CN115024218A - Method for improving autotetraploid inductivity of black-bone cabbage and identifying ploidy of black-bone cabbage - Google Patents
Method for improving autotetraploid inductivity of black-bone cabbage and identifying ploidy of black-bone cabbage Download PDFInfo
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Abstract
The invention relates to the field of vegetable chemical control, and provides a method for improving autotetraploid inductivity of black-bone cabbage, which comprises the following steps: (1) soaking diploid black-bone vegetable seeds in warm water for accelerating germination; (2) after the seed coat is broken and white, soaking the seeds in a colchicine-Tween 80 mixed solution for 1h for mutagenesis treatment, treating for 2 times every day, and continuously treating for 1-3 days; (3) sowing the treated black-cabbage seeds in a plug tray for seedling. The invention also provides a method for identifying the ploidy of the black-boned vegetable. The invention fills the blank of the cabbage polyploid breeding at home and abroad, provides a method for the homologous tetraploid mutagenesis of the black cabbage, which is simple to operate and effective, and creates a new quality and high-yield tetraploid germplasm of the black cabbage; meanwhile, the method for identifying the ploidy of the black-bone mustard is also improved, and a basis are provided for large-scale and efficient identification.
Description
Technical Field
The invention relates to the field of vegetable chemical control, in particular to a method for improving autotetraploid inductivity of black-bone cabbage and identifying ploidy of the black-bone cabbage.
Background
Wucai (Brassica campestris L.ssp. chinensis var. rosella Tsen et Lee), also known as Wuta-tsai, Hei-cai, and Berberian mustard, is a variety of Brassica campestris subspecies belonging to Brassicaceae Brassica. The black cabbage mutton is cold-resistant and not high-temperature-resistant, is mainly widely planted in Yangtze river basin in China in autumn and winter, is sweet and delicious after frost and snow, has the name of 'snowy black cabbage mutton match', and is deeply loved by consumers. The breeding of the black cabbage is generally carried out by adopting the conventional traditional crossbreeding technology so far, but the conventional breeding is time-consuming and labor-consuming, and a plurality of excellent traits cannot be improved by the method. Ploidy breeding is increasingly becoming an effective method to provide polyploid plants with desirable traits and to increase their tolerance to abiotic stress. Therefore, the creation of new germplasm and the enrichment of germplasm resources by utilizing a polyploid breeding means and the cultivation of new varieties with excellent yield, high quality and strong stress resistance are urgently needed.
At present, the colchicine is used for inducing polyploidy, which is a common ploidy breeding mode, and remarkable results are obtained, but the defects of high mortality rate and low mutagenesis rate still exist. Meanwhile, because the research on the cabbage polyploid germplasm resources at home and abroad is less, the existing technology for identifying the cabbage polyploids is not complete enough. Therefore, at present, a method for improving the induction rate of the brassica rapa autotetraploid and a method for identifying the ploidy of the induced brassica rapa autotetraploid are urgently needed to be researched and researched.
Disclosure of Invention
The invention aims to provide a method for improving the autotetraploid inductivity of black-bone cabbage and identifying the ploidy of the black-bone cabbage.
The invention adopts the following technical scheme to solve the technical problems:
a method for improving induction rate of homologous tetraploid of lindera aggregate comprises the following steps:
(1) soaking diploid black-bone vegetable seeds in warm water for accelerating germination;
(2) after the seed coat is broken and white, soaking the seeds in a colchicine-Tween 80 mixed solution for 1h for mutagenesis treatment, treating for 2 times every day, and continuously treating for 1-3 days; after mutagenesis treatment, washing with clear water for 4-5 times; wherein, in the colchicine-Tween 80 mixed solution, the mass fraction of the colchicine is 0.1 to 0.3 percent, and the mass fraction of the Tween 80 is 0.5 to 1.5 percent;
(3) sowing the treated black-cabbage seeds in a plug tray for seedling.
As one preferable mode of the invention, in the step (1), the selected black-boned seeds are full in shape and uniform in size.
In a preferred embodiment of the present invention, in the step (1), the period of time for performing the warm water seed soaking and germination acceleration treatment on the diploid lindera aggregata seeds is 12 hours.
