DOI: 10.5061/dryad.bg79cnpmw
title: Knockdown of BmorCPR67 gene disrupts prepupal–pupal transition of silkworm Bombyx mori by thinning the endocuticle

description: The cuticle of insects serves as a crucial organ for preserving body
 composition, protecting against pathogen invasion, and retaining moisture
 in their bodies. Cuticular proteins (CPs) are the main constituents of
 insect cuticles and interact with chitin to form cuticle structural
 framework and mechanical properties. In this study, we investigated the
 role of a cuticular protein with R&R consensus (CPR), BmorCPR67, a
 member of the RR-2 subfamily, during the prepupal-to-pupal transition in
 Bombyx mori. The BmorCPR67 gene exhibited high expression levels during
 the prepupal stage, with the highest expression detected in the epidermis
 of the day-1 pupa in B. mori. And the expression of BmorCPR67 gene was
 induced by 20-hydroxyecdysone (20E). Chitin-binding assays indicated that
 the BmorCPR67 protein selectively binds to crystalline chitin and chitosan
 but not to amorphous chitin. Silencing BmorCPR67 gene disrupted the
 molting process from prepupa to pupa, resulting in silkworms mortality.
 Furthermore, knockdown of BmorCPR67 altered the expression profiles of key
 genes involved in chitin metabolism. Notably, significant thinning of the
 endocuticle was observed 48 to 96 h after siRNA injection in
 BmorCPR67-silenced silkworms. These findings highlight the critical role
 of BmorCPR67 in cuticle development during the prepupal-to-pupal
 transition in B. mori, contributing to our understanding of the functions
 of CPs in insect metamorphosis.
description: # Knockdown of BmorCPR67 gene disrupts prepupal–pupal transition of
 silkworm Bombyx mori by thinning the endocuticle
 [https://doi.org/10.5061/dryad.bg79cnpmw](https://doi.org/10.5061/dryad.bg79cnpmw) ## Description of the data and file structure Knockdown of BmorCPR67 gene disrupts prepupal–pupal transition of silkworm Bombyx mori by thinning the endocuticle This dataset contains data and analysis code related to the research on the role of BmorCPR67 gene disrupts prepupal–pupal transition of silkworm Bombyx mori. The BmorCPR67 gene exhibited high expression levels during the prepupal stage, with the highest expression detected in the epidermis of the day-1 pupa in B. mori. And the expression of BmorCPR67 gene was induced by 20-hydroxyecdysone (20E). Chitin-binding assays indicated that the BmorCPR67 protein selectively binds to crystalline chitin and chitosan but not to amorphous chitin. Silencing BmorCPR67 gene disrupted the molting process from prepupa to pupa, resulting in silkworms mortality. Furthermore, knockdown of BmorCPR67 altered the expression profiles of key genes involved in chitin metabolism. Notably, significant thinning of the endocuticle was observed 48 to 96 h after siRNA injection in BmorCPR67-silenced silkworms. These findings highlight the critical role of BmorCPR67 in cuticle development during the prepupal-to-pupal transition in B. mori, contributing to our understanding of the functions of CPs in insect metamorphosis. ### Files File: BmorCPR67 mRNA sequence.fas Description: This data is the sequence of BmorCPR67 mRNA. We designed primers and siRNAS based on this sequence. File: RR2 sequence for phylogenetic analysis.fas Description: This is the homologous sequences of BmorCPR67 sequence downloaded from the National Center for Biotechnology Information ([https://blast.ncbi.nlm.nih.gov/Blast.cgi](https://blast.ncbi.nlm.nih.gov/Blast.cgi)). Based on the these sequence, we constructed a phylogenetic tree using the Neighbour-joining method in MEGA 6.0 software. File: BmorCPR71 mRNA sequence.fas Description: This data is the sequence of BmorCPR71 mRNA. File: BmHR38 CDS.fas Description: This data is the sequence of BmHR38 CDS. We designed dsRNA based on this sequence. FIGURE 1 Bioinformatics analysis of BmorCPR67 in Bombyx mori. (a) Structural domain of BmorCPR67 protein. Pfam:chitin-bind-4: amino acid motif known as the R&R consensus. The extensive conservation of this region led to the suggestion that it functions to bind chitin (57-109). RPT1: an internal repeat, detected by the Prospero program. (b) Phylogenetic analysis of BmorCPR67 protein with other insect cuticular proteins. FIGURE 2 The expression profiles of BmorCPR67 gene in developmental stages and various tissues of Bombyx