Genomic DNA was extracted from grapevine leaves using a commercial kit (DP320, Tiangen Biotech, China). The presence of the T-DNA insertion in VqWRKY31-overexpressing lines was evaluated using PCR and the primers 2300-flg (forward) and 2300-flg (reverse). DNA from non-transgenic, wild-type control plants served as a negative control.
Western blot analysis of transgenic lines was carried out to confirm the expression of VqWRKY31-3 × Flag protein. Fresh leaves were ground into powder using a mortar and pestle under liquid nitrogen, and ~ 300 mg powdered tissue was homogenized in extraction buffer as described [32 (link)]. Then, the samples were subjected to centrifugation at 12 000 × g for 10 minutes. The supernatant was mixed with SDS–PAGE sample loading buffer and placed in a boiling water bath for 5 minutes. Western blotting was conducted as previously described [32 (link)]. Anti-Flag and peroxidase-conjugated goat anti-mouse IgG (H + L) were used for immunoblotting.
Western blot analysis of transgenic lines was carried out to confirm the expression of VqWRKY31-3 × Flag protein. Fresh leaves were ground into powder using a mortar and pestle under liquid nitrogen, and ~ 300 mg powdered tissue was homogenized in extraction buffer as described [32 (link)]. Then, the samples were subjected to centrifugation at 12 000 × g for 10 minutes. The supernatant was mixed with SDS–PAGE sample loading buffer and placed in a boiling water bath for 5 minutes. Western blotting was conducted as previously described [32 (link)]. Anti-Flag and peroxidase-conjugated goat anti-mouse IgG (H + L) were used for immunoblotting.