RNA interference (RNAi) vector construction and plant transformation

A 340 bp specific fragment of SlIPT4 was amplified from tomato cDNA using the following primers: F 5′-GGGGTACCAAGCTTTGCTGAATTGTCAAATTCCGTGG-3′ with Kpn I and Hind III restriction sites, and R 5′-CCGCTCGAGTCTAGAATAGTGAGATGCTGCTGCCA-3′ with Xho I and Xba I restriction sites. The PCR products were cloned into the pHANNIBAL vector in the sense orientation and anti-sense orientation into the Hind III-Xba I polylinker and the Kpn I-Xho I polylinker, respectively. Then the intron-spliced hairpin construct of SlIPT4 specific fragment under the transcriptional control of constitutive CaMV35S promoter and OCS terminator was subcloned as Spe I-Sac I fragment into pCAMBIA1301 binary vector, in which the hygromycin resistance gene has been replaced by the neomycin phosphotransferase II (nptII) gene. Transgenic plants were generated by Agrobacterium tumefaciens-mediated transformation. The positive transgenic lines were checked by histochemical β-glucuronidase (GUS) straining and PCR, and the silencing efficiency of SlIPT4 gene were detected by qRT-PCR.