Green fluorescent protein-based enhancer traps for rice functional genomics

Abstract

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 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.