A reagent, kit and method for separating protoplasts of Populus alba L. guard cells

Abstract

The present invention belongs to the technical field of protoplast separation, and relates to a reagent, a kit and a method for separating populus alba guard cell protoplasts. The present invention provides a reagent for separating populus alba guard cell protoplasts, comprising enzyme solution I and enzyme solution II; the enzyme solution I comprises: 0.5% cellulase R-10, 0.02% macerozyme R-10, 0.25 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, 0.2% BSA and water; the enzyme solution II comprises: 1.5% cellulase R-10, 0.02% macerozyme R-10, 0.4 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, 0.2% BSA and water. The reagent of the present invention can achieve efficient separation of populus alba guard cell protoplasts, and the obtained protoplasts have high purity, integrity and large quantity.

Description

Technical Field

[0001] The present invention belongs to the technical field of protoplast separation, and particularly relates to a reagent, a kit and a method for separating guard cell protoplasts of Populus alba. Background Art

[0002] Poplar is a major afforestation tree species in China and plays an irreplaceable role in ecological environment construction, industrial production, house construction, furniture manufacturing, etc. In recent years, as a model tree species, the biotechnology of poplar has developed rapidly. Breeding new poplar varieties with strong resistance and fast growth rate through means such as cross-breeding and molecular biology breeding is the breeding goal of poplar. Among them, among various environmental stresses that limit plant growth, drought stress is the most common and serious. Studying the molecular mechanism of plants responding to drought stress, improving the quality of forest trees and enhancing the stress resistance of forest trees through biotechnology means have important biological significance for realizing the rapid development of forestry.

[0003] Stomata are composed of a pair of kidney-shaped guard cells and are distributed on the leaf surface. Stomata are the portals for gas exchange and water evaporation. Stomata can respond to a variety of external stimuli, especially playing an important role in responding to stress. When the plant is in a drought state, in the guard cells, ABA induces the production of H2O2. As an important second messenger, H2O2 activates the Ca 2+ channel, prompting the stomata to close. Guard cells play an important role in plants' response to drought stress. In order to exclude the interference of other cells, separating plant guard cells and obtaining guard cell protoplasts are effective methods for studying stomatal movement. Plant protoplasts refer to cells without cell walls. Guard cell protoplasts (GCPs) are important materials for studying the molecular mechanism of stomatal movement, membrane permeability and ion transport processes.

[0004] Currently, there are mature methods for separating guard cell protoplasts in the herbaceous model plant Arabidopsis thaliana, and there are also methods for separating guard cell protoplasts in other plants such as Vicia faba. However, the technology for separating guard cell protoplasts in the perennial tree species poplar has not been reported. Especially for Populus alba, Populus alba has the characteristic of brittle and soft leaves. Therefore, the conventional method of tearing leaf epidermis strips cannot obtain guard cells. In addition, the leaf epidermis microhairs on the back of Populus alba leaves are developed, covering the guard cells, which also makes it difficult to separate guard cells. Summary of the Invention

[0005] The purpose of the present invention is to provide a reagent, a kit and a method for separating guard cell protoplasts of Populus alba. The reagent of the present invention can achieve the efficient separation of guard cell protoplasts of Populus alba.

[0006] The present invention provides a reagent for separating protoplasts of Populus alba L. guard cells. The reagent includes Enzyme Solution I and Enzyme Solution II; Enzyme Solution I includes: cellulase R-10 with a mass percentage of 0.5%, macerozyme R-10 with a mass percentage of 0.02%, 0.25 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, BSA with a volume percentage of 0.2%, and water;

[0007] Enzyme Solution II includes: cellulase R-10 with a mass percentage of 1.5%, macerozyme R-10 with a mass percentage of 0.02%, 0.4 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, BSA with a volume percentage of 0.2%, and water.

[0008] Preferably, the reagent further includes a washing solution; the washing solution includes: 0.4 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, and water.

[0009] The present invention also provides a kit for separating protoplasts of Populus alba L. guard cells. The kit includes the reagent described in the above technical solution and a filtering material.

