2.2 Vector construction and genetic transformation
To generate MdHY5 overexpression constructs, the coding sequence
(CDS) of MdHY5 was cloned into the pGWB415 vector containing a
CaMV 35S promoter and 3xHA tag. For MdHY5 RNA interference
(RNAi), a specific 200-bp fragment of MdHY5 was cloned into the
RNAi vector pK7GWIWG2D (a GFP tag). Agrobacterium
tumefaciens -mediated transgenic apple calli were obtained as described
by Xie et al. (2012).
The root transformation of MdHY5 was carried out using anA. rhizogenes (K599)-mediated system with tissue-culturedM. domestica (cv. Gala) as the explant, following the protocol
used by Meng et al. (2019) with slightly modifications. Tissue-cultured
plants with 3–4 leaves were used for the root transformation. In a
clean bench, the base of each tissue-cultured plant was obliquely cut
before immersing it directly into a glass beaker containing 100 ml of
K599 A. rhizogenes suspension. The treated seedlings were
vacuumed for 15 min using a vacuum pump set to 0.08 MPa. The explants
were blot-dried and transferred to the MS media supplemented with 25
mg/L kanamycin for the selection and further culture.