【作者】 王宁；

【导师】 于志国；

【作者基本信息】 沈阳农业大学 ， 农药学， 2023， 博士

【摘要】 桃蚜（Myzus persicae）是一种全球范围分布的害虫,可以危害超过500种作物,造成了严重的经济损失。化学防治是控制桃蚜的主要手段,然而化学杀虫剂的频繁和不规范使用导致了抗药性的产生,增加了对桃蚜的防治难度。因此,研究开发高效低毒环境友好的新型生物源杀虫剂成为当今害虫防治的研究热点。天然产物是新农药发现的重要来源之一,放线菌在发现新天然产物方面发挥着重要作用。为寻求具有新颖结构并对桃蚜具有毒杀作用的天然产物,本论文对107株放线菌进行液体振荡培养,对其培养液粗提取物分别进行薄层层析检测和桃蚜胃毒活性筛选,综合两种筛选结果,选定链霉菌Streptomyces sp.SA61作为对桃蚜生物活性研究的目标菌株。通过对Streptomyces sp.SA61菌株粗提取物进行分离纯化得到单体化合物,并通过对高分辨质谱（HRESIMS）、核磁共振图谱、X-射线单晶衍射等数据的综合解析,确定其化学结构包括绝对构型。测定所得化合物strekingmycin A-F对桃蚜的杀虫活性,并测定活性化合物strekingmycin A对桃蚜体内解毒酶活性的影响以及对作物的安全性评价。具体研究结果如下:1.放线菌菌株培养液粗提取物的薄层层析以及对桃蚜杀虫活性初筛:将107株放线菌在两种培养基中通过摇瓶振荡培养得到214个培养液粗提取物。根据薄层层析检测结果,选出了33个次生代谢产物丰富的粗提取物,然后采用饲喂法测定它们对桃蚜的杀虫活性。综合薄层层析和杀虫活性筛选结果,最终选定链霉菌Streptomyces sp.SA61并对其活性成分进行研究。2.Streptomyces sp.SA61菌株振荡培养及其粗提取物的分离纯化:链霉菌Streptomyces sp.SA61在摇床中进行液体振荡培养,摇床温度为28℃,转速为180 r/min,培养7 d,共培养48 L。利用大孔吸附树脂进行固相萃取,并通过有机溶剂洗脱提取,最终得到粗提取物20 g。通过硅胶柱层析,凝胶柱层析和高效液相色谱等分离技术,从Streptomyces sp.SA61菌株粗提取物中分离得到了6个单体化合物。3.Streptomyces sp.SA61菌株次生代谢产物的结构鉴定:通过高分辨质谱、核磁共振氢谱（¹H NMR）、核磁共振碳谱(¹³C NMR)、二维谱（2D-NMR）以及X-射线单晶衍射等数据,推导出化合物1-6的化学结构,均为聚酮类化合物。通过与文献数据对比,结果表明,化合物1是本实验室前期分离到的一个化合物;化合物2-6均为新化合物,并且具有不同于1的碳骨架。将化合物1-6分别命名为strekingmycin A-F。4.化合物strekingmycin A-F对桃蚜的杀虫活性测定:通过浸叶法测定了化合物对桃蚜的毒力,结果显示化合物strekingmycin A-F均对桃蚜具有杀虫活性,96 h的LC₅₀值分别为10.29、14.57、27.81、34.47、31.57和21.04μg/m L;通过饲喂法测定了化合物对桃蚜的胃毒活性,结果显示化合物strekingmycin A-F均对桃蚜具有胃毒作用,96 h的LC₅₀值分别为12.04、15.61、16.63、17.20、12.48和23.50μg/m L;通过管膜法测定了化合物对桃蚜的触杀活性,结果显示化合物strekingmycin A-F均对桃蚜具有触杀作用,96h的LC₅₀值分别为11.58、13.30、11.97、13.68、12.80和24.57μg/m L;通过蜜露法测定了化合物对桃蚜的拒食活性,结果显示48 h后,化合物strekingmycin A-F均对桃蚜具有拒食作用,AC₅₀值分别为10.41、13.09、10.86、12.56、10.52和10.86μg/m L;通过叶碟法测定了化合物对桃蚜的趋避活性,结果显示化合物strekingmycin A-F均对桃蚜具有趋避作用,48 h后趋避率分别为82.67%、82.31%、73.33%、62.30%、54.88%和69.87%。综合以上结果可以看出化合物strekingmycin A对桃蚜的杀虫效果最优。5.活性化合物strekingmycin A对桃蚜体内解毒酶活性的影响:结果表明strekingmycin A对桃蚜体内细胞色素P450（CYP450）、羧酸酯酶（Car E）和谷胱甘肽-S-转移酶（GST）均具有抑制作用,能够降低桃蚜的解毒能力,使strekingmycin A发挥更强的毒性,导致其死亡。6.活性化合物strekingmycin A对作物的安全性评价:测定了strekingmycin A在浓度为100μg/m L时对番茄和辣椒的发芽、出苗以及生长期的影响。结果显示,所有处理组的发芽率、株高、根长、叶色以及发育程度均和对照组无明显差别,表明strekingmycin A在浓度为100μg/m L时对番茄和辣椒没有影响。以上研究结果表明,Streptomyces sp.SA61产生的聚酮类化合物,strekingmycin A-F,不仅具有结构新颖性,而且对桃蚜具有杀虫活性。其中,strekingmycin A对桃蚜体内解毒酶具有抑制作用,能够降低桃蚜的解毒能力,具有成为农药活性先导化合物的潜力。更多还原

