Amino acid derivative-rhodamine B conjugate as well as preparation method and application thereof as fluorescent tracer agent

Abstract

The invention belongs to the technical field of fluorescent tracer preparation, and particularly relates to an amino acid derivative-rhodamine B conjugate, a preparation method thereof and application of the conjugate as a fluorescent tracer. The structural formula of the amino acid derivative-rhodamine B conjugate is as follows: # imgabs0 #. After the amino acid derivative-rhodamine B conjugate obtained by the invention is directionally applied to crop plants as a fluorescent tracer, the crop plants can be labeled by fluorescence, a machine vision system is assisted to distinguish crops and weeds, precise pesticide spraying or mechanical weeding is performed according to spatial distribution of the weeds, automatic and intelligent weed control is realized, and the labor intensity is reduced. The usage amount of herbicide is reduced.

Description

Technical Field

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

[0002] During the growth of crops, weeds, as a type of harmful organisms that harm 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 of farmland weed control include physical control, chemical control, biological control, and agricultural control. Among them, chemical control is widely used in weed control due to its advantages of high efficiency, economy, and labor saving. However, at the same time, the irrational use of herbicides has also caused environmental pollution, pesticide damage, 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 technology are the only way for the development of modern agricultural plant protection. Among them, using modern information technology to distinguish between farmland crops and weeds, clarifying the spatial distribution of weeds, and then accurately applying pesticides or mechanical weeding for weeds is of great significance for effectively reducing the amount of herbicides and realizing the intelligent control of farmland weeds. At present, machine vision technology is mainly used to identify crops and weeds.

[0003] Machine vision technology uses deep learning and other methods to establish a crop and weed morphological feature database, and then identify crops / weeds. It has the advantages of being fast, low cost, and high resolution. However, the existing technology is suitable for the prevention and control of inter-row weeds, and the recognition degree of inter-plant weeds is not high enough. In complex situations (such as high weed density conditions), the recognition degree is low. It is also necessary to establish a large number of weed and crop feature databases, and the preliminary preparation work is cumbersome. In addition, as the plants grow, the morphological characteristics of crops and weeds will also change, further increasing the difficulty of extracting plant morphological features.

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

[0005] Fluorescent markers are the core and foundation of crop plant signaling technology. Existing studies have shown that the fluorescent tracers that can be absorbed and transmitted by plants are mainly coumarins (such as coumarin 120) and rhodamines (such as rhodamine B, Rh-B) (Wang, Z.; Amirkhani, M.; Avelar, SAG; Yang, D.; Taylor, AG 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 studies have shown that rhodamine B has unique fluorescence characteristics (λex / λem = 555 / 582 nm), which is highly distinguishable from the fluorescence characteristics of plant chlorophyll and can be detected under a wide range of fluorescence conditions (λex = 535 nm, λem = 580~582 nm). nm) can be distinguished from chlorophyll 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 pH adaptability, good cell membrane permeability and no toxic side effects on cells, so it has more application prospects in plant tracers.

[0006] Studies have found that rhodamine B can be absorbed by kidney bean and soybean seeds and penetrate into the seeds, or absorbed by the roots of celery, lettuce, and tomatoes, and conducted upward to the upper part of the seedlings through the xylem of the plants. However, overall, rhodamine B is mainly distributed in the hypocotyls of plants (soybeans and kidney beans), and the fluorescence intensity of rhodamine B in the epicotyl is 35% (kidney beans) and 11% (soybeans) 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 systemic conduction performance of this tracer in plants needs to be further improved.

[0007] In order to address the problem that the existing tracers have insufficient systemic conductivity, which limits the machine's ability to capture fluorescent signals, the structure of the existing tracer Rhodamine B was optimized to improve its systemic conductivity, and a fluorescent tracer for precision weed control technology was developed. Summary of the invention

[0008] The purpose of the present invention is to overcome the defects of the prior art, and a series of amino acid derivative-rhodamine B conjugates and their preparation methods and applications as fluorescent tracers are proposed. The present invention optimizes the structure of the existing fluorescent marker rhodamine B, introduces amino acid fragments 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] Amino acid derivative-rhodamine B conjugate, its structural formula is as follows:

[0011] ;

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

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

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

[0015] The present invention also provides a method for preparing the amino acid derivative-rhodamine B conjugate.

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

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

[0018] More preferably, the crops are soybean, corn and tobacco.

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

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] After the amino acid derivative-rhodamine B conjugate obtained by the present invention is used as a fluorescent tracer, the content of the tracer in the aboveground part of the crop plant is significantly increased, which is beneficial to the machine vision system for crop-weed identification, and precise spraying or mechanical weeding can be performed according to the spatial distribution of weeds, so as to realize the automation and intelligence of weed control, significantly reduce the use of herbicides, effectively reduce the impact of chemical herbicides on the ecological environment, reduce the occurrence of herbicide damage, delay the development of drug resistance, and reduce the risk of pesticide residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a fluorescence detection image in soybean plants;

[0023] Figure 2 This is a fluorescence detection image in a corn plant;

[0024] Figure 3 This is a fluorescence detection image in tobacco plants;

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

[0026] Figure 5 This is a graph of the content of fluorescent tracers in different parts of soybeans;

[0027] Figure 6 This is a graph of the content of fluorescent tracers in different parts of corn;

[0028] Figure 7 This is a graph of the content of fluorescent tracers in different parts of tobacco;

[0029] Figure 8 is the distribution ratio of the fluorescent tracer in the soybean plant;

[0030] Fig. 9 is a proportional map of the distribution of fluorescent tracers in corn plants;

[0031] Fig.10 is the distribution ratio of the fluorescent tracer in the tobacco plant;

[0032] Fig.11 This is a graph showing the effects of three fluorescent tracers on soybean plant growth;

[0033] Fig.12 This is a graph showing the effects of three fluorescent tracers on the growth of corn plants;

[0034] Fig.13 This is a graph showing the effects of three fluorescent tracers on tobacco plant growth;

[0035] Fig.14 It is a schematic diagram of the sowing location of crop and weed seeds;

[0036] Note: Figure 5 , 6 Different letters in , 7, 11, 12, and 13 indicate significant differences among treatments (P<0.05). DETAILED DESCRIPTION

[0037] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0038] Example 1

[0039] Rhodamine B and amino acid methyl ester (amino acid methyl ester general formula ) as raw materials, and the amino acid derivative-rhodamine B coupling compound is constructed by amidation and ester hydrolysis. The specific synthesis route is shown in the following reaction formula:

[0040] ;

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

[0042] Ester hydrolysis conditions b: LiOH, THF / H 2 O (V / V=2:1), rt (room temperature standard pressure);

[0043] Amino acid methyl ester general formula In the above, R is determined according to the selected amino acid. For example, the amino acid selected in the present invention is selected from 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). The reaction mixture was then 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 in sequence. 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 layer was washed alternately with saturated sodium bicarbonate aqueous solution and saturated saline aqueous solution for 2-3 times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed 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), and lithium hydroxide (0.63 g, 15 mmol) was added under an ice bath. The reaction was slowly warmed to room temperature and the reaction was continued for 1 h. After the reaction was completed, the mixture was acidified to pH = 2 with 1M hydrochloric acid solution and extracted with ethyl acetate (3×200 mL). The combined organic layer was washed alternately with saturated sodium bicarbonate aqueous solution and saturated saline aqueous solution for 2-3 times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed to obtain compound 3 (proline-rhodamine B conjugate, conjugate 8 in Table 1).

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

[0047] The amino acid derivative-rhodamine B conjugate was subjected to hydrogen spectrum and mass spectrum analysis, and the data are shown in Table 1.

[0048] Table 1 NMR 1H spectra 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 plants (taking tracers 1, 2, 7, and 8 as examples)

[0055] 1.1 Plant seedling cultivation

[0056] Add 250 g of substrate to a 15 cm diameter flower pot, sow soybean, corn and tobacco seeds at a sowing depth of 2 cm, and cultivate until the soybean true leaves are fully expanded, the first leaf of corn is perpendicular to the leaf sheath, and the tobacco is in the eight-leaf stage.

[0057] Fluorescent tracers such as rhodamine B or amino acid derivative-rhodamine B conjugates were added to the Hoagland nutrient solution to prepare a hydroponic solution containing 60 mg / L of the tracer. The roots of the cultured plant seedlings were cleaned and placed in a plastic cup containing 200 mL of the hydroponic solution. The Hoagland nutrient solution was used as the blank control. The plants were cultured at 25°C, 16 h light / 8 h dark for 4 d, and each treatment was 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 them in 1000 mL of distilled water, and sterilize them at 115℃ for 20 min.

