[Arabidopsis] question on GUS vector

Smith Chiang via arab-gen%40net.bio.net (by chiangscn from gmail.com)
Mon Jun 11 23:56:00 EST 2012


Dear colleagues,

I met some problem with the GUS vector pBI101.1, as I failed to detect
GUS staining(blue patch) for several promoters, which are 1-2kb
upstream of ATG codon(i.e. 5'UTR is included in the cloning). I knew
there is a stop codon problem with pBI101.3.

The restriction sites used for cloning promoter regions into pBI101.1
are HindIII and BamHI or XmaI(SmaI), and constucts were sequenced
before use.

Even after induced with a specific stress, which upregulated the gene
beased on qPCR data, still failed to see GUS staining.

However, I saw many papers used pBI101 vector, with good staining. Is
that really because different promoters matter?

I also saw many used pBI121 vector, with promoter-of-interest
replacing CaMV35S.

Therefore, I am trying to find a good promoterless GUS vector for
promoter study in stable transformed plants.

Previously, pCAMBIA vector was used and, frequently observed rather
strong GUS staining, maybe because of the enhancer effect, as
methioned by CAMBIA.

anyone could provide some suggestion/help or recomend a good GUS vector?

thanks so much!



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