The effect of different 3' untranslated regions on the accumulation and stability of transcripts of a gfp transgene in chloroplasts of transplastomic tobacco.
The 3' untranslated region (3' UTR) of transcripts is a major determinant of transcript stability in plastids and plays an important role in regulating gene expression. In order to compare the effect of different 3' UTRs on transgene expression in tobacco chloroplasts, the 3' UTRs from the tobacco chloroplast rbcL, psbA, petD and rpoA genes and the terminator region of the Escherichia coli rrnB operon were inserted downstream of the gfp reporter gene under the control of the psbA promoter, and the constructs were introduced into the plastid genome by particle bombardment. RNA-gel blot analysis of homoplasmic transplastomic plants identified gfp transcripts of ~1.0 and ~1.4 kb from all constructs and showed that plants expressing gfp with the rrnB terminator contained 4 times more gfp transcripts than plants expressing gfp with the rbcL and rpoA 3' UTRs. The amounts of transcripts accumulated roughly correlated with the half-life of the transcripts, determined by RNA-gel blot analysis of transcripts present in leaves treated with actinomycin D to prevent continued transcription of the chimeric gfp genes. Transcripts containing the 3' region of rrnB were most stable, with half-lives of ~43 h, considerably longer than the half-lives of the other ~1.0 kb gfp transcripts (13-26 h). Immunoblot analysis with antibodies to GFP indicated that all plants contained about the same amount of GFP (~0.2% total soluble protein), suggesting either that translation was limited by something other than the amount of transcript or that the 3' UTR was affecting translation.
Tangphatsornruang
Sithichoke
S
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK.
Birch-Machin
Ian
I
Newell
Christine A
CA
Gray
John C
JC
D020413:3' Untranslated Regions; D001483:Base Sequence; D015139:Blotting, Southern; D002736:Chloroplasts / Q000235:genetics*; D017931:DNA Primers; D049452:Green Fluorescent Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*; D019076:Transgenes
D020413:3' Untranslated Regions; D017931:DNA Primers; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins