【作者】 任莉；

【导师】 陈小军；

【作者基本信息】 扬州大学 ， 农药学， 2019， 硕士

【摘要】 螺虫乙酯(Spirotetramat)是一种能有效防治蚜虫、蓟马、粉虱等刺吸式口器害虫的杀虫剂。目前,国内外关于螺虫乙酯的研究,主要集中在合成工艺、药效、在植物体内的消解残留及在环境中的迁移转化方面。有研究发现螺虫乙酯对害虫的田间药效已有所下降,昆虫害虫会发生代谢或其他原因导致对螺虫乙酯产生抗药性,目前尚无螺虫乙酯在昆虫及哺乳动物体内的代谢研究以报道。为了研究螺虫乙酯在家蚕体内的代谢规律,本论文以螺虫乙酯为研究对象,以家蚕为材料,建立了螺虫乙酯及4种代谢物在家蚕主要组织部位中的残留分析方法;研究了螺虫乙酯对家蚕的毒性及其在家蚕中肠、丝腺、脂肪、血液、粪便中螺虫乙酯及代谢物的分布;分析了螺虫乙酯对家蚕3大解毒酶系的活性变化影响;通过qRT-PCR技术分析了螺虫乙酯对家蚕解毒酶相关基因表达的影响。主要研究结果如下:(1)采用QuEChERS样品前处理方法,结合液相色谱-串联质谱(LC-MS/MS)检测技术,以负离子扫描和多反应检测模式(MRM),建立了家蚕中肠、丝腺、脂肪中螺虫乙酯及代谢物的残留分析方法。结果表明,以甲醇为提取溶剂,经C18净化,螺虫乙酯及代谢物的添加回收率为80.2-108.7%,满足农药残留检测要求,适合家蚕各组织部位样品中残留螺虫乙酯及代谢物的多残留检测。(2)分析了各组织部位中螺虫乙酯及2种代谢物的分布及随时间的变化情况。结果表明,B-enol及B-keto能在家蚕所有组织中检测到;B-glu仅在中肠及粪便中被检测到;B-mono无法检测到。从变化趋势看,螺虫乙酯能在中肠中降解为B-enol、B-keto及B-glu,且B-glu能通过粪便快速排出体外。经过血液的流动,螺虫乙酯及B-enol能在丝腺中积累,含量升高,且不易被降解转移,而在脂肪中,B-enol含量不断增高,说明螺虫乙酯在脂肪内被有效降解为B-enol,B-keto在中肠、丝腺、脂肪中都呈下降趋势,说明螺虫乙酯被快速降解为B-keto,随后B-keto慢慢被转移并排出。(3)螺虫乙酯暴露后,测定家蚕各组织部位(中肠、血液、丝腺和脂肪)3种解毒酶的活性变化。研究发现,在中肠中,处理组3种酶活性均高于对照组,中肠是家蚕的主要消化器官,螺虫乙酯在中肠中能被快速代谢,并被快速排出体外,说明中肠可能是代谢螺虫乙酯的主要场所,且3种酶均有参与螺虫乙酯的代谢。在丝腺中,仅GST活性有所上升,螺虫乙酯能够在丝腺中少量代谢,但大量的螺虫乙酯及代谢物在丝腺中积累。在脂肪中,P450活性较高,为对照组的1.87-2.44倍,螺虫乙酯在脂肪中大多能被代谢,但B-enol持续升高,说明P450可能参与螺虫乙酯的代谢,但脂肪不能将B-enol代谢并排出体外。在血液中,仅GST活性上升,说明血液对有毒物质较小的代谢作用可能与GST有关。(4)以解毒酶 7 个相关基因CYP9a19 CYP9a22、CYP6AB4、GST01、GSTe2、Bm CarE-11和BmCarE8-V1为研究对象,研究螺虫乙酯处理后家蚕解毒基因的表达情况。结果表明在中肠中,P450 3种基因的上调说明P450参与了家蚕对螺虫乙酯的代谢过程。GST相关基因上调水平较低,最高上调了 1.7-1.8倍。说明GST不是中肠主要的解毒酶。相比中肠中7种基因较高的表达量,脂肪中基因表达量较低,且随时间无显著变化趋势,这可能是由于螺虫乙酯对于脂肪合成的抑制作用,影响了解毒酶基因的表达,从而调控解毒酶活性。通过螺虫乙酯在家蚕体内的代谢研究,以明确其在家蚕体内的代谢产物;了解家蚕的主要代谢途径;阐明解毒酶的作用及相关基因的调控作用。因此,此研究为螺虫乙酯的合理使用和安全性评价提供了理论依据。更多还原

