CN117384820A - Preparation method and application of arabidopsis silique protoplast - Google Patents
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
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- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
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- 238000000605 extraction Methods 0.000 claims abstract description 6
- 108010059892 Cellulase Proteins 0.000 claims abstract description 5
- 229940106157 cellulase Drugs 0.000 claims abstract description 5
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Abstract
The invention belongs to the technical field of plant cell biology, and discloses a preparation method of arabidopsis thaliana silique protoplast and application thereof in single-cell transcriptome sequencing, wherein the preparation method comprises the following steps: s1: carrying out enzymolysis on the long-horn fruits of the arabidopsis thaliana by using an enzymolysis liquid to obtain an extraction liquid of protoplasts of the long-horn fruits of the arabidopsis thaliana; the enzymolysis liquid comprises cellulase R10, pectase Y23, eductase R10, hemicellulase, crashing enzyme, mannitol, BSA, caCl 2 KCl and MES; s2: and (2) adding a buffer solution into the extracting solution of the arabidopsis thaliana silique protoplast obtained in the step (S1) to terminate the reaction to obtain a reaction solution, and purifying the reaction solution to obtain the suspension of the arabidopsis thaliana silique protoplast. Compared with the prior art, the preparation method of the arabidopsis silique protoplast provided by the invention is simple, convenient and feasible, has high extraction efficiency, and provides sufficient materials for the subsequent single-cell transcriptome sequencing.
Description
Technical Field
The invention belongs to the technical field of plant cell biology, and particularly relates to a preparation method and application of arabidopsis thaliana silique protoplast.
Background
Plant protoplasts refer to cell clusters that have their cell walls removed but remain viable. Due to the lack of protection and support of cell walls, protoplasts can absorb macromolecular substances such as microorganisms, organelles, nucleic acids and the like, which makes the protoplasts an important multi-purpose cell system in basic theoretical research, and has application in plant virus, plant physiology and crop breeding research. At present, a protoplast separation system is established in plants such as arabidopsis thaliana, rice, corn, potato, tobacco, citrus, ginkgo and the like, but most of plant materials used by the protoplast separation system are tender plant tissues. For example, young rosette leaves are used for preparing arabidopsis leaf protoplast, yellow leaves of rice are used for preparing rice leaf protoplast under dark culture, and regeneration leaves formed by in vitro tissue culture and differentiation of seed embryo of ginkgo are used for preparing protoplast.
Plants are composed of diverse cell types, and specific transcription programs between different cell types produce unique biological functions between each cell type. Single cell transcriptome sequencing technology is a new generation of research means that has emerged in recent years and was successfully developed and applied in model plants in 2003. The technology can be used for capturing, marking and sequencing single cells after plant tissues are dispersed into the single cells, so that the in-situ gene expression and positioning information of plants can be deeply revealed. Single cell transcriptome sequencing technology has been mature, but protoplast isolation and purification of high quality plant tissue is still a major bottleneck for single cell transcriptome sequencing as a basis for single cell transcriptome sequencing. In addition, the tissue and cell composition of the silique as a mature tissue are much more complex than those of organs such as leaves and roots, and the protoplast preparation is very difficult, so that a method for preparing the protoplast of the silique suitable for single-cell sequencing is lacking.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a preparation method of the arabidopsis thaliana silique protoplast, which has simple steps and can improve the extraction rate of the arabidopsis thaliana silique protoplast.
The invention also provides the arabidopsis thaliana silique protoplast prepared by the method.
The invention also provides application of the arabidopsis thaliana silique protoplast.
According to a first aspect of the present invention, there is provided a method for preparing an Arabidopsis thaliana silique protoplast, comprising the steps of:
s1: carrying out enzymolysis on the long-horn fruits of the arabidopsis thaliana by using an enzymolysis liquid to obtain an extraction liquid of protoplasts of the long-horn fruits of the arabidopsis thaliana; the enzymolysis liquid comprises 1-2wt% of cellulase R10, 0.1-4wt% of pectase Y23, 0.5-2wt% of educing enzyme R10, 0-2wt% of hemicellulase, 1-2.5wt% of crashing enzyme, 0.2-0.5mol/L mannitol, 0.1-0.5wt% of BSA and 0.1-0.2mol/LCaCl 2 0.01-0.05mol/L KCl and 0.01-0.06mol/L MES;
s2: and (2) adding a buffer solution into the extracting solution of the arabidopsis thaliana silique protoplast obtained in the step (S1) to terminate the reaction to obtain a reaction solution, and purifying the reaction solution to obtain the suspension of the arabidopsis thaliana silique protoplast.
