A method for simultaneously preparing exosomes and cell nuclei from Arabidopsis leaf tissue and sequencing them simultaneously

Abstract

The present invention discloses a method for simultaneously extracting nuclei and exosomes from Arabidopsis leaf tissue and sequencing the method, including reagent preparation, sample processing, nucleus suspension preparation, and exosome purification. The method of the present invention fills this technical gap in the plant kingdom, promotes the research of plant exosomes and the application of single-cell nuclear sequencing technology in plant tissues, and is a major technological breakthrough in the field of plant research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for simultaneously preparing Arabidopsis thaliana leaf tissue exosomes and cell nuclei from Arabidopsis thaliana tissue and sequencing the exosomes and cell nuclei from the tissue. Background Art

[0002] Single-cell transcriptome sequencing is a technology that sequences transcriptomes at the single-cell level. It analyzes the expression patterns of single-cell transcriptomes, allowing us to monitor biological life activities in depth into the cellular field, revealing cellular heterogeneity that traditional sequencing technologies cannot resolve, and is a major breakthrough in the field of life sciences. Plant single-cell sequencing requires breaking through the limitations of the cell wall and preparing protoplasts. However, protoplasts without cell wall protection are often very easy to rupture and difficult to prepare. Plant single-cell nuclear sequencing breaks through the limitations of protoplasts. Research in the past two years has shown that plant single-cell nuclear sequencing can obtain similar cellular heterogeneity as plant single-cell sequencing and reduce bias.

[0003] Exosomes are small vesicle structures containing RNA and protein information, with a diameter of 30-150nm, and are involved in intercellular communication. As early as 1965, similar vesicle structures were found in plants, but it was not until 2009 that exosome-like vesicles were isolated from plants. While animal exosomes were developing rapidly, research on plant exosomes stagnated for a long time. Until 2018, it was discovered that Arabidopsis can transfer sRNA to pathogens by secreting exosomes, inducing the silencing of key pathogenic genes and participating in plant immunity. In recent years, due to its potential medicinal value, plant exosomes have developed rapidly. It has been proven that small RNAs in plant exosomes can regulate the expression of mRNA in humans, animals and microorganisms, and have therapeutic effects in colitis, inflammation, cancer, neural differentiation and other aspects. Sequencing small RNAs in plant exosomes can construct a plant's autoimmune map and mine micRNAs with medicinal value.

[0004] Life activities are a complex process, and a simple life phenomenon may involve the interaction of thousands of cells. The combined analysis of single-cell sequencing technology and exosome sequencing technology can deeply reveal the molecular interactions and communication networks between biological cells, analyze the upstream molecular mechanisms of cross-border communication of plant exosomes, and help us deeply study the complex molecular patterns behind a certain life phenomenon.

[0005] At present, there are no reports on the simultaneous sequencing of exosomes and single-cell nuclei in the field of plants. Arabidopsis thaliana is the first higher plant to complete whole genome sequencing in the world and is the most widely used model plant in the world. It is known as the "fruit fly" or "white mouse" of the plant world. For decades, researchers have been enthusiastic about studying Arabidopsis thaliana. To achieve simultaneous sequencing of exosomes and single-cell nuclei in plant tissues, we should not leave this most classic model plant. Summary of the invention

[0006] In order to fill the gap in single cell nucleus + exosome sequencing technology in the field of plants, the present invention aims to establish a method suitable for simultaneously preparing exosomes and cell nuclei from Arabidopsis leaf tissue and sequencing them.

[0007] The technical difficulties that need to be overcome in the process of simultaneously preparing Arabidopsis leaf tissue exosomes and cell nuclei in the present invention mainly include the following three aspects:

[0008] Technical difficulty 1: To prepare cell nuclei and exosomes simultaneously based on the same tissue, in order to avoid the destruction of the vesicle structure by the lysis solution and the degradation of exosomal RNA and nuclear RNA, a large number of experiments are needed to improve the quality of exosomes and cell nuclei, so as to achieve the simultaneous exosomal small RNA sequencing and single cell nucleus sequencing.

[0009] Technical difficulty 2: In the field of plant research, the two technical processes of exosome preparation and single-cell nuclear sequencing are not mature in themselves. There are few studies on exosomes in Arabidopsis leaves in the prior art, which adopts the apoplast fluid method, that is, dialyzing the leaves in the culture medium and then extracting the vesicles precipitated in the culture medium. However, this method is not suitable for the present invention. The precipitated exosomes are only a very small part of the tissue and cannot be used to build an interactive communication network. Based on this, it is difficult to prepare a sufficient amount of exosomes in the prior art.

[0010] Technical Difficulty 3: Regarding the preparation of cell nuclei, there are a lot of chloroplast fragments in leaf tissues. The only way at present is to use a cytometer for fluorescence sorting, but the fluorescence sorting technology is expensive and the fluorescence signal will affect the subsequent single cell nucleus library construction. Therefore, it is difficult to meet the requirements of single cell nucleus sequencing by removing chloroplast fragments using existing technology.

[0011] The present invention provides a method for simultaneously preparing Arabidopsis leaf tissue exosomes and cell nuclei from Arabidopsis leaf tissue, and the steps are as follows:

[0012] 1) Prepare reagents: prepare enzymatic solution, lysis buffer, washing buffer, purification buffer, and PBS buffer respectively, and pre-cool the prepared solutions on ice.

[0013] 2) Sample processing: tissues were broken and then enzymatically hydrolyzed; PBS buffer was used for dilution and transfer, centrifugation was performed, and the supernatant and precipitate were separated. The supernatant was used to extract exosomes and the precipitate was used to prepare cell nuclei.

[0014] 3) Cell nuclear extraction and purification

[0015] 3.1) Lysis: Add lysis buffer to the precipitate from step 2), mix well, and lyse on ice for 5-10 min;

[0016] 3.2) Sieving: After diluting the lysate with washing buffer, filter it into a new centrifuge tube using a cell sieve, centrifuge, and discard the supernatant;

[0017] 3.3) Purification: Add purification buffer and perform differential centrifugation to obtain purified cell nucleus precipitate.

[0018] 3.4) Microscopic examination: Resuspend the cell nucleus pellet with PBS buffer to obtain a single cell nucleus suspension for microscopic examination.

[0019] 4) Exosome preparation and purification: The supernatant retained in step 2) is used for exosome preparation and purification.

[0020] There is no restriction on the order of step 2) and step 3).

[0021] Preferably, the composition and formula of the enzymatic hydrolysate in step 1) are: 1% to 1.5% pectinase Y23, 1.5% to 2% hemicellulase, 0.5% to 1% snail enzyme, 1.5% to 2% cellulase R10, 0.4% to 0.6% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0022] Preferably, the composition and formula of the lysis buffer in step 1) are: 0.8% to 1.2% NP40, 0.2% to 0.4% Triton X-100, 0.8% to 1.2% digitonin, 0.4M sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1mMDTT, 1×proteaseinhibitor, 0.4U / μlRNaseinhibitor.

[0023] Preferably, the composition and formula of the washing buffer in step 1) are: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ulRNaseinhibitor.

[0024] Preferably, the composition and formula of the purification buffer in step 1) are: 0.1% TritonX-100, 0.4U / ul RNase inhibitor, and PBS buffer.

[0025] In step 1), the components were purchased from cellulase R10 (solarbio / C8260-10g), macerate R10 (solarbio / M8190-1g), pectinase (solarbio / P8181-1g), hemicellulase (solarbio / H8110-15KU), snail enzyme (solarbio / S8280-5g), MES (solarbio / M8010-10g), CaCl 2 (Sigma / C5670-100G), NaCl (Thermo / AM9760G), TritonX-100 (solarbio / T8200-100ml), NP40 (Thermo / 85124), DTT (solarbio / D8220), sucrose (Shanghai Test / 10021418), Tris-HCl pH 8.0 (Solarbio / T1150-500ml), MgCl 2 (Shanghai Test / 10012818), protease inhibitor (Sigma / P8340-1ML), RNase inhibitor (Thermo / AM2694), PBS buffer (Thermo / 10010072).

[0026] Preferably, in step 2), the sample processing method is tissue grinder liquid nitrogen disruption + enzymatic hydrolysis.

[0027] Preferably, step 2) grinding uses a 2 ml centrifuge tube and 1-2 sterilized steel balls with a diameter of 3-5 mm.

[0028] Preferably, in step 2), the sample is placed in a semiconductor plate of a tissue grinder, immersed in liquid nitrogen for more than 60 seconds, and quickly placed on a tissue grinder at a frequency of 30 to 40 Hz for 60 seconds.

[0029] Preferably, in step 2), the enzymolysis time is 10 min and the enzymolysis temperature is 25-37°C; preferably, the enzymolysis time is 10 min and the enzymolysis temperature is 37°C.

[0030] Preferably, in step 2), the centrifugal force is 1000-3000 g, and the centrifugation time is 10 min; preferably, the centrifugal force is 1000 g, and the centrifugation time is 10 min.

[0031] In step 2), the consumables and instruments were purchased from 2 ml centrifuge tubes (axygen / MCT-200-C), steel beads (EASYBIO / BE6638), and tissue grinder (Shanghai Cebo / CEBO-48).

[0032] Preferably, the lysis time in step 3-1) is 5-10 min; preferably, the lysis time is 8 min.

[0033] Preferably, step 3-2) is filtered twice, the first time using a 70um cell sieve and the second time using a 30um filter column.

[0034] Preferably, the purification in step 3-3) uses a purification buffer containing 0.1% TritonX-100 + differential centrifugation.

[0035] Preferably, in step 3-3), a total of three centrifugations are performed, the first centrifugation is 10g, 5min, the precipitate is discarded and the supernatant is retained, the second centrifugation uses the supernatant of the previous step at 300g, 10min and the supernatant is discarded, and the third centrifugation uses the purification buffer to resuspend the precipitate of the previous step at 100g, 10min and the supernatant is discarded.

[0036] Preferably, in step 3-3), after one centrifugation, a MACS column is used for filtration.

[0037] Preferably, in step 3-4), DAPI solution is selected for staining, with a final concentration of 0.5 ug / ul.

[0038] In step 3), the consumables and reagents were purchased from 70um cell sieve (Falcon / 352350), 30um filter column (Miltenyi / 130-098-458), MACS sorting column (Miltenyi / 130--096-334), and DAPI solution (Solarbio / C0060).

[0039] Preferably, in step 4), Qiagen's exoEasyMaxiKit is used to purify exosomes.

[0040] Preferably, in step 4), differential centrifugation before the kit is used to remove other membrane structure fragments other than exosomes.

[0041] Preferably, the differential centrifugation steps in step 4) are: 3000g for 10 min, 10000g for 20 min, and 16000g for 30 min.

[0042] Preferably, all centrifugations are performed using a refrigerated centrifuge at 4°C.

[0043] The present invention uses 6 new Arabidopsis thaliana leaves (about 0.3g sample) to prepare a total of 1.42-1.46 million nuclei, a clumping rate of 5%-6%, a fragmentation rate of 4%-6%, a total exosome RNA of 314-322ng, and 260 / 2801.98-2.01.

[0044] The present invention also provides a method for simultaneously preparing exosomes and cell nuclei of Arabidopsis leaf tissue and simultaneously sequencing exosomes and single cell nuclei of Arabidopsis, the specific steps of which are as follows:

[0045] a) performing single cell nucleus sequencing on the cell nucleus suspension prepared above;

[0046] b) Extracting RNA from the exosomes prepared above and performing small RNA sequencing.

[0047] Preferably, in step a), single cell nucleus sequencing is performed using Chromium Single Cell 3' Reagent Kits of the 10x Genomics platform.

[0048] Preferably, in step b), RNA is extracted using Norgen's Exosomal RNA Isolation Kit, and small RNA library is constructed using NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB).

[0049] The core technical features of the present invention include: (1) adding an enzymatic hydrolysis step after sample crushing greatly improves the yield of exosomes and cell nuclei and reduces some inadequately crushed fragmented tissues; (2) there is currently no special kit for preparing plant exosomes, and the literature mostly uses the method of ultracentrifugation + sucrose gradient centrifugation. In the present invention, Qiagen's exoEasyMaxiKit is used for the first time to purify plant tissue exosomes, saving a lot of time. Compared with purification using sucrose gradient centrifugation, the purity is higher and the recovery rate is higher, and the cost of using the kit is lower for laboratories without ultracentrifuges; (3) In the preparation of cell nuclei, a complete set of processes including three filtrations + low-concentration TrionX-100 washing + differential centrifugation is used for purification. Compared with fluorescence sorting technology, the cost is greatly reduced and the influence of fluorescence signals on subsequent library construction is avoided.

