Broad complex Z2 regulates the group Ⅳ chitinase gene HaCHT4 in the midgut of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Broad complex Z2 regulates the group Ⅳ chitinase gene HaCHT4 in the midgut of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Broad complex Z2 regulates the group Ⅳ chitinase gene HaCHT4 in the midgut of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) Deqin Hu, Yuan Li, Jingang Xie, Hongsheng Pan, Xiaoning Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8610350/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Apr, 2026 Read the published version in BMC Genomics → Version 1 posted 13 You are reading this latest preprint version Abstract Background Chitinases and ecdysone are both crucial for insect growth and development, yet the regulatory interplay between them remains poorly understood. Our previous research demonstrated that the chitinase gene HaCHT4 critically regulates the content of chitin and peritrophic membrane’s (PM) structure in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Results Here, a 1186 bp promoter region of HaCHT4 was cloned to investigate its regulation mechanism. Bioinformatics analysis predicted the presence of several 20-hydroxyecdysone (20E) related cis-regulatory elements (CREs) within this promoter, including Broad-Complex Zinc-Finger isoforms (BRCs), GATA, NF-AT1, and POU1F1. Notably, genome-wide identification and characterization revealed that the H. armigera BRC gene exhibits the highest similarity with that of Bombyx mori . Molecular docking and EMSA demonstrated the specific binding between HaBRC Z2 and HaCHT4 . Expression analysis showed concomitant upregulation of HaBRC Z2 and HaCHT4 during the late (4th to 5th ) instar stages, and were also strongly induced by 20E. In addition, RNA interference (RNAi) experiments further supported this regulatory relationship, a substantial decrease in the transcript levels of both HaBRC Z2 and HaCHT4 were observed after HaBRC Z2 silencing for 24, 48, 72, and 96 h. The knockdown of HaBRC Z2 not only stunted larval growth, evidenced by reduced body length and weight, but also confirmed its role as an activator of HaCHT4 transcription during the larval transition. Conclusion These findings reveal a critical regulatory relationship between chitinase and ecdysone, underscoring the significance of HaBRC Z2 as promising targets for informing future pest control research. Helicoverpa armigera Broad complex gene Molecular mechanism RNAi growth and development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), a polyphagous pest, inflicts substantial annual economic losses on cotton by primarily attacking its buds and bolls [ 1 , 2 ]. The effective management of this pest remains a challenge due to its wide host range, polyphagous nature, high adaptability, and widespread pesticide resistance [ 3 ]. Although chemical pesticides have been widely used, they bring unintended consequences like environmental contamination, human health threats, and devastation of beneficial non-target species [ 4 , 5 ]. Another key strategy for H. armigera management is the cultivation of Bt cotton, though the evolution of resistance in H. armigera diminish the benefits of Bt cotton [ 6 ]. Therefore, exploring effective and durable eco-friendly control strategies and molecular targets is crucial for delaying resistance evolution and advancing sustainable pest management. Previous work from our team demonstrated the critical role of the HaCHT4 in modulating chlorantraniliprole sensitivity in H. armigera , achieved through its regulation of chitin and architecture of the peritrophic membrane (PM) [ 7 ]. HaCHT4 , which mediates chitin degradation, represents a candidate target for biorational insecticide discovery. Its functional relevance in insects, combined with the absence of chitin systems in vertebrates, offers a strategic avenue for developing pest management solutions with enhanced environmental compatibility. Chitin, a fundamental structural polymer in the PM, epidermis, intestine, and trachea, is precisely regulated by molecular interactions [ 8 – 10 ]. Among these regulatory factors, chitinase and ecdysone hormone 20-hydroxyecdysone (20E) play central roles [ 11 , 12 ]. In insects, chitinases catalyze the degradation of high molecular weight chitin into low molecular weight oligosaccharides [ 13 , 14 ], thus fulfilling a critical function in insect growth and developmental processes [ 15 , 16 ]. The relative expression level of the chitinase gene has been found to increase significantly before molting (late stages) and return to normal or lower levels after molting (early stages) in insects, fluctuating with the physiological state similar to the changing trend of 20E titer [ 17 – 19 ]. The transcription levels of chitinase genes in Manduca sexta [ 20 ], Locusta migratoria [ 21 ] and Acyrthosiphon pisum [ 15 ] were induced by 20E. In B. mori , metamorphosis is marked by 20E upregulating chitinase 5 expression via the transcription factor BmBRC-Z4 [ 22 ]. The expression level of HaCHT4 was lower in the early stages of 4th (4E) to 6th (6E) instar larvae, but significantly elevated in the late stages (4L, 6L) in H. armigera [ 7 ]. This implicates a specific 20E pathway in regulating chitinases during molting, although the precise mechanistic details remain to be fully elucidated. The functional diversity of the 20E responsive Broad-Complex (BRC) transcription factor stems from its alternative splicing, which generates isoforms with distinct C-terminal zinc finger domains [ 23 , 24 ]. This gene activated directly by the EcR-USP dimer, possesses a conserved BTB domain alongside its variable DNA binding domain [ 25 , 26 ]. Functional studies reveal isoform specific roles across insects [ 4 , 27 , 28 ]. In D. melanogaster , BRC Z1 and BRC Z2 have been shown to regulate appendage elongation (legs/wings) and eversion, whereas BRC Z3 is dedicated to the process of disc fusion [ 29 ]. In B. mori , BRC Z2 not only affects yolk protein synthesis and oocyte formation, but also regulates cuticle protein gene [ 30 – 32 ]. The function of BRC Z3 remains poorly characterized, and BRC Z4 mainly regulates genes encoding insect cuticular proteins and those genes related to chitin metabolism in B. mori [ 33 ]. Furthermore, RNAi-mediated silencing of LdBRC in Lymantria dispar resulted in severely impaired chitinase gene expression, causing developmental defects and increased mortality, underscoring its essential role [ 34 ]. We proved that the midgut specific chitinase gene HaCHT4 is involved in chitin degradation of H. armigera [ 7 ]. To elucidate its transcriptional regulation, this study has cloned and analyzed CREs within the HaCHT4 promoter region. The transcriptional factor HaBRC Z2 was identified targeting HaCHT4 in H. armigera , and proposing a preliminary molecular mechanism of 20E - HaBRC Z2 - HaCHT4 regulation axis by EMSA, RT-qPCR and RNAi technologies. These findings delineate a preliminary 20E - HaBRC Z2 - HaCHT4 regulatory axis, which enhances our understanding of the interplay between chitinases and 20E, and for devising novel pest management strategies aimed at chitin metabolism. Materials and methods Insect rearing A laboratory-reared strain of H. armigera , with no history of any insecticide or toxins exposure, was maintained under controlled conditions of 26 ± 2°C, 65 ± 5% relative humidity, and a 16:8 (L:D) photoperiod. The specific rearing details of H. armigera larvae were referred to the work of Liang et al. [ 35 ]. Cloning and characterization of 5’ UTR region of HaCHT4 Genomic DNA of H. armigera 4th instar larvae were purified with the Easy Pure Genomic Kit (TransGen Biotech, Beijing, China). The promoter region of HaCHT4 was amplified from this DNA using the Genome Walking Kit (TaKaRa, Tokyo, Japan). The cloned fragment was then inserted into pEASY-T1 cloning vector. Subsequently, the positive monoclonal was sequenced by Sangon Biotechnology (Shanghai, China). Putative CREs of HaCHT4 were performed on JASPAR ( https://jaspar.elixir.no/ ). The complete primer sequences and nested PCR reaction procedures are shown in supplementary Table S1 and S2. Identification and bioinformatics analysis of the BTB gene family We obtained the reference genome sequence of H. armigera (GCF_030705265.1) from the National Center for Biotechnology Information (NCBI) database, and employed two methods to identify potential members of the HaBTBs. First, we performed BLASTP homology alignment against the local H. armigera database using the H. armigera HaBRC Z2 protein sequence as the query. Second, a hidden markov model (HMM) for the BTB domain (PF00651) was employed for a domain-based search against the H. armigera genome using TBtools. This profile was then utilized for the identification of BTB genes from the H. armigera genomic database with the assistance of TBtools [ 36 ]. The candidate sequences obtained from both methods were validated for domain structure using Pfam, and only those sequences containing the conserved BTB domain were retained as HaBTB candidates. Protein parameters were derived from ExPASy ( https://www.expasy.org/ ). The location on chromosomal of the HaBTB genes in H. armigera were obtained from the NCBI database, along with the corresponding genome annotation files. MapChart software was used to map the chromosome position of HaBTB gene. The predicted BTB protein sequences from H. armigera , D. melanogaster , Tribolium castaneum , and B. mori aligned using ClustalW with default parameters. Phylogenetic tree was constructed in MEGA 11.0 via the neighbor-joining method, which was refined using the iTOL online tool. All Figures are assembled and finalized in Adobe Illustrator 2022. Analysis of the molecular interaction between HaCHT4 and HaBRC Z2 To explore the interaction between HaCHT4 and HaBRC Z2, the amino acid sequences of HaBRC Z2 were submitted to AlphaFold 334 ( https://alphafold.com/ ) to predict protein models. Molecular docking between the HaBRC Z2 protein and the cis-regulatory element of HaCHT4 was carried out using AutoDockFR35 software. The probe for EMSA was synthesized according to the fragment (-313 ~ -5nt, 308bp) of the potential HaBRC Z2 core binding site by DIG-High Primer DNA Labeling and Detection Starter kit II (Roche, Basel, Switzerland). The His-HaBRC Z2 fusion protein had been purified in a previous experiment [ 37 ]. For the DNA-binding assay, containing 1 µL (or 2 µL) of His-HaBRC Z2 fusion protein, 1 µL of labeled probe (500 pg/µL), 2 µL of binding buffer and supplement ddH 2 O to 10 µL, and incubated at 25°C for 30 min. Samples were electrophoresed on 5% polyacrylamide gel. After protein transferring to Nylon membrane, UV crosslinking was performed for 15 min. The membrane was treated sequentially as follows: incubation in maleic acid solution for 5 min at 25 ℃, blocking with blocking buffer for 30 min, and incubation with streptavidin-HRP conjugate for 30 min at 25°C [ 22 ]. Signal detection was performed using the Clinx Science imaging system. The expression profiles of HaBRC Z2 and HaCHT4 in H. armigera To characterize the transcript levels of HaBRC Z2 and HaCHT4 in H. armigera , midgut tissue samples were collected at both the early and late stages of the 4th and 5th instar larvae for RNA extraction. For each developmental stage, three biological replicates were prepared, with each replicate comprising a pool of midgut tissues from ten larvae. The cDNA synthesis and RT-qPCR were conducted as previously described [ 7 ]. To investigate whether 20E regulates the expression of HaBRC Z2 and HaCHT4 , 4th instar larvae were treated with 5 µL of 20E (200 ng/µL; supplied by Sangon Biotech, Shanghai, China). Midgut tissues were dissected 24 h post treatment to analyze the expression levels of the target genes. The experiment included 3 biological replicates, with each replicate comprising 30 larvae. The 2 −ΔΔCt method was used to quantified the relative expression levels of HaBRC Z2 and HaCHT4 . 3 biological and 2 technical replicates were used for all reactions. Synthesis of dsRNA and RNAi assays To investigate the function of the HaBRC Z2 gene, double-stranded RNA (dsRNA) targeting its open reading frame (ORF) was designed, green fluorescent protein (GFP) serving as a control. Subsequently, dsRNAs were synthesized in vitro employing the T7 RiboMAX™ Express RNAi System (Promega, Madison, USA). For RNAi bioassay, the artificial diet was meticulously divided into rectangular pieces measuring 4 mm×4 mm×3 mm, which were then allocated to each group. 