Hello :)
I'm studing an unknown gene(about 4kb) of Arabidopsis thaliana.
To observe the phenotype when the gene was overexpressed in plants, the pBI vector (35S-unknown gene-GFP) was transformed and passed down several generation with Kanamycin MS media. But I can't see any GFP in plants. When RT-PCR was performed to confirm the vector transformed into plants, it was confirmed that the gene was overexpressed more than twice as much as the wild type.
So, what I'm curious about is why is it expressed in RNA but not GFP expressed under a microscope? Is it possible that the gene is a IncRNA?
Thank you.
Did you sequence your plasmid before transformation? Is the GFP in the same reading frame as your gene of interest? What is the expression level of the wild type gene? Twice of almost nothing could still be too low observe
The phenomenon you're describing, where GFP (Green Fluorescent Protein) is expressed at the mRNA level in reverse transcription polymerase chain reaction (RT-PCR) but isn't visible in plants, while GFP expression may be detected at the mRNA level in RT-PCR, several factors can contribute to the lack of observable fluorescence in plants, including post-transcriptional regulation, protein stability and turnover, ineffective translation or localization, incompatibility with plant systems, low expression levels, and methodological limitations.
Robert Adolf Brinzer
My vector reading frame is good. Because I saw GFP expression in some thrichome and when checked my vector sequence, it's alright. The expression level of my gene in WT is not low because I saw the GFP expression in pollen. Thank you for your replay :)
Kais Khudhair al Hadrawi
My rna sample was seedlings of 12 days after germination (35S-interest gene-GFP). I'll have to check my interest gene as you answered. Thank you so much!
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