Hybrid potato seedling hypocotyl single cell extraction method and application thereof

Abstract

The invention provides a hybrid potato seedling hypocotyl single cell extraction method and application thereof. According to the characteristics of potato seedling hypocotyl single cells, the extraction efficiency of the protoplast is remarkably improved by adjusting the dosage of different cellulase in the enzymatic hydrolysate and combining a slight extrusion mode. The method is simple and rapid, does not need complex treatment and expensive reagents, and is convenient to popularize and apply. Besides, experiments prove that the protoplast extracted by the method can be effectively used for detection such as subsequent sequencing and the like, so that the cells extracted by the method are relatively high in activity and have a wide application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a hybrid potato seedling hypocotyl single cell extraction method and application thereof. BACKGROUND

[0002] The genetic background of potato cultivars is relatively narrow, and the common potato used in production is basically tetraploid, but the wild species is diploid. Due to the difference in ploidy level and endosperm balance number, it is difficult to transfer the high-quality genes of many wild resources to common potatoes through traditional breeding. In order to fully utilize wild germplasm resources and broaden the narrow genetic basis of cultivated potatoes, researchers have made great efforts. The use of somatic hybridization technology can effectively overcome the reproductive isolation caused by ploidy differences between cultivated potatoes and wild species, transfer high-quality genes from wild species to cultivated species for selection and improvement of varieties, and also avoid biological safety supervision problems related to genetic modification. At the same time, the hybrid F1 produced by diploid hybrid potato breeding needs to be promoted from seed to seedling, which involves seed germination and developmental biology. The seed dicotyledon emerges by relying on the elongation of hypocotyl, so extracting hypocotyl single cells can help analyze the single cell biology of hypocotyl elongation, which is of great significance to understanding the regulation of hybrid diploid potato seed germination and cotyledon emergence. SUMMARY

[0003] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a hybrid potato seedling hypocotyl single cell extraction method.

[0004] Another purpose of the present application is to provide the application of the hybrid potato seedling hypocotyl single cell extraction method.

[0005] The purpose of the present application is achieved by the following technical solutions: A hybrid potato seedling hypocotyl single cell extraction method, comprising the following steps: (1) cut the potato seedling hypocotyl tissue into thin slices, and wash the tissue slices with a pretreatment solution twice; (2) put the tissue slices into an enzyme solution ES and incubate; (3) after incubation, shake the centrifuge tube, take the upper layer of the enzyme solution, filter, and obtain the first part of protoplasts; (4) resuspend the remaining tissue by adding WB solution, filter, and slightly squeeze the filtered remaining tissue, then add WB solution to the remaining tissue to obtain the second part of protoplasts; (5) combine the first part of protoplasts and the second part of protoplasts, centrifuge, and discard the supernatant; (6) add WB solution to the centrifugal sediment, resuspend, centrifuge, and discard the supernatant; (7) A small amount of W5 solution is added to the centrifugal precipitate, and resuspended, to obtain hybrid potato seedling hypocotyl single cells.

[0006] The potato in step (1) is hybrid potato diploid.

[0007] The enzyme solution ES in step (2) comprises the following components: 1.5wt% cellulase RS, 1.0wt% cellulase RS, 1.0wt% dissociation enzyme, 0.5wt% pectinase, 0.5M mannitol, 0.1mol / L MES buffer, 0.5wt% PVP-40, 2mmol / L KCl, 1mL MS medium, 0.5wt% BSA, 10mmol / L CaCl2, 0.5mmol / L DTT, 2mmol / L MgCl2, and the balance is water.

[0008] The preparation method of the enzyme solution ES described above comprises the following steps: The cellulase RS, cellulase RS, dissociation enzyme, pectinase, mannitol, MES buffer, PVP-40, and KCl are sequentially added and mixed to obtain solution 1. Solution 1 is placed in a 55℃ water bath for 10min, and then cooled to room temperature. Then, MS liquid medium, BSA, CaCl2, DTT, MgCl2, and deionized water are sequentially added to the centrifuge tube to obtain the enzyme solution ES.

[0009] The incubation time in step (2) is 4-8h; preferably 6h.

