节食夜蛾是一种远距离迁徙的害虫,具有很强的传播能力,快速的繁殖速度和破坏性的喂养,所以很难预防和控制。拟除虫菊酯杀虫剂通常用于害虫防治,由于电压门控钠通道(VGSC)是拟除虫菊酯的主要靶标,研究该基因对害虫防治具有重要意义。VGSC是一种完整的跨膜蛋白,由神经元中发现的约2,000个氨基酸残基组成。肌细胞,内分泌细胞,和卵巢细胞,并参与可兴奋细胞动作电位的启动和传播。在这项研究中,通过快速扩增cDNA末端(RACE)从S.frugiperda中鉴定出VGSC的cDNA序列,该cDNA末端包含6,261bp的开放阅读框,编码2,086个氨基酸的蛋白质。该蛋白的分子量预计为236kDa,理论等电点为5.21。基于鳞翅目昆虫构建的系统发育树表明,frugiperda的VGSC与斜纹夜蛾的VGSC最接近。VGSC是一种高度保守的蛋白质,具有跨膜蛋白的离子通道保守结构域。qPCR显示VGSC基因在2龄幼虫表皮中高表达,其在其他组织中的表达水平较低,例如前肠和马尔皮根小管。此外,在pu前阶段也检测到VGSC,然后在进入成年阶段后逐渐增加,成年雄性在第4天达到顶峰,后来减少了。构建pSumo-mut-VGSC的重组质粒并诱导表达His标签融合的VGSC蛋白。从纯化的重组VGSC蛋白制备多克隆抗体。抗体是ELISA滴定的,免疫印迹结果显示它能特异性识别VGSC,无论是重组蛋白还是内源蛋白。这些研究结果为今后研究该基因在草本生长发育中的生理功能奠定了基础。
Spodoptera frugiperda is a long-distance migratory pest with strong dispersal ability, fast reproduction speed and destructive feeding, so it is difficult to prevent and control. Pyrethroid insecticides are commonly used in pest insects control, And since the voltage-gated sodium channel (VGSC) serves as a major target of pyrethroids, it is important to study this gene for pest control. VGSC is an integral transmembrane protein consisting of approximately 2,000 amino acid residues found in neurons, myocytes, endocrine cells, and ovarian cells and involved in the initiation and propagation of excitable cellular action potentials. In this study, the cDNA sequence of the VGSC was identified from S. frugiperda by rapid amplification of cDNA ends (RACE) which contained an open reading frame of 6,261 bp encoding a protein of 2,086 amino acids. The molecular weight of this protein was predicted to be 236 kDa, and the theoretical isoelectric point was 5.21. A phylogenetic tree constructed based on lepidopteran insects showed that the VGSC of S. frugiperda was most closely relative to that of Spodoptera litura. VGSC is a highly conserved protein with Ion channel conserved structural domains of transmembrane proteins. qPCR showed that the VGSC gene was highly expressed in the epidermis of 2nd instar larvae, and its expression level was low in other tissues, such as the foregut and Malpighian tubules. In addition, VGSC was also detected in the prepupal stage, then gradually increased in abundance after entering the adult stage, peaked at the adult males on the 4th day of pupal stage, and decreased afterwards. The recombinant plasmid of pSumo-mut-VGSC was constructed and induced to express a His tag fused VGSC protein. Polyclonal antibodies were prepared from purified recombinant VGSC protein. The antibody was ELISA-titered, and the western blotting results showed that it specifically recognized VGSC, whether it was recombinant or endogenous protein. These results have laid the foundation for future studies on the physiological function of this gene in the growth and development of S. frugiperda.