本文鉴定了一个小菜蛾的羧酸酯酶基因PxαE8，其开放阅读框共1599个核苷酸，编码532年氨基酸残基。对其时空表达谱研究表明，该基因在小菜蛾的不同发育阶段和不同部位或组织均有表达，尤其在幼虫、成虫以及幼虫的脂肪体、马氏管和血淋巴中高表达。进一步研究发现PxαE8在海南（HN）和广东（GD）两个多药抗性种群中的相对表达量分别是敏感种群的24.4-和15.5倍。利用RNAi干扰PxαE8在HN种群中的表达后再用LC₅₀的不同杀虫剂处理，发现用高效氯氰菊酯和辛硫磷处理可使其死亡率分别显著增加25.3%和18.3%。上述结果表明PxαE8的上调表达参与了小菜蛾对高效氯氰菊酯和辛硫磷的抗性，为进一步阐明小菜蛾和其他害虫对杀虫剂多药抗性的分子机制提供了基础。

Received 10 May, 2017 Accepted 9 November, 2017

    Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length cDNA of carboxylesterase pxCCE016b was cloned and exogenously expressed in Escherichia coli at the first time, which contained a 1 693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of the pxCCE016b was significantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1 293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded by pxCCE016b. All the above findings provide important information for further functional study, although we are uncertainty whether the pxCCE016b gene is actually involved in chlorantraniliprole resistance.

The insecticide chlorantraniliprole exhibits good efficacy and plays an important role in controlling the diamondback moth, Plutella xylostella Linnaeus. However, resistance to chlorantraniliprole has been observed recently in some field populations. At present study, diamondback moths with resistance to chlorantraniliprole (resistant ratio (RR) was 82.18) for biochemical assays were selected. The assays were performed to determine potential resistance mechanisms. The results showed that the selected resistant moths (GDLZ-R) and susceptible moth could be synergized by known metabolic inhibitors such as piperonyl butoxide (PBO), triphenyl phosphate (TPP) and diethyl-maleate (DEM) at different levels (1.68-5.50-fold and 2.20-2.89-fold, respectively), and DEM showed the maximum synergism in both strains. In enzymes assays, a high level of glutathione-S-transferase (GST) was observed in the resistant moth, in contrast, moths that are susceptible to the insecticide had only 1/3 the GST activity of the resistant moths. The analysis of short-term exposure of chlorantraniliprole on biochemical response in the resistant strain also showed that GST activity was significantly elevated after exposure to a sub-lethal concentration of chlorantraniliprole (about 1/3 LC50, 12 mg L-1) 12 and 24 h, respectively. The results show that there is a strong correlation between the enzyme activity and resistance, and GST is likely the main detoxification mechanism responsible for resistance to chlorantraniliprole in P. xylostella L., cytochrome P450 monooxygenase (P450) and carboxy-lesterase (CarE) are involved in to some extent.

The gene expression and activity of (+)-d-cadinene synthase during cotton development and in response to stress, as well as the spatial and temporal pattern of sesquiterpene biosynthesis, constitute one of chemical defense mechanisms in cotton plants. In order to explore the effects of omethoate on the cotton defense in relation to (+)-d-cadinene synthase and gossypol, effects of omethoate treatments on activity of (+)-d-cadinene synthase and gossypol content in cotton seedlings were investigated. Cotton seedlings treated with 400 mg L-1 omethoate exhibited a significant decrease in the specific activity of (+)-d-cadinene synthase from 12 to 120 h after treating when compared to the untreated control; significantly lower (+)-d-cadinene synthase activity was also observed in cotton seedlings treated with 200 mg L-1 omethoate from 72 to 120 h after treating; but for cotton seedlings treated with 100 mg L-1 omethoate, from 12 to 120 h after treating, no significant changes were observed for activity of (+)-d-cadinene synthase. The gossypol content in cotton seedlings treated with 100, 200 or 400 mg L-1 omethoate for different time periods showed no significant changes compared to that of the control. These results indicated that the activity of (+)-d-cadinene synthase in cotton seedlings in responses to exposure of omethoate at three concentrations for different time periods followed dose- and time-dependent responses to omethoate exposure. With omethoate as a chemical stress factor for cotton seedlings, the cotton defense in relation to the activity of (+)-d-cadinene synthase in cotton seedlings may be affected by omethoate application.