AU2020101821A4 - Method for extracting apoplastic antifreeze proteins from winter rape roots - Google Patents

Abstract

The present invention relates to an antifreeze protein extraction method and particularly relates to a method for extracting apoplastic antifreeze proteins from winter rape roots. The apoplastic antifreeze proteins are successfully extracted from cold-induced winter rape roots by utilizing the method. The method for extracting apoplastic antifreeze proteins from winter rape roots includes the following steps: (1) sampling; (2) preparing an extraction buffer solution; (3) slitting; (4) rinsing; (5) vacuumizing; and (6) centrifuging. According to the present invention, the apoplastic antifreeze proteins are successfully rapidly extracted from the cold-induced winter rape roots, and a technical support is provided for further study of a cold resistance mechanism of winter rapes. Drawings of Description 150 C 100 " Root "0 (3 "' Leaf 0 First time Second time Third time FIG. 1 1

Description

Drawings of Description

150 C

100 " Root "0 (3 "'Leaf

0 First time Second time Third time

FIG. 1

Description

METHOD FOR EXTRACTING APOPLASTIC ANTIFREEZE PROTEINS FROM WINTER RAPE ROOTS

Technical Field

The present invention relates to an antifreeze protein extraction method and particularly relates to a method for extracting apoplastic antifreeze proteins from winter rape roots. The apoplastic antifreeze proteins are successfully extracted from cold-induced winter rape roots by utilizing the method.

Background

Low temperature is one of the major environmental factors that influence normal growth and development of plants in northern China. When plants are under low-temperature stress, a series of physio-biochemical changes occur in bodies to adapt to the low-temperature stress, and multiple proteins-low-temperature-induced proteins will be synthesized and secreted. Antifreeze proteins (AFPs) belong to one category in the low-temperature-induced proteins, may be produced in different organs of the plants, such as roots, stalks and leaves, and may also exist in callus under tissue culture conditions, suspension culture cells and suspension culture solutions. The AFPs have very complex positions in cells, and may exist in multiple organelles such as intercellular space, cell wall, cell nucleus, cytomembrane and cytoplasm. At present, it is discovered that, the AFPs separated from freezing-tolerant plants have higher content in apoplast, and have an ice crystal form effect, a thermal hysteresis (TH) effect and a recrystallization inhibition effect. The three characteristics of the AFPs show that, the AFPs are directly related to formation of ice crystals at a low temperature. Therefore, the AFPs have directness on protection of the plants, and are a major material basis of cold resistance of the plants. The antifreeze proteins (AFPs) were first discovered in Tenebrio molitor

Description

larvae in 1964. Through studies for half a century, the AFPs have been discovered in multiple organisms such as fishes, insects, plants, bacteria and fungi nowadays. So far, researches on the AFPs in the fishes and insects have been very thorough and systematic, while the AFPs in the plants are discovered late. Griffith et al. obtained and partially purified the AFPs from leaf apoplast of Secalecereale that is subjected to cold acclimation and can tolerate extracellular freezing for the first time in 1992, thereby starting the researches of the AFPs in the plants. Later, the AFPs were also discovered in many plants such as Ammopiptanthus monglicus, alpine vegetation Rhodiola tangutica, Daucus carota, Polygonumviviparum and winter wheat. Winter rape is an important crop in the north. Particularly the winter rapes in the north may turn green, have bolting, flower and produce seeds after long and cold winter. Root is the unique overwintering organ of the winter rape in the winter. Therefore, researching the AFPs of the roots is one of the effective ways for identifying cold resistance of the winter rapes in the north. There is no mature method of researching the AFPs of the rapes. The present invention provides a method for extracting apoplastic antifreeze proteins from winter rape roots. The apoplastic proteins can be rapidly and effectively extracted from the winter rape roots by the method, and a technical support is provided for research of a cold resistance mechanism of the winter rapes.

Summary

A purpose of the present invention is to avoid defects in the prior art so as to provide a method for extracting apoplastic antifreeze proteins from winter rape roots. According to the present invention, the apoplastic antifreeze proteins are successfully extracted from cold-induced winter rape roots, and a technical support is provided for further study of a cold resistance mechanism of the winter rapes.

