I m trying to transfer a ~ 10 kb T-DNA (containing some gene fragments) to tobacco plant via Agrobacterium. While Screening the transgenic plants by PCR I face different results that marks the absence of a gene(s) while other gene(s) are present. How strong is the possibility of T-DNA breakage while transformation? can it be integrated in genome after any damage?
It is very well possible that truncated T-DNA's are integrated into the genome. This is known for example from the SALK T-DNA insert collection. Once one end of the T-DNA is ligated to the a genomic DNA end, the other (truncated) side will follow. The frequency of truncation and extend will depend on the binary-plasmid, Agro strain and sequences cloned between the borders. However, the truncation of complete genes is in my opinion rare and could also result from instability of the plasmid, the method of transformation to the Agro strain. Do the truncations only occur flanking the T-DNA borders, or also 'internal'?
Yes, it is very possible.
Transfer of T-DNA into plants' cells is initiated at the right border (RB) and terminated at the left border (LB). Theoretically, it should be more often to see truncated T-DNA at the LB end. Once, I had one batch of transgenic lines, with them 4 lines were with single-copied transgene, and within the four lines, 3 lines were truncated (deletion) at T-DNA LB region with various sizes. I had left only one good intact line to work with. My suggestion is that you should produce as many single-copy lines as you can for screening intact and no internal rearrangement lines for downstream experiments.
Thank you for the answers;
The missing gene is mostly near RB according to a partial PCR amplification negative results, however in one or two of the plants internal and LB side truncation is also observed.
@ Saber,
Yes, although theoretically, truncations should occur more often at the LB end of a T-DNA, as also observed in my experiments, some reports also demonstrated that truncations were observed at the RB ends as well. Such as the statement found on the p.207 (yellow highlights) of the attached book chapter:
"Afolabi et al. (2004) found that nonintact T-DNAs were present in >70% of transgenic rice lines, in most cases reflecting loss of the mid to right border portion of the T-DNA."
Title of the book chapter: Transgene Integration, Expression and Stability in Plants: Strategies for Improvements
@ Saber,
There are many possible results after a T-DNA is transferred into plant cells. Another paper attached for you.
It is a review paper about T-DNA insertion mutagenesis. See p.94 (highlighted area in yellow). It describes the possible results of T-DNA structures after it integrates into Arabidopsis genome. The article also lists some other references, and you can get more info from there.
did u check your starting plasmid DNA used for this work?
3' end truncation is caused by degeredation, if you got more transgenic, you may try all of them.
how many copy number of transgenes are in your transgenics?
and ...
thank you all for your answers;
Yes I checked the plasmid carrying genes before transformation and all the genes were confirmed by PCR.
At this step I m just doing the primary screening of transgenic candidates and not yet have detected the gene copy number.
Yes, truncation occur during T-DNA integration in plant genome. You can find in this paper: Majhi BB, Shah JM, Veluthambi K. 2014. A novel T-DNA integration in rice involving two interchromosomal translocations. Plant Cell Rep 33:929-944.
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