A method for dissociating protoplasts of root tip cells of Picea australis

Abstract

The present invention relates to a method for dissociating the protoplasts of the root tip cells of sandy spruce, comprising the following steps: step (1), tissue culture of mature sandy spruce seeds to obtain the root tip tissue of sandy spruce; step (2), cutting the root tip of sandy spruce into thin slices of about 1 mm, immersing it in an enzymatic hydrolysis solution system, and performing an enzymatic hydrolysis reaction under dark conditions; step (3), using a 40 μm sterile cell filter to sieve the enzymatic hydrolysis primary product obtained in step (2), collecting it in a new test tube, centrifuging it at a low speed, and discarding the supernatant; step (4), adding a cleaning solution to the precipitate obtained in step (3) to resuspend it, centrifuging it at a low speed, discarding the supernatant, adding the cleaning solution again to resuspend the protoplasts, and detecting the cell concentration and activity by using a microscope cell counting plate and trypan blue staining. The present invention realizes the dissociation of the root tip protoplasts of sandy spruce, and can quickly and efficiently prepare the protoplast cells of the root tissue of sandy spruce.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for dissociating protoplasts of root tip cells of Picea australis, which is suitable for single-cell transcriptome sequencing. Background Art

[0002] Picea mongolica is an evergreen tree of the genus Picea in the Pinaceae family. It is a rare plant and a native tree species unique to Inner Mongolia. It can be used as an important afforestation species in the western region and plays an important role in restoring the ecological environment and restoring vegetation in sandy land. In addition, its wood is mostly used in papermaking, furniture and other production, and its bark can be used to extract turpentine. Picea mongolica is only distributed in the southeastern part of the Hunshandake Sandy Land in Inner Mongolia. The extremely harsh living environment makes Picea mongolica have excellent stress resistance characteristics such as drought resistance, cold resistance, and resistance to barrenness. Therefore, the study of its stress resistance mechanism has always attracted the attention of researchers. The root tip of Picea mongolica is the first part to respond to abiotic stress. By exploring the developmental trajectory of the root tip cells of Picea mongolica and the molecular mechanism of responding to abiotic stress, we can provide excellent gene resources for the transgenic improvement of crops to resist abiotic stress.

[0003] Traditional sequencing technology uses the entire individual, organ or tissue as a sample, and its analysis results are the mean of the effect being studied, which cannot reveal the heterogeneity of gene expression between different types of cells. Single-cell sequencing obtains information such as genomic structural changes, gene expression, DNA methylation, etc. at the level of a single cell, and can deeply analyze the expression changes of different types of cells and certain specific cell functions. In recent years, single-cell sequencing has been widely used in the study of animal cells and has made great progress. However, due to the existence of cell walls in plant cells compared to animal cells, and the uneven thickness of cell walls of different types of cells, enzymatic hydrolysis conditions are not easy to coordinate, and there are huge differences in the size of plant cells, the application of single-cell technology in the plant kingdom is relatively slow. However, due to breakthroughs in related technologies, young herbaceous plants and Arabidopsis, tobacco, rice, corn, etc. have perfect protoplast preparation technology, and related single-cell research has shown an accelerating trend in recent years.

[0004] At present, there are few methods for preparing protoplasts of coniferous species such as spruce, and the protoplasts prepared by existing methods are only used for transient genetic transformation research. The quality of protoplasts cannot meet the requirements of 10×Genomics single-cell transcriptome sequencing, which seriously restricts the pace of 10×Genomics single-cell transcriptome sequencing of woody plants. Therefore, it is necessary to develop a protoplast preparation method that can be applied to 10×Genomics single-cell transcriptome sequencing using young roots of sandy spruce as materials, laying a foundation for conifer breeding, genetic engineering and cell physiology research. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a method for dissociating protoplasts of root tip cells of Picea australis for single-cell transcriptome sequencing, so as to fill the gap in the preparation of protoplasts of root tissues of coniferous species and provide a powerful tool for molecular biology research of woody plants.