Description

In order to realize the above purpose, the present invention adopts the following technical solutions: the method for extracting apoplastic antifreeze proteins from winter rape roots mainly includes the following steps: (1) sampling: digging out rape roots in the field from October to December or to January of the following year; putting the rape roots into an ice box within 3-5 minutes; taking the ice box back to a laboratory; and preserving the ice box in a refrigerator at 3-5°C; (2) preparing 1000 ml of an extraction buffer solution according to weight volumes as follows: 6.057 g of 0.05 M/L of Tris-HCl having a pH value regulated to 8.0; 3.72 g of 0.01 M/L of EDTA-Na2.2H 20 (ethylenediamine tetraacetic acid disodium salt); 3.52 g of 0.02 M/L of VC (vitamin c); fixing the volume to 1000 ml with ddH 20, and preserving the prepared buffer solution in an environment of 3-5°C; (3) slitting: eliminating soil carried by the rape roots; removing dry dead parts of the roots; rinsing the roots with running water for 10-12 min until sundries are cleaned off; rinsing with ddH 20twice; sucking surface moisture with filter paper; cleaning the roots; and cutting open into strips of 1-2 cm; (4) rinsing: rinsing the roots which are cut into strips with the above buffer solution; (5) vacuumizing: putting the rape roots which are cut into strips into a container with stopper (a conical flask is preferred); adding 80-100 ml of the above buffer solution; vacuumizing; and after a pressure is stabilized at 0.07-0.08 Mpa, continuously vacuumizing for 7-10 min, wherein time should not be too long; (6) centrifuging: pouring the buffer solution; sucking the solution on the root surfaces with filter paper; adding the solution into an injector; putting the injector into a 50 ml centrifuge tube; performing centrifugation at a rotation speed of

Description

6100-6200 rpm at 3-5°C for 15 min; and collecting liquid at the bottom of the centrifuge tube, that is an apoplastic extract; The method for extracting the apoplastic antifreeze proteins from winter rape roots further includes the following steps: (7) preparing a mercaptoethanol solution with a formula as follows: 12.5% of 0.5 mol/L Tris-HCl (pH=6.8) 10% of glycerin 5% of p-mercaptoethanol 72.5% of ddH20; (8) purifying: A: adding the extract in the step (6) into cold acetone in an amount of 5 times of the volume; precooling at -20°C; precipitating at -20°C for 10-20 h; performing centrifugation at a rotation speed of 10000 rpm at 4°C for 30 min; pouring out the acetone; and precipitating at -20°C to volatilize the acetone so as to obtain a freeze-dried protein sample; B: dissolving the freeze-dried protein sample in the A of the step (8) with a mercaptoethanol mixed solution; performing full dissolving; performing centrifugation at the rotation speed of 10000 rpm at 4°C for 15 min; taking the supernatant; and preserving the supernatant at -20°C for later use. The present invention has beneficial effects as follows: according to the present method, with respect to the opinions given by some scholars that the proteins cannot be extracted from the rape roots or are difficult to be extracted from the rape roots, through continuous trials and errors, the apoplastic antifreeze proteins are successfully rapidly extracted from the cold-induced winter rape roots through the present invention, and the technical support is provided for further study of the cold resistance mechanism of the winter rapes.

Description of Drawings

Description

Fig. 1 shows concentration detection of apoplastic proteins of leaves and roots of Longyou #6; Fig. 2 is an electrophoretogram of apoplastic proteins of leaves and roots of Longyou #6 in different phases; Fig. 3 shows sampling numbers of mass spectrum identification difference points; Fig. 4 shows an ice crystal growth condition of apoplastic crude extract of roots of non-cold-acclimated Longyou #6 at a constant temperature of -7Cwithin minutes observed under a loX objective lens; and Fig. 5 shows an ice crystal growth condition of apoplastic crude extract of roots of cold-acclimated Longyou #6 at a constant temperature of -7C within 40 minutes observed under a 1OX objective lens, which presents an obvious recrystallization inhibition effect.