[0010] The present invention also provides a method for separating protoplasts of Populus alba L. guard cells based on the reagent described in the above technical solution or the kit described in the above technical solution, including the following steps:

[0011] 1) Mix Populus alba L. leaves with Enzyme Solution I for the first digestion, and perform the first filtration to obtain the leaf epidermis after the first digestion treatment;

[0012] 2) Wash, incubate, and perform the second filtration on the leaf epidermis after the first digestion treatment to obtain the washed leaf epidermis;

[0013] 3) Mix the washed leaf epidermis with Enzyme Solution II for the second digestion, perform the third filtration, take the filtrate, and centrifuge to obtain the precipitate to obtain the guard cell protoplasts.

[0014] Preferably, the Populus alba L. leaves are processed into leaf strips before being mixed with Enzyme Solution I.

[0015] Preferably, the time for the first digestion is 0.5 - 1.5 h; the time for the second digestion is 1 - 3 h.

[0016] Preferably, the first digestion and the second digestion are respectively subjected to horizontal shaking, and the speeds of the horizontal shaking are respectively 40 times / min.

[0017] Preferably, the first filtration and the second filtration are through a 200-mesh sieve; the third filtration is through a 300 - 800-mesh sieve.

[0018] Preferably, the incubation time is 30 min.

[0019] Preferably, the centrifugation conditions are centrifugation at 100 g for 5 min.

[0020] The present invention provides a reagent for separating Populus alba guard cell protoplasts. The reagent of the present invention can achieve efficient separation of Populus alba guard cell protoplasts, and the obtained protoplasts have high purity, integrity (high viability) and large quantity. The test results show that compared with the ordinary enzyme solution, the reagent of the present invention can obtain guard cell protoplasts with higher purity and the protoplasts are intact. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and for those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 Analysis diagram of the digestion of leaf epidermis by different enzyme solutions I provided by the present invention;

[0023] Figure 2 Surface photo of leaf epidermis strips after incubation with the cleaning solution provided by the present invention;

[0024] Figure 3 Surface result diagram of leaf epidermis strips after digestion with enzyme solution II provided by the present invention;

[0025] Figure 4 Guard cell protoplast diagram provided by the present invention;

[0026] Figure 5 Specific gene expression result diagram of guard cell protoplasts provided by the present invention;

[0027] Figure 6 Expression situation diagram of GUS gene driven by Pascap1 gene promoter provided by the present invention;

[0028] Figure 7 Viability and specificity detection diagram of guard cell protoplasts obtained at different time points after digestion with enzyme solution II provided by the present invention;

[0029] Figure 8 Flow chart for preparing Populus alba guard cell protoplasts provided by the present invention. Detailed Description of the Invention

[0030] The present invention provides a reagent for separating protoplasts of Populus alba L. guard cells, and the reagent includes Enzyme Solution I and Enzyme Solution II; Enzyme Solution I includes: cellulase R-10 with a mass percentage content of 0.5%, macerozyme R-10 with a mass percentage content of 0.02%, 0.25 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, BSA with a volume percentage content of 0.2%, and water;

[0031] Enzyme Solution II includes: cellulase R-10 with a mass percentage content of 1.5%, macerozyme R-10 with a mass percentage content of 0.02%, 0.4 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, BSA with a volume percentage content of 0.2%, and water.

[0032] The present invention has no special limitation on the sources of the components in Enzyme Solution I and Enzyme Solution II, and conventional commercially available products can be used. In the present invention, the cellulase R-10 and macerozyme R-10 are preferably of the Yakult brand. In the present invention, Enzyme Solution I has a better effect of digesting epidermal microhairs, and the microhairs attached to the back of the leaf can be digested, and the guard cells are elliptical and clearly exposed on the surface of the leaf epidermal strip. In the present invention, after digestion with Enzyme Solution II, the guard cells can fall off well. Usually, protoplasts are combined with the patch clamp technique to study membrane potential changes; or subcellular localization analysis is carried out. Therefore, obtaining guard cell protoplasts with higher purity, intact and not easily broken protoplasts, and a relatively large number is of great significance for studying plant molecular biology. The method for separating protoplasts of Populus alba L. guard cells provided by the present invention obtains guard cell protoplasts with higher concentration and better quality through the use of two enzyme solutions. The method for separating protoplasts of Populus alba L. guard cells according to the present invention solves the problem of detecting changes in tissue-specifically expressed genes in stomata under stress such as drought, and provides a basis for studying the function of stomata in response to stress such as drought.

