The enhanced green fluorescent protein (EGFP) reporter was cloned into 26
binary vectors including enhancer traps and positive/negative controls, and
successfully used for tracing dynamic gene expression in the transformation
process of rice, and for screening enhancer trap pattern lines. GUS::EGFP and
GUSPlus::EGFP fusion reporters were also created to make good use of the
advantage of GFP as a vital marker for dynamic monitoring of gene expression
and the advantage of GUS for its high...[Show more] sensitivity in histochemical staining.
An Agrobacterium-mediated transformation system for the japonica rice variety
Millin was well established. In a case study with enhancer trap constructs
harboring GUSPlus::EGFP fusion reporter, a total of 1,021 transgenic rice lines
were obtained in a single transformation experiment starting with 1,000
scutellum-derived calli. Further improvement of the transformation efficiency
was achieved by decreasing the temperature to 22°C during co-cultivation of rice
calli with agrobacteria. The efficiency can fulfill the prerequisite for the
TransGenomics project in which large populations of transgenic lines are needed.
Several sets of enhancer traps were constructed and tested through
transformation of rice calli. GFP expression of the enhancer traps, along with
corresponding Gal4-deletion constructs, was studied extensively during the
callus stage and at plant level. Constructs carrying the CaMV 35S promoter
exhibited serious within T-DNA cis-activities, imposing significant background
problem for screening genomic enhancers being trapped. The problem was in a
great extent solved by replacing the CaMV 35S promoter with Ubi- I promoter in
the selection cassette of enhancer trap constructs. Out of 393 enhancer trap lines
obtained with the improved constructs, 129 (32.8%) lines were found GFPpositive
with diverse expression patterns, which were valuable genetic resources
for functional studies of gene of interest in rice.
Sexual crosses, between enhancer trap lines with EGFP reporter and target gene
lines harboring 6xUAS-MP-GUS, were made to verify functionality of the transactivator in the system. Clear co-expressiOn of GFP and GUS in F 1
progenies were observed, implying that the transactivator performed its functions
as theoretically expected and the system established could be used for creating
gain-of-function mutagenesis in rice. This is the first genetic testing of the Gal4-
UAS system in rice, providing solid evidences for a successful establishment of
the Transcriptional Activator-Facilitated Enhancer Trap (TAFET) system in rice.
Potential usage of the system for innovative rice breeding was also discussed.
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