[0010] The incubation condition in step (2) is 28-32℃, 60-90rpm; preferably 30℃, 75rpm.

[0011] The filtration in step (3) is filtration using a 40μm cell screen.

[0012] The WB solution in step (4) is an aqueous solution containing 0.4M mannitol and 0.5wt% BSA at a final concentration.

[0013] The centrifugation in steps (5) and (6) is centrifugation at 100-200g for 3-7min; preferably 150g for 5min.

[0014] The W5 solution in step (7) comprises the following components: 2mmol / L MES buffer, 125mmol / L CaCl2, 152mmol / L NaCl, 100mmol / L glucose, 5mmol / L KCl, and the balance is ultrapure water.

[0015] The application relates to a hybrid potato seedling hypocotyl single cell extraction method and application thereof.

[0016] The application relates to a hybrid potato seedling hypocotyl single cell extraction method and application thereof.

[0017] The application has the following advantages and effects relative to the prior art. The application relates to a hybrid potato seedling hypocotyl single cell extraction method and application thereof. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a photo graph of microscopic detection of the enzymolysis process in Example 2.

[0019] Figure 2 It is a photo graph of the protoplast obtained by using enzymolysis solution 1 and extrusion treatment under a blood cell counting plate in Example 2.

[0020] Figure 3 It is a photo graph of the protoplast obtained by using enzymolysis solution 2 and without extrusion treatment under a blood cell counting plate in Example 2.

[0021] Figure 4 It is a cell group distribution schematic diagram obtained by analyzing RNA-seq sequencing of the protoplast obtained under different extraction conditions. DETAILED DESCRIPTION

[0022] The application will be further described in detail in combination with examples and drawings, but the embodiments of the application are not limited thereto.

[0023] If specific test conditions are not indicated in the following implementation solutions, the test conditions are generally conventional test conditions or test conditions suggested by reagent companies. If not specially indicated, the used materials, reagents and the like are reagents and materials obtained from commercial channels.

[0024] Experimental materials: The cellulase RS is a product of Yakult Company with a product number L0011-5g, and the enzyme activity is 10000 U / g.

[0025] Cellulase R10 is a product of Yakult Co., Ltd. with product number L0012-5g, and the enzyme activity is 10000 U / g.

[0026] Pectinase is a product of Yakult Co., Ltd. with product number L0042-1g, and the enzyme activity is 1000 U / g.

[0027] Isolation enzyme is a product of Yakult Co., Ltd. with product number L0021-5g, and the enzyme activity is 3000 U / g.

[0028] Example 1 Preparation of extract solution 1.1 Preparation of pretreatment solution After calculating the concentration, 0.1M DTT (dithiothreitol) stock solution was taken and ultrapure water was added to prepare a DTT solution with a final concentration of 0.5mM, i.e. a pretreatment solution, ready for use.

[0029] 1.2 Preparation of enzyme solution (ES) The contents of each component in enzyme solution ES1 are shown in Table 1. According to the formula in Table 1, cellulase RS, cellulase RS, isolation enzyme, pectinase, mannitol, MES buffer, PVP-40, and KCl were sequentially added to a centrifuge tube to obtain solution 1. Solution 1 was placed in a 55°C water bath for 10 minutes and then cooled to room temperature. According to the formula in Table 2, MS, BSA, CaCl2, DTT, and MgCl2 were sequentially added to the centrifuge tube, and then ultrapure water was added to make up the total volume of the enzyme solution to 5mL to obtain enzyme solution 1 (ES1).

[0030] Table 1 Components of enzyme solution ES1 added before cooling

[0031] Table 2 Components of enzyme solution added after cooling

[0032] Referring to the above steps and formula, the concentration of cellulase R10 added in Table 1 was adjusted to 20g / L, i.e. the amount added was adjusted to 0.1g, and the concentration of cellulase RS was adjusted to 5g / L, i.e. the amount added was adjusted to 0.025g, to prepare enzyme solution 2 (ES2). In addition, the concentration of cellulase R10 added in Table 1 was adjusted to 10g / L, i.e. the amount added was adjusted to 0.050g, and the concentration of cellulase RS was adjusted to 15g / L, i.e. the amount added was adjusted to 0.075g, to prepare enzyme solution 3 (ES3). The specific amounts added are shown in Table 3.

