【摘要】 【目的】明确棉蓟马（Thrips tabaci）细胞色素P450(cytochrome P450, CYP450)基因和谷胱甘肽S-转移酶（glutathione S-transferase, GST）基因的序列结构，及相关基因在棉蓟马生长发育和甲氨基阿维菌素苯甲酸盐（简称为甲维盐）胁迫下的表达情况。【方法】基于棉蓟马不同生长发育时期的转录组数据，挖掘了CYP450基因和GST基因，设计特异性引物，采用聚合酶链式反应（polymerase chain reaction, PCR）技术以棉蓟马cDNA为模板进行扩增。利用生物信息学软件预测CYP450、GST蛋白结构特征。采用浸叶法测定甲维盐对棉蓟马成虫的室内毒力。通过实时荧光定量PCR(quantitative real time PCR, qRT-PCR)分析CYP450基因和GST基因在棉蓟马不同发育阶段和甲维盐胁迫下的表达模式。【结果】克隆了3个CYP450基因CYP4C101、CYP4C102、CYP6K1和2个GST基因GST1、GSTX1。理化分析结果表明CYP4C101、CYP4C102、CYP6K1、GST1和GSTX1蛋白分别包含507、528、513、215和207个氨基酸残基，均为亲水性蛋白。系统发育分析结果表明棉蓟马CYP4C101与褐花蓟马（Frankliniella fusca）CYP4C1同源性最高，棉蓟马的CYP4C102、CYP6K1、GST1均与西花蓟马（F. occidentalis）和褐花蓟马中的同源蛋白亲缘关系最近，棉蓟马GSTX1与西花蓟马的GSTX1同源性最高。结构域预测表明CYP4C101、CYP4C102、CYP6K1具有CYP450的保守结构域，GST1、GSTX1具有GST的保守结构域。室内毒力测定结果表明甲维盐处理48 h的亚致死浓度LC20为4.01 mg·L-1。qRT-PCR结果表明，CYP4C101、CYP4C102、CYP6K1、GST1、GSTX1基因在各个发育阶段均有表达，在成虫羽化后第9天表达水平最高。同时，在甲维盐LC20剂量胁迫24 h下，上述基因的表达量均显著上调，其中CYP4C101、CYP4C102、CYP6K1分别显著上调至4.43倍、22.91倍、8.48倍，GST1、GSTX1分别显著上调至9.06倍和5.26倍；经甲维盐LC20剂量胁迫48 h后，CYP4C102、CYP6K1表达量分别显著上调至3.84倍、1.43倍，CYP4C101、GSTX1、GST1表达量虽上调但未达到差异显著水平。【结论】本研究在棉蓟马中克隆得到的3个CYP450基因和2个GST基因，均在棉蓟马成虫羽化后第9天表达量最高。甲维盐LC20剂量胁迫下，这5个解毒基因被诱导表达的时间不同，但都可能参与棉蓟马对甲维盐胁迫的响应。研究结果可为后续CYP450、GST基因的功能研究提供线索。更多还原

【Abstract】 [Objective] This study aims to clarify the sequence structure of cytochrome P450(CYP450) gene and glutathione S-transferase(GST) gene of Thrips tabaci, and the expression of these genes at the different growth and development stages of T.tabaci and emamectin benzoate stress. [Methods] Based on the transcriptome data of different growth and development stages of T. tabaci, CYP450 genes and GST genes were mined, specific primers were designed, and polymerase chain reaction(PCR) was used to amplify the cDNA of these genes. Bioinformatics software was used to predict the structural characteristics of CYP450and GST proteins. The indoor toxicity of emamectin benzoate to adult T. tabaci was determined by leaf dipping method.Quantitative real time PCR(qRT-PCR) was used to analyze the expression patterns of CYP450 and GST genes at the different development stages of T. tabaci and under the stress of emamectin benzoate. [Results] Three CYP450 genes(CYP4C101,CYP4C102, CYP6K1) and two GST genes(GST1, GSTX1) were cloned. The results of physicochemical analysis showed that CYP4C101, CYP4C102, CYP6K1, GST1, and GSTX1 were composed of 507, 528, 513, 215 and 207 amino acid residues,respectively, all of which were hydrophilic proteins. Phylogenetic analysis showed that CYP4C101 had the highest homology with CYP4C1 of Frankliniella fusca. CYP4C102, CYP6K1 and GST1 of T. tabaci had the highest relationships with the homologous proteins from F. occidentalis and F. fusca. GSTX1 had the highest homology with F. occidentalis. Domain prediction showed that CYP4C101, CYP4C102 and CYP6K1 had conserved domains of CYP450, and GST1 and GSTX1 had conserved domains of GST. The results of indoor toxicity test showed that the LC20value of emamectin benzoate was 4.01 mg·L-1at 48 h. The results of qRT-PCR showed that CYP4C101, CYP4C102, CYP6K1, GST1, and GSTX1 genes were expressed at all development stages, and the expression levels were the highest on the 9thday of adult emergence. The expression levels of the above-mentioned genes were significantly up-regulated under the stress of emamectin benzoate LC20for 24 h. Among them,CYP4C101, CYP4C102, and CYP6K1 were significantly up-regulated to 4.43, 22.91 and 8.48 times, respectively, and GST1and GSTX1 were significantly up-regulated to 9.06 and 5.26 times, respectively. At 48 h after emamectin benzoate LC20treatment, the expression levels of CYP4C102 and CYP6K1 were significantly up-regulated by 3.84 and 1.43 times, respectively.The expression levels of CYP4C101, GSTX1, and GST1 were up-regulated but did not reach a significant difference level.[Conclusion] Three CYP450 genes and two GST genes of T. tabaci were cloned, and the expression levels of the five genes were the highest on the 9th day of adult emergence. Under the stress of emamectin benzoate LC20, although these five detoxification genes were induced to express at different times, they may be involved in the response of T. tabaci to emamectin benzoate, providing clues for subsequent functional studies of CYP450 and GST genes.更多还原