药材甲羧酸酯酶家族基因对气调胁迫的应答

Insecticidal technology based on Controlled Atmosphere（CA）has been widely applied in the management pests in stored products. Researches on both the mechanism that insects were killed by CA stress and the response mechanism of insects against CA stress have already become hot topics. Physicochemical basis of CA Stress killing target insects has been clarified, while the response mechanism of insects to CA stress is unclear. To explore this potential mechanism, transcriptome sequencing (RNA-seq) of Stegobium paniceum (L) with CA stress was carried out, and it was confirmed that a better part of genes expression of S. paniceum (L) that exposed to CA stress are suppressed or effectively activated, and that carboxylesterase, as one kind of hydrolase in insects, responded to CA stress actively. What is the molecular mechanism of that response? Through the ESTs analysis of CarE genes' differential expression of S. paniceum (L) stressed by CA, the Real Time Fluorescence Quantitative PCR (qRT-PCR) verification of the regulation of CarE genes' differential expression, we will sequence the key genes from aforementioned differential expression, analyze their functions, and verify key genes' expression regulation effect with RNA interfere technology, meanwhile, their physiological biochemical characteristics of their expression product in vitro will be taken into consideration. Above researches will be expected to reveal the molecular mechanism of CarE gene family of S. paniceum (L) responding to CA stress, and to provide the scientific basis for clarifying the responding mechanism of insects to CA stress.

气调杀虫技术已被广泛应用于储藏物害虫的管理中，有关气调胁迫对昆虫作用机制及昆虫在气调胁迫下的应答机理研究已成为国内外储藏物害虫研究的前沿课题。气调胁迫致死目标昆虫的理化基础已基本明确，而昆虫对气调胁迫应答的机制尚不清楚。为探讨这一潜在机制，我们通过气调胁迫下药材甲转录组筛选，明确了气调胁迫下药材甲的大量基因表达受到抑制或被有效激活，其中羧酸酯酶（CarE）是昆虫对气调胁迫响应明显的一类水解酶。这种应答的分子机制如何？我们将通过气调胁迫下药材甲CarE差异表达的ESTs分析，qRT-PCR验证CarE基因的差异表达调控，筛选出差异表达的关键基因，并对其进行功能分析，采用RNAi技术验证其表达调控效应，同时结合关键基因体外表达产物的生理生化特性来验证其功能。研究结果有望揭示药材甲CarE基因家族对气调胁迫应答的分子机制，为阐明昆虫在气调胁迫下的应激机制提供科学依据。

药材甲是一种世界性分布的仓储害虫，对300多种储藏物造成严重的经济损失。气调技术在仓储害虫防治中广泛应用，研究发现药材甲对CO2气调产生了不同程度的抗性，而目前有关昆虫对气调胁迫应答的分子机理尚不清楚。为了阐明这一潜在机制，本研究综合运用生物化学及分子生物学技术对气调胁迫下药材甲生理变化开展了一系列研究：（1）利用生化分析检测了CO2胁迫后药材甲体内CarE酶活性、三种能源物质含量及利用率的变化；（2）利用RNA-Seq技术进行药材甲转录组测序，组装得到28,773条unigene，并获得了大量与解毒代谢、能量相关的功能基因；（3）不同浓度CO2处理药材甲后进行数字表达谱分析，发现大量基因表达受到抑制或被激活，利用实时荧光定量PCR方法检测差异基因表达；（4）克隆获得药材甲6个CarE基因的全长序列，均具有羧酸酯酶特有的功能结构域。药材甲6个CarE在各个发育阶段均有表达，且在幼虫表达量最高。10%和70%CO2气调明显诱导SpCarE8、SpCarE9和SpCarE10基因的表达。 通过原核表达技术获得了重组蛋白SpCarE10，具有羧酸酯酶活性，而SpCarE8重组蛋白以包涵体形式存在。RNA干扰结合CO2生物测定结果发现，SpCarE8和SpCarE10在dsRNA注射后48 h的沉默效率最高，靶标基因沉默后CO2气调胁迫，RNA干扰组与对照组相比，死亡率明显提高。两个SpCarE基因同时沉默导致药材甲在CO2胁迫后死亡率明显上升，说明这两个基因在药材甲应对气调胁迫过程中起着关键作用。（5）利用Long-PCR技术获得药材甲、烟草甲线粒体基因组全长序列，为两种害虫的精确分类提供便利。（6）利用Y臂嗅觉仪分析药材甲成虫对4种中药材的嗅觉行为反应，利用气相色谱-质谱联用技术解析不同中药材的挥发物成分，为接下来高效的药材甲引诱剂研发奠定基础。本研究丰富了昆虫羧酸酯酶的理论体系，为阐明昆虫在气调胁迫下的应激机制提供科学依据。