CN113030052B - A kind of detection method of banana postharvest fruit eATP level - Google Patents

Abstract

The invention relates to a high-efficiency detection method for the eATP level of postharvest banana fruits, comprising: uniform pretreatment of green ripe banana fruits; standardized production of banana peel tissue; preparation of ATP detection working solution; Calculation; efficient determination and calculation of eATP in extracted samples. The present invention takes banana as an example, extracts eATP by making wafers in banana peel tissue, and uses the principle of chemiluminometer (Luminometer) to detect the RLU fluorescence value generated by luciferase catalyzing ATP to detect aging and stress of fruits and vegetables The eATP level in the reaction process has the advantages of high sensitivity, simple operation, good repeatability, and low measurement cost.

Description

A kind of detection method of banana postharvest fruit eATP level

technical field

The invention relates to the field of plant physiology, in particular to a method for efficiently detecting the eATP level of postharvest banana fruit.

Background technique

Adenosine triphosphate (ATP) is the energy currency for all biochemical reactions and signal transmission processes in living cells. At the same time, it can be discharged to the outside of the cell through endocytosis, exocytosis, channel proteins and other pathways to become extracellular ATP (eATP), which plays an important signal. It is involved in the growth and development of plants, and as a damage-associated molecular pattern (DAMP), it plays an important role in the process of plant stress (Choi et al., 2014; Jiang et al., 2007; Li et al., 2018) . Fruit ripening is an active process triggered by internal and environmental factors, and intracellular energy status (iATP) and extracellular eATP changes are an important feature during fruit ripening and storage. Numerous experimental studies have shown that ensuring sufficient intracellular ATP supply and appropriate concentration of fine eATP signaling can delay postharvest senescence, alleviate stress loss, and maintain quality and prolong shelf life (Aghdam et al., 2018). Exogenous ATP treatment can attenuate the effects of postharvest stress and delay postharvest senescence in many horticultural products, such as lychees (Yi et al., 2010), longan (Zhang et al., 2017), carnations (Song et al., 2014), and bean sprouts (Chen et al., 2017). , 2018). Therefore, how to maintain sufficient iATP and appropriate concentration of eATP in the cells of harvested vegetables is an important basis for the development and design of fresh-keeping technology. Although a large number of methods have been reported on how to measure ATP in plant tissue cells, there are few methods on how to measure plant eATP.

In recent years, scientists have invented a non-invasive microelectrode detection method to detect the eATP level in real time. The specific operation is to make a precise metal electrode and wrap a biological enzyme layer (such as glycerol kinase and phosphoglycerol oxidase, hexamethylene phosphate) with ATP as substrate. oxidoreductase systems such as glycokinase and glucose oxidase), by using a microscope and a microscopic operating system, the electrode is sufficiently close to the cell or tissue to be tested. In the presence of ATP, a redox reaction occurs on the surface of the electrode, resulting in electron transfer. The chemical signal is converted into an electrical signal, and the concentration of ATP is indirectly determined by detecting the change of the electrical signal (Compagnone and Guilbault, 1997; Kueng et al., 2004; Llaudet et al., 2005), mainly used in the determination of nerve cells, Xenopus embryos and other cells eATP (Wang Haoran et al., 2017). Although in 2015, Vanegas et al. applied this method to plant cells for the first time, and measured the ATP released by the spores of water fern and when the corn root was touched and damaged, but the experimental operation system was not perfect, and the operation was complicated and the repeatability was poor. , the problem of high measurement cost, and the application of this technology in the field of botanical research is very limited at present, it is difficult to use this technology for large and irregularly shaped plant tissues or organs, such as bananas and other fruits and other tissues. its physiological activities.

In fact, on the one hand, because banana is a typical climacteric fruit, its quality depends to a large extent on post-harvest and post-ripening, and during the post-ripening process, it undergoes a series of physiological and biochemical changes, including color change. Yellowing, softening, starch degradation, and aroma formation (Liu et al., 2009), and banana fruits are susceptible to stresses such as chilling injury, which are closely related to intracellular energy levels (Li et al., 2015; Liu et al., 2019; Wang et al., 2015). Therefore, banana fruit is an ideal material to study the physiological role of eATP in fruit and vegetable aging and stress response. On the other hand, D-luciferin is oxidized to emit light under the catalysis of ATP and luciferase, and the fluorescence RLU value is measured by a chemiluminometer (Luminometer), which has the advantages of high sensitivity, simple operation and low measurement cost. Moreover, so far, there have been no relevant reports and patent publications on the method for measuring eATP level based on chemiluminometer (Luminometer)/luciferase method by standardizing the production of wafers in banana peel tissue to extract eATP.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of efficient detection method of banana postharvest fruit eATP level, to overcome the defects existing in the prior art.

