Overexpression of a New Zinc Finger Protein Transcription Factor OsCTZFP8 Improves Cold Tolerance in Rice

Figure 5

Generation and identification of OsCTZFP8-overexpressing rice. (a) Schematic representation of the T-DNA region on the P_Ubi:OsCTZFP8 construct. The construct contains the bar gene and a maize ubiquitin (Ubi) promoter. The small double arrow bar represents the Ubi-OsCTZFP8 probe used for Southern blot analysis. (b) Agrobacterium-mediated rice seed transformation. The procedures included induction of callus (A), subculture (B), cocultivation and Basta screening (C), differentiation (D), rooting (E), and planet acclimatization (F). (c) PCR analyses for T₀ transgenic rice using bar gene-specific primers. PC: plasmid DNA; NT: nontransgenic control plant; 1~19: T₀ generation of independent transgenic rice. (d) LibertyLink strip detections of T₀ transgenic rice plants; NT: nontransgenic control plant; 1~10: T₀ transgenic plants; C: control line; T: test line. (e) Southern blot analysis of OsCTZFP8-overexpressing lines. HindIII-digested genomic DNA from T₁ generation was separated on agarose gel and hybridized with DIG-labeled Ubi-OsCTZFP8 probe; NT: nontransgenic control plants; OE-1~OE-8: overexpressing lines; PC: plasmid DNA. (f) mRNA expression of OsCTZFP8 in overexpressing lines. One-step RT-PCR was performed using total RNA extracted from 2-week-old rice leaves. The expression of OsActin1 was used as an internal control; NT: nontransgenic control plants; OE-1~OE-6: single-copy insertion overexpressing lines. (g) Basta resistance segregation assay. Seeds were germinated on 1/2 MS medium (30 mg/L Basta), and Basta resistance were determined at the 5th day. The experiments were performed in two repetitions; NT: nontransgenic control plants; OE-1~6 and OE-3-2: overexpressing lines.

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