Active oxygen fluorescent probe as well as preparation method and application thereof in pea seed screening

Abstract

The invention discloses an active oxygen fluorescent probe and a preparation method and application thereof in pea seed screening, and belongs to the technical field of biotechnology and material scientificity, the chemical formula of the fluorescent probe is shown in formula (I), the fluorescent probe comprises the following steps: taking tetraphenyl ethylene monoaldehyde and p-bromophenylacetonitrile as raw materials, carrying out Knoevenagel condensation reaction to obtain a cyano substituted product, and carrying out column chromatography on the cyano substituted product to obtain the active oxygen fluorescent probe. Further performing Suzuki coupling reaction with 4-pyridineboronic acid to obtain a pyridine derivative, and performing affinity substitution reaction under the condition of organic alkali to obtain the active oxygen fluorescent probe. The invention also discloses application of the active oxygen fluorescent probe in pea seed screening. The active oxygen fluorescent probe provided by the invention can be used for carrying out real-time fluorescence imaging monitoring on ClO <-> gathered in the pea germination process under different stress environments, and the concentration of ClO <-> gathered in the pea germination process is determined.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and material science technology, and in particular relates to an active oxygen fluorescent probe and a preparation method thereof and application thereof in pea seed screening. Background Art

[0002] Arable land around the world is increasingly threatened by biological and non-biological pollutants, jeopardizing crop yields and food security. Therefore, it is necessary to match tailored cultivation sites to the specific requirements of each seed variety to maximize yields and maintain the highest quality of agricultural products. Therefore, seed selection is an important step before current crop planting. It has positive significance in improving survival rate, improving soil climate adaptability, stress resistance, reducing disease risks, increasing yield, ensuring quality, maintaining and increasing genetic diversity, etc. It is of great significance to ensure healthy crop growth, food safety, improve agricultural efficiency, reduce the use of pesticides and fertilizers, and promote sustainable agricultural development. In addition, seed selection is the basis of breeding work, which helps to improve crop characteristics, cultivate new varieties, and improve the market competitiveness of agricultural products.

[0003] In a specific planting location, the selection of seeds for a certain type of crop needs to consider factors such as local climate, soil type, and pest and disease conditions. On this basis, the seeds need to be tested in the laboratory, including purity, germination rate, seed vitality and health status. Then, after disinfection and soaking, the adaptability of the seeds is tested in a small-scale test field to observe their growth performance and disease resistance. The field performance of the seeds from sowing to harvesting is recorded according to the crop growth cycle and local climatic conditions. This process also needs to consider and face various influencing factors at all levels, such as sowing density, temperature, orientation, ventilation, drainage, air permeability, and pests and diseases during flowering. Therefore, it is time-consuming, labor-intensive, and greatly affected by environmental factors. Large-scale planting is required to obtain reliable results in order to screen out seeds with strong adaptability and great growth potential, and to improve the survival rate and growth performance of plants in adversity.

[0004] One of the key factors affecting seed sowing and harvesting is the accumulation of reactive oxygen species (ROS) in the early stage of seed germination. During the germination and early seedling growth of pea (Pisum sativum L), the production of extracellular reactive oxygen species is essential for plant development and plays a key role in plant growth and development 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 short burst of oxidative activity; in the final stage of germination, synchronized with radicle elongation, a second increase in reactive oxygen production was observed, which was associated with an increase in extracellular peroxidase (ECPOX) activity. Therefore, the detection of ROS content during pea seed germination is crucial for a deep understanding and improvement of the mechanism of pea variety breeding, especially pea cultivation under environmental stress, which will not only help to improve crop yield and quality, but also help to further explore the adaptability of crops to climate change.