2nd instar larvae were deprived of food for 4 h. Two groups received 10 µL of ds HaBRC Z2 solution at concentrations of 250 ng/µL and 500 ng/µL, respectively. The dsRNA-treated diet was refreshed every 48 h intervals. Control groups were administered an equivalent volume of either ddH₂O or ds GFP solution. Each treatment was applied to 35 larvae, with three independent biological replicates established. To evaluate the silencing efficiency of HaBRC Z2 , 5 larvae from each group were sampled at 12, 24, 48, 72, and 96 h dsRNAs post-feeding. To further assess the impact of ds HaBRC Z2 on larval phenotypic traits, a separate cohort of 2nd instar larvae were fed a diet containing 10 µL of ds HaBRC Z2 at 500 ng/µL. Control groups were treated with ddH₂O or ds GFP as described above. Larval body length and weight were recorded at 12, 24, 36, 48, 72, and 96 h after treatment initiation. This experiment included 3 biological replicates, with 35 larvae in each group per replicate. Statistical analysis All date from 3 experimental replicates were represented by mean ± standard error (SE). For gene expression analysis across the 4E to 5L instars, differences between early and late stages were assessed using Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001). For post-RNAi data, one-way analysis of variance (ANOVA) coupled with Tukey’s HSD test in SPSS 22.0 was used to evaluate differences among treatment groups at identical time points, where different letters mark significant differences ( P < 0.05). Results Characterization of the 5’ UTR from the HaCHT4 To investigate the regulation of HaCHT4 , a 1186 bp fragment of the HaCHT4 promoter was amplified through genome walking technology (Fig. 1 A, B). Sequence analysis using the JASPAR online platform revealed the presence of Broad-Complex (BRC) isoforms Z1-Z4 elements related to 20E, and other elements including GATA, NF-AT1, and POU1F1 (Fig. 1 C). The BRC gene is an immediate early gene of 20E and belongs to the BTB gene family. In order to fully understand the BRC gene, we identified and characterized the BTB gene family in the H. armigera genome. The results showed that 85 BTB genes (Table S3) were identified from the H. armigera genome, and they were renamed according to their functional domain and chromosomal locations (Fig. S1 ). A collinearity plot was constructed for H. armigera , B. mori , T. castaneum , and D. melanogaster. This analysis identified 48 orthologs between H. armigera and B. mori , 2 orthologs between H. armigera and T. castaneum , and no orthologs between H. armigera and D. melanogaster. These findings suggest that these genes are relatively conserved during the evolution of lepidoptera insects and play an indispensable and important role (Fig. 2). Specifically, the BRC gene possesses both a BTB domain and a C 2 H 2 zinc finger domain, and is designated HaBTBZF1 based on its chromosomal location. Phylogenetic analysis revealed that HaBTBZF1 clusters with BmBTB ZF1 of B. mori , indicating that they are the closest orthologs (Fig. 3 ). This gene features eight exons, and can generate distinct transcripts through alternative splicing. Hence, previously reported different BRC isoforms from other insect species were downloaded. In the phylogenetic analysis, each of the BRC isoforms fell within its clade, which is consistent with the previously observed evolutionary conservation of these isoforms (Fig. 4). Figure 2. Collinearity analysis of BTB genes were investigated among H. armigera , T. castaneum , B. mori and D. melanogaster. The red lines highlighted collinear BTB gene pairs, while the gray lines in the background indicated all collinear blocks. are distinguished by branches in different colors for clarity. Figure 4. A phylogenetic tree of different BRC isforms from other species. MEGA 11.0 software was used to construct a phylogenetic tree with 1000 bootstrap replications. HaBRC Z2 protein binds to the promoter of HaCHT4 To identify the BRC isoform that may regulate HaCHT4 , the binding energy between the regulatory element and HaCHT4 was predicted through molecular docking. The results indicated a strong binding affinity between HaCHT4 and the transcription factor HaBRC Z2. The binding sites were characterized by the presence of AT-rich motifs, specifically “AACTAATT”, with a binding free energy (ΔG) of -12 kcal/mol (Fig. 5A). Moreover, a DNA fragment encompassing the HaBRC Z2 binding motif within the HaCHT4 promoter was amplified (Fig. 5B). The probe labeling efficiency was validated, showing high sensitivity 10 pg/µL (Fig. 5C). EMSA demonstrated that a shift in the band intensity upon the addition of His-HaBRC Z2 in comparison to the probe alone and the His-PET32a groups (Fig. 5D). These results support the involvement of the HaBRC Z2 CRE in regulating HaCHT4 expression. Figure 5. Molecular docking prediction and EMSA analysis of the binding of HaBR-C Z2 to the promoter region of the HaCHT4 . A) Molecular docking of HaBRC Z2 binding to the key CREs of the HaCHT4 promoter. B) The core sequence of HaBRC Z2 binding to the HaCHT4 promoter. C) Detection of probe synthesis and labeling efficiency, -, negative control; 1, probe synthesis. D) EMSA analysis of the binding of HaBRC Z2 to the HaCHT4 promoter CREs. Expression profiles of HaBRC Z2 and HaCHT4 in H. armigera We profiled the transcript levels of HaBRC Z2 and HaCHT4 across the 4th to 5th instar larval stages via RT-qPCR. Both genes exhibited a marked upregulation in the late phases of each instar compared to their early phases ( P < 0.01). Specifically, HaBRC Z2 expression increased by approximately 2.56-fold ( t = 3.497, df = 10, P = 0.0058) and 2.08-fold ( t = 4.411, df = 10, P = 0.0013) in the 4L and 5L instars, respectively (Fig. 6A), while HaCHT4 exhibited more pronounced inductions of 9.33-fold ( t = 5.299, df = 10, P < 0.001) and 2.94-fold ( t = 3.078, df = 10, P = 0.0117) at the same stages, respectively (Fig. 6B). Furthermore, significantly induced the mRNA accumulation of both HaBRC Z2 (Fig. 6C) ( t = 3.628, df = 10, P = 0.0046) and HaCHT4 ( t = 4.499, df = 10, P = 0.0011) were detected after 1.0 ug/µL 20E treatment for 24h (Fig. 6D). The coordinated expression patterns suggest that HaBRC Z2 may act as anupstream regulator of HaCHT4 . Figure 6. Relative expression profiles of HaBRC Z2 and HaCHT4 in the midgut of H. armigera . A, B) Expression profile of HaBRC Z2 and HaCHT4 during the 4th to 5th instar larvae, respectively. 4E and 5E, the early stage of the 4th and 5th instar larvae, respectively; 4L and 5L, the late stage of the 4th and 5th instar larvae, respectively. C, D) Expression profiles of HaBRC Z2 and HaCHT4 following 1.0 ug/µL 20-hydroxyecdysone (20E) treatment for 24 h, respectively. Data are presented as mean ± SE (3 biological and 2 technical replicates). Significant differences between the early and late stages within the same instar or after 20E treatment were determined by Student’s t -test (* P < 0.05, * * P < 0.01, * * * P < 0.001). Functional analysis of the HaBRC Z2 using RNAi To investigate the putative regulation of HaCHT4 by HaBRC Z2 , we performed RNAi-mediated silencing of HaBRC Z2 . While transcript levels of both genes remained unchanged at 12 h post-treatment with ds HaBRC Z2 under 250 ( F = 0.236 df = 2, P = 0.793) or 500 ng/µL ( F = 1.736, df = 2, P = 0.210), a significant suppression was observed from 24 to 96 h. The expression level of HaBRC Z2 decreased to 23.50% ( F = 131.76, df = 2, P < 0.001) of that in the ds GFP group after treatment with 250 ng/µL ds HaBRC Z2 for 24 h, and the expression of HaCHT4 decreased to 18.45% ( F = 52.30, df = 2, P < 0.001). Under 500 ng/µL for 24h treatment, the expression levels of HaBRC Z2 and HaCHT4 were reduced to 56.52% ( F = 14.76, df = 2, P < 0.001) and 40.11% ( F = 33.59, df = 2, P = 0.008) of the control group, respectively. By 96 h, the expression of Ha BRC Z2 in the 250 ng/µL treatment group had decreased to 31.70% of the ds GFP control group ( F = 55.499, df = 2, P < 0.001), and HaCHT4 expression decreased to 11.45% ( F = 67.05, df = 2, P < 0.001), the mRNA expression of HaBRC Z2 was reduced to 26.58% ( F = 54.393, df = 2, P < 0.001), and HaCHT4 expression decreased to 28.24% ( F = 60.638, df = 2, P = 0.002) after treatment with 500 ng/µL ds HaBRC Z2 (Fig. 7 ). These results support a conclusion that HaBRC Z2 acts as an upstream transcriptional activator of HaCHT4 , likely through binding to the CRE element in its promoter. Effects of HaBRC Z2 silencing on the growth and development of H. armigera We also assessed the impact of ds HaBRC Z2 on larval growth and development. The results showed that the body length and weight of H.armigera were significantly inhibited after treatment with 500 ng/µL of ds HaBRC Z2 for 48 h. The average body length after treatment with ds HaBRC Z2 , ds GFP and CK at 48 h was 7.73 ± 0.23mm, 7.60 ± 0.20mm, 6.53 ± 0.15mm; the body weight was 33.67 ± 1.15mg, 30.33 ± 1.08mg, 21.34 ± 0.58mg, respectively. With the extension of treatment time, the body weight was inhibited, and the weight was only 53.9% of the ds GFP group ( F = 58.654, d f = 2, P < 0.001) (Fig. 8 ). This indicates that HaBRC Z2 knockdown severely impaired larval development. Discussion The midgut is a primary organ for food digestion, nutrient absorption, and energy metabolism in insects, and its structure undergoes significant remodeling throughout insect development [ 38 ]. Within it lies the PM, a non-cellular, semipermeable barrier synthesized from chitin, proteins, and glycans [ 39 , 40 ]. This structure serves as the midgut’s primary defense, shielding it from mechanical abrasion and preventing invasion by pathogens and macromolecules [ 41 , 42 ]. However, this protective barrier also impedes the uptake of pesticides and entomopathogenic microorganisms in pest control. Consequently, compromising PM integrity to facilitate insecticide uptake has become a prominent research objective [ 43 ]. Given that chitin is not present in mammals or plants [ 11 ], the specific regulation of chitin synthesis and degradation pathways is developing into a new strategy for pest control. In Ostrinia furnacalis , group II chitinase gene OfChtII could degrade of the cuticular chitin and affecting the transformation from larva to pupa [ 44 ]. In Aedes albopictus , the structure of the midgut PM was destroyed or even absent after silencing CHS-2 [ 45 ]. Our previous research in H. armigera demonstrated that upregulation HaCHT4 enhances chitin degradation, leading to increased PM porosity. This structural compromise severely impairs the gut barrier, which in turn heightens insecticide susceptibility [ 7 ]. To amplify this effect, we sought to identify which transcription factor responsible for modulating the pathway. Nevertheless, the upstream signaling cascades and transcriptional regulators controlling this chitinase gene have not yet been fully elucidated. To elucidate the upstream regulatory mechanism of HaCHT4 , we cloned and analyzed its promoter region. Multiple 20E-associated cis-regulating elements were identified, including binding sites for BRC Z1-Z4 isoforms, GATA, NF-AT1, and POU1F1. The BRC gene, a 20E-responsive transcription factor within the BTB family, is a key regulator of insect growth and development [ 30 ]. Its function is exemplified in B. mori , where BRC transmits the 20E signal by directly associating with the promoter regions of genes critical for larval development [ 46 , 47 ]. Meanwhile, in Spodoptera frugiperda , the isoform BRC Z4 appears to modulate gene expression by interacting with the 5’ UTR of targets such as Ecdysone receptor and SfCht5 , potentially leading to the upregulation of these and other ecdysteroid biosynthesis genes in berberine-fed insects [ 48 ]. Building on the genomic insight that identified numerous conserved BTB homologs between H. armigera and B. mori , our functional investigation demonstrated that 20E triggers HaCHT4 expression through the transcription factor HaBRC Z2 in H. armigera . Multiple experimental results converge to support this central finding. First, molecular docking indicated that a strong binding affinity of HaBRC Z2 for the core cis-element “AACTAATT”. This sequence shares homology with the BRC Z2 consensus elements “CTA” and “TAG” characterized in B. mori [ 49 ], which preferentially bind to AT-rich regions. Subsequently, EMSA confirmed the direct binding of His-HaBRC Z2 to labeled probes containing this motif. Third, transcript levels of HaBRC Z2 and HaCHT4 peaked at the 4L and 5L larval instars compared with the early stages. Similar research on B. mori [ 22 ] and Chilo suppressalis [ 50 ] showed that the peak chitinase expression appeared in the late larval and prepupal stages. Besides, a RT-qPCR assay showed that HaBRC Z2 and HaCHT4 were induced by 20E, suggesting 20E triggers HaBRC Z2 to regulate HaCHT4 . Taken together, we propose the following regulatory pathway: 20E signaling orchestrates HaCHT4 expression by first inducing HaBRC Z2 , which subsequently binds to and transactivates the HaCHT4 promoter during molting. However, definitive evidence for a direct interaction awaits further experimental validation. RNA interference (RNAi) has become an indispensable tool for elucidating gene function and formulating targeted pest control strategies in entomological research because of its high specificity, simple technology and ability to inhibit transcription. [ 51 , 52 ]. For instance, silencing CsBRC genes in C. suppressalis increased pupal instar number and significantly prolonged the developmental duration [ 50 ]. Similarly, knockdown of LdBRC in L. dispar inhibited chitinase expression in midgut and integument tissues, causing molting arrest and 67% larval mortality [ 34 ]. In addition, our research team has employed RNAi to investigate the functions of genes associated with growth, development, and insecticide resistance in Aphis gossypii , H. armigera , and Phthorimaea absoluta , which has facilitated the identification of potential targets for the biocontrol of these pests [ 45 , 53 – 55 ]. RNAi-mediated silencing of HaBRC Z2 significantly suppressed its transcript