| 4-Hydroxy-3-methoxycinnamic acid ethyl ester(ethyl ferulate)is a kind of microorganism source herbicidal substance derived from natural product ferulic acid.It has good herbicidal activity against Digitaria sanguinalis and so forth.In order to clarify its mechanism of action and target,in this study,the 4-hydroxy-3-methoxycinnamic acid ethyl ester fluorescent label was obtained by chemical synthesis.Compound fluorescent tracing technology was used to study the localization of the compound in Arabidopsis thaliana.The results are as follows:(1)A fluorescent group(9-anthracene formaldehyde)to the non-main active site of 4-hydroxy-3-methoxycinnamic acid ethyl ester was coupled,and 4-hydroxy-3-methoxycinnamic acid ethyl ester fluorescent label LX-2 was designed and synthesised.The structure of 4-hydroxy-3-methoxycinnamic acid ethyl ester fluorescent label LX-2 was characterized and confirmed by nuclear magnetic resonance hydrogen spectroscopy and high-resolution mass spectrometry,real-time non-destructive detection of the action pathway and location of the compound in A.thaliana.(2)Using A.thaliana as the test plant,the herbicidal activity of 4-hydroxy-3-methoxycinnamic acid ethyl ester and its fluorescent label LX-2 was determined by the plate method with medicine and compared and analyzed.The results showed that both 4-hydroxy-3-methoxycinnamic acid ethyl ester and LX-2 showed good inhibitory effect on A.thaliana,with IC50 was 29.84 mg/L and 20.81 mg/L,respectively.The small cup method was used to determine the herbicidal activity of 4-hydroxy-3-methoxycinnamate and its fluorescent label LX-2 on D.sanguinalis and Amaranthus retroflexus.When the drug concentration was 200.00 mg/L,4-hydroxy-3-methoxycinnamic acid ethyl ester and its fluorescent label LX-2 inhibit these two weeds by over 50%.The above results prove that the herbicidal activity of 4-hydroxy-3-methoxycinnamic acid ethyl ester and its fluorescent label LX-2 are equivalent,and LX-2 can be used for the localization of 4-hydroxy-3-methoxycinnamic acid ethyl ester test.(3)The fluorescence spectrum of the 4-hydroxy-3-methoxycinnamic acid ethyl ester fluorescent label,the factors affecting the fluorescence intensity of the compound,the absorption kinetics curve,the elution curve,and the stability of the compound in A.thaliana were measured.After the scanning of fluorescence spectrum,the maximum excitation wavelength of the compound is 329 nm and the maximum emission wavelength is 393 nm.The factors affecting the fluorescence intensity of the compound were found to be that the fluorescence intensity of the picture collected when the light intensity was 80%was most suitable for the later stage Test.The results of the absorption kinetics curve and elution curve of the 4-hydroxy-3-methoxycinnamic acid ethyl ester fluorescent label indicated that the optimal incubation concentration of the compound is 0,05 mM,and the optimal incubation time is 72 h.The fluorescence intensity of the compound does not decrease after three elutions,indicating that it has specific binding sites in A.thaliana.(4)Observation of the root microstructure of A.thaliana showed that the A.thaliana treated with 4-hydroxy-3-methoxycinnamic acid ethyl ester and its fluorescent label LX-2 all showed swelling of the root tip,part of the root cap falling off,blackening of the vessel and lignification;observation of the submicroscopic structure of its young roots found that both 4-hydroxy-3-methoxycinnamic acid ethyl ester and LX-2 can cause the inner cell wall of A.thaliana young roots to thicken and fat Large accumulation of similar substances.The herbicidal symptoms of 4-hydroxy-3-methoxycinnamic acid ethyl ester and LX-2 are the same,indicating that LX-2 can locate 4-hydroxy-3-methoxycinnamic acid ethyl ester.(5)Observed under a fluorescence microscope,4-hydroxy-3-methoxycinnamic acid ethyl ester is located in the root tips and leaves of A.thaliana,and its root tips and leaves all show the blue-violet fluorescence of LX-2. |
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