Method for screening functional volatile matters by using tea tree cell calcium signal detection system and application thereof

Abstract

The invention provides a method for screening functional volatile matters by using a tea tree cell calcium signal detection system and application of the method, and belongs to the technical field of functional volatile matter screening. The method comprises the following steps: (1) extracting protoplast cells of tea tree leaves; (2) loading a calcium specific dye to the obtained protoplast cells to obtain a tea tree cell calcium signal detection system; and (3) mixing the tea tree volatile matters with a tea tree cell calcium signal detection system, and selecting the tea tree volatile matters with enhanced fluorescence intensity as the volatile matters with potential functions. The tea tree cell calcium signal detection system provided by the invention realizes visual detection of tea tree cell calcium signal response environmental factors and high-throughput screening of functional volatile matters, and provides reference for crops lacking a stable genetic transformation technology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of functional volatile screening, and particularly relates to a method for screening functional volatiles by using a tea tree cell calcium signal detection system and application thereof. BACKGROUND

[0002] The perception of the environment by plants usually involves calcium signals. In plants, calcium ions are used in a wider range than other known messenger molecules. Almost all signals can cause the content of cytoplasmic free calcium ions ([Ca2+]i) to rise. The change in [Ca2+]i of cells caused by stress is decoded by downstream calcium receptor proteins, causing specific physiological responses and improving the adaptation of plants to the environment. Since tea trees lack stable genetic transformation technology, stable transformation of mature calcium fluorescent probes cannot be achieved. In addition, it is difficult to achieve clear fluorescence imaging in leaves with thick wax layers (such as tea trees). The tea tree volatile library has a large number of types and great potential for agricultural production application, but only a small part of the volatiles has been reported to have functions. Therefore, it is urgent to develop an efficient and convenient method for mining functional volatiles. SUMMARY

[0003] In view of this, the purpose of the present application is to provide a method for screening functional volatiles by using a tea tree cell calcium signal detection system and application thereof. The present application uses calcium-specific dye fluo-8 / AM to load tea tree protoplasts to construct a tea tree cell calcium signal detection system, and uses the system to realize the visual detection of the calcium signal response of tea tree cells to environmental factors, thereby providing a reference for crops lacking stable genetic transformation technology.

[0004] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a method for screening functional volatiles by using a tea tree cell calcium signal detection system, comprising the following steps: (1) extracting protoplast cells from tea tree leaves; (2) loading the obtained protoplast cells with calcium-specific dye to obtain a tea tree cell calcium signal detection system; (3) mixing tea tree volatiles with the tea tree cell calcium signal detection system, and selecting tea tree volatiles with enhanced fluorescence intensity as potential functional volatiles.

[0005] Preferably, the calcium-specific dye is fluo-8 / AM.

[0006] Preferably, the concentration of the calcium-specific dye is 0.5-50 μM.

[0007] Preferably, in step (3), a full-automatic enzyme marker is used to observe whether the fluorescence intensity changes.

[0008] As preferred, the tea plant exposed to the volatile with potential function obtained in step (3) is further treated, and the cold resistance effect of the treated tea plant is verified.

[0009] The application further provides application of the method in screening of the tea plant volatile with function.

[0010] As preferred, the tea plant volatile with function is a cold-induced released tea plant volatile.

[0011] As preferred, the final concentration of the tea plant volatile with function is 0.01-0.1 μM.

[0012] The application further provides application of the tea plant volatile with function screened by the method in improving the cold tolerance of the tea plant.

[0013] As preferred, the tea plant volatile with function includes trans-2-hexenal and / or thymol.

