepancy sampling numbering;
Observe the ice-crystal growth situation of Gansu Province No. 6 root apoplast crude extracts of oil behind-7 ℃ of constant temperature 40min of not cold domestication under Fig. 4 .10X object lens;
Observe the ice-crystal growth situation of Gansu Province No. 6 root apoplast crude extracts of oil behind-7 ℃ of constant temperature 40min of cold domestication under Fig. 5 .10X object lens, show tangible recrystallization retarding effect.
Embodiment
Below principle of the present invention and feature are described, institute only gives an actual example and to be used for explaining the present invention, is not for restriction scope of the present invention.
Embodiment 1: sampling for the first time, and the extraction of No. 6 root apoplast albumen of winter rape Gansu Province oil:
(1) sampling: October 5, dig out No. 6 roots of Gansu Province oil in the field, in 3-5 minute, put into ice chest, take back the laboratory, be stored in 4 ℃ of refrigerators;
(2) extract damping fluid 1000ml and press the preparation of column weight amount volume:
0.05M/L Tris-Hcl6.057g transfers ph to 8.0;
0.01M/L the EDTA-Na2.2H2O(disodium ethylene diamine tetraacetate) 3.72g;
0.02M/LVC(3.52g vitamin c);
Be settled to 1000ml with ddH2O, the damping fluid for preparing is preserved in 4 ℃ of environment;
(3) slitting: remove soil that the rape root carries, remove the root part that thirst, flowing water flushing 10-12min, till foreign material are cleaned, with ddH2O flushing 2 times, blot surface-moisture with filter paper after, cut after root cleaned that to be cut into 1-2cm rectangular open;
(4) embathe: embathe the root of cutting into inch strips with above-mentioned damping fluid, preferably wash more than twice;
(5) vacuumize: the rape root of cutting into inch strips is put into the container of band plug, and Erlenmeyer flask is best, adds the above-mentioned damping fluid of 80-100ml, vacuumizes, be stabilized to 0.07-0.08Mpa after, continue to vacuumize 7-10min, the time is difficult for long;
(6) centrifugal: outwell damping fluid, with filter paper inhale remove the root surface solution after, put it in the syringe, syringe is put into the 50ml centrifuge tube, be 6140rpm at rotating speed, 4 ℃, centrifugal 15min collects the centrifuge tube bottom liquid, is non-plastid extract.
(7) mercaptoethanol solution preparation formula
0.5mol/L Tris-HCL(PH=6.8)12.5％
Glycerine 10%
Beta-mercaptoethanol 5%
ddH2O72.5％
(8) purify:
A. the cold acetone that step (6) extract is added 5 times of volumes ,-20 ℃ of precoolings behind-20 ℃ of precipitation 10-20h, are 10000rpm at rotating speed, and 4 ℃, 30min is centrifugal, and the acetone that inclines is deposited in-20 ℃ of volatilization acetone, obtains the lyophilized protein sample;
B. use the lyophilized protein sample of 80ul mercaptoethanol mixed solution dissolving step (8) A, fully dissolving is 10000rpm at rotating speed again, and 4 ℃, 15min is centrifugal, gets supernatant liquor, and is standby-20 ℃ of preservations.
