Roberts, Alison , Liepman, Aaron H. , Sorenson, Iben , Willats, William G T , Notis, Christine , Stein, Alexis .
Physcomitrella patens as a heterologous expression system for investigating the functions of CESA-like gene products.
Cellulose synthase-like (CSL) genes are proposed to encode glycan synthases that polymerize the backbones of non-cellulosic cell wall polysaccharides. Consistent with this hypothesis, CSLA genes are known to encode mannan synthases and a CSLF gene has been implicated in mixed-linkage beta-glucan synthesis. Members of the CSLB, CSLE, CSLG, and CSLH families have not been functionally characterized. The complete genome sequence of the moss Physcomitella patens lacks members of these CSL families. We are using P. patens as a heterologous expression system to investigate the functions of the proteins encoded by CSLB, CSLE, and CSLG genes from Arabidopsis. Transformation vectors were constructed by cloning T7-tagged ORFs of CSLB3, CSLE1 and CSLG2 from Arabidopsis into a Gateway┬« expression vector that drives transgene expression via a fragment of the rice actin1 promoter and targets transgene insertion to the 108 locus of P. patens, which can be disrupted with no effect on phenotype. Stable transformants were screened for targeting to the 108 locus and clones containing properly-targeted AtCSLB3, AtCSLE1, and AtCSLG2 transgenes were identified. Expression of the transgenes in the P. patens clones was verified by RT-PCR. Expression analysis at the protein level is now underway. Results of preliminary cell wall composition analysis using microarrays spotted with sequential cell wall extracts and probed with monoclonal antibodies (Comprehensive Microarray Polymer Profiling) are consistent with alteration in cell wall polysaccharide synthesis in the transgene-expressing lines. This project was supported in part by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number # (2003-35304-13233).
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1 - University of Rhode Island, Biological Sciences, Kingston, RI, 02881, USA
2 - Eastern Michigan University, Biology
3 - The University of Copenhagen, Institute of Molecular Biology
cellulose synthase like gene.
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM