Isolation and Characterization of CpWRKY71 Gene

Total RNA isolation and the first-strand cDNA synthesis were performed as described by Liu et al. [5 (link)]. Total genomic DNA was extracted from the wintersweet leaf using CTAB method [43 (link)]. Specific primers CpWRKY71-F/R (Table S1) were designed to clone the cDNA and DNA sequence of CpWRKY71. The PCR products were isolated from the gel using the Agarose Gel DNA extraction kit (Tiangen, Beijing, China), then cloned into the pMD19-T vector (TakaRa, Dalian, China) and sequenced by TsingKe Company (TsingKe, Chengdu, China). The multiple alignments were performed using BioEdit software. MEGA 5.0 software with the NJ method (1000 BootStrap replicates) was used to construct the phylogenetic tree of CpWRKY71 and WRKY proteins from other species.
The 5’-upstream sequence of CpWRKY71 was isolated according to the protocol of the Universal Genome Walker Kit (Clontech, USA). Specific primers GSP1 and GSP2 (Table S1) were designed according to the CpWRKY71 gene sequence to clone the promoter. The purification and sequencing of the PCR products were performed as described above. The putative cis-acting regulatory elements were searched with the PLACE database (http://www.dna.affrc.go.jp/PLACE) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [44 (link)].