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The gene encoding the rice transcription factor, REB (rice endosperm
bZIP) was cloned from a bacterial artificial chromosome library
of rice. The cloned 6,227-bp-long Reb gene is composed of six exons
and five introns and is flanked by a 1.2-kb 5' promoter and a 1.2-kb
3' terminator region. The function of the Reb gene was explored
by a transient assay by using a rice immature endosperm system.
The effector constructs containing the native gene or fusion genes
linking Reb to the rice actin (Act) or globulin (Glb) gene promoters
and the reporter gene construct Glb-beta-glucuronidase (GUS) were
used in this study. When these effector constructs were cotransferred
with the reporter uidA gene encoding GUS under the control of the
Glb promoter into immature rice endosperm cells, the Glb promoter
was activated. The transient GUS expression was 2.0 to 2.5-fold
higher with the effector construct than without. When the upstream
activation sequence containing the GCCACGT(A/C)AG motifs of the
Glb promoter was deleted, the activation by REB was abolished. On
the other hand, a gain-of-function experiment showed that inserting
the upstream activation sequence into the glutelin-1 (Gt1) promoter
made it responsive to activation by REB. When cotransformed with
Reb gene, mature transgenic rice grains containing the human lysozyme
gene driven by the Glb promoter produced 3.7-fold more lysozyme.
Accumulation of recombinant lysozyme in mature seed ranged from
30.57 to 279.61 mg·mg&mg-1 total soluble protein in individual
transformants from 30 independent transformation events. Thus, our
results show that REB is not only a transcriptional activator, it
can also be used to increase the expression of recombinant protein
in transgenic rice grains.
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