[0050] The present invention also provides a method and application thereof suitable for simultaneously extracting cell nuclei and exosomes from Arabidopsis leaf tissue and sequencing, including a method and application thereof for simultaneously preparing exosomes and cell nuclei from Arabidopsis leaf tissue, a method and application thereof for simultaneously preparing exosomes and cell nuclei from Arabidopsis leaf tissue and simultaneously sequencing plant exosomes and single cell nuclei.

[0051] The present invention also provides a kit, which can be used to prepare tissue exosomes and cell nuclei in the same Arabidopsis leaf tissue and sequence them simultaneously; the kit includes an enzymatic solution, a lysis buffer, a washing buffer, a purification buffer, and a PBS buffer.

[0052] The enzymatic solution formula is: 1% to 1.5% pectinase Y23, 1.5% to 2% hemicellulase, 0.5% to 1% snail enzyme, 1.5% to 2% cellulase R10, 0.4% to 0.6% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4 mM β-mercaptoethanol;

[0053] The composition and formula of the lysis buffer are: 0.8% to 1.2% NP40, 0.2% to 0.4% TritonX-100, 0.8% to 1.2% digitonin, 0.4Msucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1mMDTT, 1×proteaseinhibitor, 0.4U / μlRNaseinhibitor;

[0054] The composition and formula of the washing buffer are: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ulRNaseinhibitor;

[0055] The composition and formula of the purification buffer are: 0.1% TritonX-100, 0.4U / ulRNaseinhibitor, and PBS buffer.

[0056] The PBS buffer: 1xPBS buffer purchased from Thermo was used directly.

[0057] Compared with the prior art, the beneficial effects achieved by the present invention include: filling the current technical gap: the existing research on plant exosomes or single-cell sequencing focuses on one node, lacking the combination of the two. The combined analysis of small RNA of exosomes and single-cell sequencing technology provides a new feasibility of constructing a communication interaction network of different cell types. The research on plant exosomes and the application of single-cell sequencing technology in the field of plants are both breakthroughs in a deeper molecular field. The present invention provides a method suitable for the simultaneous extraction and sequencing of cell nuclei and exosomes from Arabidopsis leaf tissue, which fills the technical gap in the plant kingdom, promotes the research on plant exosomes and the application of single-cell nuclear sequencing technology in plant tissues, and is a major technical breakthrough in the field of plant research. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the result of microscopic examination of the nuclei of Arabidopsis thaliana leaves in Example 1 of the present invention;

[0059] Figure 2 This is the result of microscopic examination of the nuclei of Arabidopsis thaliana leaves in Example 2 of the present invention;

[0060] Figure 3 This is the result of microscopic examination of the nuclei of Arabidopsis thaliana leaves in Example 3 of the present invention;

[0061] Figure 4 This is the result of microscopic examination of the nuclei of Arabidopsis thaliana leaves in Example 4 of the present invention;

[0062] Figure 5 This is the microscopic examination result of Arabidopsis leaf exosomes in Example 1 of the present invention;

[0063] Figure 6 This is the microscopic examination result of Arabidopsis leaf exosomes in Example 2 of the present invention;

[0064] Figure 7 This is the microscopic examination result of Arabidopsis leaf exosomes in Example 3 of the present invention;

[0065] Figure 8 This is the microscopic examination result of Arabidopsis thaliana leaf exosomes in Example 4 of the present invention;

[0066] Fig. 9 It is a schematic diagram of the technical route of the method of the present invention. DETAILED DESCRIPTION

[0067] The present invention is further described in detail with reference to the following specific embodiments and drawings. The process, conditions, experimental methods, etc. of the present invention, except for the contents specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited in the present invention.

[0068] The technical route of the present invention is as follows Fig. 9 shown.

[0069] Example 1

[0070] In this example, the green leaves of Arabidopsis thaliana grown for 20 days were used as samples, exosomes and cell nuclei were prepared at the same time, and exosome small RNA sequencing and single cell nucleus sequencing were performed respectively.

[0071] 1. Prepare reagents

[0072] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, purification buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0073] The enzymatic solution formula is: 1% pectinase Y23, 1.5% hemicellulase, 0.5% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0074] The lysis buffer formula is: 0.8% NP40, 0.2% TritonX-100, 0.8% digitonin, 0.4Msucrose, 10mMTris-HClpH8.0, 10mMMgCl 2 , 1mMDTT, 1×proteaseinhibitor, 0.4U / μlRNase inhibitor.

[0075] The washing buffer formula is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNaseinhibitor.

[0076] Purification buffer: 0.1% TritonX-100, 0.4U / ulRNaseinhibitor, PBS buffer.

[0077] PBS buffer: Use 1x PBS buffer purchased from Thermo directly.

[0078] 2. Sample processing

[0079] 2.1 Sample preparation

[0080] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0081] 2.2 Tissue fragmentation

[0082] Add two 3 mm steel beads into the centrifuge tube and freeze it together with the semiconductor plate of the tissue grinder in liquid nitrogen for 60 seconds. Set the tissue grinder in advance to 35 Hz for 60 seconds and then quickly place it on ice.

[0083] 2.3 Enzymatic hydrolysis

[0084] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0085] 2.4 Exosome nuclear isolation

[0086] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 4 ml. Centrifuge at 4 °C, 1000 g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0087] 3. Exosome extraction and purification

[0088] Qiagen's exoEasyMaxiKit was used to extract and purify exosomes. To avoid contamination by other membrane structures, differential centrifugation was first used to remove debris (at 4°C, 3000g for 10 min, 10000g for 20 min, and 16000g for 30 min), and then subsequent steps were performed according to the instructions.

[0089] 4. Cell Nuclei Preparation

[0090] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the pellet, mix by pipetting, and place on ice for 10 min. Use pre-cooled washing buffer to transfer the nuclear suspension to a new 15 ml centrifuge tube and adjust to 6 ml.

[0091] b) Filtration: 70um cell sieve filtration, 30um cell sieve filtration, centrifugation at 1000g for 10 min at 4°C, and discard the supernatant;

[0092] c) Washing and purification: add 6 ml of purification buffer, centrifuge at 10g for 5 min at 4°C, carefully aspirate the supernatant and discard the precipitate; filter the retained supernatant using a MACS filter column, centrifuge at 300g for 10 min, discard the supernatant; resuspend in 6 ml of purification buffer, centrifuge at 100g for 10 min, discard the supernatant; resuspend in 1 ml of PBS buffer.

[0093] 5. Microscopic examination

[0094] 5.1 Exosomes

[0095] Transmission electron microscopy (Hitachi, HT-7700) and particle size analyzer (NanoFCM, N30E) were used to detect particle size and morphology.

[0096] 5.2 Nucleus

[0097] Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0098] 6. Database Construction

[0099] 6.1 Single-cell nuclear library construction

[0100] The single cell nuclear RNA library was constructed using the Chromium Single Cell 3'Library and Gel Bead Kitv3 from 10x Genomics, and the specific steps were performed according to the instructions.

[0101] 6.2 Exosomal small RNA library construction

[0102] 6.2.1 Exosome RNA extraction

[0103] The Exosomal RNA Isolation Kit from Norgen was used, and the specific steps were carried out according to the instructions.

[0104] 6.2.2 Exosomal small RNA library construction

[0105] NEB's Multipex small RNA Library Prep Set for Illumina was used to construct the library and perform NGS sequencing.

[0106] Results: See Table 11

[0107] Example 2

[0108] In this example, newly grown young leaves of Arabidopsis thaliana grown for 40 days were used as samples, exosomes and cell nuclei were prepared at the same time, and exosome small RNA sequencing and single cell nucleus sequencing were performed respectively.

[0109] The specific steps are consistent with those in Example 1 of the present invention.

[0110] Results: See Table 11

[0111] Example 3

[0112] In this example, newly grown young leaves of Arabidopsis thaliana grown for 20 days were used as samples, exosomes and cell nuclei were prepared at the same time, and exosome small RNA sequencing and single cell nucleus sequencing were performed respectively.

[0113] The composition and formula of the enzymatic hydrolyzate are: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 2% cellulase R10, 0.6% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl2, 0.1% BSA, 4mM β-mercaptoethanol.

[0114] The composition and formula of the lysis buffer are: 1.2% NP40, 0.4% TritonX-100, 1.2% digitonin, 0.4M sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor. Lysis time 5 min. The remaining steps are consistent with Example 1 of the present invention.

[0115] Results: See Table 11

[0116] Example 4

[0117] In this example, newly grown young leaves of Arabidopsis thaliana grown for 40 days were used as samples, exosomes and cell nuclei were prepared at the same time, and exosome small RNA sequencing and single cell nucleus sequencing were performed respectively.

[0118] The specific steps are consistent with those of Example 3 of the present invention.

[0119] Results: See Table 11

[0120] Comparative Example 1

[0121] This sample example refers to the cell nucleus preparation method and directly uses the lysis buffer as the tissue processing buffer, first lyses and then centrifuges to separate the supernatant and precipitate.

[0122] 1) Prepare reagents

[0123] Before preparing the samples, prepare the lysis buffer, wash buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0124] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0125] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0126] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0127] 2) Sample preparation

[0128] Six newly grown young leaves of Arabidopsis thaliana that had grown for twenty days were selected, cut with scissors, placed in a 2 ml centrifuge tube and added with two 3 mm steel balls.

[0129] 3) Sample processing

[0130] Add 0.5 ml of pre-cooled lysis buffer to the centrifuge tube to cover the plant tissue, and use a tissue grinder at 30 Hz and 30 seconds. Keep the whole process on ice.

[0131] 4) Exosome nuclear isolation

[0132] Transfer the tissue suspension to a 15 ml centrifuge tube with 3 ml lysis buffer, and adjust to 5 ml with lysis buffer. Vortex to mix, centrifuge at 1000 g for 10 min at 4 °C, and carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0133] 5) Exosome extraction

[0134] The supernatant obtained in step 4) was centrifuged at 3000 g for 20 min, 6000 g for 40 min, and 10000 g for 60 min, discarding the precipitate after each centrifugation and collecting the supernatant; exosomes were precipitated by centrifugation at 150000 g for 90 min at 4°C and resuspended in 200 ul PBS buffer.

[0135] 6) Cell nucleus preparation

[0136] Add 6 ml of pre-cooled washing buffer to the precipitate obtained in step 4), filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, discard the supernatant, and resuspend in 1 ml of PBS buffer.

[0137] 7) Microscopic examination

[0138] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0139] Exosomes: The particle size, concentration and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0140] 8) Exosome RNA extraction

[0141] The Trizol method was used to extract exosome RNA. The specific steps are as follows (note that the entire process was performed on ice and centrifugation was performed at 4°C).

[0142] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0143] b) Add 200ul of chloroform, shake to mix, place on ice for 5min, and centrifuge at 13000rpm for 15min;

[0144] c) Carefully pipette the first layer of supernatant into a new RNase-free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0145] d) Discard the supernatant and keep the precipitate, add 1 ml of 75% ethanol, shake and mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate.

[0146] e) Air-dry for 1 minute, add 10ul DEPC water, flick the tube, and centrifuge briefly in a microcentrifuge.

[0147] f) RNA quality test was performed using Agilent 4150.

[0148] Results: See Table 1

[0149] Comparative Example 2

[0150] In this sample example, PBS buffer was selected as the tissue treatment buffer according to the exosome preparation method, and the supernatant and precipitate were separated by centrifugation and then lysed.

[0151] 1) Prepare reagents

[0152] Before preparing the samples, prepare the lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0153] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0154] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0155] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0156] 2) Sample preparation

[0157] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0158] 3) Sample processing

[0159] Add 2 sterilized steel balls with a diameter of 3 mm to the centrifuge tube, add 0.5 ml of pre-cooled PBS buffer to cover the plant tissue, grind the tissue at 30 Hz, 30 seconds; transfer the tissue suspension to a 15 ml centrifuge tube with 3 ml of PBS buffer, and balance to 5 ml with PBS buffer, and oscillate to mix. Keep the tube on ice throughout the operation.

[0160] 4) Exosome nuclear isolation

[0161] Centrifuge at 4°C, 1000g for 10 min, carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0162] 5) Exosome extraction

[0163] The supernatant obtained in step 4) was centrifuged at 4°C for 20 min at 3000g, 40 min at 6000g, and 60 min at 10000g. After each centrifugation, the precipitate was discarded and the supernatant was collected. The exosomes were precipitated by centrifugation at 150000g for 90 min at 4°C and resuspended in 200ul PBS buffer.

