A reactive oxygen species fluorescent probe, its preparation method, and its application in pea seed screening.

Abstract

This invention discloses a reactive oxygen species (ROS) fluorescent probe, its preparation method, and its application in pea seed screening. Belonging to the fields of biotechnology and materials science, the fluorescent probe has the chemical formula shown in formula (I) and includes the following steps: using tetraphenylethylene monoaldehyde and p-bromophenylacetonitrile as raw materials, a cyano-substituted product is obtained through a Knoevenagel condensation reaction; further, a Suzuki coupling reaction is performed with 4-pyridineboronic acid to obtain a pyridine derivative; then, an affinity substitution reaction is carried out under organic base conditions to obtain the ROS fluorescent probe. This invention also discloses the application of this ROS fluorescent probe in pea seed screening. The ROS fluorescent probe provided by this invention can detect ClO₂ accumulated during pea germination under different stress conditions. ‑ Real-time fluorescence imaging monitoring was conducted to clarify the role of ClO2 accumulation during pea germination. ‑ concentration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology and material science, and particularly relates to an active oxygen fluorescent probe, a preparation method thereof and application of the active oxygen fluorescent probe in pea seed screening. BACKGROUND

[0002] The global arable land is increasingly threatened by biological and non-biological pollutants, endangering crop yields and food security. Therefore, it is necessary to match the cultivation site according to the specific requirements of each seed variety to maximize yield and maintain the highest quality of agricultural products. Therefore, seed selection is an important step before the current crop planting, which has positive significance in improving survival rate, improving soil climate adaptability, stress resistance, reducing disease risk, improving yield, ensuring quality, maintaining and increasing genetic diversity, etc. It has important significance for ensuring crop healthy growth, guaranteeing food safety, improving agricultural efficiency, reducing the use of pesticides and fertilizers, and promoting agricultural sustainable development. Moreover, seed selection is also the basis of breeding work, which helps to improve crop characteristics, cultivate new varieties, and improve the market competitiveness of agricultural products.

[0003] For a certain type of crop seeds, the selection of seeds needs to consider the local climate, soil type, pest and disease situation, etc. On this basis, laboratory detection of seeds is needed, including purity, germination rate, seed vigor and health status. Then, after disinfection and soaking, the adaptability of the seeds is tested in a small-scale test field, the growth performance and disease resistance are observed, and the whole process of field performance of the seeds from sowing to harvesting is recorded according to the growth cycle of the crops and the local climate conditions. This process also needs to consider and face various levels of influencing factors, such as sowing density, temperature, orientation, ventilation, drainage, ventilation, flowering pest and disease, etc. Therefore, it is time-consuming and labor-intensive and is greatly affected by environmental factors. Large-area planting is needed to obtain reliable results to screen seeds with strong adaptability and high growth potential, and to improve the survival rate and growth performance of plants in adversity.

[0004] One of the key factors affecting seed germination and harvest is the accumulation of reactive oxygen species (ROS) during the early stages of seed germination. In the germination and early seedling growth of Pisum sativum L., the production of extracellular reactive oxygen species is crucial for plant development and plays a key role in plant growth and stress response, but its specific role is not fully understood. Studies have shown that during the imbibition stage of pea seeds, there is a brief burst of oxidative activity; during the last stage of germination, a second increase in reactive oxygen production is observed, which is associated with an increase in extracellular peroxidase (ECPOX) activity. Therefore, detecting ROS levels during the germination process of pea seeds is crucial for a deeper understanding and improvement of pea breeding mechanisms, especially in response to environmental stress. This not only helps to improve crop yield and quality, but also helps to explore the adaptability of crops to climate change.

[0005] Common methods for detecting reactive oxygen species (ROS) include electrochemistry, photon emission, gene expression markers, nuclear magnetic resonance (NMR), and chemical staining techniques. The detection of ROS levels during the germination process of pea seeds can also be done through various methods, including the use of specific detection reagents (for detecting H2O2), biochemical analysis techniques (for detecting malondialdehyde MDA), spectrophotometry (for measuring extracellular superoxide O2 - and H2O2, extracellular peroxidase ECPOX), antioxidant enzyme activity analysis (indirectly assessing ROS levels by measuring the activities of superoxide dismutase SOD, catalase CAT, and ascorbate peroxidase APX), and nutritional functional component change analysis (measuring total phenol content as an indirect indicator of ROS content changes). Through these methods, researchers can better understand the dynamic changes of ROS during the germination process of peas and their impact on plant growth. Choosing the right detection method requires considering the experimental purpose, operability, and sensitivity and specificity of the detection. Among these methods, chemical staining spectrophotometry uses dyes sensitive to ROS, which react with ROS to generate fluorescent products with corresponding changes in fluorescence signal. By observing the changes in fluorescence intensity or emission band, the method is increasingly favored by researchers because of its high sensitivity, specificity, real-time monitoring capability, and ease of use. In addition, this method is versatile and can be used at various tissue and cellular levels, covering different components of plant seeds.

