棉铃虫表皮蛋白基因CP22和CP14的表达特征及其对甲氧虫酰肼的响应

Expression analysis of cuticular protein genes CP22 and CP14 in cotton bollworm Helicoverpa armigera and their response to the sublethal dose of methoxyfenozide

为揭示棉铃虫Helicoverpa armigera表皮蛋白(cuticular protein,CP)基因在其生长发育及应对药剂胁迫中的作用,克隆棉铃虫2个CP基因CP22和CP14,利用实时荧光定量PCR技术分析其在不同发育阶段和不同组织中的相对表达量,并于显微镜下观察甲氧虫酰肼亚致死剂量处理后棉铃虫3龄幼虫的表皮形态,并用实时荧光定量PCR技术测定药后CP22和CP14基因的相对表达量。结果显示,CP22和CP14的开放阅读框全长分别为570 bp和393 bp,分别编码189个和130个氨基酸;CP22和CP14都具有1个几丁质结合域,属于CPR家族RR-1亚类;CP22和CP14基因均在棉铃虫5龄幼虫表皮中表达水平高;这2个基因在棉铃虫幼虫期的表达水平高于在卵期、蛹期和成虫期的表达水平,且在4龄幼虫体内表达量最高,在蜕皮后随着日龄的增加表达量逐渐降低;甲氧虫酰肼处理后棉铃虫3龄幼虫表皮黑化、皱缩,发生蜕皮异常,显微观察显示其内外表皮分离,真皮细胞解体;甲氧虫酰肼处理后24 h和48 h,棉铃虫CP22和CP14基因的相对表达量显著上调。表明棉铃虫CP22和CP14基因参与棉铃虫幼虫蜕皮,并且响应甲氧虫酰肼胁迫,可作为防治棉铃虫的潜在靶标基因。

To explore the function of insect cuticular proteins in development and response to insecticides,two cuticular proteins genes,namely CP22 and CP14,were identified from cotton bollworm Helicoverpa armigera.The expression patterns of CP22 and CP14 were measured in different tissues and developmental stages by real-time quantitative PCR(qPCR).Then the sublethal effects of methoxyfenozide(MF)on larval cuticle were observed after hematoxylin and eosin(HE)staining,and the relative expressions of CP22 and CP14 were detected by qPCR.The results showed that CP22 contained an open reading frame(ORF)of 570 bp,encoding 189 amino acids,while CP14 contained an ORF of 393 bp,encoding 130 amino acids.The functional domain analysis showed that both CP22 and CP14 contained a chitin binding domain,which belonged to RR-1 subclass of the CPR family.qPCR revealed that these two genes were highly expressed in larval cuticle during larval stage,and their expression reached the highest level in the 4th-instar larvae and then decreased after molting.Phenotype observation revealed that the larval cuticle became wrinkled and darkened after treated with sublethal dose of MF,resulting in abnormal molting.HE staining showed the separation of exocuticle and endocuticle,as well as the decomposition of epidermis.Besides,the relative expressions of CP22 and CP14 were significantly upregulated at 24 h to 48 h after MF treatment.Our results therefore demonstrated that CP22 and CP14 participated in larvae ecdysis,responded to the disturbance of MF treatment,and could serve as potential targets for controlling H.armigera.