[0164] 6) Cell nucleus preparation

[0165] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0166] b) Washing: Add 6 ml of washing buffer, centrifuge at 1000 g for 10 min at 4°C, discard the supernatant, and resuspend in 1 ml of PBS buffer.

[0167] 7) Microscopic examination

[0168] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0169] Exosomes: The particle size, concentration and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0170] 8) Exosome RNA extraction

[0171] The exosome RNA was extracted using the Trizol method. The specific steps are as follows (note that the whole process was performed on ice and centrifugation was performed at 4°C):

[0172] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0173] b) Add 200 μl of chloroform, shake to mix, place on ice for 5 min, and centrifuge at 13,000 rpm for 15 min;

[0174] c) Carefully pipette the first layer of supernatant into a new RNase-free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0175] d) Discard the supernatant and keep the precipitate, add 1 ml of 75% ethanol, shake and mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate.

[0176] e) Air-dry for 1 minute, add 10ul DEPC water, flick the tube, and centrifuge briefly in a microcentrifuge.

[0177] f) RNA quality test was performed using Agilent 4150.

[0178] Results: See Table 1

[0179] Comparative Example 3

[0180] In this comparative example, the sample processing method is to cut into pieces with scissors.

[0181] 1) Prepare reagents

[0182] Before preparing the samples, prepare the lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0183] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0184] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0185] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0186] 2) Sample preparation

[0187] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0188] 3) Sample processing

[0189] Add a small amount of PBS to the centrifuge tube to cover the plant tissue, and use sterilized small scissors to cut it as finely as possible; transfer the tissue suspension to a 15ml centrifuge tube with 3ml PBS, and use PBS to make it equal to 5ml, shake and mix, incubate on ice for 2 minutes, and repeat the mixing operation. Keep the whole process on ice.

[0190] 4) Exosome nuclear isolation

[0191] Centrifuge at 4°C, 1000g for 10 min, carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0192] 5) Exosome extraction

[0193] At 4°C, centrifuge at 3000g for 20 min, 6000g for 40 min, and 10000g for 60 min. Collect the supernatant after each centrifugation and discard the precipitate. Centrifuge at 150000g for 90 min at 4°C to precipitate the exosomes, and resuspend them in 200ul PBS buffer.

[0194] 6) Cell nucleus preparation

[0195] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0196] b) Washing: Add 6 ml of washing buffer, centrifuge at 1000 g for 10 min at 4°C, discard the supernatant, and resuspend in 1 ml of PBS buffer.

[0197] 7) Microscopic examination

[0198] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0199] Exosomes: The particle size and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0200] 8) Exosome RNA extraction

[0201] The exosome RNA was extracted using the Trizol method. The specific steps are as follows (note that the whole process was performed on ice and centrifugation was performed at 4°C):

[0202] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0203] b) Add 200 μl of chloroform, shake to mix, place on ice for 5 min, and centrifuge at 13,000 rpm for 15 min;

[0204] c) Carefully pipette the first layer of supernatant into a new RNase-free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0205] d) Discard the supernatant and keep the precipitate, add 1 ml 75% ethanol, shake to mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate;

[0206] e) Air-dry for 1 minute, add 10ul DEPC water, flick the bottle, and centrifuge briefly in a microcentrifuge;

[0207] f) RNA quality test was performed using Agilent 4150.

[0208] Results: See Table 2

[0209] Comparative Example 4

[0210] The sample processing method of this comparative example adopts manual grinding, and the remaining steps are the same as those of comparative example 1.

[0211] 1) Prepare reagents

[0212] Before preparing the samples, prepare the lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0213] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0214] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2, 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0215] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0216] 2) Sample preparation

[0217] Six newly grown young leaves of Arabidopsis thaliana that had grown for twenty days were selected, cut with scissors, and placed in a glass mortar.

[0218] 3) Sample processing

[0219] Add 2 ml of pre-cooled PBS buffer to the mortar to cover the plant tissue, and grind slowly at a constant speed until it is homogenized; transfer the tissue suspension to a 15 ml centrifuge tube with 3 ml of PBS, and use PBS to make it equal to 5 ml, and shake to mix. Note that the whole process should be kept on ice, and the mortar should be placed on ice during the grinding process.

[0220] 4) Exosome nuclear isolation

[0221] Centrifuge at 4°C, 1000g for 10 min, carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0222] 5) Exosome extraction

[0223] At 4°C, centrifuge at 3000g for 20 min, 6000g for 40 min, and 10000g for 60 min. Collect the supernatant after each centrifugation and discard the precipitate. Centrifuge at 150000g for 90 min at 4°C to precipitate the exosomes, and resuspend them in 200ul PBS buffer.

[0224] 6) Cell nucleus preparation

[0225] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0226] b) Washing: Add 6 ml of washing buffer, centrifuge at 1000 g for 10 min at 4°C, discard the supernatant, and resuspend in 1 ml of PBS buffer.

[0227] 7) Microscopic examination

[0228] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0229] Exosomes: The particle size and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0230] 8) Exosome RNA extraction

[0231] The exosome RNA was extracted using the Trizol method. The specific steps are as follows (note that the whole process was performed on ice and centrifugation was performed at 4°C):

[0232] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0233] b) Add 200 μl of chloroform, shake to mix, place on ice for 5 min, and centrifuge at 13,000 rpm for 15 min;

[0234] c) Carefully pipette the first layer of supernatant into a new RNase free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0235] d) Discard the supernatant and keep the precipitate, add 1 ml of 75% ethanol, shake and mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate.

[0236] e) Air-dry for 1 minute, add 10ul DEPC water, flick the tube after adding, and centrifuge briefly in a microcentrifuge.

[0237] f) RNA quality test was performed using Agilent 4150.

[0238] Results: See Table 2

[0239] Comparative Example 5

[0240] This comparative example sample processing method selects tissue grinder grinding

[0241] 1) Prepare reagents

[0242] Before preparing the samples, prepare the lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0243] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2, 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0244] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0245] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0246] 2) Sample preparation

[0247] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0248] 3) Sample processing

[0249] Add 2 sterilized steel balls with a diameter of 3 mm to the centrifuge tube, add a small amount of pre-cooled PBS buffer to cover the plant tissue, grind the tissue at 30 Hz for 40 seconds; transfer the tissue suspension to a 15 ml centrifuge tube with 3 ml PBS, balance to 5 ml with PBS, and oscillate to mix. Keep the tube on ice throughout the operation.

[0250] 4) Exosome nuclear isolation

[0251] Centrifuge at 4°C, 1000g for 10 min, carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0252] 5) Exosome extraction

[0253] At 4°C, centrifuge at 3000g for 20 min, 6000g for 40 min, and 10000g for 60 min. Collect the supernatant after each centrifugation and discard the precipitate. Centrifuge at 150000g for 90 min at 4°C to precipitate the exosomes, and resuspend them in 200ul PBS buffer.

[0254] 6) Cell nucleus preparation

[0255] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0256] b) Washing: Add 6 ml of washing buffer, centrifuge at 1000 g for 10 min at 4°C, discard the supernatant, and resuspend in 1 ml of PBS buffer.

[0257] 7) Microscopic examination

[0258] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0259] Exosomes: The particle size and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0260] 8) Exosome RNA extraction

[0261] The exosome RNA was extracted using the Trizol method. The specific steps are as follows (note that the whole process was performed on ice and centrifugation was performed at 4°C):

[0262] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0263] b) Add 200 μl of chloroform, shake to mix, place on ice for 5 min, and centrifuge at 13,000 rpm for 15 min;

[0264] c) Carefully pipette the first layer of supernatant into a new RNase-free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0265] d) Discard the supernatant and keep the precipitate, add 1 ml of 75% ethanol, shake and mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate.

[0266] e) Air-dry for 1 minute, add 10ul DEPC water, flick the tube, and centrifuge briefly in a microcentrifuge.

[0267] f) RNA quality test was performed using Agilent 4150.

[0268] Results: See Table 2

[0269] Comparative Example 6

[0270] This comparative example sample processing method selects tissue grinder liquid nitrogen grinding

[0271] 1) Prepare reagents

[0272] Before preparing the samples, prepare the lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0273] The formula of cell nuclear lysis buffer is: 0.2% TritonX-100, 1mM DTT, 0.25M Sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ulRNaseinhibitor.

[0274] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0275] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0276] 2) Sample preparation

[0277] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0278] 3) Sample processing

[0279] Add 2 3mm steel beads to the centrifuge tube and freeze it in liquid nitrogen together with the semiconductor plate of the tissue grinder for 60 seconds. Set the tissue grinder in advance, 30HZ, 40s, carefully place it on the tissue grinder, and turn on the switch; use pre-cooled PBS buffer to transfer the tissue powder to a 15ml centrifuge tube, balance to 5ml, and shake to mix. Keep it on ice throughout the operation.

[0280] 4) Exosome nuclear isolation

[0281] Centrifuge at 4°C, 1000g for 10 min, carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0282] 5) Exosome extraction

[0283] At 4°C, centrifuge at 3000g for 20 min, 6000g for 40 min, and 10000g for 60 min. Collect the supernatant after each centrifugation and discard the precipitate. Centrifuge at 150000g for 90 min at 4°C to precipitate the exosomes, and resuspend them in 200ul PBS buffer.

[0284] 6) Cell nucleus preparation

[0285] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0286] b) Washing: Add 6 ml of washing buffer, centrifuge at 1000 g for 10 min at 4°C, discard the supernatant, and resuspend in 1 ml of PBS buffer.

[0287] 7) Microscopic examination

[0288] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0289] Exosomes: The particle size and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0290] 8) Exosome RNA extraction

[0291] The exosome RNA was extracted using the Trizol method. The specific steps are as follows (note that the whole process was performed on ice and centrifugation was performed at 4°C):

[0292] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0293] b) Add 200 μl of chloroform, shake to mix, place on ice for 5 min, and centrifuge at 13,000 rpm for 15 min;

[0294] c) Carefully pipette the first layer of supernatant into a new RNase-free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0295] d) Discard the supernatant and keep the precipitate, add 1 ml of 75% ethanol, shake and mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate.

[0296] e) Air-dry for 1 minute, add 10ul DEPC water, flick the tube, and centrifuge briefly in a microcentrifuge.

[0297] f) RNA quality test was performed using Agilent 4150.

[0298] Results: See Table 2

[0299] Comparative Example 7

[0300] The frequency of the tissue grinder in this comparative example is selected to be 20 Hz, and the remaining steps are the same as those in comparative example 6.

[0301] Results: See Table 3

[0302] Comparative Example 8

[0303] The frequency of the tissue grinder in this comparative example is selected to be 40 Hz, and the remaining steps are the same as those in comparative example 6.

[0304] Results: See Table 3

[0305] Comparative Example 9

[0306] The frequency of the tissue grinder in this comparative example is 50 Hz, and the remaining steps are the same as those in comparative example 6.

[0307] Results: See Table 3

[0308] Comparative Example 10

[0309] The time of the tissue grinder in this comparative example is selected to be 60s, and the remaining steps are the same as those in comparative example 6.

[0310] Results: See Table 3

[0311] Comparative Example 11

[0312] The time of the tissue grinder in this comparative example is selected to be 90s, and the remaining steps are the same as those in comparative example 6.

[0313] Results: See Table 3

[0314] Comparative Example 12

[0315] In this comparative example, an enzymatic hydrolysis step was added after tissue fragmentation, and reference was made to "Arabidopsis mesophyll protoplasts: a satellite cell system for transient gene expression analysis" using 1.5% cellulase R10, 0.4% cleavage enzyme R10, 20 mM MES (pH 5.7), 0.4 M mannitol, 20 mM KCl, and 10 mM CaCl. 2 , 0.1% BSA, and incubate in the dark at 37°C for 3 hours.

[0316] 1) Prepare reagents

[0317] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0318] The enzymatic solution formula is: 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA.

[0319] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0320] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0321] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0322] 2) Sample preparation

[0323] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0324] 3) Sample processing

[0325] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0326] 4) Enzymatic hydrolysis

[0327] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 3 hours at 37°C and 200 RPM in the dark.

[0328] 5) Exosome nuclear isolation

[0329] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0330] 6) Exosome extraction

[0331] Step 5) The retained supernatant was centrifuged at 4°C for 20 min at 3000g, 40 min at 6000g, and 60 min at 10000g. The supernatant was collected after each centrifugation and the precipitate was discarded. The exosomes were precipitated by centrifugation at 150000g for 90 min at 4°C and resuspended in 200ul PBS buffer.