[0033] Table 3 Enzyme concentrations added in different enzyme solutions

[0034] 1.3 Preparation of protoplast washing buffer (WB) Add 25 μΐ of 500x BSA and 4.75 ml of ddH2O into 5 ml of 0.8 M mannitol, and pre-cool it at 4°C to obtain the protoplast washing buffer. The specific components are shown in Table 4.

[0035] Table 4 Components of protoplast washing buffer

[0036] 1.4 Preparation of W5 solution Add the components in Table 5 into a centrifuge tube, and mix well to obtain the W5 solution. The W5 solution should be prepared immediately before use.

[0037] Table 5 Components of W5 solution

[0038] Example 2 Sample treatment and protoplast extraction 2.1 Sample treatment Use a blade to cut the potato seedling (hybrid potato diploid, variety Youshu No. 1) hypocotyl tissue into thin slices. Wash the tissue slices twice with the pretreatment solution, and reserve for use.

[0039] 2.2 Tissue enzymolysis (1) Place the tissue slices into a centrifuge tube containing ES, and then place the centrifuge tube in a shaker incubator for incubation in the dark. Set the shaker incubator to 30°C and 75 rpm.

[0040] (2) During the incubation, observe the protoplast state (including morphology, quantity, size, amount of debris, and cell activity) under a microscope to determine the degree of enzymolysis.

[0041] The observation under the microscope is shown in the following photograph: Figure 1 The experimental results show that the quantity and activity of the protoplasts reach the highest values at about 6 h of incubation, and the activity of the protoplasts decreases significantly after 6 h of incubation.

[0042] 2.3 Protoplast purification (1) After the enzymolysis is completed, briefly shake the centrifuge tube by hand to completely release the protoplasts.

[0043] (2) Use a wide-bore syringe tip to transfer the enzymolysis solution, and use a 40 μm cell strainer to filter the enzymolysis solution into a new 15 ml centrifuge tube.

[0044] (3) Add 2 mL WB to the remaining tissue and shake well, use a rubber bulb pipette to suck the remaining tissue liquid into the 40 pm cell sieve on the same tube in step 2, gently squeeze the remaining tissue with the rubber piston inside the needle tube, then slowly add 2 mL WB to the remaining tissue, slowly release the protoplasts into the centrifuge tube; in addition, set up a non-squeezing control group, omit the rubber piston squeezing step, and the remaining steps are the same.

[0045] (4) Transfer the centrifuge tube into the centrifuge and centrifuge at 150 x g for 5 min.

[0046] (5) Discard the supernatant, add 2 ml WB with a wide-bore gun head, and resuspend the protoplasts gently. Transfer the centrifuge tube into the centrifuge and centrifuge at 150 x g for 3 min, repeat 2 times.

[0047] (6) After discarding the supernatant, add an appropriate amount of W5 and mix gently.

[0048] 2.4 Viability detection (1) Take 5 µL of the protoplast suspension and mix with 5 µL of 0.4% trypan blue dye solution, use a digital microscope to observe and a hemocytometer to observe the state of the protoplasts and count the yield of protoplasts, take the average value of 3 experiments in each group to calculate the final yield.

[0049] (2) Then use W5 to adjust the concentration of the protoplast suspension to 1000-2000 cells / µL, place the cells on ice, and they can be used for 10x single cell library construction within 30 min.

[0050] The experimental results are shown in Figures 2-3 and Table 6, wherein Figure 2 is a micrograph using enzyme solution 1 and squeezing treatment, Figure 3 is a micrograph using enzyme solution 2 without squeezing treatment. It can be seen that after treatment with enzyme solution 1 and slight squeezing, the yield of protoplasts is significantly improved, and the viability reaches more than 90%, which can greatly improve the efficiency of single cell extraction and help subsequent related experiments, greatly reducing the number of experiments and the consumption of enzymes and reagents.

