Gene cloning, protein expression, and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum.

RNA interference (RNAi) is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells. However, silencing efficacy varies greatly among different insect species. Recently, we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection. The disappearance of double-stranded RNA (dsRNA) could be a potential factor that restricts RNAi efficiency. Here, we found that dsRNA can be degraded in midgut fluids, and a dsRNase of A. lucorum (AldsRNase) was identified and characterized. Sequence alignment indicated that its 6 key amino acid residues and the Mg2+ -binding site were similar to those of other insects' dsRNases. The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase. AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle, with peaks at the 4th instar ecdysis in the whole body. The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA. When comparing the substrate specificity of AldsRNase, 3 specific substrates (dsRNA, small interfering RNA, and dsDNA) were all degraded, and the most efficient degradation is dsRNA. Subsequently, immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells. Through cloning and functional study of AldsRNase, the enzyme activity and substrate specificity of the recombinant protein, as well as the subcellular localization of nuclease, the reason for the disappearance of dsRNA was explained, which was useful in improving RNAi efficiency in A. lucorum and related species.

Cloning and expression profiling of voltage-gated sodium channel gene (VGSC) from Spodoptera frugiperda.

Spodoptera frugiperda is a long-distance migratory pest with strong dispersal ability, fast reproduction speed and destructive feeding, so it is difficult to prevent and control. Pyrethroid insecticides are commonly used in pest insects control, And since the voltage-gated sodium channel (VGSC) serves as a major target of pyrethroids, it is important to study this gene for pest control. VGSC is an integral transmembrane protein consisting of approximately 2,000 amino acid residues found in neurons, myocytes, endocrine cells, and ovarian cells and involved in the initiation and propagation of excitable cellular action potentials. In this study, the cDNA sequence of the VGSC was identified from S. frugiperda by rapid amplification of cDNA ends (RACE) which contained an open reading frame of 6,261 bp encoding a protein of 2,086 amino acids. The molecular weight of this protein was predicted to be 236 kDa, and the theoretical isoelectric point was 5.21. A phylogenetic tree constructed based on lepidopteran insects showed that the VGSC of S. frugiperda was most closely relative to that of Spodoptera litura. VGSC is a highly conserved protein with Ion channel conserved structural domains of transmembrane proteins. qPCR showed that the VGSC gene was highly expressed in the epidermis of 2nd instar larvae, and its expression level was low in other tissues, such as the foregut and Malpighian tubules. In addition, VGSC was also detected in the prepupal stage, then gradually increased in abundance after entering the adult stage, peaked at the adult males on the 4th day of pupal stage, and decreased afterwards. The recombinant plasmid of pSumo-mut-VGSC was constructed and induced to express a His tag fused VGSC protein. Polyclonal antibodies were prepared from purified recombinant VGSC protein. The antibody was ELISA-titered, and the western blotting results showed that it specifically recognized VGSC, whether it was recombinant or endogenous protein. These results have laid the foundation for future studies on the physiological function of this gene in the growth and development of S. frugiperda.

Identification of heat shock cognate protein 70 gene (Alhsc70) of Apolygus lucorum and its expression in response to different temperature and pesticide stresses.

Heat shock cognate protein 70 (Hsc70) is a very important stress-resistance protein of insects against environmental stresses. We employed fluorescent real-time quantitative polymerase chain reaction and Western-blot techniques to analyze the transcriptional and translational expression profiles of AlHSC70 under extreme temperature (4°C and 40°C) or 4 pesticide stresses in Apolygus lucorum. The results showed that the expression of AlHSC70 were significantly induced by cyhalothrin or extremely high temperature (40°C) in both transcriptional and translational levels (P < 0.05), while the transcriptional and translational level of AlHSC70 decreased significantly in treatments of chlorpyrifos or extreme cold temperature (4°C) (P < 0.05). Moreover, after Apolygus lucorum treated by imidacloprid or emamectin benzoate, the expression of AlHSC70 was only up-regulated significantly at the transcriptional level (P < 0.05), although obviously up-regulated at the translational level of AlHSC70. Therefore, this study confirmed that the Alhsc70 gene played important roles in response to both temperature and pesticide stresses, especially for cyhalothrin or extremely high temperature (40°C). In addition, the significant polynomial regression correlations between temperature and the Alhsc70 expression level were shown in all the nymph and adult stages (P < 0.01), indicating temperature was an important factor to affect the relative expression of Alhsc70.