levels, and the optimal knockdown efficacy occurred at 24 h and 96 h. The transcript levels of HaBRC Z2 and HaCHT4 reached the lowest at 24 h after treatment with 250 ng/µL dsRNA. However, both genes showed a transient upregulation at 48 h, which likely reflects the developmental stage specific fluctuations in ecdysteroid titers during the larval transition. All above results establish HaBRC Z2 as a critical 20E response gene that positively regulates HaCHT4 to orchestrate molting. Conclusions In summary, our data provide evidence that 20E can induce the expression of HaBRC Z2 , and then binds to the HaCHT4 promoter to activate its transcription. Combined with previous findings, we proposed that disrupting the normal expression of HaBRC Z2 would exert a broader cascade amplification regulatory effect on HaCHT4 , ultimately compromising PM structure and barrier integrity during critical developmental stages like molting. This would increase midgut permeability to conventional insecticides, Bt toxins, pathogenic microorganisms, etc. Therefore, targeting HaBRC Z2 may act as a promising sensitizing strategy. It can potentially enhance the efficacy and reduce the required dosage by destroying the physical barrier of insects to facilitate the arrival of exogenous substances at their target sites. This study not only provides a reference for the analysis of the basic mechanism, but more importantly, it suggests that HaBRC Z2 may serve as a potential molecular target in pest control. Declarations Acknowledgements Not applicable. Authorship contribution Deqin Hu: writing the original draft, experimental design, performed experiments; Yuan Li: editing and revising the manuscript. Jingang Xie: data visualization. Hongsheng Pan: supervision, project management. Xiaoning Liu: editing and revising the manuscript, project management, funding obtained. All authors have read and agreed to the published version of the manuscript. Data availability All of the datasets supporting the results of this article are provided within the manuscript or supplementary information files. The whole genome data was used in this study have been deposited in the NCBI database under accession numbers GCA_030705265.1. Funding This work was supported by the National Natural Science Foundation of China (31972279) and Excellent Doctoral Candidate Innovative Project of Xinjiang University in 2024 (XJU2024BS071). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Zhao C, Wang L, Zhang K, Zhu X, Li D, Ji J, Luo J, Cui J. Variation of Helicoverpa armigera symbionts across developmental stages and geographic locations. Front Microbiol. 2023;14:1251627. Haile F, Nowatzki T, Storer N. Overview of pest status, potential risk, and management considerations of Helicoverpa armigera (Lepidoptera: Noctuidae) for U.S. soybean production. Integr J Pest Manage 2021, 12(1). Riaz S, Johnson J, Ahmad M, Fitt G, Naiker M. A review on biological interactions and management of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J Appl Entomol 2021, 145. Ahmad KF, Engel CK, Privé GG. Crystal structure of the BTB domain from PLZF. Proc Natl Acad Sci U S A. 1998;95(21):12123–8. Rezende-Teixeira P, Dusi RG, Jimenez PC, Espindola LS, Costa-Lotufo LV. What can we learn from commercial insecticides? Efficacy, toxicity, environmental impacts, and future developments. Environ Pollut. 2022;300:118983. Tabashnik BE, Fabrick JA, Carrière Y. Global patterns of insect resistance to transgenic Bt crops: the first 25 years. J Econ Entomol. 2023;116(2):297–309. Hu DQ, Luo SH, Abudunasier M, Cai XH, Feng MM, Liu XN, Wang DM. The effect of group IV chitinase, HaCHT4 , on the chitin content of the peritrophic matrix (PM) during larval growth and development of Helicoverpa armigera . Pest Manag Sci. 2022;78(5):1815–23. Khokhani D, Carrera Carriel C, Vayla S, Irving TB, Stonoha-Arther C, Keller NP, Ané JM. Deciphering the chitin code in plant symbiosis, defense, and microbial networks. Annu Rev Microbiol. 2021;75:583–607. Mun S, Noh MY, Geisbrecht ER, Kramer KJ, Muthukrishnan S, Arakane Y. Chitin deacetylases are necessary for insect femur muscle attachment and mobility. Proc Natl Acad Sci U S A. 2022;119(24):e2120853119. Khan A, Smagghe G, Li S, Shakeel M, Yang G, Ahmed N. Insect metamorphosis and chitin metabolism under miRNA regulation: a review with current advances. Pest Manag Sci. 2025;81(7):3437–51. Chen DD, Wang ZB, Wang LX, Zhao P, Yun CH, Bai L. Structure, catalysis, chitin transport, and selective inhibition of chitin synthase. Nat Commun. 2023;14(1):4776. Zhang C, He L, Ding B, Yang H. Identification and functional characterization of the chitinase and chitinase-like gene family in Myzus persicae (Sulzer) during molting. Pest Manag Sci. 2025;81(1):327–39. Dong W, Gao YH, Zhang XB, Moussian B, Zhang JZ. Chitinase 10 controls chitin amounts and organization in the wing cuticle of Drosophila . Insect Sci. 2020;27(6):1198–207. Chen W, Yang Q. Development of novel pesticides targeting insect chitinases: a minireview and perspective. J Agric Food Chem. 2020;68(16):4559–65. Li C, Ul Haq I, Khurshid A, Tao Y, Quandahor P, Zhou JJ, Liu CZ. Effects of abiotic stresses on the expression of chitinase-like genes in Acyrthosiphon pisum . Front Physiol. 2022;13:1024136. Wan XS, Shi MR, Xu J, Liu JH, Ye H. Interference efficiency and effects of bacterium-mediated RNAi in the fall armyworm (Lepidoptera: Noctuidae). J Insect Sci 2021, 21(5). Khajuria C, Buschman LL, Chen MS, Muthukrishnan S, Zhu KY. A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae. Insect Biochem Mol Biol. 2010;40(8):621–9. Zeng QH, Gong MF, Yang H, Chen NN, Lei Q, Jin DC. Effect of four chitinase genes on the female fecundity in Sogatella furcifera (Horváth). Pest Manag Sci. 2024;80(4):1912–23. Wu ZZ, Zhang WY, Lin YZ, Li DQ, Shu BS, Lin JT. Genome-wide identification, characterization and functional analysis of the chitianse and chitinase-like gene family in Diaphorina citri . Pest Manag Sci. 2022;78(4):1740–8. Kramer KJ, Muthukrishnan S. Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochem Mol Biol. 1997;27(11):887–900. Li D, Zhang J, Wang Y, Liu X, Ma E, Sun Y, Li S, Zhu KY, Zhang J. Two chitinase 5 genes from Locusta migratoria : molecular characteristics and functional differentiation. Insect Biochem Mol Biol. 2015;58:46–54. Zhang X, Zheng S. 20-hydroxyecdysone enhances the expression of the chitinase 5 via Broad-Complex Zinc-Finger 4 during metamorphosis in silkworm, Bombyx mori . Insect Mol Biol. 2017;26(2):243–53. Zollman S, Godt D, Privé GG, Couderc JL, Laski FA. The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila . Proc Natl Acad Sci U S A. 1994;91(22):10717–21. Luan JB, Ghanim M, Liu SS, Czosnek H. Silencing the ecdysone synthesis and signaling pathway genes disrupts nymphal development in the whitefly. Insect Biochem Mol Biol. 2013;43(8):740–6. Ijiro T, Urakawa H, Yasukochi Y, Takeda M, Fujiwara Y. cDNA cloning, gene structure, and expression of Broad-Complex (BR-C) genes in the silkworm, Bombyx mori . Insect Biochem Mol Biol. 2004;34(9):963–9. Araus V, Vidal EA, Puelma T, Alamos S, Mieulet D, Guiderdoni E, Gutiérrez RA. Members of BTB gene family of scaffold proteins suppress nitrate uptake and nitrogen use efficiency. Plant Physiol. 2016;171(2):1523–32. He Q, Chen J, Chen S, Gao X. SUMOylation modulates the dual functions of Krüppel homolog 1 in transcriptional regulation of Broad-Complex expression. J Adv Res. 2025;75:123–35. Piulachs MD, Pagone V, Bellés X. Key roles of the Broad-Complex gene in insect embryogenesis. Insect Biochem Mol Biol. 2010;40(6):468–75. Kiss I, Beaton AH, Tardiff J, Fristrom D, Fristrom JW. Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster . Genetics. 1988;118(2):247–59. Tao C, Li J, Du W, Qin X, Cao J, Liu C, Cheng T. Broad Complex-Z2 directly activates BmMBF2 to inhibit the silk protein synthesis in the silkworm, Bombyx mori . Int J Biol Macromol. 2024;277(Pt 3):134211. Ma H, Abbas MN, Zhang K, Hu X, Xu M, Liang H, Kausar S, Yang L, Cui H. 20-Hydroxyecdysone regulates the transcription of the lysozyme via Broad-Complex Z2 gene in silkworm, Bombyx mori . Dev Comp Immunol. 2019;94:66–72. Cong J, Tao C, Zhang X, Zhang H, Cheng T, Liu C. Transgenic ectopic overexpression of Broad Complex (BrC-Z2) in the silk gland inhibits the expression of slk fibroin genes of Bombyx mori . Insects. 2020;11(6):374. Deng H, Niu K, Zhang J, Feng Q. BmBR-C Z4 is an upstream regulatory factor of BmPOUM2 controlling the pupal specific expression of BmWCP4 in the silkworm, Bombyx mori . Insect Biochem Mol Biol. 2015;66:42–50. Ding N, Wang Z, Geng N, Zou H, Zhang G, Cao C, Li X, Zou C. Silencing Br-C impairs larval development and chitin synthesis in Lymantria dispar larvae. J Insect Physiol. 2020;122:104041. Liang GM, Tan WJ, Guo YY. An improvement in the technique of artificial raring cotton bollworm. 1999. Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, Liu Y, Feng J, Chen H, He Y, et al. TBtools-II: A one for all, all for one bioinformatics platform for biological big-data mining. Mol Plant. 2023;16(11):1733–42. Hu DQ, Zhang LSH, Liu YL. Cloning, prokaryotic expression and spatio-temporal expression profiling of BR-C Z2 gene in Helicoverpa armigera . J Plant Prot. 2021;48(05):10. Konno K, Mitsuhashi W. The peritrophic membrane as a target of proteins that play important roles in plant defense and microbial attack. J Insect Physiol. 2019;117:103912. Tang JW, Wang Q, Jiang YM, Jiang YR, Wang Y, Liu W. Group V chitin deacetylases are responsible for the structure and barrier function of the gut peritrophic matrix in the chinese oak silkworm Antheraea pernyi . Int J Mol Sci 2024, 26(1). Yu A, Beck M, Merzendorfer H, Yang Q. Advances in understanding insect chitin biosynthesis. Insect Biochem Mol Biol. 2024;164:104058. Liu X, Cooper AMW, Zhang J, Zhu KY. Biosynthesis, modifications and degradation of chitin in the formation and turnover of peritrophic matrix in insects. J Insect Physiol. 2019;114:109–15. Guo W, Kain W, Wang P. Effects of disruption of the peritrophic membrane on larval susceptibility to Bt toxin Cry1Ac in cabbage loopers. J Insect Physiol. 2019;117:103897. Wu H, Zhao D, Guo XC, Liu ZR, Li RJ, Lu XJ, Guo W. Group V chitin deacetylases influence the structure and composition of the midgut of beet armyworm, Spodoptera exigua . Int J Mol Sci. 2023;24(4):3076. Qu MB, Sun SP, Liu YS, Deng XR, Yang J, Yang Q. Insect group II chitinase OfChtII promotes chitin degradation during larva-pupa molting. Insect Sci. 2021;28(3):692–704. Zhang L, Wei Y, Wei L, Liu X, Liu N. Effects of transgenic cotton lines expressing ds AgCYP6CY3-P1 on the growth and detoxification ability of Aphis gossypii glover. Pest Manag Sci. 2023;79(1):481–8. Mai T, Chen S, Lin X, Zhang X, Zou X, Feng Q, Zheng S. 20-hydroxyecdysone positively regulates the transcription of the antimicrobial peptide, lebocin, via BmEts and BmBR-C Z4 in the midgut of Bombyx mori during metamorphosis. Dev Comp Immunol. 2017;74:10–8. Guo MP, Qian WL, He XC, Peng J, Wang P, Wang WN, Xia QY, Cheng DJ. Genome-wide identification of target genes for transcription factor BR-C in the silkworm, Bombyx mori . Insect Sci. 2021;28(6):1530–40. Barbole RS, Sharma S, Patil Y, Giri AP, Joshi RS. Chitinase inhibition induces transcriptional dysregulation altering ecdysteroid-mediated control of Spodoptera frugiperda development. iScience 2024, 27(3):109280. Zhang J, Xu G, Qiu B, Zhang X, Feng Q, Yang Q, Zheng S. BR-C Z4 and FoxJ interact to regulate expression of a chitin synthase gene CHSA-2b in the pupal wing discs of the silkworm, Bombyx mori . Insect Biochem Mol Biol. 2020;116:103264. Zhang ZL, Xu QY, Zhang R, Shen C, Bao HB, Luo GH, Fang JC. The irregular developmental duration mainly caused by the broad-complex in Chilo suppressalis . Pestic Biochem Physiol. 2024;204:106090. Jadhav V, Vaishnaw A, Fitzgerald K, Maier MA. RNA interference in the era of nucleic acid therapeutics. Nat Biotechnol. 2024;42(3):394–405. Lucena-Leandro VS, Abreu EFA, Vidal LA, Torres CR, Junqueira C, Dantas J, Albuquerque ÉVS. Current scenario of exogenously induced RNAi for lepidopteran agricultural pest control: from dsRNA design to topical application. Int J Mol Sci 2022, 23(24). Li Y, Kong W, Li T, Zhang L, Zhuang Z, Liu N, Liu X. Functional analysis of lactase phlorizin hydrolase in insect-plant coevolution based on deglycosylation. J Agric Food Chem. 2025;73(9):5140–9. Xu X, Li T, Zhang L, Liu X. Effect of silencing the E74B gene on the development and metamorphosis of Helicoverpa armigera . Pest Manag Sci. 2024;80(3):1435–45. Xie J, Wang S, Zhuang Z, Wang X, Lin M, Liu X. Exploring the role of CYP6AB328 in spinetoram resistance and growth and development of Phthorimaea absoluta . Pestic Biochem Physiol. 2025;208:106316. Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Supplementaryrawimages.docx Cite Share Download PDF Status: Published Journal Publication published 10 Apr, 2026 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 02 Feb, 2026 Reviews received at journal 02 Feb, 2026 Reviews received at journal 29 Jan, 2026 Reviews received at journal 28 Jan, 2026 Reviewers agreed at journal 23 Jan, 2026 Reviewers agreed at journal 22 Jan, 2026 Reviewers agreed at journal 22 Jan, 2026 Reviewers agreed at journal 22 Jan, 2026 Reviewers invited by journal 21 Jan, 2026 Editor invited by journal 21 Jan, 2026 Editor assigned by journal 18 Jan, 2026 Submission checks completed at journal 18 Jan, 2026 First submitted to journal 15 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8610350","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":579629615,"identity":"853df244-e84f-46fa-be08-5337e0c4148a","order_by":0,"name":"Deqin Hu","email":"","orcid":"","institution":"XinjiangUniversity","correspondingAuthor":false,"prefix":"","firstName":"Deqin","middleName":"","lastName":"Hu","suffix":""},{"id":579629616,"identity":"c9bb6657-fd59-4089-b653-7ad6245a2c6d","order_by":1,"name":"Yuan Li","email":"","orcid":"","institution":"XinjiangUniversity","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Li","suffix":""},{"id":579629617,"identity":"c9ec2b58-1f57-45d8-94a7-72ad7f1cc64c","order_by":2,"name":"Jingang Xie","email":"","orcid":"","institution":"XinjiangUniversity","correspondingAuthor":false,"prefix":"","firstName":"Jingang","middleName":"","lastName":"Xie","suffix":""},{"id":579629618,"identity":"e8f372ed-a35f-4cdd-b43e-43d6a25298be","order_by":3,"name":"Hongsheng Pan","email":"","orcid":"","institution":"Xinjiang Uygur Autonomous Region Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hongsheng","middleName":"","lastName":"Pan","suffix":""},{"id":579629619,"identity":"251de595-73e7-4acb-96aa-324e4e999a1b","order_by":4,"name":"Xiaoning Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACPmYQaSDBI8/ewGAAZDI2ENLCBtZSYSFn2HOAWC1g8kyFMcONBDCTCC3sPGbShW0SiY0z3xgU8zDYyG44wPzsAX6H8RgbzwRqaZdOSzDmYUgz3nCAzdyAgBbDx7wgW2YnHwBqOZy44QAPmwQBLQaHQVoabh5sAGr5T5QWw8c8ZySA3mcG2XKAGC1sxcY8FRLAQE5LMJxjkGw88zCbGV4t/PyHt0nzGNQBo/KMmcGbCjvZvuPNz/BqQbHRAByZzMSqB6l9QILiUTAKRsEoGEEAADKQPGgn/bK1AAAAAElFTkSuQmCC","orcid":"","institution":"XinjiangUniversity","correspondingAuthor":true,"prefix":"","firstName":"Xiaoning","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-01-15 12:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8610350/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8610350/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-026-12806-8","type":"published","date":"2026-04-10T15:58:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101398096,"identity":"301f0d27-2d54-4e0e-94a2-59a8fce8dabc","added_by":"auto","created_at":"2026-01-29 09:39:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":703900,"visible":true,"origin":"","legend":"\u003cp\u003eAmplification and analysis of the \u003cem\u003eHaCHT4\u003c/em\u003epromoter sequence. M, Marker; A) Amplification of the \u003cem\u003eHaCHT4\u003c/em\u003e promoter sequence. 1-3, PCR products of Primer SP1 from round 1 to round 3; 4-6, PCR products of Primer SP2 from round 1 to round 3. B) pEASY-T1-\u003cem\u003eHaCHT4\u003c/em\u003emonoclonal PCR. 1, negative control; 2-6: different monoclonal samples. C) Sequence of the \u003cem\u003eHaCHT4\u003c/em\u003e promoter showing regulatory elements using the JASPR website.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/981694f23603f5c4716e8fc1.jpg"},{"id":101321053,"identity":"18c2faa6-495a-4626-bb1f-91af3424b02c","added_by":"auto","created_at":"2026-01-28 12:58:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53225,"visible":true,"origin":"","legend":"\u003cp\u003eCollinearity analysis of BTB genes were investigated among \u003cem\u003eH. armigera\u003c/em\u003e, \u003cem\u003eT. castaneum\u003c/em\u003e, \u003cem\u003eB. mori\u003c/em\u003e and \u003cem\u003eD. melanogaster. \u003c/em\u003eThe red lines highlighted collinear BTB gene pairs, while the gray lines in the background indicated all collinear blocks.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/06450f94a6046d60337ce3ee.jpg"},{"id":101321063,"identity":"52dbd1df-9520-4a57-a0e6-268fe95a2412","added_by":"auto","created_at":"2026-01-28 12:58:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":294963,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of BTB proteins from \u003cem\u003eH. armigera, B. mori, T. castaneum \u003c/em\u003eand\u003cem\u003e D. melanogaster\u003c/em\u003e. MEGA 11.0 software was used to construct a neighbor-joining phylogenetic tree with 1000 bootstrap replications. The four groups \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cbr\u003e\n are distinguished by branches in different colors for clarity.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/d297aef57acb045e9f22b129.jpg"},{"id":101321054,"identity":"8b1a3596-28af-4742-b2ef-7eebf693fa24","added_by":"auto","created_at":"2026-01-28 12:58:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":176391,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic tree of different BRC isforms from other species. MEGA 11.0 software was used to construct a phylogenetic tree with 1000 bootstrap replications.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/0b8f3f576699866e03d40804.jpg"},{"id":101398315,"identity":"b36d6632-8601-473d-9c22-bea87f1d18d7","added_by":"auto","created_at":"2026-01-29 09:40:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":467204,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking prediction and EMSA analysis of the binding of HaBR-C Z2 to the promoter region of the \u003cem\u003eHaCHT4\u003c/em\u003e. A) Molecular docking of HaBRC Z2 binding to the key CREs of the \u003cem\u003eHaCHT4\u003c/em\u003epromoter. B) The core sequence of HaBRC Z2 binding to the \u003cem\u003eHaCHT4\u003c/em\u003epromoter. C) Detection of probe synthesis and labeling efficiency, -, negative control; 1, probe synthesis. D) EMSA analysis of the binding of HaBRC Z2 to the \u003cem\u003eHaCHT4\u003c/em\u003e promoter CREs.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/2494afda54cab7297447dc49.jpg"},{"id":101398337,"identity":"1d596df1-4004-451d-82cf-f08afc4e3788","added_by":"auto","created_at":"2026-01-29 09:41:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":275957,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression profiles of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e in the midgut of \u003cem\u003eH. armigera\u003c/em\u003e. A, B) Expression profile of \u003cem\u003eHaBRC Z2 \u003c/em\u003eand\u003cem\u003e HaCHT4\u003c/em\u003e during the 4\u003csup\u003eth\u003c/sup\u003e to 5\u003csup\u003eth\u003c/sup\u003e instar larvae, respectively. 4E and 5E, the early stage of the 4\u003csup\u003eth\u003c/sup\u003e and 5\u003csup\u003eth\u003c/sup\u003e instar larvae, respectively; 4L and 5L, the late stage of the 4\u003csup\u003eth\u003c/sup\u003e and 5\u003csup\u003eth\u003c/sup\u003e instar larvae, respectively. C, D) Expression profiles of\u003cem\u003e HaBRC Z2 \u003c/em\u003eand\u003cem\u003e HaCHT4 \u003c/em\u003efollowing 1.0 ug/µL 20-hydroxyecdysone (20E) treatment for 24 h, respectively. Data are presented as mean ± SE (3 biological and 2 technical replicates). Significant differences between the early and late stages within the same instar or after 20E treatment were determined by Student’s \u003cem\u003et\u003c/em\u003e-test (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, * * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * * *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/20425cbdcc14fb3098bbd36d.jpg"},{"id":101321059,"identity":"552ebe24-7695-4b0b-876b-409eae8e0cfa","added_by":"auto","created_at":"2026-01-28 12:58:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":342886,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing efficiency of \u003cem\u003eHaBRC Z2 \u003c/em\u003ein \u003cem\u003eH. armigera\u003c/em\u003e treated with ds\u003cem\u003eHaBRC Z2\u003c/em\u003e and its effect on \u003cem\u003eHaCHT4\u003c/em\u003e. A, B) The relative expression level of the \u003cem\u003eHaBRC Z2 \u003c/em\u003eafter treatment with 250 ng/μL and 500 ng/μLds\u003cem\u003eHaBRC Z2\u003c/em\u003e, respectively. C, D) The relative expression level of \u003cem\u003eHaCHT4 \u003c/em\u003eafter treatment with 250 ng/μL and 500 ng/μL\u003cem\u003eHaBRC Z2\u003c/em\u003e, respectively. Data are shown as mean ± SE (3 biological and 2 technical replicates). Different letters above the bars represent significant differences (one-way ANOVA followed by Tukey’s test,\u003cem\u003e P\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/50e4baae051698761fa5b45d.jpg"},{"id":101321057,"identity":"3e445cc6-387b-427c-9aa6-964cc87a83bc","added_by":"auto","created_at":"2026-01-28 12:58:23","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":315321,"visible":true,"origin":"","legend":"\u003cp\u003eAverage body weight and body length of \u003cem\u003eH. armigera\u003c/em\u003e larvae after following 500 ng/µL ds\u003cem\u003eHaBRC Z2\u003c/em\u003e treatment. A) Average body weight. B) Average body length. Bars represent mean ± SE (3 biological replicates). Significant differences at each time point were determined by one-way ANOVA followed with Tukey’s HSD (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/a59be8429b80c8a40acac82c.jpg"},{"id":106809290,"identity":"7a7fb3db-82c9-4aeb-93d2-a282cfabcb43","added_by":"auto","created_at":"2026-04-13 16:09:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3565994,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/17341579-6645-4bd5-81c4-82afd2daea3a.pdf"},{"id":101397927,"identity":"9845cf00-f6ad-48af-a581-c0512dca14ca","added_by":"auto","created_at":"2026-01-29 09:38:11","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":468792,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/1aeb3548b85e8e454002e7e1.docx"},{"id":101321060,"identity":"12970e61-8080-4851-a1f7-1f8b997c83da","added_by":"auto","created_at":"2026-01-28 12:58:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3419741,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryrawimages.docx","url":"https://assets-eu.researchsquare.com/files/rs-8610350/v1/1b0ffcedf006cc0e53502e94.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Broad complex Z2 regulates the group Ⅳ chitinase gene HaCHT4 in the midgut of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eHelicoverpa armigera\u003c/em\u003e (H\u0026uuml;bner) (Lepidoptera: Noctuidae), a polyphagous pest, inflicts substantial annual economic losses on cotton by primarily attacking its buds and bolls [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The effective management of this pest remains a challenge due to its wide host range, polyphagous nature, high adaptability, and widespread pesticide resistance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although chemical pesticides have been widely used, they bring unintended consequences like environmental contamination, human health threats, and devastation of beneficial non-target species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Another key strategy for \u003cem\u003eH. armigera\u003c/em\u003e management is the cultivation of Bt cotton, though the evolution of resistance in \u003cem\u003eH. armigera\u003c/em\u003e diminish the benefits of Bt cotton [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, exploring effective and durable eco-friendly control strategies and molecular targets is crucial for delaying resistance evolution and advancing sustainable pest management. Previous work from our team demonstrated the critical role of the \u003cem\u003eHaCHT4\u003c/em\u003e in modulating chlorantraniliprole sensitivity in \u003cem\u003eH. armigera\u003c/em\u003e, achieved through its regulation of chitin and architecture of the peritrophic membrane (PM) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eHaCHT4\u003c/em\u003e, which mediates chitin degradation, represents a candidate target for biorational insecticide discovery. Its functional relevance in insects, combined with the absence of chitin systems in vertebrates, offers a strategic avenue for developing pest management solutions with enhanced environmental compatibility.\u003c/p\u003e \u003cp\u003eChitin, a fundamental structural polymer in the PM, epidermis, intestine, and trachea, is precisely regulated by molecular interactions [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among these regulatory factors, chitinase and ecdysone hormone 20-hydroxyecdysone (20E) play central roles [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In insects, chitinases catalyze the degradation of high molecular weight chitin into low molecular weight oligosaccharides [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], thus fulfilling a critical function in insect growth and developmental processes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The relative expression level of the chitinase gene has been found to increase significantly before molting (late stages) and return to normal or lower levels after molting (early stages) in insects, fluctuating with the physiological state similar to the changing trend of 20E titer [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The transcription levels of chitinase genes in \u003cem\u003eManduca sexta\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003eLocusta migratoria\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] were induced by 20E. In \u003cem\u003eB. mori\u003c/em\u003e, metamorphosis is marked by 20E upregulating chitinase 5 expression via the transcription factor \u003cem\u003eBmBRC-Z4\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The expression level of \u003cem\u003eHaCHT4\u003c/em\u003e was lower in the early stages of 4th (4E) to 6th (6E) instar larvae, but significantly elevated in the late stages (4L, 6L) in \u003cem\u003eH. armigera\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This implicates a specific 20E pathway in regulating chitinases during molting, although the precise mechanistic details remain to be fully elucidated.