[0006] To achieve the above object, the present invention provides a method for dissociating protoplasts of root tip cells of Picea australis, the dissociation method comprising the following steps:

[0007] Step (1), tissue culturing Picea australis seeds to obtain root tip tissue of Picea australis;

[0008] Step (2), cutting the root tips of Picea mongolica into thin slices of about 1 mm, immersing them in an enzymatic hydrolysis solution system, and performing an enzymatic hydrolysis reaction under dark conditions, wherein the enzymatic hydrolysis solution system comprises: 3% (w / v) macerate, 1.5% (w / v) cellulase R-10, 1% (w / v) pectinase, 0.5 mol / L mannitol, and the solvent is sterilized ddH2O;

[0009] Step (3), using a 40 μm sterile cell filter to sieve the enzymatic primary product obtained in step (2), collect it into a new test tube, centrifuge it at a low speed, and discard the supernatant;

[0010] Step (4), adding the washing solution to resuspend the precipitate obtained in step (3), centrifuging at low speed, discarding the supernatant, adding the washing solution again to resuspend the protoplasts, and detecting the cell concentration and activity using a microscope cell counting plate and trypan blue staining.

[0011] Preferably, in step (1), the step of obtaining the root tip tissue of Picea australis comprises: taking full and healthy Picea australis seeds, sterilizing them and placing them in MS culture medium for germination and growth; sampling when the root length grows more than 2 cm, and taking tissue about 1 cm from the root tip.

[0012] Preferably, the enzymatic hydrolysis reaction conditions in step (2) are: the enzymatic hydrolysis reaction temperature is 28° C., and the culture is statically cultured in the dark for 5 to 7 hours.

[0013] Preferably, the low-speed centrifugation conditions in steps (3) and (4) are 4° C. and 400 rpm for 3 to 5 min.

[0014] Preferably, the formula of the cleaning solution in step (4) is: 0.1 mol / L KCl, 4 mmol / L MES, 0.04% (w / v) BSA, 0.4 mol / L mannitol, and the solvent is sterilized ddH2O.

[0015] Preferably, the concentration of the trypan blue staining solution in step (4) is 0.4% (w / v), and the ratio of the protoplast suspension to the trypan blue staining solution is 1:1.

[0016] Another object of the present invention is to provide a method for dissociating protoplasts of root tip cells of Picea australis and use the method in constructing a single-cell transcriptome library.

[0017] Based on the above technical solution, the advantages of the present invention are:

[0018] The method for dissociating the protoplasts of the root tip cells of the sandy spruce of the present invention realizes the dissociation of the protoplasts of the root tip of the sandy spruce, and can quickly and efficiently prepare the protoplast cells of the root tissue of the sandy spruce. The concentration of the isolated protoplasts is as high as 440 cells / μL, the background is relatively clean, and the cell activity is higher than 93%. The result meets the protoplast sample concentration (100-1200 cells / μL) and activity rate requirements (higher than 85%) of 10×Genomics single-cell plant transcriptome sequencing, and the cell diameter is less than 40 μm, and single-cell sequencing can be directly performed on the machine, which provides basic conditions for exploring the developmental trajectory of the root tip cells of the sandy spruce and the molecular mechanism of responding to abiotic stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 This is a picture of mature cones of sand spruce;

[0021] Figure 2 This is a picture of a 10-day-old Picea mongolica seedling;

[0022] Figure 3 The protoplast counts and activities were prepared from the root tips under a 10× field of view;

[0023] Figure 4 The morphology of protoplasts prepared from the root tip under a 40× field of view. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments.

[0025] The present invention provides a method for dissociating the protoplasts of root tip cells of Picea australis. Figure 1 to Figure 4 As shown, a preferred embodiment of the present invention is shown.

[0026] The method for dissociating the protoplasts of the root tip cells of Picea australis comprises the following steps:

[0027] The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention. The invention relates to a method for preparing the present invention.

[0028] The detailed operations are as follows:

[0029] (1) Preparation of materials:

[0030] like Figure 1 As shown, take the mature cones of sand spruce, peel off the scales and select 50 healthy and plump sand spruce seeds, disinfect them with 2% NaClO for 15 minutes on a clean bench, wash them with sterile distilled water 8 to 10 times, put them on sterile filter paper to dry the water, and then put them in germination medium. After vernalization in a dark refrigerator at 4℃ for 4 days, move them into a light incubator. The culture conditions are: temperature 25℃, humidity 65%, 16h light / 8h light. Sampling is taken when the root length exceeds 2cm around 10 days after seed germination, such as Figure 2 shown.

