Evaluation of expression cassettes in developing rice endosperm using a transient expression assay

In order to evaluate rapidly the potential of various expression cassettes for transgene expression in rice grain, a transient expression assay was developed using rice immature endosperms. The promoters of several seed storage protein genes including the rice glutelins ( Gt3 , GluB-1 , GluB-2 ), rice prolamins ( PG5a , RP6 ), rice α-globulin ( Glb ) and wheat glutenin ( Bx7 ), were examined for their expression levels in rice immature endosperms isolated from the caryopses at 7–9 days after pollination. In this promoter comparison, the wheat Bx7 promoter showed the highest transcription activity, followed by the promoters of the Glb , GluB-2 , and Gt3 genes. Based on this result, further studies were conducted using the Glb and Bx7 promoters. We examined the effects of the addition of a second promoter fragment, the alfalfa mosaic virus (AMV) 5′untranslated leader sequence (UTL), and various introns and terminators from different genes. Although the addition of a second promoter fragment and the AMV 5′UTL had no effect on GUS expression levels, the insertion of the rice waxy first intron increased GUS expression of the Glb and Bx7 constructs 3 and 6-fold, respectively. The maize adh1 first intron also showed a 2–3-fold increase in the GUS expression levels of both the Glb and Bx7 reporter gene constructs. A comparison of terminator sequences revealed the nopaline synthase gene terminator from Agrobacterium and the rice waxy gene terminator were the most effective in contributing to elevated expression levels.