mori. (a) Expression of BmorCPR67 from the 5th instar day-3 to the day-2 of the pupal stage in B. mori. (b) Expression of BmorCPR67 gene in tissues of B. mori prepupal stage. (c) Expression of BmorCPR67 gene in tissues of B. mori day-1 of pupal stage. The expression of BmorCPR67 mRNA in tissues was determined by qRT-PCR. B. mori 18S rRNA was used as the internal control gene. Bars represent mean ± S.E. (n = 3). Bars labeled with different letters are significantly different (P &lt; 0.05). FIGURE 3 Effect of 20E on BmorCPR67 gene expression. (a) Expression profiles of BmorCPR67 after 20E injection in vivo. 5 μg 20-ecdysterone (5 μg/μL) was injected into the 5th instar day-3 of B. mori larvae. The same volume of 5% DMSO was injected as the control. Each treatment was three replicates. After 6 h, 12 h, 24 h or 48 h injection, whole larvae of B. mori were collected, respectively. The expression levels of BmorCPR67 gene induced by 20E were analyzed by qRT-PCR. (b) Expression profiles of BmorCPR67 in epidermic after 20E incubation in vitro. The epidermic from 5th instar day-3 larvae was dissected and incubated in Grace’s medium. The 20-hydroxyecdysone was added to final concentration of 2 μg/ml, and the same volume of 5% DMSO was added as control. The expression level of BmorCPR67 gene in epidermic after 20E injection at 6 h, 12 h,24 h and 48 h was analyzed by RT-qPCR. (c) Expression profiles of BmorCPR67 after BmHR38 gene knockdown. The gene expressions of BmorCPR67 were detected by RT-qPCR at 24 and 48 h after dsBmHR38 injection. Bars represent mean ± S.E. (n = 3). Bars labeled with asterisk are significantly different (t-test; *, P &lt;0.05; **, P &lt; 0.01; ***, P &lt; 0.001). FIGURE 4 Expression of BmorCPR67 protein and its binding to chitin (a) Prokaryotic expression and purification of BmorCPR67 protein. The vector used for BmorCPR67 protein expression is pET28a(+). M: Protein molecular weight standards; Lane 1: pET28a(+) empty vector; Lane 2: withou IPTG; Lane 3: 0.1 mM IPTG; Lane 4: 0.2 mM IPTG; Lane 5: 0.6 mM IPTG; Lane 6: 0.8 mM IPTG; Lane 7: 1 mM IPTG; Lane 8: Supernatant protein; Lane 9: Sediment protein; Lane 10: Purified BmorCPR67 protein. (b) BmorCPR67 binds to crystalline chitin and amorphous chitin. M: Protein molecular weight standards; C: BmorCPR67 protein; Crystal Chitin-FT: flow through indicating crystalline chitin unbound proteins; Crystal Chitin-E: elution indicating crystalline chitin bound proteins; Amorphous Chitin-FT: flow through indicating amorphous chitin unbound proteins; Amorphous Chitin-E: elution indicating amorphous chitin bound proteins. (c) Binding of BmorCPR67 to chitosan. The combination experiment was carried out with 1 mg/ml chitosan and 0.5mg/ml BmorCPR67. 1 mg/ml chitosan and 0.5 mg/ml BmorCPR67 were as control. FIGURE 5 Knockdown of BmorCPR67 gene at the wandering stage disrupts prepupal–pupal transition of Bombyx mori (a) Expression levels of BmorCPR67 gene in epidermis after three siBmorCPR67s injection. (b) Expression levels of BmorCPR71 gene in epidermis after siBmorCPR67-1 injection. (c) Body weight of B. mori after silencing BmorCPR67. (d) Phenotype of B. mori after silencing BmorCPR67. (e) Mortality rate of B. mori after silencing BmorCPR67. (f) Chitin content of B. mori epidermis after injection of BmorCPR67-1. Bars represent mean ± S.E. (n = 3). Bars labeled with asterisk are significantly different (t-test; *, P &lt;0.05; **, P &lt; 0.01; ***, P &lt; 0.001). FIGURE 6 Effects of BmorCPR67 silencing at the wandering stage on the chitin metabolic pathway gene expressions The mRNA expressions in the chitin metabolism genes (Tre, G6PI, PGM, GFAT, GPN, Chs, CHT, NAG, Chi-h and CDA) were detected by RT-qPCR at 24 h or 48 h or 72 h after siBmorCPR67-1 injection. Bars represent mean ± S.E. (n = 3). Bars labeled with asterisk are significantly different (t-test; *, P &lt;0.05; **, P &lt; 0.01; ***, P &lt; 0.001). FIGURE 7 Observation of epidermis formation after silencing BmorCPR67 by epidermal paraffin sections After injection of siBmorCPR67-1 to knockdown the BmorCPR67 gene, the epidermis from the day-1 to the day-3 of pupal stage of B. mori were collected, and the epidermal paraffin sections were prepared and stained by HE. Observe the formation of B. mori pupal epidermis under microscope. siEGFP injection was used as control. Table 1 Sequence of PCR primers and siRNAs used in this study.