For achieving the above object, the technical scheme of the present invention is: a kind of efficient detection method of banana postharvest fruit eATP level, realizes according to the following steps:

Step S1: unified pretreatment to banana green ripe fruit;

Step S2: standardized production of banana peel tissue;

Step S3: the extraction process of banana peel tissue eATP;

Step S4: preparation of ATP detection working solution;

Step S5: preparation of eATP standard curve;

Step S6: determination and calculation of eATP in the sample.

In the step S1, bananas ( Musa acuminata , AAA group, cv. Brazil) are selected to be harvested on a sunny day, and the fruit plumpness is 7 to 80% during harvesting. The fruit was soaked in 0.5% NaClO solution for 3 minutes and then quickly blotted with paper towels. 60 single fruits with uniform size, no pests and mechanical damage were selected and divided into 2 groups.

In the step S2, use a 1.2 cm diameter hole punch to take the small discs on each banana fruit, place them in a glass container, and record the weight as m, add ultrapure water to immerse the bananas, and gently shake and wash for 3 times. After that, remove the ATP released by the broken cells, then add 150 mL of sterile ultrapure water for vacuum osmosis treatment at a pressure of 75 kPa and maintain for 5 min, so that all the banana peel tissue discs are immersed in the solution and placed in the Shake at 50 rpm at room temperature (25 °C) and low temperature (6 °C), take 1 mL of the solution from the glass container in a sterilized 1.5 mL centrifuge tube at 3 and 6 h, and repeat 3 times.

In the step S3, the centrifuge tube containing the solution was inserted into a floating foam plate, boiled in boiling water at 100 °C for 5 min (to inactivate ATP hydrolase and other kinases and prevent the loss of ATP), and stored in a -20 °C refrigerator for Post detection. Take it out from the -20 ℃ refrigerator, put it on ice to dissolve, centrifuge at 12,000 g for 5 min, and take the supernatant into a new centrifuge tube, which is the ATP extract, which can be temporarily placed on ice.

In the step S4, an appropriate ATP detection working solution is prepared according to the ratio of 100 μL of the detection working solution required for each sample or standard. Dissolve the reagent to be used on ice. The preparation method of ATP detection working solution is to take an appropriate amount of ATP detection reagent (luciferase concentrated mother solution) and detection diluent, and dilute the ATP detection reagent into ATP according to the ratio of 1:9. The detection working solution can be temporarily stored on ice.

The preparation method of the concentrated luciferase mother liquor is: 0.05 mol/L glycyl-glycine buffer, and the specific preparation method is: weigh 0.6606 g of glycyl-glycine and dissolve it in 60 mL of ultrapure water, use 0.2 mol/L potassium hydroxide solution was adjusted to pH 7.6, then 100 mg of bovine serum albumin, 120.4 mg of MgSO ₄ , and 33.6 mg of EDTA-Na ₂ were added, and the volume was adjusted to 100 mL with ultrapure water.

In the step S5, the standard curve is measured and calculated.

In the step S6, rapid determination and calculation of eATP in the extraction sample are performed.

In an embodiment of the present invention, the step S5 further includes the following steps:

Step S51: ATP standard solution (0.5 mM) is diluted to 40, 80, 120, 160, 200 nmol/L according to the measurement range of eATP, and can be temporarily stored on ice.

Step S52: Slowly add 100 μL of ATP detection working solution to the detection well of a white opaque 96-well plate. Place at room temperature for 5 min, so that all the local ATP is consumed, so that the ATP that reduces the background is completely consumed, thereby reducing the influence of the background.

Step S53: Slowly add 100 μL of ATP standard solution or eATP extraction solution to the detection well to prevent air bubbles.

