A heptamethine cyanine-based compound, and a preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescent probes, and particularly relates to a compound based on heptamethine cyanine and a preparation method and application thereof. A chemical structural formula of the compound based on heptamethine cyanine is shown as formula (I). The compound of heptamethine cyanine provided by the application can be induced to form J-aggregates by multivalent anions, and can be used to evaluate the content of multivalent anions in salt stress Arabidopsis thaliana models. The salt concentration of the root system of Arabidopsis thaliana can be successfully quantified by using the dual-channel imaging function of a confocal fluorescence microscope.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fluorescent probes, and particularly relates to a compound based on heptamethine cyanine as well as a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] When plants grow in a high-salt environment, they are affected by high osmotic potential, which is called salt stress. With the increase of the degree of salt stress, the photosynthesis of plants is affected by ion injury, osmotic injury, feedback inhibition caused by sugar accumulation and other pathways, and the growth and development and yield of crops are greatly limited. Therefore, it is of great significance to develop an effective fluorescent probe for monitoring the salt stress of plants.

[0004] Common methods for detecting salt stress of crops are mostly based on destructive sampling research, which generally reflects the response of crops to salt stress by quantitatively analyzing the changes of physiological and biochemical parameters of crops, such as the response of photosynthesis process of crops to salt stress, the changes of osmotic adjustment substances in crop leaves, the changes of ion content in crop leaves, etc. to achieve the purpose of detecting salt stress. These traditional methods can reflect the response of crops to salt stress to some extent, but they are time-consuming and laborious and require destructive sampling, which cannot efficiently detect salt stress and also limits their application in living systems. Therefore, an efficient and non-destructive method is needed to monitor salt stress. In comparison, the fluorescence imaging technology related to fluorescent probes has become an ideal tool for studying living systems because of its non-invasive, real-time and dynamic visualization characteristics.

[0005] In order to realize the identification of low-osmotic Arabidopsis thaliana and accurately report the degree of salt stress suffered by plants, detecting the salt content of the root tip tissue of Arabidopsis thaliana is a simple and feasible method. The fluorescence intensity of the fluorescent probe used in the prior art depends on the concentration of the fluorescent probe itself and the concentration of the detected substance. When the concentration of the fluorescent probe in the tissue changes, the fluorescence intensity also changes, which will cause errors in the quantitative detection results of the salt content. SUMMARY

[0006] In order to solve the problems of the prior art, the present application aims to provide a compound based on heptamethine cyanine as well as a preparation method and application thereof. The compound of heptamethine cyanine provided by the present application can be induced to form J-aggregates by multivalent anions, and can be used to evaluate the content of multivalent anions in a salt-stressed Arabidopsis thaliana model. The salt concentration of the root system of Arabidopsis thaliana can be successfully quantified by using the dual-channel imaging function of a confocal fluorescence microscope.

[0007] In order to achieve the above-mentioned object, the present application is realized by the following technical scheme:

[0008] In a first aspect, the present application provides a heptamethine cyanine-based compound, whose chemical structural formula is shown as formula (I):

[0009]

[0010] wherein, the naming of JC-OH is 5,5',6,6'-tetrachloro-1,1'-diethyl-3,3'-dihydroxyethyl benzimidazyl carbocyanine iodide, and the naming of JC-C8 is 5,5',6,6'-tetrachloro-1,1'-diethyl-3,3'-dioctyl benzimidazyl carbocyanine iodide.

[0011] In a second aspect, the present application provides a preparation method of the heptamethine cyanine-based compound of the first aspect, comprising the following steps:

[0012] S1, dissolving 5,6-dichloro-2-methylbenzimidazole and 2-iodoethanol or iodo octane in acetonitrile, heating and reacting under an inert atmosphere, cooling to room temperature after the reaction is completed, precipitating a solid, filtering and recrystallizing to obtain an intermediate;

[0013] S2, dissolving the intermediate obtained in step S1, iodoform and DBU in acetonitrile, reacting under an inert atmosphere and in a dark environment, separating and recrystallizing by column chromatography after the reaction is completed to obtain the heptamethine cyanine-based compound.

