CN111999235A - Method for rapidly detecting number of viable edible fungus protoplasts by using flow cytometry - Google Patents
Method for rapidly detecting number of viable edible fungus protoplasts by using flow cytometry Download PDFInfo
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Abstract
The invention provides a method for rapidly detecting the number of protoplasts of viable edible fungi by using flow cytometry, which comprises the following steps: filtering the edible fungus protoplast cells, adding CFSE fluorescent dye for dyeing as a sample, or adding equivalent dimethyl sulfoxide for dyeing as a negative control; the stained protoplast cells were then analyzed by flow cytometry using a 488nm laser, setting the FSC threshold to 50 on an FSC/SSC scattergram, and adjusting the voltages of FSC and SSC, in which the desired cell population is circled (gate P1); plotting a scatter plot FL1/SSC, determining the negative cell population position with the negative control tube, FL1 abscissa 10 on FL1/SSC scatter plot2All circles at the other positions are set as a P2 gate. Within the P1 phylum are total protoplast cells and within the P2 phylum are viable protoplast cells. The detection method can rapidly and accurately obtain the number of the viable protoplasts in a period of time, and provides a rapid and effective method for the preparation and regeneration of the protoplasts of the edible fungi.
Description
Technical Field
The invention belongs to the technical field of edible fungus breeding, and particularly relates to a method for rapidly detecting the number of viable edible fungus protoplasts by using a flow cytometer.
Background
At present, domestic fungus varieties suitable for industrial cultivation are lacked, and little is known about the growth and cultivation mechanism of the edible fungus, so that the research on the genetic breeding and molecular mechanism of the edible fungus is urgent.
Because the edible fungi are fungi, the outer layer of the edible fungi is wrapped by hard cell walls, and the preparation of protoplasts can not be separated when the edible fungi are subjected to genetic manipulation and molecular modification. Obtaining and accurately counting viable protoplasts has important reference value for genetic breeding and molecular mechanism research of edible fungi.
The traditional counting method of protoplasts is a blood cell counting method, which has large error and cannot distinguish between viable protoplasts and non-viable protoplasts. Flow cytometry is used as an efficient counting means, is firstly applied to the technical field of microorganisms, and is then widely applied to the field of cellular immunology. Flow cytometry detects fluorescent signals and therefore requires staining of the cells to detect them. In the current commonly used dyes, PI staining solution only can stain dead cells, but can not directly detect live cells, and the detection result may have larger deviation; although SYBR Green I and SYT90 dye solutions can directly dye living cells, the dye solution pair can affect the activity of protoplasts and further affect the regeneration rate of the protoplasts.
The size difference of the protoplast cells of different edible fungi is large, for example, the diameter of the protoplast cells of morchella esculenta is 9.16-13.2 mu m, the diameter of the protoplast cells of the Agrocybe aegerita is 5.90-7.18 mu m, the diameter of the protoplast cells of the Hypsizygus marmoreus is small, and the diameter of the protoplast cells of the Hypsizygus marmoreus is 2.10 +/-5.02 mu m; in addition, the preparation process of the edible fungus protoplast shows that the size of the protoplast cells of the same material also has difference. The difference in cell size also has some effect on the count of protoplasts. Therefore, a method capable of rapidly detecting the number of protoplast cells of viable edible fungi without being affected by various factors is highly desired.
Disclosure of Invention
The invention provides a method for rapidly detecting the number of viable edible fungus protoplasts by using flow cytometry, which is simple to operate, high in efficiency and accurate in detection, and the used CFSE dye does not influence the viability and the regeneration rate of the edible fungus protoplast cells.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a method for rapidly detecting the number of protoplasts of viable edible fungi by using a flow cytometer, which comprises the following steps:
(1) sample preparation and staining: filtering the edible fungus protoplast cells obtained by the enzymolysis of the edible fungi by a screen, and adding a CFSE fluorescent staining solution for staining to obtain stained edible fungus protoplast cells;
(2) analysis and determination by flow cytometry:
a. analysis was performed using a flow cytometer: placing the dyed edible fungus protoplast cells into a flow cytometer, and respectively drawing an FSC/SSC scatter diagram and an FL1/SSC scatter diagram; setting the FSC threshold value of an FSC/SSC scatter diagram to be 50 so as to remove edible fungus protoplast cell fragments, adjusting the voltage of the FSC and SSC so that the dyed edible fungus protoplast cell clusters are completely displayed and positioned at the central position of the picture, and then setting a P1 gate to completely circle out the cell clusters, wherein the cells displayed in the P1 gate are total edible fungus protoplast cells; edible fungus protoplast cells within the P1 gate are shown on FL1/SSC scatter plots, followed by FL1 signal greater than 10 on FL1/SSC scatter plots2The cytoencystment gate of (P2), the cells shown in the P2 gate are viable edible fungus protoplast cells;
b. calculating the cell concentration of the edible fungus protoplast according to the flow rate and the collection time of the flow cytometer;
c. and counting the total number of the edible fungus protoplast cells in the P2 door and the total number of the edible fungus protoplast cells in the P1 door, and calculating the activity rate of the edible fungus protoplast.
