Molecular cloning of a PcOKS cDNA

Extraction of total RNA from roots of P. cuspidatum and reverse transcription were carried out as described before (Guo et al. 2013). The core fragment of the PcOKS cDNA was amplified using degenerate primers derived from conserved regions of plant type III PKSs, Dp-S and Dp-A (Supplementary Table S1), and the PCR program 1 (Supplementary Table S2). The PCR product was gel-purified and subcloned into the pMD18-T vector (TaKaRa, Beijing, China) for sequencing.

The 3′ and 5′ ends of the PcOKS cDNA were obtained by rapid amplification of cDNA ends (RACE). Two 3′ end gene-specific primers, GSP3-1 and GSP3-2, and three 5′ end gene-specific primers, GSP5-1, GSP5-2 and GSP5-3, were designed based on the core sequence obtained. The PCR program 2 was used for amplification.

The ORF of the PcOKS cDNA was amplified using the primers PcOKS-S and PcOKS-A and the PCR program 3. The amplified DNA was digested with NdeI and XhoI, subcloned into the pMD18-T vector (TaKaRa) and sequenced.

Genomic DNA extraction from young leaves of P. cuspidatum was carried out as described before (Guo et al. 2011). A full-length gene was amplified using the gene-specific primers PcOKS gene-S and PcOKS gene-A and the PCR program 4. The gel-purified PCR product was ligated into the pMD18-T vector (TaKaRa) and sequenced.