Department of Biological Sciences, Stanford University, Stanford, CA 94305- 5020, USA
One of our interests has been the optimization of gene expression at the level
of translation in rice. To determine those features of an mRNA which are
important, we introduced beta-glucuronidase (GUS) mRNA which had been
synthesized in vitro using a T7-based promoter, into rice protoplasts (Taipei
309) via electroporation.The rice protoplasts are approximately 20 um in
diameter and were subjected to 700 V/cm in the electroporation treatment using
a Pro-mega Biotec machine. The presence of 25A residues at the 3'-end of the
message resulted in a 15 to 20 fold increase in the level of GUS activity
detected. When the untranslated leader sequence (omega) from tobacco mosaic
virus, which is known to increase translational efficiency in dicots, was
present at the 5'-end of the GUS mRNA, a 3 to 4 fold increase in GUS activity
was measured. The means by which an omega sequence and a poly A tail enhance
GUS expression operate independently, and therefore the resulting level of GUS
expression when both are present is the multiplication of their individual
contributions.