[0014] Preferably, in step S1, the molar ratio of 5,6-dichloro-2-methylbenzimidazole to 2-iodoethanol or iodo octane is 4.3-4.4:6.5-6.6, the heating and reaction temperature is 89-91℃, and the time is 23-25h.

[0015] Preferably, in step S2, the molar ratio of the intermediate, iodoform and DBU is 1.5:0.67-0.69:10.4-10.6, the reaction temperature is room temperature, and the time is 11-13h.

[0016] In a third aspect, the present application provides a composition comprising the heptamethine cyanine-based compound of the first aspect or a pharmaceutically acceptable salt thereof.

[0017] In a fourth aspect, the present application provides a preparation comprising the heptamethine cyanine-based compound of the first aspect and a pharmaceutical excipient.

[0018] In a fifth aspect, the present application provides the use of the heptamethine cyanine-based compound of the first aspect and / or the composition of the third aspect and / or the preparation of the fourth aspect in uniformly dyeing Arabidopsis thaliana root tip tissue.

[0019] Compounds JC-OH and JC-C8 are sensitive to multivalent anions. In vitro spectral tests found that compounds JC-OH and JC-C8 can be induced to produce J-aggregation by multivalent anions.

[0020] Compound JC-OH uniformly stains Arabidopsis root apical tissue in a short staining time. At a staining concentration of 4 μM and a staining time of 30 minutes, uniform, bright green fluorescence is observed in the root apex, with good overlap with the bright-field image, and almost no fluorescence in the red channel. This demonstrates that JC-OH has excellent staining properties for Arabidopsis.

[0021] In a sixth aspect, the present invention provides use of the heptamethine cyanine-based compound as described in the first aspect and / or the composition as described in the third aspect and / or the preparation as described in the fourth aspect in monitoring the degree of salt stress in Arabidopsis thaliana.

[0022] In the seventh aspect, the present invention provides a method for monitoring the degree of salt stress in Arabidopsis thaliana, using the heptamethine cyanine-based compound as described in the first aspect and / or the composition as described in the third aspect and / or the preparation as described in the fourth aspect to stain the root tip tissue of Arabidopsis thaliana, and using dual-channel imaging of a microscope to detect the stained Arabidopsis thaliana, and monitoring the degree of salt stress in Arabidopsis thaliana by changes in fluorescence intensity.

[0023] Arabidopsis plants were treated with varying concentrations of Na2CO3 or Na2HPO4 solutions to establish a salt stress model. Root apical tissue was then stained with JC-OH and observed using the dual-channel imaging function of a confocal microscope. The results showed that under non-salt stress conditions, Arabidopsis plants emitted bright green fluorescence, while the red channel exhibited almost no fluorescence. As salt concentration gradually increased, the green channel fluorescence gradually weakened, while the red channel fluorescence gradually increased. Therefore, the fluorescence changes in the red and green channels can reflect the changing trend of salt content in the root apex of Arabidopsis, which may provide advantages for agricultural production and crop breeding.

[0024] In an eighth aspect, the present invention provides the use of the heptamethine cyanine-based compound as described in the first aspect and / or the composition as described in the third aspect and / or the preparation as described in the fourth aspect in detecting the alleviation of salt stress levels in Arabidopsis thaliana by spermine treatment.

[0025] When Arabidopsis thaliana under salt stress was treated with spermine to alleviate the salt stress, it was found that compared with the fluorescence emission intensity of the probe under salt stress, the green channel fluorescence brightness of the probe was greatly enhanced after spermine treatment, while the red channel fluorescence intensity was significantly weakened, which was similar to the emission of the probe under non-salt stress environment.

