Plasmid construction and genetic transformation
The ORF of SmGRAS5 was amplified and cloned into the Noc I
and Spe I restriction sites of the pCAMBIA1304 in sense and
antisense orientations with the CaMV35S promoter. PCR and restriction
enzyme digestion were used to confirm the positive clones. And then the
plasmids were transformed into ATCC15834 . The combination of
cefotaxime (Sigma, USA)
and
hygromycin B (MP Bio, USA) was used to screen the transformants. PCR
identification and the positive transgenic lines screening use four
primer pairs, rolB , rolC , hptII , 35S forward primer
(35S-F) and GRAS5 reverse primer (GRAS5-R). All the expression
vector construction and the PCR identification primers are listed in
Supplementary Table S2.