Embodiment 2: sampling for the second time, and the extraction of No. 6 root apoplast albumen of Gansu Province oil:
(1) sampling: November 15, dig out No. 6 roots of Gansu Province oil in the field, in 3-5 minute, put into ice chest, take back the laboratory, be stored in 4 ℃ of refrigerators;
(2) extract damping fluid 1000ml and press the preparation of column weight amount volume:
0.05M/L Tris-Hcl6.057g transfers ph to 8.0;
0.01M/L the EDTA-Na2.2H2O(disodium ethylene diamine tetraacetate) 3.72g;
0.02M/LVC(3.52g vitamin c);
Be settled to 1000ml with ddH2O, the damping fluid for preparing is preserved in 4 ℃ of environment;
(3) slitting: remove soil that the rape root carries, remove the root part that thirst, flowing water flushing 10-12min, till foreign material are cleaned, with ddH2O flushing 2 times, blot surface-moisture with filter paper after, cut after root cleaned that to be cut into 1-2cm rectangular open;
(4) embathe: embathe the root of cutting into inch strips with above-mentioned damping fluid;
(5) vacuumize: the rape root of cutting into inch strips is put into the container that band is filled in, and Erlenmeyer flask is best, adds the above-mentioned damping fluid of 80-100ml, vacuumizes, and after pressure is stabilized to 0.07-0.08Mpa, continues to vacuumize 7-10min, and the time is difficult for long;
(6) centrifugal: outwell damping fluid, with filter paper inhale remove the root surface solution after, put it in the syringe, syringe is put into the 50ml centrifuge tube, be 6140rpm at rotating speed, 4 ℃, centrifugal 15min collects the centrifuge tube bottom liquid, is non-plastid extract.
(7) mercaptoethanol solution preparation formula
0.5mol/L Tris-HCL(PH=6.8)12.5％
Glycerine 10%
Beta-mercaptoethanol 5%
ddH2O72.5％
(8) purify:
A. the cold acetone (20 ℃ of precoolings) that step (6) extract is added 5 times of volumes behind-20 ℃ of precipitation 10-20h, is 10000rpm at rotating speed, and 4 ℃, 30min is centrifugal, and the acetone that inclines is deposited in-20 ℃ of volatilization acetone, obtains the lyophilized protein sample;
B. use the lyophilized protein sample of 80ul mercaptoethanol mixed solution dissolving step (8) A, fully dissolving is 10000rpm at rotating speed again, and 4 ℃, 15min is centrifugal, gets supernatant liquor, and is standby-20 ℃ of preservations.
Embodiment 3: sampling for the third time, and the extraction of No. 6 roots of Gansu Province oil and blade apoplast albumen:
(1) sampling: December 1, dig out the rape root in the field, in 3-5 minute, put into ice chest, take back the laboratory, be stored in 4 ℃ of refrigerators;
(2) extract damping fluid 1000ml and press the preparation of column weight amount volume:
0.05M/L Tris-Hcl6.057g transfers ph to 8.0;
0.01M/L the EDTA-Na2.2H2O(disodium ethylene diamine tetraacetate) 3.72g;
0.02M/LVC(3.52g vitamin c);
Be settled to 1000ml with ddH2O, the damping fluid for preparing is preserved in 4 ℃ of environment;
(3) slitting: remove soil that the rape root carries, remove the root part that thirst, flowing water flushing 10-12min, till foreign material are cleaned, with ddH2O flushing 2 times, blot surface-moisture with filter paper after, cut after root cleaned that to be cut into 1-2cm rectangular open;
(4) embathe: embathe the root of cutting into inch strips with above-mentioned damping fluid;
(5) vacuumize: the rape root of cutting into inch strips is put into the container (Erlenmeyer flask is best) of band plug, add the above-mentioned damping fluid of 80-100ml, vacuumize, after pressure is stabilized to 0.07-0.08Mpa, continues to vacuumize the 7-10min(time and be difficult for long);
(6) centrifugal: outwell damping fluid, with filter paper inhale remove the root surface solution after, put it in the syringe, syringe is put into the 50ml centrifuge tube, be 6140rpm at rotating speed, 4 ℃, centrifugal 15min collects the centrifuge tube bottom liquid, is non-plastid extract.
(7) mercaptoethanol solution preparation formula
0.5mol/L Tris-HCL(PH=6.8)12.5％
Glycerine 10%
Beta-mercaptoethanol 5%
ddH2O72.5％
(8) purify:
A. the cold acetone (20 ℃ of precoolings) that step (6) extract is added 5 times of volumes behind-20 ℃ of precipitation 10-20h, is 10000rpm at rotating speed, and 4 ℃, 30min is centrifugal, and the acetone that inclines is deposited in-20 ℃ of volatilization acetone, obtains the lyophilized protein sample;
B. use the lyophilized protein sample of 80ul mercaptoethanol mixed solution dissolving step (8) A, fully dissolving is 10000rpm at rotating speed again, and 4 ℃, 15min is centrifugal, gets supernatant liquor, and is standby-20 ℃ of preservations.