[0006] Existing chemical staining spectrophotometry for ROS detection commonly uses traditional coplanar dyes. However, most of these dyes undergo concentration quenching, meaning the fluorescence signal decreases as the dye concentration increases to a certain level. Therefore, these dyes may cause false negatives when ROS concentrations continue to rise. In contrast, aggregation-induced luminescence (AIEgens) typically exhibit a positive correlation between fluorescence signals and dye concentration over a wider concentration range, potentially making them an effective alternative for detecting ROS changes. However, to date, AIEgens have not been used for qualitative and quantitative tracking of ROS during plant seed germination. Therefore, developing a rationally designed structure that can provide accurate and reliable information on ROS changes during pea seed germination is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes a reactive oxygen species fluorescent probe, its preparation method, and its application in pea seed screening.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A reactive oxygen species fluorescent probe, with the chemical formula shown below:

[0010]

[0011] (I);

[0012] Wherein, X is independently selected from anions;

[0013] R1 is independently selected from electron-donating groups of aromatic ring derivatives;

[0014] R2 is independently selected from 4-boronic acid phenyl and / or 4-methylphenylboronic acid pinacol ester.

[0015] Preferred, symbol Represents Ph- (phenyl), vinyl, styrene, etc.

[0016] Preferably, the anion includes iodide ions (I). - Bromide ions (Br) - OH- ions - nitrate NO3 - and hexafluorophosphate ions PF6 - One or more of them.

[0017] Preferably, the electron-donating group of the aromatic ring derivative includes one or more of the following: diethylaminophenyl, dimethylaminophenyl, carbazolephenyl, carbazoleyl, phenthiazinyl, phenoxazinyl, 9,10-dihydro-9,9-dimethylacridinyl, 9,10-dihydro-9,9-diphenylacridinyl, 10-H-spiro[acridin-9,9'-fluorene]yl, diphenylamino, triphenylamino, cycloocta-tetrathiophene, diphenylaminothiophene, bithiophene, fused thiophene, thienocyclopentadienyl, naphthamidophenyl, or dipyridinylamino.

[0018] More preferably, the reactive oxygen species fluorescent probe specifically includes the following structure:

[0019] .

[0020] A method for preparing a reactive oxygen species fluorescent probe includes the following steps:

[0021] Using aldehydes and bromine-substituted aromatic cyano groups with AIE properties as raw materials, a cyano-substituted product is obtained through a Knoevenagel condensation reaction. This product is then further coupled with basic aromatic boric acid via a Suzuki coupling reaction to obtain a basic aromatic derivative. Subsequently, an affinity substitution reaction is carried out under organic base conditions, followed by a reaction with a metal salt to obtain the active oxygen fluorescent probe.

[0022] Preferably, the aldehyde group with AIE properties includes tetraphenylethylene monoaldehyde;

[0023] The bromine-substituted aromatic cyano group includes p-bromophenylacetonitrile;

[0024] The basic aromatic boric acid includes 4-pyridineboronic acid.

[0025] Preferably, the organic base includes hexahydropyridine, pyridine, and potassium tert-butoxide.

[0026] Preferably, the preparation method of compound E includes the following steps:

[0027] Using tetraphenylethylene monoaldehyde and p-bromophenylacetonitrile as raw materials, the corresponding cyano-substituted products were obtained via Knoevenagel condensation. Further Suzuki coupling reaction with 4-pyridineboronic acid yielded the corresponding pyridine derivatives. Then, under organic base conditions, affinity substitution reaction with 4-bromomethylphenylboronic acid or the corresponding pinacol ester yielded reactive oxygen species probes.

