Expression of Two Carboxylesterase Genes(001f and 001g) from Helicoverpa armigera Using Baculovirus Expression Vector System(BEVS) and Analysis of Their Enzymatic Activity
Abstract：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.