As one preferable mode of the present invention, in the step (2), the colchicine mass fraction of the colchicine-tween 80 mixed solution is specifically 0.1%, and the tween 80 mass fraction is 1%; soaking the seeds in the colchicine-Tween 80 mixed solution for 1h for mutagenesis treatment, treating for 2 times per day, and continuously treating for 2 days.
In a preferred embodiment of the present invention, in the step (2), the mutagenesis treatment is performed once each day at 7 am and 5 pm.
In a preferred embodiment of the present invention, in the step (3), the temperature of the environment is controlled during the seedling raising period, so that the seedling growth temperature is 24 to 28 ℃ in the daytime, 18 to 22 ℃ at night, the photoperiod is 14h of light/10 h of darkness, and the light intensity is 300 μmol · m -2 ·s -1 And the relative humidity is 70-80%.
As one of the preferable modes of the invention, the method also comprises the step (4) of carrying out ploidy identification on the obtained black-boned plants.
A method for identifying ploidy of caulis et folium Brassicae Capitatae is provided, which is used for identifying ploidy of autotetraploid plant of caulis et folium Brassicae Capitatae obtained by the method, and comprises the following steps:
(1) morphological identification:
using appearance phenotype characteristics of slow growth, deepened leaf color, thickened leaf and increased flower organ appearance as selection standards, primarily screening each cultured black cabbage plant to obtain primarily screened suspected tetraploid black cabbage;
(2) photosynthetic determination:
respectively measuring the net photosynthetic rate of the suspected tetraploid black-bone vegetables screened primarily under different light intensities by using a portable photosynthetic determinator; taking the determination result of the diploid black-bone mustard tuber plant as a contrast, and obtaining the suspected tetraploid black-bone mustard tuber after secondary screening when the net photosynthetic rate of the suspected tetraploid black-bone mustard tuber after primary screening is obviously higher than that of the diploid black-bone mustard tuber plant;
(3) flow cytometry identification:
weighing 0.2-0.3 g of suspected tetraploid black-bone cabbage fresh leaves subjected to secondary screening, and placing the suspected tetraploid black-bone cabbage fresh leaves in a culture dish precooled in advance; adding 2mL of dissociation liquid to ensure that the whole blade is completely immersed in the dissociation liquid and the cell nucleus can be fully dissociated; then, cutting the leaves into long strips by using a single-sided blade, sucking the dissociation solution in the culture dish after cutting the strips so as to enable the cell nucleuses in the leaves to enter the dissociation solution, and adding 3mL of dissociation solution for dilution; finally, propidium iodide staining solution is added, the mixture is stained for 2 hours in a dark place, and then the filtrate is transferred into a flow cell test tube and is measured on a machine.
In a preferred embodiment of the present invention, in the step (2), the net photosynthetic rate per unit area of the leaves under different light intensities is measured by using a portable photosynthetic analyzer at 9 am to 11 am on a fine day, and the measurement is repeated three times for 3 leaves each time.
In a preferred embodiment of the present invention, in the step (3), the dissociation solution has a formula of: 20 mmol. L - 1 MgSO 4 ·7H 2 O，100mmol·L -1 KCl，10mmol·L -1 HEPES, volume fraction 0.5% Triton-X, 2% PVP.
Design thought and principle:
colchicine, originally extracted from colchicine of Liliaceae plant, can inhibit mitosis effectively, make chromosome double, but utilize colchicine to induce polyploid death rate high at present on the market, the mutagenicity rate is lower; tween 80 is a nonionic surfactant, has good emulsification solubilization and strong hydrophilicity, and can increase the solubility of medicinal components by adding Tween 80, thereby improving the permeability of plant tissue, making medicine easily enter into plant cells, and improving the doubling effect.
Compared with the prior art, the invention has the advantages that:
(1) the invention fills the blank of the cabbage polyploid breeding at home and abroad, provides a method for the homologous tetraploid mutagenesis of the black cabbage, which is simple to operate and effective, and creates a new quality and high-yield tetraploid germplasm of the black cabbage;
(2) the method for identifying the ploidy of the black-boned vegetable is also improved, and a method for determining the net photosynthetic rate of leaves is added in morphological identification, so that suspected tetraploid black-boned vegetable can be screened more accurately and preliminarily, and the workload of subsequent identification is reduced; further groping out a system for identifying the tetraploid of the black-bone cabbage by the flow cytometry, so that the operation is simple, the peak is fast, and a basis are provided for large-scale and efficient identification.
