1 Genesis Research and Development Corporation, 1 Fox Street, Parnel, Auckland, New Zealand 2 Akkadix Corporation, 11099 North Torrey Pine Road, Suite 160, La Jolla, CA 92037
A strong, constitutive promoter to drive expression of transgenes is an important tool for the development of transgenic trees. Few gymnosperm promoters have been cloned, and those derived from angiosperms often function poorly in gymnosperms. High-throughput sequencing of Pinus radiata ESTs associated with high-throughput promoter cloning have allowed us to identify a number of putative promoter candidates. One of the most abundant of the constitutively expressed sequences was a ubiquitin gene, that we have named "Super-Ubiquitin". Although the ubiquitin coding region is highly conserved, the 5' un-translated region (UTR) is not conserved between different ubiquitin genes in pine. Differences in the 5'UTRs of members of the ubiquitin gene family allowed us to classify the ESTs that we isolated into different subgroups. This and in silico tissue expression then allowed us to identify the most highly expressed ubiquitin gene in the Radiata pine EST database. A 2.2 Kb promoter from this "Super-Ubiquitin" gene was cloned, sequenced and characterized. This promoter contains the 5' UTR of the super-ubiquitin gene, including a 900bp intron. An intron in the 5' UTR is a common feature of plant polyubiquitin genes and may be involved in determining gene expression levels. In silico, slot-blot, northern and medium-density gene array data confirmed that the super ubiquitin gene is highly expressed in almost all Pinus and Eucalyptus tissues. Several promoter reporter gene constructs were made to detect the activity of the pine super ubiquitin promoter in Pine, Arabidopsis and tobacco. In all cases, reporter gene expression (either GFP, GUS, or Luciferase ) was equal or higher with these new constructs than with those based on the widely used 35S promoter from cauliflower mosaic virus. Thus, we suggest that the gymnosperm super ubiquitin promoter provides a superior constitutive plant promoter for use in woody plants as well as in other plant species.
Plant & Animal Genome VIII Conference Plant & Animal Genome VIII Conference