The sense RNAi fragment (RNAi1) of OslecRK were subcloned into pKANNIBAL vectors between two restriction sites (XbaI and HindIII).
The antisense fragment (RNAi2) were subcloned using EcoRI with XhoI . The new construct was digested with NotI , cloned into the pART27 vector, then digested with
SalI and cloned into the pCAMBIA1301 vector to generate the NOS:Ri transgenic plasmid. The whole length of OslecRK was cloned into the pGBKT7 vector at the
EcoRI site as the bait construct. The domain derivatives of OslecRK were fused with
Gal4 DNA-BD via the EcoRI site . OsADF was cloned into pGADT7 via the EcoRI and BamHI sites as the prey construct. The domain derivatives and whole length of
OslecRK were cloned into pSPYCE-35S vector via the XbaI site. OsADF was cloned into pSPUNE-35S vector at the XbaI site to generate an OsADF:Myc expressing plasmid.
Genomic DNA was extracted from rice using cetyltrimethyl ammonium bromide (CTAB) and treated with
RNase (Sigma). 10 μg samples of purified DNA were fully digested with the restriction enzymes Bam HI, Dra I, EcoR I,
EcoR V and Hind III . The digested DNA was then separated on a 0.8% agarose gel and transferred onto a nylon membrane (Amersham). After hybridization with the labeled probe, autoradiography was performed in the dark.