Expression of two carboxylesterase genes (001f and 001g) from Helicoverpa armigera using baculovirus expression vector system (BEVS) and analysis of their enzymatic activity | 两个棉铃虫酯酶基因(001f和001g)在杆状病毒表达载体系统(BEVS)中的表达及酶活分析

Chinese. 昆虫羧酸酯酶(carboxylesterases, CarEs)对有机磷、拟除虫菊酯、氨基甲酸酯等三大类化学杀虫剂的代谢解毒作用是昆虫代谢抗性产生的一种重要机制。为明确棉铃虫(Helicoverpa armigera)两个酯酶基因001f和001g在代谢抗性中的作用，本研究基于杆状病毒表达载体系统(BEVS)在草地夜蛾(Spodoptera frugiperda)Sf9细胞系中成功表达了酯酶基因001f和001g，表达产物经非变性聚丙烯酰胺凝胶电泳和α-乙酸萘酯染色，结果表明，两个酯酶的条带染色很深，染色条带主要分布在相对迁移率(Rm)值为0.27~0.36和 0.37~0.48之间的两个区域。进一步采用酶标仪测定了表达产物对α-乙酸萘酯(α-naphthyl acetate)和4-硝基苯基乙酸酯(para-nitrophyl acetate)的酶促代谢动力学特性，结果表明，两个酯酶对底物均有极强的亲和力和非常高效的催化效率，其对α-乙酸萘酯的亲和系数(Km值)分别为(3.9±0.6)和(3.3±0.4) μmol/L，速率常数(kcat/Km值)高达(95±9.3)和(26.5±1.9) μmol-1•s-1•L；相比较而言对于4-硝基苯基乙酸酯的kcat/Km值较低(分别为(4.47±0.33)和(12.8±0.10) μmol-1•s-1•L)。上述结果在生化水平上证实了两个酯酶均具有较强代谢作用，预示着这两个酯酶基因与棉铃虫的代谢抗性可能相关，并可能对有机磷、拟除虫菊酯等化学杀虫剂也具有一定代谢解毒作用。

English. Metabolic detoxification of carboxylesterases (CarEs) is a major mechanism for the development of resistance in insects to organophosphates (OPs), synthetic pyrethroids (SPs) and carbamates (CBs). In order to elucidate the role of CarE genes from Helicoverpa armigera in metabolic resistance, two CarE genes, 001f and 001g，were successfully expressed in the Sf9 insect cell lines using the Baculovirus Expression Vector System (BEVS). Both of the expressed CarEs were subjected to native PAGE and stained with artificial substrate α-naphthyl acetate (α-NA), and showed high activity against α-NA according to the darker stained bands. The resulting stained bands (closely together) indicated that the relative mobility (Rm) of CarEs fell in two zones (0.27~0.36 and 0.37~0.48). The kinetic assays with α-NA and para-nitrophyl acetate (p-NA) using a spectrophotometer showed the two CarEs had high affinity to the former substrate (Km=(3.9±0.6) and (3.3±0.4) μmol/L, respectively). They also produced a considerably high rate constant (kcat/Km=(26.5±1.9) and (95±9.3) μmol-1•s-1•L, respectively) agianst α-NA, but a relatively lower rate constant against p-NA (kcat/Km=(4.47±0.33) and (12.8±0.10) μmol-1•s-1•L, respectively) compared with that of α-NA. It's showed that the two CarEs had high hydrolytic activity against the artificial substrates. Thus, 001F and 001G esterases are probabaly associated with metabolic resistance of insecticides, and may have relatively high detoxification activity against some traditional chemical insecticdes such as OPs, SPs and CBs.