35S:miR-168a and 35S:rSlAGO1 vector construction and tomato
transformation
Pre-SlmiR-168a was prepared using gene-specific primers. The
sequence-confirmed polymerase chain reaction (PCR) fragment was cloned
into pCAMBIA3301/Luc, which harboured two 35S Cauliflower mosaic virus
promoters, the marker gene for kanamycin resistance, phosphinothricin,
and luciferase. The bacterial colonies were screened for positive
recombinant plasmids by colony PCR, restriction enzyme digestion, and
sequence analysis. Binary vectors containing the expected insert were
subsequently transferred into Agrobacterium tumefaciens GV3101
cells by electroporation. The competent cells harbouring the vector were
transformed into JZ18 tomatoes using a tomato genetic transformation
system. The obtained T1 transformants and their corresponding T2
families were assessed for the expression of the target gene by
quantitative real-time PCR (qRT-PCR) and evaluation of the presence of
the kanamycin marker gene. All primers used in this study are listed in
Supplementary Table S1. To generate rSlAGO1 (theSlmiR-168a -resistant construct), mutations in theSlmiR-168a target site of SlAGO1 were inserted using
two-step PCR mutagenesis. The 35S:SlAGO1 transformants were
obtained using the same method as described above.