{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T04:59:03Z","timestamp":1785560343131,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T00:00:00Z","timestamp":1785369600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"crossref","award":["2022YFD1700200"],"award-info":[{"award-number":["2022YFD1700200"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Postgraduate Research Practice Innovation Program of Jiangsu Province","award":["KYCX23_0816"],"award-info":[{"award-number":["KYCX23_0816"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["31972346"],"award-info":[{"award-number":["31972346"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["31972346"],"award-info":[{"award-number":["31972346"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>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\u2032 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.<\/jats:p>","DOI":"10.3390\/genes17080899","type":"journal-article","created":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:38:54Z","timestamp":1785411534000},"page":"899","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification of Cis-Regulatory Elements Involved in Mutually Exclusive Alternative Splicing of Exon 3 in SfGluCl from Spodoptera frugiperda"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-8416-4626","authenticated-orcid":false,"given":"Kaixiang","family":"Lin","sequence":"first","affiliation":[{"name":"State Key Laboratory of Agricultural and Forestry Biosecurity, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Key Laboratory of Integrated Pest Management on Crops in East China, Ministry of Agriculture and Rural Affair, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1906-8091","authenticated-orcid":false,"given":"Zijiao","family":"Song","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural and Forestry Biosecurity, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Key Laboratory of Integrated Pest Management on Crops in East China, Ministry of Agriculture and Rural Affair, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiutang","family":"Huang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural and Forestry Biosecurity, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Key Laboratory of Integrated Pest Management on Crops in East China, Ministry of Agriculture and Rural Affair, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0054-8861","authenticated-orcid":false,"given":"Ali","family":"Hasnain","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural and Forestry Biosecurity, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Key Laboratory of Integrated Pest Management on Crops in East China, Ministry of Agriculture and Rural Affair, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3128-673X","authenticated-orcid":false,"given":"Tao","family":"Tang","sequence":"additional","affiliation":[{"name":"Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4019-5140","authenticated-orcid":false,"given":"Wei","family":"Xu","sequence":"additional","affiliation":[{"name":"School of Agricultural Sciences, Murdoch University, Perth 6150, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-8796-7742","authenticated-orcid":false,"given":"Priscilla","family":"Amponsah","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural and Forestry Biosecurity, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Key Laboratory of Integrated Pest Management on Crops in East China, Ministry of Agriculture and Rural Affair, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7942-488X","authenticated-orcid":false,"given":"Chunqing","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural and Forestry Biosecurity, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"},{"name":"Key Laboratory of Integrated Pest Management on Crops in East China, Ministry of Agriculture and Rural Affair, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1016\/S2095-3119(20)63367-6","article-title":"Biology, invasion and management of the agricultural invader: Fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)","volume":"20","author":"Jing","year":"2021","journal-title":"J. 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