I am working on characterizing the function of a gene under stress. The T-DNA lines were obtained from SALK. The miRNA was prepared in lab and it shows very insignificant expression of my gene. Further, the 35S:over-expression lines were also generated in own lab with expression many fold to be qualified as a successful OX-lines. Currently, I am using the single T-DNA line (insertion at intron) and 3 each miRNA and Overexpression lines. I see a stress phenotype in all of them, but not in WT. Why is this weird phenotype? I understand the fact that sometimes due of presence of promoter enhancing sequences in the T-DNA, the T-DNA lines act as ox-line. But, the qRT data shows very low gene expression in my T-DNA mutant, removing the ambiguity of over-expression. Please provide your useful insight.
Further, the miRNA can be distinguished from my OX-lines by different in root length on 1/2 MS media under normal conditions. miRNA is significantly longer than the OX-expression, WT and the T-DNA line. WT and T-DNA almost same root length. The Over-expression lines shows the shortest root lengths.
Thanks!
I would check what controls you are using for each. The small manipulations that we make to the plants can induce subtle phenotypes that are not directly linked with the genes we are investigating.
If the T-DNA lines are heterozygous, try growing them without selection and then screening them with a PCR to find "wild type" lines from the same lineage. This will be a better control than your lab WT.
For the miRNA lines do you have a negative control miRNA for comparison.
For the OX-lines, are you comparing them to plants that have been transformed with an empty vector?
@Ben Peter ! Thanks for your valuable insight.
My T-DNA lines are homozygous. Further, I am not using any control for miRNA. For OX-lines, I am comparing them with WT and 3 independent miRNA lines. Is it possible that the phenotype I am looking is redundantly regulated by my gene; but may be influenced by its homologous genes. Say: Over-expression line switching on other genes and miRNA showing non-specific binding to closely related homologues. Is there any evidence showing OFF-Target effect (even if the result of qRT shows specificity for a particular gene in mutants, miRNA and OX-lines)?
Thanks!
Dear @Manu ! Thanks for your valuable comment. My all lines are homozygous (OX and miRNA are T3 lines) and T-DNA line is knock down mutant, not knock out. My all plants looks very healthy both in soil and on 1/2 MS media. Further, the qRT PCR data of the different lines are within the expected range.
Thanks!
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