cDNA cloning and characterization of the carboxylesterase 1 pxCCE016b from the diamondback moth, Plutella xylostella L. Abstract 10 Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, 11 carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced 12 carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM).

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... ver, the 13 related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length cDNA of 14 carboxylesterase pxCCE016b was cloned and exogenously expressed in Escherichia coli at the first time, which contained 15 contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that 16 this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced 17 amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B 18 carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The 19 real-time quantitative PCR (qPCR) analysis showed that the expression level of the pxCCE016b was significantly higher in 20 the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016b was highly expressed in the 21 midgut and epidermis of the DMB larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, 22 alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of 23 pxCCE016b was observed only in the group treated by chlorantraniliprole. In additional, recombinant vector 24 pET32a-pxCCE016b was constructed with the most coding region (1293 bp) and large number of soluble recombinant 25 proteins (less than 48kDa) was expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded 26 by pxCCE016b. All the above findings provide important information for further functional study, although we are 27 uncertainty that the pxCCE016b gene is actually involved in chlorantraniliprole resistance. 28 29 的 ORF 阅读框,能 42 编码 542 个氨基酸,其预测的蛋白分子量为 61.56kDa,等电点为 5.78。经比对,该序列编码的氨基 43 酸具有典型的羧酸酯酶结构特征:type-B 羧酸酯酶结构 2(EDCLYLNVYTK), type-B 羧酸酯酶丝氨酸 44 活性位点(FGGDPENITIFGESAG)和催化三联体结构(Ser186, Glu316 和 His444) 。实时荧光定量 PCR 45 (qPCR)分析表明,该基因在小菜蛾氯虫苯甲酰胺抗性品系中的相对表达量要显著高于敏感品系, 46 同时在小菜蛾抗性品系幼虫的中肠和表皮组织中表达量相对较高。为探讨该基因对氯虫苯甲酰胺的 47 响应,本研究进行了药剂诱导表达试验,结果表明当小菜蛾 3 龄幼虫曝露在阿维菌素、高效氯氰菊 48 酯、氯虫苯甲酰胺、多杀菌素、溴虫腈和茚虫威 6 种田间常用药剂的亚致死剂量时,只有氯虫苯甲 49 酰胺能诱导该基因的上调表达。此外,本研究选取了 ORF 阅读框 1293bp 的基因序列,成功构建了重 50 组表达载体 pET32a-pxCCE016b,并大量表达出可溶性重组蛋白(蛋白分子量小于 48kDa)。 Western 51 blot 分析表明表达出的蛋白为目标特异蛋白。虽然目前尚不能确定该基因是否确实与小菜蛾抗氯虫 52 苯甲酰胺有关,但上述系列基础研究将为下一步功能研究提供理论参考。 53 关键词:小菜蛾,羧酸酯酶,氯虫苯甲酰胺,杀虫剂抗药性,pxCCE016b 54 55 activities analysis and biochemical response test to insecticide-induced stress, detoxifying 76 enzymes dependent resistance is observed in resistant strains of DBM from China (Wang et al. 77 2012; Hu et al. 2014a). Our previous studies on the transcriptome analysis in DBM also suggested 78 that detoxifying enzymes were the major metabolic factors responsible for chlorantraniliprole 79 resistance (Lin et al. 2013). Based on transcriptome analysis, we identified a novel P450 gene: 80 CYP321E1 (GenBank Assession No: KC 626090) from DBM, RNA interference (RNAi) indicated 81 that this gene was related to chlorantraniliprole resistance (Hu et al. 2014b). Meanwhile, an 82 up-regulation carboxylesterase gene cDNA fragment: Unigene35058_yong_A was also obtained 83 from DBM EST database. The tag-based digital gene expression (DGE) analysis gave us an 84 opinion that this gene may also play role in chlorantraniliprole resistance of DBM (Lin et al. 2013). 85 86 Esterase is one of the major detoxifying enzymes in insects. Insecticide resistance mediated by 87 which has been found in many different insects (Zhang et al. 2010). As reported, the mainly 88 molecular basis of this kind of resistance mechanism to some normal used insecticides is gene 89 amplification, up-regulation or main coding sequence mutation (Li et al. 2007). Among them, 90 up-regulation of esterase genes has been found in Myzus persicae, Nilaparvata lugens and Aphis 91 gossypii (Bizzaro et al. 2005; Small and Hemingway 2000; Cao et al. 2008). In DBM, no relative 92 resistant gene has been cloned and characterized although studies suggested that 93 carboxylesterases may be involved in chlorantraniliprole resistance. 94 95 In this paper, a full-length cDNA of pxCCE016b from DBM was identified and analyzed. 96 Expression pattern of pxCCE016b and exposure experiments to several normal used insecticides 97 were investigated. In addition, recombinant protein of pxCCE016b cDNA was expressed in 98 Escherichia coli and then western blotting analysis was conducted. Our findings here provide