Amino acid derivative-rhodamine B conjugate, its preparation method and application as a fluorescent tracer

Abstract

The present invention belongs to the technical field of preparation of fluorescent tracers, and particularly relates to an amino acid derivative-rhodamine B conjugate, a preparation method thereof, and an application as a fluorescent tracer. The structural formula of the amino acid derivative-rhodamine B conjugate is as follows:. After the amino acid derivative-rhodamine B conjugate obtained in the present invention is directionally applied to crop plants as a fluorescent tracer, the crop plants can be fluorescently labeled, assisting the machine vision system to distinguish crops from weeds, and performing precise spraying or mechanical weeding according to the spatial distribution of weeds, realizing the automation and intelligence of weed control and reducing the usage amount of herbicides.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of fluorescent tracers, and particularly relates to an amino acid derivative-rhodamine B conjugate, a preparation method thereof, and an application as a fluorescent tracer. Background Art

[0002] During the growth process of crops, weeds, as a type of pest harmful to farmland, compete with crops for resources such as sunlight, water, air, and nutrients, greatly affecting the growth and yield of crops. At present, the common methods for controlling farmland weeds include physical control, chemical control, biological control, and agricultural control, etc. Among them, chemical control is widely used in weed control due to its advantages such as high efficiency, economy, and labor saving. However, the unreasonable use of herbicides has also caused problems such as environmental pollution, phytotoxicity, weed resistance, and pesticide residues. Therefore, improving the effective utilization rate of pesticides, reducing the amount of pesticides used, and developing precise and efficient intelligent plant protection machinery and application technologies are the only way for the development of modern agricultural plant protection. Among them, using modern information technology to distinguish farmland crops from weeds, clarify the spatial distribution law of weeds, and then carry out precise spraying or mechanical weeding for weeds is of great significance for effectively reducing the amount of herbicides used and realizing the wisdom of farmland weed control. At present, machine vision technology is mainly used to identify crops and weeds.

[0003] Machine vision technology uses methods such as deep learning to establish a morphological feature database of crops and weeds, and then identifies crops / weeds, which has the advantages of being fast, low-cost, and high-resolution. However, the existing technology is applicable to the control of inter-row weeds, and the recognition degree of in-row weeds is not high enough, and the recognition degree is relatively low in complex situations (such as under high weed density conditions). A large number of weed and crop feature databases also need to be established, and the preliminary preparation work is more cumbersome; in addition, as plants grow, the morphological features of crops and weeds will also change, further increasing the difficulty of extracting plant morphological features.

[0004] The crop plant signal technology applies an exogenous fluorescent marker to the crop through means such as seed treatment, so that the crop is marked by the fluorescent substance, while the weeds do not contain the marker. By using a CCD camera to capture the characteristic spectrum of the marker, the crop and weeds can be accurately distinguished. Therefore, the crop plant signal technology helps to improve the efficiency and accuracy of farmland automatic weeding, and can establish a high-spatial-resolution precise positioning distribution map of crops / weeds in real time. Compared with the vision technology based on computer learning algorithms, the crop plant signal technology has the advantages of simplicity and accuracy.

[0005] Fluorescent markers are the core and foundation of crop plant signal technology. Existing research has shown that the fluorescent tracers that can be absorbed and conducted by plants are mainly coumarins (such as coumarin 120) and rhodamines (such as rhodamine B, Rh-B) (Wang, Z.; Amirkhani, M.; Avelar, S.A.G.; Yang, D.; Taylor, A.G. Systemic Uptake of Fluorescent Tracers by Soybean (Glycine max (L.) Merr.) Seed and Seedlings. Agriculture 2020, 10, 248. https: / / doi.org / 10.3390 / agriculture10060248). Further research has shown that rhodamine B has unique fluorescence properties (λex / λem = 555 / 582 nm), with a high differentiation degree from the fluorescence properties of plant chlorophyll. It can be distinguished from chlorophyll under a relatively wide range of fluorescence conditions (λex = 535 nm, λem = 580 - 582 nm) and can be used for fluorescence imaging detection. Compared with other fluorescent tracers, rhodamine B has the advantages of low cost, strong photostability, high fluorescence yield, wide wavelength range, and wide pH adaptation range. Moreover, it has good cell membrane permeability and no toxic side effects on cells, making it more promising in plant tracers.

