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两种酰胺类除草剂在水稻中强化降解效应的研究
Enhanced Degradation of Two Amide Herbicides in Rice
【作者】 李娜;
【导师】 杨红;
【作者基本信息】 南京农业大学 , 化学, 2020, 硕士
【摘要】 酰胺类除草剂是目前使用较为普遍的一类芽前除草剂,广泛应用于水稻、玉米、花生等农作物,防除一年生禾本科杂草,提高农作物的产量。施用酰胺类除草剂之后,特别是不合理不规范的施用会在环境中大量残留,使农作物产生药害,影响农作物的正常生长发育,进而影响农产品质量安全。针对这些问题,近些年来,有关酰胺类除草剂环境残留降解和生物修复技术的研究越来越多。糖基转移酶是植物体内一种重要的酶系,参与植物内有毒物质的解毒与代谢作用。本文利用糖基转移酶的解毒代谢特性展开研究。首先,考查了丁草胺和毒草胺对水稻幼苗毒性的影响。结果表明,不同浓度的丁草胺(0-1 mg/L)和毒草胺(0-0.5 mg/L)处理水稻幼苗6d,水稻幼苗的生长显著受到抑制,伸长量和干重量随着丁草胺和毒草胺处理浓度的加大而明显减少。电解质渗透率(EL%)和硫代巴比妥酸反应物(TBARS)的含量随着处理浓度的增加呈先上升的趋势,当达到一定的浓度时,反而呈下降趋势,说明在丁草胺和毒草胺胁迫下,水稻幼苗组织细胞的膜通透性和膜脂过氧化程度受到显著的影响,而高浓度的农药胁迫使得水稻植物组织遭到破坏,使其难以恢复自身的抵御机制。为了探究水稻糖基转移酶缓解农药对水稻的毒害作用,本文选择了实验室前期筛选得到的在农药胁迫下表达量上调的糖基转移酶OsGT1基因作为研究对象,通过生物技术手段获得了该基因的3个过表达纯合水稻株系,OsOE-1、OsOE-2和OsOE-6。研究结果表明,在无农药胁迫下,不同生物型水稻的伸长量、干重量、电解质渗透率和硫代巴比妥酸反应物的含量等生理指标无显著性差异,说明该基因的过表达对水稻的正常生长无影响;当0.5 mg/L的丁草胺和0.05 mg/L的毒草胺处理水稻6 d,与野生型水稻相比,过表达水稻株系地上部分和根部的伸长量和干重量有显著性的增加;而电解质渗透率和硫代巴比妥酸反应物的含量减少,说明农药对过表达水稻组织细胞膜的损害程度较弱。进一步测定水稻体内糖基转移酶活力,在无农药胁迫时,过表达水稻株系糖基转移酶的活力显著高于野生型水稻,在丁草胺和毒草胺胁迫下,过表达水稻株系中糖基转移酶活力比野生型水稻中的酶活力增加的百分比明显高于未加药处理。结果表明,该基因在农药诱导下表达量上调,说明该基因可通过调控糖基转移酶的作用来缓解外源农药对水稻的生长毒害。为了进一步探究该基因缓解农药对水稻的毒害作用,采用气相色谱(GC)和高效液相色谱(HPLC)分别测定了野生型和过表达水稻中毒草胺和丁草胺的积累量,以及生长介质中的残留量。结果表明,过表达水稻中两种农药的积累量低于野生型水稻,且生长介质中的农药残留量也显著性降低。在模拟大田水稻实际生长环境的实验中,丁草胺的胁迫浓度为0.02mg/L,分别测定了 30d,60 d,90 d水稻植株不同部位的积累量和120 d成熟可食籽粒中的积累量,发现过表达水稻株系中丁草胺的积累量始终低于野生型水稻。说明OsGT1基因在植物体内的过表达,能增加糖基转移酶的活力,减少丁草胺在水稻中的积累,进而缓解农药对水稻的毒害作用。最后采用HRLC-Q-TOF-MS/MS质谱技术,检测并鉴定了过表达水稻OsOE-6和野生型水稻中丁草胺的代谢产物,共发现了 16种降解产物和7种结合物,其中3种降解产物和5种结合物只有在过表达OsOE-6水稻中鉴定发现。通过对代谢产物的相对量分析得知,丁草胺在水稻中大部分降解产物和结合物的相对含量都是OsOE-6水稻高于野生型,说明糖基转移酶增强了丁草胺在水稻中的代谢作用,进一步证明OsGT1参与水稻中农药代谢与解毒的机制。
【Abstract】 Amide herbicide was a kind of pre bud herbicide widely used at present.It was mainly used in rice,corn,peanut and other crops to control annual grass weeds and improved the yield of crops.However,the use of amide herbicide,especially a large number of unreasonable use,a large number of residues were left in the environment,which would produce pesticide damage to crops,affecting the normal growth of crops.In recent years,there are more and more researches on the degradation of amide herbicides and bioremediation technology.Glycosyltransferase was an important enzyme of detoxification and degradation for toxic substances in plants.In this paper,the detoxification and degradation characteristics of glycosyltransferase were studied.First,the effects of butachlor and propachlor on the growth toxicity of rice seedlings were studied.The results showed that the growth of rice seedlings treated with butachlor(0-1 mg/L)and propachlor(0-0.5 mg/L)for 6 days was obviously inhibited,and the elongation and dry weight decreased with the increase of butachlor and propachlor concentration.The electrolyte leakage and thiobarbituric acid reactant content increased with the increase of two herbicides concentration,but decreased when the concentration reached a certain level.The results showed that the membrane permeability and membrane lipid peroxidation of rice seedling tissue were significantly affected under the stress of butachlor and propachlor.The high concentration of two herbicides could destroy the rice tissue