CN110257354B - Efficient purification method of apple stylobate S-RNase - Google Patents
Efficient purification method of apple stylobate S-RNase Download PDFInfo
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Abstract
The invention relates to an efficient purification method of apple style S-RNase, which comprises the steps of picking flowers from the big balloon period to the full-bloom period of apples, picking and quickly freezing the flower style tissues, placing the flower style tissues in a 1.5ml EP tube, immersing acetone, grinding the flower style tissues by a glass pestle under the condition of avoiding air contact, removing pigments, carrying out protein extraction Buffer heavy suspension on precipitates, filtering impurities by a 0.22 mu m nitrocellulose membrane, then loading on a cation exchange column, washing the cation exchange column by a Buffer A, and then carrying out high-salt gradient elution, thus obtaining the high-purity S-RNase protein. The invention provides a method for efficiently obtaining the endogenous S-RNase protein of an apple stylobate, which shortens the purification time and improves the protein yield. The method provided by the invention can improve the research efficiency of the self-incompatibility molecular mechanism of the apple, promote the research process of the self-incompatibility of the apple and finally serve the breeding of the apple.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a high-efficiency purification method of apple flower column S-RNAse.
Background
The apple is the first fruit in China, has high nutritive value, is popular with consumers, but due to the self-incompatibility of the apple, a variety different from the S gene of a main cultivated variety needs to be configured as a pollinated variety in the actual production process, and the yield can be ensured only by pollination through flower visiting insects or artificial pollination in the flowering phase, so that the production cost is increased, and the management difficulty is increased. The cultivated apple variety with self-bearing capability is an effective means for realizing simplified cultivation of fruit trees. To achieve this goal, it is necessary to have an in-depth knowledge of the molecular mechanism of apple self-incompatibility. Compared with model plants, the research on the molecular mechanism of apple self-incompatibility is lagged, but the style S-RNase protein is a core factor for controlling the self-incompatibility. Almost all apple self-incompatibility related studies are developed around S-RNase at the present stage, so obtaining high-purity S-RNase protein with nuclease activity is one of the keys for developing apple self-incompatibility studies.
Although a style S-RNase protein purification system is initially established as early as 2005, the purification process consumes long time and has low product yield, and the research speed of apple self-incompatibility is severely limited. Through years of research, two factors are found to influence the purification efficiency of the S-RNase protein, on one hand, the apple flower column contains impurities such as pigment and the like, on the other hand, the S-RNase protein has glycosylation modification and can be oxidized and promote the degradation of the S-RNase when contacting air, and on the basis, the purification method is improved and simplified, so that the extraction time is shortened from 3 to 4 days to 1 day, and the protein yield is improved by 5 to 10 times. Greatly improving the purification efficiency.
The invention hopes to promote the research of the self-incompatibility mechanism of the apple by providing the efficient purification method of the style S-RNase, and provide help for the breeding and research of the apple.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for efficiently purifying apple flower column S-RNase.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:
an efficient purification method of apple style S-RNase comprises the following steps:
s1 collecting style tissues: collecting apple flowers from the big balloon period to the full bloom period, removing the flower pillar tissues by using forceps, immediately putting the removed flower pillar tissues into liquid nitrogen for quick freezing, and finally storing the flower pillar tissues in an ultra-low temperature refrigerator at minus 80 ℃.
S2 extraction of crude protein from the floral tissue:
s21 Add 600. mu.l of precooled acetone into a 1.5ml EP tube, add style tissue to just immerse the style tissue, typically 20 to 25 apple styles, and crush the style tissue in acetone to avoid exposing the style tissue to air and degrading S-RNase.
S22 the crushed flower column tissue is shaken for 30S, ice-cooled for 15min, and centrifuged at 15000rpm at 4 deg.C for 15min to remove pigment from the material and prevent contamination of the cation exchange column.
S23 discarding the supernatant, and air-drying the precipitate in a fume hood or a clean bench.
S24 protein extraction Buffer was added and shaken for 30S for resuspension of the pellet, ice-cooled for 15min, and centrifuged at 15000rpm for 15min at 4 ℃.
S25 the supernatant was aspirated and filtered through acetate membrane into a fresh clean EP tube to obtain a crude protein extract sample.
S3 sample extraction using cation exchange column crude protein:
s31, the cation exchange column is connected to the protein purification system, the tube A of the pump of the connexin purification system A is inserted into Buffer A, the tube B of the pump of the connexin purification system B is inserted into Buffer B, 2ml of Buffer A is injected into the cation exchange column, 20% ethanol in the cation exchange column is discharged, and the cation exchange column is filled with Buffer A.
