| 吴怡蓓，夏鹏亮，张廷伟，严毅，杨文佳,2023,烟草甲细胞色素P450还原酶基因的分子特征与表达模式分析[J].环境昆虫学报,45(1):124-132 |
| 烟草甲细胞色素P450还原酶基因的分子特征与表达模式分析 |
| Molecular characterization and expression profiles of a cytochrome P450 reductase gene in Lasioderma serricorne (Coleoptera: Anobiidae) |
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| DOI： |
| 中文关键词: 烟草甲 细胞色素P450还原酶 基因克隆 表达模式 甲酸乙酯 |
| 英文关键词:Lasioderma serricorne NADPH-cytochrome P450 reductase gene cloning expression pattern ethyl formate |
| 基金项目:铜仁市科技局项目（铜市科研[2018]20号）；贵州省科技厅重点实验室项目（黔科合平台人才[2020]2003号）；中国烟草总公司科技项目（110202102040）；湖北省烟草公司科技项目（027Y2020-006） |
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| 中文摘要: |
| 烟草甲Lasioderma serricorne是一种重要的仓储害虫，长期化学防治导致烟草甲已对多种传统熏蒸剂产生抗性，但其对新型熏蒸剂甲酸乙酯仍处于敏感水平。因此明确其体内细胞色素P450还原酶（cytochrome P450 reductase, CPR）对甲酸乙酯的代谢解毒功能，对开展该药剂的抗性监测及延缓抗性的发生发展具有重要意义。本研究旨在克隆烟草甲LsCPR基因，分析其分子特征和表达特性，为进一步明确其在烟草甲对甲酸乙酯解毒代谢过程中的作用奠定基础。利用RT-PCR技术克隆烟草甲LsCPR基因的开放阅读框（open reading frame, ORF）；利用生物信息学软件分析LsCPR编码蛋白的结构、特征和系统进化关系。采用实时定量PCR技术检测LsCPR在烟草甲不同发育阶段（低龄幼虫、高龄幼虫、蛹、低龄成虫、高龄成虫）、幼虫不同组织（表皮、肠道、脂肪体和马氏管）以及甲酸乙酯熏蒸胁迫后的表达模式。烟草甲LsCPR基因的ORF为2 046 bp（GenBank登录号：MZ423209），编码681个氨基酸，具有典型的昆虫CPR基因FMN区域、NADPH区域和FAD等保守结构域。系统发育分析表明，烟草甲LsCPR与鞘翅目昆虫聚为一支，且与赤拟谷盗Tribolium castaneum CPR亲缘关系最近。LsCPR在烟草甲不同发育阶段均有表达，在高龄幼虫期的表达水平较高；在幼虫体内的表达部位主要在肠道，其次为脂肪体和马氏管，而在表皮的表达水平最低。LC10、LC30和LC50 3种浓度的甲酸乙酯处理24 h后，烟草甲LsCPR表达量随着胁迫浓度升高而上调且显著高于对照；甲酸乙酯LC50处理烟草甲不同时间（3、6、12、24和48 h）后，LsCPR基因上调表达，24 h时达到表达高峰。推测LsCPR是参与烟草甲代谢甲酸乙酯的候选基因。 |
| 英文摘要: |
| Lasioderma serricorne is an important storage insect pest. Resistance of the traditional fumigant has developed quickly because of long-term chemical control, and it is still at a sensitive level to the new fumigant ethyl formate. Thus, to clarify the metabolism and detoxification function of cytochrome P450 reductase (CPR)in the body of ethyl formate is of great significance for the development of resistance monitoring of this agent and delaying the development of resistance. The objectives of this study are to clone the cDNA sequence of L.serricorne cytochrome P450 reductase gene (designated LsCPR), analyze its molecular characteristic and expression profiles, and providing information that contribute to clarify the role of LsCPR in the detoxification of ethyl formate. The full-length open reading frame (ORF)of LsCPR was cloned using RT-PCR. The deduced protein structure, molecular charateristic, and phylogenetic relationship of LsCPR were predicted using different bioinformatics softwares. The expression profiles of LsCPR in different developmental stages (early larvae, late larvae, pupae, early adults, and late adults)and different larval tissues (integument, gut, fat body, and Malpighian tubules), and exposed to ethyl formate fumigation were detected by quantitative real-time PCR (qPCR). The ORF of LsCPR was 2 046 bp (GenBank accession number: MZ423209), which encodes 681 amino acid protein. LsCPR had typical features of insect CPRs, including FMN region, an NADPH and a FAD conserved domains. Phylogenetic analyses revealed that LsCPR was clustered with CPR of other coleopteran insects, and LsCPR was closely related to the CPR of Tribolium castaneum. LsCPR was constitutively expressed in all the tested stages, and had relatively higher expression level in the late larvae. Tissue-specific expression showed that the highest expression was observed in the gut, followed by fat body and Malpighian tubules, and the expression in integument was the lowest. In L. serricorne larvae exposed to three different concentrations of ethyl formate (LC10, LC30, and LC50)for 24 h, the expression levels of LsCPR were gradually increased with the exposure concentration compared to that of the control. When the larvae were exposed to LC50 concentration of ethyl formate for different times (3, 6, 12, 24, and 48 h), the expression of LsCPR significantly increased and reached the highest level at 24 h after treatment. LsCPR is a candidate gene involved in the metabolism of ethyl formate in L. serricorne. |
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