[0005] Common methods for detecting reactive oxygen species (ROS) include electrochemistry, photon emission, gene expression labeling, nuclear magnetic resonance (NMR), and chemical staining techniques. The detection of ROS content during pea seed germination can also be carried out by a variety of methods, including the use of specific detection reagents (detection of H2O2), biochemical analysis techniques (detection of malondialdehyde MDA), spectrophotometry (determination of extracellular superoxide O 2- and H2O2, extracellular peroxidase ECPOX), antioxidant enzyme activity analysis (indirectly evaluating ROS levels by measuring the activities of superoxide dismutase SOD, catalase CAT and ascorbate peroxidase APX) and nutritional functional component change analysis (determining total phenolic content as an indirect indicator to evaluate changes in ROS content). Through these methods, researchers can better understand the dynamic changes of ROS during pea germination and its impact on plant growth. The selection of appropriate detection methods requires consideration of experimental purpose, operability, and sensitivity and specificity of detection. Among these methods, chemical staining spectrophotometry uses dyes that are sensitive to ROS, which react with ROS to generate fluorescent products with corresponding changes in fluorescence signals, which are evaluated by observing changes in fluorescence intensity or emission bands. Researchers are increasingly favoring this method because of its high sensitivity, specificity, real-time monitoring capabilities, and ease of use. In addition, this method is versatile and can be used at various tissue and cell levels, covering different components of plant seeds.

[0006] The existing chemical dyeing spectrophotometry that can be used to detect ROS often uses dyes with traditional coplanar structures. However, most of them will experience concentration quenching effects, and the fluorescence signal will decrease when the dye concentration increases to a certain level. Therefore, these dyes may cause false negative signals when the ROS concentration continues to increase to a certain level. In contrast, the fluorescence signal of aggregation-induced luminescence (AIEgens) usually responds positively to the dye concentration over a wider concentration range, which may be an effective alternative for detecting ROS changes. However, to date, AIEgens have not been used to qualitatively and quantitatively track ROS during plant seed germination. Therefore, how to develop a reasonable structural design that can provide accurate and reliable information on ROS changes during pea seed germination is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0007] In order to solve the above technical problems, the present invention proposes an active oxygen fluorescent probe and a preparation method thereof and an application in pea seed screening.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A reactive oxygen species fluorescent probe, the chemical formula is as follows:

[0010]

[0011] Wherein, the X is independently selected from anions;

[0012] The R1 is independently selected from an aromatic ring derivative electron-donating group;

[0013] R2 is independently selected from 4-phenyl borate and / or 4-methylphenylboronic acid pinacol ester.

[0014] Preferably, the symbol represents Ph-(phenyl), vinyl, styryl, and the like.

[0015] Preferably, the counter anion comprises an iodide ion I - 、Br - 、hydroxyl ion OH - , nitrate NO 3- , and hexafluorophosphate ion PF 6- One or more of the .

[0016] Preferably, the electron-donating group of the aromatic ring derivative includes one or more of diethylaminophenyl, dimethylaminophenyl, carbazolephenyl, carbazole, phenothiazine, phenoxazine, 9,10-dihydro-9,9-dimethylacridinyl, 9,10-dihydro-9,9-diphenylacridinyl, 10-H-spiro[acridine-9,9'-fluorene]yl, diphenylamine, triphenylamine, cyclooctatetrathienyl, diphenylaminethienyl, bithienyl, fusedthienyl, thienocyclopentadienyl, naphthylaminophenyl or dipyridinylamine.

[0017] More preferably, the reactive oxygen species fluorescent probe specifically comprises the following structure:

[0018]

[0019] A method for preparing an active oxygen fluorescent probe comprises the following steps:

[0020] Using aldehyde groups and bromine-substituted aromatic cyano groups with AIE properties as raw materials, a cyano-substituted product is obtained through a Knoevenagel condensation reaction, and then a Suzuki coupling reaction is carried out with a basic aromatic boronic acid to obtain a basic aromatic derivative, and then an affinity substitution reaction is carried out under organic base conditions, and then the active oxygen fluorescent probe is obtained by reacting with a metal salt.

[0021] Preferably, the aldehyde group having AIE performance comprises tetraphenylethylene aldehyde;

[0022] The bromine-substituted aromatic cyano group includes p-bromophenylacetonitrile;

[0023] The basic aromatic boronic acid includes 4-pyridineboronic acid.

[0024] Preferably, the organic base includes piperidine, pyridine, and potassium tert-butoxide.