[0031] (2) Preparation of consumables:

[0032] Prepare sterile sharp blades, culture dishes, hemocytometers, filter paper, tweezers, centrifuge tubes (50mL, 2mL), 40μm cell sieves, 10ul / 100 / 1000μL pipette tips with low adsorption filters (RANase free), pipettes of different specifications, ddH2O, etc.

[0033] (3) Preparation of enzymatic hydrolysate:

[0034] The enzymatic solution system is: 3% (w / v) cleavage enzyme, 1.5% (w / v) cellulase R-10, 1% (w / v) pectinase, 0.5 mol / L mannitol, and the solvent is sterilized ddH2O. After preparation, filter and sterilize using a 0.22 μm organic filter on a clean bench. The enzyme solution must be prepared and used immediately, and should not be stored for a long time to prevent the loss of enzyme activity.

[0035] (4) Dissociation of protoplasts:

[0036] When the root length exceeds 2 cm, the sample is taken, and the tissue about 1 cm from the root tip is taken. In the clean bench, the sand spruce seedlings are taken out with tweezers and placed on sterile filter paper. 20 root tips are cut, about 1.0 cm long, and cut into 1 mm thick filaments with a sharp blade, and quickly placed in a centrifuge tube containing 10 mL of enzymatic solution. Incubate in a constant temperature incubator at 28°C in the dark for 5 to 7 hours, during which the protoplast release can be monitored under a microscope.

[0037] (5) Filtration and collection of protoplasts:

[0038] The enzymatic primary product obtained in the previous step was filtered through a sterile 40 μm cell sieve to remove the residue, and then the cell sieve was rinsed with 5 mL of pre-cooled cleaning solution, centrifuged at 4°C and 400 rpm for 5 min, and the filtrate was collected in a 50 mL centrifuge tube; the supernatant was gently removed with a 5 mL pipette, and 3 mL of pre-cooled cleaning solution was added to resuspend the protoplasts, and centrifuged at 4°C and 400 rpm for 3 min. The resuspending and centrifugation were repeated twice, and the supernatant was removed to obtain a cell precipitate; finally, the precipitate was resuspended with 2 mL of pre-cooled cleaning solution to prepare a protoplast suspension.

[0039] The formula of the cleaning solution is: using sterilized ddH2O as solvent, adding 0.1 mol / L KCl, 4 mmol / L MES, 0.04% (w / v) BSA, and 0.4 mol / L mannitol, dissolving them completely and mixing them evenly.

[0040] (6) Microscopic examination of protoplasts:

[0041] Take 50 μL of protoplast suspension and add 50 μL of 0.4% trypan blue dye solution, mix slowly and evenly, and let stand at room temperature for 2-3 minutes in the dark. Place a drop of the mixed solution on a cell counting plate and examine it under an ordinary optical microscope. A large number of protoplasts of the roots of Picea australis can be observed, with a diameter of less than 40 μm. The cell viability results of the mixture of protoplast suspension and 0.4% trypan blue dye solution under a 10x field of view are as follows: Figure 3 As shown, the background is clean, the number of tissue or cell fragments is small, the protoplasts with high vitality are not stained, and the protoplasts with low vitality are stained blue.

[0042] The cell viability results of the protoplast suspension and 0.4% trypan blue dye mixture under 40x field of view are as follows Figure 4 As shown, the protoplasts were kept in a very complete state and appeared round and translucent under microscopic examination. Finally, the cell number and cell viability were counted and calculated.

[0043] Preparation of 0.4% Trypan blue dye solution: Weigh 4 g of Trypan blue and dissolve it in 100 mL of sterilized ddH2O. After filtering the solution through filter paper, sterilize it by suction filtration on a clean bench and store it in a 4°C refrigerator. When using, dilute it to 0.4% with PBS solution.