Step S54: After waiting for 5 seconds, use a luminometer to measure the RLU value, and the detection time is 10 seconds.

Step S55: Draw a standard curve according to the concentration of ATP and the determined RLU value.

In the step S6, it also includes the following steps:

Step S61: Add 100 μL of ATP detection working solution to the detection well of a white opaque 96-well plate. Place at room temperature for 5 minutes, so that the local ATP is completely consumed, thereby reducing the background ATP is completely consumed, thereby reducing the impact of the background.

Step S62: Slowly add 100 μL of eATP extract to the detection well of the white opaque 96-well plate to prevent air bubbles.

Step S63: After waiting for 5 seconds, use a luminometer to measure the RLU value, and the detection time is 10 seconds.

Step S64: Calculate the eATP content y in the sample extract according to the standard curve in step 3, and calculate the formula y=13304×RLU-34750. Excel 2016 was used for data sorting and statistics, and Grapgpad Prism 8 software was used for One-Way ANOVA analysis and plotting.

The beneficial effects of the present invention are as follows: the present invention takes banana as an example, extracts eATP by making a wafer in banana peel tissue, and uses a chemiluminometer (Luminometer) to detect the principle of RLU fluorescence value generated by luciferase catalyzing ATP, It has the advantages of high sensitivity, simple operation, good repeatability, and low cost of determination. The method in this study was operated at a low temperature of 6 °C, and the results were stable within a week, which could truly reflect the level of eATP.

Description of drawings

Fig. 1 is a schematic diagram of an efficient detection method for the eATP level of banana postharvest fruit in the present invention.

Fig. 2 is a schematic diagram of the tissue standardized production of banana peel discs in the present invention.

Figure 3 is a schematic diagram of the eATP extraction and measurement of banana peel tissue in the present invention.

Figure 4 is a standard curve diagram of eATP in the present invention.

Fig. 5 is the difference of the eATP level of banana peel tissue at different temperatures in the present invention.

Figure 6 is the difference of eATP levels in low temperature banana peel tissue in the present invention.

Detailed ways

The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings.

The invention provides an efficient detection method for the eATP level of banana postharvest fruit. Unified pretreatment of postharvest fruits; standardized production of peel tissue; preparation of ATP detection working solution; determination and calculation of eATP standard curve; efficient determination and calculation of eATP in extracted samples. The present invention takes banana as an example, extracts eATP by making wafers in banana peel tissue, and uses the principle of detecting the RLU fluorescence value generated by luciferase catalyzing ATP by using a chemiluminometer to detect aging and stress of fruits and vegetables The eATP level in the reaction process has the advantages of high sensitivity, simple operation, good repeatability, and low measurement cost.

Example 1 Efficient detection method of banana postharvest eATP level

It includes the following steps:

Step S1: unified pretreatment to banana green ripe fruit;

Step S2: standardized production of banana peel tissue;

Step S3: the extraction process of banana peel tissue eATP;

Step S4: preparation of ATP detection working solution;

Step S5: preparation of eATP standard curve;

Step S6: determination and calculation of eATP in the sample.

In order for those skilled in the art to further understand the method proposed by the present invention, the specific implementation method of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. In this embodiment, the standardized preparation of the original peel tissue, the extraction process of eATP, and the method for rapid determination of eATP have universality. The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

In this embodiment, as shown in FIG. 1 , it is a flowchart of the method.

In step S1, choose a sunny day to harvest bananas ( Musa acuminata , AAA group, cv. Brazil), the fullness of the fruit is 7 to 80% when harvested, and after being combed, it is divided into individual banana fingers, washed with water, and then used 0.5% Immerse the fruit in NaClO solution for 3 minutes and then quickly dry it with a paper towel. 60 single fruits with uniform size, no pests and mechanical damage were selected and divided into 2 groups.

In step S2, as shown in Figure 2, use a 1.2 cm diameter hole punch to take a small round piece on each banana fruit, only take the peel tissue, place it in a glass container, record the weight as m, and add ultrapure water. Submerge the banana, shake it gently for 3 times, remove the ATP released by the broken cells, then add 150 mL of sterilized ultrapure water, carry out vacuum osmosis treatment, the pressure is 75 kPa, and maintain for 5 minutes, so that all the banana peel tissue discs are Immerse in the solution, shake at 50 rpm at normal temperature (25 °C) and low temperature (6 °C), respectively, and extract 1 mL of the solution from the glass container for 3 and 6 hours in a sterilized 1.5 mL centrifuge tube. , repeat 3 times.