[0332] 7) Cell nucleus preparation

[0333] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0334] b) Washing: Add 6 ml of washing buffer, centrifuge at 1000 g for 10 min at 4°C, discard the supernatant, and resuspend in 1 ml of PBS buffer.

[0335] 8) Microscopic examination

[0336] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0337] Exosomes: The particle size and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0338] 9) Exosome RNA extraction

[0339] The exosome RNA was extracted using the Trizol method. The specific steps are as follows (note that the whole process was performed on ice and centrifugation was performed at 4°C):

[0340] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0341] b) Add 200 μl of chloroform, shake to mix, place on ice for 5 min, and centrifuge at 13,000 rpm for 15 min;

[0342] c) Carefully pipette the first layer of supernatant into a new RNase-free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0343] d) Discard the supernatant and keep the precipitate, add 1 ml of 75% ethanol, shake and mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate.

[0344] e) Air-dry for 1 minute, add 10ul DEPC water, flick the tube after adding, and centrifuge briefly in a microcentrifuge.

[0345] f) RNA quality test was performed using Agilent 4150.

[0346] Results: See Table 4

[0347] Comparative Example 13

[0348] In this comparative example, after tissue disruption, 1.5% cellulase RS, 0.03% pectinase Y23, 5 mM MES, 0.5 mM CaCl were used with reference to "Global Dynamic Molecular Profiling of Stomatal Lineage Cell Development by Single-Cell RNA Sequencing". 2 , 0.5 mM MgCl 2 , 0.25% BSA, incubate in the dark at 37°C for 4 hours, and the remaining steps are the same as those in Comparative Example 12.

[0349] Results: See Table 4

[0350] Comparative Example 14

[0351] In this comparative example, after tissue disruption, 1.25% cellulase R10, 0.3% macerate R10, 0.4 M mannitol, 20 mM MES, 20 mM KCl, 10 mM CaCl were used according to "Single-Cell Resolution of Lineage Trajectories in the Arabidopsis Stomatal Lineage and Developing Leaf". 2 , 1M Tris-HCl pH 7.5, incubate at 37°C in the dark for 2 hours, and the remaining steps are the same as those in Comparative Example 12.

[0352] Results: See Table 4

[0353] Comparative Example 15

[0354] In this comparative example, 6 mM β-mercaptoethanol was added to the enzymatic hydrolysis solution, and the enzymatic hydrolysis time was reduced to 30 min. The remaining steps were the same as those in comparative example 12.

[0355] Results: See Table 4

[0356] Comparative Example 16

[0357] In this comparative example, 6 mM β-mercaptoethanol was added to the enzymatic hydrolysis solution, and the enzymatic hydrolysis time was reduced to 20 min. The remaining steps were the same as those in comparative example 12.

[0358] Results: See Table 4

[0359] Comparative Example 17

[0360] In this comparative example, 2 mM β-mercaptoethanol was added to the enzymatic hydrolysis solution, and the enzymatic hydrolysis time was reduced to 10 min. The remaining steps were the same as those in comparative example 12.

[0361] Results: See Table 4

[0362] Comparative Example 18

[0363] In this comparative example, 4 mM β-mercaptoethanol was added to the enzymatic hydrolysis solution, and the enzymatic hydrolysis time was reduced to 10 min. The remaining steps were the same as those in comparative example 12.

[0364] Results: See Table 4

[0365] Comparative Example 19

[0366] In this comparative example, 2 mM β-mercaptoethanol was added to the enzymatic hydrolysis solution, and the enzymatic hydrolysis time was reduced to 5 min. The remaining steps were the same as those in comparative example 12.

[0367] Results: See Table 4

[0368] Comparative Example 20

[0369] This comparative example increases the concentration of the enzymatic solution, and the enzymatic solution formula is 2% cellulase R10, 0.6% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0370] Results: See Table 5

[0371] Comparative Example 21

[0372] In this comparative example, 1.5% cellulase R10 was replaced with 1.5% cellulase RS, and the enzymatic solution formula was 1.5% cellulase RS, 0.4% cleavage enzyme R10, 20 mM MES (pH 5.7), 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2, 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0373] Results: See Table 5

[0374] Comparative Example 22

[0375] The enzymatic solution formula of this comparative example is 1% pectinase Y23, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0376] Results: See Table 5

[0377] Comparative Example 23

[0378] The enzymatic solution of this comparative example is formulated as 2% hemicellulase, 1.5% cellulase R10, 0.4% macerate R10, 20 mM MES (pH 5.7), 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0379] Results: See Table 5

[0380] Comparative Example 24

[0381] The enzymatic solution of this comparative example is formulated as 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20 mM MES (pH 5.7), 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0382] Results: See Table 5

[0383] Comparative Example 25

[0384] The enzymatic solution of this comparative example is formulated as follows: 1% lysase, 1.5% cellulase R10, 0.4% lysase R10, 20 mM MES (pH 5.7), 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0385] Results: See Table 5

[0386] Comparative Example 26

[0387] The enzymatic solution of this comparative example is formulated as follows: 1% pectinase Y23, 2% hemicellulase, 1.5% cellulase R10, 0.4% macerate R10, 20 mM MES (pH 5.7), 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0388] Results: See Table 5

[0389] Comparative Example 27

[0390] The enzymatic solution formula of this comparative example is 1% pectinase Y23, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0391] Results: See Table 5

[0392] Comparative Example 28

[0393] The enzymatic solution formula of this comparative example is 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0394] Results: See Table 5

[0395] Comparative Example 29

[0396] The enzymatic solution formula of this comparative example is 1% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0397] Results: See Table 5

[0398] Comparative Example 30

[0399] The enzymatic solution formula of this comparative example is 0.5% pectinase Y23, 1% hemicellulase, 0.2% snail enzyme, 1% cellulase R10, 0.2% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0400] Results: See Table 6

[0401] Comparative Example 31

[0402] The enzymatic solution formula of this comparative example is 1% pectinase Y23, 1.5% hemicellulase, 0.5% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0403] Results: See Table 6

[0404] Comparative Example 32

[0405] The enzymatic solution formula of this comparative example is 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 2% cellulase R10, 0.6% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol, and the remaining steps are the same as those in Comparative Example 18.

[0406] Results: See Table 6

[0407] Comparative Example 33

[0408] The method for preparing the cell nucleus suspension in this comparative example refers to "Single-nucleus full-length RNA profiling in plants incorporates isoform information to facilitate cell type identification", and the specific steps are as follows:

[0409] 1) Prepare reagents

[0410] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0411] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0412] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 0.4M sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1mM DTT, 1×protease inhibitor, 0.4U / μl RNase inhibitor.

[0413] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0414] 2) Sample preparation

[0415] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0416] 3) Sample processing

[0417] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0418] 4) Enzymatic hydrolysis

[0419] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0420] 5) Exosome nuclear isolation

[0421] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0422] 6) Cell nucleus preparation

[0423] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, mix by pipetting, filter through a 20 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0424] b) Washing: Add 6 ml of lysis buffer, centrifuge at 2000 g for 5 min at 4°C, discard the supernatant, repeat 3 times, and resuspend in 1 ml of PBS buffer.

[0425] 7) Microscopic examination

[0426] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0427] Results: See Table 7

[0428] Comparative Example 34

[0429] The method for preparing the cell nucleus suspension in this comparative example is based on "Single-nuclei RNA-sequencing of plant tissues", and the specific steps are as follows:

[0430] 1) Prepare reagents

[0431] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0432] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0433] The formula of cell nuclear lysis buffer is: 0.5% Triton X-100, 2.5% Ficoll 400, 5% Dextran T40, 0.4M sucrose, 10mM MgCl 2 , 1μM DTT, 1×Protease Inhibitor, 0.4U / μl RiboLock, 25mM Tris-HCl pH7.4.

[0434] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0435] 2) Sample preparation

[0436] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0437] 3) Sample processing

[0438] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0439] 4) Enzymatic hydrolysis

[0440] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0441] 5) Exosome nuclear isolation

[0442] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0443] 6) Cell nucleus preparation

[0444] Cells were lysed using gentleMACS Dissociator, filtered at 70 μm, centrifuged at 1000 g for 6 min, carefully resuspended in 1 ml PBS buffer, and filtered through a 35 μm filter;

[0445] 7) Microscopic examination

[0446] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0447] Results: See Table 7

[0448] Comparative Example 35

[0449] This comparative example uses multiple filtration and multiple washing methods to purify the cell nucleus suspension, and the specific steps are as follows:

[0450] 1) Prepare reagents

[0451] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0452] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0453] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0454] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0455] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0456] 2) Sample preparation

[0457] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0458] 3) Sample processing

[0459] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0460] 4) Enzymatic hydrolysis

[0461] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0462] 5) Exosome nuclear isolation

[0463] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0464] 6) Cell nucleus preparation

[0465] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, filter through a 30 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0466] b) Washing: add 6 ml of washing buffer, centrifuge at 1000 g for 10 min at 4°C, repeat 3 times; resuspend in 3 ml of washing buffer, filter using a MACS column, centrifuge at 1000 g for 10 min, discard the supernatant, and resuspend in 1 ml of PBS buffer.

[0467] 7) Microscopic examination

[0468] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0469] Results: See Table 7

[0470] Comparative Example 36

[0471] This comparative example uses differential centrifugation to reduce centrifugal force to purify the cell nucleus suspension, and the specific steps are as follows:

[0472] 1) Prepare reagents

[0473] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0474] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0475] The formula of cell nuclear lysis buffer is: 0.2% TritonX-100, 1mM DTT, 0.25M Sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ulRNaseinhibitor.

[0476] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0477] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0478] 2) Sample preparation

[0479] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0480] 3) Sample processing

[0481] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0482] 4) Enzymatic hydrolysis

[0483] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0484] 5) Exosome nuclear isolation

[0485] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0486] 6) Cell nucleus preparation

[0487] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0488] b) Washing and purification: add 6 ml washing buffer, centrifuge at 10 g for 5 min at 4°C, remove the precipitate; centrifuge at 300 g for 10 min, remove the supernatant; resuspend in 6 ml washing buffer, centrifuge at 100 g for 10 min, remove the supernatant; resuspend in 1 ml PBS buffer.

[0489] 7) Microscopic examination

[0490] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0491] Results: See Table 7

[0492] Comparative Example 37

[0493] This comparative example uses 0.1% Triton X-100 to purify the cell nucleus suspension, and the specific steps are as follows:

[0494] 1) Prepare reagents

[0495] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0496] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0497] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0498] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0499] The formula of cell nucleus purification buffer is: 0.1% Triton X-100, 0.4U / ul RNase inhibitor, PBS buffer.

[0500] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0501] 2) Sample preparation

[0502] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0503] 3) Sample processing

[0504] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0505] 4) Enzymatic hydrolysis

[0506] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0507] 5) Exosome nuclear isolation

[0508] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0509] 6) Cell nucleus preparation

[0510] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0511] b) Purification: Add 6 ml of purification buffer, centrifuge at 1000 g for 5 min at 4°C, remove the supernatant, repeat 3 times, and resuspend in 1 ml of PBS buffer.

[0512] 7) Microscopic examination

[0513] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0514] Results: See Table 7

[0515] Comparative Example 38

[0516] This comparative example uses sucrose gradient centrifugation to purify the cell nucleus suspension, and the specific steps are as follows:

[0517] 1) Prepare reagents

[0518] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0519] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0520] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0521] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0522] Sucrose buffer formula: 1.7M Sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0523] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0524] 2) Sample preparation

[0525] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0526] 3) Sample processing

[0527] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0528] 4) Enzymatic hydrolysis

[0529] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0530] 5) Exosome nuclear isolation

[0531] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0532] 6) Cell nucleus preparation

[0533] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the precipitate, pipette and mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, balance to 6 ml, filter through a 70 um cell sieve, centrifuge at 1000 g for 10 min at 4°C, and discard the supernatant;

[0534] b) Washing: add 6 ml of washing buffer, centrifuge at 1000 g for 10 min at 4°C, repeat twice, and discard the supernatant;

[0535] c) Purification: Take a new 1.5ml centrifuge tube and add 500ul pre-cooled sucrose solution. Resuspend the cell nucleus pellet with 500ul pre-cooled sucrose solution. Slowly add it along the tube wall into the centrifuge tube containing 500ul pre-cooled sucrose solution. Centrifuge at 4℃, 16000g for 10min. Carefully aspirate the supernatant as much as possible and keep the pellet. Resuspend in 1ml PBS.

