Integrated Full-Length Transcriptomics, RNA-Seq and Peptidomics Define an Isoform-Resolved Skin Defense Repertoire in the Chinese Spiny Frog (Quasipaa spinosa)

Amphibian skin relies on secreted effectors and inducible immune pathways, but short-read RNA sequencing cannot reliably resolve the transcript isoforms and precursor structures underlying this defense system. We generated the first full-length skin transcriptome for the Chinese spiny frog (Quasipaa spinosa) by integrating pooled PacBio Iso-Seq data with six Illumina RNA-seq libraries and six skin peptidomic libraries from control and Proteus mirabilis-challenged frogs. Because Iso-Seq RNA was pooled across conditions, the long-read data were used to define transcript structure rather than condition-specific isoform changes. The reference contained 58,388 transcripts from 34,247 genes, including 32,215 non-reference models, and 5907 of 19,143 reconstructed loci had multiple isoforms. Among 108,147 predicted ORFs, 17,169 carried an N-terminal secretion signal, and 72 secretory and immune-effector ORFs from 46 genes were prioritized. Gene-level RNA-seq identified 72 differentially expressed genes at Benjamini–Hochberg-adjusted p < 0.05 and |log2FC| ≥ 1; 27 had higher expression in challenged skin and 45 in control skin. No GO term remained significant after correction, whereas three KEGG pathways did. Among these 72 genes, 60 (83.3%) had at least two full-length models, 53 (73.6%) contained at least one complete ORF, and 4 (5.6%) had peptide-sequence support. Reported peptide sequences matched 1029 entries across the full-length catalog, but shared peptide matches were not treated as ORF-specific evidence. Together, these data establish an isoform-resolved skin-defense resource for Q. spinosa and a framework for prioritizing secretory and immune candidates for isoform-specific and functional validation.