【作者】 豆威；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2006， 硕士

【摘要】 谷胱甘肽S-转移酶（Glutathione S-transferases）是生物体内广泛存在的一类重要解毒酶。它能够催化还原性谷胱甘肽的硫醇基与各种亲电化合物相结合，增加后者的可溶性从而有利于其从细胞内排出。作为杀虫剂抗性的一个重要机制，昆虫GSTs的研究主要基于杀虫剂抗性中的解毒代谢。本研究在教育部新世纪优秀人才支持计划（NECT-04-0854）资助下，以重要的储藏物害虫嗜卷书虱Liposcelis bostrychophila Badonnel为试验材料，通过室内饲养并培育出嗜卷书虱的DDVP和PH₃抗性品系，较为系统地对两种抗性品系（DDVP-R和PH₃-R）与敏感品系体内的GSTs进行比较研究，主要结果如下： 首先，采用微量滴度酶标板法，对嗜卷书虱敏感品系和两个抗性品系GSTs的生化及毒理学特性进行了较为系统的研究。比较发现，两个抗性品系GSTs的活力和比活力都显著高于敏感品系（P＜0.05），同时两抗性品系之间GSTs的活力和比活力也都达到了显著水平（P＜0.05）。酶动力学特性研究表明，以CDNB为底物时，DDVP-R和PH₃-R品系中GSTs的K_m与敏感品系相比都显著下降（P＜0.05），说明GSTs在抗性品系中发生了质变，导致其与CDNB的结合能力大幅提高，此外，DDVP-R品系中的V_max与敏感品系相比也有显著提高（P＜0.05）。相对而言，以GSH为底物时，两个抗性品系K_m值与敏感品系相比均显著提高，且抗性品系间差异也达到显著水平；同时，DDVP-R品系中V_max大约是敏感品系的4倍，说明该抗性品系中GSTs对GSH的亲和力虽然下降，但是其可以通过V_max的提高来弥补亲和力下降所造成的损失。这些研究表明，与敏感品系相比，GSTs在DDVP-R和PH₃-R品系中都不同程度地过量表达。这种过表达是杀虫剂和熏蒸剂持续不断选择压力下筛选的结果。对嗜卷书虱GSTs的杀虫剂离体抑制研究表明，在所选用的四种杀虫剂中，对氧磷和敌敌畏对各品系嗜卷书虱GSIs都有明显的离体抑制作用（P＜0.05）。说明在嗜卷书虱体内，GSTs是有机磷类杀虫剂敌敌畏和对氧磷的重要作用靶标，而且与敏感品系相比，两种抗性品系GSTs对敌敌畏表现为更不敏感。而氯氰菊酯对嗜卷书虱的抑制作用则相对不太明显。此外，低浓度毒死蜱（0.0002-0.0228μM）对各品系嗜卷书虱的GSTs均有明显离体激活作用，但高浓度毒死蜱（2.2825-22.8245μM）则对嗜卷书虱GSTs表现为一定的抑制作用。 其次，从分子水平上对嗜卷书虱GSTs的cDNA克隆进行了初步探讨。根据已发表物种Delta类GSTs氨基酸的保守序列，设计合成了针对Delta类GSTs的简并引物，以期进行RT-PCR，从而获得嗜卷书虱GSTs的cDNA克隆。结果得到一个cDNA片段：KTPGQAACLQVDDLSSMLNRRSLQGLVGTDIQASTDPRTICLRTVVISRNNDVQLAPT，对这段氨基酸残基用BLAST进行同源性分析发现，Blackcurrant reversion virus内有两个基因与本实验的扩增片段有33％同源性，比对中没有出现其他物种的GSTs基因序列。测序结果表明，扩增条带可能不是GSTs片段。说明引物的设计存在一定的问题，可能与GSTs在各个物种间更多还原

【Abstract】 Glutathione S-transferases （GSTs） are a supergene family of detoxification enzymes found ubiquitously in organisms. They could catalyse the conjugation of the thiol group of reduced glutathione （GSH） with electrophilic compounds, generally making the resultant products more water soluble and excretable than the non-GSH conjugated substrates. Interest in insects GSTs has primarily focused on their imporatant roles in insecticide resistance. To elucidate the role of GSTs and the mechanism of GSTs-mediated resistance, the biochemical and toxicological characterizations and gene cloning of GSTs were investigated in the resistant （DDVP-R and PH₃-R） and susceptible strains of Liposcelis bostrychophila Badonnel and the study was supported by the program for New Century Excellent Talents in University （NCET-04-0854）, Ministry of Education, China.The systematic investigation on biochemical and toxicological characterizations of GSTs in L. bostrychophila were conducted by the microplate reader. The biochemical results showed that compared to their susceptible counterparts, the activities per insect and specific activities of GSTs in DDVP-R and PH₃-R were significantly higher （P < 0.05）. Apparent K_m values for CDNB were significantly lower for DDVP-R and PH₃-R compared with the susceptible strain （P < 0.05）, indicating GSTs might have changed in quality. In contrast, the catalytic activity of GSTs toward GSH in the susceptible strain was significantly higher than those in resistant strains. Meanwhile, V_max values were approximately four times as high for DDVP-R compared with the susceptible strain. These results suggested compared to their susceptible counterparts, GSTs were overexpressed to various extents in DDVP-R and PH₃-R. And the continuous selection pressure of insecticides and fumigants contributed to the overexpression of GSTs. The inhibition kinetics of insecticides to GSTs in vitro revealed that dichlorvos and paraoxon possessed excellent inhibition effects on GSTs. These suggested that an elevated detoxification ability of GSTs developed in the resistant strains. In contrast, the inhibition effect of cypermethrin on GSTs was quite poor. It was noticeable that for chlorpyrifos, there was some facilitated effect of chlorpyrifos with a lower concentration （0.0002-0.0228 μM） on GSTs activities, while higher concentration （2.2825-22.8245 μM） possessed some inhibitory impact.A fragment was amplified with the degenerate primers from the conseverd peptide sequences of Delta class GSTs in insect species by reverse transcriptase-polymerase chain reaction （RT-PCR） method and the corresponding cDNA was as follows: KTPGQAACLQVDDL SSMLNRRSLQGLV GTDIQASTDPRTICLRTWISRNNDVQLAPT. The homologous analysis with BLAST software更多还原