【作者】 李鑫；

【导师】 张利辉；

【作者基本信息】 河北农业大学 ， 农药学， 2020， 硕士

【摘要】 4-羟基-3-甲氧基肉桂酸乙酯是一种微生物源除草物质,对马唐等杂草具有良好的除草活性。为了明确其作用机理以及作用靶标,本研究采用化学合成的方法得到了 4-羟基-3-甲氧基肉桂酸乙酯的荧光标记物,并通过小分子化合物荧光示踪技术,研究该化合物在拟南芥内的定位情况。研究结果如下:(1)在4-羟基-3-甲氧基肉桂酸乙酯非主要活性位点上偶联荧光基团(9-蒽甲醛),设计合成4-羟基-3-甲氧基肉桂酸乙酯荧光标记物LX-2,其结构采用核磁共振氢谱和高分辨质谱进行表征确证,实现了实时无损检测该化合物在拟南芥中作用通路和部位。(2)以拟南芥为试验植物,通过带药平皿法测定了 4-羟基-3-甲氧基肉桂酸乙酯及其荧光标记物LX-2的除草活性并对其进行了比较分析。结果发现,4-羟基-3-甲氧基肉桂酸乙酯和LX-2均对拟南芥表现出较好的的抑制效果,IC50分别为29.84 mg/L和20.81 mg/L。采用小杯法测定了 4-羟基-3-甲氧基肉桂酸乙酯及其荧光标记物LX-2对马唐和反枝苋的除草活性发现,当药物浓度为200.00 mg/L时,4-羟基-3-甲氧基肉桂酸乙酯及其荧光标记物LX-2对这两种杂草的抑制率均在50%以上。以上结果证明,4-羟基-3-甲氧基肉桂酸乙酯与其荧光标记物LX-2的除草活性相当,LX-2可以用于4-羟基-3-甲氧基肉桂酸乙酯的定位试验。(3)测定了 4-羟基-3-甲氧基肉桂酸乙酯荧光标记物的荧光光谱,影响该化合物荧光强度的因素,吸收动力学曲线,洗脱曲线以及该化合物在拟南芥内的稳定性。扫描其荧光光谱后得到,该化合物的最大激发波长为329nm,最大发射波长为393 nm;测定影响该化合物荧光强度的因素发现,当光照强度为80%时采集的图片荧光强度最合适后期试验;4-羟基-3-甲氧基肉桂酸乙酯荧光标记物的吸收和洗脱曲线结果表明,该化合物最适宜的孵育浓度为0.05 mM,最适宜的孵育时间为72 h,经3次洗脱后该化合物的荧光强度不再下降,说明其在拟南芥内存在特异性结合位点。(4)对拟南芥根部显微结构观察发现,经4-羟基-3-甲氧基肉桂酸乙酯及其荧光标记物LX-2处理的拟南芥均出现根尖膨大,部分根冠脱落,导管变黑和木质化的现象;对其幼根亚显微结构观察发现,4-羟基-3-甲氧基肉桂酸乙酯和LX-2均可导致拟南芥幼根内细胞壁加厚,脂类物质大量蓄积。4-羟基-3-甲氧基肉桂酸乙酯和LX-2的除草症状一致,说明LX-2可以对4-羟基-3-甲氧基肉桂酸乙酯进行定位。(5)在荧光显微镜下观察发现,4-羟基-3-甲氧基肉桂酸乙酯定位在拟南芥的根尖和叶片部位,拟南芥的根尖和叶片均表现出LX-2的蓝紫色荧光。更多还原

【Abstract】 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.更多还原