Drawings
FIG. 1 shows the comparison of the differences in the plant, leaf and petal between diploid and tetraploid Brassica oleracea in example 4 (in the figure, A represents the plant, B represents the leaf, and C represents the petal);
FIG. 2 is the graph of the photo response of the photosynthesis of the leaves of the diploid and tetraploid Brassica oleracea plants in example 4;
FIG. 3 is a graph showing the relative DNA content of diploid and tetraploid black cabbage in example 4.
Detailed Description
The following examples are given for the detailed implementation and the specific operation procedures, but the scope of the present invention is not limited to the following examples.
Example 1
The method for improving the autotetraploid inductivity of the lindera aggregate comprises the following steps:
(1) diploid black-cabbage seeds with full shapes and uniform sizes are selected, and the seeds are soaked in warm water for pregermination for 12 hours.
(2) After the seed coat is swelled, broken and white, soaking the seeds in a colchicine-Tween 80 mixed solution for 1h for mutagenesis treatment, carrying out mutagenesis treatment once at 7 am and 5 pm every day, and continuously treating for 3 days, wherein 100 seeds are treated each time; after mutagenesis treatment, washing the seeds for 4-5 times by using clear water, so that toxic action of medicament residues on the seeds and pollution to the environment are avoided; wherein, in the colchicine-Tween 80 mixed solution, the mass fraction of the colchicine is 0.2 percent, and the mass fraction of the Tween 80 is 1.5 percent.
(3) After water is wiped off, sowing the treated black-bone mustard seeds in a 72-hole plug tray for seedling culture; controlling the environmental temperature during seedling raising, covering a sunshade net in sunny days when the illumination is strong to avoid direct sunlight, so that the seedling growth temperature is 24 ℃ in the daytime, 18 ℃ at night, the photoperiod is 14h/10h (illumination/darkness), and the illumination intensity is 300 mu mol.m -2 ·s -1 And the relative humidity is 70%, so that excessive humidity is avoided to cause excessive growth of seedlings.
(4) And after 4-5 true leaves grow out from the black-boned plant, performing ploidy identification on the black-boned plant.
Example 2
The method for improving the induction rate of the autotetraploid of the lindera aggregate comprises the following steps:
(1) diploid black-cabbage seeds with full shapes and uniform sizes are selected, and the seeds are soaked in warm water for pregermination for 12 hours.
(2) After the seed coat is swelled, broken and white, soaking the seeds in a colchicine-Tween 80 mixed solution for 1h for mutagenesis treatment, performing mutagenesis treatment once at 7 am and 5 pm every day for 1 day, and treating 100 seeds each time; after mutagenesis treatment, washing the seeds for 4-5 times by using clear water, so that toxic action of medicament residues on the seeds and pollution to the environment are avoided; wherein, in the colchicine-Tween 80 mixed solution, the mass fraction of the colchicine is 0.3 percent, and the mass fraction of the Tween 80 is 0.5 percent.
(3) After water is wiped off, sowing the treated black-bone vegetable seeds in a 72-hole plug tray for seedling culture; controlling the environmental temperature during seedling growing period, covering a sunshade net to prevent direct sunlight when the illumination is strong in sunny days, so that the temperature of the seedling growing in the daytime is 28 ℃, the temperature at night is 22 ℃, the photoperiod is 14h/10h (illumination/darkness), and the illumination intensity is 300 mu mol.m -2 ·s -1 And the relative humidity is 80%, so that excessive humidity is avoided to cause excessive growth of seedlings.
(4) And after 4-5 true leaves grow out from the black-boned plant, performing ploidy identification on the black-boned plant.
Example 3
The method for improving the autotetraploid inductivity of the lindera aggregate comprises the following steps:
(1) diploid black-cabbage seeds with full shapes and uniform sizes are selected, and the seeds are soaked in warm water for pregermination for 12 hours.
(2) After the seed coat is swelled, broken and white, soaking the seeds in a colchicine-Tween 80 mixed solution for 1h for mutagenesis treatment, carrying out mutagenesis treatment once at 7 am and 5 pm every day, and continuously treating for 2 days, wherein 100 seeds are treated each time; after mutagenesis treatment, washing the seeds for 4-5 times by using clear water, so that toxic action of medicament residues on the seeds and pollution to the environment are avoided; wherein, in the colchicine-Tween 80 mixed solution, the mass fraction of the colchicine is 0.1 percent, and the mass fraction of the Tween 80 is 1 percent.