Further, the screen in the step (1) is 200 meshes.
Further, the addition amount of the CFSE fluorescent dye solution in the step (1) is 1% (v/v).
Further, the dyeing condition of the CFSE fluorescent dye solution in the step (1) is room temperature and dark, and the dyeing time is 5-10 minutes.
Further, the concentration of the protoplast cells of the edible fungi obtained in the step (1) is 1.0 multiplied by 104～1.0×108one/mL.
Further, the flow cytometer in step (2) has a 488nm laser.
Furthermore, the method is provided with a negative control, namely 1% (v/v) of dimethyl sulfoxide is added into the protoplast cells of the edible fungi, and the protoplast cells are placed for 5 to 10 minutes at room temperature in a dark place.
Further, in the step (2), before setting the P2 gate, the negative control protoplast cell population is adjusted to 10 of the abscissa of FL1 by using a negative control tube1Within, FL1/SSC scattergram is then plotted on FL1 abscissa 102All the other positions are circled to be P2 gates, and the FL1 signal is greater than 102A positive signal.
Furthermore, the protoplast cell size of the edible fungi is 2.1-13.2 μm.
Furthermore, the viability of the edible fungus protoplasm is equal to (the number of edible fungus protoplasm cells in P2 door/the number of edible fungus protoplasm cells in P1 door) × 100%.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the method for rapidly detecting the number of the protoplasts of the viable edible fungi by using the flow cytometer has the advantages of simple operation, rapidness, high efficiency, high result accuracy and good repeatability, and can obtain results within 15 minutes only by operating at room temperature.
(2) The detection method can obtain the number of the protoplasts of the viable edible fungi and the total number of the protoplasts, and calculate the viability rate of the protoplasts, and has important guiding significance for improving the yield and the regeneration rate of the protoplasts.
(3) The dye CFSE used by the invention has almost no influence on the vitality and the regeneration rate of the protoplast of the edible fungus, and avoids the damage of the fluorescent dye on the protoplast and the influence of the vitality and the regeneration rate.
(4) The method is not influenced by the size of the protoplast, provides a quick and effective method for the preparation and regeneration process research of the edible fungus protoplast, and has better application prospect in the aspect of edible fungus protoplast fusion breeding.
Drawings
FIG. 1 is a FSC/SSC dot plot, in which the total number of protoplast cells of edible fungi is in the P1 gate.
FIG. 2 is a FL1/SSC scatter plot wherein the FL1 signal is greater than 102The cell granules of (A) are CFSE positive cells, and the P2 gate is a viable edible fungus protoplast cell.
FIG. 3 shows the results of the measurement of the protoplast amount of edible fungi at different dilution times.
FIG. 4 shows the results of the measurement of the total number of protoplast cells of different edible fungi and the number of protoplast cells of viable edible fungi.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the experimental reagents and materials used are commercially available without specific reference.
Example 1
A method for rapidly detecting the number of protoplasts of viable edible fungi by using flow cytometry comprises the following steps:
(1) sample preparation and staining: carrying out enzymolysis or other methods on edible fungi to obtain protoplast cells, then filtering the protoplast cells by a 200-mesh screen, adding 1% (v/v) CFSE fluorescent dye solution, and dyeing for 5-10 minutes at room temperature in a dark place to obtain dyed edible fungi protoplast cells; and adding 1% (v/v) of dimethyl sulfoxide into the same amount of the edible fungus protoplast cells, and placing for 5-10 minutes at room temperature in a dark place to serve as a negative control.
Wherein the protoplast cell concentration is 1.0 × 104～1.0×108one/mL.
