Fluorescent probe for detecting magnaporthe oryzae and method for detecting magnaporthe oryzae through fluorescence imaging

Abstract

The invention belongs to the technical field of analysis and detection, and discloses a fluorescent probe for detecting magnaporthe oryzae and a method for detecting the magnaporthe oryzae through fluorescence imaging. A fluorescent probe used in the method is N-(ethyl) 10-(16 alkyl formate)-anthracene carboximide synthesized through multi-step modification on the basis of fluorescence characteristics of anthracycline dyes, and the N-(ethyl) 10-(16 alkyl formate)-anthracene carboximide can be used for carrying out specific detection on magnaporthe oryzae on the surfaces of rice leaves. After the fluorescent probe reacts with the magnaporthe oryzae, the color of the fluorescence is changed from original bright green to yellow, so that the method can be used as a method for detecting the magnaporthe oryzae. The detection method provided by the invention has good stability, and has the characteristics of good specificity, high accuracy and good stability when being used for detecting the magnaporthe oryzae.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a fluorescent probe for detecting rice blast fungi and a method for detecting rice blast fungi through fluorescent imaging. Background Art

[0002] Rice blast fungus, scientifically known as Fusarium gracilis, is a pathogenic fungus that causes rice blast and belongs to the phylum Deuteromycetes. It is one of the most serious pathogens of rice, causing serious economic losses to rice production. Fusarium gracilis has a complex life cycle and infection mechanism. Its life cycle includes two types of spores: conidia and ascospores. Conidia are the main form of transmission of Fusarium gracilis, which can be transmitted to healthy rice plants through wind, raindrops, insects and other means.

[0003] Fusarium gracilis needs to go through a series of steps during the infection process. First, conidia land on the surface of rice plants and form an adsorption structure. Then, it presses itself into the plant epidermal cells by producing branched tubes to form an invasion structure. Next, the pathogen further invades the plant cells by forming an infector and forms hyphae inside the cells. Finally, Fusarium gracilis forms spore piles in plant tissues, thus completing its life cycle and spreading to other plants through spores. The infection process of Fusarium gracilis is closely related to the disease resistance of rice plants. During the infection process, it secretes a series of toxins and enzymes to suppress the plant's immune response and promote its own infection. At the same time, rice plants also resist the invasion of Fusarium gracilis by producing disease-resistant related proteins and antioxidants. Therefore, in order to effectively control the invasion of rice blast fungus, the study of rapid detection technology for rice blast fungus is of great significance to the protection of rice.

[0004] One of the traditional detection methods for rice blast disease is to observe the disease symptoms on rice plants to determine whether they are infected with rice blast fungi. After infection with rice blast fungi, gray spots will appear on rice leaves, and gradually expand and fuse, causing the leaves to turn yellow and wither. This method is simple and intuitive, but symptom observation may be subjective and interfered by external environmental factors. Enzyme-linked immunosorbent assay (ELISA): ELISA is a commonly used immunological method that can be used for rapid detection of rice blast fungi. This method is based on the binding reaction between rice blast fungi-specific antigens and antibodies, and the presence of rice blast fungi is determined by color changes. ELISA has high sensitivity and specificity and can provide quantitative test results. Polymerase chain reaction (PCR): PCR is a molecular biology technique based on DNA amplification that can be used to detect the DNA of rice blast fungi. DNA is extracted from plant tissues, and the DNA of rice blast fungi is amplified using specific primers, and then the amplified products are detected by gel electrophoresis and other methods. PCR is highly sensitive and specific, and can detect the presence of rice blast fungi at the early infection stage. Real-time quantitative PCR (qPCR): qPCR is a modified PCR technique that can monitor the process of amplification reaction in real time. It combines PCR and fluorescent probe technology to quantitatively detect the number of rice blast fungi.

[0005] Although there are various existing detection technologies, many detection methods can accurately and quickly detect rice blast, but these detection methods also have many disadvantages. For example, the observation method will be interfered by environmental factors; ELISA usually requires a long operation time, including sample processing, reagent reaction and result reading, which may lead to a relatively long detection cycle and cannot meet the needs of real-time monitoring; PCR technology requires strict experimental operation and requires high experimental skills and experience, especially in designing primers and controlling reaction conditions. Therefore, the shortcomings of these detection methods are not conducive to their actual application in monitoring rice diseases. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art in the field of rice blast detection, such as being greatly affected by environmental factors, requiring high professionalism, and having expensive operating equipment, the primary purpose of the present invention is to provide a fluorescent probe for detecting rice blast fungi.