[0025] Preferably, the preparation method of compound E comprises the following steps:

[0026] Using tetraphenylethylene aldehyde and p-bromophenylacetonitrile as raw materials, the corresponding cyano-substituted product is obtained by Knoevenagel condensation reaction. The corresponding pyridine derivative is further obtained by Suzuki coupling reaction with 4-pyridineboronic acid. Then, under the condition of organic base, the reactive oxygen probe is obtained by affinity substitution reaction with 4-bromomethylphenylboronic acid or the corresponding pinacol ester.

[0027] Furthermore, the preparation method of tetraphenylethylene monoaldehyde is as follows: triphenylethylene bromide and 4-formylphenylboronic acid are used as starting materials, tetrahydrofuran is used as solvent, potassium carbonate is used to provide an alkaline environment, tetrakis(triphenylphosphine)palladium is used as a catalyst, and heating reaction is carried out under the protection of inert gas to obtain it.

[0028] The present invention introduces a rotational / vibrational electron donor into the tetraphenylethylene structural unit to construct a strong charge transfer (CT) state while suppressing the intermolecular π-π stacking effect of the molecules in the aggregated state, thereby giving the material significant long-wavelength emission and AIE properties. In addition, a strong electron acceptor (such as pyridine) is introduced into the other side of the corresponding derivative to act as an active oxygen such as ClO - The detection group reacts with it and undergoes molecular rearrangement, and finally The probe exists in the form of pyridine derivatives, which can achieve fluorescence emission spectrum shift and intensity change, thus realizing high selectivity and high sensitivity detection of ClO - Furthermore, the fluorescent probe provided by the present invention can accurately analyze the accumulation and expression of active oxygen in the germination process of pea seeds under stress environment through fluorescent imaging, and has good application prospects in the field of pea seed screening under stress environment.

[0029] Application of a reactive oxygen species fluorescent probe in pea seed screening.

[0030] Preferably, the pea seed screening is to analyze the accumulation and expression level of reactive oxygen species during germination of pea seeds under stress environment by fluorescence imaging.

[0031] Preferably, the active oxygen comprises ClO - .

[0032] Preferably, the stress environment includes one or any combination of salt stress, alkali stress, heavy metal stress and simulated drought.

[0033] Compared with the prior art, the present invention has the following advantages and technical effects:

[0034] The present invention synthesizes a new active oxygen fluorescence probe material system with AIE performance, overcomes the fluorescence aggregation quenching effect of the prior art, and proposes a selection process for semi-quantitative prediction of the optimal pea planting variety under different stress environments. The probe provided by the present invention has a simple synthesis method, readily available raw materials, high yield, and stable structure. It can quickly detect ClO at room temperature and pressure. - The active oxygen fluorescence probe provided by the present invention can detect ClO accumulated during pea germination under different stress environments. - In addition, the present invention reveals the mechanism of active oxygen fluorescent probe materials for pea seed selection under different stress environments, and clarifies the ClO accumulated during pea germination. - concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0036] Figure 1 A is the UV-visible absorption spectrum of TPEN in DMSO-H2O systems with different water contents, B is the fluorescence emission spectrum of TPEH in DMSO-H2O systems with different water contents, C is the line graph of the fluorescence emission peak position of TPEH in DMSO-H2O systems with different water contents, and D is the line graph of the relative fluorescence intensity (I / I0) in DMSO-H2O systems with different water contents and the photograph under 365nm UV light;

[0037] Figure 2 A is the UV-visible absorption spectrum of TPEN (10M) containing 5.0 equivalents of analyte; B is the relative fluorescence intensity of TPEN (10M) solution containing 5.0 equivalents of analyte; C is the relative fluorescence intensity of TPEN (10M) solution containing 0-5.0 equivalents of ClO - UV-visible absorption spectrum of TPEH solution (10 M); D is the fluorescence emission spectrum;

[0038] The insets in A and B are the corresponding fluorescence emission spectra, and the insets in C and D are the fluorescence spectra of ClO added under natural light (C) and UV light (D), respectively. - Visual changes of TPEN solution after TPEN treatment, and linear fitting graph of fluorescence emission intensity ratio (I / I0); the numbers 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 - ;

