Cloning and Overexpression of Transcription Factor Genes in P. ussuriensis

We synthesized the first‐stranded complementary DNA (cDNA) using PrimeScript RT First Strand cDNA Synthesis kit (Takara, Dalian, China) and mRNA from all the time points. Following that, the PuMYB40, PuWRKY75, PuLRP1, and PuERF003 were amplified by PCR with KOD DNA Polymerase (TOYOBO, Osaka, Japan), the gene‐specific primers, and P. ussuriensis cDNAs. The full‐length coding sequences of PuMYB40, PuWRKY75, PuLRP1, and PuERF003 were cloned into pBI121 vector under the control of cauliflower mosaic virus 35S promoter (35S) for overexpression. A 27‐bp DNA sequence encoding the SUPERMAN repression domain X (Hiratsu et al., 2003 (link)) was fused in‐frame to the 3’ ends of the PuMYB40, PuWRKY75, PuLRP1, and PuERF003, which were then cloned into the pBI121‐GFP to construct repression vector. Transgenic lines were generated using leaf disc method as described earlier (Maheshwari and Kovalchuk, 2016 (link)). All transgenic lines were verified by PCR and RT‐qPCR analysis. The primers used in vector construction were listed in Table S4.