[0026] The beneficial effects achieved by the one or more technical solutions of the application are as follows:

[0027] 1. The compound JC-OH and JC-C8 provided by the application are obtained by reacting 5,6-dichloro-2-methylbenzimidazole with 2-iodoethanol or iodo octane, and then reacting with triiodomethane. The molecules of the compound JC-OH and JC-C8 can form J-aggregates under the induction of external multivalent anions, so that the fluorescence performance changes obviously. The molecule JC-OH with better water solubility can uniformly dye the root tip tissue of Arabidopsis thaliana, and the content of multivalent anions in the salt-stressed Arabidopsis thaliana model is evaluated through double-channel imaging.

[0028] 2. The compound JC-OH provided by the application has a sensitive response to external multivalent anions when dyeing Arabidopsis thaliana.

[0029] 3. The compound JC-OH provided by the application can uniformly dye the root tip tissue of Arabidopsis thaliana when dyeing Arabidopsis thaliana.

[0030] 4. The compound JC-OH provided by the application can detect the concentration of external multivalent anions when dyeing Arabidopsis thaliana.

[0031] 5. The compound JC-OH provided by the application can evaluate the degree of salt stress of Arabidopsis thaliana through the change of double-channel fluorescence intensity when dyeing Arabidopsis thaliana.

[0032] In summary, the compound JC-OH and JC-C8 provided by the application can produce J-aggregation through intermolecular van der Waals force under the induction of multivalent anions, and are not dependent on the concentration of the dye itself. Moreover, the fluorescence probe JC-OH can uniformly dye the root tip tissue of Arabidopsis thaliana, and can detect the change of the concentration of multivalent anions in Arabidopsis thaliana caused by external factors. Most importantly, the response of JC-OH to multivalent anions makes it possible to monitor the degree of salt stress of Arabidopsis thaliana through the fluorescence intensity of double-channel imaging.

[0033] Based on the characteristics that multivalent anions can induce J-aggregation of dyes to produce ratio fluorescence emission, as a necessary application case, the content of multivalent anions in the salt-stressed Arabidopsis thaliana model is evaluated by using the cyanine dye JC-OH. In this case, the salt concentration of the root system of Arabidopsis thaliana is quantified by using double-channel fluorescence imaging of a confocal microscope. The above experimental results make JC-OH a powerful tool for monitoring the salt stress of Arabidopsis thaliana, and it is of great significance for evaluating the salt concentration in agricultural production and crop breeding. BRIEF DESCRIPTION OF DRAWINGS

[0034] The drawings constituting a part of the specification of the application serve to provide a further understanding of the application, and the illustrative embodiments of the application and their description serve to explain the application, and do not constitute an improper limitation on the application.

[0035] Figure 1 Test verification chart for J-aggregation of JC-OH and JC-C8 in Example 3 of the present application, a, b are UV absorption spectrum and fluorescence emission spectrum of JC-OH in different solvents, respectively, c is normalized fluorescence emission spectrum of low concentration of JC-OH in water and PBS, d is the ratio of fluorescence emission intensity of JC-OH in different kinds of ion solution in aggregated state / monomer state, e, f are UV absorption spectrum and fluorescence emission spectrum of JC-C8 in different solvents, respectively, g is normalized fluorescence emission spectrum of low concentration of JC-C8 in water and PBS, h is the ratio of fluorescence emission intensity of JC-C8 in different kinds of ion solution in aggregated state / monomer state;

[0036] Figure 2 Staining chart of probe JC-OH on Arabidopsis root tip tissue under different incubation concentrations (4 μM, 8 μM, 16 μM, 32 μM) in Example 4 of the present application;

[0037] Figure 3 Staining chart of JC-OH with a concentration of 4 μM on Arabidopsis root tip tissue under different incubation times (0 min, 15 min, 30 min, 60 min) in Example 5 of the present application;

[0038] Figure 4 Test result chart for monitoring the degree of salt stress on Arabidopsis by using JC-OH in Example 6 of the present application, a, b, c are two-channel fluorescence imaging charts of 4 μM JC-OH after staining root tip tissue for 30 min after Arabidopsis was not treated or treated with different concentrations (5 mM, 10 mM, 50 mM, 100 mM) of Na2CO3 or Na2HPO4, d is a statistical chart of fluorescence intensity change of green channel and red channel, e is a schematic chart of fluorescence change of the probe on Arabidopsis root under salt stress;

[0039] Figure 5 Fluorescence imaging chart of root tip tissue stained with 4 μM JC-OH under confocal microscope after Arabidopsis was treated in three different ways in Example 7 of the present application. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples and comparative examples.