[0536] 7) Microscopic examination

[0537] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0538] Results: See Table 7

[0539] Comparative Example 39

[0540] This comparative example uses multiple filtration + differential centrifugation to purify the nuclei, and the specific steps are as follows:

[0541] 1) Prepare reagents

[0542] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0543] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0544] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0545] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0546] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0547] 2) Sample preparation

[0548] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0549] 3) Sample processing

[0550] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0551] 4) Enzymatic hydrolysis

[0552] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0553] 5) Exosome nuclear isolation

[0554] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0555] 6) Cell nucleus preparation

[0556] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the pellet, pipette to mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, and balance to 6 ml;

[0557] b) Filtration: 70um cell sieve filtration, 30um cell sieve filtration, centrifugation at 1000g for 10 min at 4°C, and discard the supernatant;

[0558] c) Washing and purification: add 6 ml of washing buffer, centrifuge at 10 g for 5 min at 4°C, and remove the precipitate; filter the retained supernatant using a MACS filter column, centrifuge at 300 g for 10 min, and remove the supernatant; resuspend in 6 ml of washing buffer, centrifuge at 100 g for 10 min, and remove the supernatant; resuspend in 1 ml of PBS buffer.

[0559] 7) Microscopic examination

[0560] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0561] Results: See Table 7

[0562] Comparative Example 40

[0563] In this comparative example, the cell nucleus purification simultaneously uses multiple filtration + differential centrifugation + 0.1% Triton X-100, and the specific steps are as follows:

[0564] 1) Prepare reagents

[0565] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0566] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0567] The formula of nuclear lysis buffer is: 0.2% Triton X-100, 1mM DTT, 0.25M sucrose, 10mMTris-HCl pH 8.0, 10mM MgCl 2 , 1×protease inhibitor, 0.4U / ul RNase inhibitor.

[0568] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0569] The formula of cell nucleus purification buffer is: 0.1% Triton X-100, 0.4U / ul RNase inhibitor, PBS buffer.

[0570] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0571] 2) Sample preparation

[0572] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0573] 3) Sample processing

[0574] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0575] 4) Enzymatic hydrolysis

[0576] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0577] 5) Exosome nuclear isolation

[0578] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0579] 6) Cell nucleus preparation

[0580] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the pellet, pipette to mix, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, and balance to 6 ml;

[0581] b) Filtration: 70um cell sieve filtration, 30um cell sieve filtration, centrifugation at 1000g for 10 min at 4°C, and discard the supernatant;

[0582] c) Washing and purification: add 6 ml of purification buffer, centrifuge at 10 g for 5 min at 4°C to remove the precipitate; filter the retained supernatant using a MACS filter column, centrifuge at 300 g for 10 min, and remove the supernatant; resuspend in 6 ml of purification buffer, centrifuge at 100 g for 10 min, and remove the supernatant; resuspend in 1 ml of PBS buffer.

[0583] 7) Microscopic examination

[0584] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0585] Results: See Table 7

[0586] Comparative Example 41

[0587] The formula of the cell nuclear lysis solution in this comparative example is 1% Triton X-100, 0.4M sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1mM DTT, 1× protease inhibitor, 0.4U / μl RNaseinhibitor. The specific steps are as follows:

[0588] 1) Prepare reagents

[0589] Before preparing the samples, prepare the enzymatic solution, lysis buffer, washing buffer, and PBS buffer, and pre-cool the prepared solutions on ice.

[0590] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0591] The formula of nuclear lysis buffer is: 1% Triton X-100, 0.4M sucrose, 10mM Tris-HCl pH8.0, 10mM MgCl 2 , 1mM DTT, 1×protease inhibitor, 0.4U / μl RNase inhibitor.

[0592] The formula of nuclear washing buffer is: 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1×proteaseinhibitor, 0.4U / ul RNase inhibitor.

[0593] The formula of cell nucleus purification buffer is: 0.1% Triton X-100, 0.4U / ul RNase inhibitor, PBS buffer.

[0594] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0595] 2) Sample preparation

[0596] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0597] 3) Sample processing

[0598] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 30HZ and 60s, carefully place it on the tissue grinder and turn on the switch.

[0599] 4) Enzymatic hydrolysis

[0600] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0601] 5) Exosome nuclear isolation

[0602] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 5 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0603] 6) Cell nucleus preparation

[0604] a) Lysis: Add 1.5 ml of nuclear lysis buffer to the pellet, mix by pipetting, let stand on ice for 5-10 min, transfer to a new 15 ml centrifuge tube with pre-cooled washing buffer, and balance to 6 ml;

[0605] b) Filtration: 70um cell sieve filtration, 30um cell sieve filtration, centrifugation at 1000g for 10 min at 4°C, and discard the supernatant;

[0606] c) Washing and purification: add 6 ml of purification buffer, centrifuge at 10 g for 5 min at 4°C to remove the precipitate; filter the retained supernatant using a MACS filter column, centrifuge at 300 g for 10 min, and remove the supernatant; resuspend in 6 ml of purification buffer, centrifuge at 100 g for 10 min, and remove the supernatant; resuspend in 1 ml of PBS buffer.

[0607] 7) Microscopic examination

[0608] Cell nuclei: Use DAPI solution for staining and observe under a fluorescence microscope. Add 1ul of DAPI to 9ul of cell nucleus suspension. Count the number of cell nuclei and observe their chloroplast content, debris, impurities, and agglomeration ratio.

[0609] Results: See Table 8

[0610] Comparative Example 42

[0611] The formula of the cell nuclear lysis solution in this comparative example is 1% NP40, 0.2% TritonX-100, 0.4M Sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2, 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor, and the remaining steps are consistent with Comparative Example 41.

[0612] Results: See Table 8

[0613] Comparative Example 43

[0614] The formula of the cell nuclear lysis solution in this comparative example is: 1% digitonin, 0.2% TritonX-100, 0.4Msucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor, and the remaining steps are consistent with Comparative Example 41.

[0615] Results: See Table 8

[0616] Comparative Example 44

[0617] The formula of the cell nuclear lysis solution in this comparative example is: 1% NP40, 1% digitonin, 0.2% TritonX-100, 0.4Msucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor, and the remaining steps are consistent with Comparative Example 41.

[0618] Results: See Table 8

[0619] Comparative Example 45

[0620] The formula of the cell nuclear lysis solution in this comparative example is: 0.5% NP40, 0.1% TritonX-100, 0.5% digitonin, 0.4Msucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor, and the remaining steps are consistent with Comparative Example 41.

[0621] Results: See Table 8

[0622] Comparative Example 46

[0623] The formula of the cell nuclear lysis solution in this comparative example is: 0.8% NP40, 0.2% TritonX-100, 0.8% digitonin, 0.4Msucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor, and the remaining steps are consistent with Comparative Example 41.

[0624] Results: See Table 8

[0625] Comparative Example 47

[0626] The formula of the cell nuclear lysis solution in this comparative example is: 1.2% NP40, 0.4% TritonX-100, 1.2% digitonin, 0.4Msucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor, and the remaining steps are consistent with Comparative Example 41.

[0627] Results: See Table 8

[0628] Comparative Example 48

[0629] The formula of the cell nuclear lysis solution in this comparative example is: 1.5% NP40, 0.5% TritonX-100, 1.5% digitonin, 0.4Msucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor, and the remaining steps are consistent with Comparative Example 41.

[0630] Results: See Table 8

[0631] Comparative Example 49

[0632] In this comparative example, the cell nuclear precipitate is added with the lysis solution, mixed by pipetting and allowed to stand on ice for 5 minutes, and the remaining steps are the same as those in comparative example 46.

[0633] Results: See Table 8

[0634] Comparative Example 50

[0635] In this comparative example, the cell nuclear precipitate is added with lysis solution, mixed by pipetting and allowed to stand on ice for 10 minutes, and the remaining steps are the same as those in comparative example 46.

[0636] Results: See Table 8

[0637] Comparative Example 51

[0638] In this comparative example, the cell nuclear precipitate is added with the lysis solution, mixed by pipetting and allowed to stand on ice for 15 minutes, and the remaining steps are the same as those in comparative example 46.

[0639] Results: See Table 8

[0640] Comparative Example 52

[0641] Referring to "Plant-Derived Exosomal MicroRNAs Shape the Gu Microbiota", this comparative example uses ultracentrifugation to prepare exosomes and sucrose gradient centrifugation for purification.

[0642] 1) Prepare reagents

[0643] Before preparing the samples, prepare the enzymatic solution, PBS buffer, and gradient sucrose solution, and pre-cool the prepared solutions on ice.

[0644] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0645] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0646] Gradient sucrose solution: 8, 30, 45 and 60% sucrose, 20 mM Tris-HCl pH 7.4.

[0647] 2) Sample preparation

[0648] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0649] 3) Sample processing

[0650] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 35HZ for 60 seconds and then quickly place them on ice.

[0651] 4) Enzymatic hydrolysis

[0652] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0653] 5) Exosome nuclear isolation

[0654] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 6 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0655] 6) Exosome extraction

[0656] a) Extraction: 3000g, 20 min, 6000g, 40 min, 10000g, 60 min at 4°C, collect the supernatant after each centrifugation and discard the precipitate; 150000g, 40 min at 4°C to precipitate the exosomes, and resuspend in 1 ml PBS buffer.

[0657] b) Purification: The crude exosome suspension was transferred to a gradient solution of 20 mM Tris-HCl, pH = 7.2, containing 8, 30, 45, and 60% sucrose, and centrifuged at 150,000 g for 90 min at 4°C. The band between 30% and 45% was collected and diluted with 1x PBS.

[0658] 7) Microscopic examination

[0659] Exosomes: The particle size and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0660] 8) Exosome RNA extraction

[0661] The exosome RNA was extracted using the Trizol method. The specific steps are as follows (note that the whole process was performed on ice and centrifugation was performed at 4°C):

[0662] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0663] b) Add 200 μl of chloroform, shake to mix, place on ice for 5 min, and centrifuge at 13,000 rpm for 15 min;

[0664] c) Carefully pipette the first layer of supernatant into a new RNase free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0665] d) Discard the supernatant and keep the precipitate, add 1 ml of 75% ethanol, shake and mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate.

[0666] e) Air-dry for 1 minute, add 10ul DEPC water, flick the tube after adding, and centrifuge briefly in a microcentrifuge.

[0667] f) RNA quality test was performed using Agilent 4150.

[0668] Results: See Table 9

[0669] Comparative Example 53

[0670] Referring to "A cost-effective polyethylene glycol-based method for the isolation of functional edible nanoparticles from ginger rhizomes", this comparative example uses the PEG precipitation method to prepare exosomes, and sucrose gradient centrifugation is used for purification.

[0671] 1) Prepare reagents

[0672] Before preparing the samples, prepare the enzymatic solution, PBS buffer, and gradient sucrose solution, and pre-cool the prepared solutions on ice.

[0673] The enzymatic solution formula is: 1.5% pectinase Y23, 2% hemicellulase, 1% snail enzyme, 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0674] PBS buffer: Use 1x PBS buffer purchased from Thermo Fisher directly.

[0675] Gradient sucrose solution: 8, 30, 45 and 60% sucrose, 20 mM Tris-HCl pH 7.4.

[0676] 2) Sample preparation

[0677] Select 6 newly grown young leaves of Arabidopsis thaliana that have grown for 20 days, cut them with scissors, and put them into a 2 ml centrifuge tube.

[0678] 3) Sample processing

[0679] Add two 3mm steel beads to the centrifuge tube, load them into the semiconductor plate of the tissue grinder and put them into liquid nitrogen for freezing for 60 seconds. Set the tissue grinder in advance to 35HZ for 60 seconds and then quickly place them on ice.

[0680] 4) Enzymatic hydrolysis

[0681] Add 1.5 ml of enzymatic solution to the centrifuge tube, place it on a constant temperature mixer, and digest for 10 minutes at 37°C and 200 RPM in the dark.

[0682] 5) Exosome nuclear isolation

[0683] Use pre-cooled PBS buffer to transfer the enzymatic hydrolysate obtained in step 4) to a new centrifuge tube and balance to 6 ml. Centrifuge at 4°C, 1000g for 10 min. Carefully pipette the supernatant into a new 15 ml centrifuge tube. The supernatant is used to extract exosomes, and the precipitate is used to prepare cell nuclei.