In some embodiments of the present invention, the amounts of the components in the enzymatic hydrolysate in step S1 are as follows: 1.5-1.8wt% cellulase R10, 0.1-3wt% pectase Y23, 1-1.5wt% educt enzyme R10, 0.5-2wt% hemicellulase, 1.5-2wt% crashase, 0.1-0.5mol/L mannitol, 0.1-0.4wt% BSA, 0.1-0.14mol/L CaCl 2 0.02-0.03mol/L KCl and 0.02-0.05mol/L MES.
In some preferred embodiments of the present invention, the following components are used in the enzymatic hydrolysate in step S1: 1.8wt% cellulase R10, 3wt% pectinase Y23, 1.5wt% educt enzyme R10, 1.5wt% hemicellulase, 2wt% crashase, 0.2mol/L mannitol, 0.4wt% BSA, 0.14mol/L CaCl 2 0.03mol/L KCl and 0.05mol/LMES.
In some embodiments of the invention, the silique of step S1 is transected and slit prior to enzymatic hydrolysis.
In some embodiments of the present invention, the solvent of the enzymatic hydrolysate in step S1 comprises distilled water.
In some embodiments of the invention, the conditions for the enzymolysis in step S1 are: placing in a dark environment at 24-32deg.C on a shaking table for enzymolysis for 1.5-3.5 hr; the rotating speed of the shaking table is 35-55r/min.
In some preferred embodiments of the present invention, the conditions for the enzymolysis in step S1 are: placing in a dark environment at 26-30deg.C, and performing enzymolysis for 2-3 hr; the rotating speed of the shaking table is 40-50r/min.
In some more preferred embodiments of the present invention, the conditions for the enzymolysis in step S1 are: placing the mixture on a shaking table for enzymolysis for 2.5 hours at 28 ℃ in a dark environment; the rotation speed of the shaking table is 45r/min.
In some embodiments of the invention, the buffer of step S2 comprises a W5 solution.
In some preferred embodiments of the invention, the W5 solution comprises 54mmol/L NaCl,125mmol/LCaCl 2 25mmol/L KCl and 2mmol/L MES.
In some embodiments of the invention, the W5 solution has a pH of 5 to 7.
In some preferred embodiments of the invention, the W5 solution has a pH of 5.7.
In some embodiments of the invention, the method of purification described in step S2 comprises sequentially filtering and centrifuging.
In some preferred embodiments of the invention, the filtration is performed using a cell strainer with a pore size of 20-60 μm.
In some more preferred embodiments of the invention, the filtration uses a 30-50 μm cell strainer.
In some more preferred embodiments of the invention, the filtration uses a 40 μm cell strainer.
In some preferred embodiments of the invention, the centrifugation conditions are: centrifuging at 650-750r/min for 8-12min.
In some more preferred embodiments of the invention, the centrifugation conditions are: centrifuging at 680-720r/min for 9-11min.
In some more preferred embodiments of the invention, the centrifugation conditions are: centrifuging at 700r/min for 10min.
According to a second aspect of the present invention, there is provided an Arabidopsis thaliana silique protoplast prepared by the method according to the first aspect of the present invention.
According to a third aspect of the invention, there is provided the use of an Arabidopsis thaliana silique protoplast according to the second aspect of the invention in single cell transcriptome sequencing.
In some embodiments of the invention, the platform employed for sequencing comprises a 10X Genomics Chromium Next GEM Chip platform.
According to a fourth aspect of the invention, there is provided the use of an Arabidopsis thaliana silique protoplast according to the second aspect of the invention in research of cell populations in Arabidopsis seed development.
