| 季彩宏，张楠，孟祥坤，王建军,2021,二化螟CsCncC和CsKeap1基因的克隆与mRNA表达分析[J].环境昆虫学报,43(6):1504-1514 |
| 二化螟CsCncC和CsKeap1基因的克隆与mRNA表达分析 |
| Cloning and mRNA expression analysis of CsCncC and CsKeap1 from Chilo suppressalis |
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| DOI： |
| 中文关键词: 二化螟 CncC Keap1 氯虫苯甲酰胺 基因克隆 定量PCR |
| 英文关键词:Chilo suppressalis CncC Keap1 chlorantraniliprole gene cloning quantitative PCR |
| 基金项目:国家自然科学基金（31871974） |
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| 中文摘要: |
| 核因子E2相关因子2（Nrf2）-Kelch样环氧氯丙烷相关蛋白-1（Keap1）信号通路可调控抗氧化酶和代谢酶基因的表达，在机体适应氧化和化学胁迫过程中具有重要作用，克隆二化螟Chilo suppressalis CsCncC和CsKeap1基因可为研究二化螟的代谢抗性机制奠定基础。本研究采用RT-PCR和RACE技术克隆了二化螟CsCncC和CsKeap1基因全长cDNA，利用生物信息学方法对其结构特征进行了分析，并利用实时定量PCR检测了基因表达模式。结果表明，CsCncC包含一个长度为2 160 bp的开放阅读框，编码719个氨基酸；CsKeap1包含一个长度为1 923 bp的开放阅读框，编码640个氨基酸。序列分析发现，CsCncC和CsKeap1都具有保守性结构域和特征性基序。表达分析发现，CsCncC和CsKeap1分别在蛹末期和成虫初期表达水平最高，CsCncC在中肠和马氏管中的表达水平最高，CsKeap1在脂肪体中的表达水平最高。进一步研究发现，在0.47 mg/L氯虫苯甲酰胺处理36 h后，二化螟 3龄幼虫的CsCncC和CsKeap1的表达量显著上调，但在0.092 mg/L处理36 h后CsCncC无明显变化，CsKeap1的表达量显著下调，表明诱导效应具有剂量依赖性。本文研究结果表明，CsCncC和CsKeap1参与的信号通路在二化螟适应化学胁迫过程中具有重要作用。 |
| 英文摘要: |
| Nuclear factor erythroid 2-related factor (Nrf2) -Kelchlike ECH-associated protein 1(Keap1) signaling pathway regulates the gene expression of antioxidant and metabolic enzymes, which plays an important role in the adaptation of organisms to oxidative and chemical stresses. The cloning of CsCncC and CsKeap1 genes of Chilo suppressalis will lay a foundation for the study of metabolic resistance mechanisms of C. suppressalis. RT-PCR and RACE techniques were used to clone the full-length cDNAs of CsCncC and CsKeap1, the structural features of CsCncC and CsKeap1 were characterized using bioinformatics methods, and the expression patterns were detected using reverse transcription quantitative PCR. CsCncC contained an open reading frame (ORF) of 2 160 bp, which encoded a protein of 719 amino acid residues. CsKeap1 contained an ORF of 1 923 bp, which encoded a protein of 640 amino acid residues. Sequence analysis revealed that both CsCncC and CsKeap1 had conserved domains and characteristic motifs. Expression pattern analysis found that the expression levels of CsCncC and CsKeap1 were the highest at the end of pupal stage and at the beginning of adult stage, respectively. CsCncC showed the highest expression levels in midgut and Malpighian tube of C. suppressalis. The highest expression level of CsKeap1 was observed in fat body. Further study revealed that CsCncC and CsKeap1 were significantly up-regulated in 3rd instar larvae at 36 h after treatment with 0.47 mg/L chlorantraniliprole, exposure of 0.092 mg/L chlorantraniliprole to 3rd instar larvae for 36 h had not effect on CsCncC expression, but significantly up-regulated CsKeap1, suggesting that the induction effects of chlorantraniliprole on CsCncC and CsKeap1 were dose-dependent. Our results indicated that CncC-Keap1 pathway plays an important role in the adaptation of C. suppressalis to the chemical stress. |
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