The preparation method of the CFSE fluorescent dye solution comprises the following steps: add 10. mu.L of CFSE stock solution into 990. mu.L of dimethyl sulfoxide, mix well, and store at-20 ℃ in the dark. 10. mu.L of CFSE fluorescent dye solution was added to 1mL of the cell fluid during staining.
(2) Analysis and determination by flow cytometry:
a. the stained edible fungus protoplast cells were analyzed using a flow cytometer containing a 488nm laser: placing the dyed edible fungus protoplast cells into a flow cytometer, and respectively drawing an FSC/SSC scatter diagram and an FL1/SSC scatter diagram; setting the FSC threshold value of an FSC/SSC scatter diagram to be 50 so as to remove edible fungus protoplast cell fragments, adjusting the voltage of the FSC and SSC so that the dyed edible fungus protoplast cell clusters are completely displayed and positioned at the center of the picture, and then setting a P1 gate to completely circle out the cell clusters (as shown in figure 1), wherein the cells displayed in the P1 gate are total edible fungus protoplast cells; protoplast cells within the P1 gate are shown on FL1/SSC dot plots, and the negative control cell population is adjusted to 10 on the FL1 abscissa using the negative control tube2Within, 10 are then plotted on a FL1/SSC scatter plot2All the other positions are circled to be P2 gates, and the FL1 signal is greater than 102Positive signal, the cells displayed in the P2 gate were viable edible fungus protoplast cells (as shown in fig. 2);
b. calculating the cell concentration of the edible fungus protoplast according to the flow rate and the collection time of a flow cytometer, counting the total number of the edible fungus protoplast cells in a P2 gate and the total number of the edible fungus protoplast cells in a P1 gate, and representing the protoplast activity rate by the percentage of the viable protoplast to all the protoplasts, namely the activity rate of the instant edible fungus protoplast is (the number of the dyed edible fungus protoplasts/the total number of the edible fungus protoplasts) multiplied by 100 percent; can also be expressed as: the activity rate of the edible fungus protoplast is (total number of edible fungus protoplast cells in P2 door/total number of edible fungus protoplast cells in P1 door) multiplied by 100%.
Wherein the protoplast cell size of the edible fungi is 2.1-13.2 μm.
Example 2: morchella protoplast counting and viability detection
The size of protoplast cells of the morchella esculenta subjected to enzymolysis is 9.16-13.2 microns. Counting the protoplast by blood cell counting under a common optical microscope to obtain the protoplast with the number of 5.2 × 108one/mL.
Filtering the protoplast cells of the morchella esculenta by a 200-mesh screen, adding 1% (v/v) CFSE fluorescent dye solution, and dyeing for 5-10 minutes at room temperature in a dark place; and adding 1% (v/v) of dimethyl sulfoxide into 500 mu L of the protoplast cells of the morchella esculenta, and placing for 5-10 minutes at room temperature in a dark place to serve as a negative control.
The stained protoplast cells were diluted 10-fold, 100-fold, 1000-fold, and 10000-fold with a phosphate buffer containing 0.6M mannitol, respectively, and the corresponding protoplast concentration was 5.2X 10 according to the statistics of the blood cell count method8～5.2×104Per mL, which was then detected and analyzed using a flow cytometer: excited with a 488nm argon ion laser in a flow cytometer configuration. Drawing an FSC/SSC scatter diagram and an FL1/SSC scatter diagram, setting an FSC threshold value to be 50 on the FSC/SSC scatter diagram to remove cell fragments, adjusting the voltage of the FSC and SSC to enable cell clusters to be completely displayed and positioned in the center of the image, setting a P1 gate to completely circle out the cell clusters, operating in an HIRUN mode, setting the flow rate to be 1 mu L/s, stopping collection after setting 40000 cells to be collected, automatically counting and displaying the number of detected cells per second in the data collecting process, and calculating the concentration of the protoplast cells (the number of detected cells per second/flow rate); cells within the P1 gate were displayed on FL1/SSC dot plots, and the negative control cell population was adjusted to 10 on the FL1 abscissa using the negative control tube2Within, then set up P2 gate on FL1/SSC scattergram (will 10)2All other positions are circled, the FL1 signal is greater than 102Positive signal), viable Morchella esculenta protoplast cells are shown in the P2 gate, and the viability rate of Morchella esculenta protoplasts is calculated according to the formula (number of protoplast cells in P2 gate/number of protoplast cells in P1 gate) × 100%.