【Abstract】 The green peach aphid（Myzus persicae）is a globally distributed agricultural pest which is detrimental to more than 500 plant species and cause severe economic losses.Chemical insecticides are the main means to control M.persicae,but the frequently and unregulated use of chemical insecticides has led to the development of resistance and increased the difficulty of controlling M.persicae.In response,the research and development of efficient,low toxicity,and environmentally friendly new biological insecticides has become a research hotspot in pest control.Natural products are one of the important sources of new pesticide discovery.Actinomycetes play an important role in discovering novel Natural products.In order to seek natural products with novel structures and virulence activity against M.persicae,107 strains of actinomycetes were cultivated by shaking,and the crude extracts were screened for detection of thin layer chromatography and stomach toxicity activity against M.persicae.Based on the two screening results,Streptomyces sp.SA61 was selected as the targ strain to study the inhibiting activity against M.persicae.Secondary metabolites of Streptomyces sp.SA61 were separated and purified to obtain monomer compounds.The nuclear magnetic resonance spectroscopy,and X-ray single crystal diffraction were analyzed to determine their chemical structures including the absolute configurations.Insecticidal activity of stringmycin A-F against M.persicae,effect of the active compound stringmycin A on activity of the detoxification enzymes in M.persicae and its safety evaluation the on crops were determined.The specific research results are as follows:1.Screening of target strains:107 strains of actinomycetes were cultured in two kinds of culture medium by shaking flask to obtain 214 crude extracts.According to the results of thin layer chromatography,33 crude extracts with rich metabolites were selected,and their stomach toxicity activities against M.persicae were detected by feeding method.Based on the results of thin layer chromatography and insecticidal activity screening,Streptomyces sp.SA61 was finally selected and its active components were studied.2.Shaking culture of Streptomyces sp.SA61 and isolation and purification of crude extract:Streptomyces sp.SA61 was subjected to culture in a shaking table at a temperature of 28℃and a rotational speed of 180 r/min for 7 days,with a total of 48 L.Solid phase extraction was performed using macroporous adsorption resin and eluted with organic solvents to obtain 20 g of crude extract.Six pure compounds were isolated from the secondary metabolites of Streptomyces sp.SA61 by silica gel column chromatography,gel column chromatography and HPLC.3.Identification of secondary metabolites of Streptomyces sp.SA61:by analyzing data of high-resolution mass spectrometry（HRESIMS）,¹H NMR,¹³C NMR,2D-NMR and X-ray single crystal diffraction,the chemical structure of compounds 1-6 were derived as polyketides.By comparing with literature data,the results indicated that compound 1 was previously isolated in our laboratory;compounds 2-6 were novel compounds with different carbon skeletons from 1.Compounds 1-6 were named as strekingmycin A-F.4.Determination of insecticidal activity of strekingmycin A-F against M.persicae:the toxicity against M.persicae was determined by leaf soaking method,and the results showed that strekingmycin A-F had insecticidal activity against M.persicae,with LC₅₀ of 10.29,14.57,27.81,34.47,31.57,and 21.04μg/m L at 96 h,respectively.The stomach toxicity activity against M.persicaes was determined by feeding method,and the results showed that strekingmycin A-F had stomach toxicity activity against M.persicaes,with LC₅₀ of 12.04,15.61,16.63,17.20,12.48,and 23.50μg/m L at 96 h,respectively.The contact toxicity activity against M.persicaes was determined by tube membrane method,and the results showed that strekingmycin A-F had contact toxicity activity against M.persicaes,with LC₅₀of 11.58,13.30,11.97,13.68,12.80 and 24.57μg/m L at 96 h,respectively.The antifeedant activity against the M.persicae was determined by honeydew method,and the results showed that strekingmycin A-F had antifeedant activity against the M.persicae,with AC₅₀ of 10.41,13.09,10.86,12.56,10.52,and 10.86μg/m L at 96 h,respectively.The repellent activity against M.persicaes was determined by leaf plate method,and the results showed that strekingmycin A-F had repellent activity against M.persicaes.After 48 h,the repellent rates were 82.67%,82.31%,73.33%,62.30%,54.88%and 69.87%,respectively.The results showed that strekingmycin A has the best insecticidal effect.5.The effect of the active compound stringmycin A on the activity of detoxification enzymes in M.persicaes:the results showed that stringmycin A inhibited the detoxification enzymes in M.persicaes,including cytochrome P450（CYP450）,carboxylesterase（Car E）and glutathione-S-transferase（GST）,which reduced their detoxification ability,and made stringmycin A more toxic to M.persicaes.6.Safety evaluation of strekingmycin A on crops:the effects of strekingmycin A at 100μg/m L on the germination,emergence,and growth period of tomatoes and peppers were measured.The results showed that there were no significant differences in germination rate,plant height,root length,leaf color,and development degree between all treatment groups and the control group,indicating that strekingmycin A had no effect on tomatoes and peppers at 100μg/m L.The above experimental results indicated that the polyketides produced by Streptomyces sp.SA61,strekingmycin A-F,not only have structural novelty,but also have insecticidal activity M.persicae.Strekingmycin A can inhibit the detoxification enzymes activity,and reduce the detoxification ability of M.persicae which showed the potential to become a lead compound of the insecticide.更多还原