Method for detecting subcellular localization by using toona ciliata leaf protoplast

Abstract

The invention discloses a method for detecting subcellular localization by using toona ciliata leaf protoplast, on the basis of a toona ciliata protoplast separation system, the subcellular localization conditions of toona ciliata TcDXS1 and TcDXS5 are detected by using a toona ciliata protoplast transient expression system, and the method is simple, convenient, efficient, rapid and cost-saving. The exogenous gene can be quickly and efficiently transiently expressed in the toona ciliata leaf protoplast, so that the method is conveniently and quickly applied to subcellular localization of various gene expression products of the toona ciliata, and a convenient technology is provided for the function research of the toona ciliata gene.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and particularly relates to a method for detecting subcellular localization using protoplasts from toon leaves. Background Art

[0002] Toona sinensis is a high-quality, fast-growing species of the genus Toona in the Meliaceae family. Its wood is excellent, vibrant, and of high economic value. It is a Class II protected wild plant in my country. Currently, research on genetic transformation of Toona sinensis primarily focuses on tissue culture and stable genetic systems. Studies on transient expression are rare. Although established genetic transformation systems are relatively stable, they still suffer from unavoidable drawbacks such as long cycles, low transformation efficiency, and demanding operational requirements. Therefore, an efficient and simple transient transformation system for Toona sinensis is urgently needed to expand Toona sinensis genetic research and accelerate the development of genetic improvement and gene function research.

[0003] During the young stage of toon (Centella chinensis) forests, the terminal buds are highly susceptible to feeding damage from the neem borer, which bores into the trunk, causing hollowing, breakage, and even death of branches, seriously affecting the growth of the tree. Currently, chemical control is the most common preventive measure against the neem borer, while physical control methods are relatively less studied and applied. Chemical control still has certain shortcomings, and this method cannot be widely promoted in toon breeding, resulting in huge human and material resources consumption. Therefore, genetic engineering research on toon (Centella chinensis) needs to be continuously deepened to actively and vigorously promote the sustainable development of Centella chinensis.

[0004] Previous studies have shown that when the volatile monoterpene compound α-pinene emitted by toona chinensis exists in a certain ratio with other compounds (such as nonanal, decanal, and α-terpineol), the presence and content of α-pinene is highly likely to act as a signaling agent for the neem borer. Based on the development of a whole-genome sequencing platform for toona chinensis, transcriptome analysis of leaves from 3-month-old (non-infested) and 2-year-old (severely infested) toona chinensis plants identified a number of differentially expressed genes in the terpenoid biosynthesis pathway. The most significant difference was found in the gene encoding the rate-limiting enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). As the first, rate-limiting enzyme in the monoterpene biosynthesis pathway, DXS is generally believed to play a key regulatory role in catalyzing the synthesis of terpenoid precursors and influencing the ratio of other terpenoids to final products. If we can clearly understand the subcellular localization of TcDXSs, the key rate-limiting enzyme in the synthesis pathway of monoterpenoid substances in C. redus, it will provide a basis for the subsequent identification of the function and mechanism of TcDXSs in C. redus, and greatly promote the process of directed genetic improvement of C. redus. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for detecting subcellular localization using protoplasts from Toona chinensis leaves.

[0006] The object of the present invention is to provide a method for detecting subcellular localization using protoplasts from toon leaves, comprising the following steps:

[0007] S1. Select young leaves from two-month-old sterile seedlings of Toona chinensis and cut them into thin strips perpendicular to the main veins. Immerse the leaves in a 0.6 M mannitol solution for 20 minutes. Remove the strips, blot dry, and then soak them in the enzymatic solution. Enzyme hydrolysis was performed at 50 rpm in the dark for 4 hours.

[0008] S2. Add an equal volume of W5 solution to the enzymatic hydrolyzate from step S1, filter through a 200-mesh cell sieve into a centrifuge tube, centrifuge, remove the supernatant, add 5 mL of W5 solution, and centrifuge again; after centrifugation, add an appropriate amount of W5 solution to resuspend until the protoplasts are evenly distributed, and let stand on ice for 30 minutes to allow the protoplasts to sink to the bottom of the centrifuge tube. Remove the supernatant, add MMG solution, and resuspend to obtain a protoplast suspension;

