Hybrid potato seedling embryo axis single cell extraction method and application thereof

Abstract

The application provides a hybrid potato seedling hypocotyl single cell extraction method and application thereof. According to the characteristics of the potato seedling hypocotyl single cell, the application significantly improves the extraction efficiency of protoplast by adjusting the amount of different cellulases in the enzyme solution and combining a slight extrusion mode. The method is simple and fast, does not need to use complex treatment and expensive reagents, and is convenient for popularization and application. In addition, the protoplast extracted by the method can be effectively used for subsequent sequencing and detection, and it is proved that the cell activity extracted by the method is high, and the method has wide application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for extracting single cells from hypocotyls of hybrid potato seedlings and its application. Background Technology

[0002] The genetic background of cultivated potatoes is relatively narrow. Common potatoes used in production are primarily tetraploid, while wild varieties are diploid. Due to differences in ploidy levels and endosperm equilibrium numbers, many superior genes from wild resources are difficult to transfer to common potatoes through traditional breeding. Researchers have made significant efforts to fully utilize wild germplasm resources and broaden the narrow genetic base of cultivated potatoes. The use of somatic cell hybridization technology can effectively overcome reproductive isolation caused by ploidy differences between cultivated and wild potato varieties, transferring superior genes from wild varieties to cultivated varieties for selection and improvement, while also avoiding biosafety regulatory issues related to genetically modified organisms (GMOs). Furthermore, for the heterozygous F1 generation produced by diploid hybrid potato breeding to be widely adopted, the most crucial step is from seed to seedling. This involves seed germination and developmental biology. The emergence of dicotyledons from the seed relies on the elongation of the hypocotyl. Therefore, extracting single cells from the hypocotyl can help elucidate the single-cell biology of hypocotyl elongation, which is of great significance for understanding the regulation of seed germination and cotyledon emergence in hybrid diploid potatoes. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for extracting single cells from hypocotyls of hybrid potato seedlings.

[0004] Another objective of this invention is to provide an application of the above-mentioned method for extracting single cells from hypocotyls of hybrid potato seedlings.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for extracting single cells from hypocotyls of hybrid potato seedlings includes the following steps:

[0007] (1) Cut the hypocotyl tissue of potato seedlings into thin slices and wash the tissue slices twice with pretreatment solution;

[0008] (2) Place the tissue slices into the enzyme digest ES and incubate;

[0009] (3) After incubation, shake the centrifuge tube, take the upper enzyme hydrolysate and filter it to obtain the first part of protoplasts;

[0010] (4) Add WB solution to the remaining tissue, resuspend, filter, and gently squeeze the filtered remaining tissue. Then add WB solution to the remaining tissue to obtain the second part of protoplasts.

[0011] (5) Combine the first and second parts of protoplasts, centrifuge, and discard the supernatant;

[0012] (6) Add WB solution to the centrifuged precipitate, resuspend, centrifuge, and discard the supernatant;

[0013] (7) Add a small amount of W5 solution to the centrifuged precipitate and resuspend it to obtain a single hypocotyl cell of the hybrid potato seedling.

[0014] The potato mentioned in step (1) is a hybrid potato diploid.

[0015] The enzymatic hydrolysate ES mentioned in step (2) comprises the following components:

[0016] 1.5 wt% cellulase RS, 1.0 wt% cellulase RS, 1.0 wt% analyte, 0.5 wt% pectinase, 0.5 M mannitol, 0.1 mol / L MES buffer, 0.5 wt% PVP-40, 2 mmol / L KCl, 1 mL MS medium, 0.5 wt% BSA, 10 mmol / L CaCl2, 0.5 mmol / L DTT, 2 mmol / L MgCl2, balance water.

[0017] The preparation method of the above-mentioned enzymatic hydrolysate ES includes the following steps:

[0018] Cellulase RS, cellulase RS, analyte, pectinase, mannitol, MES buffer, PVP-40, and KCl were added sequentially to obtain solution 1. Solution 1 was incubated in a 55°C water bath for 10 minutes and then cooled to room temperature. Subsequently, MS liquid culture medium, BSA, CaCl2, DTT, and MgCl2 were added to the centrifuge tube in sequence, and deionized water was added to make up the volume to obtain enzyme hydrolysate ES.