[0033] In the present invention, the reagent preferably further includes a washing solution; the washing solution includes: 0.4 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, and water. Using the washing solution of the present invention for washing and incubation can, by regulating the cell osmotic pressure, be conducive to obtaining a relatively large number and higher purity of guard cell protoplasts.

[0034] The present invention also provides a kit for separating protoplasts of Populus alba L. guard cells, and the kit includes the reagent described in the above technical solution and a filtering material. In the present invention, the filtering material preferably includes a nylon mesh, and more preferably includes a double-layer nylon mesh.

[0035] The present invention also provides a method for isolating Populus alba guard cell protoplasts based on the reagent or the kit described in the above technical solution, comprising the following steps:

[0036] 1) Mix the Populus alba leaves with Enzyme Solution I for the first digestion and the first filtration to obtain the leaf epidermis after the first digestion treatment;

[0037] 2) Wash, incubate, and perform the second filtration on the leaf epidermis after the first digestion treatment to obtain the washed leaf epidermis;

[0038] 3) Mix the washed leaf epidermis with Enzyme Solution II for the second digestion and the third filtration, take the filtrate, centrifuge to obtain the precipitate, and obtain the guard cell protoplasts.

[0039] In the present invention, the Populus alba leaves are mixed with Enzyme Solution I for the first digestion and the first filtration to obtain the leaf epidermis after the first digestion treatment. In the present invention, before being mixed with Enzyme Solution I, the Populus alba leaves are preferably processed into leaf strips. The present invention preferably takes Populus alba tissue culture seedlings growing for 5 - 8 weeks, cuts the leaves with smooth growth and good condition, places them in sterile double-distilled water for standby, cuts off the leaf tips, leaf veins, and edge parts of the leaves, and cuts the remaining parts into leaf epidermis strips with a width of 1 mm. In the present invention, the time for the first digestion is preferably 0.5 - 1.5 h, more preferably 1 h. In the present invention, the first digestion is preferably carried out with horizontal oscillation, and the speed of the horizontal oscillation is preferably 40 times / min. In the present invention, the first filtration is preferably through a 200-mesh sieve. In the present invention, the sieve is preferably a double-layer nylon mesh.

[0040] After obtaining the leaf epidermis after the first digestion treatment, the present invention washes, incubates, and performs the second filtration on the leaf epidermis after the first digestion treatment to obtain the washed leaf epidermis. The present invention uses the cleaning solution to fully wash the residual enzyme solution on the surface of the leaf epidermis strips. In the present invention, the second filtration is preferably through a 200-mesh sieve. In the present invention, the sieve is preferably a double-layer nylon mesh. In the present invention, the incubation time is 30 min. After incubation, the present invention can obtain enlarged and round stomata.

[0041] After obtaining the cleaned leaf epidermis, the present invention mixes the cleaned leaf epidermis with Enzyme Solution II for secondary digestion, followed by tertiary filtration. The filtrate is taken, and the precipitate is obtained by centrifugation to acquire guard cell protoplasts. In the present invention, the time for the secondary digestion is 1 - 3 h, more preferably 2 h. In the present invention, the secondary digestion is preferably carried out with horizontal shaking, and the speed of the horizontal shaking is preferably 40 times / min. In the present invention, the tertiary filtration is preferably through a 300-mesh and / or 800-mesh sieve, more preferably through a 300-mesh sieve. The function of the tertiary filtration in the present invention is to separate impurities and mesophyll cell protoplasts. In the present invention, the sieve is preferably a double-layer nylon mesh. In the present invention, the centrifugation conditions are centrifugation at 100 g for 5 min.

[0042] The present invention uses a two-step enzymatic hydrolysis method to isolate Populus alba guard cell protoplasts, obtaining a large number of guard cell protoplasts with high purity and high viability.

