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灰茶尺蠖CYP4G15基因克隆分析及对茚虫威胁迫的表达响应

Cloning, Expression, and Association with Indoxacarb Stress Response of CYP4G15 in Ectropis grisescens

  • 摘要:
    目的 克隆灰茶尺蠖(Ectropis grisescens Warren)细胞色素P450基因CYP4G15,并对其进行生物学信息和表达特征分析,为进一步研究灰茶尺蠖解毒代谢和抗性机理提供依据。
    方法 采用RT-PCR技术克隆CYP4G15基因的编码区。利用生物信息学软件分析该基因的生物学信息、序列特征及与其他近缘物种的P450基因的系统进化关系。采用RT-qPCR技术测定该基因在灰茶尺蠖不同发育阶段以及茚虫威胁迫后的表达特征。
    结果 克隆获得了灰茶尺蠖CYP4G15基因,该序列开放阅读框全长1572 bp,编码523个氨基酸,预测氨基酸序列相对分子质量为59.85 KD,理论等电点为8.85,预测蛋白结构存在细胞色素P450家族的保守结构域,该基因与夜蛾科的草地贪夜蛾(Spodoptera frugiperda)、甜菜夜蛾(Spodoptera exigua)等亲缘关系较近。RT-qPCR分析发现,CYP4G15在3龄、4龄幼虫中表达最高,卵表达最低,且雄性蛹和成虫表达量显著高于雌性。3龄灰茶尺蠖幼虫在茚虫威LC20和LC50剂量处理下,CYP4G15均被诱导表达,且在48 h后表达量最高,分别较对照提高了7.16倍和3.65倍。
    结论 CYP4G15基因可能在灰茶尺蠖对茚虫威解毒过程中发挥重要作用,为今后灰茶尺蠖解毒代谢研究和抗虫新靶点筛选提供依据。

     

    Abstract:
    Objective The cytochrome P450 gene from Ectropis grisescens Warren, CYP4G15, was cloned for bioinformatics and expression analysis in relation to the detoxification metabolism and resistance mechanism toward the pesticide indoxacarb of the most destructive defoliators at tea plantations in China.
    Method The coding region of CYP4G15 was cloned using RT-PCR, the bioinformatics, gene sequence, and phylogenetic relationships with the P450 genes of other closely related species analyzed by software, and the gene expressions at developmental stages and after an indoxacarb induction of E. grisescens determined by RT-qPCR.
    Result CYP4G15 was successfully cloned to show a full-length with an open reading frame of 1572 bp encoded with 523 amino acids. It had a predicted molecular weight of 59.85 kD and a theoretical isoelectric point at pH 8.85. The protein appeared to contain a conserved domain of the cytochrome P450 family closely related to Lepidoptera Noctuidae insects, such as Spodoptera frugiperda and Spodoptera exigua. CYP4G15 was highly expressed in the 3rd and 4th instar larvae, lowly in the eggs, and significantly higher in the male than in the female pupae and adults. The 3rd instar larvae of E. grisescens treated by indoxacarb at LC20 and LC50 doses had its CYP4G15 reached the highest expressions that were 7.16-fold and 3.65-fold, respectively, of control 48h after the artificial induction.
    Conclusion CYP4G15 appeared to play a significant role in the detoxification of E. grisescens against indoxacarb. This study unveiled a plausible new venue to advance the research on control of the debilitating pest in tea plantations.

     

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