In step S3, as shown in Figure 3, the centrifuge tube containing the solution was inserted into the floating foam plate, boiled in boiling water at 100 °C for 5 min (to inactivate ATP hydrolase and other kinases and prevent the loss of ATP), and stored at -20 ℃ refrigerator for later testing. Take it out from the -20 ℃ refrigerator, put it on ice to dissolve, centrifuge at 12,000 g for 5 min, and take the supernatant into a new centrifuge tube, which is the ATP extract, which can be temporarily placed on ice.

In step S4, an appropriate ATP detection working solution is prepared according to the ratio of 100 μL of detection working solution required for each sample or standard. Dissolve the reagent to be used on ice. The preparation method of ATP detection working solution is to take an appropriate amount of ATP detection reagent (luciferase concentrated mother solution) and detection diluent, and dilute the ATP detection reagent into ATP according to the ratio of 1:9. The detection working solution can be temporarily stored on ice.

The ATP detection diluent is 0.05 mol/L glycyl-glycine buffer. The specific preparation method is as follows: Weigh 0.6606 g of glycyl-glycine, dissolve it in 60 mL of ultrapure water, and adjust it with 0.2 mol/L potassium hydroxide solution. The pH was adjusted to 7.6, and 100 mg of bovine serum albumin, 120.4 mg of MgSO ₄ , and 33.6 mg of EDTA-Na ₂ were added, and the volume was made up to 100 mL with ultrapure water.

Luciferase concentrated stock solution configuration: powder luciferase is the same as the above buffer at 30 mg/mL.

In step S5, the standard curve is measured and calculated.

ATP standard solution (0.5 mM) was diluted with ultrapure water to 40, 80, 120, 160, 200 nmol/L according to the measurement range of eATP, which can be temporarily stored on ice; slowly add 100 μL of ATP detection working solution to white opaque96 in the detection hole of the orifice plate. Place at room temperature for 5 minutes, so that all the local ATP is consumed, thereby reducing the background ATP is completely consumed, thereby reducing the impact of the background; slowly add 100 μL ATP standard solution or eATP extraction solution to the detection well to prevent There are bubbles; after waiting for 5 seconds, use a chemiluminometer (Luminometer) to measure the RLU value, the detection time is 10 seconds, as shown in Table 1; according to the concentration of ATP and the measured RLU value, draw the ATP concentration in excel A scatter plot with RLU values, then adding a trend line and calculating the formula and R ² , can be drawn into a standard curve graph, as shown in Figure 4.

Table 1 Standard ATP concentrations and measured fluorescence RLU values.

In step S6, rapid determination and calculation of eATP in the extraction sample.

Use a pipette to add 100 μL of ATP detection working solution to the detection well of a white opaque 96-well plate, as shown in Figure 3F; leave it at room temperature for 5 minutes, so that all the local ATP is consumed, thereby reducing the background ATP is completely consumed. , so as to reduce the influence of the background; slowly add 100 μL of eATP extract to the detection well of the white opaque 96-well plate to prevent air bubbles; after waiting for 5 seconds, use a chemiluminometer (Luminometer) to measure the RLU value, the detection time is 10 seconds; calculate the eATP content y in the sample extract according to the standard curve in step 3, the calculation formula is y=(13304×RLU-34750)÷m, the unit is nmol ∙ g ^-1 FW. Excel 2016 was used for data sorting and statistics, and Grapgpad Prism 8 software was used for One-Way ANOVA analysis and plotting. As shown in Figure 5 and Figure 6, the extraction effect of eATP at a low temperature of 6 °C is better than that at a normal temperature of 25 °C, and the extraction time at low temperature has little effect on the concentration. Significant impact. As shown in Figure 6, the concentration of eATP increased under low temperature stress, and showed a trend that the longer the stress time, the higher the concentration of eATP. The above data results show that the method in this study operates at a low temperature of 6 °C, and the results are stable within a week, which can truly reflect the level of eATP.

