【作者】 谢苗；

【导师】 尤民生；

【作者基本信息】 福建农林大学 ， 农药学， 2011， 博士

【摘要】 小菜蛾(Plutella xylostella L.)是一种世界性的十字花科蔬菜主要害虫,它对化学杀虫剂和生物杀虫剂都能很快地产生抗性。目前杀虫剂的使用仍然是控制小菜蛾的重要手段。由于杀虫剂的过度使用,小菜蛾已对多种药剂产生了不同程度的抗性。近年来对杀虫剂的研究已经涉及生理、生化以及分子多个方面,但小菜蛾对杀虫剂的胁迫响应尚缺系统的研究。为了能使杀虫剂持久地发挥作用,保证靶标位点的敏感性,系统研究杀虫剂作用的有关机制是很有必要的。氟虫腈(Fipronil)是首个用于有害生物防治的苯基吡唑类杀虫剂,其作用靶标是Y-氨基丁酸受体(GABA),起到阻断由GABA控制的神经膜氯离子通道的作用,对包括小菜蛾在内的多种农业害虫有防治作用。本研究以小菜蛾为材料,采用LC5o剂量的氟虫腈进行处理,从解毒酶系、蛋白质和基因等方面研究了小菜蛾响应杀虫剂氟虫腈胁迫的生理生化及分子机制,初步揭示了可能参与到小菜蛾响应氟虫腈胁迫的蛋白,克隆了与机体免疫反应和能量代谢相关的4个基因,并明确了这4个编码基因的表达动态,为进一步研究小菜蛾对氟虫腈的响应机制奠定了基础。1.小菜蛾对氟虫腈的生理生化响应在小菜蛾幼虫对氟虫腈的短期响应中,体内的多功能氧化酶(MFO)、酯酶(EST)和谷胱甘肤-S-转移酶(GSTs)的活性均显著高于对照组,在24h内,随着处理时间的延长,各解毒酶的活性也逐渐提高。在3种解毒酶中,MFO对外界的刺激更加敏感,受诱导程度最高,在响应初期活性就快速升高。2.小菜蛾对氟虫腈响应蛋白的分析鉴定利用双向电泳和质谱技术的差别蛋白质组学技术,研究氟虫腈处理后,小菜蛾在Oh、8h、16h和24h时蛋白质组差异,观察到20个蛋白发生了差异表达,其中9个蛋白点为下调表达,11个蛋白点为上调表达,其中5个蛋白点仅在处理组表达。在鉴定成功的17蛋白点,包括了免疫反应和各种代谢途径参与蛋白,还有细胞骨架蛋白、分子伴侣等。这些蛋白参与了昆虫体内的多个生理过程,反映了小菜蛾对杀虫剂的响应会在细胞骨架、代谢水平等多方面发生综合的改变,说明昆虫在应对杀虫剂的响应是一个网络系统。3.小菜蛾响应氟虫腈相关蛋白的基因克隆与分析本研究克隆得到小菜蛾在响应氟虫腈过程中与免疫相关的硫氧还蛋白过氧化物酶(thioredoxin peroxidase,TPx)和凝集素(C-type lectin)、与能量代谢相关的ATP合酶亚基d(ATP synthase unit d)、以及腺苷酸转移酶(adenine nucleotide translocase, ANT)这4个基因的cDNA全长序列,利用生物信息学方法分析了这4个基因的结构和所编码蛋白质的结构特征及其与其他昆虫的同源性,构建了所编码蛋白质的3D结构模型。采用实时荧光定量PCR在mRNA水平上分析了这4个基因的表达特性。结果表明这4个基因在敏感品系小菜蛾的生长发育过程都能检测到,并且有表达量上的差异；在对氟虫腈胁迫的短期响应中,4个基因的表达量也会由于杀虫剂的胁迫而发生相应的变化。更多还原

【Abstract】 The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an economically important pest of cruciferous crops throughout the world. This insect pest is difficult to control as it can rapidly develop its tolerance to many synthetic insecticides and bioinsecticides. When DBM is exposed to insecticides, it can lead to various behavioral and metabolic responses to insecticides. The responses may invovle multiple physiological changes, which cannot be assigned to only a single gene or protein.In recent years, studies on insceticides have developed at different levels of physiological, biochemical and molecular mechanisms, but information on the biochemical and molecular mechanism of P. xylostella in response to fipronil, a phenylpyrazole insecticide, which blocks the inhibitory GABA-gated chloride channel and is used to protect crops, is still limited.To better understand the DBM’s response mechanisms, we observed the various levels of biochemical, proteomic and molecular response in P. xylostella that was exposed to fipronil stress. The proteins that may be involved in the response to fipronil were investigated using differential proteomics. The full-length cDNAs encoding thioredoxin peroxidase, C-type lectin, adenine nucleotide translocase and ATP synthase subunit d were all cloned from P.xylostella and their gene expressions were analyzed at the mRNA level. The major results are summarized in the following sections.1. The biochemical response of DBM to fipronil-induced stressThe short-term biochemical response of P.xylostella to fipronil was studied by examining the activities of detoxifying enzymes. We founded significantly higher activities of the detoxifying enzymes, mixed-function oxidase (MFO), esterase (EST) and glutathione-S-transferases (GSTs), in the DBM larvae that were treated with an LC50 dose of fipronil, compared to the larvae in fipronil-free controls. The activities of these detoxifying enzymes gradually increased over 24h, with MFO being more sensitive to external stimuli and showing higher induced activity than either EST or GSTs. 2. The proteomic response of DBM to fipronil-induced stressThe proteomic response of the DBM to fipronil was examined using two-dimensional electrophoresis (2-DE) and mass spectrometry at 8,16 and 24 h following fipronil treatment. Twenty proteins showed differential expression, including nine down-regulated spots, eleven up-regulated spots, of which five spots expressed only in the treatment group. Seventeen protein spots were successfully identified and include proteins that participate in the immune response and in other metabolic pathways, cytoskeletal proteins, and molecular chaperones. Differences in the expression of these proteins suggest that P. xylostella could create general changes in the cytoskeleton, metabolic level, and many other characteristics in response to the inesticide stress.3. Cloning and expression analysis of genes involved in the DBM response to fipronilThe full-length cDNA encoding thioredoxin peroxidase, C-type lectin, adenine nucleotide translocase and ATP synthase subunit d were cloned from P.xylostella by reverse transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). These four gene sequences and their encoding proteins were then analyzed, and a phylogenetic tree were contructed based on the amino acid sequences to reveal the relationship between DBM and other insects. In addition,3D protein structure models were developed using SWISS-MODEL.The mRNA levels of these four genes were measured using real-time PCR. These genes could be detected during the growth and development of DBM and were differentially expressed. However, the expression levels of these genes were also variable in the larvae treated with an LC50 value of fipronil.更多还原