\u003c/p\u003e \u003cp\u003eThe functional diversity of the 20E responsive Broad-Complex (BRC) transcription factor stems from its alternative splicing, which generates isoforms with distinct C-terminal zinc finger domains [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This gene activated directly by the EcR-USP dimer, possesses a conserved BTB domain alongside its variable DNA binding domain [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Functional studies reveal isoform specific roles across insects [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In \u003cem\u003eD. melanogaster\u003c/em\u003e, \u003cem\u003eBRC Z1\u003c/em\u003e and \u003cem\u003eBRC Z2\u003c/em\u003e have been shown to regulate appendage elongation (legs/wings) and eversion, whereas \u003cem\u003eBRC Z3\u003c/em\u003e is dedicated to the process of disc fusion [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In \u003cem\u003eB. mori\u003c/em\u003e, \u003cem\u003eBRC Z2\u003c/em\u003e not only affects yolk protein synthesis and oocyte formation, but also regulates cuticle protein gene [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The function of \u003cem\u003eBRC Z3\u003c/em\u003e remains poorly characterized, and \u003cem\u003eBRC Z4\u003c/em\u003e mainly regulates genes encoding insect cuticular proteins and those genes related to chitin metabolism in \u003cem\u003eB. mori\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, RNAi-mediated silencing of \u003cem\u003eLdBRC\u003c/em\u003e in \u003cem\u003eLymantria dispar\u003c/em\u003e resulted in severely impaired chitinase gene expression, causing developmental defects and increased mortality, underscoring its essential role [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe proved that the midgut specific chitinase gene \u003cem\u003eHaCHT4\u003c/em\u003e is involved in chitin degradation of \u003cem\u003eH. armigera\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To elucidate its transcriptional regulation, this study has cloned and analyzed CREs within the \u003cem\u003eHaCHT4\u003c/em\u003e promoter region. The transcriptional factor \u003cem\u003eHaBRC Z2\u003c/em\u003e was identified targeting \u003cem\u003eHaCHT4\u003c/em\u003e in \u003cem\u003eH. armigera\u003c/em\u003e, and proposing a preliminary molecular mechanism of 20E - \u003cem\u003eHaBRC Z2\u003c/em\u003e - \u003cem\u003eHaCHT4\u003c/em\u003e regulation axis by EMSA, RT-qPCR and RNAi technologies. These findings delineate a preliminary 20E - \u003cem\u003eHaBRC Z2\u003c/em\u003e - \u003cem\u003eHaCHT4\u003c/em\u003e regulatory axis, which enhances our understanding of the interplay between chitinases and 20E, and for devising novel pest management strategies aimed at chitin metabolism.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInsect rearing\u003c/h2\u003e \u003cp\u003eA laboratory-reared strain of \u003cem\u003eH. armigera\u003c/em\u003e, with no history of any insecticide or toxins exposure, was maintained under controlled conditions of 26\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, and a 16:8 (L:D) photoperiod. The specific rearing details of \u003cem\u003eH. armigera\u003c/em\u003e larvae were referred to the work of Liang et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning and characterization of 5\u0026rsquo; UTR region of\u003c/b\u003e \u003cb\u003eHaCHT4\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenomic DNA of \u003cem\u003eH. armigera\u003c/em\u003e 4th instar larvae were purified with the Easy Pure Genomic Kit (TransGen Biotech, Beijing, China). The promoter region of \u003cem\u003eHaCHT4\u003c/em\u003e was amplified from this DNA using the Genome Walking Kit (TaKaRa, Tokyo, Japan). The cloned fragment was then inserted into pEASY-T1 cloning vector. Subsequently, the positive monoclonal was sequenced by Sangon Biotechnology (Shanghai, China). Putative CREs of \u003cem\u003eHaCHT4\u003c/em\u003e were performed on JASPAR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jaspar.elixir.no/\u003c/span\u003e\u003cspan address=\"https://jaspar.elixir.no/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The complete primer sequences and nested PCR reaction procedures are shown in supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentification and bioinformatics analysis of the BTB gene family\u003c/h3\u003e\n\u003cp\u003eWe obtained the reference genome sequence of \u003cem\u003eH. armigera\u003c/em\u003e (GCF_030705265.1) from the National Center for Biotechnology Information (NCBI) database, and employed two methods to identify potential members of the HaBTBs. First, we performed BLASTP homology alignment against the local \u003cem\u003eH. armigera\u003c/em\u003e database using the \u003cem\u003eH. armigera\u003c/em\u003e HaBRC Z2 protein sequence as the query. Second, a hidden markov model (HMM) for the BTB domain (PF00651) was employed for a domain-based search against the \u003cem\u003eH. armigera\u003c/em\u003e genome using TBtools. This profile was then utilized for the identification of BTB genes from the \u003cem\u003eH. armigera\u003c/em\u003e genomic database with the assistance of TBtools [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The candidate sequences obtained from both methods were validated for domain structure using Pfam, and only those sequences containing the conserved BTB domain were retained as HaBTB candidates. Protein parameters were derived from ExPASy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.expasy.org/\u003c/span\u003e\u003cspan address=\"https://www.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The location on chromosomal of the HaBTB genes in \u003cem\u003eH. armigera\u003c/em\u003e were obtained from the NCBI database, along with the corresponding genome annotation files. MapChart software was used to map the chromosome position of HaBTB gene. The predicted BTB protein sequences from \u003cem\u003eH. armigera\u003c/em\u003e, \u003cem\u003eD. melanogaster\u003c/em\u003e, \u003cem\u003eTribolium castaneum\u003c/em\u003e, and \u003cem\u003eB. mori\u003c/em\u003e aligned using ClustalW with default parameters. Phylogenetic tree was constructed in MEGA 11.0 via the neighbor-joining method, which was refined using the iTOL online tool. All Figures are assembled and finalized in Adobe Illustrator 2022.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the molecular interaction between\u003c/b\u003e \u003cb\u003eHaCHT4\u003c/b\u003e \u003cb\u003eand HaBRC Z2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the interaction between \u003cem\u003eHaCHT4\u003c/em\u003e and HaBRC Z2, the amino acid sequences of HaBRC Z2 were submitted to AlphaFold 334 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.com/\u003c/span\u003e\u003cspan address=\"https://alphafold.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to predict protein models. Molecular docking between the HaBRC Z2 protein and the cis-regulatory element of \u003cem\u003eHaCHT4\u003c/em\u003e was carried out using AutoDockFR35 software. The probe for EMSA was synthesized according to the fragment (-313 ~ -5nt, 308bp) of the potential \u003cem\u003eHaBRC Z2\u003c/em\u003e core binding site by DIG-High Primer DNA Labeling and Detection Starter kit II (Roche, Basel, Switzerland). The His-HaBRC Z2 fusion protein had been purified in a previous experiment [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For the DNA-binding assay, containing 1 \u0026micro;L (or 2 \u0026micro;L) of His-HaBRC Z2 fusion protein, 1 \u0026micro;L of labeled probe (500 pg/\u0026micro;L), 2 \u0026micro;L of binding buffer and supplement ddH\u003csub\u003e2\u003c/sub\u003eO to 10 \u0026micro;L, and incubated at 25\u0026deg;C for 30 min. Samples were electrophoresed on 5% polyacrylamide gel. After protein transferring to Nylon membrane, UV crosslinking was performed for 15 min. The membrane was treated sequentially as follows: incubation in maleic acid solution for 5 min at 25 ℃, blocking with blocking buffer for 30 min, and incubation with streptavidin-HRP conjugate for 30 min at 25\u0026deg;C [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Signal detection was performed using the Clinx Science imaging system.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression profiles of\u003c/b\u003e \u003cb\u003eHaBRC Z2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eHaCHT4\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eH. armigera\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo characterize the transcript levels of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e in \u003cem\u003eH. armigera\u003c/em\u003e, midgut tissue samples were collected at both the early and late stages of the 4th and 5th instar larvae for RNA extraction. For each developmental stage, three biological replicates were prepared, with each replicate comprising a pool of midgut tissues from ten larvae. The cDNA synthesis and RT-qPCR were conducted as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To investigate whether 20E regulates the expression of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e, 4th instar larvae were treated with 5 \u0026micro;L of 20E (200 ng/\u0026micro;L; supplied by Sangon Biotech, Shanghai, China). Midgut tissues were dissected 24 h post treatment to analyze the expression levels of the target genes. The experiment included 3 biological replicates, with each replicate comprising 30 larvae. The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was used to quantified the relative expression levels of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e. 3 biological and 2 technical replicates were used for all reactions.\u003c/p\u003e\n\u003ch3\u003eSynthesis of dsRNA and RNAi assays\u003c/h3\u003e\n\u003cp\u003eTo investigate the function of the \u003cem\u003eHaBRC Z2\u003c/em\u003e gene, double-stranded RNA (dsRNA) targeting its open reading frame (ORF) was designed, green fluorescent protein (GFP) serving as a control. Subsequently, dsRNAs were synthesized in vitro employing the T7 RiboMAX\u0026trade; Express RNAi System (Promega, Madison, USA).\u003c/p\u003e \u003cp\u003eFor RNAi bioassay, the artificial diet was meticulously divided into rectangular pieces measuring 4 mm\u0026times;4 mm\u0026times;3 mm, which were then allocated to each group. 2nd instar larvae were deprived of food for 4 h. Two groups received 10 \u0026micro;L of ds\u003cem\u003eHaBRC Z2\u003c/em\u003e solution at concentrations of 250 ng/\u0026micro;L and 500 ng/\u0026micro;L, respectively. The dsRNA-treated diet was refreshed every 48 h intervals. Control groups were administered an equivalent volume of either ddH₂O or ds\u003cem\u003eGFP\u003c/em\u003e solution. Each treatment was applied to 35 larvae, with three independent biological replicates established. To evaluate the silencing efficiency of \u003cem\u003eHaBRC Z2\u003c/em\u003e, 5 larvae from each group were sampled at 12, 24, 48, 72, and 96 h dsRNAs post-feeding.\u003c/p\u003e \u003cp\u003eTo further assess the impact of ds\u003cem\u003eHaBRC Z2\u003c/em\u003e on larval phenotypic traits, a separate cohort of 2nd instar larvae were fed a diet containing 10 \u0026micro;L of ds\u003cem\u003eHaBRC Z2\u003c/em\u003e at 500 ng/\u0026micro;L. Control groups were treated with ddH₂O or ds\u003cem\u003eGFP\u003c/em\u003e as described above. Larval body length and weight were recorded at 12, 24, 36, 48, 72, and 96 h after treatment initiation. This experiment included 3 biological replicates, with 35 larvae in each group per replicate.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll date from 3 experimental replicates were represented by mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). For gene expression analysis across the 4E to 5L instars, differences between early and late stages were assessed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (* \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). For post-RNAi data, one-way analysis of variance (ANOVA) coupled with Tukey\u0026rsquo;s HSD test in SPSS 22.0 was used to evaluate differences among treatment groups at identical time points, where different letters mark significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of the 5\u0026rsquo; UTR from the\u003c/b\u003e \u003cb\u003eHaCHT4\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the regulation of \u003cem\u003eHaCHT4\u003c/em\u003e, a 1186 bp fragment of the \u003cem\u003eHaCHT4\u003c/em\u003e promoter was amplified through genome walking technology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Sequence analysis using the JASPAR online platform revealed the presence of Broad-Complex (BRC) isoforms Z1-Z4 elements related to 20E, and other elements including GATA, NF-AT1, and POU1F1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The BRC gene is an immediate early gene of 20E and belongs to the BTB gene family. In order to fully understand the BRC gene, we identified and characterized the BTB gene family in the \u003cem\u003eH. armigera\u003c/em\u003e genome. The results showed that 85 BTB genes (Table S3) were identified from the \u003cem\u003eH. armigera\u003c/em\u003e genome, and they were renamed according to their functional domain and chromosomal locations (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A collinearity plot was constructed for \u003cem\u003eH. armigera\u003c/em\u003e, \u003cem\u003eB. mori\u003c/em\u003e, \u003cem\u003eT. castaneum\u003c/em\u003e, and \u003cem\u003eD. melanogaster.