RNA interference (RNAi) induced through double-stranded RNA (dsRNA) has been used widely to study gene function in insects. In this paper we demonstrate the efficacy of RNAi in the cotton bollworm, Helicoverpa armigera. Using CYP6B6 as the target gene, which is expressed in the fat baby and midgut of the lepidopteran pest H. armigera, we constructed the vector which expressed dsRNA of CYP6B6. Northern blot analysis showed that dsRNA expressed in the Escherichia coli (HT115) was target gene. The results also showed that the gene expression level and protein expression level of H. armigera larvae fed with dsRNA expressed by E. coli were significantly lower than those of all controls, but the gene expression level was more obvious than that at the protein level; significant lethality differences were also found between HT115 bacteria containing L4440-dsC1 treatment and HT115 bacteria containing L4440 vector or CK (ddH₂O) in instar larvae on 4 day when continuous feeding, 32.45% mortality was recorded in the group of feeding HT115 bacteria containing L4440-dsC1 on 10 day. Our results suggest that the RNAi pathway can be exploited to control insect pests.

Insects have developed a complex network of enzymatic antioxidant systems for handling reactive oxygen species (ROS) generated during stress. Superoxide dismutases (SODs) play a determinant role in balancing ROS in insect. However, studies devoted to SODs functions in insects under cold stress are limited. In the present study, we attempted to identify and characterize a mitochondrial manganese SOD (mMn-SOD) from the desert beetle Micordera punctipennis (denoted as MpmMn-SOD) and explore its protective effects on bacteria cells under cold stress. MpmMn-SOD is composed of 202 amino acids with conserved domains required for metal ions binding and enzyme activity. RT-qPCR experiments revealed that the expression of MpmMn-SOD was ubiquitous but tissue-specific and was induced by cold stress. An E. coli (BL21) system was applied to study the function of MpmMn-SOD. The MpmMn-SOD gene was cloned into the prokaryotic expression vector pET-32a to generate a recombinant plasmid pET-32a(MpmMn-SOD). After transformation of the plasmid into E. coli BL21, the fusion protein Trx-His-MpmMn-SOD was overexpressed and identified by SDS-PAGE and Western blotting. Antioxidant activity assay showed that the death zones of the transformed bacteria BL21 (pET32a-mMn-SOD) were smaller in diameter than the control bacteria BL21 (pET32a). Survival curves under −4 °C showed that BL21 (pET32a-mMn-SOD) had significant enhanced cold resistance compared to BL21 (pET32a). Its SOD activity under −4 °C had a significant negative correlation (r = − 0.995) with superoxide anion O₂^•− content. Accordingly, under cold stress BL21 (pET32a-mMn-SOD) had lower electric conductivity and malondialdehyde (MDA) content than BL21 (pET32a). Taken together, our results showed that cold stress stimulated the expression of MpmMn-SOD in M. punctipennis. The E. coli cells that overexpress MpmMn-SOD increase their resistance to cold stress by scavenging ROS, and mitigate potential cell damage caused by ROS under cold conditions.

Exogenous double-stranded RNA (dsRNA) is a potent initiator of gene silencing in diverse organisms. In the present study, we used genetically engineered bacterial strain HT115 to express dsRNA corresponding to CYP6B6 of Helicoverpa armigera Hübner, which is an economical way to produce large quantities of dsRNA. After we investigated the effect of pH, saline solution, hemolymph plasma, and time on the stability of the dsRNA from the bacteria in vitro, we tested the effect of the exogenous dsRNA on the transcription and translation of larval CYP6B6 , larval growth, development, and the insecticide tolerance of the cotton bollworm after ingestion of the engineered bacteria. The stability analysis showed that the dsRNA from the engineered bacteria remained unchanged for 24 h in pH 7.0 KH2PO4/K2HPO4 buffer at room temperature. Both the qPCR and immunohistochemistry results showed that obvious decrease in CYP6B6 decreased compared with the corresponding controls, and the larval growth and development were significantly retarded, the rate of pupation declined, and insecticide tolerance decreased. Thus, the data show that CYP6B6 silencing can disturb the growth and development of cotton bollworm and also reduce its insecticide tolerance. These results provide a good foundation for applying RNAi strategies targeting insect P450 genes by transforming cotton or other plants for protection against the cotton bollworm.

Eukaryote transcription is controlled by regulatory DNA sequences and transcription factors, so transcriptional control of gene plays a pivotal role in gene expression. In this study, we identified the region of theCYP6B6gene promoter ofHelicoverpa armigerawhich responds to the plant secondary toxicant 2-tridecanone. Transient transfection assay results from five of stepwise deletion fragments linked to the luciferase reporter gene revealed that the promoter activity of eachCYP6B6fragment was significantly higher than that of their basal activity after the Sf9 cells were treated with 2-tridecanone. Among all, the fragment spanning −373 to +405 bp of theCYP6B6promoter showed an obviously 2-tridecanone inducibility (P<0.0001), which might have the 2-tridecanone responsive element based on promoter activity. Electrophoretic mobility shift assays revealed that the nuclear protein extracted from midgut of the 6th instar larva ofH. armigera, reared on 10 mg 2-tridecanone per gram artificial diet for 48 h, could specifically bind to the active region from −373 to 21 bp of theCYP6B6promoter. The combination feature also appeared when using a shorter fragment from −292 to −154 bp of theCYP6B6promoter. Taken together, we found a 2-tridecanone core responsive region between −292 and −154 bp of theCYP6B6promoter. This may lead us to a better understanding of transcriptional mechanism of P450 gene and provide very useful information for the pest control.