[0009] S3. After the plasmid containing the subcellular localization gene to be detected is mixed evenly with the protoplast suspension, it is allowed to stand at room temperature for 10 minutes; then PEG-Ca 2+ solution, mixed evenly, and incubated at room temperature in the dark for 30 minutes; after the incubation, W5 solution was added to terminate the reaction, mixed evenly, and centrifuged, and the supernatant was removed; W5 solution was added again, mixed evenly, and centrifuged, and the supernatant was removed; finally, 500 μL of W5 solution was added, mixed evenly, and incubated at 25°C in the dark for 48 hours; after the dark incubation, the protoplast pellet was enriched by centrifugation, and only an appropriate amount of supernatant was retained to mix the protoplast pellet to obtain a protoplast solution into which the subcellular localization gene to be detected was transferred;

[0010] S4. Take an appropriate amount of protoplast solution into which the subcellular localization gene to be detected has been transferred for observation of subcellular localization.

[0011] Preferably, the enzymatic hydrolysate in step S1 is prepared as follows per 10 mL of the system: first mix 1.5% vol cellulase, 1.5% vol macerator, 0.6 M mannitol, 20 mM MES at pH 5.7, 50 mM KCl, and 7.5 mL ddH2O; treat at 55°C for 10 min; after cooling to room temperature, add 10 mM CaCl2 and 0.1 wt% BSA, make up to 10 mL with ddH2O, and then filter and sterilize with a 0.45 μm filter membrane.

[0012] Preferably, in step S2, the composition of the W5 solution is: 2 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl with a pH of 5.7, and the solvent is water; the composition of the MMG solution is: 4 mM MES, 0.6 M mannitol, 15 mM MgCl2 with a pH of 5.7, and the solvent is water.

[0013] Preferably, the centrifugation in step S2 is performed at 940 rpm and 4° C. for 2 min.

[0014] Preferably, the density of the protoplast suspension obtained in step S2 is 2×10 5 pieces / mL.

[0015] Preferably, the step S3 is to mix 20 ng of the plasmid containing the subcellular localization gene to be detected with 100 μL of the protoplast suspension, and then let it stand at room temperature for 10 minutes; then add 110 μL of PEG-Ca 2+ The solution was mixed evenly and incubated in the dark at room temperature for 30 min;

[0016] Preferably, the plasmid containing the subcellular localization gene to be detected in step S3 is pCAMBIA1300-35s-TcDXS1-GFP or pCAMBIA1300-35s-TcDXS5-GFP; the nucleotide sequence of TcDXS1 is shown in SEQ ID NO.1, and the nucleotide sequence of TcDXS5 is shown in SEQ ID NO.2.

[0017] Preferably, the PEG-Ca in step S3 2+ The solution comprises the following components: 40 wt% PEG4000, 0.6 M mannitol, 100 mM CaCl2, and the solvent is water.

[0018] Preferably, the centrifugation in step S3 is performed at 500 rpm for 5 min.

[0019] Beneficial effects of the present invention:

[0020] (1) The transient transformation method of toon protoplasts for subcellular localization can also be used for gene function verification, protein interaction analysis, and genome editing, providing efficient and convenient technical support for the improvement of toon breeding and genetic systems.

[0021] (2) The subcellular localization method established in the present invention adopts a protoplast transient expression method, which can be quickly applied to the subcellular localization of various gene expression products of Toona chinensis, providing a convenient technology for the study of gene function of Toona chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are images of the subcellular localization of TcDXS1 in C. rubrum. From left to right, they represent green fluorescence observed at an excitation wavelength of 484 nm, bright field, chloroplast spontaneous red fluorescence observed at an excitation wavelength of 546 nm, and superimposed field.

[0023] Figure 2 These are subcellular localization images of TcDXS5 in C. rubrum. From left to right, they represent green fluorescence observed at an excitation wavelength of 484 nm, bright field, chloroplast spontaneous red fluorescence observed at an excitation wavelength of 546 nm, and superimposed field. DETAILED DESCRIPTION

[0024] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0025] The following are the main reagent formulas used in the examples:

[0026] W5 solution: 2 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl with a pH of 5.7, the solvent is water, sterilized by filtration through a 0.2 μm filter membrane, and stored at -4°C until use.

[0027] MMG solution: 4 mM MES, 0.6 M mannitol, 15 mM MgCl2 with a pH of 5.7, the solvent is water, sterilized by filtration through a 0.2 μm filter membrane, and stored at -4°C until use.