[0019] The incubation time in step (2) is 4 to 8 hours; preferably 6 hours.

[0020] The incubation conditions described in step (2) are 28–32°C and 60–90 rpm; preferably 30°C and 75 rpm.

[0021] The filtration described in step (3) is filtration using a 40μm cell sieve.

[0022] The WB solution in step (4) is an aqueous solution containing mannitol with a final concentration of 0.4 M and 0.5 wt% BSA.

[0023] The centrifugation in steps (5) and (6) is performed at 100-200g for 3-7 minutes; preferably at 150g for 5 minutes.

[0024] The W5 solution described in step (7) comprises the following components:

[0025] 2 mmol / L MES buffer, 125 mmol / L CaCl2, 152 mmol / L NaCl, 100 mmol / L glucose, 5 mmol / L KCl, with the remainder being ultrapure water.

[0026] The above-mentioned method for extracting single-cell hypocotyls from hybrid potato seedlings is applied to the extraction of diploid single-cell protoplasts from hybrid potatoes.

[0027] The above-mentioned method for extracting single cells from hypocotyls of hybrid potato seedlings is applied to single-cell RNA-seq sequencing analysis.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] This invention provides a method for extracting single cells from hypocotyls of hybrid potato seedlings and its application. Taking into account the characteristics of single cells from hypocotyls of potato seedlings, this invention significantly improves the extraction efficiency of protoplasts by adjusting the amount of different cellulases in the enzymatic hydrolysate and combining it with a gentle squeezing method. This method is simple and rapid, requiring no complex processing or expensive reagents, making it easy to promote and apply. Furthermore, the protoplasts extracted by this method have been experimentally verified to be effective for subsequent sequencing and other detection methods, demonstrating that the cells extracted by this method have high viability and broad application value. Attached Figure Description

[0030] Figure 1 This is a photograph taken under a microscope during the enzymatic digestion process in Example 2.

[0031] Figure 2 This is a photograph of protoplasts obtained using enzymatic hydrolysate 1 and extrusion treatment in Example 2 on a hemocytometer under a microscope.

[0032] Figure 3 This is a photograph of protoplasts obtained using enzymatic hydrolysate 2 and without compression treatment on a hemocytometer under a microscope, as shown in Example 2.

[0033] Figure 4 This is a schematic diagram of cell population distribution obtained by RNA-seq sequencing analysis of protoplasts extracted under different extraction conditions. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0035] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0036] Experimental materials:

[0037] The cellulase RS is a product from Yakult, catalog number L0011-5g, with an enzyme activity of 10000 U / g.

[0038] Cellulase R10 is a product from Yakult, catalog number L0012-5g, with an enzyme activity of 10000 U / g.

[0039] The pectinase is a product from Yakult, catalog number L0042-1g, with an enzyme activity of 1000 U / g.

[0040] The ionizing enzyme is a product from Yakult, catalog number L0021-5g, with an enzyme activity of 3000 U / g.

[0041] Example 1: Preparation of the extract

[0042] 1.1 Preparation of pretreatment solution

[0043] After calculating the concentration, take 0.1M DTT (dithiothreitol) stock solution and add ultrapure water to prepare a DTT solution with a final concentration of 0.5mM, which is the pretreatment solution, for later use.

[0044] 1.2 Preparation of Enzyme Solution (ES)

[0045] The contents of each component in the enzymatic hydrolysate ES1 are shown in Table 1. Cellulase RS, cellulase RS, dissociative enzyme, pectinase, mannitol, MES buffer, PVP-40, and KCl were added to the centrifuge tube in sequence according to the formula in Table 1 to obtain solution 1. Solution 1 was placed in a water bath at 55℃ for 10 min and cooled to room temperature. MS, BSA, CaCl2, DTT, and MgCl2 were added to the centrifuge tube in sequence according to the formula in Table 2. Ultrapure water was then added to bring the total volume of the enzymatic hydrolysate to 5 mL to obtain enzymatic hydrolysate 1 (ES1).

[0046] Table 1. Components of the enzyme hydrolysate ES1 added before cooling.