[0051] Table 6 Extraction effect of different enzyme solutions and squeezing treatment

[0052] Example 3 Single cell RNA-seq sequencing In order to verify whether the quality of the extracted protoplasts can be used for related experimental detection, the diploid hybrid potato hypocotyl single cells were extracted under light / dark conditions according to the method of Example 2 (enzyme solution 1 + squeezing), and the cell type was analyzed after RNA-seq sequencing.

[0053] The experimental results are shown in Table 7 and Figure 4 As can be seen from Table 7 and

[0054] Table 7 RNA-seq sequencing results and cell type classification

[0055] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for extracting single cells from the hypocotyls of hybrid potato seedlings, characterized by It comprises the following steps: (1) cut the hypocotyl tissue of potato seedling into slices, and wash the tissue slices twice with pretreatment solution; (2) put the tissue slices into enzyme solution ES for incubation; (3) after the incubation, shake the centrifuge tube, take the upper enzyme solution for filtration to obtain the first part of protoplasts; (4) resuspend the remaining tissue with WB solution, filter, and squeeze the filtered remaining tissue, then add WB solution to the remaining tissue to obtain the second part of protoplasts; (5) combine the first part of protoplasts and the second part of protoplasts, centrifuge, and discard the supernatant; (6) add WB solution to the centrifugal sediment, resuspend, centrifuge, and discard the supernatant; (7) add W5 solution to the centrifugal sediment, resuspend, and obtain the hybrid potato seedling hypocotyl single cell. The enzyme solution ES in step (2) comprises cellulase RS, cellulase R-10, pectinase, and dissociative enzyme.

2. The hybrid potato seedling hypocotyl single cell extraction method according to claim 1, wherein the enzyme solution ES in step (2) comprises the following components: 1.5wt% cellulase RS, 1.0wt% cellulase RS, 1.0wt% dissociative enzyme, 0.5wt% pectinase, 0.5M mannitol, 0.1mol / L MES buffer, 0.5wt% PVP-40, 2mmol / L KCl, 1mL MS medium, 0.5wt% BSA, 10mmol / L CaCl2, 0.5mmol / L DTT, 2mmol / L MgCl2, and the rest is water.

3. The hybrid potato seedling hypocotyl single cell extraction method according to claim 2, wherein the preparation method of the enzyme solution ES comprises the following steps: add cellulase RS, cellulase RS, dissociative enzyme, pectinase, mannitol, MES buffer, PVP-40, and KCl in sequence to obtain solution 1, heat solution 1 in a 55℃ water bath for 10min, cool to room temperature, then add MS liquid medium, BSA, CaCl2, DTT, MgCl2 to the centrifuge tube in sequence, and add deionized water to constant volume to obtain enzyme solution ES.

4. The hybrid potato seedling hypocotyl single cell extraction method according to claim 1, wherein the incubation time in step (2) is 4-8h; The incubation condition in step (2) is 28-32℃ and 60-90rpm.

5. The hybrid potato seedling hypocotyl single cell extraction method according to claim 2, wherein the filtration in step (3) is filtration using a 40μm cell sieve.

6. The hybrid potato seedling hypocotyl single cell extraction method according to claim 2, wherein the WB solution in step (4) is an aqueous solution containing 0.4M mannitol and 0.5wt% BSA.

7. The hybrid potato seedling hypocotyl single cell extraction method according to claim 2, wherein ​ ​ ​ ​ ​ The centrifugation in steps (5) and (6) is 100-200g for 3-7 min. 8.The hybrid potato seedling hypocotyl single cell extraction method according to claim 2, characterized in that: The W5 solution in step (7) comprises the following components: 2 mmol / L MES buffer, 125 mmol / L CaCl2, 152 mmol / L NaCl, 100 mmol / L glucose, 5 mmol / L KCl, and the balance is ultrapure water. 9.The use of the hybrid potato seedling hypocotyl single cell extraction method according to any one of claims 1-8 in extracting hybrid potato diploid single cell protoplasts. 10.The use of the hybrid potato seedling hypocotyl single cell extraction method according to any one of claims 1-8 in single cell RNA-seq sequencing analysis.

Images