[0041] The Arabidopsis samples used in each example were cultivated by the following method:

[0042] Using wild-type Arabidopsis, immerse the seeds in 1 mL of 75% alcohol for 10 minutes. Remove excess alcohol with sterile water, then wash the seeds 4 times with sodium hypochlorite solution (200 μM), and wash the seeds several times with sterile water until no sodium hypochlorite solution remains. The dry seeds are cultured in a basal medium containing 50% vitamins (MS), 12% sucrose, and 3.2% agar. The pH value of the culture medium is set to 5.8. Wrap it in tin foil and place it in a refrigerator at 4 degrees Celsius for two days. The culture medium is then transferred to an incubator and further cultivated for 7 days (24±1°C, 60% humidity, 16 / 8h day and night). The 7-day-old Arabidopsis obtained is used for the next experiment.

[0043] Example 1

[0044] The preparation process of compound JC-OH is as follows:

[0045]

[0046] Synthesis of Compound C-OH: 20 mL of acetonitrile, 0.52 mL (6.55 mmol) of 2-iodoethanol, and 1.0 g (4.39 mmol) of 5,6-dichloro-2-methylbenzimidazole were added to a Shrek tube. The mixture was heated to 90°C under nitrogen and allowed to react for 24 hours. After the reaction, the mixture was cooled to room temperature, whereupon a white solid precipitated. After filtration, the solid was recrystallized from ethanol to yield 90% (1.58 g, 3.95 mmol).

[0047] C-OH: 1 H NMR (400MHz, DMSO-d6): δ (ppm): 8.25 (t, 1H, J=

[0048] 13.4Hz),8.01(s,2H),7.94(s,2H),5.87(d,2H,J=13.4Hz),5.22(t,2H,J=5.1Hz),4.33(d ,8H,J=6.9Hz),3.86(d,4H,J=4.7Hz),1.36(t,6H,J=7.1Hz).HRMS:m / zcalcd.for[C-OH-I] + :273.0556,found:273.0554.

[0049] Synthesis of JC-OH: In a Schlenk tube, 25 mL of acetonitrile, 0.6 g (1.5 mmol) of compound C-OH, 0.27 g (0.68 mmol) of iodoform and 0.15 ml (10.5 mmol) of DBU were added. The reaction was stirred at room temperature for 12 h under nitrogen protection in the dark. After the reaction was completed, the final product was separated and purified by column chromatography using a mixture of dichloromethane / ethanol as the developing agent, and then the compound was purified by recrystallization in ethanol. The yield was 10% (0.05 g, 0.073 mmol).

[0050] JC-OH: 1 H NMR (400 MHz, DMSO-d6): δ (ppm): 8.25 (t, 1H, J = 13.4 Hz), 8.01 (s, 2H), 7.94 (s, 2H), 5.87 (d, 2H, J = 13.4 Hz), 5.22 (t, 2H, J = 5.1 Hz), 4.33 (d, 8H, J = 6.9 Hz), 3.86 (d, 4H, J = 4.7 Hz), 1.36 (t, 6H, J = 7.1 Hz).

[0051] 13.4 Hz), 8.01 (s, 2H), 7.94 (s, 2H), 5.87 (d, 2H, J = 13.4 Hz), 5.22 (t, 2H, J = 5.1 Hz), 4.33 (d, 8H, J = 6.9 Hz), 3.86 (d, 4H, J = 4.7 Hz), 1.36 (t, 6H, J = 7.1 Hz). 13 C NMR (101 MHz, DMSO-d6): δ 149.93, 143.48, 133.30, 132.10, 126.20, 126.07, 112.38, 111.42, 86.02, 59.13, 47.67, 13.75. HRMS: m / z calcd for [JC-OH-I] + : 557.0859, found: 557.0870.