Detailed Description

Principles and features of the present invention are described below. Listed embodiments are merely used for illustrating the present invention, rather than limitating the scope of the present invention. Embodiment 1: first sampling, and extracting apoplastic proteins from roots of winter rapes Longyou #6: (1) sampling: roots of Longyou #6 were dug out in the field on October 5; the roots were put into an ice box within 3-5 minutes; the ice box was taken back to a laboratory; and the ice box was preserved in a refrigerator at 4°C; (2) 1000 ml of extraction buffer solution was prepared according to weight volumes as follows: 6.057 g of 0.05 M/L of Tris-HCl having a pH value regulated to 8.0; 3.72 g of 0.01 M/L of EDTA-Na2.2H 20 (ethylenediamine tetraacetic acid disodium salt); 3.52 g of 0.02 M/L of VC (vitamin c);

Description

the volume was fixed to 1000 ml with ddH 20, and the prepared buffer solution was preserved in an environment of 4°C; (3) slitting: soil carried by the rape roots was eliminated; dry dead parts of the roots were removed; the roots were rinsed with running water for 10-12 min until sundries were cleaned off; the roots were rinsed with ddH 20 twice; surface moisture was sucked with filter paper; and the roots were cleaned, and cut open into strips of 1-2 cm; (4) rinsing: the roots which were cut into strips were rinsed with the above buffer solution, wherein the roots were preferably rinsed for more than two times; (5) vacuumizing: the rape roots which were cut into strips were put into a container with a stopper; a conical flask was preferred; 80-100 ml of the above buffer solution was added; the container was vacuumized; and after a pressure was stabilized at 0.07-0.08 Mpa, the container was vacuumized continuously for 7-10 min, wherein time should not be too long; (6) centrifuging: the buffer solution was poured; the solution on the root surfaces was sucked with filter paper; the solution was added into an injector; the injector was put into a 50 ml centrifuge tube; centrifugation was performed at a rotation speed of 6140 rpm at 4°C for 15 min; and liquid at the bottom of the centrifuge tube was collected, that is an apoplastic extract; (7) a mercaptoethanol solution with a following formula was prepared: 12.5% of 0.5 mol/L Tris-HCl (pH=6.8) 10% of glycerin 5% of p-mercaptoethanol 72.5% of ddH20; (8) purifying: A: the extract in the step (6) was added into cold acetone in an amount of 5 times of the volume; the mixture was precooled at -20°C, precipitated at -20°C for -20 h, and centrifuged at a rotation speed of 10000 rpm at 4°C for 30 min; the

Description

acetone was poured out; and precipitation was performed at -20°C to volatilize the acetone so as to obtain a freeze-dried protein sample; B: the freeze-dried protein sample in the A of the step (8) was dissolved with pl of mercaptoethanol mixed solution, was fully dissolved and centrifuged at the rotation speed of 10000 rpm at 4°C for 15 min; and the supernatant was taken, and preserved at -20°C for later use. Embodiment 2: second sampling, and extracting apoplastic proteins from roots of Longyou #6: (1) sampling: roots of Longyou #6 were dug out in the field on November 15; the roots were put into an ice box within 3-5 minutes; the ice box was taken back to a laboratory; and the ice box was preserved in a refrigerator at 4°C; (2) 1000 ml of an extraction buffer solution was prepared according to weight volumes as follows: 6.057 g of 0.05 M/L of Tris-HCl having a pH value regulated to 8.0; 3.72 g of 0.01 M/L of EDTA-Na2.2H 20 (ethylenediamine tetraacetic acid disodium salt); 3.52 g of 0.02 M/L of VC (vitamin c); the volume was fixed to 1000 ml with ddH20, and the prepared buffer solution was preserved in an environment of 4°C; (3) slitting: soil carried by the rape roots was eliminated; dry dead parts of the roots were removed; the roots were rinsed with running water for 10-12 min until sundries were cleaned off; the roots were rinsed with ddH 20 twice; surface moisture was sucked with filter paper; and the roots were cleaned, and cut open into strips of 1-2 cm; (4) rinsing: the roots which were cut into strips were rinsed with the above buffer solution; (5) vacuumizing: the rape roots which were cut into strips were put into a container with a stopper; a conical flask was preferred; 80-100 ml of the above

