Construction Of User-Friendly Plant Expression Vectors Using Rice Promoters

Abstract

This project (3 months duration) was embedded within our ongoing projects on “Rice Functional Genomics”. A PhD student, Andrew Eamens was employed in this project to continue work on the development of user friendly plant expression vectors based on rice promoters. This work was started towards the end of Andrew’s PhD studentship. Using reporter genes containing a minimal promoter (enhancer trap) or intron splice acceptors (gene trap) in T-DNA or transposon tagging systems, several promoter sequences were identified by Andrew during his doctoral research and were used to produce plant expression vectors with tissue specific expression. The previously developed double right boarder (DRB) vector technology was used to construct a small group of user-friendly plant expression vectors with tissue-specific expression promoters. A new base binary vector construct (PDRB12dn) was constructed during this project period. The binary vector contained a promoterless reporter gene (sgfpS65T) mounted between the second right border (RB2) and the T-DNA left border (LB). The reporter gene is flanked upstream by a multiple cloning site (MCS) containing several unique restriction enzyme (RE) cleavage sites for easy cloning of putative promoter fragments. A total of 12 promoter fragments were also amplified by the polymerase chain reaction (PCR), ready for addition to the base vector. Cloning of individual promoter fragments is now in progress. The plant expression constructs being produced will enable the production of selectable marker free transgenic plants expressing GOIs in specific cells, tissues or organs.