[0028] PEG-Ca 2+ Solution: 40 wt% PEG 4000, 0.6 M mannitol, 100 mM CaCl2, in water. Prepare the PEG solution immediately before use, half an hour before the start of the transformation experiment. Dissolve in a shaker or water bath, and sterilize by filtration through a 0.45 μm filter.

[0029] The kit used for plasmid purification was Plasmid Mini KitⅠ was purchased from OMEGA, product number is D6943-02.

[0030] Cellulase (Cellulase R-10, product number L0012) and macerase (Macerozyme R-10, product number L0021) were both produced by Yakult, Japan, and purchased from Guangzhou Baijing Biotechnology Co., Ltd.

[0031] Example 1

[0032] (1) The transient expression vector pCAMBIA1300-35s-MCS-GFP (a vector without the target gene) and its recombinant vectors (pC1300-TcDXS1-GFP and pC1300-TcDXS5-GFP) of C. rubrum were constructed, transformed into Escherichia coli, and identified to obtain the correctly sequenced bacterial suspension (pCAMBIA1300-35s-TcDXS1-GFP and pCAMBIA1300-35s-TcDXS5-GFP), which were then stored at -80°C.

[0033] Specifically, the E. coli transformation steps are as follows: Place E. coli DH5α on ice and aliquot. Immediately add the ligation product of TcDXS1 (its nucleotide sequence is shown in SEQ ID NO. 1) and TcDXS5 (its nucleotide sequence is shown in SEQ ID NO. 2) to the DH5α tube. Gently tap the tube to mix thoroughly, and then place on ice for 30 minutes. Heat shock at 42°C for 90 seconds and immediately place on ice for 2 minutes. Add 700 μL of blank LB liquid medium on a sterile operating table and incubate at 37°C on a shaker at 200 rpm for 1-2 hours. Centrifuge at 6000 rpm for 2 minutes to allow the E. coli to settle to the bottom of the tube. Aspirate 500 μL of the supernatant on a sterile operating table and mix thoroughly by pipetting. Incubate the tube overnight at 37°C on an LB medium plate containing kanamycin. Select a single intact colony and add it to 200 μL of LB liquid medium containing kanamycin and shake at 37°C on a shaker at 200 rpm for 1-2 hours. Take 2 μL of bacterial solution as a template and perform colony PCR identification using universal primers M13F / R (primer sequence: M13-F: GTAAAACGACGGCCAGT, SEQ ID NO.3; M13-R: CAGGAAACAGCTATGAC, SEQ ID NO.4). After obtaining the correct bacterial solution for sequencing, store it at 4°C.

[0034] (2) Add 1 mL of the stored target strain to 100-200 mL of LB liquid culture medium containing kanamycin and culture overnight at 37°C on a shaker at 200 rpm.

[0035] (3) Plasmid purification;

[0036] The specific steps are as follows:

[0037] ① Collect the cultured bacterial solution in a 50mL centrifuge tube and centrifuge at 8000rpm for 3min at 4℃. Sediment the bacterial solution in one centrifuge tube and remove the supernatant as much as possible.

[0038] ② Pour off the supernatant and leave the bacterial pellet. Wipe dry with absorbent paper or drain the inner wall of the centrifuge tube. Add 8 mL of Buffer P1 and use a pipette or oscillator to thoroughly suspend the pellet.

[0039] ③ After adding 8 mL of P2 to the centrifuge tube obtained in step ②, use a pipette to immediately and gently invert the tube 6-8 times to fully lyse the cells. Let it stand at room temperature for 5 minutes.

[0040] ④ After adding 8 mL of P4 to the centrifuge tube prepared in step ③, gently invert the tube 6-8 times to mix the solution thoroughly until a white flocculent precipitate forms. Then, place the tube at room temperature for 10 minutes. Centrifuge at 8000 rpm for 5 minutes to separate the white flocculent precipitate to the bottom of the tube. Then, slowly and gently introduce the entire solution into the filter CS1. Gently push the handle to filter the solution. Collect the filtrate into a clean 50 mL tube prepared in advance.

[0041] ⑤ Wet the equilibrated column with 2.5 mL of buffer, let it stand for 2 minutes, and then centrifuge at 6000 rpm for 2 minutes.