[0028] Furthermore, the preparation method of tetraphenylethylene monoaldehyde is as follows: using triphenylbromoethylene and 4-formylphenylboronic acid as starting materials, tetrahydrofuran as solvent, potassium carbonate as an alkaline environment, and tetra(triphenylphosphine)palladium as a catalyst, the reaction is carried out under inert gas protection by heating.

[0029] This invention introduces rotational / vibrational electron donors into tetraphenylethylene structural units, constructing a strong charge transfer (CT) state while suppressing intermolecular π-π stacking interactions in the aggregated state, thereby endowing the material with significant long-wavelength emission and AIE properties. Furthermore, introducing 4-boronic acid benzyl into the other side of the corresponding derivative, which is a strong electron acceptor (such as pyridine), can act as a reactive oxygen species such as ClO. - The detection group interacts with it, and after molecular rearrangement, it finally... The probe, existing as a pyridine derivative, departs from its original form, achieving a shift and intensity change in the fluorescence emission spectrum, thus enabling highly selective and sensitive detection of ClO. - Furthermore, the fluorescent probe provided by this invention can accurately analyze the accumulation and expression levels of reactive oxygen species in pea seeds during germination under stress conditions through fluorescence imaging, and has good application prospects in the field of pea seed screening under stress conditions.

[0030] Application of a reactive oxygen species fluorescent probe in pea seed screening.

[0031] Preferably, the pea seed screening involves fluorescence imaging analysis of the accumulation and expression levels of reactive oxygen species during the germination process of pea seeds under stress.

[0032] Preferably, the active oxygen includes ClO - .

[0033] Preferably, the stress environment includes one or more of salt stress, alkali stress, heavy metal stress, and simulated drought.

[0034] Compared with the prior art, the present invention has the following advantages and technical effects:

[0035] This invention synthesizes a novel reactive oxygen species (ROS) fluorescent probe material system with AIE performance, overcoming the fluorescence aggregation quenching effect of existing technologies, and proposes a semi-quantitative prediction selection process for optimal pea varieties under different stress conditions. The probe provided by this invention has a simple synthesis method, readily available raw materials, high yield, and stable structure, and can rapidly detect ClO₂ under ambient temperature and pressure. - The highly sensitive and selective recognition response has a detection limit as low as 26 nmol / L. The reactive oxygen species fluorescent probe provided by this invention can detect ClO₂ accumulated during pea germination under different stress conditions. - Real-time fluorescence imaging monitoring was performed. Furthermore, this invention reveals the mechanism by which reactive oxygen species fluorescent probe materials are used for seed selection in peas under different stress conditions, clarifying the mechanism based on the accumulation of ClO₂ during pea germination. - concentration. Attached Figure Description

[0036] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0037] Figure 1 In the image, A represents the UV-Vis absorption spectrum of TPEN in DMSO-H2O systems with different water contents; B represents the fluorescence emission spectrum of TPEH in DMSO-H2O systems with different water contents; C represents the fluorescence emission peak position graph of TPEH in DMSO-H2O systems with different water contents; and D represents the relative fluorescence intensity (I / I0) graph and photograph under a 365 nm UV lamp in DMSO-H2O systems with different water contents.

[0038] Figure 2 In the image, A represents the UV-Vis absorption spectrum of TPEN (10 M) containing 5.0 equivalents of the analyte; B represents the relative fluorescence intensity of a solution containing 5.0 equivalents of TPEN (10 M); and C represents the relative fluorescence intensity of a solution containing 0-5.0 equivalents of ClO₂. - UV-Vis absorption spectrum of TPEH solution (10 M); D is fluorescence emission spectrum;

[0039] In the figures, the insets in A and B are the corresponding fluorescence emission spectra, and the insets in C and D are the fluorescence spectra under natural light (C) and ultraviolet light (D) respectively, after the addition of ClO₂. - Visual changes after applying TPEN solution, and linear fitting of fluorescence emission intensity ratio (I / I0); the labels in Part B are: 0, blank; 1, PO4. 3- ;2,I - 3, NO3 - 4, AcO - ;5,SO4 2- 6, ClO3 - 7, SCN - ;8, CO3 2- ;9,PF6 - ;10,Br - ;11,Cl - ;12,1O2;13,·OH;14,ONOO - ;15, H2O2;16, ClO - ;

[0040] Figure 3 With or without the mitigating effect of Bacillus subtilis, (A) pure water, (B) NaCl, (C) Na2CO3, (D) PEG simulating drought, and (E) heavy metal Pb 2+ Fluorescence imaging of pea germination 72 hours after induced oxidative stress;

