|Turley, Rickie - Rick|
Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/10/2006
Publication Date: 8/5/2006
Citation: Turley, R.B., Vaughn, K.C. Expression of pectin methylesterase in cotton fiber. American Society of Plant Biologists Annual Meeting. Plant Physiology 1415 supplemental: 193. Abstract #P18035 Interpretive Summary:
Technical Abstract: Cotton (Gossypium hirsutum L.) fibers provide a model system to study cell elongation and wall deposition. The walls of elongating fiber cells are bilayered with the outer layer enriched in de-esterified polygalacturonic acids (PGA) and the inner layer enriched in xyloglucans and cellulose. This bilayer, and the de-esterified PGAs, are conspicuously absent in the cell walls of the ovule epidermal cells adjacent to fiber cells. Additionally, evaluation of fiber mutants Li1 and Li2 (short fiber, approximately 1/10th the length of wildtype fiber) indicates a reduction in de-esterified PGA’s in their respective cell walls but not a reduction in pectin methylesterase (PME) activity. In a continued effort to understand the role of these de-esterified PGAs in fiber elongation, a PME gene and four PME inhibitor (PMEI) family genes were isolated, sequenced and ligated into the vector pPICZ alpha A for heterologous protein expression in Pichia pastoris. Three PMEI’s have been successfully expressed and purified. PME and one PMEI, however, were expressed only in trace amounts. After three attempts to express these proteins in Pichia, we theorize the problems of expression resides in the codon usage of these genes. Two Arginine (Arg) codons in cotton CGG and CGC appear to be the problem in producing both PME and PMEI. Using PCR and site directed mutagenesis these Arg codons are being modified so that functional proteins may be produced. The Champion pET directional TOPO expression system is being used as a contingency to express these proteins in bacteria. As monospecific polyclonal antibodies are produced and verified, these will be used to assess the isoforms and quantity of PME and the activity of each PMEI in wildtype and mutant fibers.