[0043] To further illustrate the present invention, the following describes in detail a reagent, kit, and method for isolating Populus alba guard cell protoplasts provided by the present invention in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0044] Example 1

[0045] Analysis of the digestion of leaf epidermal strips by Enzyme Solution I

[0046] 1. Select 5 - 8-week-old Populus alba tissue culture seedlings, cut the fully expanded and well-grown leaves and collect them in a 500 mL beaker, and add sterile deionized water to soak the leaves to prevent the leaves from wilting due to water loss.

[0047] 2. Use a surgical blade to cut off the base, tip, and veins of the leaves, evenly cut the remaining middle part of the leaves into thin strips about 1 mm wide, and quickly transfer them to a petri dish containing 100 mL of Enzyme Solution I. To explore the digestion effect of different Enzyme Solution I on Populus alba leaves, four concentration gradients of macerozyme R-10 are set, as shown in Table 1. The components of Enzyme Solution I are:

[0048] Table 1 Enzyme Solution I containing different concentrations of macerozyme

[0049]

[0050] Note: The preparation method of Enzyme Solution I is as follows: Dissolve cellulase R-10, macerozyme R-10, and D-mannitol in deionized water respectively, sequentially add KCl and MES stock solutions, then water bath at 55 °C for 10 min. After the solution is placed at room temperature, then add CaCl2 and BSA and make up the volume.

[0051] 3. Repeat step 2 to cut the leaves and accumulate the amount required for the experiment. Incubate the petri dish containing the leaf strips horizontally at room temperature (20 - 24 °C) for 1 h with a shaking speed of 40 times / min. Figure 1 Figure for analyzing the digestion of leaf epidermis by different Enzyme Solutions I. Observe the leaf epidermis strips and find that the enzymatic hydrolysis effect of Enzyme Solution I-1 is the worst, and there are still many epidermal microhairs attached to the surface of the leaf epidermis strips ( Figure 1 A in Figure 1 ); some epidermal microhairs are digested by Enzyme Solution I-2, and the effect of Enzyme Solution I-3 in digesting epidermal microhairs is better. It can be observed that the microhairs attached to the back of the leaf are digested, and the epidermal microhairs are free in the enzyme solution, and the guard cells are oval and clearly exposed on the surface of the leaf epidermis strips ( Figure 1 B and C in

[0052] Example 2

[0053] Effect of incubation with cleaning solution on stomatal aperture

[0054] 1. Collect the digested leaf epidermis strips on a 200-mesh nylon net and rinse the epidermis strips with cleaning solutions containing different concentrations of D-mannitol three times repeatedly. The collected epidermis strips are continued to be used for separating guard cells.

[0055] The components of the cleaning solutions are shown in Table 2:

[0056] Table 2 Cleaning solutions containing different concentrations of mannitol

[0057] Component Washing Solution - 1 Washing Solution - 2 Washing Solution - 3 D - Mannitol 27.325 g (0.3 M) 36.435 g (0.4 M) 45.54 g (0.5 M) <![CDATA[1MCaCl2]]> 5 ml (10 mM) 5 ml (10 mM) 5 ml (10 mM) 2 M KCl 5 ml (20 mM) 5 ml (20 mM) 5 ml (20 mM) 0.2 M MES 50 ml (20 mM) 50 ml (20 mM) 50 ml (20 mM) Volume made up to 500 mL Volume made up to 500 mL Volume made up to 500 mL

[0058] Note: Preparation method of the cleaning solution: Weigh D-mannitol powder and dissolve it in deionized water, add CaCl2, KCl and MES stock solutions in sequence, and finally make up the volume.

