Plasmid construction and plant transformation
The coding sequence of each gene was first cloned into pDONR221 vector
by BP reaction following the manufacture’s instruction (Life
Technologies, cat. #11789-020). These entry vectors were recombined
into destination vectors by LR reactions (Life Technologies, cat.
#11791-020). For Crispr-cas9 constructs, two guide RNAs, target 1 and
2, or Target 3 and 4 were designed following the instruction in the
website www.skl.scau.edu.cnand included in primers for PCR amplification (Xie et al., 2017). DNA
fragments amplified using the plasmid pCBC-DT1T2.2 as a template were
ligated into pG3K-U6SC vector using Golden Gate Assembly method, and all
constructs were transformed into Agrobacterium strains LBA4404
harboring the helper plasmids pVS1-VIR2 (Zhang et al., 2019). Transgenic
Arabidopsis seedlings with overexpression constructs were generated
throughAgrobaterium -mediated
floral dipping method (Clough & Bent, 1998). Transgenic tomato plants
using cultivar ‘Heinz 1706’ were generated throughAgrobaterium -mediated transformation as previously described (H.
J. Sun, Uchii, Watanabe, & Ezura, 2006).