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

[0060] A stereoscopic fluorescence microscope was used to detect the distribution of fluorescent tracers in various parts of the plant, and whether the tracer could be conducted to the stems and leaves of the plant was used as the criterion for evaluating its systemic conduction performance (compared with rhodamine B). Soybean seedlings treated with 1.1 were divided into six parts: trifoliate, true leaf, epicotyl, cotyledon, hypocotyl, and root; corn was divided into three parts: root, leaf sheath, and leaf; tobacco was divided into three parts: root, stem, and leaf; and the RFP detection module (excitation wavelength 541-551 nm, detection wavelength 590 nm) was used for screening.

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

[0062] The aboveground parts of soybean plants (trifoliate leaves, true leaves, epicotyls, cotyledons, and hypocotyls) were observed using a stereofluorescence microscope. Figure 1 As shown, compared with the control tracer rhodamine B, the fluorescence intensity of tracers 7 and 8 in the aerial part of soybean plants was enhanced, and the fluorescence intensity of tracer 2 in the true leaves was 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 corn plants

[0064] Fluorescence detection of corn plants by stereo fluorescence microscope, such as Figure 2 As shown, compared with the control agent Rhodamine B, the fluorescence intensity of tracers 1, 2, 7 and 8 in the aboveground parts of corn plants (leaves and leaf sheaths) was enhanced, indicating that coupling with amino acids such as isoleucine and proline can improve the systemic conduction performance of Rhodamine B in corn plants.

[0065] 1.2.3 Evaluation of the conduction performance of fluorescent tracers in tobacco plants

[0066] Tobacco seedlings treated with tracers were examined under a stereo fluorescence microscope. Figure 3 As shown, compared with the control agent Rhodamine B, the fluorescence intensity of tracers 1, 7 and 8 in the aboveground parts of tobacco plants (leaves and stems) was enhanced, indicating that coupling with amino acids such as isoleucine and proline can improve the systemic conduction performance of Rhodamine B in tobacco plants.

[0067] Overall, the fluorescence intensity of the amino acid derivative-rhodamine B conjugate in the aboveground part of the plant is higher than that of rhodamine B, indicating that after coupling with rhodamine B, amino acids such as isoleucine and proline can significantly improve the systemic conduction performance of rhodamine B in the plant, especially in monocotyledonous plants (corn).

[0068] 2 Quantitative analysis of fluorescent tracers in different parts of plants

[0069] 2.1 Test methods

[0070] Using tracers No. 7 and 8 as representatives, liquid chromatography was used to further detect the content of fluorescent tracers in various parts of the plant.

[0071] (1) Sample processing

[0072] Take 0.5 g of different parts of soybean, corn and tobacco seedlings treated with rhodamine B, 7 and 8 tracers in treatment 1.1, grind them, add 4 mL of acetonitrile, and extract them by ultrasonic for 40 min. After immersion for 1 day, concentrate to 1 mL, filter with 0.22 μm organic microporous filter membrane, and perform high performance liquid chromatography analysis to determine the content of fluorescent tracers in different parts of soybean, corn 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, the specific ratio is shown in Table 2:

[0074] Table 2 Ratio of three tracer mobile phases

[0075]

[0076] (2) Preparation of standard solution and drawing of standard curve

[0077] Weigh 5.0 mg of rhodamine B standard, 7 and 8 fluorescent tracers respectively, dissolve them in acetonitrile to 10 mL to obtain 500 mg / L standard stock solution, then dilute them step by step with acetonitrile, measure the absorption peak area of ​​each standard solution, and draw a standard curve.

[0078] (3) Addition recovery test

[0079] Standard solutions at three levels of 100.0000, 33.3333, and 11.1111 mg / L were added to blank samples of soybean, corn, and tobacco parts, respectively. Each addition level was repeated 3 times. Sample pretreatment and analysis were carried out according to the method, and the addition recovery rate and relative standard deviation were calculated.

[0080] 2.2 Test results

[0081] 2.2.1 Construction and validation of HPLC analysis method

[0082] The standard curve was drawn according to the concentration gradient. The correlation coefficients of the three tracers, rhodamine B, No. 7 and No. 8, were 0.9996, 0.9990 and 0.9935, respectively. Within the concentration range of the standard curve, the mass concentration and the corresponding response value showed a good linear relationship ( Figure 4 Note: A. Rhodamine B; B. Tracer No. 7; C. Tracer No. 8). As shown in Table 3, the average recoveries of Rhodamine B, Tracer No. 7 and Tracer No. 8 were between 81% and 109%, and the relative standard deviations of the three were all less than 0.65%, indicating that the method met the analytical requirements.

[0083] Table 3 Validation of three tracer analysis methods

[0084]

[0085] 2.2.2 Content of fluorescent tracer in different parts of soybean

[0086] From the distribution of tracers in the aboveground part of soybean ( Figure 5 As shown in Figure A, the content of tracer No. 7 in soybean hypocotyls, true leaves, and trifoliate leaves was significantly higher than that of rhodamine B, and the content of tracer No. 8 in true leaves was significantly higher than that of rhodamine B. From the distribution of tracers in the underground part of soybean ( Figure 5 As shown in Figure B), the content of rhodamine B in soybean roots was significantly higher than that of tracers 7 and 8, while there was no significant difference between 7 and 8, indicating that the soybean root system (underground part) has a strong absorption capacity for rhodamine B. However, based on the content of the three tracers in the aboveground part of soybean, the content of tracer 7 was higher than that of rhodamine B and tracer 8, indicating that the systemic conduction performance of tracer 7 in soybean seedlings was significantly improved compared with rhodamine B.

[0087] 2.2.3 Fluorescent tracer content in different parts of corn

[0088] From the distribution of tracers in different parts of corn plants ( Figure 6 , A. aboveground part; B. root part), the contents of tracers 7 and 8 in leaf sheaths and leaves were significantly higher than that of rhodamine B. The content of tracer 8 in roots was significantly higher than that of tracer 7 and rhodamine B, indicating that the maize root system had the strongest absorption capacity for tracer 8. Overall, the contents of tracers 7 and 8 in the aboveground part of maize were higher than that of rhodamine B, indicating 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 Fluorescent tracer content in different parts of tobacco

[0090] From the distribution of tracers in different parts of tobacco plants ( Figure 7 , A. aboveground part; B. root part), the content of tracer No. 8 in tobacco stems and leaves was significantly higher than that of tracer No. 7 and rhodamine B, and the content of tracer No. 7 in tobacco leaves was significantly higher than that of rhodamine B. Overall, the contents of tracers No. 7 and No. 8 in tobacco aboveground parts were higher than those of rhodamine B, indicating that the conduction performance of tracers No. 7 and No. 8 in tobacco seedlings was better than that of rhodamine B.

[0091] 2.2.5 Distribution ratio of fluorescent tracers in three plants

[0092] In the three crop plants, the three tracers were all in the root with the highest proportion. In the distribution of the aboveground part of soybean (the sum of the five parts of hypocotyl, cotyledon, epicotyl, true leaf, and trifoliate), tracer No. 7 was the highest, followed by tracer No. 8, and rhodamine B was the lowest ( Figure 8 ); In the distribution of the aboveground parts of corn plants (the sum of leaf sheaths and leaves), tracer No. 7 was higher than rhodamine B ( Fig. 9 ), combined with the results of 2.2.3, the contents of tracers 7 and 8 in the aboveground part of corn were significantly higher than that of rhodamine B; in the distribution of the aboveground part of tobacco plants (stems and leaves combined), the distribution of tracer 8 in the aboveground part was significantly higher than that of tracer 7 and rhodamine B ( Fig.10 ).

[0093] In summary, although the tracers Rhodamine B and tracers 7 and 8 were mainly distributed in the roots of soybeans, corn and tobacco, the contents of tracers 7 and 8 in the aboveground parts of the three crops were significantly higher than that of Rhodamine B, indicating that the systemic conductivity of tracers 7 and 8 in soybeans, corn and tobacco was better than that of Rhodamine B, indicating that the introduction of amino acid groups into the Rhodamine B molecule can significantly improve its upward conduction ability.

[0094] 3 Safety evaluation of fluorescent tracers for plants

[0095] 3.1 Test methods

[0096] (1) Effects of fluorescent tracers on seed germination of soybean and other three crops: The paper method was used to study the effects of three tracers on seed germination of soybean, corn and tobacco. Dissolve in acetonitrile and dilute with deionized water to prepare 200 mL of rhodamine B, 7 and 8 tracer solutions 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] Soak the soybean seeds in the above solution for 30 min, select the seeds with intact epidermis, dry them in the shade, place them on two layers of germination paper, and cover them with one germination paper. Repeat 3 times for each group of 50 seeds, with distilled water treatment as the control, and culture them in the dark at 25°C and 80% humidity. Record the number of germinations on the 5th and 8th days according to the requirements of the Association of Official Seed Analysts (AOSA).

