Molecular Characterization of Spodoptera frugiperda Heme Oxygenase and Its Involvement in Susceptibility to Chlorantraniliprole

Abstract

The mammalian heme oxygenase (HO) plays an important role in cytoprotection against oxidative-stress-induced cell damage; however, functional characterization of insect HO is still limited. In this study, cDNA encoding a HO, named Sf HO, was cloned from Spodoptera frugiperda. Analysis of the transcription level and enzymatic activity showed that exposure of the LC 30 concentration of chlorantraniliprole to the third instar larvae significantly upregulated both the mRNA level and enzymatic activity of Sf HO at 24 h after treatment. Further injection of the HO activator, hemin, into the third instar larvae led to the upregulation of Sf HO as well as decreased susceptibility of S. frugiperda to chlorantraniliprole. Consistently, overexpression of Sf HO increased the Sf9 cell viability under chlorantraniliprole treatment. Strikingly, both RNAi and the dual-luciferase reporter assay in Sf9 cells revealed that, unlike mammalian HO that is regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), Sf HO was not subject to the regulation by cap 'n' collar isoform C (CncC), the Nrf2 homologue in insects. These data provide insights into the function and regulatory mechanism of insect HOs and had applied implications for the control of S. frugiperda.

Involvement of Glutamate Cysteine Ligase Genes in Tolerance to Emamectin Benzoate in Spodoptera frugiperda and Their Putative Regulatory Mechanisms

“…EMB treatment was performed as described previously. , Briefly, EMB was dissolved in DMSO and diluted with distilled water. Insecticide-incorporated diets were prepared as described above.…”

“…A Bio-Rad CFX 96 real-time PCR system (Bio-Rad, Hercules, CA, USA) was used for qRT-PCR reactions with SYBR PrimeScript RT-PCR Kit II (TaKaRa, Dalian, China) and gene-specific primers (Table ). Reactions were carried out as described previously, and standard curves and melting curve analysis were used to assess PCR efficiencies and primer specificity, respectively. The stably expressed genes encoding elongationfactor1-alpha (EF1-α) and ribosomal protein L27 (RPL27) were used for normalization of mRNA abundance in FAW larvae and Sf9 cells, respectively, with the 2 –ΔΔ CT method .…”

“…The Sf Gclc and Sf Gclm proteins were expressed in the bac-tobac baculovirus expression system as previously described. 21,27 Briefly, the ORFs of Sf Gclc and Sf Gclm were amplified using primers containing BamHI and EcoRI restriction sites (Table 1) and subcloned into the pFastBac-HTb vector (Thermo Fisher Scientific, USA) using the ClonExpress II one step Cloning Kit (Vazyme, Nanjing, China). The recombinant pFastBac-HTb-Sf Gclc and pFastBac-HTb-Sf Gclm were transformed into competent E. coli DH10 Bac cells (Tsingke Biotechnology Co., Ltd.) to obtain the recombinant bacmid.…”

“…The Sf-900 III medium was removed upon the formation of monolayers (∼24 h), and transfection with dsRNAs was carried out with the Cellfectin II reagent (Gibco) as previously described. 21 Cells were harvested at 72 h for total RNA isolation after treatment.…”

“…The expression vectors pIZT-Sf Cncc and of pIZT-Sf Maf were constructed as previously described. 21 The putative regulatory regions of Sf Gclc (from −1927 to −334 bp) and Sf Gclm (from −1939 to −4 bp) were amplified from genomic DNA by PCR with specific primers listed in Table 1 and subcloned into the linearized pGL3-promoter reporter vector and fused using the Trelief SoSoo Cloning Kit according to the manufacturer's instruction.…”