赤拟谷盗钠离子通道重复基因的功能比较研究

The insect voltage-gated sodium channels are the primary targets of pyrethroids and novel efficient insecticides including indoxacarb and metaflumizone. Gene duplication provides raw genetic material for generating novel eukaryotic genes and causes functional differentiation of duplicated genes. While only one sodium channel gene is found in most of the insect genomes, our previous studies have found the occurrence of sodium channel gene duplication in the genome of Tribolium castaneum, which generated two genes, TcNav1 and TcNav2. On the basis of this finding, we will comparatively analyze the spatiotemporal expression patterns of TcNav1 and TcNav2 at different developmental stages and tissues as well as the functional differentiation of these two duplicated genes by using molecular biology techniques such as quantitative PCR, RNA interference and in vitro expression. The results will not only assist in understanding the function and evolution of insect sodium channels, but also provide important information for the development of novel insecticides.

昆虫钠离子通道是拟除虫菊酯类杀虫剂以及新型高效杀虫剂茚虫威和氰氟虫腙的作用靶标。基因重复是真核生物新基因起源和基因功能分化的重要途径。目前大多数昆虫基因组中只发现一个钠离子通道基因，但我们前期的研究发现赤拟谷盗基因组中钠离子通道发生基因重复，产生了TcNav1和TcNav2两个基因。本项目拟在此基础上，利用定量PCR、RNA干扰和体外表达等分子生物学技术分析TcNav1和TcNav2在赤拟谷盗不同组织、不同发育阶段的表达模式及其药理学、生物学功能差异。研究结果在提高对昆虫钠离子通道功能与进化认识的同时，也为新型杀虫剂的创制提供理论依据。

昆虫钠离子通道是拟除虫菊酯类杀虫剂以及新型高效杀虫剂茚虫威和氰氟虫腙的作用靶标。目前大多数昆虫基因组中只发现一个钠离子通道基因。本项目是基于前期发现赤拟谷盗体内存在钠离子通道基因重复现象的基础上开展研究工作的。克隆并鉴定了赤拟谷盗体内的两个钠离子通道基因TcNav1和TcNav2，分别编码2045和2037个氨基酸残基。TcNav1和TcNav2氨基酸序列的一致性为86%。TcNav1和TcNav2与para氨基酸序列的一致性分别为76.1%和75.5%。系统发生分析也表明，TcNav1和TcNav2与其它昆虫钠离子通道基因聚为一支，进一步证实了赤拟谷盗钠离子通道基因确实发生了重复。基因组结构的比较分析表明，TcNav1和TcNav2分别含有26和27个外显子。选择性剪接位点分析表明TcNav1存在3个选择性剪接位点：a、i、j，而TcNav2存在4个选择性剪接位点：a、f、i、j。表达谱分析显示，TcNav1的表达量要显著高于TcNav2。TcNav1和TcNav2均在头部的表达水平最高。TcNav1和TcNav2基因在头部的表达有显著性差异，但在其他组织中无显著性差异。RNA干扰介导的TcNav1和TcNav2基因敲除对成虫羽化产生不利影响，并显著降低对溴氰菊酯的敏感性。注射靶向TcNav1而非TcNav2的siRNA后，化蛹率和对高效氯氰菊酯的敏感性显著降低。综上所述，我们提供的证据表明，钠离子通道基因在赤拟谷盗体内发生了重复，从而产生了多样化的发育和毒理学功能。