Total RNA was isolated from the leaves of P. alba var. pyramidalis saplings using Plant Mini Kit (Qiagen, Germany). First-strand cDNA was synthesized from 2 μg of total RNA in a 20-μl reaction mixture using the RT-AMV transcriptase kit (TaKaRa, Dalian, China). The coding sequences of PalbHLH1 and PalMYB90, were amplified using gene-specific primers (Table S1 in Datasheet 1) designed based on the PalbHLH1/PalMYB90 gene sequences respectively, in the P. alba var. pyramidalis genome (Ma et al., 2018 (link)). PCR was carried out with Pfu DNA polymerase (TaKaRa) in a total volume of 50 μl with a thermal cycler program of 98°C for 2 s, 38 cycles of 98°C for 10 s, 55°C for 5 s, and 72°C for 2 min, and a final extension step at 72°C for 10 min. The amplification products were inserted into the plant binary vector pCAMBIA1305 through intermediate vectors pMD19 and pCXSN using the enzyme digestion–linked cloning system to produce 35S::PalbHLH1 and 35S::PalMYB90 constructs (Figure 2A). Positive clones were verified by DNA sequencing and aligned with sequences from the P. alba var. pyramidalis genome (Ma et al., 2018 (link)).
Free full text: Click here