氯虫苯甲酰胺和氟虫腈对黏虫细胞色素P450基因表达的影响
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摘要
为筛选出黏虫Mythimna separata参与杀虫剂解毒代谢的主效细胞色素P450基因,采用叶片浸渍法测定了用于处理黏虫3龄幼虫的亚致死浓度,通过构建转录组测序文库并结合数字基因表达(digital gene expression,DGE)对不同处理的黏虫进行测序,并运用实时荧光定量PCR(realtime quantitative polymerase chain reaction,RT-qPCR)技术验证12个P450基因的表达情况。结果表明,用于处理黏虫的氯虫苯甲酰胺和氟虫腈亚致死浓度LC_(10)分别为0.15、13.66 mg/L;对照样品、氟虫腈处理样品和氯虫苯甲酰胺处理样品分别获得59 521 504、64 838 148和41 722 990个原始序列数据,分别获得57 441 216、62 368 912和40 285 164个过滤后的序列数据;过滤后的序列长度分别为8.62、9.36和6.04 G;碱基错误率均为0.02%;Phred数值大于20、30的碱基占总碱基的百分比均高于90.59%;鸟嘌呤+胞嘧啶(guanine cytosine,GC)含量分别为47.16%、48.94%和47.55%,表明转录组测序质量较高;黏虫受氯虫苯甲酰胺胁迫后,29个P450基因表达量上调,27个P450基因表达量下调;黏虫受氟虫腈胁迫后,23个P450基因表达量上调,26个P450基因表达量下调;12个P450基因表达量的RT-qPCR技术检测结果与DGE测序文库显示的结果基本一致。
To identify the main cytochrome P450 genes involved in insecticide metabolic regulation, the leaf dipping method was used to determine the sublethal concentration(LC_(10)) of chlorantraniliprole and fipronil for controlling the armyworm; RNA sequencing together with digital gene expression(DGE) libraries and expression verification of 12 P450 genes by real-time quantitative PCR(RT-qPCR) were performed. The results showed that the sublethal concentrations of chlorantraniliprole and fipronil(LC_(10)) against the armyworm were 0.15 and 13.66 mg/L, respectively. Based on Illumina RNA sequencing, the raw reads(59 521 504, 64 838 148 and 41 722 990), clean reads(57 441 216, 62 368 912 and 40 285 164), clean bases(8.62, 9.36 and 6.04 G), error of basic group(0.02%), the ratio of basic group of Phred value >20, 30 to total basic group(>95.59%) and content of GC(47.16%, 48.94% and47.55%) were obtained for the control sample, fipronil-treated sample and chlorantraniliprole-treated sample, respectively, showing that the quality of transcriptome sequencing was high. In addition, 29 P450 genes were up-regulated and 27 P450 genes were down-regulated in the armyworm in response to chlorantraniliprole stress; Twenty-three P450 genes were up-regulated and 26 P450 genes were up-regulated in the armyworm in response to fipronil stress. The DGE library was validated by RT-qPCR analysis of 12 P450 genes.
To identify the main cytochrome P450 genes involved in insecticide metabolic regulation, the leaf dipping method was used to determine the sublethal concentration(LC_(10)) of chlorantraniliprole and fipronil for controlling the armyworm; RNA sequencing together with digital gene expression(DGE) libraries and expression verification of 12 P450 genes by real-time quantitative PCR(RT-qPCR) were performed. The results showed that the sublethal concentrations of chlorantraniliprole and fipronil(LC_(10)) against the armyworm were 0.15 and 13.66 mg/L, respectively. Based on Illumina RNA sequencing, the raw reads(59 521 504, 64 838 148 and 41 722 990), clean reads(57 441 216, 62 368 912 and 40 285 164), clean bases(8.62, 9.36 and 6.04 G), error of basic group(0.02%), the ratio of basic group of Phred value >20, 30 to total basic group(>95.59%) and content of GC(47.16%, 48.94% and47.55%) were obtained for the control sample, fipronil-treated sample and chlorantraniliprole-treated sample, respectively, showing that the quality of transcriptome sequencing was high. In addition, 29 P450 genes were up-regulated and 27 P450 genes were down-regulated in the armyworm in response to chlorantraniliprole stress; Twenty-three P450 genes were up-regulated and 26 P450 genes were up-regulated in the armyworm in response to fipronil stress. The DGE library was validated by RT-qPCR analysis of 12 P450 genes.
引文
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