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Plant Science, Vol. 161 (6) (2001) pp. 1107 - 1116
© 2001 Elsevier Science Ireland Ltd. All rights reserved.
Evaluation of expression cassettes in developing rice endosperm using a transient expression assay
Yong-Sic Hwang, Cass McCullar and Ning Huang

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 a-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. © 2001 Elsevier Science Ireland Ltd. All rights reserved.