[0014] Compared with the prior art, the application has the following beneficial effects: the application provides a method for screening of volatile with function by using a tea plant cell calcium signal detection system and application thereof. The application constructs a tea plant cell calcium signal detection system by loading tea plant protoplast with calcium-specific dye fluo-8 / AM, and realizes visual detection of the calcium signal response of the tea plant cell to environmental factors by using the system, thereby providing a reference for crops lacking stable genetic transformation technology. The application realizes detection of the calcium signal response of the tea plant to released volatile, realizes high-throughput screening, and provides specific small molecule compounds for agricultural production. By verifying the screened volatile with function, it is found that the volatile with function can be successfully screened by using the method of the application. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The tea plant protoplast cell is isolated and obtained; Figure 2 The tea plant protoplast cell is stained with FDA for activity analysis; Figure 3 The cell background calcium ion fluorescence is observed by laser confocal; Figure 4 The volatile capable of inducing the change of the cytoplasmic calcium ion content of the tea plant cell is screened in high throughput; Figure 5 The volatile exposed to the cell calcium response improves the cold tolerance of the tea plant, wherein a is a chlorophyll fluorescence image of the tea plant after cold treatment, the color bar represents the maximum photosynthetic efficiency Fv / Fm value, the blue-violet color represents the normal state of the tea plant, and the yellow color represents the damage of photosystem II caused by cold; b is the maximum photosynthetic efficiency Fv / Fm value; c is the activity of superoxide dismutase (SOD); and d is the activity of peroxidase (POD). DETAILED DESCRIPTION

[0016] The application provides a method for screening functional volatiles by using a tea tree cell calcium signal detection system, comprising the following steps: (1) extracting tea tree leaf protoplast cells; (2) loading the obtained protoplast cells with calcium-specific dyes to obtain a tea tree cell calcium signal detection system; (3) mixing tea tree volatiles with the tea tree cell calcium signal detection system, and selecting tea tree volatiles with enhanced fluorescence intensity as potential functional volatiles.

[0017] In the application, the protoplast cells are loaded with calcium-specific dyes to obtain a tea tree cell calcium signal detection system; fluo-8 / AM is added to the protoplast cells obtained in step (1), and the protoplast cells are treated at 37 DEG C for 30 min, and the cells are gently blown every 10 min to ensure that the fluo-8 / AM is in full contact with the cells, so as to obtain a tea tree cell calcium signal detection system; the volume ratio of the protoplast cells to the fluo-8 / AM is 1 mL: 1 μL, the fluo-8 / AM is used at a concentration of 0.5-50 μM, preferably 1-25 μM, and further preferably 5 μM.

[0018] In the application, tea tree volatiles are mixed with the tea tree cell calcium signal detection system, and tea tree volatiles with enhanced fluorescence intensity are selected as potential functional volatiles. The tea tree volatiles are added to the tea tree cell calcium signal detection system at a final concentration of 0.01-0.1 μM, and the fluorescence intensity of the tea tree cells after adding the volatiles is recorded by using a full-automatic enzyme marker every 1 min, and tea tree volatiles with enhanced fluorescence intensity are selected as potential functional volatiles.

[0019] In the application, the potential functional volatiles obtained in step (3) are used to treat tea trees, and the cold resistance of the treated tea trees is verified.

[0020] The application further provides an application of the method in screening tea tree functional volatiles.

[0021] In the application, the tea tree functional volatiles are cold-induced release volatiles of tea trees.

[0022] In the application, the tea tree functional volatiles are cold-induced release volatiles of tea trees.

[0023] The application further provides an application of the tea tree functional volatiles screened by the method in improving the cold tolerance of tea trees.

[0024] In the present application, the tea tree functional volatile comprises trans-2-hexenal and / or thymol, and the final concentration of the tea tree functional volatile is 0.01-0.1 μM.

[0025] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0026] Examples

[0027] Cold-induced released tea tree volatiles: trans-2-hexenal ((E)-2-hexenal), cis-3-hexenol ((Z)-3-hexenol), 1-hexanol, trans-3-hexenyl acetate ((E)-3-hexenyl acetate), hexyl acetate, 1,2,6-hexanetriol, nonanal, cis-3-hexenyl 3-methylbutanoate ((Z)-3-hexenyl 3-methylbutanoate), thymol, and α-farnesene were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. and Thermo Fisher Scientific. E Z E Z