[0684] 6) Exosome extraction

[0685] a) Extraction: 4°C, 3000g, 20min, 6000g, 40min, 10000g, 60min, collect the supernatant after each centrifugation and discard the precipitate; add PEG6000 to the supernatant to a final concentration of 10%, mix well and incubate at 4°C overnight, centrifuge at 8000g for 30min at 4°C, discard the supernatant as much as possible, resuspend the precipitate with 1ml PBS to obtain a crude exosome suspension.

[0686] b) Purification: The crude exosome suspension was transferred to a gradient solution of 20 mM Tris-HCl, pH = 7.2, containing 8, 30, 45, and 60% sucrose, and centrifuged at 150,000 g for 90 min at 4°C. The band between 30% and 45% was collected and diluted with 1x PBS.

[0687] 7) Microscopic examination

[0688] Exosomes: The particle size and morphology were detected using a transmission electron microscope (Hitachi, HT-7700) and a particle size analyzer (NanoFCM, N30E).

[0689] 8) Exosome RNA extraction

[0690] The exosome RNA was extracted using the Trizol method. The specific steps are as follows (note that the whole process was performed on ice and centrifugation was performed at 4°C):

[0691] a) Add 1 ml of Trizol lysis buffer to the exosome solution, shake and mix for 1 min using an oscillator, and place on ice for 5 min;

[0692] b) Add 200 μl of chloroform, shake to mix, place on ice for 5 min, and centrifuge at 13,000 rpm for 15 min;

[0693] c) Carefully pipette the first layer of supernatant into a new RNase-free centrifuge tube (be careful not to suck up the precipitate or the second layer of solution), add an equal volume of isopropanol, shake to mix, let stand at -20°C for 30 minutes, and centrifuge at 13,000 rpm for 15 minutes;

[0694] d) Discard the supernatant and keep the precipitate, add 1 ml of 75% ethanol, shake and mix, incubate at 13000 rpm, 4°C, 5 min, discard the supernatant and keep the precipitate. Centrifuge briefly and carefully aspirate the liquid, being careful not to aspirate the precipitate.

[0695] e) Air-dry for 1 minute, add 10ul DEPC water, flick the tube, and centrifuge briefly in a microcentrifuge.

[0696] f) RNA quality test was performed using Agilent 4150.

[0697] Results: See Table 9

[0698] Comparative Example 54

[0699] This comparative example uses Qiagen's exoEasy Maxi Kit developed based on the membrane binding method. The exosome extraction and purification steps are carried out according to the instructions, and the remaining steps are the same as those of comparative example 52. (Because its principle can adsorb membrane structures indiscriminately, before the exosome extraction, differential centrifugation at 3000g for 10min, 10000g for 20min, and 16000g for 30min at 4°C is performed to remove debris and avoid contamination of other membrane structures)

[0700] Results: See Table 9

[0701] Comparative Example 55

[0702] This comparative example uses umibio developed based on the precipitation principle. UR52121 Exosome Extraction and Purification Kit (Cell Supernatant) 20T, the exosome extraction steps were carried out according to the instructions, and the remaining steps were the same as those in Comparative Example 52. (In the experiment, we found that PBS buffer was not suitable for this kit, so we used buffer A, which is closer to the cell environment, for exosome extraction)

[0703] Buffer A composition formula: 2mM MES, 5mM KCl, 125mM CaCl 2 , 154mM NaCl.

[0704] Results: See Table 9

[0705] Comparative Example 56

[0706] This comparative example uses Norgen's Cell Culture Media Exosome Purification Mini Kit developed based on silicon carbide technology. The exosome extraction step is carried out according to the instructions, and the remaining steps are the same as comparative example 52.

[0707] Results: See Table 9

[0708] Comparative Example 57

[0709] When using the Trizol method to precipitate RNA for exosome RNA extraction, 1 / 10 volume of 3M sodium acetate, 2.5 volumes of anhydrous ethanol and 20ul of nucleic acid precipitation agent (Solabo / SA1020) were used to precipitate RNA, and the remaining steps were consistent with Comparative Example 54.

[0710] Results: See Table 10

[0711] Comparative Example 58

[0712] Exosome RNA was extracted using Qiagen's miRNeasy Serum / Plasma Kit, and the specific steps were carried out according to the instructions. The remaining steps were consistent with Comparative Example 54.

[0713] Results: See Table 10

[0714] Comparative Example 59

[0715] Exosome RNA was extracted using SBI's ExoQuick Exosome RNA Column Purification Kit, the exosome RNA extraction step was carried out according to the instructions, and the remaining steps were consistent with Comparative Example 54.

[0716] Results: See Table 10

[0717] Comparative Example 60

[0718] Exosome RNA was extracted using Norgen's Exosomal RNA Isolation Kit, and the exosome RNA extraction steps were carried out according to the instructions, and the remaining steps were consistent with Comparative Example 54.

[0719] Results: See Table 10

[0720] The comparative examples mentioned in the present invention are as follows:

[0721] The establishment of the Arabidopsis leaf tissue exosome + single cell nucleus sequencing process first considers how to combine the two processes, whether they can be combined, and at which step the combination point should be set. Through the analysis of the technical routes of the two processes, it was found that the preparation of exosomes is to use the supernatant for subsequent extraction steps after centrifugation of tissue fragmentation, while the preparation of nuclei is to use the precipitate for subsequent extraction steps after centrifugation of tissue fragmentation. This is due to the great difference in sedimentation coefficients in the same medium caused by the great structural size difference between exosomes and nuclei. After tissue fragmentation and centrifugation, using the supernatant to prepare exosomes and using the precipitate to prepare nuclei is theoretically a feasible combination point. The published plant nucleus and exosome preparation processes have all used the homogenization method for tissue fragmentation. After tissue fragmentation, the nucleus preparation process will first use the lysis buffer to lyse the cells and then centrifuge. The exosome preparation process does not require cell lysis and directly uses PBS or other buffers close to the cell growth environment for centrifugation. According to this difference, the present invention firstly carried out a preliminary feasibility verification, that is, in Comparative Example 1, the cell lysis solution was used for homogenization and centrifugation, and the supernatant was used to prepare exosomes and the precipitate was used to prepare the nucleus; in Comparative Example 2, the PBS buffer without lysis components was used for homogenization and centrifugation, and the supernatant was used to prepare exosomes, and the precipitate was added with lysis solution to prepare the nucleus. The results showed that in Comparative Example 1, the number of nuclei was 3.67 million and the total amount of exosome RNA was 26.6 ng, and in Comparative Example 2, the number of nuclei was 3.54 million and the total amount of exosome RNA was 129 ng. In both cases, the nucleus and exosomes can be prepared simultaneously. The number of exosome RNA obtained by lysis first and then centrifugation is very small, which is far less than the number of exosome RNA obtained by centrifugation with a buffer without lysis components. It is proved that it is feasible to prepare exosomes and nuclei simultaneously in the same Arabidopsis tissue; but the lysis buffer will affect the membrane structure of the vesicles while destroying the cell membrane, resulting in a large amount of degradation of the vesicles. In summary, the present invention determines the basic technical route of first separating the supernatant and the precipitate with a PBS buffer without lysis components, and then using the supernatant to prepare exosomes and the precipitate to prepare the nucleus.

[0722] Table 1 Comparative Examples 1 to 2 Results

[0723]

[0724]

[0725] According to the results of comparative examples 1 to 2, the technical route of first centrifuging the supernatant and the precipitate with a buffer solution containing no lysis components after tissue crushing and then lysing the cells to prepare exosomes and nuclei separately was determined. The first step of this technical route is tissue crushing. The present invention first tried several commonly used tissue crushing methods and conducted comparative experiments 3 to 5. The number of nuclei and exosome RNA obtained by scissor crushing in comparative example 3 was very small, almost none. It may be that the scissor crushing method is difficult to destroy the cell wall components of plant tissues, and only a small amount of nuclei and exosomes are released from the incision; the tissue grinder in comparative example 5 can obtain more nuclei and exosome RNA and less impurities than manual grinding in comparative example 4. Further considering that manual grinding is too laborious, time-consuming, and multiple samples cannot be crushed at the same time, the present invention chooses to use a tissue grinder for tissue crushing. Plant tissues are often crushed by liquid nitrogen grinding, which can be applied to a variety of leaves, stems, roots, skins, seeds, etc. that are not easy to homogenize. It has a strong crushing ability and avoids RNA degradation during the homogenization process. On the basis of using a tissue grinder, the present invention adds the element of liquid nitrogen, puts the sample into the semiconductor sample plate of the tissue grinder and soaks it in liquid nitrogen for more than 60 seconds, sets the instrument frequency and running time in advance, so that it can be quickly broken in an ultra-low temperature environment similar to liquid nitrogen for a short time. The liquid nitrogen grinding of Comparative Example 6 has 380,000 more total amount of homogenized cell nuclei in the buffer solution of Comparative Example 5, 17 ng more exosome RNA and less impurities. The comparison of the above-mentioned Comparative Examples 3 to 6 verifies that the present invention chooses to use liquid nitrogen grinding of a tissue grinder as a tissue crushing method.

[0726] Table 2 Comparative Examples 3 to 6 Results

[0727]

[0728] Through the verification of comparative examples 3 to 6, the present invention chooses to use liquid nitrogen grinding with a tissue grinder as a tissue crushing method, but the frequency of the tissue grinder is too high and the time is too long, which will break the centrifuge tube wall and may also damage the cell nucleus structure. The frequency is too low and the time is too short to achieve the effect of fully crushing the tissue. Therefore, in the next step, we use several different frequencies for comparative verification. The results of comparative examples 6 to 11 show that the total RNA of the nucleus and exosomes obtained under 30HZ and 40HZ conditions is the largest and the fragments are the least; based on this result, we used different grinding times under 30HZ conditions, and the results showed that the total RNA of the nucleus and exosomes obtained under 60s conditions is the largest and the fragments and impurities are the least. It shows that the oscillation frequency of the tissue grinder is preferably 30HZ to 40HZ, and the time is preferably 60s.

[0729] Table 3 Comparative Examples 7 to 11 Results

[0730]

[0731] Plant tissues are limited by cell walls. At present, all methods for preparing cell nuclei and exosomes rely on mechanical force to physically break the cell walls while breaking the tissues. However, mechanical breaking alone cannot completely and fully destroy the cell walls. During the microscopic examination of the cell nuclei, we observed a large number of multi-cell blocks connected by unbroken cell walls. The exosome and nuclear RNA information in these multi-cell blocks cannot be extracted. Sufficient RNA information can provide more basis for building a complete communication network in the later stage. Therefore, on the basis of mechanical breaking, the present invention innovatively adds an enzymatic hydrolysis step to further digest the unbroken cell wall structure. First, we refer to "Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis" and use 1.5% cellulase R10, 0.4% macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, 10mM CaCl 2 , 0.1% BSA, incubated in the dark at 37°C for 3 hours, the results showed that the number of cell nuclei was 3.2 million, the proportion of clumps was 15%, the proportion of fragments was 61%, the number of exosome RNA was 52ng, and the RNA was severely degraded; referring to "Global Dynamic Molecular Profiling of Stomatal Lineage Cell Development by Single-Cell RNA Sequencing", 1.5% cellulase RS, 0.03% pectinase Y23, 5mM MES, 0.5mM CaCl 2 , 0.5 mM MgCl 2 , 0.25% BSA incubated in the dark at 37°C for 4 hours. The results showed that the number of nuclei was 2.4 million, the proportion of agglomerates was 17%, the proportion of fragments was 62%, the number of exosome RNA was 40ng, and the RNA was severely degraded; referring to "Single-Cell Resolution of Lineage Trajectories in the Arabidopsis Stomatal Lineage and Developing Leaf", 1.25% cellulase R10, 0.3% maceration enzyme R10, 0.4M mannitol, 20mM MES, 20mM KCl, 10mM CaCl 2, 1MTris-HClpH7.537℃ incubated in the dark for 2 hours, the results showed that the number of nuclei was 3.4 million, the proportion of clusters was 13%, the proportion of fragments was 60%, the number of exosome RNA was 73ng, and the RNA was severely degraded. Comparative Examples 12 to 14 used the above three methods of Arabidopsis tissue enzymatic hydrolysis to obtain the number of nuclei and exosome RNA, which did not increase but decreased, and there were problems such as low nuclear activity and RNA degradation, indicating that the existing methods suitable for Arabidopsis leaf enzymatic hydrolysis are not suitable for the present invention.