The apoplast protein concentration detects:
Measure the above-mentioned apoplast protein concentration that extracts for three times, result such as table 1 and Fig. 1 with the coomassie brilliant blue staining method.In root and leaf, extracted apoplast albumen as can be seen from the chart respectively, and along with the reduction of temperature, the apoplast protein content increases gradually in the rape body.
No. 6 blades of table 1 Gansu Province oil and root apoplast protein concentration detect
The detection of apoplast antifreeze protein
1.SDS-PAGE electrophoresis
Apoplast antifreeze protein with embodiment extracts for 1 to 3 time carries out protein electrophorese, and the SDS-PAGE of non-plastid albumen adopts the separation gel of 4% concentrated glue and 15%.Applied sample amount is every hole 10-15 μ g albumen, and during electrophoresis, concentrating glue voltage is 80V, treat the bromjophenol blue indicator enter separation gel after voltage transfer to 150V, open condenser system, electrophoresis after about 3 hours indicator near the forward position, the end electrophoresis takes off the gel on the sheet glass, uses ddH
2Behind 0 electrode buffer that rinses out on the gel, the Xylene Brilliant Cyanine G R-250 1.5h that dyes, decolouring is spent the night.
See Fig. 2, among the figure 1. embodiment 1 leaf-October 2. embodiment 2 leaf-November 3. embodiment 3 leaf-December 4. embodiment 1 root-October 5. embodiment 2 root-November 6. embodiment 3 root-December.
Wherein: S is beta-1,3-glucanase, has proved a kind of antifreeze protein in winter rye.
2. mass spectroscopy:
Discrepancy after electrophorogram is chosen processing sampling numbering is seen Fig. 3, cuts glue and gets and a little carry out MALDI-TOF/TOF mass spectrum evaluation (Shanghai Bo Yuan bio tech ltd).
Discrepancy mass spectrum result
Table 2 discrepancy mass spectrum result
From table 2, see, 1,2,3 and 4 differential proteins are respectively glyceraldehyde 3-phosphate dehydro-genase, glyceraldehyde 3-phosphate dehydrogenation, basic dextranase enzyme and β-1, the 3-dextranase, β-1 wherein, the 3-dextranase has had the people to prove antifreeze protein in rye, and other three whether antifreeze protein is still waiting further checking.Below be respectively the mass spectrum result of basic dextranase enzyme and beta-1,3-glucanase;
3. ice crystal is modified effect detection: see Fig. 4, and Fig. 5,
Whether have freeze proof activity for detecting apoplast albumen, adopt the improved phase microscope observation observation root in this laboratory and blade apoplast albumen to have the growth characteristics of ice crystal down.
Observe the ice-crystal growth situation of Gansu Province No. 6 root apoplast crude extracts of oil behind-7 ℃ of constant temperature 40min of not cold domestication under Fig. 4 .10X object lens;
Observe the ice-crystal growth situation of Gansu Province No. 6 root apoplast crude extracts of oil behind-7 ℃ of constant temperature 40min of cold domestication under Fig. 5 .10X object lens, show tangible recrystallization retarding effect.
