*2.1. Cloning and Analysis of the I1AP2 Sequence*

Based on transcriptome data, primers were designed to obtain the full-length ORF sequence of IlAP2. The ORF of *IlAP2* was 1296 bp and encoded 432 amino acids. Several proteins with high similarity to IlAP2 in other plant species were selected using Blast on the NCBI website, and their amino acid sequences were analyzed using ClustalX software (Figure 1a). The alignment results showed that IlAP2 contained two conserved AP2 domains (Figure 1a). A phylogenetic tree was constructed using the neighbor-joining method to further analyze the evolutionary relationship between the *IlAP2* and *AP2* genes in other plants. The results showed that *IlAP2* was most closely related to the genes of *Phoenix dactylifera* (Figure 1b).

To further understand the cytological function of the IlAP2 protein, we inserted *IlAP2* into a pCAMBIA expression vector with an eGFP tag and transgenically transformed *A. thaliana* protoplasts to observe the subcellular distribution of IlAP2 proteins. The *IlAP2*–GFP fusion protein was only expressed in the nucleus, while the empty GFP vector was expressed in all parts of the cell, which indicates that *IlAP2* is a nuclear-localized transcription factor (Figure 2).

**Figure 1.** Analysis of the AP2 sequence: (**a**) alignment result of IlAP2 amino acid sequence (the red boxes represent functional domains, '\*' indicates positions which have a single, fully conserved residue); and (**b**) phylogenetic tree of AP2 proteins from *I. lactea* (IlAP2) and other species.

**Figure 2.** Subcellular localization of IlAP2 in *A. thaliana* leaf cells, with GFP as control. Green fluorescence protein (GFP), chlorophyll autofluorescence (Auto) and merged images (Merged 1 and Bright Merged 2) are shown. Scale bar: 10 μm.