\u003c/em\u003e This analysis identified 48 orthologs between \u003cem\u003eH. armigera\u003c/em\u003e and \u003cem\u003eB. mori\u003c/em\u003e, 2 orthologs between \u003cem\u003eH. armigera\u003c/em\u003e and \u003cem\u003eT. castaneum\u003c/em\u003e, and no orthologs between \u003cem\u003eH. armigera\u003c/em\u003e and \u003cem\u003eD. melanogaster.\u003c/em\u003e These findings suggest that these genes are relatively conserved during the evolution of lepidoptera insects and play an indispensable and important role (Fig.\u0026nbsp;2). Specifically, the BRC gene possesses both a BTB domain and a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e zinc finger domain, and is designated HaBTBZF1 based on its chromosomal location. Phylogenetic analysis revealed that HaBTBZF1 clusters with BmBTB ZF1 of \u003cem\u003eB. mori\u003c/em\u003e, indicating that they are the closest orthologs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This gene features eight exons, and can generate distinct transcripts through alternative splicing. Hence, previously reported different BRC isoforms from other insect species were downloaded. In the phylogenetic analysis, each of the BRC isoforms fell within its clade, which is consistent with the previously observed evolutionary conservation of these isoforms (Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2.\u003c/b\u003e Collinearity analysis of BTB genes were investigated among \u003cem\u003eH. armigera\u003c/em\u003e, \u003cem\u003eT. castaneum\u003c/em\u003e, \u003cem\u003eB. mori\u003c/em\u003e and \u003cem\u003eD. melanogaster.\u003c/em\u003e The red lines highlighted collinear BTB gene pairs, while the gray lines in the background indicated all collinear blocks.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e are distinguished by branches in different colors for clarity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4.\u003c/b\u003e A phylogenetic tree of different BRC isforms from other species. MEGA 11.0 software was used to construct a phylogenetic tree with 1000 bootstrap replications.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHaBRC Z2 protein binds to the promoter of\u003c/b\u003e \u003cb\u003eHaCHT4\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify the BRC isoform that may regulate \u003cem\u003eHaCHT4\u003c/em\u003e, the binding energy between the regulatory element and \u003cem\u003eHaCHT4\u003c/em\u003e was predicted through molecular docking. The results indicated a strong binding affinity between \u003cem\u003eHaCHT4\u003c/em\u003e and the transcription factor HaBRC Z2. The binding sites were characterized by the presence of AT-rich motifs, specifically \u0026ldquo;AACTAATT\u0026rdquo;, with a binding free energy (ΔG) of -12 kcal/mol (Fig.\u0026nbsp;5A). Moreover, a DNA fragment encompassing the HaBRC Z2 binding motif within the \u003cem\u003eHaCHT4\u003c/em\u003e promoter was amplified (Fig.\u0026nbsp;5B). The probe labeling efficiency was validated, showing high sensitivity 10 pg/\u0026micro;L (Fig.\u0026nbsp;5C). EMSA demonstrated that a shift in the band intensity upon the addition of His-HaBRC Z2 in comparison to the probe alone and the His-PET32a groups (Fig.\u0026nbsp;5D). These results support the involvement of the HaBRC Z2 CRE in regulating \u003cem\u003eHaCHT4\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5.\u003c/b\u003e Molecular docking prediction and EMSA analysis of the binding of HaBR-C Z2 to the promoter region of the \u003cem\u003eHaCHT4\u003c/em\u003e. A) Molecular docking of HaBRC Z2 binding to the key CREs of the \u003cem\u003eHaCHT4\u003c/em\u003e promoter. B) The core sequence of HaBRC Z2 binding to the \u003cem\u003eHaCHT4\u003c/em\u003e promoter. C) Detection of probe synthesis and labeling efficiency, -, negative control; 1, probe synthesis. D) EMSA analysis of the binding of HaBRC Z2 to the \u003cem\u003eHaCHT4\u003c/em\u003e promoter CREs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression profiles of\u003c/b\u003e \u003cb\u003eHaBRC Z2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eHaCHT4\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eH. armigera\u003c/b\u003e\u003c/p\u003e \u003cp\u003e We profiled the transcript levels of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e across the 4th to 5th instar larval stages via RT-qPCR. Both genes exhibited a marked upregulation in the late phases of each instar compared to their early phases (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Specifically, \u003cem\u003eHaBRC Z2\u003c/em\u003e expression increased by approximately 2.56-fold (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.497, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0058) and 2.08-fold (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.411, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0013) in the 4L and 5L instars, respectively (Fig.\u0026nbsp;6A), while \u003cem\u003eHaCHT4\u003c/em\u003e exhibited more pronounced inductions of 9.33-fold (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.299, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 2.94-fold (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.078, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0117) at the same stages, respectively (Fig.\u0026nbsp;6B). Furthermore, significantly induced the mRNA accumulation of both \u003cem\u003eHaBRC Z2\u003c/em\u003e (Fig.\u0026nbsp;6C) (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.628, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0046) and \u003cem\u003eHaCHT4\u003c/em\u003e (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.499, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0011) were detected after 1.0 ug/\u0026micro;L 20E treatment for 24h (Fig.\u0026nbsp;6D). The coordinated expression patterns suggest that \u003cem\u003eHaBRC Z2\u003c/em\u003e may act as anupstream regulator of \u003cem\u003eHaCHT4\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 6.\u003c/b\u003e Relative expression profiles of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e in the midgut of \u003cem\u003eH. armigera\u003c/em\u003e. A, B) Expression profile of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e during the 4th to 5th instar larvae, respectively. 4E and 5E, the early stage of the 4th and 5th instar larvae, respectively; 4L and 5L, the late stage of the 4th and 5th instar larvae, respectively. C, D) Expression profiles of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e following 1.0 ug/\u0026micro;L 20-hydroxyecdysone (20E) treatment for 24 h, respectively. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (3 biological and 2 technical replicates). Significant differences between the early and late stages within the same instar or after 20E treatment were determined by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (* \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, * * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, * * *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFunctional analysis of the\u003c/b\u003e \u003cb\u003eHaBRC Z2\u003c/b\u003e \u003cb\u003eusing RNAi\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the putative regulation of \u003cem\u003eHaCHT4\u003c/em\u003e by \u003cem\u003eHaBRC Z2\u003c/em\u003e, we performed RNAi-mediated silencing of \u003cem\u003eHaBRC Z2\u003c/em\u003e. While transcript levels of both genes remained unchanged at 12 h post-treatment with ds\u003cem\u003eHaBRC Z2\u003c/em\u003e under 250 (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.236 \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.793) or 500 ng/\u0026micro;L (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.736, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.210), a significant suppression was observed from 24 to 96 h. The expression level of \u003cem\u003eHaBRC Z2\u003c/em\u003e decreased to 23.50% (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;131.76, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of that in the ds\u003cem\u003eGFP\u003c/em\u003e group after treatment with 250 ng/\u0026micro;L ds\u003cem\u003eHaBRC Z2\u003c/em\u003e for 24 h, and the expression of \u003cem\u003eHaCHT4\u003c/em\u003e decreased to 18.45% (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;52.30, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Under 500 ng/\u0026micro;L for 24h treatment, the expression levels of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e were reduced to 56.52% (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14.76, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 40.11% (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;33.59, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) of the control group, respectively. By 96 h, the expression of Ha\u003cem\u003eBRC Z2\u003c/em\u003e in the 250 ng/\u0026micro;L treatment group had decreased to 31.70% of the ds\u003cem\u003eGFP\u003c/em\u003e control group (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;55.499, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and \u003cem\u003eHaCHT4\u003c/em\u003e expression decreased to 11.45% (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;67.05, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), the mRNA expression of \u003cem\u003eHaBRC\u003c/em\u003e Z2 was reduced to 26.58% (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;54.393, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and \u003cem\u003eHaCHT4\u003c/em\u003e expression decreased to 28.24% (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;60.638, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) after treatment with 500 ng/\u0026micro;L ds\u003cem\u003eHaBRC Z2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results support a conclusion that \u003cem\u003eHaBRC Z2\u003c/em\u003e acts as an upstream transcriptional activator of \u003cem\u003eHaCHT4\u003c/em\u003e, likely through binding to the CRE element in its promoter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of\u003c/b\u003e \u003cb\u003eHaBRC Z2\u003c/b\u003e \u003cb\u003esilencing on the growth and development of\u003c/b\u003e \u003cb\u003eH. armigera\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe also assessed the impact of ds\u003cem\u003eHaBRC Z2\u003c/em\u003e on larval growth and development. The results showed that the body length and weight of \u003cem\u003eH.armigera\u003c/em\u003e were significantly inhibited after treatment with 500 ng/\u0026micro;L of ds\u003cem\u003eHaBRC Z2\u003c/em\u003e for 48 h. The average body length after treatment with ds\u003cem\u003eHaBRC Z2\u003c/em\u003e, ds\u003cem\u003eGFP\u003c/em\u003e and CK at 48 h was 7.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23mm, 7.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20mm, 6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15mm; the body weight was 33.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15mg, 30.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08mg, 21.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58mg, respectively. With the extension of treatment time, the body weight was inhibited, and the weight was only 53.9% of the ds\u003cem\u003eGFP\u003c/em\u003e group (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;58.654, \u003cem\u003ed f\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This indicates that \u003cem\u003eHaBRC Z2\u003c/em\u003e knockdown severely impaired larval development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe midgut is a primary organ for food digestion, nutrient absorption, and energy metabolism in insects, and its structure undergoes significant remodeling throughout insect development [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Within it lies the PM, a non-cellular, semipermeable barrier synthesized from chitin, proteins, and glycans [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This structure serves as the midgut\u0026rsquo;s primary defense, shielding it from mechanical abrasion and preventing invasion by pathogens and macromolecules [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, this protective barrier also impedes the uptake of pesticides and entomopathogenic microorganisms in pest control. Consequently, compromising PM integrity to facilitate insecticide uptake has become a prominent research objective [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Given that chitin is not present in mammals or plants [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], the specific regulation of chitin synthesis and degradation pathways is developing into a new strategy for pest control. In \u003cem\u003eOstrinia furnacalis\u003c/em\u003e, group II chitinase gene \u003cem\u003eOfChtII\u003c/em\u003e could degrade of the cuticular chitin and affecting the transformation from larva to pupa [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In \u003cem\u003eAedes albopictus\u003c/em\u003e, the structure of the midgut PM was destroyed or even absent after silencing \u003cem\u003eCHS-2\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our previous research in \u003cem\u003eH. armigera\u003c/em\u003e demonstrated that upregulation \u003cem\u003eHaCHT4\u003c/em\u003e enhances chitin degradation, leading to increased PM porosity. This structural compromise severely impairs the gut barrier, which in turn heightens insecticide susceptibility [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To amplify this effect, we sought to identify which transcription factor responsible for modulating the pathway. Nevertheless, the upstream signaling cascades and transcriptional regulators controlling this chitinase gene have not yet been fully elucidated.