[0006] It was found that rhodamine B can be absorbed by kidney bean and soybean seeds and penetrate into the seeds, or be absorbed by the roots of celery, lettuce, and tomato, and conduct upward through the xylem of the plant to the upper part of the seedlings. However, overall, rhodamine B is mainly distributed in the hypocotyls of plants (soybean, kidney bean). The fluorescence intensity of rhodamine B in the epicotyl is 35% (kidney bean) and 11% (soybean) of that in the hypocotyl, while the fluorescence intensity of rhodamine B in the leaves is only about 8% of that in the hypocotyl, indicating that the upward conduction ability of rhodamine B is weak and the endosmotic conduction performance of this tracer in the plant needs to be further improved.

[0007] Aiming at the problem that the insufficient endosmotic conduction of the existing tracer limits the machine to capture the fluorescence signal, the structure of the existing tracer rhodamine B is optimized to improve its endosmotic conduction performance, and a fluorescent tracer for precision weeding technology is developed. Summary of the Invention

[0008] The object of the present invention is to overcome the defects of the prior art, and a series of amino acid derivative-rhodamine B conjugates, their preparation methods and applications as fluorescent tracers are proposed. On the basis of the existing fluorescent marker rhodamine B, the present invention optimizes the structure, introduces an amino acid fragment into the molecular structure of rhodamine B, constructs a novel fluorescent marker of amino acid derivative-rhodamine B, and develops a fluorescent marker suitable for crop plant signal technology.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The amino acid derivative-rhodamine B conjugate has the following structural formula:

[0011] ;

[0012] Among them, the coupling bond in the above structure is an amide bond formed by the amino group of the amino acid derivative and the carboxyl group of rhodamine B.

[0013] The group R is selected from , , , , , , , , , one of them.

[0014] Further preferably, the group R is selected from or .

[0015] The present invention also provides a preparation method of the above amino acid derivative-rhodamine B conjugate.

[0016] The present invention further provides an application of the above amino acid derivative-rhodamine B conjugate as a fluorescent tracer.

[0017] Preferably, the application of the amino acid derivative-rhodamine B conjugate as a fluorescent tracer in the identification of crops and weeds.

[0018] Further preferably, the crops are soybeans, corn and tobacco.

[0019] The amino acid derivative-rhodamine B conjugate in the present invention includes a conjugate formed by an amino acid methyl ester and rhodamine B and a conjugate formed by an acidified amino acid and rhodamine B.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] After the amino acid derivative-rhodamine B conjugate obtained in the present invention is used as a fluorescent tracer, the content of the tracer in the above-ground part of the crop plants increases significantly, which is conducive to the crop-weed identification by the machine vision system. Precise spraying or mechanical weeding can be carried out according to the spatial distribution of weeds, realizing the automation and intelligence of weed control, significantly reducing the usage amount of herbicides, effectively reducing the impact of chemical herbicides on the ecological environment, alleviating the occurrence of herbicide phytotoxicity, delaying the development of drug resistance, and reducing the risk of pesticide residues. Description of the Drawings

[0022] Figure 1 It is the fluorescence detection diagram in soybean plants;

[0023] Figure 2 It is the fluorescence detection diagram in corn plants;

[0024] Figure 3 It is the fluorescence detection diagram in tobacco plants;

[0025] Figure 4 It is the standard curve of three fluorescent tracers;

[0026] Figure 5 It is the content diagram of the fluorescent tracer in different parts of soybean;

[0027] Figure 6 It is the content diagram of the fluorescent tracer in different parts of corn;

[0028] Figure 7 It is the content diagram of the fluorescent tracer in different parts of tobacco;

[0029] Figure 8 It is the distribution ratio diagram of the fluorescent tracer in soybean plants;

[0030] Figure 9 It is the distribution ratio diagram of the fluorescent tracer in corn plants;

[0031] Figure 10 It is the distribution ratio diagram of the fluorescent tracer in tobacco plants;

[0032] Figure 11 It is the influence diagram of three fluorescent tracers on the growth of soybean plants;

[0033] Figure 12 It is the influence diagram of three fluorescent tracers on the growth of corn plants;

[0034] Figure 13 It is the influence diagram of three fluorescent tracers on the growth of tobacco plants;

[0035] Figure 14 It is the schematic diagram of the sowing positions of crop and weed seeds;

[0036] Note:Figure 5 , 6 Among 7, 11, 12, and 13, different letters indicate significant differences between treatments (P < 0.05). Specific implementation mode

[0037] The technical solution of the present invention will be described in detail below in conjunction with the drawings and embodiments.