[0028] Enzymatic hydrolysate system: 20 mL of enzymatic hydrolysate contained 0.4 M mannitol (Sigma, Germany), 0.5% of dissociated enzyme R10 (Yakult, Tokyo, Japan), 0.7% of snail enzyme (YEASEN, Shanghai, China), 1.5% of cellulase R10 (Yakult, Japan), 20 mM 4-morpholine ethanesulfonic acid (MES, pH=5.7), 20 mM KCl (Sigma, Germany), 10 mM CaCl2 (Sigma), 10 mM β-thioethanol (Macklin, Shanghai, China), and 0.1% bovine serum albumin (Sigma). After the enzymatic hydrolysate was dissolved thoroughly, it was filtered with a 0.45 µm filter membrane to remove bacteria.

[0029] Cell culture medium: 0.4 M mannitol solution contained 1 mM MES / KOH, pH 5.7.

[0030] ​​​​W5 solution formula: 4.5g NaCl, 9.2g CaCl2·2H2O, 0.185g KCl, 0.45g glucose, 0.15g MES 4-morpholineethanesulfonic acid, dissolved in 500mL water, 1M KOH to adjust pH to 5.6~5.8.

[0031] Protoplast cell acquisition: take 2g of healthy and fresh tea tree leaves, wash once with 75% alcohol, wash twice with PBS, and reserve. Remove the leaf edge and leaf vein (usually remove the leaf edge by 0.2mm), and then cut the leaves into 0.2~0.5mm wide strips, as thin as possible, which helps to release the cells. The filtered enzyme solution is placed in a glass culture dish (90mm), and the treated leaf strips are placed in the enzyme solution, 20mL of enzyme solution is mixed, and vacuum is applied for 5 minutes. Place the culture dish in a 25℃ incubator in the dark, and enzymolysis for 6 hours. Shake the culture dish gently every hour to ensure that the enzyme solution and the leaves are in full contact.

[0032] After the enzymolysis is completed, 10mL of W5 solution is added to terminate the enzymolysis reaction. Enzymolysis cells, 40µm nylon mesh screening treatment, collect the cell liquid, 100g centrifugation for 2 minutes, acceleration and deceleration settings are 2 (3-18KS, Sigma, Germany), carefully aspirate the supernatant, add 10mL of cell culture solution to resuspend, wash twice, and obtain the tea tree leaf protoplast cell liquid, see Figure 1 .

[0033] As can be seen from Figure 1 , the above method can effectively obtain the tea tree leaf protoplast cells.

[0034] Add fluorescein diacetate (FDA) to the tea tree leaf protoplast cell liquid to a final concentration of 2µg / mL, obtain the stained tea tree leaf protoplast cells, and directly observe the stained tea tree leaf protoplast cells using a confocal laser scanning microscope (Lecia DMi8, Germany), see Figure 2 . Cell quality extraction requirements: tea tree leaf protoplast with cell viability greater than 90% and impurity fragments less than 10% is placed in a 50mL centrifuge tube for reserve.

[0035] As can be seen from Figure 2 , the protoplast cells of the tea tree leaves obtained by the method of the present application have high activity and can be used for subsequent experiments.

[0036] Construction of tea plant cell calcium signal detection system (tea plant cell-specific calcium dye loading): Tea plant leaf protoplast cells were loaded with calcium-specific dye fluo-8 / AM (ATT Bioquest, USA). 10 mL of cell solution was added with 10 µL of calcium-specific dye. The concentration gradient of the calcium-specific dye was set as: 0 µM, 0.5 µM, 1 µM, 5 µM, 25 µM, and 50 µM. The tea plant cell calcium signal detection system was obtained after 30 min of treatment at 37 °C. During the treatment of the tea plant leaf protoplast cells with the calcium-specific dye, the cells were gently blown every 10 min to ensure that the calcium-specific dye and the cells were in sufficient contact. The optimal concentration of the calcium-specific dye was screened.