[0007] Another object of the present invention is to provide a method for detecting rice blast fungi by fluorescence imaging using the above-mentioned fluorescent probe; this method uses fluorescence imaging to detect the presence of rice blast fungi through changes in color signals, which is simple, fast, efficient, and low-cost.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A fluorescent probe for detecting rice blast fungus, wherein the fluorescent probe is N-(ethyl) 10-(16-alkyl formate)-anthracenecarboximide, and has a structure shown in the following formula (I):

[0010]

[0011] A method for detecting rice blast fungi by fluorescence imaging using the above-mentioned fluorescent probe, wherein N-(ethyl) 10-(16-alkylcarboxylate)-anthracenecarboximide is used as a fluorescent probe. After the fluorescent probe reacts with rice blast fungi, the fluorescent color changes from bright green to yellow, thereby detecting the presence of rice blast fungi.

[0012] The method specifically follows the following steps:

[0013] (1) dissolving N-(ethyl)-10-(1,6-alkylcarboxylate)-anthracenecarboximide as a fluorescent probe in an ethanol solution or a methanol solution to obtain a 10 μM probe solution;

[0014] (2) evenly coating the probe solution obtained in step (1) on the surface of rice leaves by spraying;

[0015] (3) The rice blast fungus on the rice leaves was detected under the irradiation of a handheld ultraviolet lamp at room temperature. Yellow fluorescence color observed by naked eyes indicated the presence of the rice blast fungus on the rice leaves, while bright green fluorescence color indicated the absence of the rice blast fungus on the rice leaves.

[0016] 4. The method for detecting rice blast fungi by fluorescence imaging according to claim 3, characterized in that: the time of detecting rice blast fungi on rice leaves in step (3) is 16 hours after the probe solution is applied to the surface of the rice leaves.

[0017] Principle of the present invention:

[0018] The anthracene ring is a single fluorophore ratiometric fluorescent probe with a long analytical wavelength. The photophysical properties of the 1,2-anthracenyl fluorophore depend on the electron-donating ability of the 10th substituent of the anthracene ring. After the probe reacts with the ester group on the 10th substituent, the electron-donating ability of the 10th substituent is changed, thereby significantly changing the photophysical properties of the probe and changing the color of the probe from green to yellow. The probe was used to detect rice blast fungus.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] (1) The fluorescent probe used in this method is based on the fluorescent properties of naphthylimide dyes. N-(ethyl) 10-(16-alkylcarboxylate)-anthracenecarboximide synthesized through multi-step modification has the ability to specifically detect rice blast fungi on the surface of rice leaves. When the fluorescent probe reacts with rice blast fungi, the color of the fluorescence changes from the original bright green to yellow. Therefore, this method can be used as a method for detecting rice blast fungi.

[0021] (2) The detection method of the present invention has good stability and has the characteristics of good specificity, high accuracy and good stability for the detection of rice blast fungus.

[0022] (3) The present invention attaches fluorescent probes to the surface of rice by spraying for the detection of rice blast fungi. The operation is very simple and the results can be directly observed with the naked eye. The detection process from spraying the probe to the handheld UV analyzer only takes a few minutes and is very efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the structure diagram of N-ethyl-6-bromo-anthracenimide.

[0024] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of N-ethyl-6-bromo-anthracenimide.

[0025] Figure 3 This is the mass spectrum of N-ethyl-6-bromo-anthracenimide.

[0026] Figure 4 This is the structure diagram of N-ethyl-6-methoxy-anthracenimide.

[0027] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of N-ethyl-6-methoxy-anthracenimide.

[0028] Figure 6 This is the mass spectrum of N-ethyl-6-methoxy-anthracenimide.

[0029] Figure 7 This is the structure diagram of N-ethyl-6-hydroxy-anthracenimide.

[0030] Figure 8 This is the hydrogen nuclear magnetic resonance spectrum of N-ethyl-6-hydroxy-anthracenimide.

[0031] Fig. 9 This is the mass spectrum of N-ethyl-6-hydroxy-anthracenimide.

[0032] Fig.10 This is the structural diagram of N-(ethyl) 10-(16-alkylcarboxylate)-anthracenecarboximide.

[0033] Fig.11This is the hydrogen nuclear magnetic resonance spectrum of N-(ethyl) 10-(16-alkylcarboxylate)-anthracenecarboximide.

[0034] Fig.12 This is the mass spectrum of N-(ethyl) 10-(16-alkylcarboxylate)-anthracenecarboximide.

[0035] Fig.13 The picture shows the fluorescent probe under UV analyzer and after adding rice blast fungus.

