Role of Col1a2 in Collagen Deposition in the Carapace of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis): From Molecular Evolution to Expression Profile and Then to Function Validation

The carapace of the Chinese soft-shelled turtle (Pelodiscus sinensis) is rich in collagen and stands as a crucial economic trait for assessing its quality, as well as a key indicator for selective breeding. However, current studies on the mechanisms underlying collagen deposition in the carapace remain severely limited, significantly hindering progress in selective breeding. Here, the Col1a2 gene of P. sinensis was molecularly characterized for the first time. Analysis of gene structure, phylogenetic tree, and amino acid sequence homology revealed that Col1a2 is relatively conserved among tetrapods but divergent from fishes. Collinearity analysis identified the BET1-COL1A2-CASD1-SGCE gene block shared across all 14 representative vertebrates and found that the Col1a2 is located on the Z chromosome of Thamnophis elegans. Tissue expression analysis showed that Col1a1 was highly expressed in the heart, gonad, and lung. Additionally, Col1a1 expression levels markedly increased during carapace development, exhibiting a strongly positive correlation with the changes in collagen content of the carapace. In situ hybridization results revealed strong signal for the Col1a2 transcripts in fibroblasts of the dermal layer of P. sinensis carapace. Knockdown of the Col1a2 gene in the carapace cells of P. sinensis significantly reduced collagen content. Transcriptome analysis following Col1a2 knockdown identified several differentially expressed genes associated with collagen deposition, including Fbln2, IL-11, and Rspo4, as well as significantly enriched pathways such as the JAK-STAT signaling pathway, the apelin signaling pathway, and the Hippo signaling pathway. Our findings offer a molecular basis for elucidating the mechanisms of collagen deposition in the carapace of P. sinensis, while also supplying a potential target for the selective breeding of collagen-rich strains of P. sinensis.