[0052] Example 2

[0053] The preparation process of compound JC-C8 is shown as follows:

[0054]

[0055] Synthesis of compound C8: In a Schlenk tube, 20 mL of acetonitrile, 1.06 ml (6.58 mmol) of iodoctane and 1.0 g (4.39 mmol) of 5,6-dichloro-2-methylbenzimidazole were added. It was heated to 90 °C under nitrogen protection and reacted for 24 h. After the reaction was completed, the mixture was cooled to room temperature and white solid was precipitated. After filtration, it was purified by recrystallization in ethanol and ethyl acetate, and the yield was 85% (1.75 g, 3.73 mmol).

[0056] C8: 1H NMR (400 MHz, DMSO-d6): δ (ppm): 8.56 (s, 2H), 4.56 - 4.39 (m, 4H), 2.92 (s, 3H), 1.75 (p, 2H, J = 7.8 Hz), 1.31 (dt, 13H, J 2 = 5.7 Hz), 0.91 - 0.80 (m, 3H). HRMS: m / z calcd for [C8-I] + : 341.1551, found: 341.1546.

[0057] Synthesis of JC-C8: In a Schlenk tube was added 25 mL of acetonitrile, 0.6 g (1.5 mmol) of compound C8, 0.27 g (0.68 mmol) of iodoform and 0.15 ml (10.5 mmol) of DBU. The reaction was stirred at room temperature for 12 hours under nitrogen protection in the dark. After the reaction was completed, the final product was separated and purified by column chromatography with a mixture of dichloromethane / methanol as the developing agent, and then the crude product was purified by recrystallization in ethanol. The yield was 5% (0.03 g, 0.037 mmol).

[0058] JC-C8: 1 H NMR (400 MHz, DMSO-d6): δ (ppm): 8.56 (s, 2H), 4.56 - 4.39 (m, 4H), 2.92 (s, 3H), 1.75 (p, 2H, J = 7.8 Hz), 1.31 (dt, 13H, J 2 = 5.7 Hz), 0.91 - 0.80 (m, 3H). HRMS: m / z calcd for [C8-I]

[0059] 3.3 Hz), 7.89 - 7.79 (m, 1H), 5.91 (d, 2H, J = 13.3 Hz), 4.32 (dt, 8H, J = 35.4, 7.2 Hz), 1.73 (p, 4H, J = 7.3 Hz), 1.44 - 1.18 (m, 26H), 0.87 - 0.76 (m, 6H). 13 C NMR (101 MHz, DMSO-d6): δ 149.20, 142.36, 132.62, 132.21, 126.45, 126.40, 111.95, 111.74, 86.04, 44.78, 31.65, 29.15, 29.06, 28.10, 26.30, 22.51, 14.37, 13.73. HRMS [JC-C8-I] + : calcd.: 693.2838, found: 693.2838.

[0060] Example 3

[0061] Response test of JC-OH and JC-C8 to polyvalent anions.

[0062] UV absorption spectra of JC-OH and JC-C8 in different solvents (a, e in Fig. 1) were obtained using a HITACHI U-2910 UV spectrophotometer with 10 μM JC-OH and JC-C8 solutions prepared in different solvents (CH2Cl2, THF, MeOH, DMSO, Gly, H2O, PBS); fluorescence emission spectra of JC-OH and JC-C8 in different solvents (b, f in Fig. 1) were obtained using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 488 nm excitation; fluorescence emission spectra of low concentration JC-OH (1 μM) and JC-C8 (0.1 μM) (c, g in Fig. 1) were obtained using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 488 nm excitation; fluorescence emission spectra of 2 μM JC-OH and JC-C8 in different ionic solutions (d, h in Fig. 1) were obtained using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 488 nm excitation, and the ratios of fluorescence intensities of aggregates to monomers were analyzed. Figure 1 Figure 1 Figure 1 Figure 1