[0059] 2. Divide the leaf epidermis strips collected on the nylon net into three equal parts and place them in different cleaning solutions to adjust the cell osmotic pressure. After incubating for 30 min, observe the guard cells of the leaf epidermis strips under the microscope. When most of the stomata become round, transfer to the next experiment. It is found that after incubating in Cleaning Solution 2 ( Figure 2 A in Figure 2 ) for 0.5 h, the stomata become large and round, while the stomata in Cleaning Solution 1 ( Figure 2 B in Figure 2 , photos of the leaf epidermis strip surface after incubation with the cleaning solution) and Cleaning Solution 3 (

[0060] C in

[0061] Enzyme solution II digests the leaf epidermis strips to obtain guard cell protoplasts

[0062] 1. Collect the epidermis strips in the cleaning solution with a 200-mesh nylon net, and place the epidermis strips in a petri dish containing 100 mL of enzyme solution II. Perform horizontal shaking digestion at room temperature with a shaking speed of 40 times / min.

[0063] Table 3 Composition of Enzyme Solution II

[0064] Component Enzyme Solution II Cellulase R - 10 1.5g(1.5％) Macerozyme R - 10 0.02g(0.02％) D - Mannitol 7.287 g (0.4 M) 2 M KCl 1 ml (20 mM) 0.2 M MES 10 ml (20 mM) Water bath at 55 °C for 10 min 2% BSA 10ml(0.2％) <![CDATA[1MCaCl2]]> 1 ml (10 mM) Volume made up to 100 mL

[0065] Note: The preparation method of enzyme solution II is as follows: Dissolve cellulase R-10, macerozyme R-10, and D-mannitol in deionized water respectively. Add KCl and MES stock solution successively and then incubate in a water bath at 55 °C for 10 min. After the solution is placed at room temperature, then add CaCl2 and BSA and make up the volume.

[0066] 2. Every 1 h, gently stir the epidermis strips in the enzyme solution II several times with a 1 mL pipette tip (axygen) to release the guard cell protoplasts.

[0067] 3. Take the epidermis strips and observe the leaf epidermis strips under a microscope at the time points of 1 h, 2 h, and 3 h of digestion respectively. Figure 3 It is the surface result diagram of the leaf epidermis strips after digestion with enzyme solution II. It is found that after 1 h of digestion, the stomata are oval but there are more stomata attached to the surface of the leaf epidermis strips ( Figure 3 A in, the arrows in the figure point to the stomata); after 2 h of digestion, the stomata become larger and fall off on the leaf epidermis strips, and the small holes left after the guard cells fall off can be clearly observed ( Figure 3 B in, the arrow in the figure points to the small hole left on the leaf epidermis after the guard cells are free); after 3 h of digestion, basically no oval stomata can be observed on the surface of the epidermis strips, but many small holes left after the guard cells fall off are left. At this time, stop digestion and filter to collect the protoplasts ( Figure 3 C in).

[0068] 4. The filtration in step 3 is specifically as follows: Filter through two layers of 300-mesh and 800-mesh nylon nets respectively to remove the epidermis strips, and collect the filtrate with a 50 mL centrifuge tube. Centrifuge the collected filtrate, centrifuge at 100×g for 5 min, take the precipitate, and collect the released guard cell protoplasts.

[0069] Figure 4 It is the diagram of guard cell protoplasts observed under a microscope. The amount of protoplasts, cell integrity and not easy to break can be observed ( Figure 4 , A is the filtration result diagram of guard cell protoplasts through 300-mesh; B is the filtration result diagram of guard cell protoplasts through 800-mesh).

[0070] 5. Wash the precipitate three times with the cleaning solution. Place the separated guard cell protoplasts in the dark at 2-4°C for storage for later use.

[0071] Example 4

[0072] Detection of specifically expressed genes in guard cells:

[0073] 1. RNA extraction: Take the leaf epidermal strips after digestion with Enzyme Solution II (the digested leaves, specifically the part of the leaf epidermal strips removed after filtering through two layers of 300-mesh and 800-mesh nylon nets, without protoplasts), the Populus alba protoplasts (ordinary protoplasts) obtained according to the method provided by (Sheen, 2001), and the guard cell protoplasts (Guard cell protoplast 300-mesh, Guard cell protoplast 800-mesh) obtained by filtering through 300-mesh and 800-mesh nylon nets, a total of four samples, for total RNA extraction. The RNA extraction method refers to: RNeasy plant mini kit (Qiagen, cat. no. 74904).