As shown in Table 1 and FIG. 3, the total number of protoplasts and viable protoplast cells detected continuously decreased with the increase of the dilution factor of the sample, but the change amplitude was small; when in 104～108In the detection range of each mL, the number of the protoplasm somatic cells and the number of the positive protoplasm somatic cells detected by the method are related to the dilution multiple, and the protoplasm activity rate has better consistency under different dilution multiples.
TABLE 1 Morchella protoplast number at different dilution factor
Example 3: hypsizigus marmoreus protoplast count and viability detection
The size of protoplast cells of the hypsizigus marmoreus after enzymolysis is 2.10-5.02 mu m. Counting the protoplast by blood cell counting under a common optical microscope to obtain the protoplast with the number of 2.5 × 107one/mL.
Filtering protoplast cells of hypsizygus marmoreus by a 200-mesh screen, adding 1% (v/v) CFSE fluorescent dye solution, and dyeing for 5-10 minutes at room temperature in a dark place; separately, 500. mu.L of protoplast cells of Hypsizygus marmoreus was added with 1% (v/v) dimethyl sulfoxide, and left at room temperature for 5-10 minutes in the dark to serve as a negative control.
The stained hypsizigus marmoreus protoplast cells were detected and analyzed by a flow cytometer, the specific parameters and steps were the same as in example 2, and the total number of protoplast cells detected by the flow cytometer was 5.1 × 107Number of viable protoplasts per mL of 2.4X 107The cell/mL, the protoplast viability rate was 47.06%.
Example 4: agrocybe aegerita protoplast counting and viability detection
The size of protoplast cells of the oudemansiella radicata after enzymolysis is 5.90-7.18 mu m. Counting the protoplast by blood cell counting under a common optical microscope to obtain the protoplast with the number of 4.2 × 106one/mL.
Filtering protoplast cells of the oudemansiella radicata by a 200-mesh screen, adding 1% (v/v) CFSE fluorescent dye solution, and dyeing for 5-10 minutes at room temperature in a dark place; and adding 1% (v/v) dimethyl sulfoxide into 500 mu L of the Agrocybe aegerita protoplast cells, and standing at room temperature in the dark for 5-10 minutes to serve as a negative control.
Detecting and analyzing the stained oudemansiella radicata protoplast cells by using a flow cytometer, and specifically setting parameters, steps and realityIn example 2, the total number of protoplast cells detected by flow cytometry was 5.5X 106Number of viable protoplasts per mL of 3.8X 106The cell/mL, the protoplast viability rate was 69.09%.
Example 5:
first, protoplast cells of Morchella esculenta, Hypsizygus marmoreus and Agrocybe radicata were uniformly diluted to 5.6X 104each/mL, 300. mu.L of protoplast cells were applied to solid regeneration medium 1-4, respectively, the compositions of the media are shown in Table 2, and the protoplast regeneration rates of Morchella esculenta, Hypsizygus marmoreus and Agrocybe aegerita were measured as controls.
Then, 1mL of protoplast cells of morchella esculenta, hypsizygus marmoreus and oudemansiella radicata are respectively taken, 10 muL of CFSE fluorescent dye solution is added, the mixture is placed in the dark at room temperature for 5-10 minutes, and the dyed protoplast cells are uniformly diluted to 5.6 multiplied by 104And (4) coating 300 mu L of the stained protoplast cells on a solid regeneration culture medium 1-4 respectively, detecting the regeneration rate of the protoplasts, and comparing the influence of the CFSE fluorescent dye solution on the regeneration rate of the protoplasts.
TABLE 2 solid regeneration Medium and composition thereof
The results are shown in Table 3, which show that CFSE fluorescent staining solution has no effect on the regeneration of protoplast cells of Morchella esculenta, Hypsizygus marmoreus and Agrocybe radicata.