[0063] Figure 1 From the results in Fig. 1, it can be seen that for JC-OH and JC-C8 in organic solvents, only monomer absorption and fluorescence peaks exist. In water, a new aggregate absorption peak of JC-OH appears at 574 nm, and in PBS buffer, the absorption peak of JC-OH aggregates is further enhanced. Similarly to the absorption spectra, in water, the fluorescence emission peak of JC-OH monomers is stronger than that of aggregates, while in PBS buffer, the fluorescence emission peak of aggregates is stronger than that of monomers. For JC-C8, in water and PBS, the absorption peak is significantly broadened, and there is almost no fluorescence emission peak of monomers. Low concentration of JC-OH almost exists in the form of monomers in water, while in PBS buffer, there is a certain degree of aggregation; the fluorescence emission peak of low concentration of JC-C8 aggregates is more easily captured, and is significantly stronger than that of monomers. These spectral data show that ions in PBS solution can induce the formation of J-aggregates of dyes. The ion selectivity experiments of JC-1 and JC-OH further show that only polyvalent anions can induce the formation of J-aggregates of the two dyes.

[0064] Example 4

[0065] Arabidopsis thaliana was stained with different concentrations of JC-OH.

[0066] ​​​​​To obtain the ideal image, different concentrations of JC-OH were used to stain the root tip tissue of Arabidopsis. The solubility concentration of JC-OH in water was 4 μM, 8 μM, 16 μM, 32 μM, respectively. After incubation for 30 min, the glass slide containing the root tip tissue was placed under the microscope, and the double-channel imaging mode was used for observation. When the staining concentration was 4 μM, the green channel of the root tip produced bright fluorescence, and the bright field image almost completely overlapped with the green channel. The whole root tip tissue was uniformly stained by JC-OH, and there was almost no fluorescence in the red channel. With the increase of the staining concentration of the probe, the fluorescence of the red channel gradually increased. When the staining concentration was 32 μM, the fluorescence intensity of the red channel was similar to that of the green channel.

[0067] As shown in the experimental results, it can be seen that the staining concentration of JC-OH is 4 μM, and the imaging effect of the root tip tissue of Arabidopsis is best. Figure 2

[0068] Example 5

[0069] Fluorescence imaging of JC-OH at different times.

[0070] A plurality of Arabidopsis were stained with 4 μM of JC-OH to study the effect of different staining times on the imaging performance. The incubation time was selected as 0 min, 15 min, 30 min, and 60 min, respectively. After incubation, the glass slide containing the root tip tissue was placed under the microscope, and the double-channel imaging mode was used for observation. When the incubation time was 0 min, almost no background fluorescence was observed. With the extension of the incubation time, the fluorescence signal of the root tip gradually increased. When the incubation time was 30 min, the whole root tip uniformly emitted green fluorescence, and there was no obvious fluorescence in the red channel. Compared with 30 min, there was no obvious change in the fluorescence of the root tip after incubation for 1 h.

[0071] The experimental results are shown in Figure 3 As can be seen from the experimental results, the fluorescence intensity of the root tip tissue after staining reaches a maximum value after 30 min, which is conducive to monitoring the salt stress of plants.

[0072] Example 6

[0073] Monitoring the degree of salt stress of Arabidopsis by using JC-OH.

[0074] A plurality of Arabidopsis were soaked in different concentrations (5 mM, 10 mM, 50 mM, 100 mM) of Na2CO3 or Na2HPO4 solution for 6 hours to induce different degrees of salt stress, and an Arabidopsis salt stress model was established. Then 4 μM of JC-OH was used for staining for 30 min. After incubation, the glass slide containing the root tip tissue was placed under the microscope, and the double-channel imaging mode was used for observation. The confocal image showed that in the non-salt stress environment (such as Figure 4 ​As shown in a in the figure), the root tip showed bright fluorescence only in the green channel. Figure 4 b) or Na2HPO4 (as shown in Figure 4 After treatment (as shown in c), the fluorescence signal of the red channel is enhanced and the fluorescence signal of the green channel is weakened. After that, as the salt concentration continues to increase, the fluorescence signals of the red and green channels continue to change in the opposite direction. The intensity of the fluorescence signal change is shown in Figure 4 Schematic diagram of the fluorescence changes of JC-OH in Arabidopsis roots under salt stress is shown in d. Figure 4 As shown in e.