\u003c/p\u003e \u003cp\u003eTo elucidate the upstream regulatory mechanism of \u003cem\u003eHaCHT4\u003c/em\u003e, we cloned and analyzed its promoter region. Multiple 20E-associated cis-regulating elements were identified, including binding sites for BRC Z1-Z4 isoforms, GATA, NF-AT1, and POU1F1. The BRC gene, a 20E-responsive transcription factor within the BTB family, is a key regulator of insect growth and development [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Its function is exemplified in \u003cem\u003eB. mori\u003c/em\u003e, where BRC transmits the 20E signal by directly associating with the promoter regions of genes critical for larval development [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Meanwhile, in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e, the isoform BRC Z4 appears to modulate gene expression by interacting with the 5\u0026rsquo; UTR of targets such as Ecdysone receptor and \u003cem\u003eSfCht5\u003c/em\u003e, potentially leading to the upregulation of these and other ecdysteroid biosynthesis genes in berberine-fed insects [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Building on the genomic insight that identified numerous conserved BTB homologs between \u003cem\u003eH. armigera\u003c/em\u003e and \u003cem\u003eB. mori\u003c/em\u003e, our functional investigation demonstrated that 20E triggers \u003cem\u003eHaCHT4\u003c/em\u003e expression through the transcription factor \u003cem\u003eHaBRC Z2\u003c/em\u003e in \u003cem\u003eH. armigera\u003c/em\u003e. Multiple experimental results converge to support this central finding. First, molecular docking indicated that a strong binding affinity of \u003cem\u003eHaBRC Z2\u003c/em\u003e for the core cis-element \u0026ldquo;AACTAATT\u0026rdquo;. This sequence shares homology with the BRC Z2 consensus elements \u0026ldquo;CTA\u0026rdquo; and \u0026ldquo;TAG\u0026rdquo; characterized in \u003cem\u003eB. mori\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], which preferentially bind to AT-rich regions. Subsequently, EMSA confirmed the direct binding of His-HaBRC Z2 to labeled probes containing this motif. Third, transcript levels of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e peaked at the 4L and 5L larval instars compared with the early stages. Similar research on \u003cem\u003eB. mori\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and \u003cem\u003eChilo suppressalis\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] showed that the peak chitinase expression appeared in the late larval and prepupal stages. Besides, a RT-qPCR assay showed that \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e were induced by 20E, suggesting 20E triggers \u003cem\u003eHaBRC Z2\u003c/em\u003e to regulate \u003cem\u003eHaCHT4\u003c/em\u003e. Taken together, we propose the following regulatory pathway: 20E signaling orchestrates \u003cem\u003eHaCHT4\u003c/em\u003e expression by first inducing \u003cem\u003eHaBRC Z2\u003c/em\u003e, which subsequently binds to and transactivates the \u003cem\u003eHaCHT4\u003c/em\u003e promoter during molting. However, definitive evidence for a direct interaction awaits further experimental validation.\u003c/p\u003e \u003cp\u003eRNA interference (RNAi) has become an indispensable tool for elucidating gene function and formulating targeted pest control strategies in entomological research because of its high specificity, simple technology and ability to inhibit transcription. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. For instance, silencing \u003cem\u003eCsBRC\u003c/em\u003e genes in \u003cem\u003eC. suppressalis\u003c/em\u003e increased pupal instar number and significantly prolonged the developmental duration [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Similarly, knockdown of \u003cem\u003eLdBRC\u003c/em\u003e in \u003cem\u003eL. dispar\u003c/em\u003e inhibited chitinase expression in midgut and integument tissues, causing molting arrest and 67% larval mortality [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In addition, our research team has employed RNAi to investigate the functions of genes associated with growth, development, and insecticide resistance in \u003cem\u003eAphis gossypii\u003c/em\u003e, \u003cem\u003eH. armigera\u003c/em\u003e, and \u003cem\u003ePhthorimaea absoluta\u003c/em\u003e, which has facilitated the identification of potential targets for the biocontrol of these pests [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. RNAi-mediated silencing of \u003cem\u003eHaBRC Z2\u003c/em\u003e significantly suppressed its transcript levels, and the optimal knockdown efficacy occurred at 24 h and 96 h. The transcript levels of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e reached the lowest at 24 h after treatment with 250 ng/\u0026micro;L dsRNA. However, both genes showed a transient upregulation at 48 h, which likely reflects the developmental stage specific fluctuations in ecdysteroid titers during the larval transition. All above results establish \u003cem\u003eHaBRC Z2\u003c/em\u003e as a critical 20E response gene that positively regulates \u003cem\u003eHaCHT4\u003c/em\u003e to orchestrate molting.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our data provide evidence that 20E can induce the expression of \u003cem\u003eHaBRC Z2\u003c/em\u003e, and then binds to the \u003cem\u003eHaCHT4\u003c/em\u003e promoter to activate its transcription. Combined with previous findings, we proposed that disrupting the normal expression of \u003cem\u003eHaBRC Z2\u003c/em\u003e would exert a broader cascade amplification regulatory effect on \u003cem\u003eHaCHT4\u003c/em\u003e, ultimately compromising PM structure and barrier integrity during critical developmental stages like molting. This would increase midgut permeability to conventional insecticides, Bt toxins, pathogenic microorganisms, etc. Therefore, targeting \u003cem\u003eHaBRC Z2\u003c/em\u003e may act as a promising sensitizing strategy. It can potentially enhance the efficacy and reduce the required dosage by destroying the physical barrier of insects to facilitate the arrival of exogenous substances at their target sites. This study not only provides a reference for the analysis of the basic mechanism, but more importantly, it suggests that \u003cem\u003eHaBRC Z2\u003c/em\u003e may serve as a potential molecular target in pest control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeqin Hu: writing the original draft, experimental design, performed experiments; Yuan Li: editing and revising the manuscript. Jingang Xie: data visualization. Hongsheng Pan: supervision, project management. Xiaoning Liu: editing and revising the manuscript, project management, funding obtained. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the datasets supporting the results of this article are provided within the manuscript or supplementary information files. The whole genome data was used in this study have been deposited in the NCBI database under accession numbers GCA_030705265.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31972279) and Excellent Doctoral Candidate Innovative Project of Xinjiang University in 2024 (XJU2024BS071).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao C, Wang L, Zhang K, Zhu X, Li D, Ji J, Luo J, Cui J. Variation of \u003cem\u003eHelicoverpa armigera\u003c/em\u003e symbionts across developmental stages and geographic locations. Front Microbiol. 2023;14:1251627.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaile F, Nowatzki T, Storer N. Overview of pest status, potential risk, and management considerations of \u003cem\u003eHelicoverpa armigera\u003c/em\u003e (Lepidoptera: Noctuidae) for U.S. soybean production. Integr J Pest Manage 2021, 12(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiaz S, Johnson J, Ahmad M, Fitt G, Naiker M. A review on biological interactions and management of the cotton bollworm, \u003cem\u003eHelicoverpa armigera\u003c/em\u003e (Lepidoptera: Noctuidae). J Appl Entomol 2021, 145.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad KF, Engel CK, Priv\u0026eacute; GG. Crystal structure of the BTB domain from PLZF. Proc Natl Acad Sci U S A. 1998;95(21):12123\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRezende-Teixeira P, Dusi RG, Jimenez PC, Espindola LS, Costa-Lotufo LV. What can we learn from commercial insecticides? Efficacy, toxicity, environmental impacts, and future developments. Environ Pollut. 2022;300:118983.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabashnik BE, Fabrick JA, Carri\u0026egrave;re Y. Global patterns of insect resistance to transgenic Bt crops: the first 25 years. J Econ Entomol. 2023;116(2):297\u0026ndash;309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu DQ, Luo SH, Abudunasier M, Cai XH, Feng MM, Liu XN, Wang DM. The effect of group IV chitinase, \u003cem\u003eHaCHT4\u003c/em\u003e, on the chitin content of the peritrophic matrix (PM) during larval growth and development of \u003cem\u003eHelicoverpa armigera\u003c/em\u003e. Pest Manag Sci. 2022;78(5):1815\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhokhani D, Carrera Carriel C, Vayla S, Irving TB, Stonoha-Arther C, Keller NP, An\u0026eacute; JM. Deciphering the chitin code in plant symbiosis, defense, and microbial networks. Annu Rev Microbiol. 2021;75:583\u0026ndash;607.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMun S, Noh MY, Geisbrecht ER, Kramer KJ, Muthukrishnan S, Arakane Y. Chitin deacetylases are necessary for insect femur muscle attachment and mobility. Proc Natl Acad Sci U S A. 2022;119(24):e2120853119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan A, Smagghe G, Li S, Shakeel M, Yang G, Ahmed N. Insect metamorphosis and chitin metabolism under miRNA regulation: a review with current advances. Pest Manag Sci. 2025;81(7):3437\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen DD, Wang ZB, Wang LX, Zhao P, Yun CH, Bai L. Structure, catalysis, chitin transport, and selective inhibition of chitin synthase. Nat Commun. 2023;14(1):4776.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, He L, Ding B, Yang H. Identification and functional characterization of the chitinase and chitinase-like gene family in \u003cem\u003eMyzus persicae\u003c/em\u003e (Sulzer) during molting. Pest Manag Sci. 2025;81(1):327\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong W, Gao YH, Zhang XB, Moussian B, Zhang JZ. Chitinase 10 controls chitin amounts and organization in the wing cuticle of \u003cem\u003eDrosophila\u003c/em\u003e. Insect Sci. 2020;27(6):1198\u0026ndash;207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen W, Yang Q. Development of novel pesticides targeting insect chitinases: a minireview and perspective. J Agric Food Chem. 2020;68(16):4559\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Ul Haq I, Khurshid A, Tao Y, Quandahor P, Zhou JJ, Liu CZ. Effects of abiotic stresses on the expression of chitinase-like genes in \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e. Front Physiol. 2022;13:1024136.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan XS, Shi MR, Xu J, Liu JH, Ye H. Interference efficiency and effects of bacterium-mediated RNAi in the fall armyworm (Lepidoptera: Noctuidae). J Insect Sci 2021, 21(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhajuria C, Buschman LL, Chen MS, Muthukrishnan S, Zhu KY. A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of \u003cem\u003eOstrinia nubilalis\u003c/em\u003e larvae. Insect Biochem Mol Biol. 2010;40(8):621\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng QH, Gong MF, Yang H, Chen NN, Lei Q, Jin DC. Effect of four chitinase genes on the female fecundity in \u003cem\u003eSogatella furcifera\u003c/em\u003e (Horv\u0026aacute;th). Pest Manag Sci. 2024;80(4):1912\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu ZZ, Zhang WY, Lin YZ, Li DQ, Shu BS, Lin JT. Genome-wide identification, characterization and functional analysis of the chitianse and chitinase-like gene family in \u003cem\u003eDiaphorina citri\u003c/em\u003e. Pest Manag Sci. 2022;78(4):1740\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKramer KJ, Muthukrishnan S. Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochem Mol Biol. 1997;27(11):887\u0026ndash;900.