【作者】 李修伟；

【导师】 张兴；

【作者基本信息】 西北农林科技大学 ， 农药学， 2010， 博士

【摘要】 化肥和合成农药的大量使用导致了水体富营养化和农药残留污染,影响非靶标生物,对人类和环境构成了威胁并引起了世界范围内的关注。农药残留污染物通过改变非靶标生物的酶、蛋白质的代谢途径造成不利影响,因此对水环境污染进行监测非常必要的。摇蚊是一种世界性分布的水生昆虫,其幼虫几乎遍及所有的淡水生境,是水生食物链网的重要环节,在过去几十年内,环境工作者普遍将其作为水体生物监测的指示生物。目前国内外对摇蚊幼虫生态毒理学及利用摇蚊监测水体污染等方面进行了有益的探索。为进一步了解环境污染物胁迫下摇蚊的毒理和分子反应,本研究通过对摇蚊毒理学上具有重要作用的生化解毒酶和生理上重要的血红蛋白的EST进行生物信息学分析,克隆获得摇蚊GSTs的全长cDNA序列并进行系统命名。通过测定明确了这些GSTs在不同组织和发育期特异性表达分布情况,并测试了环境中以甲草胺(alachlor)为代表药剂的农药残留污染物胁迫下摇蚊幼虫GSTs和基因表达的变化情况,评估了相关基因作为生化标记物检测环境污染的可能。此外,还针对水体富营养化引起的摇蚊爆发带来的公害,本研究还测定了雷公藤总生物碱对摇蚊幼虫的杀灭活性以及雷公藤总生物碱对摇蚊GSTs酶活性和基因表达的影响,探讨了利用天然产物雷公藤总生物碱作为植物源摇蚊控制剂的可能,并对其提取工艺和制剂加工进行了系统研究。研究主要结果如下:1.借助Blast2go和NCBI blastx等工具采用生物信息学方法对根据EST序列分析结果,筛选得到毒理学重要的生化解毒酶和生理上重要的血红蛋白基因进行分析。这些基因片段包括:29个摇蚊细胞色素P450基因、11个GSTs基因、14个酯酶基因、7个乙醇脱氢酶基因、3个金属硫蛋白基因和89个血红蛋白基因。此项研究为以后进行摇蚊相关的基因等的各项研究奠定了基础。2.获得了11个GSTs cDNA的全长序列,并将这些GSTs进行序列比对和系统发育分析及命名,结果显示这11个摇蚊GSTs中有2个归类于delta家族,4个归类于sigma家族,1个归类于omega家族,根据命名法则命名这些GSTs并登录在GenBank,各接入码及GSTs名字分别为FJ851365 (CtGSTd1),FJ851366 (CtGSTd2), FJ851367 (CtGSTs1) , FJ851368(CtGSTs2) , FJ851369 (CtGSTs3) , FJ851370 (CtGSTs4) ,FJ851371(CtGSTu1),FJ851372 (CtGSTu2),FJ851373 (CtGSTu3),FJ851374(CtGSTu4)和FJ851375 (CtGSTo1)。此外还对这些GSTs的酶促结合位点和维持酶活性构象的关键氨基酸残基进行了分析。3.摇蚊GSTs基因在不同组织(唾液腺,血淋巴,中肠,马氏管,表皮,脂肪体)和不同发育期(卵,一龄,二龄,三龄,四龄幼虫,蛹,成虫)表达分布存在在差异,其在不同组织的分布和不同发育期的表达差异可能与其所行使的不同功能有关。4.甲草胺能显著抑制摇蚊4龄幼虫GSTs活性且抑制存在剂量依赖关系。RT-PCR测定结果显示在11个摇蚊GSTs基因中,分属delta家族和sigma家族的CtGSTd1,CtGSTs2和CtGSTs3被诱导上调表达且表达量存在着剂量依赖关系,实时定量PCR测定显示与对照相比10,100,1000μg/L浓度甲草胺处理摇蚊72h后,摇蚊CtGSTd1上调表达1.38,1.56和2.06倍,而CtGSTs2上调表达1.36,2.10和2.83倍,CtGSTs3上调表达1.28,2.11和4.30倍。这些结果显示GSTs基因可以用作生化标记物检测环境污染。5.雷公藤总生物碱对摇蚊幼虫具有较高的杀灭活性,其摇蚊4龄幼虫24h,48h和72h的致死中浓度分别为33.08,19.09和16.76μg/L。亚致死剂量(5,10,15μg/L)的雷公藤总生物碱处理摇蚊4龄幼虫72h后能显著抑制GSTs活性,以CDNB为底物测定,与同期对照相比,酶比活力分别降低了2.37,2.82和3.77倍。以DCNB为底物测定,与同期对照相比,酶比活力分别降低了2.34,2.79和3.73倍。RT-PCR测定结果显示CtGSTu3和CtGSTu4被诱导上调表达且表达量存在着剂量依赖关系。这些结果显示雷公藤总生物碱具有较高的摇蚊杀灭活性,具有开发为摇蚊控制剂的可能。6.采用实验室小试和工厂中试相结合的方法对雷公藤根皮中生物碱的提取工艺进行了探讨,最终获得较优的提取工艺条件为:以乙酸乙酯为浸提溶剂,浸提温度50-60℃,浸提5次的罐组式逆流提取工艺。初次提取时间8h,其余提取时间为4h。该优化工艺条件不仅可减少成本,降低能耗,且能提高雷公藤总生物碱的提取率,在实际生产中具有较好的应用价值。7.研制出1.0%雷公藤总生物碱微乳剂的制剂配方,系统测试显示该制剂各项理化指标均合格。1.0%雷公藤总生物碱微乳剂在田间对菜青虫具有较好的防治效果,值得在生产中推广应用。更多还原

【Abstract】 Heavy uses of chemical fertilizers and synthetic chemical pesticides have led to significant water eutrophication and pesticide residue contaminations, which have not only affected non-target organisms in the ecosystem, but also harmed to the residents’ health through water and food, and become a growing global concern. Environmental pesticides can affect non-target organisms harmfully through modifying many metabolic pathways involving many enzymes and proteins. As an ecologically important bioindicator species due to the significant role in food webs and occurrence in various aquatic habitats worldwide, the aquatic midge (Chironomus tentans) is a well recognized and widely used insect species for studying the impact of environmental pollutants in aquatic systems in the past decades. Although the application of the aquatic midge as bioindicator species has been explored by domestic and international scientists, glutathione S-transferases as an important phase II detoxification enzyme family have not been characterized