[0041] Figure 4To simulate drought and (D) heavy metal Pb in different (A) NaCl, (B) Na2CO3, (C) PEG. 2+ The relationship between fluorescence signal intensity and root length in five pea varieties under stress conditions;

[0042] Figure 5 (A) is the fluorescence signal intensity captured in the fluorescence image of peas 72 hours after germination under pure water and alkali stress; (B) is the vigor index of peas 10 days after germination under pure water and alkali stress; (C) is the relative value of pea planting and harvesting parameters under pure water and alkali stress, including pod wet weight, bean wet weight, and dry weight of 1,000 peas; (D) is the fluorescence signal intensity captured in the fluorescence image of crisp peas 72 hours after germination under pure water and alkali stress; (E) is the vigor index of crisp peas 10 days after germination under pure water and alkali stress; (F) is the relative value of crisp pea planting and harvesting parameters under pure water and alkali stress, including pod wet weight, bean wet weight, and dry weight of 1,000 peas.

[0043] Figure 6 TPEH fluorescence imaging signals captured in 10 different pea seeds under alkaline stress (A) and no alkaline stress (B) environments;

[0044] Figure 7 (A) The relative values ​​of fluorescence intensity captured 72 hours after germination of 10 pea seeds under alkali stress conditions; (B) The relative values ​​of germination rate of 10 pea seeds captured on the 10th day of growth under natural or alkali stress conditions; (C) The relative values ​​of pea vigor index under different conditions. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] In the embodiments of this invention, room temperature or normal temperature refers to 25±3℃.

[0048] All raw materials used in the embodiments of this invention were purchased through commercial channels.

[0049] Example 1

[0050] A method for preparing a reactive oxygen species fluorescent probe (TPEN), the structural formula of which is shown below:

[0051]

[0052] The preparation method of this reactive oxygen species fluorescent probe (TPEN) includes the following steps:

[0053] (1) Synthesis of intermediate TPE-CHO:

[0054] The synthesis route is as follows:

[0055]

[0056] Specifically, the following steps are included:

[0057] Triphenylbromoethylene (2.00 g, 5.97 mmol), 4-formylphenylboronic acid (1.07 g, 7.16 mmol), and 45 mL of tetrahydrofuran solution were added to a 250 mL round-bottom flask. The mixture was stirred at room temperature, and then 15 mL of potassium carbonate aqueous solution (2 mol / L) was added. Tetra(triphenylphosphine)palladium (138 mg, 119.32 μmol) was added under N2 protection to initiate the reaction. The reaction temperature was controlled at 65 °C and refluxed for 12 h. After the reaction was complete, the solution was cooled to room temperature and extracted with saturated brine and dichloromethane. The resulting extract was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness. Column chromatography [eluent: V(dichloromethane) / V(petroleum ether) = 1:1] yielded 1.61 g of a pale yellow solid, with a yield of approximately 75%.

[0058] FT-IR (KBr) ν (cm -1 ): 3047, 2821, 2724, 1951, 1698, 1601, 1476, 1440, 1301, 1215, 1161, 1075, 1032, 855, 828, 776, 763, 747, 731, 700, 627. 1 HNMR (400 MHz, DMSO-d) 6 ): 6.98-7.00 (m, 6H), 7.14-7.20 (m, 11H), 7.67 (d, J = 8.24 Hz, 2H), 9.88 (s, 1H). 13 C NMR (100 MHz, DMSO-d) 6) δ: 127.42, 127.56, 128.41,128.56, 129.59, 131.12, 131.20, 131.94, 134.69, 140.14, 142.84, 142.99,143.14, 150.21, 193.05.

[0059] (2) Synthesis of intermediate TPEA:

[0060] The synthesis route is as follows:

[0061]

[0062] Specifically, the following steps are included:

[0063] In a 100 mL round-bottom flask, TPE-CHO (800 mg, 2.22 mmol), 4-bromophenylacetonitrile (522 mg, 2.66 mmol), potassium tert-butoxide (498 mg, 4.44 mmol), and 20 mL of ethanol obtained in step (1) were added and reacted at 80 °C under reflux for 3 h. After the reaction was completed, a solid precipitated out. The solid was filtered while hot and washed with ethanol to obtain a crude product. The crude product was then subjected to column chromatography [eluent: V (petroleum ether) / V (dichloromethane) = 2:1] to obtain approximately 0.84 g of a yellow-green solid, with a yield of 70%.