[0098] Corn seeds were soaked in a dark environment at 25℃ for 12 h, washed, dried in the shade, and placed in a sterile culture dish (18 cm) covered with two layers of moist filter paper, with 30 seeds placed in each dish, and each group was repeated 3 times, with distilled water as the control. Cultured at 25℃, the number of germinations on the 4th and 7th days was recorded according to AOSA requirements.

[0099] Select tobacco seeds with full grains, soak them in the dark at 25℃ for 24 h, wash them and dry them in the dark at 20℃, and use distilled water as the control. Place the seeds in a 9 cm culture dish covered with 2 layers of moist filter paper and culture them at 25℃, 16 h light and 8 h dark. Each treatment was repeated 3 times, with 50 seeds in each group. Record the number of germinations on the 7th and 14th days according to AOSA requirements. Calculate the germination potential and germination rate.

[0100] (2) Effects of fluorescent tracers on the growth of soybean and other three crops: The fresh weights of the three leaves, true leaves, epicotyls, cotyledons, hypocotyls and roots of soybean seedlings treated in 1.1 were weighed; the fresh weights of the roots, sheaths and leaves of corn seedlings; and the fresh weights of the roots, stems and leaves of tobacco seedlings were weighed to study the effects of rhodamine B, 7 and 8 tracers on the growth of the three crops under these concentration conditions.

[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 difference significance analysis.

[0103] 3.3 Test results

[0104] 3.3.1 Effects of fluorescent tracers on plant seed germination

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

[0106] Table 4 Effects of three tracer concentrations 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] There was no significant difference in the fresh weight of each part of soybean, corn and tobacco plants treated with 60 mg / L rhodamine B, 7 and 8 tracers for 4 days compared with the blank control treatment ( Figure 11-13 ), indicating that at this concentration, the three fluorescent tracers had no significant effect on the growth of soybean, corn and tobacco seedlings.

[0111] In summary, rhodamine B, 7 and 8 tracers had no significant effects on seed germination and seedling growth of soybean, corn and tobacco, indicating that rhodamine B, 7 and 8 tracers are highly safe for soybean, corn and tobacco.

[0112] 4. Validation of the identification of fluorescent tracers between crops and weeds

[0113] The potted plant method was used to test the distribution of fluorescent tracers in crops and weed plants after seed treatment to evaluate the recognition of crops and weeds by fluorescent tracers.

[0114] The specific method is as follows:

[0115] 4.1 Seed treatment.

[0116] The seeds of crops such as soybean, corn, tobacco and weeds such as quinoa, barnyard grass, foxtail grass and Amaranthus retroflexus were used as test objects. 0.01g of fluorescent tracer, 0.01g of binder (sodium carboxymethyl cellulose) and 0.48g of deionized water were mixed with a vortex oscillator and evenly attached to the surface of 10g of crop or weed seeds, and dried in the shade at room temperature for later use. 10g of crop or weed seeds treated with 0.5g of deionized water were used as controls.

[0117] 4.2 Matrix treatment

[0118] The soil retrieved from the teaching farm of Inner Mongolia Agricultural University was dried in the shade and sieved (2 mm pore size) to remove large particles of stones and other interferences. 1500 g of soil was placed in a flower pot with a diameter of 8 cm, compacted and leveled, and 300 mL of deionized water was added to keep the soil moisture content at 20%.

[0119] 4.3 Seeding

[0120] Sow a treated crop seed in the center of the flowerpot, and with the crop seed as the center, sow 8 weed seeds 2cm and 4cm away from the crop seed (2 seeds for each weed, see the sowing positions of crop and weed seeds for details). Fig.14 ), seed depth 3cm, 4 pots of each crop seed were planted (4 times repeated). The flower pots were placed in an artificial climate chamber (25℃, humidity 40%, light / dark: 16 / 8h), and the distribution of fluorescent tracers in each plant (crop, weed) was detected when corn was in the 3-4 leaf stage, soybean was in the 2nd leaf stage, and tobacco was in the 4th leaf stage.

[0121] 4.4 Detection of the distribution of fluorescent tracers in crops and weeds

[0122] Liquid chromatography was used to test the content of fluorescent tracers in roots, stems (leaf sheaths) and leaves of various crops and weed plants. The extraction and testing methods were the same as those in “2. Quantitative analysis of fluorescent tracers in different parts of plants”.