[0037] Cell background calcium ion fluorescence observation: The tea plant cells loaded with fluo-8 / AM were recovered at room temperature for 1 h to restore the background calcium to a resting state. Then, the laser confocal (Lecia DMi8, Germany) was used to observe the local calcium ion fluorescence of the cells. The excitation light was 488 nm, and the emission light was 520-550 nm. All experiments were performed at room temperature (22-24 °C). The fluorescence intensity was analyzed using ImageJ software (https: / / imagej.net / ij / index.html). To quantify the calcium dynamics, the fluorescence intensity at each time point was normalized to the baseline fluorescence intensity, and the relative calcium change was calculated as ( F ) / F0 . F-F0 F0 The results are shown in Figure 3 .

[0038] As can be seen from Figure 3 , as the concentration of fluo-8 / AM dye increases, the cells have obvious fluorescence at 5 µM dye loading. To facilitate the subsequent observation of the changes in the calcium ion content of the cells induced by environmental factors and to reduce the toxic effects of the dye on the cells, 5 µM was selected as the loading concentration of the calcium-specific dye.

[0039] Calcium response ability detection of tea plant volatiles: 100 µL of the tea plant cell calcium signal detection system was added to a 96-well plate, and 200 µL of cold-induced released tea plant volatile solution was automatically injected into the wells (final concentration of 0.1 µM and 0.01 µM). The fluorescence intensity of the cells was recorded every 1 min after sample addition using a fully automatic enzyme marker. The results are shown in Figure 4 .

[0040] As can be seen from Figure 4 , four kinds of volatiles ( Z )-3-hexenol, thymol, ( E ​α-2-hexenal and α-farnesene significantly induced an increase in cellular calcium ion content.

[0041] Example of effect

[0042] To evaluate the effectiveness of the functional volatiles screened using the method of this invention, examples of the screened ( Z 3-hexenol, thymol, and 1-hexanol and hexyl acetate (which were not screened out) were used as samples of tea plant volatiles released by cold induction to verify the stress tolerance of tea plants.

[0043] 100 µL of cold-induced released tea plant volatiles solution was dropped onto defatted cotton balls and placed in 5-liter glass containers (ensuring the concentrations of cold-induced released tea plant volatiles in the glass containers were 0.1 µM and 0.01 µM, respectively). Healthy tea plants were placed in the glass containers, and the treatment time was 8 hours. The volatiles were dissolved in dimethyl sulfoxide (DMSO), with pure DMSO used as a control. After exposure, the tea plants were treated in a -5°C incubator for 2 hours. Phenotypic observation was performed after treatment, and the results are shown below. Figure 5 .

[0044] Depend on Figure 5 It can be seen that the cold-induced release of tea plant volatiles screened by the method of the present invention can effectively improve the stress tolerance of tea plants, indicating that the method of the present invention can effectively screen out functional volatiles with effects.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for screening functional volatiles using a tea tree cell calcium signal detection system, characterized in that, The method comprises the following steps: (1) extracting protoplast cells from tea leaves; (2) loading the obtained protoplast cells with calcium-specific dye to obtain a tea cell calcium signal detection system; (3) mixing tea volatiles with the tea cell calcium signal detection system, and selecting tea volatiles with enhanced fluorescence intensity as potential functional volatiles.

2. The method of claim 1, wherein, The calcium-specific dye is fluo-8 / AM.

3. The method of claim 1, wherein, The concentration of the calcium-specific dye is 0.5-50 μM.

4. The method of claim 1, wherein, In step (3), a full-automatic enzyme marker is used to observe whether the fluorescence intensity changes.

5. The method of claim 1, wherein, The method further comprises exposing the potential functional volatiles obtained in step (3) to tea plants to verify the cold resistance of the treated tea plants.

6. The use of the method according to any one of claims 1-5 in screening tea functional volatiles.

7. Use according to claim 6, characterized in that, The tea functional volatiles are cold-induced tea volatiles.

8. Use according to claim 7, characterized in that, The final concentration of the tea functional volatiles is 0.01-0.1 μM.

9. The use of the tea functional volatiles screened by the method according to any one of claims 1-5 in improving the cold tolerance of tea plants.

10. The use according to claim 10, characterized in that, The tea functional volatiles comprise trans-2-hexenal and / or thymol.

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