Plb simple

DsRNA fragments were isolated from N. oryzae strain 9-1 according to the methodology reported previously with a slight modification [50 (link)], CF-11 cellulose being replaced with Sigmacell cellulose (S6790, Sigma, Steinheim, Germany). The molecular cloning of dsRNA was performed according to the previous report [50 (link)]. Briefly, the dsRNA fragment was mixed with Primer A (5′-PO4-TCTTCGGGTGTCCTTCCTCG-NH2-3′, Sangon, Shanghai, China), T4 RNA ligase (NEB) and T4 DNA ligase (NEB). The mixture was incubated at 37 °C for 3 hr. The partial duplex was repaired with 2×Taq PCR StarMix (Genstar, Nanjin, China) at 68 °C for 30 min and precipitated by the addition of 2.5 volumes of ethanol with 10% 3M sodium acetate (pH 5.4). Following denaturation of the tailed dsRNA at 99 °C for 3 min and ice incubation for 5 min, first-strand cDNA was synthesized using the complementary Primer B (5′-CGAGGAAGGACACCCGAAGA-3′) and SuperScript^TM III reverse transcriptase (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. The full-length cDNA was obtained by a regular PCR using Primer B and Q5 High-Fidelity DNA Polymerase (NEB). The resultant cDNA was cloned into the vector pLB-simple (Tiangen, Beijing, China) for sequencing.

The full-length cDNAs of PnbHLH [22 ] and PnMYB1 [21 ] were amplified and identified as described previously. The full-length coding sequence of PnMYB4 was amplified using the first-strand cDNA of P. notoginseng rootlets with primers designed according to the P. notoginseng transcriptomic dataset. After amplification, the amplicons were inserted into pLB-simple (Tiangen, Beijing) and transferred into Trans1-T1. Sanger sequencing was carried out by BGI-Beijing. The amino acid alignments and the phylogenetic tree of R2R3-MYB proteins were constructed with DNAMAN software and the MEGA 11.0 program, respectively, following previous reports [28 (link)].