[0074] 2. Fluorescent quantitative PCR: Take 800 ng of total RNA and perform cDNA synthesis and dilution according to the instructions of PrimeScript TM RT reagent Kit (Takara cat. no. RR037A).

[0075] Primers used for qRT-PCR:

[0076] Pascap1-F: GAGCAAGCCTTGAAGTGTCCAAGATGTGAGTC (SEQ ID NO.1);

[0077] Pascap1-R: GACTCCAAAGACTCCAGTGGCTCCTCAGAGA (SEQ ID NO.2);

[0078] PaACTIN-F: GACCTTCAATGTGCCTGCAA (SEQ ID NO.3);

[0079] PaACTIN-R: CGAAGGATGGCATGTGGAAG (SEQ ID NO.4);

[0080] Use TB Green Premix Ex Taq TMPerform qRT-PCR reactions with II (Takara cat.no.RR820Q). The experimental results show that the Pascabp1 gene specifically expressed in guard cells has the lowest expression level in the digested leaves, and its expression level in ordinary protoplasts is about 7 times that in the digested leaves; while Pascap1 has the highest expression level in guard cell protoplasts, and filtering with a double-layer nylon mesh of 300 meshes and 800 meshes has no obvious effect on the expression level of Pascap1, indicating that the filtering effects of the double-layer nylon mesh of 300 meshes and 800 meshes on guard cell protoplasts are the same ( Figure 5 , results of specific gene expression in guard cell protoplasts), and the relative expression level of Pascap1 in guard cell protoplasts filtered with 300 meshes and 800 meshes is 40-50 times higher than that of Pascabp1 in the digested leaves.

[0081] The sequence of the Pascap1 gene on the genome:

[0082] ATGGTGCCCCCGGATAATCTTGATCAGGAGAAACAGACCTCCAAAGGCGACAACCAAAGCTCTTCTAGTAGCCGTAAAACAGCCACGACAAGGCCACAAGAGCAAGCCTTGAAGTGTCCAAGATGTGAGTCACCCAACACAAAATTCTGCTACTACAACAATTACAGTTTAACACAGCCCAGACATTTTTGCAAGACCTGTAGAAGGTACTGGACTAAAGGAGGAGCCTTGCGCAGCGTACCCATCGGCGGTGGCTGTAGAAAGAACAAGAAGATGAAGTCATCTTCAAGGCTCTCAAGTGACTCCAAAGACTCCAGTGGCTCCTCAGAGATCGCTGGATTCAGCTTCTTTCATGGACTCTCCCCAACCGTTGATTTTAATCTTGGTGGGTTATCATTTCCTAGGCTAAACCCTTCACAAAATGGCTTATATAACCATTTCCCTTCATTTGGGGATATTTCAGCAACTTCAGCAGCTGCAGCTACTGTTACTAGTCCTTGTTTCACTCTTGATCCATCTGTGAGCTCCACTGGTTCTTGTTCCTTGATGGGGTTTAATTATCCACTTACTTCGGTTGCTAGTGGGTTTGGTGGTGCAATTCAGGAGAACATTGGTGGTGCTTCAATGAATATCAACACTAATCTCCCGTCTTCAATTGAGTCTTTGAGTTGTATAAACCAAGATTTACACTGGAAGTTGCAGCAGCAGAGATTGGCTATGCTATTTGGTACAGGAGAAAAACATAAAGATGGCAGTACTGTTTCTACAGATCCTATTGAGAACCAGGTACAGAAACCACAGCCTATTATGTTTGAAAATCTTGAAATTTCAAAACCACAAGTATGCGCAGCTGGTAATTCAAGAAAAGAAGGTGCAGCTAGTGGTGATACAGCAACAGAATGGTTCTTTGGGAATTCTTATTCTCAAGTGACTGCAACGACGACAAATACTAGCAACAATGGCAATAATGACAACACAGGCAATTGGAATGGGGTTCAAGCATGGGGTGATTTGCATCAGTATAGTGCTTTGCCCTAG(SEQ ID NO.5).