[0064] FT-IR (KBr) ν (cm -1 ):3020, 2348, 2214, 1653, 1595, 1488, 1445, 1402,1184, 1074, 1010, 909, 833, 754, 702, 640, 612. 1 HNMR (400 MHz, DMSO-d 6 ): 6.98-7.00 (m, 6H), 7.14-7.20 (m, 11H), 7.67 (d, J = 8.24 Hz, 2H), 9.88 (s, 1H). 13CNMR (100 MHz, DMSO-d 6) δ: 109.02, 118.20, 123.01, 127.34, 127.46, 128.28,128.39, 128.53, 129.35, 131.15, 131.22, 131.26, 131.81, 132.10, 132.63,133.75, 140.28, 142.38, 143.20, 143.40, 146.54.

[0065] (3) Synthesis of intermediate TBEB:

[0066] The synthesis route is as follows:

[0067]

[0068] Specifically, the following steps are included:

[0069] In a 50 mL round-bottom flask, TPEA (650 mg, 1.21 mmol), 4-pyridineboronic acid (178 mg, 1.45 mmol), and 10 mL of dioxane obtained in step (2) were added for reaction. The reaction temperature was set at 100 °C, and the mixture was stirred at room temperature. Then, 5 mL of potassium carbonate aqueous solution (2 mol / L) was added, and tetrakis(triphenylphosphine)palladium (42 mg, 36.21 μmol) was added under N2 protection. The mixture was refluxed for 12 h. After the reaction was completed, the solution was cooled to room temperature and extracted with saturated brine and dichloromethane. The extracted organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The solution was then subjected to column chromatography [eluent: V(dichloromethane) / V(ethyl acetate) = 4:1] to obtain 0.58 g of a yellow-green solid, with a yield of approximately 90%.

[0070] FT-IR (KBr) ν (cm -1 ):3026, 2358, 2207, 1593, 1487, 1442, 1403, 1076,912, 807, 739, 693, 611. 1 HNMR (400 MHz, DMSO-d 6 ): 7.02-7.08 (m, 6H), 7.10-7.15(m, 11H), 7.48 (s, 1H), 7.52 (dd, J = 4.52 Hz, 2H), 7.67-7.71 (m, 4H), 8.18 (d, J = 8.72 Hz, 2H), 8.68 (dd, J = 4.52 Hz, 2H). 13C NMR (100 MHz, DMSO-d 6 ) δ:109.83, 118.03, 121.51, 126.69, 126.86, 127.04, 127.71, 127.80, 127.98,128.02, 129.01, 131.38, 131.46, 132.10, 135.53, 138.73, 140.05, 142.49, 142.61, 143.23, 143.39, 146.98, 147.20, 150.53.

[0071] (4) Synthesis of intermediate TBEBr:

[0072] The synthesis route is as follows:

[0073]

[0074] Specifically, the following steps are included:

[0075] In a 50 mL round-bottom flask, TPEB (200 mg, 372.67 μmol), pinacol 4-bromomethylphenylboronic acid (133 mg, 447.21 μmol), potassium tert-butoxide (2 mg, 17.86 μmol), and 10 mL of chloroform obtained in step (3) were added and reacted at 60 °C under N2 protection for 8 h. After the reaction was completed, the mixture was cooled to room temperature, and the organic solvent was evaporated to dryness. Thin-layer chromatography analysis revealed unreacted TPEB in the crude product. The TPEB in the crude product was then washed away with toluene to obtain 100 mg of yellow solid, with a yield of 35.6%.

[0076] FT-IR (KBr) ν (cm -1 ):3333, 3034, 2218, 1637, 1586, 1490, 1441, 1412,1368, 1160, 1071, 1019, 823, 748, 697. 1 H NMR (400 MHz, DMSO-d 6 ): 5.82 (s, 2H), 6.94-7.00 (m, 6H), 7.09-7.14 (m, 12H), 7.47 (d, J = 6.92 Hz, 2H), 7.74 (d, J =7.52 Hz, 2H), 7.81 (d, J = 7.32 Hz, 2H), 7.94 (d, J= 7.88 Hz, 2H), 8.11 (s, 3H), 8.17 (d, J = 8.04 Hz, 2H), 8.55 (d, J = 5.20 Hz, 2H), 9.21 (d, J = 5.32 Hz, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) δ: 63.04, 108.89, 118.16, 125.45, 127.29,127.38, 127.50, 128.15, 128.41, 128.56, 129.60, 131.14, 131.26, 131.87,132.02, 134.28, 135.40, 136.61, 137.92, 140.26, 140.31, 142.51, 143.17,143.37, 144.73, 145.46, 146.93, 154.46. APCI-MS: m / z=671.29 ([M-Br - ]), calcdfor C 47 H 36 BN2O2Br: 751.51.