S32 the loading ring was washed repeatedly 4-5 times with Buffer A while 2ml of Buffer A was injected into the cation exchange column, and then the crude protein extract sample was injected into the loading ring from the loading hole.
S33 starting the purification procedure of cation exchange column in protein purification system, before B pump elution, the time for A pump to clean cation exchange column can be increased, the column washing time is 20-25 min, to reduce impurity protein pollution.
S34, collecting the corresponding eluent at the elution peak, and removing salt ions from the eluent by dialysis or a protein concentration column to obtain high-purity S-RNase;
s35 sample preservation: adding 10-20% glycerol into the high-purity S-RNase obtained in step S34, and storing in a refrigerator at-80 deg.C for use or performing SDS-PAGE electrophoresis or Western blot experiment.
S4 cleaning and storing the cation exchange column;
injecting Buffer B into the cation exchange column, washing to remove foreign protein, washing with double distilled water until the conductivity line and ultraviolet leveling are unchanged, and filling the cation exchange column with 20% ethanol.
Based on the above scheme, the step S21 of grinding the floral pillar tissue in acetone is specifically grinding the floral pillar tissue in acetone with a glass pestle.
On the basis of the above scheme, the cellulose acetate membrane in step S25 is a cellulose acetate membrane with Millipore diameter of 0.22 um.
On the basis of the above scheme, the protein extraction Buffer described in step S24 includes: 50mM PBS pH7.0, 1% protease inhibitor cocktail, 1% DTT, 0.5% NP-40.
Based on the above protocol, Buffer A in step S31 is 50mM PBS (pH 7.0), and Buffer B is a mixed solution of 50mM PBS (pH 7.0) and 1M NaCl.
Based on the above protocol, the elution peak described in step S34 generally occurs at a salt conductivity between 13 and 15.
On the basis of the scheme, the model of the cation exchange column in the step S31 is MonoS 5/50 GL; the protein purification system is an Akta system of GE company.
The invention has the beneficial effects that:
the method can efficiently obtain the apple style endogenous S-RNase with nuclease activity, has the characteristics of high yield and less time consumption, and can complete the experiment within one day. However, the method of purifying S-RNase completely referring to herbaceous plants or the method of purifying apple S-RNase without improvement is often difficult to obtain protein or the yield is only about 10% -20% of the present invention, and the experiment usually requires 3-4 days. Therefore, the method has important significance for efficiently carrying out the research on the self-incompatibility molecular mechanism of the apples.
Drawings
The invention has the following drawings:
FIG. 1(A) is a schematic diagram of centrifuge tubes and glassware used for grinding the floral pillar tissue in the purification experiment.
FIG. 1(B) is a schematic diagram showing the usage of centrifuge tubes and glassware for grinding the floral pillar tissue in the purification experiment.
FIG. 2 is a schematic diagram showing the SDS-PAGE electrophoresis of purified "Gala" apple floral pillar S-RNase.
FIG. 3 is a schematic diagram showing the results of SDS-PAGE electrophoresis of purified S-RNase from apple' S floral pillars.
FIG. 4 is a schematic diagram showing the experimental results of Western blot experiment carried out on purified Gala apple columna S-RNase.
FIG. 5 is a schematic diagram showing the experimental results of Western blot experiment carried out on the purified 'Hui' apple floral pillar S-RNase.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings 1 to 3.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The concentrations of the following components are the final concentrations of the above reagents unless otherwise specified.
Example 1 efficient purification of Gala' apple column S-RNase.
1, flower column collection: collecting Gala apple flowers from a big balloon period to a full-bloom period in spring, removing the style tissues by using forceps, immediately placing the removed style tissues into liquid nitrogen for quick freezing, and finally storing the style tissues in an ultra-low temperature refrigerator at minus 80 ℃.
2, extracting crude protein of the floral pillar tissue:
(1) add 600. mu.l of pre-cooled acetone to a 1.5ml EP tube, add the floral pillar tissue to allow the acetone to just submerge the floral pillar tissue, and crush the floral pillar tissue in acetone with a glass pestle, as shown in FIG. 1, to avoid exposing the floral pillar tissue to air for degradation of S-RNase.
(2) The ground columella tissue was shaken briefly, iced for 15min, and centrifuged at 15000rpm at 4 ℃ for 15min to remove pigments from the material and prevent contamination of the cation exchange column.
(3) The supernatant was discarded, the pellet was quickly air-dried in a fume hood or clean bench, and the pellet was resuspended in protein extraction Buffer (50mM pH7.0 PBS; 1% cocktail (Roche); 1% DTT; 0.5% NP-40).
(4) The precipitate was shaken briefly, iced for 15min, and centrifuged at 15000rpm at 4 ℃ for 15 min.