[0047]

[0048] Table 2. Composition of the enzymatic hydrolysate added after cooling

[0049]

[0050] Following the steps and formula described above, the concentration of cellulase R10 added in Table 1 was adjusted to 20 g / L, i.e., the amount added was adjusted to 0.1 g, and the concentration of cellulase RS was adjusted to 5 g / L, i.e., the amount added was adjusted to 0.025 g, thus preparing enzymatic hydrolysate 2 (ES2). In addition, the concentration of cellulase R10 added in Table 1 was adjusted to 10 g / L, i.e., the amount added was adjusted to 0.050 g, and the concentration of cellulase RS was adjusted to 15 g / L, i.e., the amount added was adjusted to 0.075 g, thus preparing enzymatic hydrolysate 3 (ES3). For specific amounts added, please refer to Table 3.

[0051] Table 3 Enzyme concentrations added to different enzymatic hydrolysates

[0052]

[0053] 1.3 Preparation of Protoplast Washing Solution (WB)

[0054] Add 25 μl of 500×BSA and 4.75 ml of ddH2O to 5 ml of 0.8 M mannitol, and pre-cool at 4 °C to obtain the protoplast washing solution. The specific components are shown in Table 4.

[0055] Table 4 Composition of Protoplast Cleansing Solution

[0056]

[0057] 1.4 Preparation of W5 solution

[0058] Add the ingredients in Table 5 to a centrifuge tube, mix well, and you will get the W5 solution. It should be prepared fresh before use.

[0059] Table 5 Composition of W5 solution

[0060]

[0061] Example 2 Sample processing and protoplast extraction

[0062] 2.1 Sample preparation

[0063] Cut the hypocotyl tissue of the potato seedling (hybrid diploid potato, variety Youshu No. 1) into thin slices with a blade. Wash the tissue slices twice with a pretreatment solution and set aside.

[0064] 2.2 Tissue enzymatic digestion

[0065] (1) Place the tissue slices into centrifuge tubes containing ES, and then place the centrifuge tubes in a shaker to incubate in the dark. The shaker is set to 30°C and 75 rpm.

[0066] (2) Microscopic observation during incubation to observe the state of protoplasts (including morphology, number, size, amount of fragments and cell activity) to determine the degree of enzymatic hydrolysis.

[0067] Microscopic observation of photographs as follows Figure 1 As shown in the figure, the experimental results show that the number and activity of protoplasts reach their highest values ​​after incubation for about 6 hours, and the activity of protoplasts decreases significantly after 6 hours.

[0068] 2.3 Protoplast purification

[0069] (1) After the enzymatic digestion is complete, shake the centrifuge tube briefly by hand to completely release the protoplasts.

[0070] (2) Transfer the enzyme digest using a wide-mouth pipette tip and filter the enzyme digest into a new 15 ml centrifuge tube using a 40 μm cell sieve.

[0071] (3) Add 2 mL of WB to the remaining tissue and shake well. Use a pipette to transfer the remaining tissue fluid to the 40 μm cell sieve on the same tube as in step 2. Gently squeeze the remaining tissue with the plunger inside the syringe. Then slowly add 2 mL of WB to the remaining tissue and slowly release the protoplasts into the centrifuge tube. In addition, a control group without squeezing was set up, and the plunger squeezing step was omitted. The other steps were the same.

[0072] (4) Transfer the centrifuge tube to the centrifuge and centrifuge at 150×g for 5 minutes.

[0073] (5) Discard the supernatant, add 2 ml of WB using a wide-mouth pipette tip, and gently resuspend the protoplasts. Transfer the centrifuge tube to a centrifuge and centrifuge at 150×g for 3 min. Repeat twice.

[0074] (6) After discarding the supernatant, add an appropriate amount of W5 and mix gently.

[0075] 2.4 Vitality Detection

[0076] (1) Mix 5µL of protoplast suspension with 5µL of 0.4% trypan blue staining solution. Use a digital microscope and a hemocytometer to observe the protoplast state and count the protoplast yield. Take the average of 3 experiments for each group and round it down to calculate the final yield.

[0077] (2) Then, use W5 to adjust the concentration of protoplast suspension to 1000-2000 cells / µL, place the cells on ice, and they can be used for 10× single-cell library construction within 30 min.