in detail. To comprehensively characterize midge’s toxicological and molecular responses to environmental pesticides and other toxic stressors, we have developed an expressed sequence tag (EST) database containing over 10,000 sequences from a C. tentans cDNA library. From the EST database, we have identified a great deal of important detoxification enzymes and physiologically important heamglobin. Specifically, a total of 11 cDNAs encoding full-length GSTs were obtained and a relatively detailed analysis of these genes was carried out. We explored molecular characteristics of the 11 GST genes, their developmental stage- and tissue-specific expression patterns, and the effect of alachlor on total GSTs activities and gene expressions. Our study provides the first insight into molecular characteristics of GSTs and their transcriptional response to alachlor exposures in C. tentans. Furthermore, to encounter public health nuisance caused by the outbreak of non-biting mosquito, we studied larvicidal ctivity of alkaloids from Tripterygium. wilfordii against aquatic midges,optimized extracting technology and micro-emulsifier(ME)formulation processing. The main results and conclusions were outlined as follows:1. EST from the aquatic midge was analyzed by using Blast2go, NCBI blastx and other online programs. A number of important ESTs putatively encoding detoxification enzymes and physiologically important heamglobin, including 29 cytochrome P450, 11 GSTs, 14 esterases, 7 alcohol dehydrogenases, 3 metallothionein and 89 hemoglobin were identified and analyzed. Those findings provided a basis for further investigation on genes of aquatic midge.2. 11 full-length GSTs cDNA sequences were obtained and phylogenetically analyzed. Phylogenetic analysis of these 11 GSTs deduced from their cDNAs revealed 7 GSTs that belong to three different cytosolic classes, including 2 in delta, 4 in sigma and 1 in omega, based on their sequence similarities to other insect GSTs, particularly those from Anopheles gambiae and Drosophila melanogaster. The remaining four GSTs were unclassified due to the lack of homologies to the currently known class. The nomenclature of these C. tentans GSTs cDNA and their deduced amino acid sequences have been deposited in GenBank with the following accession numbers: FJ851365(CtGSTd1), FJ851366(CtGSTd2), FJ851367 (CtGSTs1), FJ851368(CtGSTs2), FJ851369(CtGSTs3), FJ851370(CtGSTs4), FJ851371(CtGSTu1), FJ851372 (CtGSTu2), FJ851373 (CtGSTu3), FJ851374(CtGSTu4) and FJ851375 (CtGSTo1) Furthermore, several conserved amino acid residues which represent the catalytic pocket and binding site were analysised by sequence alignment.3. Tissue-specific expression patterns of the 11 C. tentans GSTs genes were analyzed in each of six different tissues, including salivary glands, hemolymph, midgut, Malpighian tubules, fatbodies, and cuticle, by using semi-quantitative RT-PCR. Stage-specific expression patterns of C. tentans GSTs genes were determined in eggs, four different larval instars (1st, 2nd, 3rd and 4th), pupae and adults by using semi-quantitative RT-PCR. Such tissue- and stage- specific expression patterns may reflect specific roles and functions