[0038] Example 1

[0039] Using rhodamine B and amino acid methyl ester (general formula of amino acid methyl ester ) as raw materials, an amino acid derivative-rhodamine B conjugate was constructed through amidation and ester hydrolysis. The specific synthesis route is as follows in the following reaction formula:

[0040] ;

[0041] Among them, the amidation condition a: N-methylmorpholine (NMM), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), DMAP, anhydrous dichloromethane, r.t (room temperature and standard pressure);

[0042] The ester hydrolysis condition b: lithium hydroxide, THF / H2O (V / V = 2:1), r.t (room temperature and standard pressure);

[0043] In the general formula of amino acid methyl ester , R is determined according to the selected amino acid. For example, the amino acids selected in the present invention are one of isoleucine, tyrosine, tryptophan, proline, and alanine.

[0044] Taking proline as an example, a mixture of proline methyl ester hydrochloride (2.48 g, 15 mmol) and N-methylmorpholine (NMM) (2.20 mL, 20 mmol) was dissolved in anhydrous dichloromethane (150 mL). Then the reaction mixture was cooled to 0 °C, and rhodamine B (4.79 g, 10 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (2.88 g, 15 mmol), and 4-dimethylaminopyridine (DMAP) (0.12 g, 1 mmol) were added successively. The resulting solution was stirred at room temperature for 3 h. After the reaction was completed, water was added, and the organic layer was extracted with dichloromethane (3 × 50 mL). The combined organic layers were washed alternately with saturated sodium bicarbonate aqueous solution and saturated brine solution 2-3 times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was used to obtain compound 2 (proline methyl ester-rhodamine B conjugate, conjugate 7 in Table 1).

[0045] Compound 2 (3.22 g, 5 mmol) was dissolved in a mixed solution of water (10 mL) and THF (20 mL). Lithium hydroxide (0.63 g, 15 mmol) was added under an ice bath. The reaction was slowly warmed to room temperature and continued for 1 h. After the reaction was completed, it was acidified to pH = 2 with 1 M hydrochloric acid solution and extracted with ethyl acetate (3 × 200 mL). The combined organic layers were washed alternately with saturated aqueous sodium bicarbonate solution and saturated brine solution 2 - 3 times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain Compound 3 (proline - rhodamine B conjugate, Conjugate 8 in Table 1).

[0046] Isoleucine methyl ester - rhodamine B conjugate (Conjugate 1 in Table 1), isoleucine - rhodamine B conjugate (Conjugate 2 in Table 1), tyrosine methyl ester - rhodamine B conjugate (Conjugate 3 in Table 1), tyrosine - rhodamine B conjugate (Conjugate 4 in Table 1), tryptophan methyl ester - rhodamine B conjugate (Conjugate 5 in Table 1), tryptophan - rhodamine B conjugate (Conjugate 6 in Table 1), proline methyl ester - rhodamine B conjugate (Conjugate 7 in Table 1), proline - rhodamine B conjugate (Conjugate 8 in Table 1), alanine methyl ester - rhodamine B conjugate (Conjugate 9 in Table 1), and alanine - rhodamine B conjugate (Conjugate 10 in Table 1) were synthesized respectively according to the above method.

[0047] The above amino acid derivative - rhodamine B conjugates were analyzed by hydrogen nuclear magnetic resonance and mass spectrometry, and the data are shown in Table 1.

[0048] Table 1 1H NMR and high - resolution mass spectrometry data of amino acid derivative - rhodamine B conjugates

[0049]

[0050]

[0051]

[0052]

[0053] Example 2

[0054] 1. Qualitative study on the systemic conduction performance of fluorescent tracers in plant plants (taking Tracer Nos. 1, 2, 7, and 8 as examples)

[0055] 1.1 Plant seedling cultivation

[0056] 250 g of substrate was added to a flowerpot with a diameter of 15 cm. Soybean, corn, and tobacco seeds were sown at a depth of 2 cm and cultivated until the true leaves of soybeans were fully unfolded, the first leaf of corn was perpendicular to the leaf sheath, and the tobacco was at the eight - leaf stage.