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi D, Zhang J, Wang Y, Liu X, Ma E, Sun Y, Li S, Zhu KY, Zhang J. Two chitinase 5 genes from \u003cem\u003eLocusta migratoria\u003c/em\u003e: molecular characteristics and functional differentiation. Insect Biochem Mol Biol. 2015;58:46\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Zheng S. 20-hydroxyecdysone enhances the expression of the chitinase 5 via Broad-Complex Zinc-Finger 4 during metamorphosis in silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Insect Mol Biol. 2017;26(2):243\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZollman S, Godt D, Priv\u0026eacute; GG, Couderc JL, Laski FA. The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in \u003cem\u003eDrosophila\u003c/em\u003e. Proc Natl Acad Sci U S A. 1994;91(22):10717\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuan JB, Ghanim M, Liu SS, Czosnek H. Silencing the ecdysone synthesis and signaling pathway genes disrupts nymphal development in the whitefly. Insect Biochem Mol Biol. 2013;43(8):740\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIjiro T, Urakawa H, Yasukochi Y, Takeda M, Fujiwara Y. cDNA cloning, gene structure, and expression of Broad-Complex (BR-C) genes in the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Insect Biochem Mol Biol. 2004;34(9):963\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAraus V, Vidal EA, Puelma T, Alamos S, Mieulet D, Guiderdoni E, Guti\u0026eacute;rrez RA. Members of BTB gene family of scaffold proteins suppress nitrate uptake and nitrogen use efficiency. Plant Physiol. 2016;171(2):1523\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Q, Chen J, Chen S, Gao X. SUMOylation modulates the dual functions of Kr\u0026uuml;ppel homolog 1 in transcriptional regulation of Broad-Complex expression. J Adv Res. 2025;75:123\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiulachs MD, Pagone V, Bell\u0026eacute;s X. Key roles of the Broad-Complex gene in insect embryogenesis. Insect Biochem Mol Biol. 2010;40(6):468\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiss I, Beaton AH, Tardiff J, Fristrom D, Fristrom JW. Interactions and developmental effects of mutations in the Broad-Complex of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. Genetics. 1988;118(2):247\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao C, Li J, Du W, Qin X, Cao J, Liu C, Cheng T. Broad Complex-Z2 directly activates BmMBF2 to inhibit the silk protein synthesis in the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Int J Biol Macromol. 2024;277(Pt 3):134211.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa H, Abbas MN, Zhang K, Hu X, Xu M, Liang H, Kausar S, Yang L, Cui H. 20-Hydroxyecdysone regulates the transcription of the lysozyme via Broad-Complex Z2 gene in silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Dev Comp Immunol. 2019;94:66\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCong J, Tao C, Zhang X, Zhang H, Cheng T, Liu C. Transgenic ectopic overexpression of Broad Complex (BrC-Z2) in the silk gland inhibits the expression of slk fibroin genes of \u003cem\u003eBombyx mori\u003c/em\u003e. Insects. 2020;11(6):374.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng H, Niu K, Zhang J, Feng Q. BmBR-C Z4 is an upstream regulatory factor of \u003cem\u003eBmPOUM2\u003c/em\u003e controlling the pupal specific expression of \u003cem\u003eBmWCP4\u003c/em\u003e in the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Insect Biochem Mol Biol. 2015;66:42\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing N, Wang Z, Geng N, Zou H, Zhang G, Cao C, Li X, Zou C. Silencing Br-C impairs larval development and chitin synthesis in \u003cem\u003eLymantria dispar\u003c/em\u003e larvae. J Insect Physiol. 2020;122:104041.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang GM, Tan WJ, Guo YY. An improvement in the technique of artificial raring cotton bollworm. 1999.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, Liu Y, Feng J, Chen H, He Y, et al. TBtools-II: A one for all, all for one bioinformatics platform for biological big-data mining. Mol Plant. 2023;16(11):1733\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu DQ, Zhang LSH, Liu YL. Cloning, prokaryotic expression and spatio-temporal expression profiling of BR-C Z2 gene in \u003cem\u003eHelicoverpa armigera\u003c/em\u003e. J Plant Prot. 2021;48(05):10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKonno K, Mitsuhashi W. The peritrophic membrane as a target of proteins that play important roles in plant defense and microbial attack. J Insect Physiol. 2019;117:103912.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang JW, Wang Q, Jiang YM, Jiang YR, Wang Y, Liu W. Group V chitin deacetylases are responsible for the structure and barrier function of the gut peritrophic matrix in the chinese oak silkworm \u003cem\u003eAntheraea pernyi\u003c/em\u003e. Int J Mol Sci 2024, 26(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu A, Beck M, Merzendorfer H, Yang Q. Advances in understanding insect chitin biosynthesis. Insect Biochem Mol Biol. 2024;164:104058.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Cooper AMW, Zhang J, Zhu KY. Biosynthesis, modifications and degradation of chitin in the formation and turnover of peritrophic matrix in insects. J Insect Physiol. 2019;114:109\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo W, Kain W, Wang P. Effects of disruption of the peritrophic membrane on larval susceptibility to Bt toxin Cry1Ac in cabbage loopers. J Insect Physiol. 2019;117:103897.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu H, Zhao D, Guo XC, Liu ZR, Li RJ, Lu XJ, Guo W. Group V chitin deacetylases influence the structure and composition of the midgut of beet armyworm, \u003cem\u003eSpodoptera exigua\u003c/em\u003e. Int J Mol Sci. 2023;24(4):3076.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu MB, Sun SP, Liu YS, Deng XR, Yang J, Yang Q. Insect group II chitinase \u003cem\u003eOfChtII\u003c/em\u003e promotes chitin degradation during larva-pupa molting. Insect Sci. 2021;28(3):692\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Wei Y, Wei L, Liu X, Liu N. Effects of transgenic cotton lines expressing ds\u003cem\u003eAgCYP6CY3-P1\u003c/em\u003e on the growth and detoxification ability of \u003cem\u003eAphis gossypii\u003c/em\u003e glover. Pest Manag Sci. 2023;79(1):481\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMai T, Chen S, Lin X, Zhang X, Zou X, Feng Q, Zheng S. 20-hydroxyecdysone positively regulates the transcription of the antimicrobial peptide, lebocin, via BmEts and BmBR-C Z4 in the midgut of \u003cem\u003eBombyx mori\u003c/em\u003e during metamorphosis. Dev Comp Immunol. 2017;74:10\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo MP, Qian WL, He XC, Peng J, Wang P, Wang WN, Xia QY, Cheng DJ. Genome-wide identification of target genes for transcription factor BR-C in the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Insect Sci. 2021;28(6):1530\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbole RS, Sharma S, Patil Y, Giri AP, Joshi RS. Chitinase inhibition induces transcriptional dysregulation altering ecdysteroid-mediated control of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e development. \u003cem\u003eiScience\u003c/em\u003e 2024, 27(3):109280.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Xu G, Qiu B, Zhang X, Feng Q, Yang Q, Zheng S. BR-C Z4 and FoxJ interact to regulate expression of a chitin synthase gene CHSA-2b in the pupal wing discs of the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Insect Biochem Mol Biol. 2020;116:103264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang ZL, Xu QY, Zhang R, Shen C, Bao HB, Luo GH, Fang JC. The irregular developmental duration mainly caused by the broad-complex in \u003cem\u003eChilo suppressalis\u003c/em\u003e. Pestic Biochem Physiol. 2024;204:106090.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJadhav V, Vaishnaw A, Fitzgerald K, Maier MA. RNA interference in the era of nucleic acid therapeutics. Nat Biotechnol. 2024;42(3):394\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLucena-Leandro VS, Abreu EFA, Vidal LA, Torres CR, Junqueira C, Dantas J, Albuquerque \u0026Eacute;VS. Current scenario of exogenously induced RNAi for lepidopteran agricultural pest control: from dsRNA design to topical application. Int J Mol Sci 2022, 23(24).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Kong W, Li T, Zhang L, Zhuang Z, Liu N, Liu X. Functional analysis of lactase phlorizin hydrolase in insect-plant coevolution based on deglycosylation. J Agric Food Chem. 2025;73(9):5140\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu X, Li T, Zhang L, Liu X. Effect of silencing the E74B gene on the development and metamorphosis of \u003cem\u003eHelicoverpa armigera\u003c/em\u003e. Pest Manag Sci. 2024;80(3):1435\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie J, Wang S, Zhuang Z, Wang X, Lin M, Liu X. Exploring the role of \u003cem\u003eCYP6AB328\u003c/em\u003e in spinetoram resistance and growth and development of \u003cem\u003ePhthorimaea absoluta\u003c/em\u003e. Pestic Biochem Physiol. 2025;208:106316.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Helicoverpa armigera, Broad complex gene, Molecular mechanism, RNAi, growth and development","lastPublishedDoi":"10.21203/rs.3.rs-8610350/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8610350/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChitinases and ecdysone are both crucial for insect growth and development, yet the regulatory interplay between them remains poorly understood. Our previous research demonstrated that the chitinase gene \u003cem\u003eHaCHT4\u003c/em\u003e critically regulates the content of chitin and peritrophic membrane\u0026rsquo;s (PM) structure in \u003cem\u003eHelicoverpa armigera\u003c/em\u003e (H\u0026uuml;bner) (Lepidoptera: Noctuidae).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, a 1186 bp promoter region of \u003cem\u003eHaCHT4\u003c/em\u003e was cloned to investigate its regulation mechanism. Bioinformatics analysis predicted the presence of several 20-hydroxyecdysone (20E) related cis-regulatory elements (CREs) within this promoter, including Broad-Complex Zinc-Finger isoforms (BRCs), GATA, NF-AT1, and POU1F1. Notably, genome-wide identification and characterization revealed that the \u003cem\u003eH. armigera\u003c/em\u003e BRC gene exhibits the highest similarity with that of \u003cem\u003eBombyx mori\u003c/em\u003e. Molecular docking and EMSA demonstrated the specific binding between \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e. Expression analysis showed concomitant upregulation of \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e during the late (4th to 5th ) instar stages, and were also strongly induced by 20E. In addition, RNA interference (RNAi) experiments further supported this regulatory relationship, a substantial decrease in the transcript levels of both \u003cem\u003eHaBRC Z2\u003c/em\u003e and \u003cem\u003eHaCHT4\u003c/em\u003e were observed after \u003cem\u003eHaBRC Z2\u003c/em\u003e silencing for 24, 48, 72, and 96 h. The knockdown of \u003cem\u003eHaBRC Z2\u003c/em\u003e not only stunted larval growth, evidenced by reduced body length and weight, but also confirmed its role as an activator of \u003cem\u003eHaCHT4\u003c/em\u003e transcription during the larval transition.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings reveal a critical regulatory relationship between chitinase and ecdysone, underscoring the significance of \u003cem\u003eHaBRC Z2\u003c/em\u003e as promising targets for informing future pest control research.\u003c/p\u003e","manuscriptTitle":"Broad complex Z2 regulates the group Ⅳ chitinase gene HaCHT4 in the midgut of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 12:58:18","doi":"10.21203/rs.3.rs-8610350/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-02T18:13:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T05:32:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-29T08:36:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T16:49:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30509297501523528710135437062889806093","date":"2026-01-23T07:07:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242550094991689970717789172168769307730","date":"2026-01-22T17:53:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24375671790357606277424714181525829211","date":"2026-01-22T06:58:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13229532916669232104081369956640939012","date":"2026-01-22T06:28:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-22T01:35:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-21T18:33:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-18T23:32:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-18T23:31:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2026-01-15T12:03:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b33200a-a3a4-4440-906a-5a19c5811736","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:05:04+00:00","versionOfRecord":{"articleIdentity":"rs-8610350","link":"https://doi.org/10.1186/s12864-026-12806-8","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2026-04-10 15:58:35","publishedOnDateReadable":"April 10th, 2026"},"versionCreatedAt":"2026-01-28 12:58:18","video":"","vorDoi":"10.1186/s12864-026-12806-8","vorDoiUrl":"https://doi.org/10.1186/s12864-026-12806-8","workflowStages":[]},"version":"v1","identity":"rs-8610350","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8610350","identity":"rs-8610350","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}