(5) The supernatant was aspirated and filtered through Millipore 0.22um acetate membrane into a fresh clean EP tube to obtain a crude protein extract sample.
3 purifying crude protein by using a cation exchange column to extract a sample:
(1) a cation exchange column MonoS 5/50GL (other types of cation exchange columns are also available) was inserted into a protein purification system (for example, Akta system from GE Co.), a tube A connected to a pump A was inserted into Buffer A (50mM pH7.0PBS), a tube B connected to a pump B was inserted into Buffer B (50mM pH7.0PBS +1M NaCl), 2ml of Buffer A was injected into the cation exchange column, 20% ethanol in the cation exchange column was discharged, and the cation exchange column was filled with Buffer A.
(2) While injecting 2ml of Buffer A into the cation exchange column, the loading loop was repeatedly washed with Buffer A4-5 times, after which the crude protein extract sample was injected from the loading well into the loading loop.
(3) The purification procedure corresponding to the cation exchange column is started, but the time for cleaning the cation exchange column by the pump A can be prolonged before the pump B is eluted, so that the impurity protein pollution is reduced.
(4) Eluting peak usually appears in salt conductivity between 13 and 15, collecting corresponding eluate at eluting peak, removing salt particles by dialysis or protein concentration column, adding 10-20% final concentration glycerol, and storing at-80 deg.C in refrigerator for use or SDS-PAGE electrophoresis detection or Western blot experiment.
(5) After purification, Buffer B is injected into the cation exchange column, foreign proteins are washed off, the cation exchange column is washed by double distilled water until the conductivity line and the ultraviolet leveling do not change, and finally the cation exchange column is filled with 20 percent ethanol.
By adopting the method, about 100 mu g of S-RNase protein can be extracted from about 60 flowers and columns of flowers of the Gala apple within one day.
Example 2 efficient purification of' Whiteflower apple column S-RNase.
1, collecting the floral pillar tissues: collecting 'Hui' apple flowers from a big balloon period to a full-bloom period in spring, removing the flower pillar tissues by using forceps, immediately putting the removed flower pillar tissues into liquid nitrogen for quick freezing, and finally storing the flower pillar tissues in an ultra-low temperature refrigerator at minus 80 ℃.
2, extracting crude protein of the floral pillar tissue:
(1) add 600. mu.l of pre-cooled acetone to a 1.5ml EP tube, add the floral pillar tissue to allow the acetone to just submerge the floral pillar tissue, and crush the floral pillar tissue in acetone with a glass pestle, as shown in FIG. 1, to avoid exposing the floral pillar tissue to air for degradation of S-RNase.
(2) The ground columella tissue was shaken briefly, iced for 15min, and centrifuged at 15000rpm at 4 ℃ for 15min to remove pigments from the material and prevent contamination of the cation exchange column.
(3) The supernatant was discarded, the pellet was quickly air-dried in a fume hood or clean bench, and the pellet was resuspended in protein extraction Buffer (50mM pH7.0 PBS; 1% cocktail (Roche); 1% DTT; 0.5% NP-40).
(4) The precipitate was shaken briefly, iced for 15min, and centrifuged at 15000rpm at 4 ℃ for 15 min.
(5) The supernatant was aspirated and filtered through Millipore 0.22um acetate membrane into a fresh clean EP tube to obtain a crude protein extract sample.
3 purifying crude protein by using a cation exchange column to extract a sample:
(1) a cation exchange column MonoS 5/50GL (other types of cation exchange columns are also available) was inserted into a protein purification system (for example, Akta system from GE Co.), a tube A connected to a pump A was inserted into Buffer A (50mM pH7.0PBS), a tube B connected to a pump B was inserted into Buffer B (50mM pH7.0PBS +1M NaCl), 2ml of Buffer A was injected into the cation exchange column, 20% ethanol in the cation exchange column was discharged, and the cation exchange column was filled with Buffer A.
(2) While injecting 2ml of Buffer A into the cation exchange column, the loading loop was repeatedly washed with Buffer A4-5 times, after which the crude protein extract sample was injected from the loading well into the loading loop.
(3) The purification procedure corresponding to the cation exchange column is started, but the time for cleaning the cation exchange column by the pump A can be prolonged before the pump B is eluted, so that the impurity protein pollution is reduced.
(4) Eluting peak usually appears in salt conductivity between 13 and 15, collecting corresponding eluate at eluting peak, removing salt particles by dialysis or protein concentration column, adding 10-20% final concentration glycerol, and storing at-80 deg.C in refrigerator for use or SDS-PAGE electrophoresis detection or Western blot experiment.
(5) After purification, Buffer B is injected into the cation exchange column, foreign proteins are washed off, the cation exchange column is washed by double distilled water until the conductivity line and the ultraviolet leveling do not change, and finally the cation exchange column is filled with 20 percent ethanol.
