Chloroplast Gfp Expression in Tobacco Plants Agroinfiltrated with Tobacco Mosaic Virus Based Vectors

GFP expressed from the TMV vector TMV-30B-GFP is also found in chloroplasts and we are using TMV-30B-GFP to understand how GFP is expressed inside the chloroplast. Two putative Shine-Dalgarno motifs were found immediately upstream of GFP. To find out the exact role of these motifs, we introduced point mutations in each of them. When the mutation was introduced in both the motifs, GFP expression in the chloroplast was even lesser. Most importantly, the results obtained with p35SGFP show that GFP is somehow able to gain entry to chloroplasts without the aid of any TMV sequences. My work is important because it shows that GFP is an imperfect negative control for the analysis of chloroplast transit sequences. A certain percentage of chloroplasts will express GFP even in the absence of a clearly defined chloroplast transit sequence. The key question I did not address in my thesis was whether the transcript from the 35SGFP construct was able to gain entry to the chloroplast. This information would allow me to assess whether translation of GFP from the 35SGFP transcript is even a possibility. This is undoubtedly the next piece of information to