[0078] Experimental results are as follows Figures 2-3 As shown in Table 6, Figure 2 Micrographs taken using enzymatic hydrolysate 1 and after extrusion processing. Figure 3The image shows a micrograph taken with enzyme hydrolysate 2 and without compression. It can be seen that after treatment with enzyme hydrolysate 1 and slight compression, the yield of protoplasts is significantly increased, and the viability reaches over 90%. This greatly improves single-cell extraction efficiency, facilitates subsequent experiments, significantly reduces the number of experiments, and reduces the consumption of enzymes and reagents.

[0079] Table 6. Extraction effects of different enzymatic hydrolysates and extrusion treatments

[0080]

[0081] Example 3 Single-cell RNA-seq sequencing

[0082] To verify whether the quality of the extracted protoplasts could be used for relevant experimental testing, following the method in Example 2 (enzymatic hydrolysate 1 + extrusion), single cells of hypocotyls from diploid hybrid potatoes were extracted under light / dark conditions, and their cell types were analyzed after RNA-seq sequencing.

[0083] The experimental results are shown in Table 7 and Figure 4 As shown, the cells can be roughly divided into nine categories, proving that the single cells extracted by the method in Example 2 can meet the requirements of experiments such as RNA-seq sequencing. Moreover, the cell differentiation under different conditions can be clearly distinguished from the distribution of the cell population, proving that the cell damage during the extraction process is small. The single-cell protoplasts obtained in the end not only meet the experimental requirements in quantity, but also ensure high viability.

[0084] Table 7 RNA-seq sequencing results and cell type classification

[0085]

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for extracting single cells from hypocotyls of hybrid potato seedlings, characterized by comprising the following steps: (1) Cut the hypocotyl tissue of hybrid potato seedlings into thin slices and wash the tissue slices twice with pretreatment solution; (2) Place the tissue slices into the enzyme digest ES and incubate; (3) After incubation, shake the centrifuge tube, take the upper enzyme hydrolysate and filter it to obtain the first part of protoplasts; (4) Add WB solution to the remaining tissue, resuspend, 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 and second parts of protoplasts, centrifuge, and discard the supernatant; (6) Add WB solution to the centrifuged precipitate, resuspend, centrifuge, and discard the supernatant; (7) Add W5 solution to the centrifuged precipitate and resuspend to obtain single cells of hypocotyl of hybrid potato seedlings; The pretreatment solution mentioned in step (1) is a DTT solution with a final concentration of 0.5 mM; The components of the enzymatic hydrolysate ES mentioned in step (2) are: 1.0% cellulase RS, 1.5% cellulase R10, 1.0% cleavage enzyme, 0.5% pectinase, 0.5M mannitol, 10mL MME S buffer, 0.5% PVP-40, 2mmol / L KCl, 1mL MS liquid medium, 0.5% BSA, 10mmol / L CaCl2, 0.5mmol / L DTT, 2mmol / L MgCl2, balance water; The preparation method of the enzyme hydrolysate ES includes the following steps: Cellulase RS, cellulase R10, analyte, pectinase, mannitol, MES buffer, PVP-40, and KCl were added sequentially to a centrifuge tube to obtain solution 1. Solution 1 was incubated in a 55°C water bath for 10 min and then cooled to room temperature. MS liquid culture medium, BSA, CaCl2, DTT, and MgCl2 were then added sequentially to the centrifuge tube, and ultrapure water was added to bring the total volume to 5 mL to obtain the enzymatic hydrolysate ES. The incubation time described in step (2) is 6 hours; The incubation conditions described in step (2) are 30°C and 75 rpm; The filtration described in step (3) is filtration using a 40μm cell sieve; The WB solution described in steps (4) and (6) is an aqueous solution of mannitol and 0.5% BSA with a final concentration of 0.4M; The centrifugation described in steps (5) and (6) is performed at 100-200g for 3-7 minutes; The components of the W5 solution mentioned in step (7) are: 2 mmol / L MES buffer, 125 mmol / L CaCl2, 154 mmol / L NaCl, 100 mmol / L glucose, 5 mmol / L KCl, with the remainder being ultrapure water; The hybrid potato mentioned is Youshu No.

1.

2. The application of the method for extracting single-cell hypocotyls from hybrid potato seedlings according to claim 1 in the extraction of diploid single-cell protoplasts from hybrid potatoes; The hybrid potato mentioned is Youshu No.

1.

3. The application of the single-cell extraction method of hypocotyl from hybrid potato seedlings as described in claim 1 in single-cell RNA-seq sequencing analysis.

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