If you want to transfer an exogenous gene into certain plant for overexpression, what factors should be considered when making construct, and what analyses of molecular biology should be commonly conducted on the transgenic plants
Since plant is an eukaryotic organism and there are a coupled of things which you may need to consider:
1) The exogenous gene is from what origin? Prokaryotes? Eukaryotes or?
2) Is the expressed gene product required post-transcriptional modification like spicing?
3) What is the size of the gene and what is the method you want to use to insert the gene? by what vector? technique?
4) What is the preferred method reported in any previous studies which could help in optimized the gene expression?
5) How are you going to validate whether the gene expression is successful or not?
There are still some more issues which you might need to consider and it will be quite lengthy to mention all here. You will need more studying and planning to ensure your experiment is successful.
Good luck!
Best wishes,
Chong
To add to Zx Chong's answer, you may also need to consider the promoter of your gene of interest, whether it is suitable for plants, and whether its a constitutive or inducible promoter. Sometimes the use of strong, constitutive promoters like CaMV35S may lead to gene silencing.
for molecular analysis, the "standard" is usually PCR, followed by southern blotting. You can also do northern blot or real-time PCR to determine the expression level of your transgene.
I am agree with Dr. Willy Yee as promoter is very essential for your desired gene expression. You have to decide first what gene you are targeting and for what product you want. You can use tissue specific promoter (like Viciline) for specific expression or for consecutively expression you can use CaMV.
What factors should be considered when making a DNA construct for transformation?
1. Plant for genetic transformation. Monocots or Dicots? This will decide what promoter you should use--monocot promoter or dicot promoter.
2. Method of transformation. If the construct is used for Agrobacterium-mediated genetic transformation or Agroinfiltration, your T-DNA should contain both the 'right border' (RB) and 'left border' (LB), required elements for Agrobacterium transformation. If for bombardment or protoplast (PEG-mediated) transformation, those borders are not needed.
3. Promoter. What kind of promoter you should use in your project? A constitutive promoter (ex. 2x 35S; Ubiqutin..) for overexpression, an inducible promoter (ex. heat-induced promoter), a pollen promoter (for tissue specific expression).........
4. Gene of interest (GOI). Are you planning to use cDNA or genomic DNA from the gene. cDNA is usually shorter. GOI should be sequenced before sticking into the vector.
5. GOI origin. Is the gene from prokaryotes or eukaryotes? If from prokaryotes, do you need to codon-optimize the gene to make it more suitable for expression in plants?
6. Organelle targeting. Would you like your expressed protein targets to a particular organelle? If target to nucleus, you need to add a nucleus localization signal (NLS) to your gene. Likewise, other signals are needed if the protein is targeted to an organelle such as mitochrondria or chloroplasts.
7. 'Tag' for purification. Do you need to purify your expressed protein later? If so, you might need to clone your trait gene into a expression vector with tags, such as 6x His tags, which you can use to purify your protein later.
8. Research purpose. For example, (1) you might need a fusion protein such as GFP (fuses to your protein) to see where your protein locates in the cell. (2) You need to clone in the CRISPR elements (such as Cas9 gene and sgRNA) if your research goal is doing some CRISPR experiments.
9. Plant selectable marker gene (SMG). Some selection system / antibiotic-resistant gene can be good for one plant species, but not necessary suitable for other plant species. Review publications related to your plant species transformation
10. SMG-free. Do you want to eliminate the SMG (selectable marker gene) to produce SMG-free transgenic plants? If so, you should incoporate elements of site-specific recombination system (ex. Cre-lox) or other strategy into your DNA constract to remove SMG later.
11. Positions of genes. How to arrange your genes (ex. GOI, SMG,.....) , ex. orientations, in your construct is also important. Many long repeats in your construct? It can cause unwanted gene rearrangement or deletion...
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