[0083] cDNA sequence of Pascap1:

[0084] ATGGTGCCCCCGGATAATCTTGATCAGGAGAAACAGACCTCCAAAGGCGACAACCAAAGCTCTTCTAGTAGCCGTAAAACAGCCACGACAAGGCCACAAGAGCAAGCCTTGAAGTGTCCAAGATGTGAGTCACCCAACACAAAATTCTGCTACTACAACAATTACAGTTTAACACAGCCCAGACATTTTTGCAAGACCTGTAGAAGGTACTGGACTAAAGGAGGAGCCTTGCGCAGCGTACCCATCGGCGGTGGCTGTAGAAAGAACAAGAAGATGAAGTCATCTTCAAGGCTCTCAAGTGACTCCAAAGACTCCAGTGGCTCCTCAGAGATCGCTGGATTCAGCTTCTTTCATGGACTCTCCCCAACCGTTGATTTTAATCTTGGTGGGTTATCATTTCCTAGGCTAAACCCTTCACAAAATGGCTTATATAACCATTTCCCTTCATTTGGGGATATTTCAGCAACTTCAGCAGCTGCAGCTACTGTTACTAGTCCTTGTTTCACTCTTGATCCATCTGTGAGCTCCACTGGTTCTTGTTCCTTGATGGGGTTTAATTATCCACTTACTTCGGTTGCTAGTGGGTTTGGTGGTGCAATTCAGGAGAACATTGGTGGTGCTTCAATGAATATCAACACTAATCTCCCGTCTTCAATTGAGTCTTTGAGTTGTATAAACCAAGATTTACACTGGAAGTTGCAGCAGCAGAGATTGGCTATGCTATTTGGTACAGGAGAAAAACATAAAGATGGCAGTACTGTTTCTACAGATCCTATTGAGAACCAGGTACAGAAACCACAGCCTATTATGTTTGAAAATCTTGAAATTTCAAAACCACAAGTATGCGCAGCTGGTAATTCAAGAAAAGAAGGTGCAGCTAGTGGTGATACAGCAACAGAATGGTTCTTTGGGAATTCTTATTCTCAAGTGACTGCAACGACGACAAATACTAGCAACAATGGCAATAATGACAACACAGGCAATTGGAATGGGGTTCAAGCATGGGGTGATTTGCATCAGTATAGTGCTTTGCCCTAG(SEQ ID NO.6).

[0085] Amino acid sequence of Pascap1:

[0086] MVPPDNLDQEKQTSKGDNQSSSSSRKTATTRPQEQALKCPRCESPNTKFCYYNNYSLTQPRHFCKTCRRYWTKGGALRSVPIGGGCRKNKKMKSSSRLSSDSKDSSGSSEIAGFSFFHGLSPTVDFNLGGLSFPRLNPSQNGLYNHFPSFGDISATSAAAATVTSPCFTLDPSVSSTGSCSLMGFNYPLTSVASGFGGAIQENIGGASMNINTNLPSSIESLSCINQDLHWKLQQQRLAMLFGTGEKHKDGSTVSTDPIENQVQKPQPIMFENLEISKPQVCAAGNSRKEGAASGDTATEWFFGNSYSQVTATTTNTSNNGNNDNTGNWNGVQAWGDLHQYSALP(SEQ ID NO.7).

[0087] Promoter (-3000bp to -1bp) sequence of Pascap1:

[0088]

[0089] Pascap1 Tissue Localization Analysis:

[0090] The promoter of the Pascap1 gene was fused with the GUS reporter gene for expression, and GUS staining analysis was carried out. It was found that the GUS reporter gene was specifically expressed in stomata ( Figure 6 ). By taking advantage of the specific expression of Pascap1 in guard cells, the purity of the obtained guard cell protoplasts was verified by detecting the difference in the expression level of the Pascap1 gene.

[0091] Example 5

[0092] Isolation of Guard Cell Protoplasts ( Figure 8 Taking the digestion with Enzyme Solution II for 2 h as an example, it is the flow chart for preparing Populus alba guard cell protoplasts):

[0093] 1. Take the tissue culture seedlings of Populus alba that have grown for 5 - 8 weeks, cut the leaves with smooth growth and good condition, place them in sterile double-distilled water for standby, cut off the leaf tips, leaf veins and edge parts of the leaves, and cut the remaining parts into leaf epidermis strips 1 mm wide.