【Abstract】 Spirotetramat is a insecticide which can effectively control piercing-sucking mouthparts pests such as aphids,thrips and whiteflies.At present,the researches on spirotetramat in domestic and foreign countries mainly focus on the synthesis process,the efficacy,the residue in plants and the migration and transformation in the environment.It has been reported that the field efficacy of spirotetramat declined,however,there is currently no metabolic study of spirotetramat in insects and mammals to guide its rational use.In order to study the metabolic characteristics of spirotetramat in different tissues in silkworm,this paper used spirotetramat as the research object,and established the residue analysis method of spirotetramat and four metabolites in the main parts of silkworm.This paper also studied the toxicity of spirotetramat on silkworm and the distribution of spirotetramat and metabolites in silkworm midgut,silk gland,fat,blood and feces after adding mulberry leaves;the activity of three major detoxification enzymes of silkworm,treated with spirotetramat;the expression of the enzyme-related genes was verified by RT-PCR.The main findings as follows:(1)The paper used QuEChERS sample pretreatment method,combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)detection technology,negative ion scanning and multiple reaction detection mode(MRM),established residue analysis method of spirotetramat and metabolites in silkworm midgut,silk gland,fat.The results showed that with methanol as extraction solvent and C18 purification,the recoveries rate of spirotetramat and metabolites was 80.2-108.7%,which met the requirements of pesticide residue detection and was suitable for simultaneous detection of various parts of silkworm.(2)The distribution and the changes over time of spirotetramat and two metabolites in various parts of silkworm were analyzed by LC-MS/MS.The results showed that the spirotetramat was mainly degraded into two metabolites(B-enol and B-keto)in the silkworm,which could be detected in all tissues;B-glu could be detected in the midgut and feces;B-mono could not be detected.From the trend of change,spirotetramat can be degraded into metabolites B-enol,B-keto and B-glu in the midgut,and B-glu can be quickly excreted through feces.Through the flow of blood,spirotetramat and B-enol can accumulate in the silk gland,resulting in increased content and not easily degraded and metastasized.In fat,B-enol content is continuously increased,indicating that spirotetramat is may be effectively degraded to B-enol in fat,and B-keto is declining in the midgut,silk gland and fat,indicating that the spirotetramat is rapidly degraded to B-keto and then slowly transferred and discharged.(3)The mulberry leaves soaked with 200 mg/L spirotetramat were fed to the silkworm,and the activity of three detoxifying enzymesn silkworm parts(middle intestine,blood,silk gland and fat)were measured.The study found that in the midgut,the three enzyme activities in the treatment group were higher than the control group.The midgut is the main digestive organ of silkworm,and spirotetramat can be rapidly metabolized in the midgut and quickly excreted from the body.All three enzyme activities increased,which indicated that the midgut may be the main site for the metabolism of spirotetramat and all three enzymes are involved in the metabolism of spirotetramat.In the silk gland,only GST enzyme activity increased,and the spirotetramat can be metabolized in a small amount in the silk gland,but a large amount of spirotetramat and metabolites accumulate in the silk gland.This may affect the quality of silkworm silk,which affects its economic value.In fat,P450 enzyme activity is 1.87-2.44 times of the control group.Spirotetramat is mostly metabolized in fat,but B-enol continues to rise,indicating that P450 enzyme may be involved in the metabolism of spirotetramat,but fat cannot metabolize B-enol and excrete it.In the blood,only the activity of GST enzyme increased,indicating that the smaller metabolism of blood to toxic substances may be related to GST enzyme.(4)Seven genes including YP9a19、CYP9a22、CYP6AB4、GST01、GSTe2、BmCarE-11 and BmCarE8-V1 were selected as the research objects to study the expression of detoxification genes in silkworm after treatment with spirotetramat.The results showed that in the midgut,Up-regulation of the three genes from the P450 enzyme is involved in the metabolism of the spirotetramat in silkworm.The GST enzyme related gene was up-regulated at a lower level,The GST enzyme was up-regulated by 1.7-1.8fold.This indicates that GST enzyme is not the main detoxification enzyme in the midgut.Compared with the higher relative expression of the seven genes in the midgut,the relative expression of genes in fat is lower,and there is no significant change with time.This may be due to the inhibitory effect of spirotetramat on fat synthesis,affecting the the expression of the enzyme gene,thereby regulating the detoxification enzyme activity.Through the metabolism of spirotetramat in the silkworm,the metabolites in the silkworm can be identified;the main metabolic pathways of the silkworm can be understood and the role of detoxification enzymes and the regulation of related genes can be clarified.Therefore,this study provides data support for the rational use and safety evaluation of spirotetramat.更多还原