[0057] Add fluorescent tracers such as rhodamine B or amino acid derivative - rhodamine B conjugate to Hoagland's nutrient solution to prepare a hydroponic solution containing 60 mg / L of the tracer. After cleaning the roots of the cultivated plant seedlings, place them in a plastic cup containing 200 mL of the hydroponic solution, using Hoagland's nutrient solution as the blank control, and culture for 4 days at 25°C under the conditions of 16 h of light / 8 h of darkness. Each treatment is repeated 4 times.

[0058] Preparation method of Hoagland's nutrient solution: Weigh 1.26 g of Hoagland's nutrient solution medium and 0.945 g of calcium nitrate, heat and dissolve in 1000 mL of distilled water, and sterilize at 115°C under high pressure for 20 min for standby.

[0059] 1.2 Research on the systemic conduction ability of fluorescent tracers

[0060] Use a stereoscopic fluorescence microscope to detect the distribution of fluorescent tracers in various parts of the plant, and use whether the tracer can be conducted to the stems and leaves of the plant as a criterion for evaluating its systemic conduction performance (compared with rhodamine B). Divide the soybean seedlings treated in 1.1 into six parts: trifoliate leaves, true leaves, epicotyls, cotyledons, hypocotyls, and roots; divide maize into three parts: roots, leaf sheaths, and leaves; divide tobacco into three parts: roots, stems, and leaves; screen with an RFP detection module (excitation wavelength 541 - 551 nm, detection wavelength 590 nm).

[0061] 1.2.1 Evaluation of the conduction performance of fluorescent tracers in soybean plants

[0062] Observe the above-ground parts (trifoliate leaves, true leaves, epicotyls, cotyledons, hypocotyls) of soybean plants through a stereoscopic fluorescence microscope. As Figure 1 shown, it is found that compared with the control tracer rhodamine B, the fluorescence intensities of tracers No. 7 and No. 8 in the above-ground parts of soybean plants are both enhanced, and the fluorescence intensity of tracer No. 2 in the true leaf part is higher than that of rhodamine B, indicating that coupling with amino acids such as proline can improve the systemic conduction performance of rhodamine B in soybean plants, and isoleucine can enhance the conduction ability of rhodamine B in leaves to a certain extent.

[0063] 1.2.2 Evaluation of the conduction performance of fluorescent tracers in maize plants

[0064] Detect the fluorescence of maize plants through a stereoscopic fluorescence microscope. As Figure 2 shown, it is found that compared with the control agent rhodamine B, the fluorescence intensities of tracers No. 1, No. 2, No. 7, and No. 8 in the above-ground parts (leaves, leaf sheaths) of maize plants are all enhanced, indicating that coupling with amino acids such as isoleucine and proline can improve the systemic conduction performance of rhodamine B in maize plants.

[0065] 1.2.3 Evaluation of the Conductivity Performance of Fluorescent Tracer in Tobacco Plants

[0066] The tobacco seedlings treated with the tracer were detected under a stereoscopic fluorescence microscope. As Figure 3 shown, it was found that compared with the control agent Rhodamine B, the fluorescence intensities of Tracer Nos. 1, 7, and 8 in the above-ground parts (leaves and stems) of tobacco plants were all enhanced, indicating that coupling with amino acids such as isoleucine and proline could improve the systemic absorption and conductivity performance of Rhodamine B in tobacco plants.

[0067] Overall, the fluorescence intensity of the amino acid derivative-Rhodamine B conjugate in the above-ground parts of the plant was higher than that of Rhodamine B, indicating that after coupling of amino acids such as isoleucine and proline with Rhodamine B, there was an obvious improvement effect on the systemic absorption and conductivity performance of Rhodamine B in the plant, especially in monocotyledonous plants (maize).

[0068] 2 Quantitative Analysis of Fluorescent Tracer in Different Parts of Plants

[0069] 2.1 Experimental Method

[0070] Taking Tracer Nos. 7 and 8 as representatives, a liquid chromatograph was used to further detect the content of fluorescent tracer in each part of the plant.