and make it difficult to restore its own resistance mechanism.In order to the alleviating effect of glycosyltransferase on the toxicity of herbicides to rice,OsGT1 gene was selected as the research object.Three over expressed rice lines,OsOE-1,OsOE-1 and OsOE-1,were obtained by biotechnology.The results showed that there was no significant difference in the elongation,electrolyte leakage and thiobarbituric acid reactant content of different biotypes of rice without pesticide.However,when treated with 0.5 mg/L butachlor and 0.05 mg/L propachlor for 6 days,comparing with wild-type rice,the elongation and dry weight of the shoot and root of the over expressed rice lines increased significantly.The electrolyte permeability and the content of thiobarbituric acid reactant decreased in over expressed rice lines,indicating that the damage degree of cell membrane was reduced.The activity of glucosyltransferase in over expressed rice lines was significantly higher than that in wild-type rice with/without pesticide stress.The percentage of glucosyltransferase activity in over expressed rice lines was significantly higher than that in wild-type rice with butachlor and propachlor stress.The results showed that the expression of the gene was up-regulated under pesticides stress,which indicated that the gene could alleviate the toxic efect of exogenous pesticides on the growth of rice by regulating the function of glycosyltransferase.In order to further explore the reason of the gene alleviating the toxicity of rice,the accumulation of propachlor and butachlor in overexpressed rice and wild type rice and the residue in growth medium were determined by GC and HPLC respectively.The results showed that the accumulation of over expressed rice was lower than that of wild type rice,and the residue of nutrient solution was also significantly reduced.In the experiment of simulating the actual growth environment of rice in the field,the concentration of butachlor was set to 0.02 mg/L.The accumulation amount of butachlor in different parts of rice at 30 days,60 days,90 days and 120 days was measured respectively.It was found that the accumulation amount of butachlor in overexpressed rice lines was always lower than that of wild type rice.It was suggested that the overexpression of OsGT1 gene in plants could reduce the accumulation of butachlor in rice and alleviate the toxic effect of pesticides on rice.Finally,the metabolites of butachlor in rice were detected by HRLC-Q-TOF-MS/MS.Sixteen degradation products and seven conjugates were found and confirmed,among which 3 degradation products and 5 conjugates were identified only in overexpressed rice OsOE-6.Through the relative quantitative analysis,the relative content of most the degradation products and conjugates were higher in OsOE-6 than in wild-type rice.The results showed that the overexpression of rice enhanced the metabolism of butachlor in rice,which further confirmed that OsGT1 gene was involved in the metabolism and detoxification mechanism of pesticides in rice.
【Key words】 butachlor; propachlor; overexpressed rice; accumulation; metabolization;
- 【网络出版投稿人】 南京农业大学 【网络出版年期】2022年 04期
- 【分类号】TQ457
- 【下载频次】60