Figure 4
Construction and identification of CSN6 sense (S) and antisense (A) transgenic rice lines.
(a) Using SpeI sites, CSN6 was cloned into pCAMBIA1302 with the cauliflower mosaic virus (CaMV) 35S promoter at the 3′-upstream end and GFP at the 5′-downstream end. Vectors were digested with SalI (present either at the CSN6 sequence or vector sequence) and run on an agarose gel to screen the sense and antisense recombinants. M1 represents the HindIII-digested λ phage DNA marker; M2 represents the DL2000 DNA marker. (b) Three sense (S) and three antisense (A) CSN6 transgenic rice lines were chosen to determine the relative expression levels of CSN6 by real-time PCR. Total RNA was extracted from 14-day-old CSN6 transgenic rice seedlings. (c) Protein was extracted from 14-day-old CSN6 transgenic rice seedlings and subjected to SDS-PAGE and immunoblot analysis. In sense lines, the exogenous CSN6::GFP fusion protein was detected using anti-GFP antibody. The endogenous CSN6 (En-CSN6) protein in sense and antisense lines was detected using an anti-CSN6 antibody. HSP80 was used as a loading control. (d) The histogram represents quantification of the endogenous CSN6 band using Image J. Protein levels are expressed as a ratio of CSN6 to HSP80. Error bars indicate SD (n = 3). Asterisks indicate P < 0.05 (*) and P < 0.01 (**) in Student’s t test analysis. (e) Expressions analysis of CSN5 and CUL1 in CSN6 sense (S) and antisense (A) transgenic rice lines. Proteins were extracted from 14-day-old WT and T3 generations of CSN6 transgenic rice seedlings and analysed using anti-AtCSN5 and anti-CUL1 antibodies, respectively. Equal protein loading was confirmed by immunoblotting with an antibody against HSP80 in rice.
