I'm trying to introduce some PR genes to cotton and I'm using the same vectors and transformation method (agro infection) for all of them but the results are significantly different. could you please tell me what the reason might be???
Some genes are expressed better than others, depending on where they end up in the plant genome and how toxic they are for the cell. You may be best to screen a wide range of transformants for the ones which are the best expressors.
Hi Rasi,
Many factors can affect the results of a transgenesis experiment; some of them are listed below:
1. Plant materials you use: (1) different plant species, (2) different plant varieties, etc., can result in different experimental outcomes.
2. Promoters used to drive the gene: ex. 35S (1 enhancer) vs. d35S (2 enhancers), etc.
3. Gene versions: ex. GFP vs. EGFP (enhanced GFP), etc.
4. 'Codon Usage' of the gene: two proteins with the SAME amino acid sequences, but derived from different DNA sequences (due to codon usage), the expression ability will be different
5. Position effect: depending on where the 'transgene' is integrated; for example, integration at a 'centromeric region' will most likely result in poor or no transgene expression
6. 'Copy number' effect : multiple copies of transgene insertions can induce gene silencing, resulting in no transgene product
7. Transgene integration fashion: ex. if 2 copies of transgenes land in the same locus with 'head to head' fashion, it can cause gene silencing.
8. Transgene integrity: ex. intact vs. truncated version of transgene in the plants
9. The developmental stage or tissues of transgenic plants used for analysis
10. Different type of PR-gene or even related PR-gene can have different responses at different tissues to the attack of a specific pathogen. See this attached paper about PR-2, PR-5 and PR-8's response to pathogen Erwinia amylovora in apple.
This phenomena is normal, by the different factors inherent to plant transformation (Agrobacterium tumefacien strain, age of your bacteria culture, plant cultivar, temperature, humidity, position effect, kind of promoters, etc). You have to transform several plants to establish plant transformation lines to determine the most stable phenotype (in your case most probably the phenotype that you are looking for is an enhanced resistance against a pathogen) in your population of transformants. After that, you will be able to establish your plant lines to work and to carry out your experiments without a significant variability between your plants.
Regards
I really enjoyed your interesting answers. Meanwhile, I would like to mention that I use three different kinds of PR genes family. Apart from the factor of gene, all the other factors are supposed to be the same. I wonder if different kinds of PR genes could make negetive effect on plant growth and vigore, because I assume that transform of different genes could be compeletely different.
@Rasi,
Yes, it is possible. Please refer to the attached paper: "Growth–Defense Tradeoffs in Plants: A Balancing Act to Optimize Fitness". The energy for plants to growth and defense is well controlled and balanced. If too much energy is used for defense, it will affect the overall fitness of the plants. Several research results have supported this statement. See some study cases in the paragraph "In defense of the growth tradeoff" of the paper (p.1270-71, yellow highlighted).
PR genes are usually induced by pathogen attack. If you artificially overexpress the gene in the plants (and if use a constitutive promoter, ex 35S), the gene product will be produced constitutively. As a result, the plant defense system can be constantly turned ON. In turn, the part of energy used for plant growth (fitness) will be relocated for using in 'defense', causing the undesirable plant phenotypes you observed. The 'degree' of the undesirable phenotype can depend on which PR gene you are overexpressing in plants. Expression of some types of PR genes might have more effect on the phenotype than expressing others.
This is another paper (2016): "Modulation of R-gene expression across environments".
"These results suggest that variation in R-gene expression across environments may be shaped by natural selection to reduce fitness costs of R-gene expression in permissive or predictable environments. Down-regulation (of R-gene) would follow if costs of R-gene expression negatively impact plant fitness in the absence of disease."
So, it can be a burden to the plants if you overexpressing those R-genes constantly.
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