Gene of interest refers to the genes that we’re studying. We extracted the MYB46 DNA sequence, then we increased the tiny number of genes substantially (The Polymerase Chain Reaction Technology). The gene of interest repeats to make sure when experimenting genes and expression vectors combine, the rate of combination is faster. We gained the genes from recycling; we used the Gel Electrophoresis to detect the concentration and purity of the genes.

We built a cloning vector (pEASY) which was in a cyclic form, we used it to connect to the product. pEasy was in a liquid state, and it had an unstable property. To make pEasy more stable, we transformed it into a germ which was the Escherichia Coli. The PCR product was connected with the clone vector using pEASY®-Blunt Cloning Kit, and then transformed into Trans1-T1 Chemically Competent Cell and sent to the company for sequencing. The result of sequencing is figure 3.

The constructed expression vectors pBI121 and pYES2 were transformed into Agrobacterium tumefaciens and yeast receptive cells, respectively. Then PtoMYB46 gene was identified by colony PCR.

We needed to transform the genes onto the cells of the plants, T-DNA was a fragment of the plasmids. We used Agrobacterium to make the fragment of PtoMYB46 through T-DNA vector, then transformed the fragment of the PtoMYB46 onto the genome of the recipient plant, it could replicate as the plant replicates. The construct was mobilized into Agrobacterium tumefaciens strain GV3101, which was used for poplar transformation. We found that PtoMYB46 not only positively regulates plant growth, but also promotes Pb2+ uptake (Figure 6).

We sampled some yeasts, and observed how much lead ion could they absorbed after a period of time. PtoMYB46 expression enhances Pb2+ sensitivity and increases Pb2+ content in yeast. To investigate the cellular function of PtoMYB46, the protein was expressed in BY4741 susceptible to Pb2+ excess. PtoMYB46 and empty vector-complemented BY4741 cells were grown in SD/Ura medium overnight. Cells grown overnight were used for spotting on SD/Ura agar plates supplemented with 0, 10, 20 and 30 μM (CH3COO)2Pb at indicated dilutions. The Pb2+ supplementation of the medium caused more considerable growth inhibition in yeast cells expressing PtoMYB46 than in the control (Fig. 7). We also analyzed the relative growth in liquid media in the presence of Pb2+ in yeast cells. The growth of BY4741 cells expressing PtoMYB46 were Higher than cells transformed with the empty vector (Fig. 8). The growth inhibition due to the functional PtoMYB46 in ycf1 suggested that PtoMYB46 may facilitate the import of Cd inside the yeast.

PtoMYB46 protein sequences of multiple species were collected from NCBI, and then MAGE was used to perform multiple sequence alignment and draw phylogenetic evolutionary trees. Then, positive selected amino acid sites were analyzed by EasyCodeML. And the library of potential mutants was constructed according to the positive selected amino acid sites (Table 1). We used alphafold to predict protein structure (Figure 9).

Tab.1 Positive selection analysis of PtoMYB46 gene