Description

buffer solution was added; the container was vacuumized; and after a pressure was stabilized at 0.07-0.08 Mpa, the container was vacuumized continuously for 7-10 min, wherein time should not be too long; (6) centrifuging: the buffer solution was poured; the solution on the root surfaces was sucked with filter paper; the solution was added into an injector; the injector was put into a 50 ml centrifuge tube; centrifugation was performed at a rotation speed of 6140 rpm at 4°C for 15 min; and liquid at the bottom of the centrifuge tube was collected, that is an apoplastic extract; (7) a mercaptoethanol solution with a following formula was prepared: 12.5% of 0.5 mol/L Tris-HCl (pH=6.8) 10% of glycerin 5% of p-mercaptoethanol 72.5% of ddH20; (8) purifying: A: the extract in the step (6) was added into cold acetone in an amount of 5 times of the volume (precooled at -20°C); the mixture was precipitated at -20°C for -20 h, and centrifuged at a rotation speed of 10000 rpm at 4°C for 30 min; the acetone was poured out; and precipitation was performed at -20°C to volatilize the acetone so as to obtain a freeze-dried protein sample; B: the freeze-dried protein sample in the A of the step (8) was dissolved with pl of mercaptoethanol mixed solution, fully dissolved and centrifuged at the rotation speed of 10000 rpm at 4°C for 15 min; and the supernatant was taken, and preserved at -20°C for later use. Embodiment 3: third sampling, and extracting apoplastic proteins from roots and leaves of Longyou #6: (1) sampling: rape roots were dug out in the field on December 1; the rape roots were put into an ice box within 3-5 minutes; the ice box was taken back to a laboratory; and the ice box was preserved in a refrigerator at 4°C;

Description

(2) 1000 ml of extraction buffer solution was prepared according to weight volumes as follows: 6.057 g of 0.05 M/L of Tris-HCl having a pH value regulated to 8.0; 3.72 g of 0.01 M/L of EDTA-Na2.2H 20 (ethylenediamine tetraacetic acid disodium salt); 3.52 g of 0.02 M/L of VC (vitamin c); the volume was fixed to 1000 ml with ddH20, and the prepared buffer solution was preserved in an environment of 4°C; (3) slitting: soil carried by the rape roots was eliminated; dry dead parts of the roots were removed; the roots were rinsed with running water for 10-12 min until sundries were cleaned off; the roots were rinsed with ddH 20 twice; surface moisture was sucked with filter paper; and the roots were cleaned, and cut open into strips of 1-2 cm; (4) rinsing: the roots which were cut into strips were rinsed with the above buffer solution; (5) vacuumizing: the rape roots which were cut into strips were put into a container with a stopper (a conical flask was preferred); 80-100 ml of the above buffer solution was added; the container was vacuumized; and after a pressure was stabilized at 0.07-0.08 Mpa, the container was vacuumized continuously for 7-10 min (time should not be too long); (6) centrifuging: the buffer solution was poured; the solution on the root surfaces was sucked with filter paper; the solution was added into an injector; the injector was put into a 50 ml centrifuge tube; centrifugation was performed at a rotation speed of 6140 rpm at 4°C for 15 min; and liquid at the bottom of the centrifuge tube was collected, that is an apoplastic extract; (7) a mercaptoethanol solution with a following formula was prepared: 12.5% of 0.5 mol/L Tris-HCl (pH=6.8) 10% of glycerin

Description

5% of p-mercaptoethanol 72.5% of ddH20; (8) purifying: A: the extract in the step (6) was added into cold acetone in an amount of 5 times of the volume (precooled at -20°C); the mixture was precipitated at -20°C for -20 h and centrifuged at a rotation speed of 10000 rpm at 4°C for 30 min; the acetone was poured out; and precipitation was performed at -20°C to volatilize the acetone so as to obtain a freeze-dried protein sample; B: the freeze-dried protein sample in the A of the step (8) was dissolved with ul of mercaptoethanol mixed solution, fully dissolved and centrifuged at the rotation speed of 10000 rpm at 4°C for 15 min; and the supernatant was taken, and preserved at -20°C for later use. Concentration detection of apoplastic proteins: Concentrations of the extracted apoplastic proteins in the above three times are determined by Coomassie brilliant blue staining, and results are shown in Table 1 and Fig. 1. It can be seen from the figures that, the apoplastic proteins are respectively extracted from the roots and the leaves. Moreover, as the temperature is reduced, the content of the apoplastic proteins in rape bodies is gradually increased. Table 1 Concentration detection of apoplastic proteins of leaves and roots of Longyou #6

First time (pg/g FW) Second time (pg/g FW) Third time (pg/g FW)

