【作者】 谢永坚；

【导师】 雷朝亮；

【作者基本信息】 华中农业大学 ， 农业昆虫与害虫防治， 2013， 博士

【摘要】 我国每年因白蚁危害造成的经济损失超过30亿元。目前,国内外白蚁预防和防治仍然以化学杀虫剂为主,由于有机磷和有机氯杀白蚁剂的禁用,寻求新的防治白蚁技术和方法显得十分必要。利用精油或精油中的活性物质防治白蚁是目前白蚁防治领域的研究热点之一。柳杉作为我国本土的、分布广泛的丰富天然植物资源,值得深入研究它的生物活性。因此,本研究分析了柳杉精油化学成分及其杀白蚁活性,并对精油进行纯化、分离、鉴定,从生化水平、蛋白质水平以及基因水平探讨其杀虫机理；此外,本文初步探讨了α-松油醇类似物化学结构与杀白蚁活性的关系,取得如下主要结果：1.柳杉精油化学成分及杀白蚁活性为明确柳杉和日本柳杉精油对白蚁的毒杀活性及化学成分特征,采用GC-MS法,比较分析了柳杉和日本柳杉精油的化学成分,并测试2种精油对黑胸散白蚁的毒杀活性。结果表明,不同种柳杉精油的化学成分存在一定差异,柳杉精油杀白蚁活性高于日本柳杉。本文进一步分析了昆明、雅安、武汉、恩施、合肥、南京、天目山、福州和庐山等9个国内主要分布区,不同季节、树龄,以及树皮、边材、心材和树叶4种器官的柳杉精油含量、化学成分及其杀白蚁活性。结果表明,不同产地、不同季节、树龄以及柳杉不同器官精油化学成分存在一定差异,但它们的杀虫活性差异不明显。同时,柳杉叶精油杀白蚁明显高于树皮、边材、心材。因此,柳杉叶精油可作为一种潜在的天然杀白蚁药剂。柳杉叶精油成分的鉴定与化学品系的分析,可以了解不同来源的柳杉叶精油组成分的差异与种源地之间的关系。本研究结果可以为柳杉品系的选育与精油的利用或生物活性测定提供参考依据。2.柳杉精油有效杀虫成分的分离与鉴定为进一步了解柳杉叶精油中具有杀白蚁活性成分。采用生物活性追踪法,利用硅胶柱层析,从柳杉叶精油中分离、纯化杀白蚁活性物质。通过红外光谱(IR)、质谱(MS)、紫外光谱(UV)、核磁共振(NMR)对柳杉叶精油分离得到的活性物质进行解析,经鉴定为α-松油醇,分子式为C10H18O,分子量为154.25,为单环单萜醇。α-松油醇对黑胸散白蚁具有较强的毒性,其毒力回归方程为Y=0.34+5.05x(R2=0.87),LC50为0.86mg/mL。本研究表明,α-松油醇可作为一种高效、环境友好的杀白蚁药剂。为进一步研究杀虫机理和化学结构--活性关系奠定基础,并为产业化开发提供理论依据。3.α-松油醇对白蚁的杀虫机理为探讨α-松油醇的杀白蚁作用机理。测定了α-松油醇对黑胸散白蚁体内解毒酶和抗氧化酶活性的影响。结果表明,α-松油醇对白蚁体内的解毒酶和抗氧化酶的活性均表现出较强的时间效应。LC50剂量(0.86mg/mL)的α-松油醇对黑胸散白蚁工蚁体内的乙酰胆碱酯酶(AChE)、钠钾ATP酶(Na+-K+-ATPase)、过氧化物酶(POD)和过氧化氢酶(CAT)的酶活性随处理时间的延长整体表现出抑制作用；对谷胱甘肽转移酶(GST)、超氧化物岐化酶(SOD)表现诱导增加作用。用0.2、0.4、0.6、0.8、1.0μl/mL剂量的α-松油醇处理试虫后,各剂量的α-松油醇均可抑制黑胸散白蚁体内的乙酰胆碱酯酶(AChE)和钠钾ATP酶(Na+-K+-ATPase)的酶活性。白蚁体内的谷胱甘肽转移酶(GST)、超氧化物岐化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的酶活性随α-松油醇处理剂量的增加而诱导增加。其中,α-松油醇对Na+-K+-ATPase具有较强的抑制作用,且随着中毒症状的加剧,对该酶的抑制作用增强。因此,Na+-K+-ATPase可能是α-松油醇的主要作用靶标。为了进一步探讨α-松油醇的作用机理。本文利用差异蛋白质组学技术,研究α-松油醇胁迫下黑胸散白蚁体内蛋白质的变化,观察到40个蛋白质发生了差异表达,其中7个蛋白点为上调表达,24个点为下调表达,9个蛋白点为在处理后新增或消失。质谱分析共鉴定出40个蛋白点,包括能量代谢、转运蛋白、细胞骨架蛋白等。这些蛋白参与了白蚁体内的生理生化过程,表明白蚁在应对α-松油醇的响应是一个网络系统。在α-松油醇胁迫下黑胸散白蚁体内相关的蛋白质发生表达量的变化,为更好的了解基因水平的表达差异,本研究应用RT-PCR和RACE技术,克隆得到黑胸散白蚁与能量代谢相关的ATP合成酶(ATP synthase-β-subunit)基因的大部分序列,从序列的保守性可推知ATP合成酶在不同物种中的功能比较保守。4.α-松油醇类似物杀白蚁活性研究为研究α-松油醇类似物结构与杀白蚁活性的关系,测定了单萜类、醇类、酚类、酮类、醛类、酯类和醚类等7个系列42个化合物对白蚁的熏杀效果,结果表明,含氧萜类化合物的杀白蚁活性高于萜类化合物；羟基(-OH)基团的位置强于乙酰基：酚类物质结构上羟基(-OH)位置不同,生物活性也不一样：丙烯基双键的位置对杀白蚁活性很重要：大多数酮类化合对白蚁的熏杀效果都比醇类化合物强,羰基的存在可能提高了化合物的毒效。本研究初步揭示了单萜类化合物的不同化学基团对杀白蚁活性的影响,并发现了大量活性明显高于α-松油醇的化合物。通过本论文研究,明确了不同种间、不同产地、不同季节、不同树龄及不同器官柳杉精油化学成分存在一定程度的差异,同时,这些精油对白蚁都具有较好毒杀效果,但活性差异不明显。首次从柳杉叶精油中分离得到杀白蚁活性物质a-松油醇,并对它的杀虫机理进行研究,发现α-松油醇可抑制黑胸散白蚁体内的解毒酶和诱导氧化酶系。同时,本文开展了黑胸散白蚁响应α-松油醇胁迫的蛋白质组学研究,找到了多个与α-松油醇胁迫相关的蛋白,揭示了白蚁对α-松油醇响应是一个网络反应。初步揭示了单萜类化合物的不同化学基团对杀白蚁活性的影响。本研究结果为柳杉精油的应用及杀白蚁新药的研制提供科学依据。更多还原

【Abstract】 Termites are important insect pests in the world. In China, the annual economic losses caused by termite were over3billion RMB. At present, the main termite prevention and control is still the chemical pesticides in domestic and foreign. Because of the forbidden organochlorine and organophosphate insecticides, the termite prevention and control is an embarrassing situation. Therefore, it is very necessary to search new efficiency method. Using the plant essential oil and the bioactivity compound from plant oil to control termite is one of the hotspots in the research fields of termite control. Cryptomeria fortunei Hooibrenk is the most abundant one and unique to China. It is worth to further study its biological activity. Therefore, the study identified the chemical compositions and termiticide activity of essential oil from C. fortunei. Furthermore, we also purified and isolated the activity chemical compound in essential oil, and explored its insecticidal mechanism. At the same time, we analyzed and compared the termiticide activity of analogues of a-terpineol. The results showed as follows:1. Chemical composition and antitermitic activity of C. fortunei essential oil against R. chinensisAiming at to clarify chemistry composition and antitermitic activitie of