By the method, about 90 mu g of S-RNase protein can be extracted from about 60 'Hui' apple flower columns in one day.
The above examples illustrate the efficient production of S-RNase protein from the flower column of Rosaceae fruit trees by the improved S-RNase purification method.
Those not described in detail in this specification are within the skill of the art.
Claims (3)
1. An efficient purification method of apple style S-RNase is characterized by comprising the following steps:
s1 collecting style tissues: collecting apple flowers from a big balloon period to a full-bloom period, removing a style tissue by using a forceps, immediately putting the removed style tissue into liquid nitrogen for quick freezing, and finally storing to a-80 ℃ ultra-low temperature refrigerator;
s2 extraction of crude protein from the floral tissue:
s21 adding 600 μ l precooled acetone into 1.5ml EP tube, adding style tissue to make acetone just submerge the style tissue, grinding the style tissue in acetone to avoid the style tissue from contacting air to degrade S-RNase;
s22 shaking the crushed flower column tissue for 30S, ice-cooling for 15min, and centrifuging at 15000rpm at 4 deg.C for 15min to remove pigment from the material and prevent contamination of cation exchange column;
s23, discarding the supernatant, and airing the precipitate in a fume hood or a super-clean workbench;
s24 adding protein extraction Buffer, shaking for 30S, resuspending the precipitate, ice-cooling for 15min, and centrifuging at 15000rpm at 4 deg.C for 15 min;
s25 sucking the supernatant, filtering the supernatant into a new clean EP tube by using an acetate fiber membrane to obtain a crude protein extraction sample;
s3 purification of crude protein extract samples using cation exchange column:
s31, the cation exchange column is connected to the protein purification system, the tube A of the pump of the protein purification system A is inserted into the Buffer A, the tube B of the pump of the protein purification system B is inserted into the Buffer B, 2ml of Buffer A is injected into the cation exchange column, 20% ethanol in the cation exchange column is discharged, and the cation exchange column is filled with the Buffer A;
s32, injecting 2ml of Buffer A into the cation exchange column, and repeatedly washing the sample loading ring for 4-5 times by using the Buffer A, and then injecting the crude protein extraction sample into the sample loading ring from the sample loading hole;
s33 starting the purification procedure of the cation exchange column in the protein purification system;
s34, collecting the corresponding eluent at the elution peak, and removing salt ions from the eluent by dialysis or a protein concentration column to obtain high-purity S-RNase;
s35 sample preservation: adding 10-20% of glycerol into the high-purity S-RNase obtained in the step S34, and storing in a refrigerator at-80 ℃ for later use or performing SDS-PAGE electrophoresis or Western blot experiment;
s4 cleaning and storing the cation exchange column;
injecting Buffer B into the cation exchange column, washing to remove foreign proteins, washing with double distilled water until the conductivity line and ultraviolet leveling do not change, and filling the cation exchange column with 20% ethanol;
crushing the floral pillar tissue in acetone in step S21, specifically crushing the floral pillar tissue in acetone with a glass pestle;
the protein extraction Buffer of step S24 includes: 50mM PBS pH7.0, 1% protease inhibitor cocktail, 1% DTT, 0.5% NP-40;
the acetate fiber membrane of the step S25 is an acetate fiber membrane with Millipore aperture of 0.22 um;
the Buffer A in the step S31 is 50mM PBS (pH7.0);
buffer B described in step S31 was a mixed solution of 50mM PBS (pH7.0) and 1M NaCl.
2. The method for efficiently purifying apple style S-RNase according to claim 1, wherein the elution peak of step S34 appears in a salt conductivity range of 13 to 15.
3. The method for efficiently purifying apple style S-RNase according to claim 1, wherein the cation exchange column of step S31 is MonoS 5/50 GL; the protein purification system is an Akta system of GE company.
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| CN1389567A (en) * | 2002-07-15 | 2003-01-08 | 南京农业大学 | Ribonuclease extracting process |
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| CN1389567A (en) * | 2002-07-15 | 2003-01-08 | 南京农业大学 | Ribonuclease extracting process |
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| Characterization of Ribonuclease Activity of Three S-Allele-Associated Proteins of Petunia inflata;Anuradha Singh et al.;《Plant Physiol.》;19910531;第96卷(第1期);61-68 * |
| In Vitro Function of S RNases in Lycopersicon peruvianum;Myung Hee Kim et al.;《Mol. Cells》;20011231;第12卷(第3期);329-335 * |
| 红星苹果花柱S-核酸酶的分离与纯化;李天忠 等;《农业生物技术学报》;20051030;第13卷(第5期);568-571 * |
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