[0071] (1) Sample Treatment

[0072] Respectively take 0.5 g of different parts of soybean, maize, and tobacco seedlings treated with Rhodamine B, Tracer Nos. 7 and 8 in 1.1 treatment. After grinding, add 4 mL of acetonitrile, ultrasonically extract for 40 min, concentrate to 1 mL after leaching for 1 day, and filter through a 0.22 μm organic microporous filter membrane for high-performance liquid chromatography analysis to determine the content of fluorescent tracer in different parts of soybean, maize, and tobacco.

[0073] Liquid Chromatography Detection Conditions: C 18 Chromatographic column (C18 - AQ, 5 μm, 250 mm × 4.6 mm), detection wavelength 560 nm, injection volume 10 μL, flow rate 1 mL / min, column temperature 35°C. The mobile phase is 0.1% formic acid aqueous solution and acetonitrile, and the specific ratios are shown in Table 2:

[0074] Table 2 Mobile Phase Ratios of Three Tracers

[0075]

[0076] (2) Preparation of Standard Solution and Drawing of Standard Curve

[0077] Weigh 5.0 mg of rhodamine B standard, fluorescent tracers No. 7 and No. 8 respectively, dissolve them with acetonitrile and make up the volume to 10 mL to obtain a standard stock solution of 500 mg / L. Then dilute it step by step with acetonitrile, measure the absorption peak areas of each standard solution, and plot the standard curve.

[0078] (3)Recovery test

[0079] Add standard solutions at three levels of 100.0000, 33.3333, and 11.1111 mg / L to blank samples of various parts of soybeans, corn, and tobacco respectively. Each addition level is repeated 3 times. Carry out sample pretreatment and analysis according to the method, and calculate the recovery rate and relative standard deviation of the addition.

[0080] 2.2 Test results

[0081] 2.2.1 Construction and verification of the HPLC analysis method

[0082] Plot the standard curve according to the concentration gradient. The correlation coefficients of the three tracers, rhodamine B, No. 7, and No. 8, are 0.9996, 0.9990, and 0.9935 respectively. There is a good linear relationship between the mass concentration and the corresponding response value within the concentration range of the standard curve ( Figure 4 Note: A. Rhodamine B; B. Tracer No. 7; C. Tracer No. 8). As shown in Table 3, the average recovery rates of rhodamine B, No. 7, and No. 8 tracers are between 81% and 109%, and the relative standard deviations of the three are all less than 0.65%, indicating that this method meets the analysis requirements.

[0083] Table 3 Verification of the analysis method for three tracers

[0084]

[0085] 2.2.2 Contents of fluorescent tracers in different parts of soybeans

[0086] Judging from the distribution of tracers in the above-ground parts of soybeans ( Figure 5 Figure A in it), the contents of tracer No. 7 in the hypocotyls, true leaves, and trifoliate leaves of soybeans are significantly higher than those of rhodamine B, and the content of tracer No. 8 in the true leaves is significantly higher than that of rhodamine B; judging from the distribution of tracers in the underground parts of soybeans ( Figure 5 Figure B in it), the content of rhodamine B in the roots of soybeans is significantly higher than those of No. 7 and No. 8 tracers, and there is no significant difference between No. 7 and No. 8, indicating that the absorption ability of soybean roots (underground parts) to rhodamine B is relatively strong. However, considering the contents of the three tracers in the above-ground parts of soybeans, the content of tracer No. 7 is higher than those of rhodamine B and tracer No. 8, indicating that the systemic conduction performance of tracer No. 7 in soybean seedlings is significantly improved compared with that of rhodamine B.

[0087] 2.2.3 Content of Fluorescent Tracer in Different Parts of Maize

[0088] From the distribution of the tracer in different parts of the maize plant ( Figure 6 , A. aboveground part; B. root), the contents of tracers 7 and 8 in the leaf sheath and leaves were significantly higher than that of Rhodamine B. The content of tracer 8 in the root was significantly higher than that of tracer 7 and Rhodamine B, indicating that the maize root system had the strongest absorption ability for tracer 8. Generally speaking, the contents of tracers 7 and 8 in the aboveground part of maize were higher than that of Rhodamine B, suggesting that the systemic conduction performance of tracers 7 and 8 in maize seedlings was better than that of the control agent Rhodamine B.

