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.

Alcohol dehydrogenase 5 (ADH5) is a member of medium‐chain dehydrogenase/reductase family and takes part in cellular formaldehyde and S‐nitrosoglutathione metabolic network. 2‐tridecanone (2‐TD) is a toxic compound in many Solanaceae crops to defend against a variety of herbivory insects. In the broader context of insect development and pest control strategies, this study investigates how a new ADH5 from Helicoverpa armigera (HaADH5) regulates the expression of CYP6B6, a gene involved in molting and metamorphosis, in response to 2‐TD treatment. Cloning of the HaADH5 complementary DNA sequence revealed that its 1002 bp open reading frame encodes 334 amino acids with a predicted molecular weight of 36.5 kD. HaADH5 protein was purified in the Escherichia coli Transetta (pET32a‐HaADH5) strain using a prokaryotic expression system. The ability of HaADH5 protein to interact with the 2‐TD responsive region within the promoter of CYP6B6 was confirmed by an in vitro electrophoretic mobility shift assay and transcription activity validation in yeast. Finally, the expression levels of both HaADH5 and CYP6B6 were found to be significantly decreased in the midgut of 6th instar larvae after 48 h of treatment with 10 mg/g 2‐TD artificial diet. These results indicate that upon 2‐TD treatment of cotton bollworm, HaADH5 regulates the expression of CYP6B6 by interacting with its promoter. As HaADH5 regulation of CYP6B6 expression may contribute to the larval xenobiotic detoxification, molting and metamorphosis, HaADH5 is a candidate target for controlling the growth and development of cotton bollworm.

The cotton bollworm (Helicoverpa armigera, Lepidoptera: Noctuidae) is a globally distributed agricultural pest. When conducting expression analysis of its functional genes, appropriate reference genes should be selected to ensure the reliability of the results. In this study, five algorithms including Delta Ct, GeNorm, Normfinder, BestKeeper, and RefFinder were used to evaluate the expression stability of eleven candidate reference genes under different developmental stages, larval tissues, adult sexes, plant secondary substance stresses, and insecticide treatments in H. armigera. The candidate genes included Actin, Tubulin, EF-1α, RPS3, RPS15, RPL27, RPL32, 28S, GAPDH, SOD, and TRX. The reliability of the recommended reference gene combinations was validated using the growth arrest and DNA-damage-inducible gene 45 (GADD45). The results showed that normalizing relative expression of the target gene with the combination of the two most stable reference genes is recommended. Specifically, the combination of RPS3 + RPL27 is recommended for developmental stage comparisons; RPL32 + RPL27 for larval tissues; RPS3 + RPL27 for adult sex comparisons; GAPDH + RPL32 under tannic acid stress; RPL32 + RPS3 under quercetin stress; RPS15 + RPL32 under 2-tridecanone stress; RPS3 + RPL32 under ZQ-8 stress; RPL27 + TRX following chlorantraniliprole treatment; and RPL27 + RPL32 following indoxacarb treatment. Moreover, larvae exposed to three concentrations of plant secondary substances and to sublethal doses of insecticides exhibited significant upregulation of GADD45: after 4 h of exposure to 1% tannic acid, 0.1% and 1% quercetin, 1% 2-tridecanone, and 0.05% ZQ-8; after 15 h of chlorantraniliprole treatment; and after 24 h of indoxacarb treatment. Thus, GADD45 was overexpressed in response to various plant secondary substances and insecticide treatments, indicating its involvement in the detoxification and metabolism of H. armigera. This study proves to be helpful for selecting reference genes in H. armigera under plant secondary substance and insecticide stress and lays the foundation for further research utilizing GADD45 as a molecular target for pest control.

Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, the optimal conditions for callus induction were identified, and formation of the embryogenic callus was confirmed by histological analysis. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using suitable embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5 + 1.5 mg/L 6-BA + 0.5 mg/L NAA + 30 g/L sucrose + 7 g/L agar (pH = 5.8). In this medium, the induction rate of callus achieved 97.50%, and the rate of embryogenic callus formation was 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82 × 10⁶ protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridisation and transient gene expression in this species.

Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, we identified the optimal conditions for callus induction using a full-factorial experimental design and accurately identified embryogenic callus through paraffin sectioning. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5, supplemented with 1.5 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 7 g/L agar. This medium achieved a callus induction rate of 97.50% and an embryogenic callus induction rate of 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82×10⁶ protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridization and transient gene expression in this species.

Lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) as a crucial cytokine can mediate the inflammatory and immune regulatory responses of an organism. It plays a significant mediatory role in the innate immune system of insects. Firstly, the LITAF family of cotton bollworm (Helicoverpa armigera, Lepidoptera: Noctuidae) was analyzed, and the differentially expressed genes of HaLITAFs were screened from the 2-tridecanone-treated transcriptome. Subsequently, these two differentially expressed HaLITAF genes were cloned and analyzed, and the effect of different larval stages, tissues, and plant secondary metabolites on their gene expression were detected in H. armigera. The cotton bollworm contains 13 LITAF genes, and there are no repetitive sequences among them. Upon 2-tridecanone treatment, only HaLITAF5 and HaLITAF7 were significantly upregulated in the LITAF family of cotton bollworm larvae after 6 h. The HaLITAF5 and HaLITAF7 proteins, respectively, comprised 78 and 113 amino acids, and both contained a CXXC motif, hydrophobic amino acid region and HXCPXCXXXXG motif. Both of them belong to the LITAF analogues (zf-LITAF-like) superfamily. The expressions of HaLITAF5 and HaLITAF7 were both the lowest in fourth-instar larvae and the highest in sixth-instar larvae, and both were abundantly expressed in the midgut of sixth-instar larvae. Following a 28 h treatment with 2-tridecanone, the expression levels of HaLITAF5 and HaLITAF7 peaked. HaLITAF7 exhibited the highest expression level after a 4 h exposure to tannic acid, whereas HaLITAF5 reached its peak after 28 h of tannic acid treatment. The maximum expression levels of HaLITAF5 and HaLITAF7 were observed after 28 h and 4 h of quercetin treatment, respectively. Subsequent to ZQ-8 treatment, HaLITAF5 expression peaked at 28 h, while HaLITAF7 expression peaked at 8 h. Both HaLITAF5 and HaLITAF7 were overexpressed after different kinds of plant secondary metabolite stresses, and thereby, they may be regulated in the expression of genes related to downstream detoxification metabolic processes. This provides a theoretical foundation for further studies on the functional mechanism of the LITAF gene in H. armigera.