*2.3. IlAP2 Expression Pattern Analysis*

To explore the expression pattern of *IlAP2* in response to Cd stress, semi-quantitative PCR and RT-qPCR were used to analyze the expression of *IlAP2* in *I. lactea* roots under 50 μM CdCl2 stress at different times. Semi-quantitative PCR showed up-regulated *IlAP2* expression after stress treatment, which was further demonstrated by RT-qPCR (Figure 4). There was no significant difference in *IlAP2* expression level among the different treatment times (Figure 4).

**Figure 4.** Analysis of *IlAP2* expression pattern in *I. lactea* roots at different times of Cd stress: (**a**) semiquantitative PCR and (**b**) RT-qPCR. '\*\*' shows the significance level of *p* < 0.01.

#### *2.4. Functional Verification of I1AP2 in A. thaliana*

To investigate whether *IlAP2* can improve Cd tolerance, we constructed *I1AP2* overexpressing *A. thaliana* and obtained eight homozygous T3-generation transgenic lines. Three independent transgenic lines were selected for functional verification: OE2, OE4 and OE5. After 10 days of growth (3-day-old seedlings were exposed to cadmium for 7 days), there was no significant difference in root length and growth between the WT and transgenic plants grown on normal half-MS medium. Under a 7-day Cd treatment, although the root length and growth of the WT and transgenic plants decreased continuously, the degree of decline for the transgenic plants was slower than that of the WT plants (Figure 5), suggesting that *IlAP2* improved Cd tolerance in *A. thaliana*.

**Figure 5.** Functional verification analysis of *I1AP2* in *A. thaliana*. (**a**) Root length of wild-type (WT) and transgenic *A. thaliana* under CdCl2 stress. (**b**) Root length statistics of WT and transgenic *A. thaliana* under CdCl2 stress. (**c**) Comparison of growth of WT and transgenic *A. thaliana* under CdCl2 stress. (**d**) Fresh weight of WT and transgenic *A. thaliana* under CdCl2 stress. (**e**) Agarose gel electrophoresis profile for the expression of *I1AP2* in WT and transgenic *A. thaliana* (OE2, OE4 and OE5). The + band was amplified with *I. lactea* cDNA and ORF primers of *I1AP2*, serving as positive control. '\*\*' shows the significance level of *p* < 0.01.