[0077] (5) Target compound TPEN

[0078]

[0079] Synthetic steps of the target compound TPEN

[0080] Add TPEB (100 mg, 13.3 μmol), silver nitrate (27 mg, 16 μmol), and 10 mL of acetonitrile obtained in step (4) to a 50 mL round-bottom flask. Reflux the mixture in the dark for 4 h. After the reaction is complete, filter off the precipitate, remove the solvent under reduced pressure, wash with water, and dry to obtain 87 mg of yellow solid, with a yield of 89.3%.

[0081] FT-IR (KBr) ν (cm -1 ): 3025, 2209, 1645, 1590, 1491, 1444, 1385, 1345,1188, 1073, 1024, 824, 751, 698.

[0082] Technical effects:

[0083] 1. Characterization of AIE properties of reactive oxygen species fluorescent probes

[0084] Figure 1 The figures show the UV-Vis absorption and fluorescence spectra of the reactive oxygen species (ROS) fluorescent probe obtained in Example 1 under different water content conditions. As can be seen from the figures, the ROS fluorescent probe obtained in Example 1 does not emit light in pure dimethyl sulfoxide solvent. With the addition of water, a poor solvent, the fluorescence gradually increases. When the water content increases to 99%, the fluorescence intensity of TPEN reaches its strongest state, indicating that all TPEN photosensitizers possess AIE characteristics.

[0085] 2. Reactive oxygen species fluorescent probe (TPEN) for ClO - Recognition and detection

[0086] Figure 2 Based on the reactive oxygen species fluorescent probe obtained in Example 1, the reaction of ClO - The identification and detection were conducted in a THF-H2O medium (V:V=4:1), specifically including the following steps: Adding TPEN to a TPEN-THF solution (TPEN concentration 1.0×10⁻⁶)... -5 Adding different equivalents of ClO (mol / L) - (From 0 equivalent to 5.0 equivalent), adjust the water content to V. THF V H2O = 4:1, test the UV-Vis absorption spectrum and fluorescence emission spectrum.

[0087] from Figure 2 Part A and Figure 2 As can be seen from section C, when TPEN is used to detect ClO - At that time, ClO was added to the probe (TPEN) solution. - Subsequently, the UV absorption spectrum of TPEN showed a significant blue shift, and the solution color changed from pink to yellow. This occurred after the addition of other analytes (including 1,PO4). 3- ;2,I - 3, NO3 - 4, AcO - ;5,SO4 2- 6, ClO3 - 7, SCN - ;8, CO3 2- ;9,PF6 - ;10,Br - ;11,Cl - ;12,1O2;13,·OH;14,ONOO - No similar phenomenon was observed with 15, H2O2, etc. However, when ClO was added... -As the equivalent increases from 0 to 5.0, the UV-Vis absorption spectrum exhibits a gradual blue shift, with three isoabsorption points located at 337 nm, 383 nm, and 464 nm. Simultaneously with this change in the UV-Vis absorption spectrum, the fluorescence emission spectrum (…) Figure 2 A significant increase in fluorescence intensity was also observed in the C portion, with the fluorescence emission peak shifting blue from 670 nm to 620 nm, while no similar phenomenon was observed with the addition of other analytes. With ClO - With increasing addition, the fluorescence intensity gradually increases, at a certain ClO - Within the concentration range, ClO - The concentration and intensity change showed a linear relationship, and the fitted limit of detection was 26 nmol·L⁻¹. -1 .