(3) After water is wiped off, sowing the treated black-bone vegetable seeds in a 72-hole plug tray for seedling culture; controlling the environmental temperature during seedling raising, covering a sunshade net to avoid direct sunlight when the illumination is strong in sunny days, ensuring that the temperature of the seedlings is 26 ℃ in the daytime,the light cycle is 14h/10h (light/dark) at the night temperature of 20 ℃, and the light intensity is 300 mu mol.m -2 ·s -1 And the relative humidity is 75%, so that excessive humidity is avoided to cause excessive growth of seedlings.
(4) And after 4-5 true leaves grow out from the black-boned plant, performing ploidy identification on the black-boned plant.
Example 4
The method for identifying ploidy of black-boned vegetable in this embodiment (taking the black-boned vegetable plant obtained in embodiment 3 as an example):
firstly, morphological identification
After the treated black cabbage plant grows up, the morphological identification is carried out on the treated black cabbage plant, and suspected tetraploid black cabbage is preliminarily screened. As shown in figure 1, the appearance of the tetraploid plant is mainly represented by obvious variations (diploid plants are used as a reference) such as slow growth, deepened leaf color, thickened leaf, huge flower organ appearance and the like, and the variant plants are screened by taking the appearance phenotypic characteristics as selection standards to obtain a preliminarily screened suspected tetraploid black-bone cabbage. And primarily screening the living Brassica oleracea plants to obtain suspected variant plants 278 according to the morphological variation.
Second, photosynthetic assay
For the suspected tetraploid black-bone cabbage screened primarily, the net photosynthetic rate per unit leaf area under different light intensities was measured by using a portable photosynthetic analyzer from 9 am to 11 am on a fine day, and the measurement was repeated three times with 3 leaves each time, and the obtained results are shown in fig. 2. The results in FIG. 2 show that the photosynthetic characteristics of tetraploid plants are stronger, with a net photosynthetic rate higher than that of diploids. 93 suspected tetraploid black-bone silkworms are further screened from 278 plants by the measured photosynthetic index, so that the accuracy of morphological initial identification of the tetraploid black-bone silkworms is improved, and the subsequent identification quantity is reduced.
Flow cytometry identification
Weighing 0.2-0.3 g of the primarily screened tetraploid black-bone vegetable fresh leaves, and placing the fresh leaves in a culture dish precooled in advance; 2mL of dissociation solution (20 mmol. multidot.L) was added -1 MgSO 4 ·7H 2 O，100mmol·L -1 KCl，10mmol·L -1 HEPES, volume fraction of 0.5% Triton-X, 2% PVP), whole leafPartially immersing in a dissociation liquid to make the cell nucleus fully dissociated; cutting into strips by a single-sided blade, disinfecting each sample by alcohol by the blade and tweezers used in cutting, and wiping the samples by clean paper to prevent mutual influence; after cutting, absorbing the dissociation solution in the culture dish to enable cell nuclei in the leaves to enter the dissociation solution, and then adding 3mL of dissociation solution for dilution (according to the solubility of the prepared dye solution, if the color is darker and the concentration is too high, the dissociation solution needs to be diluted for many times); finally, propidium iodide staining solution is added, after 2 hours of light-shielding staining, the filtrate is transferred to a flow cytometry tube, and the DNA content of the plant is analyzed by a flow cytometer (diploid plants are used as a control).
As can be seen from FIG. 3, the ordinate represents the relative value of the number of cells measured, the abscissa represents the channel value of fluorescence, the position of the peak reflects the ploidy of the plant, and the major peak of the relative content of diploid DNA is found to be 200, and the major peak of the relative content of tetraploid DNA is found to be 400, which is twice that of diploid.
