Construction Of User-Friendly Plant Expression Vectors Using Rice Promoters

摘要

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