A method for extracting maize endosperm nuclei suitable for ATAC-seq sequencing

Abstract

The present invention discloses a method for extracting corn endosperm kernels suitable for ATAC-seq sequencing. The method comprises the following steps: (1) removing the seed coat and embryo from corn seeds, collecting the endosperm, and storing it at -80°C for a long time; taking approximately 5 g of the endosperm and manually grinding it into powder in liquid nitrogen; (2) adding 10 mL of sucrose buffer to each powder sample, mixing it thoroughly, and then filtering it through a 100 μm pore sieve; (3) passing the filtrate from step (2) through a 38 μm pore mesh, and then centrifuging the product at 700 × g for 5 minutes to collect the supernatant; (4) filtering the supernatant through a 38 μm sieve and centrifuging it at 2000 × g for 5 minutes. The cells were centrifuged at 1000 × g for 5 minutes, the supernatant was removed, and the precipitate was resuspended in 10 mL of sucrose buffer; (5) the cells were filtered through a 38 μm sieve again, centrifuged at 2000 × g for 5-10 minutes, the supernatant was removed, and the precipitate was resuspended in 5 mL of resuspension buffer; (6) the cells were centrifuged at 1000 × g for 5 minutes, the supernatant was discarded, the precipitate was suspended in 5 mL of PBS solution containing protease inhibitors, 10-20 μL of the solution was stained with DAPI at equal volumes for 10 minutes, and the cells were observed under a fluorescence microscope. The endosperm nuclei prepared by the present invention are numerous and highly active, which can overcome the influence of high starch content in storage tissues, provide strong technical support for subsequent single-cell sequencing, CUT&TAG, ATAC-seq and other related studies, and the extraction effect will also have an important impact on the experimental data.

Description

Technical Field

[0001] The present invention belongs to the field of cell engineering technology, and particularly relates to a method for extracting nuclei from corn endosperm suitable for ATAC-seq sequencing. Background Art

[0002] The nucleus is the core of the cell and one of its most important organs. It contains the primary sources of genetic information, such as DNA and RNA. Within the nucleus, DNA sequences are woven together by the protein-based chromatin, forming the nucleus's genetic information. The nucleus plays a crucial role in corn seeds. Environmental factors such as light and temperature can influence biochemical processes within the nucleus, such as DNA replication and RNA transcription, influencing the growth and development of corn seeds and playing an irreplaceable role in seed germination and growth. Extracting plant nuclei is a complex biotechnological process that involves isolating the nucleus intact from the plant cell while removing starch granules and other organelles, such as mitochondria and chloroplasts. The specialized structures of plant cells, such as the cell wall and specialized organelles, make extracting plant nuclei more challenging than extracting animal nuclei. Nevertheless, this technique is crucial for understanding the growth, development, and genetic mechanisms of plant cells. For example, studying the nucleus of corn endosperm cells can provide insights into the molecular regulation of cells, cell proliferation and differentiation, and how these processes influence plant growth, development, and yield. Furthermore, ATAC-seq technology is increasingly being used in plant science research, providing a powerful tool for understanding the three-dimensional structure of plant genomes, chromatin accessibility, and the regulatory mechanisms of gene expression. High-throughput sequencing (ATAC-seq) analysis of transposase-accessible chromatin requires highly purified nuclei. Therefore, developing a novel method for extracting maize endosperm nuclei suitable for ATAC-seq sequencing is crucial for advancing gene function research.

[0003] The endosperm is a crucial tissue within plant seeds, located beneath the embryo and composed of the cotyledons and their bases. It is the seed's primary storage tissue, rich in nutrients such as starch, protein, and fat, providing nutrients for embryo development. The endosperm plays a crucial role in the plant life cycle. First, the endosperm is the seed's primary source of nutrition. During seed development, the mother plant, through nutrient absorption and synthesis, transfers a large amount of nutrients to the endosperm. These nutrients, including starch, protein, and fat, provide the embryo with energy and the raw materials it needs for growth. This stored nutrient supply ensures the seed's energy and material needs for growth and development under favorable environmental conditions. Second, the endosperm plays a crucial role in seed germination and initial growth. When the seed encounters favorable conditions, such as temperature, moisture, and oxygen, the nutrients in the endosperm are absorbed and utilized by the embryo. Starch in the endosperm is broken down by enzymes into glucose, which provides energy for the embryo, while protein is broken down into amino acids, providing the building blocks for embryonic development. These nutrients support embryo growth and promote seedling formation and rooting. Endosperm formation typically occurs during the early stages of seed development. During embryo formation, vegetative cells continuously divide and proliferate, accumulating nutrients. Initially, these storage materials exist in the form of protein. As the embryo develops, these cells merge to form a central core, where these rich nutrients are gradually converted into starch, fat, and other substances, ultimately forming the endosperm. Studying the endosperm is crucial for understanding the mechanisms of seed development and germination.