The invention has at least the following beneficial effects: the invention provides a preparation method of arabidopsis thaliana silique protoplast, which has simple and easy steps and high extraction efficiency. Compared with the existing preparation method for the arabidopsis leaf protoplast, the preparation method for the protoplast is based on the mature tissue of the arabidopsis, namely the silique protoplast, and the high-yield protoplast is obtained by improving the cutting mode of the silique of the arabidopsis and the formula of enzymolysis liquid. The arabidopsis silique protoplast prepared by the scheme is suitable for subsequent further single cell sequencing due to higher abundance. After single-cell transcriptome sequencing and grouping, 19 cell subsets are obtained, and the scheme has important significance for single-cell transcriptome research of cell groups in arabidopsis seed development.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram showing the different cutting methods of the Arabidopsis thaliana silique sample in example 1 of the present invention, wherein A is a transverse cutting, and B is a longitudinal cutting;
FIG. 2 is a graph showing the dimension-reduced cluster analysis of protoplasts prepared in example 7 according to the present invention by single-cell sequencing analysis.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1
The embodiment prepares an arabidopsis silique protoplast, which comprises the following specific preparation processes:
40 pieces of Arabidopsis thaliana silique are taken, and are transversely cut by a blade, the cutting method is shown in figure 1A, and the size of a sample after transverse cutting is 2-4 mm. The samples are respectively put into 5mL of enzymolysis liquid (the formula of the enzymolysis liquid is shown in table 1), and the fixed shaking table is subjected to enzymolysis for 2.5h at the temperature of 28 ℃ in the dark at the rotating speed of 45r/min. After completion of the enzymatic hydrolysis, 1mL of a W5 solution (54 mmol/L NaCl,125mmol/L CaCl) was added 2 25mmol/L KCl and 2mmol/L MES, pH 5.7). Filtering with 40 μm cell filter screen, centrifuging at 700r/min for 10min, removing supernatant, resuspending with 100 μm L W solution, and gently mixing to obtain protoplast suspension.
Example 2
This example produced an Arabidopsis thaliana silique protoplast, which was prepared by a procedure differing from example 1 only in the method of cutting the sample, using a slitter as shown in FIG. 1B.
Example 3
This example produced an Arabidopsis thaliana silique protoplast, which was prepared by a procedure differing from example 1 only in the method of cutting the sample, using transverse and longitudinal cuts as shown in FIG. 1.
Example 4
The present example prepared an Arabidopsis thaliana silique protoplast, and the specific preparation process was different from example 3 only in that the enzymatic hydrolysate was different, and the specific composition of the enzymatic hydrolysate is shown in Table 1.
Table 1: formula of different enzymolysis solutions
Example 5
This example produced an Arabidopsis thaliana silique protoplast, which differs from example 3 only in the enzymatic hydrolysate, the specific composition of which is shown in Table 1.
Example 6
This example produced an Arabidopsis thaliana silique protoplast, which differs from example 3 only in the enzymatic hydrolysate, the specific composition of which is shown in Table 1.
Test examples
The present test example examined the yield of protoplasts prepared in examples 1-6 in order to screen more optimal protoplast preparation conditions; this experimental example also isolated arabidopsis thaliana silique protoplasts using the same protoplast preparation method as in example 3 and used for single cell transcriptome sequencing.
In this test example, the protoplast yield was counted using a hemocytometer, and the plastid yield calculation formula was: protoplast yield (number/g) =number of protoplasts/mass of silique.
1) Study of the effect of different arabidopsis long angle fruit cuts on protoplast yield:
the results of the statistics of the yields of protoplasts prepared in examples 1 to 3 are shown in Table 2, and it can be seen from Table 2 that the yield of the protoplasts of Arabidopsis thaliana after transection was 1.2658X 10 5 The yield per gram after slitting is 1.9481 multiplied by 10 5 The highest yield of protoplasts per gram after transverse and longitudinal cutting was 5.9880X 10 5 Each/g. The above results demonstrate that when preparing Arabidopsis thaliana silique protoplasts, the optimal sample treatment method is cross-cut plus slit.