[0039] Figure 3 (A) pure water, (B) NaCl, (C) Na2CO3, (D) PEG-simulated drought, and (E) heavy metal Pb with or without the mitigation effect of Bacillus subtilis 2+ Fluorescence imaging of pea sprouts after 72 hours of induced oxidative stress;

[0040] Figure 4To simulate drought under different (A) NaCl, (B) Na2CO3, (C) PEG and (D) heavy metal Pb 2+ The relationship between fluorescence signal intensity and root length of five pea varieties under stress environment;

[0041] Figure 5 (A) is the fluorescence signal intensity captured in the fluorescence image after germination of peas for 72 hours under pure water and alkali stress, (B) is the vitality index of peas for 10 days after germination under pure water and alkali stress, (C) is the relative values ​​of pea planting and harvesting parameters under pure water and alkali stress conditions, including pod wet weight, bean wet weight, and 1,000 pea dry weight, (D) is the fluorescence signal intensity captured in the fluorescence image after germination of sweet snap peas for 72 hours under pure water and alkali stress, (E) is the vitality index of sweet snap peas for 10 days after germination under pure water and alkali stress, (F) is the relative values ​​of sweet snap pea planting and harvesting parameters under pure water and alkali stress conditions, including pod wet weight, bean wet weight, and 1,000 pea dry weight;

[0042] Figure 6 The TPEH fluorescence imaging signals captured in 10 different pea seeds under alkaline stress (A) and without alkaline stress (B) environments;

[0043] Figure 7 (A) shows the relative value of fluorescence intensity of 10 kinds of pea seeds captured after germination for 72 hours under alkaline stress or without alkaline stress, (B) shows the relative value of germination rate of 10 kinds of peas captured on the 10th day of growth under natural or alkaline stress conditions, and (C) shows the relative value of vitality index of peas under different conditions. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0046] The room temperature or normal temperature in the embodiments of the present invention refers to 25±3°C.

[0047] The raw materials in the examples of the present invention are all purchased from commercial sources.

[0048] Example 1

[0049] A method for preparing an active oxygen fluorescent probe (TPEN), the structural formula of the active oxygen fluorescent probe is as follows:

[0050]

[0051] The preparation method of the reactive oxygen species fluorescent probe (TPEN) comprises the following steps:

[0052] (1) Synthesis of intermediate TPE-CHO:

[0053] The synthetic route is as follows:

[0054]

[0055] The specific steps include:

[0056] Add triphenylethylene bromide (2.00 g, 5.97 mmol), 4-formylphenylboronic acid (1.07 g, 7.16 mmol) and 45 mL of tetrahydrofuran solution into a 250 mL round-bottom flask. Stir at room temperature, then add 15 mL of potassium carbonate aqueous solution (2 mol / L), add tetrakis(triphenylphosphine)palladium (138 mg, 119.32 μmol) under N2 protection to react, the reaction temperature is controlled at 65°C, and reflux for 12 hours. After the reaction is completed, the solution is cooled to room temperature, extracted with saturated brine and dichloromethane, and the obtained extract is dried over anhydrous sodium sulfate, filtered, and then spin-dried. After column chromatography [eluent: V (dichloromethane) / V (petroleum ether) = 1:1], 1.61 g of light yellow solid is obtained, and the yield is about 75%.

[0057] 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=8.24Hz,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.

[0058] (2) Synthesis of intermediate TPEA:

[0059] The synthetic route is as follows:

[0060]

[0061] The specific steps include:

[0062] In a 100 mL round-bottom flask, TPE-CHO (800 mg, 2.22 mmol) obtained in step (1), 4-bromobenzeneacetonitrile (522 mg, 2.66 mmol), potassium tert-butoxide (498 mg, 4.44 mmol) and 20 mL of ethanol were added to react at 80° C. and refluxed for 3 h. After the reaction, solids were precipitated, which were filtered while hot and washed with ethanol to obtain a crude product, which was then subjected to column chromatography [eluent: V (petroleum ether) / V (dichloromethane) = 2:1] to obtain about 0.84 g of a yellow-green solid with a yield of 70%.

[0063] 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=8.24Hz,2H),9.88(s,1H).13C NMR(100MHz,DMSO-d 6 )δ:109.02,118.20,123.01,127.34,127.46,128.28,128.39,128.53,129.35,131.15,13 1.22,131.26,131.81,132.10,132.63,133.75,140.28,142.38,143.20,143.40,146.54.

