*2.5. Analysis of Gene Structural and Conserved motifs of EgrVQ Genes*

To further understand the structural features of the *EgrVQ* genes, exon/intron structural analysis was performed and results are shown in Figure 4 and Table 1. Interestingly, 25 *EgrVQ* genes had only one exon, while the *EgrVQ7* and *EgrVQ21* genes had two exons. Subsequently, the conserved motifs of the EgrVQ proteins were studied. All of the *EgrVQ* genes contained motif1, which was the DNA-binding domain of *EgrVQ* genes (Figure 5). Notably, some motifs appeared in one sub-family of *EgrVQ*. For example, motif2, motif3, and motif17 only existed in sub-familyV-3, III, and V, respectively, which indicated that these motifs were the characteristic elements of these sub-families. Meanwhile, it was found that the same sub-family had similar motifs. For example, sub-familyV-3 genes contained motif4, motif9, motif1, motif5, motif7, and motif11, whereas sub-familyIII included motif4, motif12, motif7, motif8, motif1, motif6, and motif3. Figure 5 and Table 4 lists the details. *Int. J. Mol. Sci.* **2019**, *20*, x 6 of 17

**Figure 3.** Multiple sequence alignment of VQ proteins in *E. grandis*. Sequences were aligned using DNAMAN software. The FxxxVQxxxG motif was highly conserved. The dark blue indicated the conserved section of EgrVQs protein family. The pink indicated the four conserved motif variations of LGT, FTG, VTG, and LSG. **Figure 3.** Multiple sequence alignment of VQ proteins in *E. grandis*. Sequences were aligned using DNAMAN software. The FxxxVQxxxG motif was highly conserved. The dark blue indicated the conserved section of EgrVQs protein family. The pink indicated the four conserved motif variations of LGT, FTG, VTG, and LSG. *Int. J. Mol. Sci.* **2019**, *20*, x 7 of 17

**Figure 4.** Gene structure of *VQ* genes in *E. grandis*. Exons were indicated by yellow rectangles. Upstream/downstream's sequences of *EgrVQs* were indicated by blue lines. Gray lines connecting two exons represented introns. **Figure 4.** Gene structure of *VQ* genes in *E. grandis*. Exons were indicated by yellow rectangles. Upstream/downstream's sequences of *EgrVQs* were indicated by blue lines. Gray lines connecting two exons represented introns.

**Figure 5.** Phylogenetic relationships and conserved motifs of VQ proteins in *E. grandis*. The left panel showed the phylogenetic tree of EgrVQ, which was constructed by the neighbor-joining method based on the results of sequence alignment. Proteins were divided into seven subgroups (marked by different colors). The right panel showed the distribution of the 20 conserved motifs in the *EgrVQ* genes following analysis with Multiple Expectation Maximization for Motif Elicitation (MEME). Each specific motif was marked by a different colored box, and the motif numbers were included in the center of each box. The length of each box was proportional to the actual size of the motif.

**Figure 4.** Gene structure of *VQ* genes in *E. grandis*. Exons were indicated by yellow rectangles.

**Figure 5.** Phylogenetic relationships and conserved motifs of VQ proteins in *E. grandis*. The left panel showed the phylogenetic tree of EgrVQ, which was constructed by the neighbor-joining method based on the results of sequence alignment. Proteins were divided into seven subgroups (marked by different colors). The right panel showed the distribution of the 20 conserved motifs in the *EgrVQ* genes following analysis with Multiple Expectation Maximization for Motif Elicitation (MEME). Each specific motif was marked by a different colored box, and the motif numbers were included in the center of each box. The length of each box was proportional to the actual size of the motif. **Figure 5.** Phylogenetic relationships and conserved motifs of VQ proteins in *E. grandis*. The left panel showed the phylogenetic tree of EgrVQ, which was constructed by the neighbor-joining method based on the results of sequence alignment. Proteins were divided into seven subgroups (marked by different colors). The right panel showed the distribution of the 20 conserved motifs in the *EgrVQ* genes following analysis with Multiple Expectation Maximization for Motif Elicitation (MEME). Each specific motif was marked by a different colored box, and the motif numbers were included in the center of each box. The length of each box was proportional to the actual size of the motif.


**Table 4.** Summary of stresses inducible *cis*-elements in the promoter regions of *VQ* genes in *E. grandis*.

1 cis-acting element involved in the abscisic acid responsiveness; <sup>2</sup> cis-acting regulatory element involved in the MeJA-responsiveness; <sup>3</sup> cis-acting regulatory element involved in the MeJA-responsiveness; <sup>4</sup> cis-acting element involved in salicylic acid responsiveness; <sup>5</sup> MYB binding site involved in drought-inducibility; <sup>6</sup> cis-acting element involved in low-temperature responsiveness; <sup>7</sup> cis-acting element involved in dehydration, low-temperature, and salt stresses.
