Rt amv transcriptase kit

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)).

Total RNA was extracted from the leaves of 6-month-old P. tomentosa Carr. by using the Trizol Reagent (Tiangen, China), then revers transcribed to cDNA by using the RT-AMV transcriptase Kit (TaKaRa, Dalian, China). The PtoABCG36 specific fragment was amplified by PCR using specific primers (Supplementary Table S1). Cycling conditions were: 98 °C for 3 min followed by 34 cycles of 98 °C for 30 s, 56.6 °C for 30 s and 72 °C for 2 min 58 s, adding a final prolongation step at 72 °C for 10 min. The amplification products were cloned into the BamHI site of the plant binary vector pCAMBIA-1300-GFP [33 (link)] as well as the SpeI and XmaI sites of the yeast vector pDR196 [34 (link)], to construct pCAMBIA-1300-PtoABCG36 and pDR196-PtoABCG36.
Prediction and analysis of the structure of PtoABCG36 protein was performed with the Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de). The homologous amino acid sequences of PtoABCG36 in other species were downloaded from NCBI (http://www.ncbi.nlm.nih.gov), and aligned with DNAMAN 8.0 (Lynnon Biosoft, San Ramon, CA, USA). The phylogenetic analysis of amino acid sequences was carried out with MEGA 5.0 software by using neighbor-joining (NJ).

Total RNA was isolated from P. tomentosa Carr. using the Plant Mini Kit (Qiagen, Germany). First-strand cDNAs were synthesized from 2 μg of total RNA in 20 μl of reaction mixture using the RT-AMV transcriptase Kit (TaKaRa, Dalian, China). The coding sequence (CDS) of PtoMYB156 was amplified by gene-specific primers (Supplementary Table S1). Thermal cycler programmes were as follows: 96 °C for 3 min followed by 32 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 50 s, and a final extension step at 72 °C for 10 min. The amplification products were cloned into the plant binary vector pCXSN as previously described35 (link).
The amino acid sequences of R2R3 MYB transcription factors in other species were obtained by BLAST searchers (http://www.phytozome.com). The deduced amino acid sequences were aligned with the program DNAMAN7.0 (Lynnon Corporation, USA). Phylogenetic analysis based on amino acid sequences was preformed using the Neighbor-Joining (NJ) method through MAGE 5.036 (link).