[0064] (3) Synthesis of intermediate TBEB:

[0065] The synthetic route is as follows:

[0066]

[0067] The specific steps include:

[0068] In a 50 mL round-bottom flask, TPEA (650 mg, 1.21 mmol), 4-pyridine boronic acid (178 mg, 1.45 mmol), and 10 mL of dioxane obtained in step (2) were added to react. The reaction temperature was set to 100°C, and the mixture was stirred at room temperature. Then, 5 mL of potassium carbonate aqueous solution (2 mol / L) was added. Tetrakis(triphenylphosphine)palladium (42 mg, 36.21 μmol) was added under N2 protection, and the mixture was refluxed for 12 h. After the reaction, the solution was cooled to room temperature, extracted with saturated brine and dichloromethane, and the organic phase obtained by the extraction was dried over anhydrous sodium sulfate, filtered, and spin-dried. After column chromatography [eluent: V (dichloromethane) / V (ethyl acetate) = 4:1], 0.58 g of a yellow-green solid was obtained, and the yield was about 90%.

[0069] 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=4.52Hz ,2H),7.67-7.71(m,4H),8.18(d,J=8.72Hz,2H),8.68(dd,J=4.52Hz,2H). 13 C NMR (100 MHz, DMSO-d 6 )δ:109.83,118.03,121.51,126.69,126.86,127.04,127.71,127.80,127.98,128.02,129.01,131.3 8,131.46,132.10,135.53,138.73,140.05,142.49,142.61,143.23,143.39,146.98,147.20,150.53.

[0070] (4) Synthesis of intermediate TBEBr:

[0071] The synthetic route is as follows:

[0072]

[0073] The specific steps include:

[0074] TPEB (200 mg, 372.67 μmol) obtained in step (3), 4-bromomethylphenylboronic acid pinacol ester (133 mg, 447.21 μmol), potassium tert-butoxide (2 mg, 17.86 μmol) and 10 mL of chloroform were added to a 50 mL round-bottom flask for reaction. The reaction temperature was set at 60° C. and refluxed for 8 h under N2 protection. After the reaction was completed, the mixture was cooled to room temperature and the organic solvent was dried by spin drying. After thin layer chromatography analysis, it was found that unreacted TPEB was present in the crude product. The TPEB in the crude product was then washed away with toluene to obtain 100 mg of a yellow solid with a yield of 35.6%.

[0075] 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=6.92Hz,2H),7.74(d,J=7.52Hz,2H),7.81(d,J=7.3 2Hz, 2H), 7.94 (d, J = 7.88Hz, 2H), 8.11 (s, 3H), 8.17 (d, J = 8.04Hz, 2H), 8.55 (d, J = 5.20Hz, 2H), 9.21 (d, J = 5.32Hz, 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 - ]),calcd for C 47 H 36 BN2O2Br:751.51.

[0076] (5) Target compound TPEN

[0077]

[0078] Synthesis steps of the target compound TPEN

[0079] In a 50 mL round-bottom flask, TPEB (100 mg, 13.3 μmol) obtained in step (4), silver nitrate (27 mg, 16 μmol) and 10 mL of acetonitrile were added. The mixture was refluxed for 4 h in the dark. After the reaction, the precipitate was filtered out, the solvent was removed under reduced pressure, the mixture was washed with water and dried to obtain 87 mg of a yellow solid with a yield of 89.3%.

[0080] FT-IR(KBr)ν(cm -1 ):3025,2209,1645,1590,1491,1444,1385,1345,1188,1073,1024,824,751,698.

[0081] Technical effects:

[0082] 1. Characterization of AIE characteristics of reactive oxygen species fluorescent probe

[0083] Figure 1 The UV-visible absorption spectrum and fluorescence spectrum of the reactive oxygen fluorescent probe obtained in Example 1 under different water content conditions are shown in the figure. It can be seen from the figure that the reactive oxygen fluorescent probe obtained in Example 1 basically does not emit light in pure dimethyl sulfoxide solvent. With the addition of poor solvent water, the fluorescence begins to gradually increase. When the water content is increased to 99%, the fluorescence intensity of TPEN reaches the strongest state, indicating that TPEN photosensitizers all have AIE properties.

