Trehalose-6-phosphate synthase (TPS) is a key enzyme in the biosynthesis of trehalose, with its direct product, trehalose-6-phosphate, playing important roles in regulating whole-plant carbohydrate allocation and utilization. Genes encoding TPS constitute a multigene family in which functional divergence appears to have occurred repeatedly. To identify the crucial evolutionary amino acid sites of TPS in higher plants, a series of bioinformatics tools were applied to investigate the phylogenetic relationships, functional divergence, positive selection, and co-evolution of TPS proteins. First, we identified 150 TPS genes from 13 higher plant species. Phylogenetic analysis placed these TPS proteins into 2 clades: clades A and B, of which clade B could be further divided into 4 subclades (B1-B4). This classification was supported by the intron-exon structures, with more introns present in clade A. Next, detection of the critical functionally divergent amino acid sites resulted in the isolation of a total of 286 sites reflecting nonredundant radical shifts in amino acid properties with a high posterior probability cutoff among subclades. In addition, positively selected sites were identified using a codon substitution model, from which 46 amino acid sites were isolated as exhibiting positive selection at a significant level. Moreover, 18 amino acid sites were highlighted both for functional divergence and positive selection; these may thus potentially represent crucial evolutionary sites in the TPS family. Further co-evolutionary analysis revealed 3 pairs of sites: 11S and 12H, 33S and 34N, and 109G and 110E as demonstrating co-evolution. Finally, the 18 crucial evolutionary amino acid sites were mapped in the 3-dimensional structure. A total of 77 sites harboring functionally and structurally important residues of TPS proteins were found by using the CLIPS-4D online tool; notably, no overlap was observed with the identified crucial evolutionary sites, providing positive evidence supporting their designation. A total of 18 sites were isolated as key amino acids by using multiple bioinformatics tools based on their concomitant functional divergence and positive selection. Almost all these key sites are located in 2 domains of this protein family where they exhibit no overlap with the structurally and functionally conserved sites. These results will provide an improved understanding of the complexity of the TPS gene family and of its function and evolution in higher plants. Moreover, this knowledge may facilitate the exploitation of these sites for protein engineering applications.

海藻糖-6-磷酸合酶（TPS）是海藻糖生物合成的关键酶，其直接产物海藻糖-6-磷酸在调节全植物碳水化合物的分配和利用中发挥着重要作用。编码TPS的基因构成了一个多基因家族，其中功能分歧似乎反复发生。为了鉴定高等植物中TPS的关键进化氨基酸位点，应用一系列生物信息学工具来研究TPS蛋白的系统发育关系、功能分歧、正选择和共同进化。首先，我们从 13 种高等植物中鉴定出 150 个 TPS 基因。系统发育分析将这些TPS蛋白分为2个进化枝：进化枝A和B，其中进化枝B可进一步分为4个亚进化枝（B1-B4）。这种分类得到了内含子-外显子结构的支持，进化枝 A 中存在更多的内含子。接下来，对关键功能上不同的氨基酸位点的检测导致分离出总共 286 个位点，这些位点反映了氨基酸特性的非冗余激进转变，其中分支之间的高后验概率截止。此外，使用密码子替换模型鉴定了正选择位点，从中分离出46个氨基酸位点，显示出显着水平的正选择。此外，18 个氨基酸位点因功能分歧和正选择而被突出显示；因此，这些可能代表 TPS 家族中关键的进化位点。进一步的协同进化分析揭示了 3 对位点：11S 和 12H、33S 和 34N、以及 109G 和 110E，证明了协同进化。最后，18个关键的进化氨基酸位点被映射到3维结构中。通过使用CLIPS-4D在线工具，总共发现了77个含有功能和结构上重要的TPS蛋白残基的位点；值得注意的是，没有观察到与已确定的关键进化位点重叠，这为支持其指定提供了积极的证据。通过使用多种生物信息学工具，根据其伴随的功能分歧和正选择，总共分离出 18 个位点作为关键氨基酸。几乎所有这些关键位点都位于该蛋白质家族的 2 个结构域中，它们与结构和功能保守位点没有重叠。这些结果将有助于更好地理解 TPS 基因家族的复杂性及其在高等植物中的功能和进化。此外，这些知识可能有助于利用这些位点进行蛋白质工程应用。