Root 14.67 79.14 120.02

Leaf 13.89 92.31 113.38

Detection of apoplastic antifreeze proteins: 1. SDS-PAGE electrophoresis

Description

The apoplastic antifreeze proteins extracted in embodiments 1-3 were subjected to protein electrophoresis; 4% of spacer gel and 15% of separation gel were adopted in SDS-PAGE electrophoresis of the apoplastic proteins; the sample loading quantity was 10-15 pg protein per well; during electrophoresis, the voltage of the spacer gel was 80 V; after a bromophenol blue indicator entered the separation gel, the voltage was regulated to 150 V; a condensing system was started; within about 3 hours after electrophoresis, the indicator approached the leading edge; electrophoresis was ended, and gel on a glass plate was removed; an electrode buffer solution on the gel was washed away with ddH 20; Coomassie brilliant blue R-250 staining was performed for 1.5 h; and decolorizing was performed overnight. Referring to Fig. 2, in Fig. 1, leaf in embodiment 1- October 2; leaf in embodiment 2- November 3; leaf in embodiment 3- December 4; root in embodiment 1- October 5; root in embodiment 2- November 6; and root in embodiment 3: December. S is p-1,3-dextranase and has been proved as an antifreeze protein in Secalecereale. 2. Mass spectrometry: Treated difference point sampling numbers were selected on the electrophoretogram; referring to Fig. 3, the gel was cut to take points for performing MALDI-TOF/TOF mass spectrum identification (Shanghai Boyuan Biotechnology Co., Ltd.) Table 2 Difference point mass spectrum results

No. NCBI accession No. Protein name Species Score Sequence coverage(%)

1 gi 120675 glyceraldehyde-3-phosphate dehydrogenase Sinapis alba 433 34

2 gi 120675 glyceraldehyde-3-phosphate dehydrogenase Sinapis alba 332 32

3 gi 118763538 Basic glucanase Brassicajuncea 315 53

Description

4 gi 62361691 p-1,3-dextranase Brassicarapasubsp. chinensis 348 48

It can be seen from Table 2 that, the differential proteins 1, 2, 3 and 4 are respectively glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, basic glucanase and p-1,3-dextranase. The P-1,3-dextranase has been proved as the antifreeze protein in Secalecereale. Whether the other three proteins are the antifreeze proteins needs further validation. Mass spectrometry results of the basic glucanase and p-1,3-dextranase are respectively as follows: 1. oli e76353- Vais: 37849 Score: 888 Vatchee 8(0) Sequences: 8(?) emPAI: 2.41 bsei glwcanse EBesscajunca]

[iCheck to include -his hit in error tolerant earch

Query Observed Er(expt) Ir(calc) ppm mis Score Expect Rank linique Peptide t, I hlC1b'In83 I I%11, Y8 1516.183b -4.9b V 93 2.7bc-IJ I 1t .LY1DP8QEVLSALLC CE 1739.9294 1738.9221 1138.9478 -14.T 0 07 0.00029 1 1r .IGNLPRFDAVALTh.N T 4 3 17481.8320 , 147.6247 1747.8371 -74? B g t 5o-05 1 1 R.ISVGEVQPSDPTSL. E ~' 1783.8531 1782.8468 1782.8577 -6.68 0 131 1.2-8 1 1ECISGFPPOSCTFTPEFR. 9E 1878.9781 1877.97D8 1077.9847 -7.38 0 118 2.Ie-09 1 9 R.GSNIDLLLBVPNPDLqR.I IT,2 2290.0649 ??89.076 ??B9.0913 -14.73 0 164 3.C-14 1 g. 1.3YTLFT3PSTVVKil;SNA.N N-2_; 2334.0933 2333.3860 2333.1514 -28.03 0 29 1.2 1 U LQSPLLVNVYPTFSYINURD+ Oxidation (K) EL 15 2709.2891 27T88.78R 2788.324 -15.99 0 200 8.1-l18 2 K.7TC iSE SEWPTACGTMN8DMA~.T

2. iLLA694 Nass: 43633 Score: 554 Ratches: 80(4) Sequences: 6(4) emPAI: 1.04 beta-1, 3-clucnase (Drasica rapa subsp. chinenis] J Check to include this hit in error tolerant search