[0084] 2. Reactive oxygen species fluorescent probe (TPEN) for ClO - Identification and detection

[0085] Figure 2 Based on the reactive oxygen species fluorescent probe obtained in Example 1, ClO - The recognition and detection of TPEN was carried out in THF-H2O medium (V:V = 4:1), which specifically included the following steps: adding TPEN-THF solution (TPEN concentration was 1.0×10 -5 mol / L) added different equivalents of ClO - (from 0 equivalent to 5.0 equivalent), adjust the water content to V THF :V H2O =4:1, test UV-visible absorption spectrum and fluorescence emission spectrum.

[0086] from Figure 2 Part A and Figure 2 As can be seen in part C, when TPEN is used to detect ClO - When ClO was added to the probe (TPEN) solution -After addition, the UV absorption spectrum of TPEN shifted significantly to the blue, and the color of its solution changed from pink to yellow. 3- ,2:I - ,3:NO 3- ,4:AcO - ,5:SO4 2- ,6:ClO3 - ,7:SCN - ,8:CO3 2- ,9:PF6 - ,10:Br - ,11:Cl - ,12: 1 O2,13:·OH,14:ONOO - ,15:H2O2) had no similar phenomenon. - When the equivalent of increases from 0 to 5.0, the UV-visible absorption spectrum gradually blue-shifts, and there are three isosbestic points, located at 337nm, 383nm and 464nm. At the same time as the UV-visible absorption spectrum changes, the fluorescence emission spectrum ( Figure 2 The fluorescence intensity of the sample (part C) also showed an obvious enhancement, and the fluorescence emission peak shifted from 670nm to 620nm, while no similar phenomenon was observed when other analytes were added. - As the amount of added increases, the fluorescence intensity gradually increases. - In the concentration range, ClO - The concentration added was linearly related to the intensity change, and the lowest detection limit obtained by fitting was 26 nmol·L -1 .

[0087] 3. Imaging of germinating peas using a fluorescent probe for reactive oxygen species (TPEN)

[0088] Figure 3 The results of the experiment were as follows: (A) pure water, (B) NaCl, (C) Na2CO3, (D) PEG simulated drought and (E) heavy metal Pb under the mitigation effect of TPEN obtained in Example 1 with or without Bacillus subtilis 2+ Fluorescence imaging of pea sprouts under induced oxidative stress for 72 hours, where the concentration of Bacillus subtilis was adjusted to 1.0×10 8 CFU mL -1, imaging excitation wavelength ex; 480nm, fluorescence signal collection band em; 620±50nm. Taking Zhongwan No. 11 pea as a typical example, when pea seeds germinated in the natural environment for 72 hours, TPEN fluorescence imaging showed that the reactive oxygen species produced during the germination process mainly existed in the inner part of the seeds, and its content gradually decreased towards the edge of the seeds. When the pea seeds were exposed to salt, alkali, PEG simulated drought, heavy metal Pb 2+ In the induced oxidative stress environment, the fluorescence intensity captured by imaging was enhanced compared to the environment without stress. When each stress environment was alleviated by Bacillus subtilis, the captured fluorescence intensity was weaker than that in the environment without Bacillus subtilis alleviation, but stronger than that in the natural environment.

[0089] 4. Correlation between the imaging signal of pea sprouts using the reactive oxygen species fluorescent probe (TPEN) and the actual root length

[0090] Figure 4 For salt, alkali, simulated drought, heavy metal Pb 2+ The specific data of the correlation between the active oxygen content signal captured by the active oxygen fluorescence probe (TPEN) and the root length of pea seeds after germination for 72 hours under four different oxidative stress environments (jade pea, hemp pea, sweet crisp pea, fresh pea No. 2, medium pea No. 6 and medium pea No. 11) are listed in Table 1. Table 1 also includes salt stress + Bacillus subtilis mitigation (the concentration of Bacillus subtilis was 1.0×10 8 CFU mL -1 ) culture conditions, salt, alkali, simulated drought, heavy metal stress and salt stress + Bacillus subtilis mitigation conditions are: salt stress: 100mM NaCl solution, alkali stress: 5mM Na2CO3 solution, simulated drought stress: 5% PEG solution, heavy metal stress: 0.5mM Pb 2+ Solution, salt stress + Bacillus subtilis: 100 mM NaCl + 1.0 × 10 8 CFU mL -1 Bacillus subtilis dispersion.