[0004] ATAC-seq provides a comprehensive perspective on the spatial organization of the genome and the multi-level regulation of gene expression. The endosperm plays a crucial role in grass seed development and crop production. The nucleus contains essential genetic information and vital characteristics of the seed. Therefore, obtaining maize endosperm nuclei suitable for ATAC-seq sequencing is of paramount importance. However, there are currently no reports on the extraction of endosperm nuclei from grass crops that are suitable for ATAC-seq sequencing throughout the entire grain filling period. This is partly due to the greater difficulty in extracting plant endosperm nuclei than from animal cells. Isolating the nucleus intact from plant cells requires the removal of other organelles such as mitochondria and chloroplasts. Furthermore, the increasing starch content during endosperm development makes it difficult to remove starch while preserving the nucleus. High-throughput sequencing (ATAC-seq) for transposase-accessible chromatin analysis requires highly purified nuclei, significantly increasing the difficulty in extracting endosperm nuclei suitable for ATAC-seq. Summary of the Invention

[0005] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for extracting nuclei from corn endosperm suitable for ATAC-seq sequencing, and establishes an efficient separation system for extracting nuclei from corn endosperm for ATAC-seq sequencing. This system can be used to perform various molecular biology operations in the nuclei of grass endosperm cells.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] A method for extracting nuclei from corn endosperm suitable for ATAC-seq sequencing comprises the following steps:

[0008] Remove the seed coat and embryo from the corn seeds and collect the endosperm. They can be stored at -80℃ for a long time. Take about 5g of the endosperm and grind it into powder manually in liquid nitrogen.

[0009] Add 10 mL of sucrose buffer to each powder sample, mix thoroughly, and filter through a 100 μm sieve;

[0010] The filtrate from step (2) was passed through a 38 μm pore mesh, and the product was centrifuged at 700 × g for 5 minutes, and the supernatant was collected;

[0011] The supernatant was filtered through a 38 μm mesh and centrifuged at 2000 × g for 5 min, the supernatant was removed, and the pellet was resuspended in 10 mL of sucrose buffer;

[0012] Filter again through a 38 μm mesh, centrifuge at 2000 × g for 5-10 minutes, remove the supernatant, and resuspend the pellet in 5 mL of resuspension buffer;

[0013] After centrifugation at 1000×g for 5 minutes, the supernatant was discarded and the precipitate was suspended in 5 mL of PBS solution containing protease inhibitors. 10-20 μL was taken and stained with an equal volume of DAPI for 10 minutes and observed under a fluorescence microscope.

[0014] The sucrose buffer comprises 1M sucrose, 1M MgCl2, 0.2% TritonX100, and 2% protease inhibitors; the resuspension buffer comprises 0.2% TritonX100 and 2% protease inhibitors.

[0015] Furthermore, the corn endosperm is the endosperm 9-30 days after pollination.

[0016] Furthermore, the kernels are corn kernels.

[0017] Furthermore, the sucrose in the sucrose buffer solution must first be dissolved in 20-30 mL of PBS solution.

[0018] Furthermore, the centrifugation in step (3), step (4) and step (5) is low-temperature centrifugation at 4°C.

[0019] Furthermore, the sucrose buffer solution must be pre-cooled before use.

[0020] The beneficial effects of the present invention are:

[0021] The present invention is the optimal combination of conditions obtained after a large number of experimental optimizations. The endosperm nuclei prepared are numerous and highly active, which can overcome the influence of high starch content in storage tissues. Among them, the average nucleus yield is 6.25×107·mL-1, and the activity can reach more than 96%, which can provide strong technical support for subsequent single-cell sequencing, CUT&TAG, ATAC-seq and other related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Fresh corn ears 27 days after pollination;

[0023] Figure 2 The corn endosperm at different days after pollination; from left to right, the corn endosperm at 30, 27, 18, and 9 days after pollination;

[0024] Figure 3 The process of corn endosperm grinding and filtration; A is a fresh corn ear 27 days after pollination, B is the corn endosperm grinding process, and C is the corn endosperm filtration after grinding;

[0025] Figure 4 The supernatant extracted above was stained with DAPI;

[0026] Figure 5 These are the ATAC-seq sequencing results of maize endosperm nuclei at different days after pollination, including 9, 18, 27, and 30 days from left to right. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0028] Example 1

[0029] 1. Material collection and processing:

[0030] Corn material field planting management, bag pollination, mark the pollination date, take the corn ears 9-30 days after pollination ( Figure 1) were used for the experiment. The endosperm cells of corn 1-6 days after pollination were not fully developed and were too small to be easily picked; the endosperm cells of corn 9-30 days after pollination ( Figure 2 ) were used for the experiment. The female ears of corn were taken at 9, 18, 27 and 30 days after pollination and brought back to the laboratory for processing. The middle corn kernels with good and uniform growth were selected. The outer seed coat was peeled off with tweezers (sterilized), the nucellus was removed, and the endosperm tissue ( Figure 2 ).

