*2.2. Phylogenetic Analysis and Classification of NnDofs*

To fully comprehend the evolutionary relationship, a phylogenetic tree was constructed with the 29 identified *NnDofs* and the 36 reported *AtDofs* of Arabidopsis (Figure 1). The results indicated that 65 Dof proteins were grouped into seven major groups, with 29 NnDof proteins in each of the following categories: II, III, IV, V, VI, and VII. Group VI had nine NnDof proteins, accounting for 31.03 percent of the total number of *NnDofs*, while group V had only two, accounting for 6.90 percent. Groups II, III, VI, and VII each have five NnDof proteins.

Multiple sequence alignment analysis of the amino acids of the Dof structural domain of 29 lotus flowers was performed to investigate the sequence characteristics of the NnDof proteins. It was found that the NnDof proteins' structural domain sequences were highly conserved and all contained the typical CX2CX21CX2C motif, where one Zn2+ can covalently combine with four Cys residues (Figure 2).