[0732] First of all, in terms of experimental purpose, the existing enzymatic hydrolysis method of Arabidopsis is mainly to prepare a sufficient number of protoplasts with physiological activity, while the present invention is to digest the cell wall to separate the cells and release the exosomes between the cells and the nucleus inside the cell. Secondly, in terms of technical methods, the existing enzymatic hydrolysis method uses a blade to cut into thin strips, while the present invention uses a liquid nitrogen tissue grinder to grind. After liquid nitrogen is broken, a large amount of active substances will be released. The enzymatic hydrolysis environment is also the best working environment for these active substances. After liquid nitrogen is broken, a large amount of exosomes and nuclei that have been released will be exposed in the enzymatic hydrolysis solution. The enzymatic hydrolysis time is as long as 2 to 4 hours. In this process, the exposed exosomes and nuclei are very easy to be degraded. The enzymatic hydrolysis time used to extract protoplasts is not suitable in the present invention. Therefore, the present invention shortens the enzymatic hydrolysis time and adds β-mercaptoethanol to slow down RNA degradation. Through a series of verifications of comparative examples 15 to 19, the present invention explores the enzymatic hydrolysis time and β-mercaptoethanol concentration suitable for the present invention, and unexpectedly finds that adding β-mercaptoethanol to the enzymatic hydrolysis solution of the present invention can not only inhibit RNA degradation but also inhibit the effect of active substances on the nucleus and exosomes during the enzymatic hydrolysis process. In Comparative Example 15, 6 mM β-mercaptoethanol was added after incubation for more than 30 minutes, and the number of cell nuclei and exosomes decreased compared with Comparative Example 10, and RNA degradation was caused; in Comparative Example 16, 6 mM β-mercaptoethanol was added after incubation for 20 minutes, and the number of cell nuclei and exosomes increased significantly compared with Comparative Example 10, but RNA was still degraded; in Comparative Example 17, 2 mM β-mercaptoethanol was added and incubated for 10 minutes, and the cell nuclei increased by 170,000 exosome RNA, the fragments increased by 7 ng, and the RNA was degraded by 2% compared with Comparative Example 10; in Comparative Example 18, 4 mM β-mercaptoethanol was added and incubated for 10 minutes, and the cell nuclei increased by 240,000 exosome RNA, the fragments increased by 11 ng, and the RNA quality was good. In Comparative Example 19, 2 mM β-mercaptoethanol was added and incubated for 5 minutes, and the cell nuclei increased by 120,000 exosome RNA, the fragments increased by 2 ng, and the RNA quality was good. The enzymatic hydrolysis time is selected to be 5 to 10 minutes and the concentration of β-mercaptoethanol is selected to be 4 mM. The RNA quality is good. Considering that it takes time for the enzyme to bind to the substrate, the enzymatic hydrolysis time is preferably 10 minutes. In summary, in order to ensure that qualified cell nuclei and exosomes are prepared simultaneously in the same tissue, the enzymatic hydrolysis time is strictly controlled to 10 minutes in the present invention, and 4 mM β-mercaptoethanol is added to inhibit RNA degradation.

[0733] Table 4 Comparative Examples 12 to 19 Results

[0734]

[0735]

[0736] The cell wall structure of young Arabidopsis leaves grown in the laboratory is relatively simple. Using a combination of basic cellulase and cleavage enzyme or pectinase, a sufficient number of protoplasts can be released after 2 to 4 hours of enzymatic hydrolysis. After shortening the enzymatic hydrolysis time, the effect is minimal, and when applied to the technical field of the present invention, it cannot solve the technical problem that the present invention needs to solve. Therefore, the present invention first attempts to explore the method of changing the enzyme concentration and replacing it with a stronger cellulase on the basis of Comparative Example 18. First, it attempts to solve the technical problem to be solved by the present invention by increasing the enzyme concentration to improve the enzymatic effect. The results show that the number of nuclei and exosome RNA prepared after 10 minutes of enzymatic hydrolysis with 2% cellulase R10 and 0.6% cleavage enzyme R10 in Comparative Example 20 has no obvious increase compared with Comparative Example 18, indicating that increasing the enzyme concentration alone cannot work in such a short time; the xylanase activity contained in cellulase RS is about 3 times higher than that of cellulase R-10, and is more extensive than cellulase R-10. It can obtain protoplasts in a very short time and degrade cell walls of more plant species. Therefore, Comparative Example 21 uses 1.5% cellulase RS instead of cellulase R10. The results show that the number of nuclei and exosome RNA has almost no increase compared with Comparative Example 18, and there is no obvious effect of using cellulase RS instead of cellulase R10; the results of Comparative Examples 20 to 21 show that the cell wall cannot be digested in a short time by the original combination of the two digestive enzymes. Therefore, the present invention attempts to add a new type of enzyme on the basis of Comparative Example 18 to compare and verify the formula of the enzymatic hydrolysate.It is speculated that the cells of young leaves of Arabidopsis thaliana are mainly connected by the middle lamella, which is mainly composed of pectin. Pectin is also the matrix of cellulose and hemicellulose fibers embedded in the plant cell wall. If cellulase and hemicellulase want to react with the substrate as soon as possible and separate the cells as soon as possible, it may be necessary to digest the pectin first. In Comparative Example 22, 1% pectinase Y23 was added. The results showed that the number of cell nuclei increased by 400,000, the fragments decreased by 6%, the number of exosome RNA increased by 22ng, and the agglomeration did not change compared with Comparative Example 18; hemicellulose is one of the main components of the primary wall. In Comparative Example 23, 2% hemicellulase was added, and the number of cell nuclei increased by 230,000, the fragments decreased by 2%, the number of exosome RNA increased by 9ng, and the agglomeration did not change compared with Comparative Example 18; snail enzyme is often used to digest yeast cell walls. It is a composite enzyme with multiple biological activities and has a strong enzymatic effect. The more complex plant cell wall structure can improve the enzymatic hydrolysis efficiency by adding a small amount of snail enzyme. Compared with Comparative Example 18, the addition of 1% snail enzyme in Comparative Example 24 increased the number of cell nuclei by 100,000, reduced the proportion of fragments by 2%, increased the number of exosome RNA by 7 ng, and had no change in agglomeration; Compared with Comparative Example 18, the addition of 1% crashase in Comparative Example 25 increased the number of cell nuclei by 40,000, reduced the proportion of fragments by 1%, increased the number of exosome RNA by 2 ng, and had no change in agglomeration, with no obvious effect; Through Comparative Examples 22 to 25, the present invention found that the addition of pectinase, hemicellulase, and snail enzyme would improve the enzymatic hydrolysis efficiency and increase the number of cell nuclei and exosome RNA obtained, but a large number of undigested small pieces of tissue could still be observed during microscopy. The present invention considers further improving the enzymatic hydrolysis efficiency by adding multiple types of enzymes at the same time on the basis of these three enzymes. In Comparative Example 26, the addition of 1% pectinase Y23 + 2% hemicellulase increased the number of cell nuclei by 550,000, the proportion of fragments decreased by 8%, the number of exosome RNA increased by 35 ng, and the agglomeration did not change compared with Comparative Example 18; in Comparative Example 27, the addition of 1% pectinase Y23 + 1% snail enzyme increased the number of cell nuclei by 400,000, the proportion of fragments decreased by 6%, the number of exosome RNA increased by 24 ng, and the agglomeration did not change compared with Comparative Example 18; in Comparative Example 28, the addition of 2% hemicellulase + 1% snail enzyme increased the number of cell nuclei by 160,000, the proportion of fragments decreased by 3%, the number of exosome RNA increased by 15 ng, and the agglomeration did not change compared with Comparative Example 18; in Comparative Example 29, the addition of 1% pectinase Y23 + 2% hemicellulase + 1% snail enzyme increased the number of cell nuclei by 640,000, the proportion of fragments decreased by 12%, the number of exosome RNA increased by 52 ng, and the agglomeration did not change. In summary, the components of the enzymatic hydrolyzate of the present invention are pectinase Y23, hemicellulase, snail enzyme, cellulase R10, macerate R10, 20mM MES (pH 5.7), 0.4M mannitol, 20mM KCl, and 10mM CaCl. 2 , 0.1% BSA, 4mM β-mercaptoethanol.

[0737] Table 5 Comparative Examples 20 to 29 Results

[0738]

[0739]

[0740] After confirming the enzymatic components, the present invention further explored the concentration range of pectinase, hemicellulase, snail enzyme, cellulase, and macerate in the present invention. By comparing the verification of Examples 30 to 32, it was determined that the number of cell nuclei obtained in the concentration range of 1% to 1.5%, 1.5% to 2%, 0.5% to 1%, 1.5% to 2%, and 0.4% to 0.6% for pectinase, hemicellulase, snail enzyme, cellulase, and macerate was 5.28 to 5.3 million, the proportion of fragments was 72%, and the number of exosome RNA was 234 to 235 ng, which was the best effect.

[0741] Table 6 Comparative Examples 30 to 32 Results

[0742]

[0743] By comparing Examples 12 to 32, the present invention determines the enzymolysis solution formula and enzymolysis time that are more suitable for this technical solution. The next step is to explore and confirm the cell nucleus preparation process. Currently, the single-cell nuclear sequencing articles published mostly use fluorescence sorting technology (FACS) to screen the cell nuclei. FACS technology is expensive, time-consuming and will affect the subsequent library construction, but without using fluorescence sorting technology (FACS), there are many chloroplast fragments in the plant cell nucleus suspension, and the RNA in the chloroplast will interfere with the subsequent single-cell nuclear sequencing results. Comparative Examples 33 to 34 refer to "Single-nucleus full-length RNA profiling in plants incorporates isoform information to facilitate cell type identification" and "Single-nuclei RNA-sequencing of plant tissues". The number of cell nuclei obtained under the condition of not using FACS technology can meet the requirements of the machine, but the fragmentation rate is as high as 43% and 56%, respectively, which is far from the requirements of the machine.

[0744] The cell nucleus preparation process mainly includes: lysis, filtration, washing and purification, resuspension and microscopic examination. The cell nucleus is prepared by using the "existing technology" "Single-nucleus full-length RNA profiling in plants incorporates isoform information to facilitate cell type identification" and "Single-nuclei RNA-sequencing of plant tissues". Although the number of cell nuclei meets the requirements of single-cell sequencing, the high fragment impurities have a high risk of failure for subsequent sequencing on the machine. In order to reduce chloroplast impurities, the present invention first tried several cell nucleus purification methods. In Comparative Example 35, the number of cells was less than 2.35 million and the fragmentation rate was 32% using multiple filtration and washing methods; in Comparative Example 36, the number of cell nuclei was 1.49 million and the fragmentation rate was 24% using differential centrifugation to reduce centrifugal force; in Comparative Example 37, Trion X-100 was added to the washing solution, the number of cell nuclei was 3.23 million and the fragmentation rate was 46%; in Comparative Example 38, the number of cell nuclei was 2.8 million and the fragmentation rate was 38% using sucrose gradient centrifugation; the high fragmentation rate did not meet the requirements of the single-cell sequencing experiment. Comparative Example 39 used multiple filtration + differential centrifugation to reduce centrifugal force, resulting in 1.04 million cell nuclei and 11% fragmentation rate, which did not meet the requirements for the machine; Comparative Example 40 used multiple filtration + differential centrifugation + adding TrionX-100 to the washing solution, resulting in 800,000 cell nuclei and 7% fragmentation rate, which was low enough to meet the requirements of the single-cell nucleus sequencing experiment.

