【作者】 周耀振；

【导师】 董双林；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2008， 硕士

【摘要】 昆虫在长期进化过程中形成了高度灵敏的嗅觉系统,并以此来感受自然环境中的各种化学信息,产生相应的生理和行为反应,如寻找配偶、食物源和栖息场所、产卵行为、逃避危险或者不适合的栖息地和寄主等。将昆虫嗅觉应用于害虫防治,已成为害虫管理的一个重要手段,并将在未来害虫治理中起到越来越重要的作用。深入研究蛾类昆虫性信息素通讯的分子机制,弄清性信息素感受相关蛋白的功能,对于针对特定嗅觉蛋白设计和开发更为高效的基于两性通讯的害虫调控技术,至关重要。昆虫信息素结合蛋白(PBP)及受体蛋白(OR)被认为在性信息素感受中起重要作用,但目前为止在活体昆虫内进行该类蛋白功能的研究国内外还没有报道。在课题组前期成功克隆出甜菜夜蛾性信息素结合蛋白1(SexigPBP1)和信息素受体蛋白2(SexiOR2)全基因序列的基础上,本研究重点利用RNAi技术对SexigPBP1基因的功能进行了研究,同时对嗅觉受体蛋白SexiOR2和SlitOR2的组织分布进行了调查,主要结果如下:1)设计引物并通过RT-PCR扩增出530 bp的SexiPBP1 DNA片段,并以此为模板合成用于RNAi的双链SexiPBP1RNA。2)通过腹腔注射法将双链RNA导入到初羽化雄蛾体内,48小时后的RT-PCR和qRT-PCR的检测结果表明,处理试虫触角内SexigPBP1基因的表达量被敲减了约90%,同时处理试虫死亡率和对照相比没有明显差异。说明该方法可以有效沉默昆虫PBP基因的表达,从而进行相关功能研究。3)进一步的触角电位(EAG)检测结果表明,RNAi处理48小时后,雄蛾对雌蛾性信息素主组分Z9,E12,14:Ac的EAG反应降低了约60%,直接证明SexigPBP1在雄蛾对该组分的感受中起重要作用。这是首次应用RNA干扰技术对蛾类昆虫PBP功能的研究报道。4)通过设计特异性引物,以RT-PCR方法对嗅觉受体基因SexiOR2和SlitOR2分别在甜菜夜蛾和斜纹夜蛾中的组织表达谱进行了分析。结果表明,SexiOR2和SlitOR2基因只表达于雌雄蛾的触角中,在头、胸、腹、足、喙、翅等其他部位没有表达。更多还原

【Abstract】 In insects, the olfactory systems have been evolved to be highly sensitive, enabling insects to detect and discriminate among a diverse array of volatile chemicals. With this sensitive olfactory system, insects resopond properly to the chemical environment by different behaviors, such as mate-seeking, food and habitat locating, and oviposition-site finding. Therefore, the olfactory-based control strategies have developed to be an important means for pest control. To furter improve the olfactory-based control strategies, it is very important to gain insight on the molecular mechanism of the perception of female sex pheromone by the male. With the insights on the function of olfactory related protein, it will be facilitated for molecular designing of specific regulator targeted to these olfactory proteins, and for further development of more effective pest behaviorally interference techniques.Pheromone binding proteins (PBPs) and olfactory receptor (Ors) are thought to play critical roles in male perception of conspecific female sex pheromone. The major method to address the functions of PBP is in vitro binding assays between PBP and sex pheromone chemicals. As a robust method to study the new protein or gene functions, RNA interference (RNAi) has been used in many organisms including plants and animals. However, there is no report to date on the PBP fuction study with the employment of the RNAi method. Based on our previous molecular characterization of the PBP and OR2 genes in Spodopera exigua and S. litura, the present paper dealt with the PBP function study by using RNA interference technique. In addition, the expression pattern of SexiOR2 and SlitOR2 genes among different insect tissures of S. exigua and S. litura were inversitated using RT-PCR method. The main results were as follows.1) Fragment of 530bp was amplified with the specific primers designed accoding to known cDNA sequence of Sexig PBP1, and the dsRNA of Sexig PBP1 used for RNAi was further synthesized with above 530bp fragment as template using T7 RiboMAX Express RNAi System. 2) Abodomen injection method was tested for introducing SexigPBP1 dsRNA into the moth abdomen cavity. RT-PCR and qRT-PCR detections at two days after injection of dsRNA revealed that the mRNA expression level of SexigPBP1 in male antenna was significantly reduced by about 90%, and that the moth survival rates were not significantly different between treatments of dsRNA injected and DEPC-water injected. Therefore, the abodomen injection method was effective to be used for PBP RNAi in the moth species.3) Two days after dsRNA injection, the electroantennography (EAG) mesurment was further performed to detect the response of live moth at electrophysiolocial level. The results showed that the EAG response to the major femal sex pheromone component Z9,E12,14:Ac was reduced significantly by about 60% in dsRNA injected moths comparied to the DEPC-water injected control moths, which provided the direct evidence that SexigPBP1 played important role in the sex pheromone perception in male moths. This is the first report on investigation of insect PBP function in vivo by using RNAi approach.4) The tissure expression patterns of SexiOR2 and SlitOR2 were investigated, respectively, in S. exigua and S. litura by RT-PCR approach. As a result, the antenna of both sexes was the unique tissur to express the OR2 gene, while the head, thorax, abdomen, wing, proboscis and leg had no detected expression of the OR2 gene.更多还原