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. the extracting method of a winter rape root apoplast antifreeze protein is characterized in that including following steps:
(1) sampling: in 10 to December or in January, second, dig out the rape root in the field, in 3-5 minute, put into ice chest, take back the laboratory, be stored in 3-5 ℃ of refrigerator;
(2) extract damping fluid 1000ml and press the preparation of column weight amount volume:
0.05M/L Tris-Hcl 6.057g transfers ph to 8.0;
0.01M/L the EDTA-Na2.2H2O(disodium ethylene diamine tetraacetate) 3.72g;
0.02M/LVC(3.52g vitamin c);
Be settled to 1000ml with ddH2O, the damping fluid for preparing is preserved in 3-5 ℃ of environment;
(3) slitting: remove soil that the rape root carries, remove the root part that thirst, flowing water flushing 10-12min, foreign material are used ddH till cleaning
2O flushing 2 times, blot surface-moisture with filter paper after, cut after root cleaned that to be cut into 1-2cm rectangular open;
(4) embathe: embathe the root of cutting into inch strips with above-mentioned damping fluid;
(5) vacuumize: the rape root of cutting into inch strips is put into the container of band plug, adds the above-mentioned damping fluid of 80-100ml, vacuumize, be stabilized to 0.07-0.08Mpa after, continue to vacuumize 7-10min;
(6) centrifugal: outwell damping fluid, with filter paper inhale remove the root surface solution after, put it in the syringe, syringe is put into the 50ml centrifuge tube, is 6100-6200rpm at rotating speed, 3-5 ℃, centrifugal 14-16min collects the centrifuge tube bottom liquid, is non-plastid extract.
2. the extracting method of winter rape root apoplast antifreeze protein as claimed in claim 1 is characterized in that also including following steps:
(7) mercaptoethanol solution preparation formula
0.5mol/L Tris-HCL(PH=6.8) 12.5％
Glycerine 10%
Beta-mercaptoethanol 5%
ddH2O 72.5％
(8) purify:
A. the cold acetone that step (6) extract is added 5 times of volumes ,-20 ℃ of precoolings behind-20 ℃ of precipitation 10-20h, are 10000rpm at rotating speed, and 4 ℃, 30min is centrifugal, and the acetone that inclines is deposited in-20 ℃ of volatilization acetone, obtains the lyophilized protein sample;
B. use the lyophilized protein sample of mercaptoethanol mixed solution dissolving step (8) A, fully dissolving is 10000rpm at rotating speed again, and 4 ℃, 15min is centrifugal, gets supernatant liquor, and is standby-20 ℃ of preservations.
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| CN105628471A (en) * | 2016-01-25 | 2016-06-01 | 甘肃农业大学 | Winter rape lamina apoplast antifreeze protein standard sample preparation method and protein lysis buffer |
| CN110483616A (en) * | 2019-07-31 | 2019-11-22 | 中国农业科学院农产品加工研究所 | The method of the apoplast effect protein of pathogen secretion is separated from the plant tissue of infection pathogen |
| CN111471082A (en) * | 2019-01-24 | 2020-07-31 | 华东师范大学 | Method for extracting radish seedling apoplast antifreeze protein |
| CN114891081A (en) * | 2022-05-09 | 2022-08-12 | 河南工业大学 | Dragon juniper leaf antifreeze protein and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628471A (en) * | 2016-01-25 | 2016-06-01 | 甘肃农业大学 | Winter rape lamina apoplast antifreeze protein standard sample preparation method and protein lysis buffer |
| CN111471082A (en) * | 2019-01-24 | 2020-07-31 | 华东师范大学 | Method for extracting radish seedling apoplast antifreeze protein |
| CN110483616A (en) * | 2019-07-31 | 2019-11-22 | 中国农业科学院农产品加工研究所 | The method of the apoplast effect protein of pathogen secretion is separated from the plant tissue of infection pathogen |
| CN110483616B (en) * | 2019-07-31 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Method for separating apoplast effector protein secreted by pathogenic bacteria from plant tissue infected by pathogenic bacteria |
| CN114891081A (en) * | 2022-05-09 | 2022-08-12 | 河南工业大学 | Dragon juniper leaf antifreeze protein and preparation method thereof |
| CN114891081B (en) * | 2022-05-09 | 2024-02-23 | 河南工业大学 | Dragon cypress She Kangdong protein and preparation method thereof |
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