[0094] 2. Place the leaf epidermis strips obtained in the previous step in Enzyme Solution I for digestion for 1 h, and the oscillation speed is 40 times / min.

[0095] 3. Filter with a double-layer nylon mesh of 200 meshes and collect the leaf epidermis strips.

[0096] 4. Thoroughly rinse the residual enzyme solution on the surface of the leaf epidermis strips with the washing solution and incubate in the washing solution for 30 min. At this time, the stomata attached to the leaf epidermis strips become larger and rounder.

[0097] 5. Filter the leaf epidermis strips in the washing solution with a double-layer nylon mesh of 200 meshes.

[0098] 6. Place the collected leaf epidermis strips in Enzyme Solution II and digest for 1 h, 2 h, and 3 h respectively, and the oscillation speed is 40 times / min.

[0099] 7. Filter with a double-layer nylon mesh of 300 meshes and collect the filtrate.

[0100] 8. Place the collected filtrate in a 50 ml centrifuge tube, centrifuge at 100 g for 5 min to obtain guard cell protoplasts.

[0101] 9. By performing FDA staining on the obtained guard cell protoplasts, it was found that when digested in Enzyme Solution II for 1 h ( Figure 7 in A), 2 h ( Figure 7 in B), 3 h ( Figure 7The protoplast viability was higher after C) in; through the detection of guard cell-specific expression genes, it was found that the purity of guard cell protoplasts obtained by digesting in Enzyme Solution II for 2 h was the highest ( Figure 7 D) in.

[0102] 10. Further, by counting the guard cell protoplasts, 10 mature tissue culture seedlings of Populus alba were taken, about 50 leaves, and after digesting in Enzyme Solution II for 2 h, about 1.45×10 6 protoplasts could be obtained through cell counting.

[0103] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative labor, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for isolating Populus alba guard cell protoplasts based on reagents for isolating Populus alba guard cell protoplasts, comprising the following steps: 1) Mix Populus alba leaves with Enzyme Solution I for the first digestion, and perform the first filtration to obtain the leaf epidermis after the first digestion treatment; 2) Wash the leaf epidermis after the first digestion treatment with the washing solution, incubate, and perform the second filtration to obtain the washed leaf epidermis; 3) Mix the washed leaf epidermis with Enzyme Solution II for the second digestion, perform the third filtration, take the filtrate, centrifuge to obtain the precipitate, and obtain the guard cell protoplasts; The reagents for isolating Populus alba guard cell protoplasts include Enzyme Solution I and Enzyme Solution II; Enzyme Solution I includes: Cellulase R-10 with a mass percentage content of 0.5%, Macerozyme R-10 with a mass percentage content of 0.02%, 0.25 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, BSA with a volume percentage content of 0.2%, and water; Enzyme Solution II includes: Cellulase R-10 with a mass percentage content of 1.5%, Macerozyme R-10 with a mass percentage content of 0.02%, 0.4 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, BSA with a volume percentage content of 0.2%, and water. The reagents for isolating Populus alba guard cell protoplasts also include the washing solution; the washing solution includes: 0.4 M D-mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, and water.

2. The method according to claim 1, characterized in that Before mixing with Enzyme Solution I, the Populus alba leaves are processed into leaf strips.

3. The method according to claim 1, wherein The time for the first digestion is 0.5 - 1.5 h; the time for the second digestion is 1 - 3 h.

4. The method according to claim 1 or 3, characterized in that, The first digestion and the second digestion are respectively subjected to horizontal shaking, and the speed of the horizontal shaking is 40 times / min respectively.

5. The method according to claim 1, wherein The first filtration and the second filtration are through a 200-mesh sieve; the third filtration is through a 300 - 800-mesh sieve.

6. The method according to claim 1, wherein The incubation time is 30 min.

7. The method according to claim 1, characterized in that, The centrifugation conditions are centrifugation at 100 g for 5 min.

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