Complement C9, as a member of terminal complement component (TCC) protein, plays important roles in innate immunity. However, some complement components appear to show difference and evolutionary complexity between higher and lower vertebrates. Hence, it is essential to carry on a study of evolutionary origin and systematic function of C9 in fish and non-fish vertebrates. This study aims to explore the complement gene evolution and potential function in fish based on molecular and structural biology. Herein, we found complete divergence of C9 throughout the gene evolution. The optimal codons of C9 sequences tended to be closer to the genomes of lower vertebrates compared to higher vertebrates. Further, conserved amino acids in the C9 TMH1 region were identified, implying their potential functional association with MAC growth and pore formation. Transposons and simple repeats, as gene elements, exhibited a differential distribution in the genomic regions in different animal groups but were sparsely scattered around the sixth exon (TMH1 region). Notably, this demonstrated the regulatory complexity of the C9 gene in higher vertebrates. The negative selection pressures on fish and non-fish groups improved both the sequence conservation and similarity. Through gene/protein regulatory network and pathway analyses, the systematic function of C9 protein was showcased; thus, we could reveal the divergence of the systematic function of C9 across species from different evolutionary positions. In addition, more complicated functions of C9 in higher vertebrates could established by the altered spatial conformation of the protein. Collectively, the present study illustrates the C9 gene evolutionary process and the difference in its systematic function across multiple species. Such advances provide new insights for understanding the evolutionary and potential functions of complement C9.

补体C9作为末端补体成分（TCC）蛋白的成员，在先天免疫中起重要作用。但是，某些补体成分似乎显示出较高和较低脊椎动物之间的差异和进化复杂性。因此，有必要进行C9在鱼类和非鱼类脊椎动物中的进化起源和系统功能的研究。这项研究旨在探索基于分子和结构生物学的鱼类补体基因的进化和潜在功能。在本文中，我们发现在整个基因进化过程中C9完全不同。与高等脊椎动物相比，C9序列的最佳密码子倾向于更接近低等脊椎动物的基因组。此外，鉴定了C9 TMH1区中的保守氨基酸，这暗示了它们与MAC生长和孔形成的潜在功能关联。转座子和简单重复序列作为基因元件，在不同动物组的基因组区域表现出不同的分布，但稀疏地散布在第六个外显子（TMH1区域）周围。值得注意的是，这表明了监管的复杂性高等脊椎动物中的C9基因。对鱼类和非鱼类群体的负选择压力改善了序列保守性和相似性。通过基因/蛋白质调控网络和途径分析，展示了C9蛋白的系统功能。因此，我们可以揭示不同进化位置的物种之间C9系统功能的差异。此外，在高等脊椎动物中，C9的更复杂功能可以通过改变蛋白质的空间构象来建立。总体而言，本研究说明了C9基因的进化过程以及其跨多个物种的系统功能的差异。这些进展为理解补体C9的进化和潜在功能提供了新的见解。