[0042] ⑥ Add 0.3 times the volume of isopropanol to the filtrate, then invert it to mix thoroughly and transfer it to the adsorption column CP6 using a pipette;

[0043] ⑦ Centrifuge at 8000 rpm for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube;

[0044] ⑧ Add 10 mL of rinse solution PW to the adsorption column CP6 using a pipette, then centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and then place the adsorption column back into the collection tube;

[0045] ⑨Repeat step ⑧;

[0046] ⑩ Add 3 mL of anhydrous ethanol to the adsorption column CP6 using a pipette, centrifuge at 8000 rpm for 2 min at room temperature, and discard the waste liquid;

[0047] Place the adsorption column CP6 back into the collection tube and centrifuge at 8000 rpm for 5 minutes at room temperature to remove the remaining rinse solution in the adsorption column.

[0048] Place the adsorption column CP6 in a clean 50 mL collection tube prepared in advance, and add 1-2 mL of eluent TB dropwise to the middle of the white adsorption membrane using a rubber-tipped dropper. Let it stand at room temperature for 5 minutes, then centrifuge at 8000 rpm for 2 minutes at room temperature. Transfer all the eluate in the 50 mL centrifuge tube after centrifugation into a clean 1.5 mL centrifuge tube, measure the concentration, and store at -20°C.

[0049] (4) Obtaining high-quality protoplasts of Toona sinensis;

[0050] The specific steps are as follows:

[0051] ①Select the young leaves of two-month-old toon seedlings and cut them into thin strips parallel to the main veins for later use;

[0052] ② Preparation of enzymatic solution: First, add 1.5% vol cellulase, 1.5% vol macerate, 0.6 M mannitol, 20 mM MES (pH 5.7), and 50 mM KCl to a conical flask, and dilute to 7.5 mL with ddH2O. Incubate in a 55°C water bath for 10 min. After cooling to room temperature, add 10 mM CaCl2 and 0.1% wt% BSA, and dilute to 10 mL with ddH2O. Finally, filter and sterilize with a 0.45 μm filter.

[0053] ③ Pre-plasmolysis: Soak the leaves of Toona sinensis cut into thin strips in 0.6 M mannitol solution for 20 minutes;

[0054] ④ Enzymatic hydrolysis: After drying the thin leaf strips obtained from the pre-plasmolysis step with filter paper, place them in the enzymatic hydrolysis solution (enzymatic hydrolysis solution should be enough to cover the thin leaf strips), and enzymatic hydrolyze at 50 rpm in the dark for 4 h;

[0055] ⑤ Purification: Add an equal volume of W5 solution to the reaction solution in step ④ and filter through a 200-mesh cell sieve into a 50-mL centrifuge tube; centrifuge at 940 rpm and 4°C for 2 min, remove the supernatant, add 5 mL of W5 solution, centrifuge again (940 rpm and 4°C for 2 min), add 5 mL of W5 solution and resuspend until the protoplasts are evenly distributed. Place on ice and let stand for 30 min to allow the protoplasts to sink to the bottom of the tube, remove the supernatant, add MMG solution and resuspend.

[0056] (5) In PEG-Ca 2+ The subcellular localization of TcDXSs was carried out under transient transformation of Toona sinensis protoplasts mediated by PCR;

[0057] The specific steps are as follows:

[0058] ① Dilute the isolated and purified high-quality protoplasts of Toona sinensis with MMG solution to a density of 2×10 5 / mL;

[0059] ② Add concentrated plasmid (20 ng) and 100 μL protoplast suspension to a 2 mL centrifuge tube, flick the tube wall to mix, and let stand at room temperature for 10 min.

[0060] ③Add 110 μL PEG-Ca 2+ The solution was mixed by gently tapping the tube wall and incubated in the dark at room temperature for 30 min;

[0061] ④ Add 500 μL of W5 solution to terminate the reaction, mix well, and centrifuge at 500 rpm for 5 minutes. Carefully remove the supernatant, then add another 500 μL of W5 solution, centrifuge again at 500 rpm for 5 minutes, and remove the supernatant.

[0062] ⑤ Finally, add 500 μL of W5 solution and gently pipette to mix, then transfer to a cell culture dish and culture in the dark at 25°C for 48 h (the cell culture dish needs to be thoroughly rinsed with 5% BSA solution in advance to prevent protoplasts from sticking to the wall during culture).

