Expression and characterization of two pesticide resistance-associated serine protease genes (NYD-tr and NYD-ch) from Culex pipiens pallens for metabolism of deltamethrin.
Two deltamethrin resistance-associated serine protease genes (NYD-tr and NYD-ch) were isolated from Culex pipiens pallens in our previous study. To study the function of NYD-Tr and NYD-Ch in the metabolism of deltamethrin, we constructed the recombinant plasmid pET32a(+)/NYD-tr and pET32a(+)/NYD-ch with a 6x histidine tag. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses of the recombinant proteins revealed that the molecular weights of NYD-Tr and NYD-Ch are 42 and 50 kDa. Enzyme activity assay indicated that the recombinant NYD-Tr and NYD-Ch had the corresponding features of trypsin and chymotrypsin. Using BApNA as the substrate, NYD-Tr gave optimal activity between pH 9.0 and 10.5, while NYD-Ch was optimally active over the range of pH 8.0-11.0 using the S(Ala)(2)ProPhe-pNA as the substrate. Then, we investigated the metabolism of deltamethrin by NYD-Tr and NYD-Ch. Our results showed that NYD-Tr and NYD-Ch could hydrolyze deltamethrin. The acute oral toxicity of the metabolite to Wistar rats was much lower than deltamethrin.
Yang
Qinggui
Q
Department of Pathogen Biology, Jiangsu Province Key Laboratory of Modern Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China.
Zhou
Dan
D
Sun
Lixin
L
Zhang
Donghui
D
Qian
Jin
J
Xiong
Chunrong
C
Sun
Yan
Y
Ma
Lei
L
Zhu
Changliang
C
D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D003465:Culex / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000378:metabolism* / Q000633:toxicity; D007700:Kinetics; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism* / Q000633:toxicity; D011722:Pyrethrins / Q000378:metabolism* / Q000633:toxicity; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D007306:Insecticides; D009570:Nitriles; D011722:Pyrethrins; D011994:Recombinant Proteins; C017180:decamethrin; D001586:Benzoylarginine Nitroanilide; D012697:Serine Endopeptidases