I used 35s promoter to over express gene in rice. But when i test over expression gene expression level in several T1 generation plants, I found the expression level did not increase remarkably(my colleague over express other gene in rice using 35s promoter get hundred times increase in expression level) . Did somedody used the 35s promoter in rice? I want to know why the expression level did not increase remarkerably in my transgenetic plant. Thank you very much.
Did you use regular 35S (1x) promoter, or double-enhancer 35S (2x) promoter. The results can be different.
Also, although 35S promoter is presumed to be a constitutive promoter and should expressed in all tissues, reports have showed that in some experiments, 35S-driven gene expression is developmental regulated and tissue-specific. See this paper: "Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP." [ http://www.ncbi.nlm.nih.gov/pubmed/12369622 ]
1. Are you overexpressing a screenable marker gene (such as GFP, GUS,....) or plant genes?
2. You said, "not increased remarkably", or it did not increase at all STATISTICALLY. Was your construct correct?
1. Or, maybe it is because of 'position effect' of the insertion sites. How many plants you have checked for expression level?
2. Multiple transgene insertion can also cause gene silencing. Did you know the copy numbers of transgenes in those T1 plants you checked??
Attached is a good review paper about plant promoters, including the discussion of 35S promoter. At page #3, it says (see the highlighted yellow part):
"In other cases, transgenic chrysanthemum expressing a GUS gene under the control of the 35S promoter exhibited weak transgene expression levels."
**I think the Journal is published by your University.
I used hpt gene as a selective marker, and selected the positive transegentic plant by PCR. I planted the T1 lines in the field, and identified the positive plants by PCR. The selective marker segregation ratio near 3:1 in T1 lines were chosen for farther analysis. So they can be seen as just single copy. I chosen 4 single copy inserted T1 lines, the expression level only increased five fold in transgenetic lines.
Hi Th,
1. First of all, segregation rate near 3:1 doesn't warrant that a single copy transgene is inserted. For example, you have 2 transgenes integrated in the genome, and Copy #1 has an intact construct, and Copy #2 has a 'truncated' construct. If the truncated part of the Copy #2 is on the hpt gene (lost function of hpt gene), then this line will have a 3:1 segregation rate (under hygromycin selection), behaving like a 'single' copy line. And, at the same time, you will have 2 copies of un-truncated GOI (Gene-of-Interest). Besides, multiple copies integrate at the same locus can also a problem.
However, you said you have used 4 independent events ('single' copy line) for expression level test, so this probably is not the cause of the problem.
Another thing you need to consider too, is the 'Protein Turnover'.
Although you and your colleague used the same vector and the same promoter (35S), if the protein species you were overexpressing were different, the expression results (i.e. amount in the cells) could be different when you measure them. This can be due to their different 'degradation rate'. The half-lives of a protein can be minutes, others can be days. See the website below, and the article attached (P.272; yellow highlighted part).
http://www.ncbi.nlm.nih.gov/books/NBK9957/
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