of the GSTs genes within different tissue and during the midge development.4. After treatment with alachlor for 72h at 10, 100, 1,000μg/L, GSTs activity of fourth-instar midges were reduced by 1.3-, 1.7-, and 2.2-fold, respectively, compared with the control when CDNB were used as a substrate. Similarly, alachlor at 10, 100, 1,000μg/L reduced the GSTs activity by 1.3-, 1.7-, and 1.8-fold, respectively, when DCNB was used as a substrate. Semi-quantitative RT-PCR results clearly showed that alachlor significantly increased the mRNA levels of the CtGSTd1, CtGSTs2 and CtGSTs3 genes in an alachlor concentration-dependent manner in the midges exposed to alachlor. These results suggested that alachlor can induce the expression of some genes in at least two classes of the GSTs gene family, including delta (CtGSTd1) and sigma (CtGSTs2, CtGSTs3). Real-time quantitative PCR results showed the exposure of forth-instar larvae to alachlor at 1,000μg/L for 72 h increased the CtGSTd1, CtGSTs2 and CtGSTs3 mRNA levels by 2.1-, 2.8- and 4.3-fold, respectively. Those GST genes can be used as biochemical marker for environment pollution monitoring.5. The Tripterygium wilfordii alkaloids showed a high toxicity to fourth-instar larvae of the aquatic midge with the LC50 values of 33.08,19.09 and 16.76μg/L at 24 , 48 and 72 h, respectively. After the treatments with T. wilfordii alkaloids at 5,10,15μg/L for 72h, GSTs activity of fourth-instar midges were reduced by 2.37-, 2.82-, and 3.77-fold, respectively, compared with the control when CDNB were used as a substrate. Similarly, the GSTs activity was reduced by 2.34-, 2.79-, and 3.73-fold, respectively, when DCNB was used as a substrate. Semi-quantitative RT-PCR results clearly showed that alachlor significantly increased the mRNA levels of the CtGSTu3 and CtGSTu4 genes in a T. wilfordii alkaloid concentration-dependent manner in the midges exposed to T. wilfordii alkaloids. These results indicated that T. wilfordii alkaloids were highly toxic to aquatic midges and can be potentially used to develop a mosquito control agent.6. The extracting techniques for the alkaloids from the root bark of T. wilfordii by using ethyl acetate were evaluated both at small laboratory experimental and pilot-plant scales. The optimal extraction conditions were obtained as follows: root bark powder 30 mesh, temperature 50-60oC, duration of extraction 8 h for first time and 4h for another 4 tiems. Applications of these extracting conditions can reduce the costs of production, decrease the energy consuming, and increase the alkaloid yields. This extraction technique appears to be feasiable and practicable.7. The alkaloids extracted from the root bark of T. wilfordii were formulated to micro-emulsifier (ME) by the way of combining researches in physical and chemical characters with laboratory bioassay and field trial. The solvent and emulsifier which suited 1.0% T. wilfordii alkaloids ME had been screened out and successfully developed. Its quality index met the standard of a commercial pesticide. The 1.0% T. wilfordii alkaloids ME possesses excellent insecticidal activities, and has a great potential for its development and application.更多还原