[0745] Table 7 Comparative Examples 33 to 40 Results

[0746]

[0747]

[0748] Through a series of comparative examples 35 to 40, the purified cell nucleus suspension is basically free of chloroplast impurities, but the number of cell nuclei is also greatly reduced. Considering that the plant cell lysate and lysis steps currently used in the present invention mainly refer to the cell nucleus preparation method in the article "Single-nucleus full-length RNA profiling in plants incorporates isoform information to facilitate cell type identification", the prepared cell nucleus suspension is directly used for fluorescence sorting to avoid the loss during the purification process and requires less number of cell nuclei. The entire experimental process and reagent components have room for optimization to meet the requirements of the number of purified cell nuclei. Therefore, the present invention further explores and optimizes the lysis step. The surfactant in the lysis solution not only lyses the cell membrane, but also separates a large number of high-concentration cells without agglomeration. Direct filtration without sufficient reaction is very likely to filter out a large number of unlysed cells. The low concentration of 0.2% TrionX-100 has a weak lysis strength. The original method is for the roots of Arabidopsis thaliana. The number of cell nuclei in young leaf tissue of the same weight is much higher than that in root tissue. Comparative Example 41 increases the concentration of TrionX-100. Using 1% TrionX-100, the number of cell nuclei obtained was 940,000 and the agglomeration rate was 12%. Although the high concentration of TrionX-100 has a high lysis intensity, it also puts pressure on the cell nucleus. Most detergents destroy the cell membrane non-specifically, and the effect on the nuclear membrane is relatively weak, but too high a concentration will also destroy the nuclear membrane structure. Therefore, the present invention still uses a low concentration of TrionX-100 and adds a new, milder lysis component that has less impact on the nuclear membrane, so as to avoid damage to the cell nucleus while lysing the cell membrane. NP40 is one of the commonly used lysate components, and has a weak effect on the nuclear membrane. In Comparative Example 42, adding 1% NP40 to the lysate resulted in 1.12 million nuclei and a 7% agglomeration rate. Digital saponin can specifically lyse the cell membrane and has a very low effect on the nuclear membrane. In Comparative Example 43, adding 1% digitonin to the lysate resulted in 900,000 nuclei and a 4% agglomeration rate. From the results of Comparative Examples 42 to 43, it seems that the lysis effect of NP40 is stronger than that of digitonin, but the effect of digitonin is milder than that of NP40. Adding digitonin has almost no effect on the state of the nucleus. Therefore, it is attempted to add digitonin on the basis of adding NP40, which can ensure the lysis intensity without excessively damaging the nuclear membrane. The results of Comparative Example 44 show that the number of nuclei obtained by adding NP40 and digitonin to the lysate at the same time is 1.2 million and a 7% agglomeration rate, proving that adding digitonin can improve the lysis efficiency and has a very low effect on the nuclear membrane, but the lysis effect is also relatively limited.By comparing Examples 41 to 44, the present invention confirms that the lysate components used are: NP40, TritonX-100, digitonin, 0.4Msucrose, 10mMTris-HClpH8.0, 10mMMgCl2, 1mMDTT, 1×protease inhibitor, 0.4U / μlRNaseinhibitor. If the concentration of the lysate is too low, the lysis effect is not good. If the concentration is too high, the situation of Comparative Example 41 will occur, and the nuclear membrane will be destroyed to cause the cell nucleus to rupture or agglomerate. The present invention further conducts a series of explorations on the working concentrations of TrionX-100, NP40 and digitonin. The results of Comparative Examples 45 to 48 show that the number of cell nuclei obtained when the working concentrations of TrionX-100, NP40 and digitonin are between 0.2% to 0.4%, 0.8% to 1.2%, and 0.8% to 1.2% respectively is 1.18 to 1.23 million, and the agglomeration rate is 6% to 7%, which is the best effect. The lysate of the present invention has the following formula: 0.8%-1.2% NP40, 0.2%-0.4% TritonX-100, 0.8%-1.2% digitonin, 0.4Msucrose, 10mM Tris-HCl pH 8.0, and 10mM MgCl. 2 , 1mMDTT, 1×proteaseinhibitor, 0.4U / μlRNaseinhibitor.

[0749] The lysis step of the present invention mainly refers to the article "Single-nucleus full-length RNA profiling in plants incorporates isoform information to facilitate cell type identification". After adding the lysis solution, the filtration and centrifugation are directly performed. The lysis solution and the tissue are not given time to fully react, and a large number of cells that have not reacted with the lysis solution may be directly filtered. The present invention adds a step of mixing and standing on ice after adding the lysis solution. Comparative Example 49 adds lysis buffer and mixes and stands on ice for 5 minutes to obtain 1.42 million cell nuclei and a 6% agglomeration rate; Comparative Example 50 adds lysis buffer and mixes and stands on ice for 10 minutes to obtain 1.45 million cell nuclei and a 7% agglomeration rate; Comparative Example 51 adds lysis buffer and mixes and stands on ice for 15 minutes to obtain 1.43 million cell nuclei and a 12% agglomeration rate. Too long a lysis time affects the quality of the cell nucleus. The lysis time should be controlled within 5 to 10 minutes.

[0750] In summary, the cell nucleus preparation process includes a lysis solution formula of: 0.8% to 1.2% NP40, 0.2% to 0.4% TritonX-100, 0.8% to 1.2% digitonin, 0.4Msucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl 2 , 1mM DTT, 1× protease inhibitor, 0.4U / μl RNase inhibitor; lysis time is 5-10min; purification method is: multiple filtration + differential centrifugation + adding 0.1% TrionX-100 in the washing solution; the number of cell nuclei suspension obtained is 1.42 million to 1.45 million, the clumping rate is 6% to 7%, and the fragments account for 5% to 6%, which meets the requirements for the machine.

[0751] Table 8 Comparative Examples 41 to 50 Results

[0752]

[0753]

[0754] The formal research time of plant exosomes is relatively short, and no systematic rules and systems have been formed. Many preparation methods are still in the trial stage, and there are no exosome extraction kits for plant samples on the market. In this case, the present invention compares two methods used for plant exosome extraction and purification: ultracentrifugation + sucrose gradient centrifugation and PEG precipitation + sucrose gradient centrifugation. The amount of total exosome RNA obtained by PEG precipitation + sucrose gradient centrifugation is much lower than that of ultracentrifugation + sucrose gradient centrifugation, and there is no large difference in purity. The precipitation method commonly used to extract animal-derived exosomes may not be suitable for preparing plant exosomes. Considering that the operation time of ultracentrifugation + sucrose gradient centrifugation is too long, the steps are complicated and the purity is not high, the present invention further selected three kits based on different principles on the market (due to the great possibility of differences between the surface proteins of plant exosomes and animal exosomes, we gave up using kits based on the principle of surface protein immunity) to explore whether they can be applied to the extraction of plant exosomes. The results showed that Qiagen's exoEasy Maxi Kit and Umibio The total amount of exosome RNA prepared by UR52121 Exosome Extraction and Purification Kit (cell supernatant) 20T is the largest, which is higher than the traditional ultracentrifugation + sucrose purification method, but the RNA obtained by Qiagen's exoEasy Maxi Kit is purer, and the total amount of exosome RNA obtained by Norgen's Cell Culture Media ExosomePurification Mini Kit is even lower than that of ultracentrifugation. Umibio relies on precipitation method, and Norgen relies on SiC technology combined with the modification of exosome surface proteins. Compared with the other two, Qiagen's unique membrane binding method can bind to the membrane structure non-specifically. Whether it is plant exosomes or animal exosomes, its biological membrane simulation characteristics will not change, which greatly avoids the preference caused by sample specificity. After comparison, we chose Qiagen's exoEasy MaxiKit to prepare exosomes.

[0755] Table 9 Comparative Examples 52 to 56 Results

[0756]

[0757]

[0758] There is currently no recognized better method for extracting exosome RNA. The present invention compares several commonly used methods for extracting exosome RNA. The results show that adding a precipitation aid can improve the extraction efficiency of the Trizol method. The total RNA amount obtained is not significantly different from that of the kit, but the RNA purity obtained using the kit is higher. Based on downstream sequencing considerations, the present invention selected Norgen's Exosomal RNA Isolation Kit with higher purity.

[0759] Table 9 Comparative Examples 57 to 60 Results

[0760]

[0761] Through all the embodiments described above and not shown in the present invention, we determined the specific operating steps of the entire process, and used Arabidopsis tender green rosette leaves grown for 20 days and 40 days as samples for verification. The results showed that the total amount of prepared nuclei was 1.42 to 1.46 million, the agglomeration rate was 5% to 6%, the fragmentation rate was 4% to 6%, the total amount of exosome RNA was 314 to 322 ng, and the 260 / 2801.98 to 2.01. The present invention can simultaneously perform exosome small RNA sequencing and single cell nucleus sequencing in Arabidopsis leaf tissues at two growth periods, filling the current gap in plant tissues in exosome + single cell nucleus RNA sequencing and achieving a new technological breakthrough.

[0762] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

[0763] Table 11 Results of Examples 1 to 4

[0764]

[0765]

Claims

1. A method for simultaneously preparing exosomes and cell nuclei from Arabidopsis leaf tissue, characterized in that: The specific steps are as follows: 1) Prepare reagents: prepare enzymatic solution, lysis buffer, washing buffer, purification buffer, and PBS buffer respectively, and pre-cool the prepared solutions on ice in advance; the enzymatic solution formula is: 1%~1.5% pectinase Y23, 1.5%~2% hemicellulase, 0.5%~1% snail enzyme, 1.5%~2% cellulase R10, 0.4%~0.6% macerator R10, 20 mM MES at pH 5.7, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 0.1% BSA, 4 mM β-mercaptoethanol; The composition and formula of the lysis buffer are: 0.8%~1.2% NP40, 0.2%~0.4% Triton X-100, 0.8%~1.2% digitonin, 0.4 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor; 2) Sample processing: tissue crushing, enzymatic hydrolysis, diluting with PBS buffer and transferring to a centrifuge tube, centrifuging to obtain a supernatant rich in exosomes and a precipitate of cell nuclei; in the step 2), a tissue grinder is used to crush the plant tissue; after the tissue crushing, an enzymatic hydrolysis step is further included; the centrifugal force is 1000-3000g, and the time is 10min; before crushing, the sample and the semiconductor plate of the tissue grinder are immersed in liquid nitrogen for more than 60s to quickly crush them at ultra-low temperature; the frequency of the tissue grinder is 30~40HZ, and the time is 60s; the enzymatic hydrolysis time is 10min, and the temperature is 25~37℃; 3) Preparation of cell nucleus suspension: preparing and purifying the cell nuclei of the precipitate; the step 3) includes the following specific steps: 3-1) Lysis: Add lysis solution to the precipitate and let stand for 5-10 minutes; 3-2) Filtration: Dilute and filter with washing buffer, centrifuge, and discard the supernatant; 3-3) Purification and washing: add purification buffer and perform differential centrifugation; 3-4) Microscopic examination: Resuspend the cell nuclear pellet in 1 x PBS to obtain a single cell nuclear suspension for microscopic examination; 4) Exosome purification: extracting and purifying exosomes from the supernatant; There is no order restriction between the steps 3) and 4).

2. The method according to claim 1, characterized in that In the step 1), The composition and formula of the washing buffer are: 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1× protease inhibitor, 0.4U / ul RNase inhibitor; The composition and formula of the purification buffer are: 0.1% Triton X-100, 0.4U / ul RNase inhibitor, and PBS buffer.

3. The method according to claim 1, characterized in that The steps 3-2) and 3-3) include three filtrations, wherein the first filtration uses a 70um cell sieve, the second filtration uses a 30um filter column, and the third filtration uses a MACS sorting column; The step 3-3) includes three centrifugations, wherein the first centrifugation is 10 g and the precipitate is discarded and the supernatant is retained, the second centrifugation uses the supernatant of the first centrifugation 300 g and the supernatant is discarded and the precipitate is retained, and the third centrifugation uses purification buffer to resuspend 100 g and the supernatant is discarded and the precipitate is retained, wherein the first centrifugation time is 5 min, and the second and third centrifugation times are 10 min respectively.

4. The method according to claim 3, characterized in that The third filtration is performed after centrifugation in step 3-3).

5. The method according to claim 1, characterized in that: In step 4), Qiagen's exoEasy MaxiKit is used to purify exosomes.

6. Use of the method for simultaneously preparing Arabidopsis leaf tissue exosomes and cell nuclei as claimed in claim 1 in sequencing plant exosomes and single cell nuclei.

7. A kit, characterized in that: The kit is used to prepare tissue exosomes and cell nuclei in the same Arabidopsis leaf tissue and sequence them simultaneously; the kit includes an enzyme solution, a lysis buffer, a washing buffer, a purification buffer, and a PBS buffer; wherein, The composition and formula of the enzymatic hydrolysate are: 1% to 1.5% pectinase Y23, 1.5% to 2% hemicellulase, 0.5% to 1% snail enzyme, 1.5% to 2% cellulase R10, 0.4% to 0.6% macerate R10, 20 mM MES at pH 5.7, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 0.1% BSA, and 4 mM β-mercaptoethanol; The composition and formula of the lysis buffer are: 0.8%~1.2% NP40, 0.2%~0.4% Triton X-100, 0.8%~1.2% digitonin, 0.4 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 mM DTT, 1× protease inhibitor, 0.4 U / μl RNase inhibitor; The composition and formula of the washing buffer are: 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1× protease inhibitor, 0.4U / ul RNase inhibitor; The composition and formula of the purification buffer are: 0.1% Triton X-100, 0.4U / ul RNase inhibitor, and PBS buffer.

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