Heterologous Expression of the Porphyra yezoensis CesA gene in Physcomitrella patens

Author(s)

Virginia Lai, University of Rhode IslandFollow

Advisor

Roberts, Alison [Faculty Advisor, Department of Biological Sciences]

Date

5-2010

Comments

Opens with Windows Picture & Fax Viewer

Keywords

plant cell wall, cellulose microfibrils, terminal complex

Abstract

Cellulose is an essential component of plant cell walls and occurs in the form of long, thread-like structures known as cellulose microfibrils. The cellulose microfibrils are produced by terminal complexes in the plasma membrane, which are aggregates of proteins encoded by CESA genes. These terminal complexes extrude cellulose microfibrils as they move freely in the plasma membrane. In land plants, the proteins assemble into circular rosette terminal complexes that synthesize rod-shaped microfibrils. Other organisms, including some algae, have linear terminal complexes that synthesize ribbon-shaped microfibrils. Differences in the CESA gene sequences are proposed to be the cause of differences found in the structure of terminal complexes and consequently, the shape of cellulose microfibrils. Porphyra yezoensis, a red alga, has linear terminal complexes producing ribbon-like microfibrils. In contrast Physcomitrella patens, a land plant from the moss group, has rosette shaped terminal complexes that produce rod-like microfibrils like all other land plants. The ultimate purpose of this study is to genetically modify P. patens so that it will produce the ribbon-like microfibril structure seen in Porphyra yezoensis.

In this study the CESA gene found in P. yezoensis was inserted into the genome of wild type P. patens and a mutant line of the moss that was lacking one of its CESA genes and was unable to produce leafy shoots as a result. Genotypic analyses were then performed to ensure proper integration of the P. yezoensis CESA gene into the nuclear genome of P. patens. Transcription of mRNA was tested through RT-PCR and immunofluorescence was used to visualize localization of the protein. Sixteen stable lines have been established that show integration, and mRNA and protein expression.