I have found dwarf phenotype, cosegregate to T-DNA insertion. My problem is:
1. T_DNA insertion (pGA2715 vector) locates between 2 genes(A and B), upstream of A around 82bp, expression level of A in homo up around 2 fold, expression level of B in homo up around 4 fold (but expression level of B very low, around 0.004).
3. However, if phenotype is caused by activation of the gene, the hetero plants do not show any differences to wild type plants.
3. I have over-expression of A gene, it does not show any dwarf phenotype (looks like wild type).
Phenotypes might be caused by activation of gene B, but hetero plants do not show phenotypes and this makes me really confused, moreover B protein is a kind of hypothetical protein.
Does anyone have experience with this, and can you confirm what caused this phenotype?
First of all you have to be sure about the presence of only one T-DNA insertion by Q-PCR or Southern blot, the phenotype could be provoked by a different insertion linked to the one that you are working with. Lack of phenotype in hetero and over expression, low increase in the gene expression (two fold) makes me wonder that could be a different insertion.
What is the distance between A and B? Are you sure that you're not in an intron of the A+B gene? Or is it possible that your genes are expressed but the transcript and consequently the protein sequence is altered and thus non functional? Or is it possible that your primers are not specific for that locus (I had a similar problem with genes that differ in the coding sequence only by one or two bases...)
Dear Jose, actually I run tail-PCR to detect again T-DNA position, and only one insertion, moreover, phenotype co-segregate with T-DNA insertion. Thank you for your opinion.
Dear Francesco, distance between them is 1.1kbp. From annotation source, they are absolutely independent genes. I consider more about "transcript and protein sequence is altered". But I still wonder, it is really possible like T-DNA make my transcript change sequence? Phenotype co-segregate with T-DNA insertion well. Thank for your help.
Of course, TAIL-PCR is a system to isolate T-DNA insertions border plant sequences but the big problem is that works with canonical left/right borders and that implies the loss of information about partial integrations, self-recombination etc. Usually T-DNA is associated with translocations, duplications, short inverted repeats and DNA methylation in the proximity of the insertions being impossible to detect such modifications with TAIL-PCR. The point here is that your phenotype co-segregates with the T-DNA but in my opinion doesn’t mean the T-DNA adjacent genes are the responsible of the phenotype your enhancer could affect other neighbour genes in hidden longer promoters, the extremely packed Arabidopsis genome is always confusing. Moreover, any change in less than 1 cM around your insertion will co-segregates with your enhancer in 99.999 % of your progeny and you have to take in account that in Arabidopsis 1 cM, on average, corresponds to 200 kb. Long distance expression alterations as far as 200 kb were previously described (http://pcp.oxfordjournals.org/content/48/1/169.full.pdf).
The lack of phenotype in the hemizygous is really odd because only dominant mutants in the transformation are selected in this type of approach. Anyway, in activation tagging a dominant mutation is almost always expected if that is not the case indicates that T-DNA is probably provoking a recessive loss of function in a specific genomic region dependent manner (chromatin rearrangement and other aspects of 35S enhancers were also described elsewhere). With time and money an array or RNA Seq could give you some cues.
Good luck!
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