[0075] The experimental results show that as the salt concentration gradually increases, the fluorescence of the green channel is gradually quenched, while the fluorescence of the red channel is gradually enhanced. Therefore, the degree of salt stress in Arabidopsis thaliana can be monitored by observing the changes in the fluorescence of the red and green channels of JC-OH.

[0076] Example 7

[0077] Detect the changes in salt stress levels in Arabidopsis thaliana after spermine treatment.

[0078] Arabidopsis plants grown from the same batch were divided into three groups: the first group received no treatment and served as a control; the second group was treated with 100 mM Na₂CO₃ to induce salt stress; and the third group was treated with 100 mM Na₂CO₃ and 1 mM spermine. After all three treatments, the plants were stained with 4 μM JC-OH for 30 minutes and observed using dual-channel imaging. In the first group, the root tip tissue emitted bright green fluorescence, with no significant red channel fluorescence. In the second group, the root tip tissue had a strong red channel fluorescence signal and a weak green channel fluorescence intensity. In the third group, the red channel fluorescence in the root tip tissue almost disappeared, while the green channel fluorescence was greatly enhanced, similar to the control group.

[0079] The results are as follows Figure 5 As shown in the figure, the experimental results show that spermine can alleviate the salt stress level of plants, which is in contrast to other experiments, indicating the potential and reliability of JC-OH in the visualization of salt stress monitoring in living plants.

[0080] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An application of a compound based on heptamethine cyanine in uniformly staining Arabidopsis root tip tissue, characterized in that: Its chemical structure is shown below; 、 。 2. Use of a composition comprising a heptamethine cyanine-based compound or a pharmaceutically acceptable salt thereof for uniformly staining Arabidopsis root tip tissue; The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。 3. Use of a preparation comprising a heptamethine cyanine-based compound and a pharmaceutical excipient for uniformly staining Arabidopsis root tip tissue; The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。 4. Application of a compound based on heptamethine cyanine in monitoring salt stress in Arabidopsis thaliana; Its chemical structural formula is shown below; 、 。 5. Use of a composition comprising a heptamethine cyanine-based compound or a pharmaceutically acceptable salt thereof in monitoring the degree of salt stress in Arabidopsis thaliana; The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。 6. Use of a formulation comprising a heptamethine cyanine-based compound and a pharmaceutical excipient in monitoring the degree of salt stress in Arabidopsis thaliana; The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。 7. A method for monitoring the degree of salt stress in Arabidopsis thaliana, characterized in that: The root tip tissue of Arabidopsis thaliana was stained with a compound based on heptamethine cyanine, and the stained Arabidopsis was detected using dual-channel imaging of a microscope to monitor the degree of salt stress in Arabidopsis thaliana by changes in fluorescence intensity. The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。 8. A method for monitoring the degree of salt stress in Arabidopsis thaliana using a composition comprising a heptamethine cyanine-based compound or a pharmaceutically acceptable salt thereof; The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。 9. A method for monitoring the degree of salt stress in Arabidopsis thaliana using a formulation comprising a heptamethine cyanine-based compound and a pharmaceutical excipient; The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。 10. Application of a heptamethine cyanine-based compound in detecting the effect of spermine treatment on the alleviation of salt stress in Arabidopsis thaliana; Its chemical structure is shown below; 、 。 11. Use of a composition comprising a heptamethine cyanine-based compound or a pharmaceutically acceptable salt thereof in detecting the effect of spermine treatment on the alleviation of salt stress in Arabidopsis thaliana; The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。 12. Use of a preparation comprising a heptamethine cyanine-based compound and a pharmaceutical excipient in detecting the effect of spermine treatment on the alleviation of salt stress in Arabidopsis thaliana; The chemical structural formula of the heptamethine cyanine based compound is shown below; 、 。

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