Query Observed Ireapt) Er(cslc) ppa iss Score Expect Rank Unique Peptide 2 857.4740 850.4267 856.4079 22.0 1 27 3-6 1 9 LFIDEYRW 3EJ 1431.7091 1430.7018 1430.6976 2.9b 0 102 1.1e-a7 t 11 L YGPyPDALVL8.G L 1447.6872 1446.6797 1446.6925 -0.86 0 (24) 7 1 3 LETGPNPDALALLC + Oxidation (2) 101 1948.8916 194.0$843 1947.9037 -9.94 0 151 1.1e-12 1 0 K.VSTTTFIXAFABTYPPSLG E 10 1966.9033 1965.8960 1965.9669 -35.52 0 40 0.13 1 0 LSPLLVNIYTYFCYER.D + Oxidation (1) I OKI 7841.407? 2840,3999 2840.4490 -17.29 0 113 4e-09 1 I .YTST0UEV1PGEPGAAALANQITDLA L 17 2867.033 2856.3760 2856.4440 -23.78 0 (13) 36 1 11 .ToSxCIPPAALIQANQ31D2.A +0idation (1) M 17 2874.3423 2873.3350 2873.3944 -20.66 0 121 6.6e-10 1 9 L DVSLQFALLQFESNEEFTDPNQLR. T

3. Detection of ice crystal modification effect: see Fig. 4 and Fig. 5. In order to detect whether the apoplastic proteins have antifreeze activity, an improved phase contrast microscope observation method in the laboratory is adopted for observing growth characteristics of ice crystals in presence of the apoplastic proteins of the roots and leaves. Fig. 4 shows the ice crystal growth condition of apoplastic crude extract of roots of non-cold-acclimated Longyou #6 at a constant temperature of -7°C within minutes observed under a lOX objective lens. Fig. 5 shows the ice crystal growth condition of apoplastic crude extract of roots of cold-acclimated Longyou #6 at a constant temperature of -7°C within 40

Description

minutes observed under a loX objective lens, which presents an obvious recrystallization inhibition effect. The above only describes preferred embodiments of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (2)

Claims

1. A method for extracting apoplastic antifreeze proteins from winter rape roots, comprising the following steps: (1) sampling: digging out rape roots in the field from October to December or to January of the following year; putting the rape roots into an ice box within 3-5 minutes; taking the ice box back to a laboratory; and preserving the ice box in a refrigerator at 3-5°C; (2) preparing 1000 ml of an extraction buffer solution according to weight volumes as follows: 6.057 g of 0.05 M/L of Tris-HCl having a pH value regulated to 8.0; 3.72 g of 0.01 M/L of EDTA-Na2.2H 20 (ethylenediamine tetraacetic acid disodium salt); 3.52 g of 0.02 M/L of VC (vitamin c); fixing the volume to 1000 ml with ddH 20, and preserving the prepared buffer solution in an environment of 3-5°C; (3) slitting: eliminating soil carried by the rape roots; removing dry dead parts of the roots; rinsing the roots with running water for 10-12 min until sundries are cleaned off; rinsing with ddH 20twice; sucking surface moisture with filter paper; cleaning the roots; and cutting open into strips of 1-2 cm; (4) rinsing: rinsing the roots which are cut into strips with the above buffer solution; (5) vacuumizing: putting the rape roots which are cut into strips into a container with a stopper; adding 80-100 ml of the above buffer solution; vacuumizing; and after a pressure is stabilized at 0.07-0.08 Mpa, continuously vacuumizing for 7-10 min; (6) centrifuging: pouring the buffer solution; sucking the solution on the root surfaces with filter paper; adding the solution into an injector; putting the injector into a 50 ml centrifuge tube; performing centrifugation at a rotation speed of

Claims

6100-6200 rpm at 3-5°C for 14-16 min; and collecting liquid at the bottom of the centrifuge tube, that is an apoplastic extract.

2. The method for extracting the apoplastic antifreeze proteins from winter rape roots according to claim 1, further comprising the following steps: (7) preparing a mercaptoethanol solution with a formula as follows: 12.5% of 0.5 mol/L Tris-HCl (pH=6.8) 10% of glycerin 5% of p-mercaptoethanol 72.5% of ddH20; (8) purifying: A: adding the extract in the step (6) into cold acetone in an amount of 5 times of the volume; precooling at -20°C; precipitating at -20°C for 10-20 h; performing centrifugation at a rotation speed of 10000 rpm at 4°C for 30 min; pouring out the acetone; and precipitating at -20°C to volatilize the acetone so as to obtain a freeze-dried protein sample; B: dissolving the freeze-dried protein sample in the A of the step (8) with a mercaptoethanol mixed solution; performing full dissolving; performing centrifugation at the rotation speed of 10000 rpm at 4°C for 15 min; taking the supernatant; and preserving the supernatant at -20°C for later use.