Figure 4.

Expression of ribozyme R(−) and nonfunctional mutant mR(−) in transgenic potato plants. (a) Detection of PSTVd isolated from transgenic potato lines. Lanes: c, transformed with empty vector pROK2; TR-1–3, transformed with pROK2–R(−)D; and TmR-1–4, transformed with pROK2–mR(−)D. Total RNA (10 μg) isolated from stems and leaves of individual plant was loaded in each lane of 5% polyacrylamide gel, using return gel electrophoresis. Gel was stained by silver staining (35). (b and c) Northern blot analysis of RNA isolated from transgenic potato lines. Lanes: IN, PSTVd inoculum (0.1 μg); c, control plant transformed with empty vector pROK2; TR-1, -2, -8, and -10, transgenic plants transformed with pROK2–R(−)D; TmR-2 and -4, transgenic plants transformed with pROK2–mR(−)D; and R(−), in vitro-transcripted ribozyme R(−)M as a marker. Total RNA (4 μg) isolated from the transgenic plant was loaded in each lane of 5% polyacrylamide gel and electroblotted onto an S & S Nytron Nylon membrane (Schleicher & Schuell). Blotted RNAs were hybridized with a ³²P-labeled PSTVd cDNA fragment probe. The PSTVd, ribozyme R(−), and mutant mR(−) are indicated by arrows.