[0091] The fluorescence intensity of peas germinated in pure water (naturally) was defined as 1.0, and its corresponding root length was defined as 1.0.

[0092] Combination Figure 4 From the analysis of Table 1, it can be seen that the imaging signal of germinating pea captured by the reactive oxygen fluorescent probe (TPEN) is negatively correlated with the actual root length of germination, indicating that after 72 hours of germination, the total amount of reactive oxygen accumulated during the germination process has had an adverse effect on seed germination.

[0093] Table 1

[0094]

[0095] *y: relative value of fluorescence intensity of pea under salt stress with / without the relief of Bacillus subtilis, x: root length of pea under salt stress with / without the relief of Bacillus subtilis, the number of samples is 6.

[0096] 5. Correlation between the imaging signal of peas 72 hours after germination using the reactive oxygen species fluorescent probe (TPEN) and the harvest parameters of the actual plants after half a year of planting

[0097] Conventional pea planting was used to obtain harvest parameters, which were then comprehensively analyzed with the imaging signal of the pea reactive oxygen fluorescent probe (TPEN) at 72 hours after germination.

[0098] The conventional planting objects were hemp pea and sweet snap pea, which were planted in natural soil (control group) and alkaline stress soil respectively. The vitality index of pea seedlings cultivated in the plant incubator for 10 days was first investigated, and then the pea seedlings were transplanted into the soil of the experimental field, with 9 seedlings transplanted in each group, and conventional field management, prevention of winter frostbite and spring pests and diseases were carried out. After 6 months of planting, sweet snap peas bloomed in April of the following year and were harvested in May, and hemp peas bloomed in May of the following year after the apical effect was removed and harvested in June.

[0099] The results showed that the harvest of pea plants under alkaline stress was superior to that of the control group in the natural soil environment. The plant height of pea plants subjected to alkaline stress increased by 1.43 times, the number of harvested pods increased by 2.89 times, the average wet weight of pods increased by 1.39 times, the average wet weight of beans increased by 1.43 times, the total harvest yield increased by 4.25 times, and the dry weight of each thousand seeds reached 134.18±5.15 grams, which increased by 1.50 times compared with the natural soil environment (control group).

[0100] Compared with the improvement of plant yield of pea under alkaline stress, the yield of sweet snap pea under alkaline stress was worse than that of the control group in natural soil environment. The dry weight of 1,000 seeds in the control group was 79.67±14.05 g. Under alkaline stress, the plant height of sweet snap pea 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 1,000-seed dry weight were 93.73%, 48.47%, 62.73%, 56.55%, 8.65%, and 87.69% of those in the control group, respectively.

[0101] The results of the planting and harvesting, which lasted a total of 228 days, showed that alkaline soil had a significant negative impact on the growth of sweet snap peas, while hemp peas showed effective resistance to this oxidative stress. This planting experiment showed that the harvest results of the same pea variety under stress and natural environments were compared with the TBP fluorescence signal detection results observed in the early stage of pea germination. The change trend was completely consistent, but the analysis of the fluorescence signal detection results only took 73 hours, which greatly saved the experimental time while ensuring the consistency of the result trend. The experimental process was simple and effective.

[0102] 6. The imaging signal of pea reactive oxygen fluorescent probe (TPEN) at 72 hours after germination is used to infer the planting conditions under stress environment and is promoted and applied in 10 types of peas.

