Hello everyone, I am intended to over express one gene in arabidopsis. I just wondering to know whether cloning gene (with UTR regions without introns) into vector through TA cloning and transforming into arabidopsis through agrobacterium mediated transformation by floral dip method would suffice my needs or it has to have only coding region from ATG to stop codon ?? Your response is appreciated.
Thanks in advance
Uday
Hello Uday Sir,
Use strong promoter (CaMV 35S) for over expression. You can use cDNA version of your gene of interest as cloning requisite.
Good Luck
In most cases simply using the cDNA will be sufficient. The CaMV 35S promoter is very strong, so even when UTR regions and/or introns promote mRNA stability, you should get phenotype caused by overexpression. If you have any information regarding your gene, ie expression levels from microarray analysis (eFP browser) or information from family members this could help to design the best cloning strategy.
1. I have used gene ORF (from start codon to stop codon) for transgene overexpression driven by 35S promoter, the results of different phenotypes between the transgenic and non-transgenic lines are clear. It works well for me.
2. Most studies on 5' UTRs have focused on elucidating the importance of viral leader sequences (see attached paper for more description). People have used these viral 'leader sequences' to enhance gene expression. Several plant 5'UTRs have also seen to increase the transgene expression. However, if you use 35S promoter, especially the 'double 35S' (with 2 enhancers) promoter for transgene overexpression, you will get very strong transgene expression anyway, given that the transgene is integrated into a 'good' spot and no gene-silencing caused by multiple transgene integration.
Dear Uday
you can take pCAMBIA1305.1 for over expression of your gene. Take coding region (ATG to Stop codon) but check translational fusion I means check whether your gene sequences are in frame with promoter sequences. otherwise it may get problem what I face during promoter isolation.
All the best
Only the coding region will work fine. UTRs and introns are not involved in the translation of the mRNA into a protein, so you don´t need them…
For over-expression (using CaMV35S promoter) is recommended only the use of the coding region (ATG to Stop codon). Only if you will not determine any differences among WT and your putative over-expressing lines, you could use the coding region plus the 3'-UTR to improve your mRNA stability and hence to achieve a phenotype.
Regards
@ Stefano,
I am interested in what you said "Using the whole sequence could cause RNA processing problems due to the high amount of transcripts". Because I was thinking about which one will be a better version for using as a transgene in overexpress under 35S promoter:(1) the cDNA version (no introns) or (2) the full gene (introns + exons) [which will go through RNA process]. I am interested to know if any papers out there support the theory of 'high amount of transcripts' and why. Thanks.
I would prefer to use the whole genomic sequence of your interest gene. And also, if you get its expression under its native promoter will be more preferable, just in case of the dominant negtive effect caused by the extopic expression caused by the 35s promoter driven expression.
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