[0089] 2.2.4 Content of Fluorescent Tracer in Different Parts of Tobacco

[0090] From the distribution of the tracer in different parts of the tobacco plant ( Figure 7 , A. aboveground part; B. root), the content of tracer 8 in the stem and leaves of tobacco was significantly higher than that of tracer 7 and Rhodamine B, and the content of tracer 7 in the leaves of tobacco was significantly higher than that of Rhodamine B. Generally speaking, the contents of tracers 7 and 8 in the aboveground part of tobacco were higher than that of Rhodamine B, indicating that the conduction performance of tracers 7 and 8 in tobacco seedlings was better than that of Rhodamine B.

[0091] 2.2.5 Distribution Ratio of Fluorescent Tracer in Three Plants

[0092] Among the three crop plants, the proportion of the three tracers was the highest in the roots. In the distribution proportion of the aboveground part of soybean (the sum of hypocotyl, cotyledon, epicotyl, true leaf, and trifoliate leaf), the proportion of tracer 7 was the highest, followed by tracer 8, and Rhodamine B was the smallest ( Figure 8 ); in the distribution proportion of the aboveground part of maize plant (the sum of leaf sheath and leaves), the proportion of tracer 7 was higher than that of Rhodamine B ( Figure 9 ), and combined with the results of 2.2.3, the contents of tracers 7 and 8 in the aboveground part of maize were significantly higher than that of Rhodamine B; in the distribution proportion of the aboveground part of tobacco plant (the sum of stem and leaves), the distribution of tracer 8 in the aboveground part was significantly higher than that of tracer 7 and Rhodamine B ( Figure 10 ).

[0093] Generally speaking, although the tracer Rhodamine B and tracers 7 and 8 were mainly distributed in the roots of soybean, maize, and tobacco, the contents of tracers 7 and 8 in the aboveground parts of the three crop plants were significantly higher than that of Rhodamine B, indicating that the systemic conduction of tracers 7 and 8 in soybean, maize, and tobacco was better than that of Rhodamine B, suggesting that introducing amino acid groups into the Rhodamine B molecule could significantly improve its upward conduction ability.

[0094] 3 Safety Evaluation of Fluorescent Tracer on Plants

[0095] 3.1 Test Method

[0096] (1)Effect of fluorescent tracers on the germination of seeds of three crops such as soybean: The paper method was used to study the effects of three tracers on the germination of soybean, corn, and tobacco seeds. Dissolved in acetonitrile and diluted with deionized water, prepare 200 mL of rhodamine B, tracer solutions No. 7 and No. 8 with concentrations of 3.75 mg / L, 7.50 mg / L, 15.00 mg / L, 30.00 mg / L, and 60.00 mg / L.

[0097] Soybean seeds were soaked in the above solutions for 30 min. Select seeds with intact epidermis, air-dry them, and place them on 2 layers of germination paper, covered with 1 layer of germination paper, 50 seeds in each group, repeated 3 times. Use distilled water treatment as the control, and culture in the dark at 25°C and 80% humidity. Record the number of germinated seeds on the 5th day and the 8th day according to the requirements of the Association of Official Seed Analysts (abbreviated as AOSA).

[0098] Corn seeds were soaked in the dark at 25°C for 12 h, washed, air-dried, and placed in a sterile petri dish (18 cm) lined with 2 layers of moist filter paper. 30 seeds were placed in each dish, repeated 3 times for each group, with distilled water as the control. Cultured at 25°C, record the number of germinated seeds on the 4th day and the 7th day according to the requirements of AOSA.

[0099] Select plump tobacco seeds, soak them in the dark at 25°C for 24 h, wash them, and place them in the dark at 20°C to air-dry, with distilled water treatment as the control. Place the seeds in a 9 cm petri dish lined with 2 layers of moist filter paper, and culture at a constant temperature of 25°C in an environment of 16 h light and 8 h dark. Each treatment has 3 replicates, 50 seeds in each group. Record the number of germinated seeds on the 7th day and the 14th day according to the requirements of AOSA. Calculate the germination potential and germination rate.

[0100] (2)Effect of fluorescent tracers on the growth of three crops such as soybean: Weigh the fresh weights of the trifoliate leaves, true leaves, epicotyls, cotyledons, hypocotyls, and roots of the soybean seedlings in 1.1 treatment; the fresh weights of the roots, leaf sheaths, and leaves of the corn seedlings; the fresh weights of the roots, stems, and leaves of the tobacco, and study the effects of rhodamine B, tracer solutions No. 7 and No. 8 on the growth of the seedlings of the three crops under this concentration condition.