[0044] (7) Protoplast counting method:

[0045] This example adopts the cell counting method to count the total number of root tip protoplasts and the number of dead cells under a 10× visual field, and calculate the protoplast concentration and protoplast survival rate (%). The calculation formula is:

[0046] Protoplast number / μL = 10 × total number of protoplasts in the field of view × 10000 × dilution factor;

[0047] Protoplast activity (%) = (total number of protoplasts - dead protoplasts) / total number of protoplasts × 100;

[0048] Calculated by the above formula, the protoplast concentration was 440 cells / μL; the survival rate of the protoplasts was counted by trypan blue staining, and the results showed that the protoplast vitality was 93%.

[0049] (8) Preservation of protoplasts:

[0050] Place on ice and perform 10x on-machine operation. Avoid leaving the tube on ice for more than 30 minutes.

[0051] The present invention also provides a method for dissociating protoplasts of root tip cells of Picea australis and its application in constructing a single-cell transcriptome library. Based on the dissociation method, a protoplast preparation method that can be applied to 10×Genomics single-cell transcriptome sequencing is obtained, which will lay a foundation for breeding of Picea australis, the development of genetic engineering and cell physiology research.

[0052] The method for dissociating the protoplasts of the root tip cells of the sandy spruce of the present invention realizes the dissociation of the protoplasts of the root tip of the sandy spruce, and can quickly and efficiently prepare the protoplast cells of the root tissue of the sandy spruce. The concentration of the isolated protoplasts is as high as 440 cells / μL, the background is relatively clean, and the cell activity is higher than 93%. The result meets the protoplast sample concentration (100-1200 cells / μL) and activity rate requirements (higher than 85%) of 10×Genomics single-cell plant transcriptome sequencing, and the cell diameter is less than 40 μm, and single-cell sequencing can be directly performed on the machine, which provides basic conditions for exploring the developmental trajectory of the root tip cells of the sandy spruce and the molecular mechanism of responding to abiotic stress.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A method for dissociating protoplasts of root tip cells of Picea australis, characterized in that: The dissociation method comprises the following steps: Step (1), tissue culture of mature Picea australis seeds to obtain root tip tissue of Picea australis; Step (2), sampling when the root length grows more than 2 cm, cutting the root tip of the mongolica spruce into 1 mm slices, immersing the slices in an enzymatic solution system, and performing an enzymatic reaction under dark conditions, wherein the enzymatic solution system comprises: 3% cleavage enzyme (w / v), 1.5% cellulase R-10 (w / v), 1% pectinase (w / v), 0.5 mol / L mannitol, and the solvent is sterilized ddH2O; Step (3), using a 40 μm sterile cell filter to sieve the enzymatic primary product obtained in step (2), collect it into a new test tube, centrifuge it at low speed, and discard the supernatant; wherein the low-speed centrifugation condition is 4°C and 400 rpm for 3 to 5 minutes; Step (4), adding a cleaning solution to resuspend the precipitate obtained in step (3), centrifuging at a low speed, discarding the supernatant, adding the cleaning solution again to resuspend the protoplasts, and detecting the cell concentration and activity using a microscope cell counting plate and trypan blue staining, wherein the concentration of the separated protoplasts reaches 440 cells / μL, the cell activity is higher than 93%, and the cell diameter is less than 40 μm; wherein the formula of the cleaning solution is: 0.1 mol / L KCl, 4 mmol / L MES, 0.04% BSA (w / v), 0.4 mol / L mannitol, and the solvent is sterilized ddH2O; the low speed centrifugation conditions are 4°C, 400 rpm centrifugation for 3-5 min.

2. The dissociation method according to claim 1, characterized in that: In step (1), the step of obtaining the root tip tissue of Picea australis includes: taking full and healthy Picea australis seeds, sterilizing them and placing them in MS culture medium for germination and growth, sampling when the root length exceeds 2 cm, and taking tissue 1 cm from the root tip.

3. The dissociation method according to claim 1, characterized in that: The enzymatic hydrolysis reaction conditions in step (2) are: the enzymatic hydrolysis reaction temperature is 28° C., and the culture is statically incubated in the dark for 5 to 7 hours.

4. The dissociation method according to claim 1, characterized in that: The concentration of the trypan blue staining solution in step (4) is 0.4% (w / v), and the ratio of the protoplast suspension to the trypan blue staining solution is 1:1.

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