Identification of Cis-Regulatory Elements Involved in Mutually Exclusive Alternative Splicing of Exon 3 in SfGluCl from Spodoptera frugiperda

Background/Objectives: Glutamate-gated chloride channels (GluCls) are essential inhibitory neurotransmitter receptors in insects and key targets of several insecticides. In Spodoptera frugiperda (FAW), mutually exclusive alternative splicing (MEAS) of exon 3 in SfGluCl produces three splice variants (SfGluCl 3A, SfGluCl 3B and SfGluCl 3C), yet the cis-regulatory elements involved in this splicing event remain unknown. This study aimed to identify candidate cis-regulatory regions associated with exon 3 MEAS and characterize their effects on exon 3 selection in a Sf9 cell-based minigene system. Methods: Semi-quantitative RT-PCR was used to assess the expression levels of three splice variants of exon 3 in SfGluCl across developmental stages. Comparative sequence analysis, combined with intron-deletion and exon-exchange assays, were performed to identify candidate cis-regulatory regions. A total of 27 constructs (23 intron-deletion and 4 exon-exchange constructs) were generated and analyzed using a minigene splicing assay in Sf9 cells. Results: Three splice variants were consistently detected across all developmental stages. In the Sf9 cell-based minigene system, two candidate cis-regulatory regions affecting SfGluCl 3B inclusion were identified: a 20 bp intronic region spanning nucleotides +13 to +32 downstream of the 5′ splice junction in the intron between exons 3B and 3C, corresponding to the region deleted in ExonB-C (Exon 3B-exon 3C)-Intdel52, and an 11 bp region within exon 3B containing six nucleotide substitutions in Ex3B (Exchanged exon 3B)-SubA2. Deleting the ExonB-C-Intdel52 or modifying the Ex3B-SubA2 significantly increased the SfGluCl 3B inclusion. Conclusions: These candidate intronic and exonic regions can influence exon 3B inclusion in the Sf9 cell-based minigene system. Although their functional effects and in vivo relevance remain to be validated in further study, our findings still provide a basis for future studies of GluCl splice variant regulation, receptor biology, and insecticide response in insects.