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 (<i>Pelodiscus sinensis</i>) 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 <i>Col1a2</i> gene of <i>P. sinensis</i> was molecularly characterized for the first time. Analysis of gene structure, phylogenetic tree, and amino acid sequence homology revealed that <i>Col1a2</i> 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 <i>Col1a2</i> is located on the Z chromosome of <i>Thamnophis elegans</i>. Tissue expression analysis showed that <i>Col1a1</i> was highly expressed in the heart, gonad, and lung. Additionally, <i>Col1a1</i> 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 <i>Col1a2</i> transcripts in fibroblasts of the dermal layer of <i>P. sinensis</i> carapace. Knockdown of the <i>Col1a2</i> gene in the carapace cells of <i>P. sinensis</i> significantly reduced collagen content. Transcriptome analysis following <i>Col1a2</i> knockdown identified several differentially expressed genes associated with collagen deposition, including <i>Fbln2</i>, <i>IL-11</i>, and <i>Rspo4</i>, 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 <i>P. sinensis</i>, while also supplying a potential target for the selective breeding of collagen-rich strains of <i>P. sinensis</i>.