Table 2: protoplast yield from different arabidopsis long angle fruit cuts
2) Study of the effect of different enzymatic liquids on protoplast yield:
examples 3-6 protoplasts were prepared using enzymatic solutions of different formulations, respectively, and the other preparation conditions were the same, and the protoplast yields of examples 3-6 are shown in Table 3. The results in Table 3 show that the protoplast isolated in example 3 gives a yield of 6.2333X 10 5 The protoplast yield isolated in example 4 was 0.9585X 10 per gram 5 Each g, example 5 was isolatedThe protoplast yield of (C) was 2.4823X 10 5 The protoplast yield isolated in example 6 was 3.3123X 10 per gram 5 Each/g. The only difference between example 3 and example 6 is the amount of mannitol added, 0.2mol/L for example 3 and 0.4mol/L for example 6. It is known that mannitol with high concentration has the main function of promoting separation of plant cytoplasm walls in the enzymolysis process, so that enzymes in the enzymolysis liquid can better play a role. However, example 3 of the present invention achieves higher yields by using lower concentrations of mannitol.
Table 3: protoplast yield of different enzymatic hydrolysate formulations
3) Test of the suitability of arabidopsis silique protoplasts in single cell transcriptome sequencing:
30 Arabidopsis thaliana siliques with different maturity degrees are collected, and protoplasts of the Arabidopsis thaliana siliques are obtained by separation by adopting the same protoplast preparation method as in example 3 and are used for single cell transcriptome sequencing. The total collection of 33240 effective protoplast cells by using 10X Genomics Chromium Next GEM Chip is used for sequencing, 761983365reads of 23158 cells are actually obtained, QC treatment is carried out on the data, the treated data comprise 96.5% of reads of effective Barcode, and the comparison rate of base quality Q30 and reads is 93.5%. And performing UMAP dimension reduction treatment and clustering analysis to finally obtain 19 cell subsets shown in figure 2. The cell marker genes used in the cluster analysis are shown in Table 4.
Table 4: cell marker gene
As can be seen from the results of FIG. 2, the yield of the Arabidopsis thaliana silique protoplast prepared by the method is high, the Arabidopsis thaliana silique protoplast can be directly used for single cell transcriptome sequencing, and different cell groups in Arabidopsis thaliana seed development can be accurately positioned, so that the cell composition of different development stages in the Arabidopsis thaliana seed development process is further explored.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The preparation method of the arabidopsis silique protoplast is characterized by comprising the following steps of:
s1: carrying out enzymolysis on the long-horn fruits of the arabidopsis thaliana by using an enzymolysis liquid to obtain an extraction liquid of protoplasts of the long-horn fruits of the arabidopsis thaliana; the enzymolysis liquid comprises 1-2wt% of cellulase R10, 0.1-4wt% of pectase Y23, 0.5-2wt% of educing enzyme R10, 0-2wt% of hemicellulase, 1-2.5wt% of crashing enzyme, 0.2-0.5mol/L mannitol, 0.1-0.5wt% of BSA and 0.1-0.2mol/LCaCl 2 0.01-0.05mol/L KCl and 0.01-0.06mol/L MES;
s2: and (2) adding a buffer solution into the extracting solution of the arabidopsis thaliana silique protoplast obtained in the step (S1) to terminate the reaction to obtain a reaction solution, and purifying the reaction solution to obtain the suspension of the arabidopsis thaliana silique protoplast.
2. The method of claim 1, wherein the arabidopsis thaliana silique in step S1 is subjected to cross cutting and slitting prior to enzymatic hydrolysis.
3. The method according to claim 1, wherein the conditions for the enzymolysis in step S1 are: placing in a dark environment at 24-32deg.C on a shaking table for enzymolysis for 1.5-3.5 hr; the rotating speed of the shaking table is 35-55r/min.
4. The method of claim 1, wherein the buffer in step S2 comprises a W5 solution; preferably, the W5 solution comprises 54mmol/L NaCl and 125mmol/L CaCl 2 25mmol/L KCl and 2mmol/L MES.
5. The method of claim 1, wherein the purification in step S2 comprises sequential filtration and centrifugation.
6. The method according to claim 5, wherein the filtration is performed using a cell strainer having a pore size of 20 to 60 μm; and/or, the centrifugation conditions are: centrifuging at 650-750r/min for 5-15min.
7. An arabidopsis thaliana silique protoplast prepared by the method of any one of claims 1-6.
8. Use of the arabidopsis thaliana silique protoplast according to claim 7 in single cell transcriptome sequencing.
9. The use of claim 8, wherein the platform for sequencing comprises a 10X Genomics Chromium Next GEM Chip platform.
10. Use of the arabidopsis thaliana silique protoplast according to claim 7 in a cell population study in arabidopsis thaliana seed development.
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