2.3 Vector construction and tobacco transformation
For RNAi vector construction, the different CDS fragments ofNtMYB12a and MtMYB12b genes were amplified with gene
specific primers containing attB adaptor, the correct fragments were
then cloned into pHellsgate 2 vectors via BP reaction. The complete open
reading frame of NtMYB12a gene without the stop codon was
amplified and digested with the endonucleases Kpn I and Spe I
(Supplementary Figure S6a), and then cloned into the pCAMBIA1300 vector
to obtain the fusion NtMYB12a-GFP, under the control of the CaMV35S
promoter (Supplementary Figure S6b). Similarly, the NtMYB12a CDS
sequence was also amplified and digested with the endonucleases XhoI and
ApaI, and then cloned into pGreen-35S-GR (GLUCOCORTICOID RECEPTOR) to
obtain an in frame fusion of NtMYB12a-GR
(Chen et al., 2014). For Cas9/sgRNA
vector construction, 20 nucleotides located in the first exon ofNtMYB12a gene was selected and used for sgRNA generation via
overlapping PCR method as described previously
(Xie et al., 2017). The sgRNA was then
subcloned into the pORE-Cas9 binary vector. The predicted promoter
fragment (2 kb) of NtMYB12a gene was amplified and digested with
Hind III and BamH I, and then cloned into pBI121 to construct theProNtMYB12a: GUS vector. All the primers used in
the vector constructions are listed in Supplementary Table S3.
The recombinant plasmids were transformed into Agrobacterium
tumefaciens strain GV3101, which was used for the transgenic
manipulation via leaf disks method
(Palmgren, Mattson, & Okkels, 1993). The
positive transgenic plants were selected with hygromycin or basta,
confirmed by DNA and RNA analyses (Supplementary Figure S6c), and then
self-pollinated twice to generate T2 transgenic progeny.
To detect the mutations generated by Cas9/sgRNA, an about 250 bp
fragment of NtMYB12a gene containing the target sites was
amplified and used for Hi-TOM sequencing analysis as described in
previous study (Q. Liu et al., 2019). The
individual plants with >90% mutations were chosen for the
subsequent studies (Supplementary Figure S6d).