Figure 3.

Confirmation of 3-HKT dsRNA transcription in Chlamydomonas chloroplast transformants. CC4147 chloroplast transformants 13 and 15 were tested for expression of 3-HKT dsRNA by RT-PCR. psbD, a chloroplast gene was used as control in the experiment. Absence of any band in the control PCR experiments for psbD (lanes 2 to 4) and 3-HKT (lanes 9 and 10) indicates that all the RNA samples were free of DNA contamination. Amplification of psbD in lanes 5 to 7 indicates that cDNA synthesis was successful and the absence of the 3-HKT band in CC4147 control (lane 11) was real as it lacks 3-HKT. Presence of the 3-HKT band in CC4147/pCVAC108 clones 13 and 15 confirms that these are transgenics and they transcribe the 3-HKT dsRNA. Loading pattern: 1:100 bp ladder, 2: Parent strain CC-4147 no RT, PCR with psbD primers, 3:108-13 no RT, PCR with psbD primers, 4: CC4147/pCVAC108-15 no RT, PCR with psbD primers , 5: Parent strain CC4147 cDNA, PCR with psbD primers, 6: CC4147/pCVAC108-13 cDNA, PCR with psbD primers, 7: CC4147/pCVAC108-15 cDNA, PCR with psbD primers, 8: Parent strain CC4147 no RT, PCR with 3-HKT primers, 9: CC4147/pCVAC108-13 no RT, PCR with 3-HKT primers, 10: CC4147/pCVAC108-15 no RT, PCR with 3-HKT primers, 11: Parent strain CC4147 cDNA, PCR with 3-HKT primers, 12: CC4147/pCVAC108-13 cDNA, PCR with 3-HKT primers,13: CC4147/pCVAC108-15 cDNA, PCR with 3-HKT primers, 14: 100 bp ladder.