[0063] ⑥ After 48 hours of dark culture, the protoplasts were enriched by horizontal centrifugation at 500 rpm for 5 minutes. After removing the upper half of the supernatant, about 100 μL of the supernatant was left and mixed with the protoplast pellet; 30-50 μL of the protoplast solution was placed on a slide, covered with a coverslip, and its subcellular localization was observed under a laser confocal microscope. The results are as follows Figure 1 and Figure 2 As shown, the two fluorescence signals overlap, indicating that the expression products of the toon genes TcDXS1 and TcDXS5 are both located in the chloroplast.

Claims

1. A method for detecting subcellular localization using protoplasts of Toona chinensis leaves, characterized in that: The following steps are involved: S1. Select young leaves of 2-month-old sterile seed seedlings of Toona chinensis, cut the leaves into thin strips perpendicular to the main veins, immerse them in 0.6M mannitol solution for 20 minutes, take out the thin strips, absorb the water, and then soak them in the enzymatic solution, and enzymolyze them for 4 hours at 50rpm in the dark; S2. Add an equal volume of W5 solution to the enzymatic hydrolyzate of step S1, filter through a 200-mesh cell sieve into a centrifuge tube and centrifuge, remove the supernatant, add 5 mL of W5 solution, and centrifuge again; after the centrifugation, add an appropriate amount of W5 solution to resuspend until the protoplasts are evenly distributed, and place on ice for 30 minutes to allow the protoplasts to sink to the bottom of the centrifuge tube, remove the supernatant, add MMG solution and resuspend to obtain a protoplast suspension; S3. After the plasmid containing the subcellular localization gene to be detected is mixed evenly with the protoplast suspension, it is allowed to stand at room temperature for 10 minutes; then PEG-Ca 2+ solution, mixed evenly, and incubated at room temperature in the dark for 30 minutes; after the incubation, W5 solution was added to terminate the reaction, mixed evenly and centrifuged, and the supernatant was removed; W5 solution was added again, mixed evenly and centrifuged, and the supernatant was removed; finally, 500 μL of W5 solution was added, mixed evenly and incubated at 25° C. in the dark for 48 hours; after the dark incubation, the protoplast precipitate was enriched by centrifugation, and only an appropriate amount of supernatant was retained to mix the protoplast precipitate to obtain a protoplast solution into which the subcellular localization gene to be detected was transferred; S4. Take an appropriate amount of protoplast solution into which the gene to be detected for subcellular localization has been transferred, and use it to observe the subcellular localization.

2. The method according to claim 1, characterized in that The enzymatic hydrolysate in step S1 is prepared as follows per 10 mL system: first mix 1.5% vol cellulase, 1.5% vol macerator, 0.6 M mannitol, 20 mM MES at pH 5.7, 50 mM KCl, and 7.5 mL ddH2O; treat at 55°C for 10 min; after cooling to room temperature, add 10 mM CaCl2 and 0.1 wt% BSA, make up to 10 mL with ddH2O, and then filter and sterilize with a 0.45 μm filter membrane.

3. The method according to claim 1, characterized in that In the step S2, the composition of the W5 solution is: 2 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl with a pH of 5.7, and the solvent is water; The composition of the MMG solution is: 4 mM MES at pH 5.7, 0.6 M mannitol, 15 mM MgCl2, and the solvent is water.

4. The method according to claim 1, characterized in that: The centrifugation in step S2 is performed at 940 rpm and 4° C. for 2 min.

5. The method according to claim 1, characterized in that: The density of the protoplast suspension obtained in step S2 is 2×10 5 Pieces / mL.

6. The method according to claim 1, characterized in that Step S3 is to mix 20 ng of the plasmid containing the subcellular localization gene to be detected with 100 μL of the protoplast suspension, and then let it stand at room temperature for 10 minutes; then add 110 μL of PEG-Ca 2+ The solution was mixed well and incubated at room temperature in the dark for 30 min.

7. The method according to claim 1, characterized in that The plasmid containing the subcellular localization gene to be detected in step S3 is pCAMBIA1300-35s-TcDXS1-GFP or pCAMBIA1300-35s-TcDXS5-GFP; the nucleotide sequence of TcDXS1 is shown in SEQ ID NO.1, and the nucleotide sequence of TcDXS5 is shown in SEQ ID NO.

2.

8. The method according to claim 1, characterized in that The PEG-Ca in step S3 2+ Its composition is: 40wt% PEG4000, 0.6M mannitol, 100mM CaCl2, and the solvent is water.

9. The method according to claim 1, characterized in that: The centrifugation in step S3 is performed at 500 rpm for 5 min.

Images