[0088] 3. Imaging of germinating peas using a reactive oxygen species fluorescent probe (TPEN)

[0089] Figure 3 Based on the material TPEN obtained in Example 1, with or without the mitigating effect of Bacillus subtilis, (A) pure water, (B) NaCl, (C) Na2CO3, (D) PEG simulated drought, and (E) heavy metal Pb were used to simulate drought. 2+ Fluorescence imaging of pea germination 72 hours under induced oxidative stress, with the concentration of Bacillus subtilis adjusted to 1.0 × 10⁻⁶. 8 CFU mL -1 The imaging excitation wavelength (ex) was 480 nm, and the fluorescence signal collection band (em) was 620 ± 50 nm. Taking the Zhongwan 11 pea variety as a typical example, after 72 hours of germination in a natural environment, TPEN fluorescence imaging showed that the reactive oxygen species (ROS) produced during germination were mainly located in the seed center, with their content gradually decreasing towards the seed edge. When pea seeds were exposed to salt, alkali, PEG-simulated drought, and heavy metal Pb... 2+ In induced oxidative stress environments, the fluorescence intensity captured by imaging is enhanced compared to environments without stress. When each stress environment is alleviated with Bacillus subtilis, the captured fluorescence intensity is weaker than that in environments without Bacillus subtilis, but stronger than that in natural environments.

[0090] 4. Correlation between the imaging signal of germinating peas and the root length of germinating plants using the reactive oxygen species fluorescent probe (TPEN).

[0091] Figure 4 For salt, alkali, simulated drought, heavy metal Pb 2+Table 1 lists the correlation data between the reactive oxygen species (ROS) content signal captured by the reactive oxygen species fluorescent probe (TPEN) and the root length of peas after 3 days of hydroponics under four different oxidative stress environments (jade pea, mottled pea, sweet and crisp pea, tender No. 2 pea, medium pea No. 6 pea, and medium pea No. 11 pea) 72 hours after germination. Table 1 also includes data on salt stress relief combined with Bacillus subtilis (Bacillus subtilis concentration 1.0 × 10⁻⁶). 8 CFU mL -1 The specific conditions for the culture of *Bacillus subtilis*, including salt, alkali, simulated drought, heavy metal stress, and salt stress relief with *Bacillus subtilis*, are as follows: Salt stress: 100 mM NaCl solution; Alkali stress: 5 mM Na2CO3 solution; Simulated drought stress: 5% PEG solution; Heavy metal stress: 0.5 mM Pb. 2+ Solution, salt stress + Bacillus subtilis: 100 mM NaCl + 1.0 × 10⁻⁶ 8 CFU mL -1 Bacillus subtilis dispersion.

[0092] The fluorescence intensity of peas germinated naturally under pure water conditions is defined as 1.0, and the corresponding root length is defined as 1.0.

[0093] Combination Figure 4 As shown in Table 1, the imaging signal of germinating peas captured by the reactive oxygen species fluorescent probe (TPEN) is negatively correlated with the root length of the germinating seed, indicating that after 72 hours of germination, the total amount of reactive oxygen species accumulated during the germination process has already had an adverse effect on seed germination.

[0094] Table 1

[0095]

[0096] y: Relative fluorescence intensity of peas under salt stress with / without Bacillus subtilis relief; x: Root length of peas under salt stress with / without Bacillus subtilis relief; Sample size: 6.

[0097] 5. Correlation between the imaging signal of peas 72 hours after germination and harvest parameters after six months of actual planting using the reactive oxygen species fluorescent probe (TPEN).

[0098] We used conventional pea cultivation methods to obtain harvest parameters, and then performed a comprehensive analysis with the imaging signal from the pea reactive oxygen species fluorescent probe (TPEN) 72 hours after germination.

[0099] The subjects of the conventional planting study were peas and crisp peas, which were planted in natural soil (control group) and alkaline-stressed soil, respectively. First, the vigor index of pea seedlings cultivated in plant culture boxes for 10 days was examined. Then, the pea seedlings were transplanted into the experimental field soil, with 9 seedlings transplanted per group. Conventional field management was implemented, along with prevention of winter frost damage and spring pests and diseases. After 6 months of planting, crisp peas flowered in April of the following year and were harvested in May, while peas, after apical effect removal, flowered in May of the following year and were harvested in June.

[0100] The results showed that pea plants under alkali stress were superior to the control group in the natural soil environment in terms of harvest. Pea plants subjected to alkali stress had a plant height that increased by 1.43 times, a number of harvested pods that increased by 2.89 times, an average wet weight of pods that increased by 1.39 times, an average wet weight of beans that increased by 1.43 times, and a total harvest yield that increased by 4.25 times. The dry weight of 1,000 seeds reached 134.18±5.15 grams, which was 1.50 times higher than that in the natural soil environment (control group).

