Figure 2.

Reduction in phototropin levels by using the RNAi technique. (A) The plasmid used for transformation. (B) Phot construct generated for a reduction of in vivo phototropin levels. Black boxes represent exons, white boxes represent introns, and the cDNA is indicated by a stippled line. The orientations of the gene segments are indicated by arrows. The predicted structure of the resulting RNA is given below the construct. (C) Relative amounts of phototropin in different transformants as compared with wild-type strain CC-124. For this assay, an Ab directed against Chlamydomonas phototropin (8) was used. For a loading control, an Ab that reacts with the cochaperone CGE1 (47) was used. (D) Assay of transformants that exhibited reduced levels of phototropin for pregamete to gamete conversion. Pregametes of strain CC-124 and various transformants of CC-124 harboring the RNAi construct were generated by incubation in TAP-N for 16 h in the dark. Pregametes were then irradiated for 90 min with white light at a fluence rate of 0.64 μmol⋅m⁻²⋅s⁻¹. Then the cells were mated and the percentage of gametes was calculated as described in Materials and Methods.