TABLE 3 influence of CFSE dye liquor on the regeneration rate
The protoplast cells of the edible fungi have larger size difference, and the prepared protoplast cells have larger concentration difference and are thinThe difference in cell size also has some effect on the count of protoplasts. However, as can be seen from the above examples and FIG. 4, the flow cytometry combined with CFSE fluorescent dye can remove the influence of the cell size on the counting result, and can distinguish between live cells and dead cells, the present invention has a cell size of 2.1-13.2 μm and a cell concentration of 104～108The protoplast cell count of each/mL has better repeatability, and the viability rate of the protoplast has better consistency under different dilution times. Compared with the traditional blood counting method, the detection method provided by the invention has the advantages that the better consistency is kept on the magnitude order, the speed is higher, the efficiency is higher, and the result can be obtained within 15 minutes. The CFSE is used as a tracing dye of living cells, the activity and the regeneration rate of protoplast cells of the edible fungi are not influenced, the concentration of the CFSE cells is continuously reduced along with the division of the cells, and the existence of the CFSE dye cannot be observed in the finally formed fruiting body.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A method for rapidly detecting the number of protoplasts of viable edible fungi by using flow cytometry is characterized by comprising the following steps:
(1) sample preparation and staining: filtering the edible fungus protoplast cells obtained by the enzymolysis of the edible fungi by a screen, and adding a CFSE fluorescent staining solution for staining to obtain stained edible fungus protoplast cells;
(2) analysis and determination by flow cytometry:
a. analysis was performed using a flow cytometer: placing the stained protoplast cells of the edible fungi into a flow cytometer, and respectively drawingMaking an FSC/SSC scatter diagram and a FL1/SSC scatter diagram; setting the FSC threshold value of an FSC/SSC scatter diagram to be 50 so as to remove edible fungus protoplast cell fragments, adjusting the voltage of the FSC and SSC so that the dyed edible fungus protoplast cell clusters are completely displayed and positioned at the central position of the picture, and then setting a P1 gate to completely circle out the cell clusters, wherein the cells displayed in the P1 gate are total edible fungus protoplast cells; edible fungus protoplast cells within the P1 gate are shown on FL1/SSC scatter plots, followed by FL1 signal greater than 10 on FL1/SSC scatter plots2The cell circle gate of (1) is set as a P2 gate, and viable edible fungus protoplast cells are displayed in a P2 gate;
b. calculating the cell concentration of the edible fungus protoplast according to the flow rate and the collection time of the flow cytometer;
c. and counting the total number of the protoplast cells of the edible fungi in the P2 gate and the total number of the protoplast cells of the edible fungi in the P1 gate, and calculating the activity rate of the protoplasts of the edible fungi.
2. The method for rapidly detecting the number of the protoplasts of the viable edible fungi according to claim 1, wherein the screen in the step (1) is 200 meshes.
3. The method for rapidly detecting the number of the protoplasts of the viable edible fungi by using the flow cytometry as claimed in claim 1, wherein the addition amount of the CFSE fluorescent staining solution in the step (1) is 1% v/v.
4. The method for rapidly detecting the number of the protoplasts of the viable edible fungi by using the flow cytometry as claimed in claim 1, wherein the staining condition of the CFSE fluorescent staining solution in the step (1) is room temperature and dark, and the staining time is 5-10 minutes.
5. The method for rapidly detecting the number of viable protoplasts of edible fungus by using flow cytometry as claimed in claim 1, wherein the cell concentration of the protoplasts of edible fungus obtained in the step (1) is 1.0 x 104~1.0×108one/mL.
6. The method for rapidly detecting the number of protoplasts of viable edible fungus by using flow cytometry as claimed in claim 1, wherein the flow cytometer in the step (2) is provided with a 488nm laser.
7. The method for rapidly detecting the number of the viable edible fungus protoplasts by using the flow cytometry as claimed in claim 1, wherein a negative control is set in the method, namely, an equal amount of 1% v/v of dimethyl sulfoxide is added into the edible fungus protoplast cells, and the mixture is placed at room temperature in a dark place for 5-10 minutes.
8. The method for rapid detection of protoplast count of viable edible fungus by flow cytometry as claimed in claim 7, wherein in step (2), before setting the P2 gate, negative control protoplast cell population is adjusted to 10 of FL1 abscissa using negative control tube2Within, FL1/SSC scattergram is then plotted on FL1 abscissa 102All the other positions are circled to be P2 gates, and the FL1 signal is greater than 102A positive signal.
9. The method for rapidly detecting the number of viable edible fungus protoplasts by using flow cytometry as claimed in claim 1, wherein the cell size of the edible fungus protoplasts is 2.1-13.2 μm.
10. The method for rapid detection of the number of viable edible fungus protoplasts by flow cytometry as claimed in claim 1, wherein the viability rate of the edible fungus protoplasts = (total number of edible fungus protoplast cells in P2 gate/total number of edible fungus protoplast cells in P1 gate) x 100%.
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