[0101] Compared to the improved harvest of *Vigna arvensis* under alkali stress, the harvest of *Vigna arvensis* under alkali stress was worse than that of the control group in natural soil environment. The dry weight of 1000 seeds in the control group was 79.67 ± 14.05 g. Under alkali stress, the plant height of *Vigna arvensis* decreased by 69.19%, and the number of harvested pods, average wet weight per pod, total number of seeds, total wet weight of seeds, total dry weight, and dry weight of 1000 seeds were 93.73%, 48.47%, 62.73%, 56.55%, 8.65%, and 87.69% of those in the control group, respectively.

[0102] The planting and harvesting results, spanning a total of 228 days, showed that alkaline soil had a significant negative impact on the growth of crisp peas, while bromelain peas exhibited effective resistance to this oxidative stress. This planting experiment revealed that the harvest results of the same pea variety under stress and natural conditions showed a completely consistent trend compared to the TBP fluorescence signal detection results observed in the early stages of pea germination. However, analysis using the fluorescence signal detection results only required 73 hours, significantly saving experimental time while ensuring consistent results. The experimental process was simple and effective.

[0103] 6. Imaging signals of reactive oxygen species (TPEN) in peas 72 hours after germination were used to infer the planting conditions under stress, and their application was promoted in 10 pea varieties.

[0104] The changes in the 72-hour germination reactive oxygen species fluorescent probe (TPEN) imaging signal of peas under stress were compared with the actual planting and harvest results and then extended to 10 other pea species. The 10 pea species are: Vitex aegypti, Vitex rescuingii, Vitex arvensis, white pea, Taiwan longevity pea, Rongding longevity pea, Gaoliu No. 1, Delicious pea sprouts, Sweet and crisp pea No. 2, and Longevity sweet pea. Figure 6 Images A and B show TBP fluorescence images captured 72 hours after seed germination of 10 different pea species under alkali-free (A) and alkali-free (B) environments, respectively. The images also include photographs of the 10 pea species cultivated for ten days. The 10 pea species are: 1. Vitex acutum, 2. Vitex negundo, 3. Pea shoots, 4. White pea, 5. Taiwan longevity pea, 6. Rongding longevity pea, 7. Gaoliu No. 1 pea, 8. Delicious pea shoots, 9. Crisp pea 2#, and 10. Longevity sweet pea. The changes in fluorescence intensity are shown in the figures. Figure 7 Part A, Figure 6 The images in C, D, and E respectively show the seedlings of 10 pea seeds after 10 days of germination under alkali-free (C) and alkali-free (D) stress environments. The changes in germination rate and vigor index are displayed. Figure 7 In section BC, the values ​​of each pea variety under natural conditions serve as a control.

[0105] Based on the experimental results of these 10 pea varieties, and the experimental results of the aforementioned 5 pea varieties studied in detail (pea pods, sweet and crisp peas, fresh and tender No. 2, medium pea No. 6, and medium pea No. 11), it can be concluded that the accuracy of predicting the seed germination rate under stress conditions using the AIE molecule fluorescence signal in pea seeds 72 hours after germination reaches 93.8%, and the accuracy of predicting the vigor index reaches 87.5%.

[0106] The seed selection method under stress conditions provided by this invention is crucial for selecting suitable cultivars in various soil environments and determining the optimal planting conditions for new varieties. This AIEgen-based selection strategy is simpler, more convenient, and more reliable than traditional methods, saving significant time.

[0107] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. Use of a reactive oxygen species fluorescent probe in pea seed screening, characterized in that, The active oxygen fluorescent probe has the following chemical formula: ； The pea seeds are screened for the accumulation and expression of active oxygen in the germination process of pea seeds under alkaline stress environment through fluorescence imaging analysis; The pea seeds are one or more of the following: hairy pea, sweet crisp pea, widely distributed wild pea, wild pea for emergency food, pea tip without whiskers, white pea, Taiwan long-lived benevolence, delicious pea seedling, long-lived sweet pea.

2. Use according to claim 1, characterized in that, The preparation method of the active oxygen fluorescent probe comprises the following synthesis route: 。 3. Use according to claim 1, characterized in that, The active oxygen includes CIO - .

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