[0036] Fig.14 This is the fluorescence spectrum of the fluorescent probe and after adding rice blast fungus.

[0037] Fig.15 This is an image of rice under natural light.

[0038] Fig.16 Fluorescence image of rice sprayed with fluorescent probes under a handheld UV analyzer.

[0039] Fig.17 Fluorescence images of the fluorescent probe and different rice blast fungus concentrations under a handheld UV analyzer.

[0040] Fig.18 This is a comparison of the fluorescence images of rice leaves (containing fluorescent probes) and rice leaves (containing fluorescent probes and rice blast fungi) under a handheld UV analyzer. DETAILED DESCRIPTION

[0041] The present invention is further described below with reference to specific embodiments, but they should not be construed as limiting the present invention.

[0042] Embodiment 1:

[0043] A fluorescent probe for detecting rice blast fungus is prepared according to the following steps:

[0044] (1) Under nitrogen protection, 7-bromo-1,2-anthracene dicarboxylic anhydride (0.9 g, 27.5 mmol) and ethylamine (643 μl, 10 mmol) were dissolved in ethanol (about 55 ml), and the reaction temperature was raised to 78°C and reacted for 8 hours. After cooling, the solvent was removed by rotary evaporator, and then extracted with dichloromethane and deionized water. The organic phase was collected and further rotary evaporated to remove dichloromethane. After purification by silica gel column chromatography (dichloromethane), brown powder N-ethyl-6-bromo-anthracene imide (0.44 g, yield 49%) was obtained, with the structure shown below: Figure 1The product was characterized by hydrogen nuclear magnetic resonance spectrum (1H NMR (500MHz, Chloroform-d) δ9.71 (s, 1H), 8.34 (s, 1H), 8.22 (s, 1H), 8.04 (s, 1H), 7.68 (s, 1H), 7.62 (s, 1H), 7.53 (s, 1H), 4.09 (s, 2H), 1.35 (s, 3H).), and the results are as follows Figure 2 As shown. In addition, mass spectrometry was used to assist in the proof, and the results are as follows Figure 3 shown.

[0045] (2) Under nitrogen protection, the above-obtained N-ethyl-6-bromo-anthracenimide (0.44 g, 1.2 mmol), copper sulfate pentahydrate (8.4 mg, 0.036 mmol), and sodium methoxide (0.44 g, 8 mmol) were dissolved in methanol (20 mL), and stirred and refluxed at 71°C for 10 hours. After cooling, the methanol solvent was removed by rotary evaporator, and then extracted with dichloromethane and deionized water. After collecting the organic phase, the dichloromethane was further removed by rotary evaporation. After purification by silica gel column chromatography (dichloromethane: methanol = 18: 1), yellow powder N-ethyl-6-methoxy-anthracenimide (0.30 g, yield 80%) was obtained, and the structure is as follows Figure 4 The product was characterized by hydrogen nuclear magnetic resonance spectrum (1H NMR (500MHz, Chloroform-d) δ9.30 (s, 1H), 8.30 (d, J = 4.8Hz, 2H), 8.16 (s, 1H), 7.71 (s, 1H), 7.58 (s, 1H), 4.09 (s, 2H), 3.92 (s, 3H), 1.35 (s, 3H).), and the results are as follows Figure 5 As shown. In addition, mass spectrometry was used to assist in the proof, and the results are as follows Figure 6 shown.

[0046] (3) Under nitrogen protection, the above-obtained N-ethyl-6-methoxy-anthracenimide (0.30 g, 1.3 mmol) was added to 10 mL of hydroiodic acid (57%), and the temperature was raised to 135° C. for reaction for 10 hours. After the solution was cooled to room temperature, the pH value of the solution was adjusted to acidic with sodium hydroxide solution (2 M), filtered and washed with water, and then purified by silica gel chromatography (dichloromethane: methanol = 8: 1) to obtain a red powder solid N-ethyl-6-hydroxy-anthracenimide (0.15 g, yield 50%), the structure of which is shown below: Figure 7The product was characterized by hydrogen nuclear magnetic resonance spectrum (1H NMR (500MHz, Chloroform-d) δ9.00 (s, 1H), 8.31 (s, 1H), 8.23 ​​(s, 1H), 8.15 (s, 1H), 7.71 (s, 1H), 7.49 (s, 1H), 6.98 (s, 1H), 4.09 (s, 2H), 1.35 (s, 3H).), and the result is shown in 8. In addition, mass spectrometry was used for auxiliary proof, and the result was as shown in Fig. 9 shown.

