【作者】 周青燕；

【导师】 章永松；

【作者基本信息】 浙江大学 ， 环境修复与资源再生， 2010， 博士

【摘要】 农业有机化合物广泛存在于环境中,其手性对映异构体在生物有效性、环境安全性等方面存在不可忽视的差异性,已越来越引起人们的重视。作为我国除草剂支柱产品的咪唑啉酮类除草剂是典型的具有手性结构的农药,其对映体对靶标酶乙酰乳酸合成酶(ALS)的离体活性表现出对映体选择性抑制。然而,酶活抑制的手性差异性机理尚未被揭示,对映体对植株的差异性抑制研究也尚未见报道。为此,本文首先建立了最具代表性的咪唑啉酮除草剂咪唑乙烟酸(IM)对映体在高效液相色谱(HPLC)上的手性分离条件。在得到对映体制备样的基础上,通过对咪唑乙烟酸的对映异构体以及外消旋体作用于玉米幼苗和拟南芥幼苗的研究,一方面观察作为土施农药,其对首要作用部位根系的生长形态、亚显微结构和根毛发生发育的选择性影响,以及植物体内生长调节物质的响应与根毛的形成、发育之间的相互关系；另一方面,研究其对靶标酶乙酰乳酸合成酶活性抑制的对映体选择性差异及机理。从这两方面探明和揭示IM对映体对植物的对映体选择性抑制及机理。本文取得的主要研究结果如下在高效液相色谱上,采用Chiralpak OJ手性柱,以正己烷/乙醇/乙酸(75/25/0.5,v/v/v)为流动相,基线分离得到IM的对映体。并采用HPLC-CD联用技术,确定对映体的出峰次序和CD光谱图。通过八区律确定了IM两个对映体的绝对构型分别为S-(+)-IM和R-(-)-IM。同时以所建立的对映体分离方法制备了对映体标样。通过除草剂IM对映体(浓度为100,200,400和800μgL-1)作用于玉米和拟南芥幼苗的试验发现,IM显著抑制植株地上部和根系的生长,使植株表现出明显的毒害症状,包括叶子萎黄、生长停滞以及根尖溃烂等。IM对生长的抑制作用呈对映体选择性,R-(-)-IM的毒害作用最强,S-(+)-IM的生长抑制作用最弱,外消旋的抑制能力介于左右旋之间。当处理浓度为100,200,400和800μgL-1,IM对玉米根系形态呈对映体选择性影响,根直径增粗,而根表面积和体积显著减小,侧根数也显著减少,根系活力显著下降。200和400μgL-1IM对映体分别选择性抑制玉米和拟南芥根毛的生长,根毛长度明显变短,数量显著减少,根毛细胞破损。通过转基因拟南芥DR5：GFP观察根部的生长素水平发现,IM处理降低了生长素含量,在受抑制强烈的R-(-)-IM中,根毛中荧光强度比s-(+)-IM处理的弱,表明生长素含量低。添加外源生长素处理,可缓解根毛生长的抑制状况。根中生长素含量的降低是IM毒害根毛生长的重要原因。400μgL-1 IM处理对根冠细胞器亚显微结构具有对映体选择性毒害作用,导致根尖细胞核呈不规则形状,而且核仁消失,其他细胞器数量减少,个体变小其中,线粒体膜解体,线粒体嵴也变得模糊不清；内质网膜断裂；高尔基体膜囊肿胀,小液泡消失。IM处理还导致细胞膜和细胞壁增粗,纤维化,失去弹性,其中平衡石的亚显微结构的破坏导致根系横向生长。施入土壤或残留的除草剂对植株的对映体选择性毒害首先表现在对根系的快速抑制,其通过对根系形态结构的改变、对根细胞内部结构的破坏、对根系和根毛生长的抑制以及生理活性抑制,最终导致对植株的毒害。IM对离体和活体ALS酶活性均存在对映体选择性抑制,R-(-)-对映体的抑制作用最强,S-(+)-对映体的抑制作用最弱,(±)-IM介于左右旋之间。离体酶活性抑制情况比较复杂,随着处理浓度变化,左右旋抑制差异呈规律性变化,当IM浓度为40μg L-1时,R-(-)-对映体对离体酶活的抑制率达到5.6%,而S-(+)-对映体对酶活仅有微弱的抑制,抑制率为0.2%,右旋和左旋抑制能力相差25倍之多。随着浓度的升高,抑制作用越加显著,左右旋的差异也逐渐减少。浓度升高至200μg L-1,R-(-)-IM的抑制率为28.9%,S-(+)-和(±)-IM为3.8%和19.8%。R-(-)-对映体和S-(+)-对映体的抑制率差异降低至7倍。当IM浓度低于1 mg L-1,S-(+)-IM的抑制作用上升缓慢,而R-(-)-IM的抑制却已很强烈,1 mg L-1时已经达到70.4%。随着的浓度呈倍数升高,S-(+)-对映体的抑制作用也显著提高,在5mg L-1和25 mg Lq,S-(+)-,R-(-)-和(±)-IM三者都对酶活有很高的抑制,左右旋的差异仅为1倍多。；100,200,400和800μg L-1的IM处理后,左右旋对活体酶活抑制的差异在2倍左右,与植株生长抑制情况一致。表明活体酶活与生长情况有更大的相关性。分子对接实验表明IM对映体与ALS相互作用存在立体选择性.,R-(-)-IM才是与ALS结合的优势构型。因此R-(-)-IM比S-(+)-IM对ALS的抑制作用更强。农药的手性研究、单一光学活性异构体生产上的突破,是减少生态环境污染和推行绿色农业的必然要求。为此,在手性层面上深入研究对映异构体的活性、作用机理等问题具有重要现实意义。本文通过对咪唑乙烟酸的对映体以及外消旋体作用于玉米和拟南芥幼苗根系及靶标酶的研究,探明和揭示IM对映体对植物的对映体选择性抑制及机理,为客观评价咪唑啉酮类除草剂的生物活性、生产上如何防治或降低残留除草剂对后茬作物的毒害以及开发新型单一旋光性除草剂提供理论依据。更多还原

