RNAi-Mediated Knockdown of Catalase Causes Cell Cycle Arrest in SL-1 Cells and Results in Low Survival Rate of <i>Spodoptera litura</i> (Fabricius)

Deregulated reactive oxygen species (ROS) production can lead to the disruption of structural and functional integrity of cells as a consequence of reactive interaction between ROS and various biological components. Catalase (CAT) is a common enzyme existing in nearly all organisms exposed to oxygen, which decomposes harmful hydrogen peroxide, into water and oxygen. In this study, the full length sequence that encodes CAT-like protein from Spodoptera litura named siltCAT (GenBank accession number: JQ_663444) was cloned and characterized. Amino acid sequence alignment showed siltCAT shared relatively high conservation with other insect, especially the conserved residues which defined heme and NADPH orientation. Expression pattern analysis showed that siltCAT mRNA was mainly expressed in the fat body, midgut, cuticle and malpighian tube, and as well as over last instar larvae, pupa and adult stages. RNA interference was used to silence CAT gene in SL-1 cells and the fourth-instar stage of S. litura larvae respectively. Our results provided evidence that CAT knockdown induced ROS generation, cell cycle arrest and apoptosis in SL-1 cells. It also confirmed the decrease in survival rate because of increased ROS production in experimental groups injected with double-stranded RNA of CAT (dsCAT). This study implied that ROS scavenging by CAT is important for S. litura survival.失调的活性氧（reactive oxygen species, ROS）生成可因ROS与多种生物组分发生反应性相互作用，导致细胞结构与功能完整性遭到破坏。过氧化氢酶（Catalase, CAT）是几乎所有需氧生物中普遍存在的常见酶类，可将有害的过氧化氢分解为水与氧气。本研究克隆并鉴定了来自斜纹夜蛾（Spodoptera litura）的CAT样蛋白编码全长序列，将其命名为siltCAT（GenBank登录号：JQ_663444）。氨基酸序列比对结果显示，siltCAT与其他昆虫的同源序列保守性较高，尤其是参与界定血红素与NADPH结合方位的保守氨基酸残基。表达模式分析表明，siltCAT mRNA主要在脂肪体、中肠、表皮与马氏管中表达，同时在末龄幼虫、蛹及成虫阶段均有分布。本研究分别利用RNA干扰（RNA interference）技术沉默SL-1细胞与斜纹夜蛾四龄幼虫体内的CAT基因。实验结果证实，敲低CAT基因可诱导SL-1细胞内ROS生成、细胞周期阻滞与细胞凋亡；同时也证实，注射CAT双链RNA（dsCAT）的实验组中，宿主存活率因ROS生成增多而下降。本研究表明，CAT介导的ROS清除对于斜纹夜蛾的存活具有重要意义。