Claims (2)

1. an efficient detection method of banana postharvest fruit eATP level, is characterized in that, comprises the following steps: Step S1: unified pretreatment to banana green ripe fruit; Step S2: standardized production of banana peel tissue; Step S3: the extraction process of banana peel tissue eATP; Step S4: preparation of ATP detection working solution; Step S5: preparation of eATP standard curve; Step S6: determination and calculation of eATP in the sample; The green ripe fruit of banana described in step S1 is that the fullness of the fruit is 7 to 80% during harvesting, and is divided into a single banana finger after being combed; the pretreatment is specifically: clean with clear water, and then soak the fruit with 0.5% NaClO solution 3 After a few minutes, quickly blot dry with paper towels; select 60 single fruits with uniform size, no pests and mechanical damage, and divide them into 2 groups; The standardized production of the banana peel disc tissue described in step S2 includes the following steps: using a puncher to take a small disc on each banana fruit, placing it in a glass container, adding ultrapure water to immerse the banana, and gently shaking and washing 3 times After that, remove the ATP released by the broken cells, then add sterile ultrapure water, and carry out vacuum infiltration treatment, so that all the banana peel tissue discs are immersed in the solution, and shake at 50 rpm at room temperature and low temperature, respectively. 3. Take 1 mL of the solution from the glass container in 6 h into a sterilized 1.5 mL centrifuge tube, and repeat 3 times; the diameter of the hole punch is 1.2 cm, the pressure of the vacuum infiltration treatment is 75 kPa, and the pressure is maintained at 5 min; the normal temperature is 25 °C, and the low temperature is 6 °C; The extraction of eATP from banana pericarp tissue described in step S3 includes the following steps: insert a centrifuge tube containing the solution into a floating foam plate, and cook in boiling water at 100° C. for 5 min to inactivate ATP hydrolase and other kinases to prevent the loss of ATP. 12000 g, centrifuged for 5 min, take the supernatant in a new centrifuge tube, which is the eATP extract; The preparation of the ATP detection working solution in step S4 includes the following steps: mixing the ATP detection reagent and the ATP detection diluent in a volume ratio of 1:9 to prepare the ATP detection working solution; the ATP detection reagent is luciferase Concentrate mother liquor; Described detection diluent is 0.05 mol/L glycyl-glycine buffer; The compound method of described 0.05 mol/L glycyl-glycine buffer is: take 0.6606 g of glycyl-glycine and dissolve it in In 60 mL of ultrapure water, adjust the pH to 7.6 with 0.2 mol/L potassium hydroxide solution, then add 100 mg bovine serum albumin, 120.4 mg MgSO ₄ , and 33.6 mg EDTA-Na2, and make up to 100 mL with ultrapure water The preparation method of described luciferase concentrated mother liquor is: powder luciferase is dissolved in above-mentioned buffer solution at 30 mg/mL; The making of the described eATP standard curve of step S5 comprises the following steps: (1) The 0.5 mM ATP standard solution was diluted to 40, 80, 120, 160, 200 nmol/L according to the measurement range of eATP; (2) Slowly add 100 μL of ATP detection working solution to the detection well of the white opaque 96-well plate; leave it at room temperature for 5 minutes, so that all the ATP in the background is consumed, thereby reducing the influence of the background; (3) Slowly add 100 μL ATP standard solution to the detection well to prevent air bubbles; (4) After waiting for 5 seconds, use a chemiluminometer to measure the RLU value, and the detection time is 10 seconds; (5) According to the concentration of ATP and the measured RLU value, draw a standard curve; The measurement and calculation of eATP in the sample described in step S6 includes the following steps: (1) Add 100 μL of ATP detection working solution to the detection well of a white opaque 96-well plate; leave it at room temperature for 5 minutes, so that all the ATP in the background is consumed, thereby reducing the influence of the background; (2) Slowly add 100 μL of eATP extract to the detection well of a white opaque 96-well plate to prevent air bubbles; (3) After waiting for 5 seconds, use a chemiluminometer to measure the RLU value, and the detection time is 10 seconds; (4) Calculate the eATP content y in the sample extract according to the standard curve in step S5, the calculation formula y=13304×RLU-34750, the unit is nmol. 2. a method as claimed in claim 1 detects the application of banana postharvest fruit eATP level.

Images