【Abstract】 The enantioselectivety of the chiral agricultural organic compounds widespread in the environment has received more and more extensive understanding and became one of the focus researches. Imidazolinones, as a pillar of China’s herbicide products, are a class of pesticides with typical chiral structure. It has been reported that the imidazolinones enantioselectively inhibited the acetolactate synthase (ALS), however, the mechanism of the enantioselective inhibition on anzyme has not been revealed; and the enantioselective phytotoxicity has not yet reported. In this study, imazethapyr (IM), one of the imidazolinones, which is chiral and is number one among all of the imidazolinones in sales, was chosed in this article as a model herbicide. Enantiomeric pure IM was eperated by High Performance Liquid Chromatography (HPLC). On this basis, enantioselective phytotoxicity of IM on plant roots growth were determined by measuring changes in the root morphology, subcellular structure and root hair growth of tender maize and Arabidopsis thaliana seedlings. As IM is a soil-applied herbicide, the radicle is the first organ to contact the herbicide in the soil. Exploring the relationship between plant growth regulators responses and root hair development and the mechanism of enantioselective inhibition of IM on acetolactate synthase activity so as to explore and reveal the mechanism of enantioselective phytotoxicities. The main findings of the paper are as follows:The IM enantiomers were baseline separated by HPLC on chiral column Chiralpak OJ with the mobile phase of hexane/ethanol/acetic acid solution (75/25/0.5 by volume). Circular dichroism (CD) detectors were used to determine the elution order and CD spectra of the enantiomers. The absolute configuration of IM enantiomers was identified as S-(+)-IM and R-(-)-IM by the octant rule from force-field calculations and CD spectra.Plant growth was enantioselectively inhibited by IM enantiomers at the concentration of 100,200,400 and 800μg L-1. Visible crop injury symptoms were observed, including chlorosis of leaves, reduced plant growth, and putrescence on the root tips.R-(-)-IM was the most effective inhibitor in the damage of maize and Arabidopsis thaliana growth in comparison to the S-(+)-IM and racemate mixtures at equal concentrations. The inhibition ability of racemate was between the R and S enantiomer.The maize root morphology was enantioselectively affected by the IM at the concentration of 100,200,400 and 800μg L-1. Root diameter was increased; the number of root tips, the root volume and surface area were significantly decreased. IM also enantioselectively decreased root activity.IM Enantioselectively inhibited the growth of root hairs of maize and Arabidopsis at 200 and 400μg L-1, respectively. Root hair length was shortened, the number of root hair significantly was reduced, and cell membranes were damaged. The observation of hormone levels in transgenic Arabidopsis DR5::GFP showed that the auxin content was involved in root hair growth inhibition rate. Exogenous auxin treatment alleviated the growth inhibition of root hair.Ultrastructural studies revealed that IM at the concentration of 400μgL-1 had adverse effects on cell organelles in maize root caps:cell nucleuses were irregular, and nucleoluses were disappeared; other cell organelles were damaged by a reduction in number and size; mitochondrial membranes were disintegrated, mitochondrial cristae became blurred; endoplasmic reticulum membranes were ruptured; the dictyosomers were severely swollen, small vacuoles were disappeared; membranes and cell walls became thicker and showed the phenomenon of fibrosis, and lacked flexibility. The ultrastructure of the statocyte seriously damaged, releasing the starch grains into the cytoplasm, so that the root cap would no longer be able to respond correctly to gravity, resulting in a transverse growth of the root.Enantioselective toxicity of herbicide on the plants after applied into the soil first reflects in the rapid inhibition of root. The changing of root morphology, the destruction of the internal structure of root cells, root hair growth suppression and physical activity inhibition ultimately lead to plant toxicity.IM enantioselectively suppressed the in vitro and in vivo ALS activity of maize leaves. R-(-)-IM was more active than S-(+)-IM. The in vivo ALS activity study showed only a 2-fold difference between R-(-)-IM and S-(+)-IM at the concentration of 100,200,400 and 800μg L-1 which is accordance with the previous established difference in retarding the plant growth of maize. Quite different from the in vivo study, the in vitro study showed that the difference in inhibition between the enantiomers fell sharply as concentration increased. At the lowest concentration of 40μg L-1,R-(-)-IM appeared 25 times more active than S-(+)-IM, but only 7 times at 200μg L-1. At the highest concentration of 25 mg L-1, in vitro ALS activity was almost completely inhibited by S-(+)-IM, there was only 1.1times differences between S-(+)-IM and R-(-)-IM. In the study of molecular docking, the combination of the IM with ALS shows chiral discrimination. R-(-)-IM could bind to ALS in its preferred orientation. The different interaction modes of the R-(-)-and the S-(+)-IM with ALS obtained from molecular docking provide a structural explanation for the more potent activity of the R-(-)-IM in contrast to the S-(+)-IM.Because of the very different biological properties that selectively interacting with biological systems, enantiomers of chiral coumpounds can be regarded as different substances. The enantioselectivity in efficacy, environmental safety and other aspects can not be ignored. Realization of the major breakthrough of chiral pesticides researches and production of a single optically active isomer are the inevitable requirements for reducing environmental pollution and the implementation of green agriculture. The study provides indicative values for the objective evaluation of imida.zolinone herbicides in environmental effects, for the prevention of the succeeding crop from the poisoning of residual herbicides, for directing research into the manufacture of sole optically active herbicides with high efficiency and low side-effects.更多还原