[0031] 2. Grinding and filtration of endosperm cells:

[0032] The formula of sucrose buffer is shown in Table 1. Sucrose buffer was prepared using a 50 mL round-bottom EP tube. 1 g of PBS was dissolved in ultrapure water to make 100 mL of solution for later use. Sucrose was first dissolved in 20-30 mL of PBS solution, and then MgCl2, Triton X100, and protease inhibitors were added. Finally, the volume was made up to 50 mL with PBS solution. Approximately 5 g of endosperm was taken and manually ground into powder in liquid nitrogen ( Figure 3 B); add 10 mL of sucrose buffer to each powder sample, mix thoroughly, and filter through a 100 μm sieve ( Figure 3 C); the filtrate of step (2) was filtered through a 38 μm mesh again, and the product was centrifuged at 700 × g for 5 minutes, and the supernatant was collected; the supernatant was filtered through a 38 μm mesh and centrifuged at 2000 × g for 5 minutes, the supernatant was removed, and the precipitate was resuspended in 10 mL of sucrose buffer; it was filtered through a 38 μm mesh again, centrifuged at 2000 × g for 5-10 minutes, the supernatant was removed, and the precipitate was resuspended in 5 mL of resuspension buffer, the resuspension buffer formula is shown in Table 2, and finally the volume was made up to 50 mL with PBS solution.

[0033] Table 1 Sucrose buffer

[0034]

[0035] Table 2 Resuspension Buffer

[0036]

[0037] 3. DAPI staining:

[0038] After centrifugation at 1000×g for 5 minutes, the supernatant was discarded and the precipitate was suspended in 5 mL of PBS solution containing protease inhibitors. 10-20 μL of the supernatant was added to DAPI stain with a final concentration of 1 ng / mL. The staining was performed for 10 minutes and the cells were observed under a fluorescence microscope.

[0039] 4. ATAC-seq sequencing:

[0040] ATAC-seq was performed on maize endosperm nuclei at different days after pollination.

Claims

1. A method for extracting nuclei from corn endosperm suitable for ATAC-seq sequencing, characterized in that: The following steps are involved: (1) Remove the seed coat and embryo from the corn seeds, collect the endosperm, and store it at -80°C for a long time. Take about 5 g of the endosperm and grind it into powder manually in liquid nitrogen; (2) Add 10 mL of sucrose buffer to each powder sample, mix thoroughly, and filter through a 100 μm sieve; (3) The filtrate from step (2) was passed through a 38 μm pore mesh, and the product was centrifuged at 700 × g for 5 minutes, and the supernatant was collected; (4) The supernatant was filtered through a 38 μm mesh and centrifuged at 2000 × g for 5 min. The supernatant was removed and the pellet was resuspended in 10 mL of sucrose buffer. (5) Filter again through a 38 μm mesh, centrifuge at 2000 × g for 5–10 min, remove the supernatant, and resuspend the pellet in 5 mL of resuspension buffer; (6) After centrifugation at 1000 × g for 5 minutes, the supernatant was discarded and the precipitate was suspended in 5 mL of PBS solution containing protease inhibitors. 10-20 μL was taken and stained with an equal volume of DAPI for 10 minutes and observed under a fluorescence microscope; The sucrose buffer comprises 1M sucrose, 1M MgCl2, 0.2% TritonX100, and 2% protease inhibitors; the resuspension buffer comprises 0.2% TritonX100 and 2% protease inhibitors.

2. The method for extracting nuclei from corn endosperm suitable for ATAC-seq sequencing according to claim 1, characterized in that: The corn endosperm is the endosperm 9-30 days after pollination.

3. The method for extracting nuclei from corn endosperm suitable for ATAC-seq sequencing according to claim 1, characterized in that: The sucrose in the sucrose buffer should first be dissolved in 20-30 mL of PBS solution.

4. The method for extracting nuclei from corn endosperm suitable for ATAC-seq sequencing according to claim 1, characterized in that: The centrifugation in step (3), step (4) and step (5) is performed at a low temperature of 4°C.

5. The method for extracting nuclei from corn endosperm suitable for ATAC-seq sequencing according to claim 1, characterized in that: The sucrose buffer solution should be pre-cooled before use.

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