essential ols from C. fortunei and C. japonica, the dissertation analyzed and compared the chemical composition of essential oil from the C. fortunei and C. japonica with GC-MS, and antitermitic activities of2essential oils against R. chinensis. The results also indicated that the genus of Cryptomeria had a considerable variation in the essential oil compositions, and the antitermitic activity of leaf essential oil of C. fortunei was stronger than that of C. japonica.Furthermore, the chemical compositions of essential oils were analyzed with GC-MS and antitermitic activities were tested against R. chinensis, from9locations in China: Kunming, Yaan, Wuhan, Enshi, Hefei, Nanjing, Mt Tianmu, Fuzhou, Mt Lu, and in different seasons and age, and from4organs:bark, sapwood, heartwood and leaf. The results showed that the essential oil exist a degree of differences in chemical composition from different locations, seasons and age, and organs. However, there were no significant differences in antitermitic activities of essential oils from different locations, seasons and age. However, the leaf essential oil of C. fortunei posses significant antitermitic activity, which promotes the isolation and purification of the main active constituents for further study. The results of this study showed that the leaf essential oil of C. fortunei might be considered as a potential source for fine natural termiticide. Through the identification the composition of C. fortunei leaf essential oil and chemotypes analysis, we can understand the relationship between the composition of C. fortunei leaf essential oil and the provenance of C. fortunei. The results of this study can provide a reference for C. fortunei breeding lines and the C. fortunei leaf essential oil usage or biological activity measurement.2. Bioassay-guided isolation and identification of antitermitic active compound from C. fortuneiIn order to further study the termiticidal components in leaf essential oil of C. fortunei, bioactivity-directed fractionation and purification processes were employed to identify the termiticidal components by silica gel column chromatography. The active compound, a-terpineol with a molecular formula of C10H18O, and molecular weight of154.25, which was responsible for the antitermitic property of C. fortunei leaf, was obtained and identified by IR, MS, UV, and NMR. For a-terpineol, the linear equation Y=0.34+5.05x (R2=0.87) was derived from the regression analysis of probit mortality of R. chinensis bioassay in test solution. The calculated LC50was0.86mg/mL with the95%confidence interval of0.76-0.98mg/mL. The results of this study showed that a-terpineol might be considered as a potent source for the production of environmental safe termiticide. The study layed the foundation for further studying the mechanism of insecticide and the chemical structure--activity relationships and provided a theoretical basis for industrial development.3. Insecticidal mechanism of a-terpineol to R. chinensisTo revealed the insecticidal mechanism of a-terpineol. The effect of a-terpineol on activities of detoxification enzymes and antioxidant enzymes to R. chinensis, were determined. The result showed that the Acetylcholinesterase (AChE), Na+-K+-ATPase, peroxidase (POD) and catalase (CAT) activitives were inhibited by a-terpineol with the prolonging of exposure time at0.86mg/mL. In addition, the activitives of glutathione S-transferase (GST) and superoxide dismutase (SOD) were induced by a-terpineol at the same doage. When R. chinensis work was treated with a-terpineol at0.2,0.4,0.6,0.8,1.0μl/mL doages, the activites of AChE and Na+-K+-ATPase were inhibited at various concentration a-terpineol. The glutathione S-transferase (GST), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activitives were induced with the increasing of α-terpineol dosage at24h. Among, α-terpineol had strong inhibition on activity of Na+-K+-ATPase. With poisoning symptom exacerbating, the inhibition rates were gradually increased. Therefore, Na+-K+-ATPase is likely the target of α-terpineol against R. chinensis.To studied insecticidal mechanism of α-terpineol. The proteomic response of the R. chinensis to α-terpineol was examined using differential proteomics technologies at LC10, LC50, and LC90following α-terpineol treatment. Forty proteins showed differential expression, including seven up-regulated spots, twenty-four down-regulated, of which eight spots newly expressed or disappeared in R. chinensis after exposure to α-terpineol. MS analysis and database searching helped us to successfully identify forty protein spots, including metabolisms, cytoskeletal, and transporter proteins. We found several proteins related with α-terpineol stress proteins, revealing the α-terpineol response in termites was a network response.Treated with α-terpineol, the expression level of some related proteins in R. chinensis were changed. In order to study the gene expression difference in transcription level, using RT-PCR and RACE technology, we cloned the most sequences of ATP synthase-β-subunit in R. chinensis, associated with energy metabolism. From the conserved sequences of ATP synthase in different species we can infer the functions of ATP synthase in different species are conservative.4. Antitermitic activity of analogues of α-terpineol against R.chinensisAiming to clarify relationship between chemical construction and antitermitic activities of analogues of α-terpineol, The fumigation toxicities of7series42compounds, including Hydrocarbons, Alcohols, Phenols, Ketones, Aldehydes, Acetates and Oxides were determined against subterranean termite, R. chinensis. This result indicated that the antitermitic activities of oxygenated hydrocarbon compounds were higher than the hydrocarbon group; the position of hydroxyl (-OH) groups were stronger than those with an acetyl group; the position of the hydroxyl group (-OH) is the only difference in the chemical structures of the two phenols; the position of the hydroxyl group is very important in antifungal and insecticidal activity; the position of the double bond of the propenyl group is also very important for antitermitic activity; some ketones were more effective fumigants than alcohols, the presence of a carbonyl group augments toxicity. The research preliminarily clarified the effects on antitermitic activities when attached different chemical group in monoterpenes, and found a number of active compounds significantly higher than a-terpineol.Our research showed chemical composition of C. fortunei oil from different species, different origin, different seasons, different age or different organs had a certain degree of variation, these oils all had better killing effects against R.chinensis, but the termiticidal activity of them were not significantly different. Our study was first to isolate the termiticidal active substance, a-terpineol, from C. fortunei leaf oil, and explore its termiticidal mechanism. Meanwhile, our study was also carry out the proteomics research on R. chinensis under the stress response of a-terpineol. We found several proteins related with a-terpineol stress proteins, revealing the a-terpineol response in termites was a network response. The research preliminarily clarified the effects on antitermitic activities when attached different chemical group in monoterpenes. The results of our study provided scientific basis for the C. fortunei essential oil application and the new termiticidal drugs development.更多还原