甜菜夜蛾嗅觉受体分子克隆及功能研究

项目名称： 甜菜夜蛾嗅觉受体分子克隆及功能研究

项目编号： No.30800725

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 修伟明

作者单位： 农业部环境保护科研监测所

项目金额： 19万元

中文摘要： 本项目采用荧光定量PCR、RNA干扰等多种研究手段对甜菜夜蛾OR83b 类化感蛋白（Sexi\Orco）进行了系统研究。取得以下主要结果：（1）、鉴定了甜菜夜蛾Sexi\Orco基因。cDNA全序列为1906 bp，开放阅读框473 个氨基酸；（2）、明确了Sexi\Orco基因exon-intron结构。全基因含10个外显子和9个内含子，并且内含子具有典型的GT-AG结构，长度差异较大；（3）、明确了Sexi\Orco基因的时空表达。Sexi\Orco基因在卵、幼虫、蛹、成虫四个阶段均有表达。成虫仅触角和喙内有表达，羽化后36h的雄虫触角Sexi\Orco基因表达量最大，约为同期雌虫的5.5倍；在喙内表达量极低；（4）、RNA干扰结果表明：触角内Sexi\Orco基因与对照相比表达量被抑制了90%以上，明显影响了触角对性信息素的反应，且dsRNA长度对RNAi作用没有明显影响；（5）、系统发育分析表明：昆虫的OR83b类嗅觉受体明显被分为三个组：鳞翅目组、双翅目组、膜翅目和鞘翅目组。Sexi\Orco位于鳞翅目组。上述结果将为进一步研究甜菜夜蛾嗅觉通讯的分子机制和绿色防控奠定基础。

中文关键词： 甜菜夜蛾;非典型嗅觉受体;表达谱;RNA干扰

英文摘要： We studied an unusually highly conserved olfactory receptor Sexi\Orco(orthologue to the Drosophila melanogaster DOR83b) from Spodoptera exigua using various means, such as Real-time quantitative PCR and RNAi. We found：(1)Sexi\Orco gene consisted of nucleotide sequence of 1906, and both with deduced amino acid sequences of 473 residues.(2)Sexi\Orco gene contains 10 extrons and 9 introns.The ends of the introns have a typical GT-AG structure.(3)Sexi\Orco was expressed in egg, larva, pupae and adult. During the adult stage, Sexi\Orco was expressed in antennae and proboscis of male and female moths.The expression level of Sexi\Orco was very low in proboscis.The expression level was the highest in male antennae 36 h after emergence, and was 5.5-fold higher than female antennae.(4)RNAi of Sexi\Orco in S.exigua Abodomen injection approach has been applied to introduce dsRNA molecules for Sexi\Orco into the moth abdomen cavity. RT-PCR and qRT-PCR detections at 24 hours after injecting of dsRNA molecules revealed that the mRNA expression level of Sexi\Orco in male and female antenna was significantly reduced by about 90%，and that the moth survival rates were not significantly different between treatments of dsRNA injected, dsEGFP and DEPC-water injeeted. (5) In this Neighbor joining tree the insect OR83b orthologue sequences clearly formed three clades, illustrating the diversity of this multigenic family: Lepidopteran and Dipteran OR83b orthologues formed one clade, respectively; Hymenopteran and Coleopteran orthologues formed the third clade.These data provide a the basis for further study on molecular mechanisms of olfactory communication in S. exigua.

英文关键词： Spodoptera exigua; atypical olfactory receptor;expression profile ; RNAi