棉酚胁迫下棉铃虫 HarmCYP6AE 家族的表达响应及 HarmCYP6AE19 的功能解析

Expression response of HarmCYP6AE family in Helicoverpa armigera under gossypol stress and functional analysis of HarmCYP6AE19

摘要

[目的]本研究旨在明确棉酚对棉铃虫(Helicoverpa armigera)幼虫生长发育的影响，鉴定 HarmCYP6AE 家族基因对棉酚的解毒功能，为阐明昆虫与寄主植物协同进化机制及开发新型害虫防控策略提供理论依据。[方法]采用生物测定分析不同浓度棉酚对棉铃虫发育历期、死亡率及体重的影响；通过 RT-qPCR 检测 HarmCYP6AE 家族基因表达模式；利用碳量子点(CQD)介导的 RNA 干扰(RNAi)技术沉默 HarmCYP6AE19，评估其对幼虫生长发育的作用。[结果]棉酚对棉铃虫生长发育呈浓度依赖性影响：低浓度(≤0.0075%)显著延长发育历期，降低存活率及体重(P<0.05)；高浓度(≥0.3000%)缩短发育历期，增加 5 龄幼虫体重(P<0.05)。HarmCYP6AE 家族 10 个成员含保守基序及 SRS 位点，序列相似性 48.94%~91.97%，聚类为 4 个分支。该家族基因表达受棉酚浓度特异性影响，其中，HarmCYP6AE17 和 HarmCYP6AE19 受高浓度棉酚诱导显著上调表达，HarmCYP6AE20 受低浓度棉酚诱导显著上调表达，HarmCYP6AE24 只在高浓度棉酚处理下特异性高表达(P<0.05)。RNAi 沉默幼虫中肠组织中 HarmCYP6AE19 的表达(P<0.05)，造成取食棉酚后幼虫化蛹率降低 20.76%~40.51%，蛹重增加 3.57%~33.70%(P<0.05)。[结论]棉酚以浓度依赖性影响棉铃虫生长发育，HarmCYP6AE 家族基因对棉酚表现出差异化的表达响应，其中 HarmCYP6AE19 在高浓度棉酚胁迫下显著上调，沉默该基因导致棉铃虫化蛹率显著下降，暗示其可能在棉铃虫响应棉酚胁迫中发挥重要作用。

Abstract

[Objectives] This study aimed to clarify the effects of gossypol on the growth and development of Helicoverpa armigera larvae, unravel the gossypol detoxification function of the midgut HarmCYP6AE gene family, and provide a theoretical basis for elucidating the co-evolutionary mechanisms between insects and host plants, as well as for developing novel pest management strategies. [Methods] Bioassays were performed to assess the effects of different gossypol concentrations on the developmental duration, mortality rate, and body weight of H. armigera. RT-qPCR was employed to determine the expression patterns of the HarmCYP6AE family. Carbon quantum dot (CQD)-mediated RNAi was utilized to silence HarmCYP6AE19, and its impacts on larval growth and pupation were evaluated. [Results] Gossypol exerted a concentration-dependent effect on the growth and development of H. armigera: low concentrations (≤0.0075%) significantly prolonged the developmental duration and reduced the survival rate and body weight (P<0.05), while high concentrations (≥0.3000%) shortened the developmental duration and increased the body weight of 5th instar larvae (P<0.05). The 10 members of the HarmCYP6AE family contained conserved motifs and substrate recognition sites (SRS), with amino acid sequence identity ranging from 48.94% to 91.97%, and were clustered into 4 phylogenetic clades. Genes of this family exhibited concentration-specific expression profiles: HarmCYP6AE17 and HarmCYP6AE19 were up-regulated under high gossypol concentrations, HarmCYP6AE20 was up-regulated under low concentrations, and HarmCYP6AE24 showed specific high expression under high concentrations (P<0.05). After silencing HarmCYP6AE19, the expression level of this gene in the larval midgut was significantly downregulated (P<0.05), the pupation rate was reduced by 20.76%-40.51%, and the pupal weight increased by 3.57%-33.70% (P<0.05). [Conclusions] Gossypol regulates the growth and development of H. armigera in a concentration-dependent manner. Members of the HarmCYP6AE family exhibit differential expression responses to gossypol. Specifically, HarmCYP6AE19 was significantly up-regulated under high gossypol concentrations, and its silencing led to a marked decrease in pupation rate, suggesting its crucial role in the H. armigera's response to gossypol stress, though whether it directly participates in gossypol detoxification remains to be elucidated.

关键词

植物次生代谢物质 / 棉酚 / 棉铃虫 / CYP6AE / RNA 干扰

Key words

plant secondary metabolites / gossypol / Helicoverpa armigera / CYP6AE / RNA interference

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基金资助

新疆维吾尔自治区科技计划项目-重点研发专项(2024B02005)

新疆维吾尔自治区自然科学基金项目(2024D01C26)