[0103] The changes in the imaging signal of the active oxygen fluorescence probe (TPEN) of peas 72 hours after germination under stress environment were compared with the actual planting and harvesting conditions, and then extended to the other 10 types of peas. The 10 types of peas are the widespread wild pea, the wilderness pea, the beardless pea tip, the white pea, the Taiwan longevity kernel, the Rongding longevity kernel, the Gaoliu No. 1, the delicious pea seedlings, the sweet crisp pea No. 2, and the longevity sweet pea, which can be planted in various continents and different soil environments. Figure 6 Figures AB show the TBP fluorescence images of 10 different pea seeds captured after germination for 72 hours in an environment without alkali stress (A) and with alkali stress (B), and the actual photos of 10 peas cultivated for ten days. The 10 peas are: 1: Widespread Wild Pea, 2: Saving Wilderness Pea, 3: Whiskerless Pea Tip, 4: White Pea, 5: Taiwan Longevity Kernel, 6: Rongding Longevity Kernel, 7: Gaoliu No. 1 Pea, 8: Delicious Pea Tip, 9: Sweet and Crisp Pea 2#, 10: Longevity Sweet Pea. The changes in the fluorescence intensity signals are shown in Figure 7 Part A, Figure 6 Figures C and D show the seedlings of 10 pea seeds germinated 10 days after germination under no alkali stress (C) and alkali stress (D). The changes in germination rate and vitality index are shown in Figure 7 In the CD parts, the values ​​of each pea in its natural state are used as a control.

[0104] Combining the experimental results of these 10 types of peas with the experimental results of the above-mentioned 5 types of peas studied in detail (numb pea, sweet crisp pea, fresh and tender No. 2, medium pea No. 6 and medium pea No. 11), it can be analyzed that the accuracy of seed germination rate under stress environment can be estimated by using the fluorescence signal of AIE molecules in pea seeds 72 hours after germination, and the accuracy of estimated vitality index can reach 93.8%, and 87.5%.

[0105] The plant seed selection method under stress environment provided by the present invention is crucial for selecting suitable cultivars in various soil environments and determining the optimal planting conditions for new varieties. Compared with traditional methods, this AIEgen-based seed selection method strategy is simpler, more convenient, has reliable results, and can save a lot of time.

[0106] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A fluorescent probe for active oxygen, characterized in that: The chemical formula is as follows: Wherein, the X is independently selected from anions; The R1 is independently selected from an aromatic ring derivative electron-donating group; R2 is independently selected from 4-phenyl borate and / or 4-methylphenylboronic acid pinacol ester.

2. The reactive oxygen species fluorescent probe according to claim 1, characterized in that: The counter anion includes I - 、Br - OH - 、NO3 - 、SO3H - and PF6 - One or more of the .

3. The reactive oxygen species fluorescent probe according to claim 1, characterized in that: The electron-donating group of the aromatic ring derivative includes one or more of diethylaminophenyl, dimethylaminophenyl, carbazolephenyl, carbazole, phenothiazine, phenoxazine, tetraphenylethylene, 9,10-dihydro-9,9-dimethylacridinyl, 9,10-dihydro-9,9-diphenylacridinyl, 10-H-spiro[acridine-9,9'-fluorene]yl, diphenylamine, triphenylamine, cyclooctatetrathienyl, diphenylaminethienyl, bithienyl, fusedthienyl, thienocyclopentadienyl, naphthylaminophenyl or dipyridinylamine.

4. A method for preparing an active oxygen fluorescent probe according to any one of claims 1 to 3, characterized in that: The following steps are involved: Using aldehyde groups and bromine-substituted aromatic cyano groups with AIE properties as raw materials, a cyano-substituted product is obtained through a Knoevenagel condensation reaction, and then a Suzuki coupling reaction is carried out with a basic aromatic boronic acid to obtain a basic aromatic derivative, and then an affinity substitution reaction is carried out under organic base conditions, and then the active oxygen fluorescent probe is obtained by reacting with a metal salt.

5. Use of an active oxygen fluorescent probe as described in any one of claims 1 to 3 in pea seed screening.

6. The use according to claim 5, characterized in that: The pea seed screening is to analyze the accumulation and expression of active oxygen in the pea seed germination process under stress environment by fluorescence imaging.

7. The use according to claim 5, characterized in that: The active oxygen includes ClO - .

8. The use according to claim 5, characterized in that: The stress environment includes one or any combination of salt stress, alkali stress, heavy metal stress and simulated drought.

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