[0123] 4.5 Results

[0124] Liquid chromatography was used to detect the distribution of amino acid derivative-rhodamine B conjugates 7, 8 and rhodamine B in soybean, corn, tobacco and 4 weed seedlings, as shown in Tables 5 to 7. The study found that the fluorescent tracer was mainly present in the roots of the crops. Although the content in the aboveground parts (stems and leaves) was lower than that in the roots, it was obviously distributed in the stems and leaves of the crops and could be used for fluorescence detection; the fluorescent tracer was only detected in the roots of weeds 2 cm away from the crops (the content was less than 0.4 mg / L), and no fluorescent tracer distribution was detected in the aboveground parts of the weeds, indicating that the amino acid derivative-rhodamine B conjugates 7, 8 and rhodamine B had less lateral migration in the soil. Therefore, the fluorescent tracer developed by the present invention has a higher recognition between crops and weeds.

[0125] Table 5 Fluorescent tracer content in different parts of soybean and weeds (mg / L)

[0126]

[0127] Table 6 Fluorescent tracer content in different parts of corn and weeds (mg / L)

[0128]

[0129] Table 7 Fluorescent tracer content in different parts of tobacco and weeds (mg / L)

[0130]

[0131] The above studies show that after hydroponic treatment, the content of amino acid derivative-rhodamine B conjugate in the aboveground parts (stems and leaves) of soybean, corn and tobacco was significantly higher than that of the control agent rhodamine B, especially the proline-containing conjugates No. 7 and No. 8, whose upward conduction ability in different plants was significantly higher than that of rhodamine B, indicating that the improved conjugate has more excellent systemic conduction performance and has no adverse effects on plant seed germination and seedling growth. After seed treatment, the fluorescent tracer has a high degree of distinction between crops and weeds and can be used for crop / weed identification.

[0132] The process parameters (such as temperature, time, etc.) of the present invention can realize the method by taking upper and lower limits and interval values, and the embodiments are not listed one by one here.

[0133] Any contents not described in detail in the present invention can be based on the conventional technical knowledge in the art.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art 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 should be included in the scope of the claims of the present invention.

Claims

1. An amino acid derivative-rhodamine B conjugate, characterized in that: The structural formula of the amino acid derivative-rhodamine B conjugate is as follows: ； Wherein, the coupling bond in the amino acid derivative-rhodamine B coupling compound is formed by the amine group of the amino acid derivative and the carboxyl group of rhodamine B, which is an amide bond; The group R is selected from , , , , , , , , , one of the.

2. The amino acid derivative-rhodamine B conjugate according to claim 1, characterized in that: The group R is selected from or .

3. A method for preparing an amino acid derivative-rhodamine B conjugate, the method comprising the following steps: A mixture of amino acid methyl ester hydrochloride and N-methylmorpholine is dissolved in anhydrous dichloromethane, cooled to 0°C, and rhodamine B, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine are added in sequence; the resulting solution is stirred at room temperature for amidation reaction, and after the reaction is completed, extraction, washing, drying, filtering, concentrating under reduced pressure, and column chromatography is performed to obtain an amino acid methyl ester-rhodamine B coupling compound; The amino acid methyl ester-rhodamine B conjugate is dissolved in a mixed solution of water and THF, lithium hydroxide is added in an ice bath, the temperature is slowly raised to room temperature, after the hydrolysis reaction is completed, the pH is acidified to 2, extraction, washing, drying, filtering, concentrating under reduced pressure, and column chromatography to obtain the amino acid-rhodamine B conjugate; The amino acid derivative-rhodamine B conjugate includes amino acid methyl ester-rhodamine B conjugate and amino acid-rhodamine B conjugate.

4. The preparation method according to claim 3, 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.

5. The preparation method according to claim 3, characterized in that: The molar ratio of amino acid methyl ester-rhodamine B coupling compound to lithium hydroxide is 5:15, the reaction temperature is room temperature, and the reaction time is 1-2 hours.

6. The preparation method according to claim 3, characterized in that: The amino acid methyl ester hydrochloride is selected from one of isoleucine methyl ester hydrochloride, tyrosine methyl ester hydrochloride, tryptophan methyl ester hydrochloride, proline methyl ester hydrochloride and alanine methyl ester hydrochloride.

7. Use of the amino acid derivative-rhodamine B conjugate according to claim 1 as a fluorescent tracer.

8. The use according to claim 7, characterized in that: Application of amino acid derivative-rhodamine B conjugates as fluorescent tracers in crop and weed identification.

9. The use according to claim 8, characterized in that: The crops are soybean, corn and tobacco.

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