[0047] (4) Under nitrogen protection, add N-ethyl-6-hydroxy-anthracenimide (0.15 g, 0.5 mmol) and hexadecanoyl chloride (0.14 g, 0.5 mmol) to 20 mL of acetone, stir and add 10 mL of ethylenediamine. Stir the reaction at room temperature for 15 hours. After cooling, remove the solvent with a rotary evaporator, extract with dichloromethane and deionized water, collect the organic phase and continue to rotary evaporate to remove dichloromethane. After purification by silica gel column chromatography (dichloromethane: methanol = 20: 1), the powder obtained is the fluorescent probe N-(ethyl) 10-(16-alkylcarboxylate)-anthracenimide, with the structure as shown Fig.10 The product was characterized by hydrogen nuclear magnetic resonance spectrum (1H NMR (500MHz, Chloroform-d) δ9.31 (s, 0H), 8.31 (s, 0H), 8.16 (s, 0H), 7.94 (s, 0H), 7.71 (s, 0H), 7.58 (s, 0H), 7.49 (s, 0H), 4.09 (s, 1H), 2.55 (s, 1H), 1.63 (s, 1H), 1.35 (s, 1H), 1.32-1.24 (m, 6H), 1.27 (s, 5H), 0.89 (s, 1H).), and the result is shown in Figure 11. In addition, mass spectrometry was used for auxiliary proof, and the result was as shown in Figure 11. Fig.12 shown.

[0048] Will Fig.13 As shown in the figure, the fluorescent probe obtained above was irradiated with 400nm excitation light, and the probe had a maximum absorption peak at 513nm, and the fluorescence was bright green. When the rice blast fungus was added, the electron donating ability of the 6-position substituent of the anthracene ring on the probe changed, the emission peak at 513nm became smaller, and a new emission peak appeared at 592nm. Fig.14 As shown, the probe color changes from green to yellow.

[0049] Example 2

[0050] Fig.15 This is an image of rice under natural light.

[0051] The probe N-(ethyl) 10-(16-alkylcarboxylate)-anthracenecarboximide obtained in Example 1 was dissolved in an ethanol solution to prepare a probe solution with a concentration of 10 μM. Then, the probe solution was sprayed on the surface of rice leaves. Fig.16 The fluorescent probe was sprayed on rice, and the fluorescence image under the handheld UV analyzer was taken; then the rice blast fungus was also sprayed on the surface of the rice leaves; at room temperature, the fluorescent color changes of the rice leaves were observed with a handheld UV analyzer at 2 hours, 8 hours, 16 hours, 24 hours, 30 hours, and 36 hours. The results showed that Fig.17 As shown, with the increase of time, the number of rice blast fungi on the surface of rice leaves increases. After 16 hours, it is obviously visible to the naked eye that the color of the fluorescent probe on the surface of rice leaves changes from bright green to yellow, indicating that the method of the present invention can indeed detect rice blast fungi on the surface of rice leaves quickly and conveniently under realistic conditions.

[0052] Fig.18 This is a comparison of the fluorescence images of rice leaves (containing fluorescent probes) and rice leaves (containing fluorescent probes and rice blast fungi) under a handheld UV analyzer.

[0053] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting rice blast fungus, characterized in that: The fluorescent probe is N-(ethyl) 10-(16-alkylcarboxylate)-anthracenecarboximide, having a structure shown in the following formula (I):

2. A method for detecting rice blast fungus by fluorescence imaging using the fluorescent probe according to claim 1, characterized in that: The method uses N-(ethyl) 10-(16-alkyl formate)-anthracenecarboximide as a fluorescent probe. After the fluorescent probe reacts with rice blast fungus, the fluorescent color changes from bright green to yellow, thereby detecting the presence of rice blast fungus.

3. The method for detecting rice blast fungus by fluorescence imaging according to claim 2, characterized in that: The method specifically follows the following steps: (1) dissolving N-(ethyl)-10-(1,6-alkylcarboxylate)-anthracenecarboximide as a fluorescent probe in an ethanol solution or a methanol solution to obtain a 10 μM probe solution; (2) evenly coating the probe solution obtained in step (1) on the surface of rice leaves by spraying; (3) The rice blast fungus on the rice leaves was detected under the irradiation of a handheld ultraviolet lamp at room temperature. Yellow fluorescence color observed by naked eyes indicated the presence of the rice blast fungus on the rice leaves, while bright green fluorescence color indicated the absence of the rice blast fungus on the rice leaves.

4. The method for detecting rice blast fungus by fluorescence imaging according to claim 3, characterized in that: The time for detecting the rice blast fungus on the rice leaves in step (3) is 16 hours after the probe solution is applied to the surface of the rice leaves.

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