[0101] 3.2 Data Processing

[0102] IBM SPSS statistics 25 software was used for data statistical analysis, and Duncan's new multiple range method was used for significant difference analysis.

[0103] 3.3 Test Results

[0104] 3.3.1 Effects of Fluorescent Tracers on the Germination of Plant Seeds

[0105] By comparing the germination potential and germination rate of soybean, corn, and tobacco seeds under different treatments, it was found that the germination rate and germination percentage of some tracer treatments were slightly higher than those of the blank control. However, there were no significant differences in the germination potential and germination rate between all treatments and the control (Table 4), indicating that each tracer had no obvious effect on the germination of soybean, corn, and tobacco seeds.

[0106] Table 4 Effects of Different Concentrations of Three Tracers on Seed Germination

[0107]

[0108] Note: The data in the table are expressed as mean ± standard error. Different letters in the same column indicate significant differences (P<0.05).

[0109] 3.3.2 Effects of Fluorescent Tracers on Plant Growth

[0110] After 4 days of treatment with 60 mg / L rhodamine B, Tracer No. 7, and Tracer No. 8, there were no significant differences in the fresh weights of various parts of soybean, corn, and tobacco plants compared with the blank control treatment ( Figure 11 - 13 ), indicating that at this concentration, these three fluorescent tracers had no obvious effect on the seedling growth of soybean, corn, and tobacco.

[0111] Overall, rhodamine B, Tracer No. 7, and Tracer No. 8 had no obvious effect on the seed germination and seedling growth of soybean, corn, and tobacco, indicating that rhodamine B, Tracer No. 7, and Tracer No. 8 had high safety for soybean, corn, and tobacco.

[0112] 4 Verification Study on the Discrimination of Fluorescent Tracers between Crops and Weeds

[0113] The potting method was used to evaluate the discrimination of fluorescent tracers between crops and weeds by testing the distribution of fluorescent tracers in crop and weed plants after seed treatment.

[0114] The specific method is as follows:

[0115] 4.1 Seed Treatment.

[0116] Taking the seeds of crops such as soybean, corn, and tobacco and weeds such as Chenopodium album, Echinochloa crusgalli, Setaria viridis, and Amaranthus retroflexus as test objects, 0.01 g of fluorescent tracer, 0.01 g of binder (sodium carboxymethyl cellulose), and 0.48 g of deionized water were mixed with a vortex oscillator and evenly attached to the surface of 10 g of crop or weed seeds, and then air-dried at room temperature for later use. 10 g of crop or weed seeds treated with 0.5 g of deionized water were used as the control.

[0117] 4.2 Substrate Treatment

[0118] Air-dry the soil retrieved from the teaching farm of Inner Mongolia Agricultural University and sieve it through a sieve with a 2-mm aperture to remove interfering substances such as large gravel. Take 1,500 g of soil and place it in a flower pot with a diameter of 8 cm. Compact and level the soil, and add 300 mL of deionized water to keep the soil moisture content at 20%.

[0119] 4.3 Sowing

[0120] Directly sow 1 pre-treated crop seed at the center of the flower pot. Taking the crop seed as the center, directly sow 8 weed seeds at 2 cm and 4 cm away from the crop seed respectively (2 seeds of each type of weed seed. For the sowing positions of the crop and weed seeds, see Figure 14 ). The depth of the seeds is 3 cm. Plant 4 pots of each type of crop seed (4 replicates). Place the flower pots in an artificial climate chamber for cultivation (25 °C, humidity 40%, light / dark: 16 / 8 h). When the corn has 3 - 4 leaves, the soybean has two unfolded leaves, and the tobacco has 4 leaves, detect the distribution of the fluorescent tracer in each plant (crop, weed).

[0121] 4.4 Detection of the Distribution of Fluorescent Tracer in Crops and Weeds

[0122] Using high performance liquid chromatography, measure the contents of the fluorescent tracer in the roots, stems (leaf sheaths), and leaves of each crop and weed plant respectively. The extraction method and the measurement method are the same as those in "2. Quantitative Analysis of Fluorescent Tracer in Different Parts of Plants".

