基于DIA蛋白质组学解析菲律宾蛤仔壳色分化的多通路协同调控机制

Multi-Pathway Coordinated Regulatory Mechanisms Underlying Shell Color Differentiation in Ruditapes philippinarum Revealed by DIA-Based Proteomics

摘要: 为揭示菲律宾蛤仔(Ruditapes philippinarum)同一壳体黑白壳区域色泽分化的分子机制，本研究采用数据非依赖采集(DIA)蛋白质组学技术，对黑壳(B组)与白壳(W组)区域对应的外套膜组织进行差异蛋白筛选及功能解析。通过主成分分析(PCA)验证样本重复性，结合P值(<0.05)与差异倍数(FC ≥ 1.5或≤1/1.5)筛选显著差异蛋白，同步开展GO功能富集与KEGG通路富集分析。结果显示，共筛选获得1500个显著差异表达蛋白，其中下调蛋白数量(1117个)约为上调蛋白(383个)的2.9倍，提示蛋白表达抑制在壳色分化中占主导。KEGG富集分析发现，差异蛋白显著富集于溶酶体、SNARE、Spliceosome三条核心通路：溶酶体通路中CTSL、CTSS蛋白下调抑制壳基质更新；SNARE通路Syntaxin、SNAP-25等蛋白下调阻碍囊泡运输与色素分泌；Spliceosome通路SF3B/U2复合体、PRPF/SF蛋白下调影响转录后RNA加工效率。三条通路与能量代谢通路协同作用，形成“能量代谢–物质分泌–转录后加工”的调控网络，共同介导蛤仔壳色差异。本研究从蛋白质组层面揭示了蛤仔壳色分化的多通路协同调控机制，填补了贝类壳色形成中RNA加工层面调控的研究空白，为蛤仔壳色性状的分子育种提供理论依据。

Abstract: To elucidate the molecular mechanisms underlying color differentiation between black and white shell regions within the same shell of the Manila clam (Ruditapes philippinarum), this study employed data-independent acquisition (DIA) proteomics to identify and functionally characterize differentially expressed proteins in mantle tissues corresponding to black (B group) and white (W group) shell areas. Sample reproducibility was validated by principal component analysis (PCA). Significantly differentially expressed proteins were screened using a P value (<0.05) combined with fold-change criteria (FC ≥ 1.5 or ≤1/1.5), followed by Gene Ontology (GO) functional enrichment and KEGG pathway enrichment analyses. The results identified a total of 1,500 significantly differentially expressed proteins were identified, among which downregulated proteins (1117) were approximately 2.9 times more abundant than upregulated proteins (383), indicating that suppression of protein expression plays a dominant role in shell color differentiation. KEGG enrichment analysis revealed that these proteins were significantly enriched in three core pathways: lysosome, SNARE, and spliceosome. In the lysosome pathway, downregulation of CTSL and CTSS inhibited shell matrix turnover; in the SNARE pathway, reduced expression of proteins such as syntaxin and SNAP-25 impeded vesicle transport and pigment secretion; and in the spliceosome pathway, downregulation of the SF3B/U2 complex and PRPF/SF proteins affected the efficiency of post-transcriptional RNA processing. These three pathways act synergistically with energy metabolism pathways to form an integrated regulatory network of “energy metabolism - material secretion - post-transcriptional processing”, collectively mediating shell color differences in the Manila clam. At the proteomic level, this study reveals a multi-pathway coordinated regulatory mechanism underlying shell color differentiation, fills a knowledge gap regarding RNA-processing-level regulation in molluscan shell coloration, and provides a theoretical basis for molecular breeding of shell color traits in clams.