To determine the exact subcellular localization of my gene within plant cell. GFP gene was fused in frame with my gene.
Then i performed transient expression analysis by introducing fusion gene into onion epidermal cells by particle bombardment.
Three images were taken
1) GFP control (30X)
2) GFP fused to my gene (20X)
3)GFP fused to my gene (10X)
can anyone help me out to read this images? As i am doing this GFP localization experiment first time.
Thanks and regards
Harshraj
Hi Harshraj,
1. I think it would be better to take 2 kinds of pictures of a treatment, one for GFP and another one under regular light. It will be easy to see the 'borders' of cells. (see attached sample picture from web, also GFP on onion cells).
2. From your second picture, it looks like the GFP is all over inside a single cell?! Does your gene include a known (or unknown) 'signal peptide' for subcellular compartment targeting?
3. In the fig.3, isn't the background one the real onion epidermis? I am not sure the 'round thing' on the foreground...
Hi sir,
Are you using tobacco or onion epidermal cells? You can take images using confocal microscope.
Thank you dear. i did onion epidermal cell bombardment using gene gun and i took images using confocal only.
i am growing tobacco too soon i will try agroinfiltration also
Hi Harshraj,
I am curious, after Agroinfiltration, what are you going to do to check out the subcellular localization of your fused protein?
@ Harshraj,
1. I think that to better observe the results of 'subcellular localization' of your fused genes, 'protoplasts' (plant cells without cell walls) should be a better material for this purpose. Their cell walls are digested and removed by using cell-wall digesting enzymes.
2. You can simply isolate protoplasts from tobacco leaves (protocol is pretty rutine), then transform your plasmids into the protoplasts through PEG-mediated transformation for transient assay. Then, observe the results with a confocal microscope.
3. There should be many papers out there for your references. I attach one exemplary paper for you (see attachment). Check out their Fig. 4. Their fused gene was targeted to tonoplast.
@ Harshraj,
1. For using GFP to investigate gene subcellular localization, I attach a paper for you. It is a bit old, but it reviews some results published by researchers. It may be useful for you to find some important info in doing this kind of experiments (attachment 1).
2. I also attach another paper for using tobacco protoplasts for experiments involved in subcellular localization of some fused genes (attachment 2). See their Fig. 1.
Since you are studying protein subcellular localization, these two website can be useful for you. Company with the websites, I attach a few papers related to them for you.
(1) SABA3 database ( http://suba3.plantenergy.uwa.edu.au/ )
SUBA provides a powerful tool to investigate subcellular localization in Arabidopsis through the unification of disparate datasets and through the provision of a web accessible interface for the construction of powerful user based queries resulting in a one-stop-shop for protein localization in this model plant. SUBA3 houses large scale proteomic and GFP localization sets from cellular compartments of Arabidopsis. It also contains precompiled bioinformatic predictions for protein subcellular localizations. Protein-protein interaction data allow the search for interacting protein pairs.
(2) CropPAL ( http://croppal.org/ )
The compendium of crop Proteins with Annotated Locations (cropPAL) collates more than 550 data sets from previously published fluorescent tagging or mass spectrometry studies and eight pre-computed subcellular predictions for barley, wheat, rice and maize proteomes.
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