[0123] 4.5 Results

[0124] Using high performance liquid chromatography, the distributions of amino acid derivative - rhodamine B conjugates 7, 8, and rhodamine B in soybean, corn, tobacco, and 4 types of weed seedlings were detected. The specific results are shown in Tables 5 - 7. The study found that the fluorescent tracer mainly exists in the roots of the crops. Although the content in the above-ground parts (stems and leaves) is relatively lower than that in the roots, there is an obvious distribution in the crop stems and leaves, which can be used for fluorescence detection. However, the fluorescent tracer was only detected in the roots of the weeds 2 cm away from the crops (the content is lower than 0.4 mg / L), and no distribution of the fluorescent tracer was detected in the above-ground parts of the weeds, indicating that the lateral migration of amino acid derivative - rhodamine B conjugates 7, 8, and rhodamine B in the soil is less. Therefore, the fluorescent tracer developed in the present invention has a high recognition degree between crops and weeds.

[0125] Table 5 Contents of Fluorescent Tracer in Different Parts of Soybean and Weeds (mg / L)

[0126]

[0127] Table 6 Contents of Fluorescent Tracer in Different Parts of Corn and Weeds (mg / L)

[0128]

[0129] Table 7 Contents of Fluorescent Tracer in Different Parts of Tobacco and Weeds (mg / L)

[0130]

[0131] The above research shows that after hydroponic treatment, the contents of amino acid derivative-rhodamine B conjugates in the above-ground parts (stems and leaves) of three plants, soybean, corn and tobacco, are significantly higher than those of the control agent rhodamine B. Especially for the 7th and 8th conjugates containing proline, their upward conduction abilities in different plants are significantly higher than that of rhodamine B, indicating that the modified conjugates have more excellent systemic conduction performance and have no adverse effects on plant seed germination and seedling growth. After seed treatment, the fluorescent tracer has a high discrimination degree between crops and weeds and can be used for crop / weed identification.

[0132] The upper and lower limit values and interval values of the process parameters (such as temperature, time, etc.) of the present invention can all implement this method, and the examples are not listed one by one here.

[0133] The content not detailed in the present invention can all adopt the conventional technical knowledge in the art.

[0134] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that any modification or equivalent replacement of the technical solutions of the present invention does not depart from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. An amino acid derivative-rhodamine B conjugate having the conductive property of the above-ground part of a plant plant, characterized in that, The structural formula of the amino acid derivative-rhodamine B conjugate is as follows: ； Among them, the coupling bond in the amino acid derivative-rhodamine B conjugate is formed by the amino group of the amino acid derivative and the carboxyl group of rhodamine B, and it is an amide bond; The group R is selected from , one of them.

2. The preparation method of the amino acid derivative-rhodamine B conjugate according to claim 1, wherein the preparation method comprises the following steps: Dissolve a mixture of amino acid methyl ester hydrochloride and N-methylmorpholine in anhydrous dichloromethane, cool it to 0 °C, and sequentially add rhodamine B, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine; stir the obtained solution at room temperature for amidation reaction, after the reaction is completed, extract, wash, dry, filter, concentrate under reduced pressure, and perform column chromatography to obtain the amino acid methyl ester-rhodamine B conjugate; Dissolve the amino acid methyl ester-rhodamine B conjugate in a mixed solution of water and THF, add lithium hydroxide under ice bath, slowly raise the temperature to room temperature, after the hydrolysis reaction is completed, acidify to pH = 2, extract, wash, dry, filter, concentrate under reduced pressure, and perform column chromatography to obtain the amino acid-rhodamine B conjugate; The amino acid derivative-rhodamine B conjugate includes the amino acid methyl ester-rhodamine B conjugate and the amino acid-rhodamine B conjugate.

3. The preparation method according to claim 2, characterized in that, The molar ratio of amino acid methyl ester hydrochloride, N-methylmorpholine, rhodamine B, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine is 15:20:10:15:1, the reaction temperature is room temperature, and the reaction time is 2-4 hours.

4. The preparation method according to claim 2, wherein The molar ratio of the amino acid methyl ester-rhodamine B conjugate and lithium hydroxide is 5:15, the reaction temperature is room temperature, and the reaction time is 1-2 hours.

5. The application of the amino acid derivative-rhodamine B conjugate according to claim 1 as a fluorescent tracer in the identification of crops and weeds; The crops are soybeans, corn, and tobacco.

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