Example 5
This example is used to verify the effect of "different colchicine and tween 80 mutagenesis concentrations" and "different mutagenesis treatment times" on the induction results of the autotetraploid brassica oleracea:
the test method comprises the following steps: and (3) selecting 7-2 black cabbage seeds with full shapes and uniform sizes as test materials, and soaking the seeds in warm water for accelerating germination for 12 hours. After the seed coat is broken and white, the seed coat is respectively immersed in a mixed solution of colchicine (the mass fractions are 0.1%, 0.2% and 0.3%) and tween 80 (the mass fractions are 0%, 0.5%, 1% and 1.5%) for 1h, and the seed coat is treated once at 7 am and 5 pm every day, and treated for 1d, 2d and 3d continuously, and 100 seeds are treated for each time. And after the soaking of the standby medicament is finished, the standby medicament is washed for 4-5 times by using clear water, so that the toxic action of medicament residues on seeds and the pollution to the environment are avoided. After water is wiped off, the treated black cabbage seeds are sown in a 72-hole plug tray for seedling culture, the environmental temperature is controlled during seedling culture, a sun-shading net is required to be covered under strong illumination in sunny days to avoid direct sunlight, so that the seedling can grow at the temperature of 26 ℃ in the daytime, at the temperature of 20 ℃ at night, the light cycle is 14h/10h (illumination/darkness), and the illumination intensity is 300 mu mol.m -2 ·s -1 Relative to each otherHumidity 75%. After 4-5 true leaves of the black-boned plant grow, the ploidy identification method of the black-boned plant is adopted to carry out ploidy identification on the black-boned plant in the embodiment 4.
Survival (%) × 100 (number of surviving plants/number of treated plants);
variation rate (%) × 100 (number of variant plants/number of treated plants);
addition rate (%) × 100 (number of tetraploid plants/number of treated plants).
And (3) test results: the induction results of the brassica rapa autotetraploid under different colchicine concentrations and different mutagenesis treatment times are shown in table 1, and the induction results of the brassica rapa autotetraploid under different colchicine and tween 80 concentrations and different mutagenesis treatment times are shown in table 2.
TABLE 1 tetraploid induction results of Brassica Oleracea with different colchicine concentrations and different treatment times
As can be seen from Table 1, under the conditions of different colchicine concentrations (0.1%, 0.2%, 0.3%) and different treatment times (2, 4, 6), the autotetraploid black cabbage can be obtained, but the doubling effect is different. When the drug concentration is low and the number of treatments is small, the survival rate is high, but the doubling effect is poor. If the drug concentration is too high and the number of treatments is large, the survival rate is low and the doubling rate of the number of surviving plants is not high. Within a certain range, the survival rate and the doubling rate of the black-boned vegetable are increased along with the increase of the concentration and the treatment times of the colchicine, but when the concentration reaches 0.2 percent and the treatment times are too long, the survival rate and the mutation rate start to be obviously reduced, which can cause the low germination rate of seeds and the reduction of the survival rate of seedlings because the influence of the toxicity of the colchicine on the black-boned vegetable seeds is too large. The mutagenesis effect is different according to the treatment frequency and the colchicine concentration. As can be seen from Table 1, the optimum colchicine treatment concentration is 0.2%, the treatment times are 6 times, under the condition, the multiplying power of the black-cabbage plant reaches the maximum, namely 5.78%, and the optimum mutagenesis effect can be obtained.
TABLE 2 tetraploid induction results of Brassica oleracea with different concentrations of colchicine and Tween 80 and different treatment times
Combining tables 1 and 2, it can be seen that the doubling effect of the chromosome is very low when the lindera aggregata is treated by using colchicine, and the solubility of the medicament components can be increased by adding tween 80, so that the permeability of the plant tissues is improved. As can be seen from Table 2, the application of Tween 80 can effectively promote colchicine to enter plant cells, thereby shortening the treatment time and improving the doubling effect; and when the concentration of the Tween 80 is 1 percent and the concentration of the colchicine is 0.1 percent, the maximum multiplying power can be reached after the treatment for 4 times. Thus, when colchicine is used for treating plants, the concentration and the use times of the colchicine can be reduced by applying the Tween 80 externally, the toxic action of the Tween on the plants is reduced, the variation rate and the doubling rate of the plants can be improved, and the colchicine has better effect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for improving the induction rate of autotetraploid of black-bone cabbage is characterized by comprising the following steps:
(1) soaking diploid black-bone vegetable seeds in warm water for accelerating germination;
(2) after the seed coat is broken and white, soaking the seeds in a colchicine-Tween 80 mixed solution for 1h for mutagenesis treatment, treating for 2 times every day, and continuously treating for 1-3 days; after mutagenesis treatment, washing for 4-5 times by using clear water; wherein, in the colchicine-Tween 80 mixed solution, the mass fraction of the colchicine is 0.1 to 0.3 percent, and the mass fraction of the Tween 80 is 0.5 to 1.5 percent;
(3) sowing the treated black-cabbage seeds in a plug tray for seedling.
2. The method for improving autotetraploid induction rate of Brassica campestris as claimed in claim 1, wherein in step (1), the selected Brassica campestris seeds are plump in shape and uniform in size.
3. The method for improving autotetraploid induction rate of lindera aggregate according to claim 1, wherein in the step (1), the time for soaking diploid lindera aggregate seeds in warm water for germination acceleration is 12 h.
4. The method for improving autotetraploid inductivity of Brassica campestris as claimed in claim 1, wherein in step (2), the colchicine mass fraction of colchicine-Tween 80 mixture is specifically 0.1%, and Tween 80 mass fraction is 1%; soaking the seeds in the colchicine-Tween 80 mixed solution for 1h for mutagenesis treatment, treating for 2 times per day, and continuously treating for 2 days.
5. The method for increasing the autotetraploid induction rate of Brassica oleracea as claimed in claim 1, wherein in step (2), the mutagenesis is performed once each day at 7 am and 5 pm.
6. The method for increasing autotetraploid induction rate of lindera aggregata as claimed in claim 1, wherein in the step (3), the environmental temperature is controlled during seedling raising, so that the seedling growth temperature is 24-28 ℃ in the daytime, 18-22 ℃ at night, the photoperiod is 14h of light/10 h of dark, and the light intensity is 300 μmol-m -2 ·s -1 And the relative humidity is 70-80%.
7. The method for improving autotetraploid induction rate of Brassica rapa Linne as claimed in claim 1, further comprising the step (4) of performing ploidy identification on the obtained Brassica rapa Linne plant.
8. A method for identifying the ploidy of the black-cabbage is characterized by identifying the ploidy of the autotetraploid plant of the black-cabbage obtained by the method of any one of claims 1 to 7 by the method, which comprises the following steps:
(1) morphological identification:
using appearance phenotype characteristics of slow growth, deepened leaf color, thickened leaf and increased flower organ appearance as selection standards, primarily screening each cultured black cabbage plant to obtain primarily screened suspected tetraploid black cabbage;
(2) photosynthetic determination:
respectively measuring the net photosynthetic rate of the suspected tetraploid black-bone vegetables screened primarily under different light intensities by using a portable photosynthetic determinator; taking the determination result of the diploid black-bone cabbage plant as a contrast, and obtaining the suspected tetraploid black-bone cabbage subjected to secondary screening when the net photosynthetic rate of the suspected tetraploid black-bone cabbage subjected to primary screening is higher than that of the diploid black-bone cabbage plant;
(3) flow cytometry identification:
weighing 0.2-0.3 g of suspected tetraploid black-bone cabbage fresh leaves subjected to secondary screening, and placing the suspected tetraploid black-bone cabbage fresh leaves in a culture dish precooled in advance; adding 2mL of dissociation liquid to ensure that the whole blade is completely immersed in the dissociation liquid and the cell nucleus can be fully dissociated; then, cutting the leaves into long strips by using a single-sided blade, sucking the dissociation solution in the culture dish after cutting the strips so as to enable the cell nucleuses in the leaves to enter the dissociation solution, and adding 3mL of dissociation solution for dilution; finally, propidium iodide staining solution is added, the mixture is stained for 2 hours in a dark place, and then the filtrate is transferred into a flow cell test tube and is measured on a machine.
9. The method for ploidy determination of Wucai dish as claimed in claim 8, wherein in step (2), the net photosynthetic rate per unit area of leaves under different light intensity is measured by portable photosynthetic apparatus at 9-11 am of fine day, and the measurement is repeated three times for 3 leaves each time.
10. The method for ploidy identification of lindera strychnifolia according to claim 8, wherein said step is carried outIn the step (3), the formula of the dissociation liquid is as follows: 20 mmol. L -1 MgSO 4 ·7H 2 O，100mmol·L -1 KCl，10mmol·L -1 HEPES, volume fraction 0.5% Triton-X, 2% PVP.
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