| Cotton is one of the most important economic crops in the world and is the most prevalent natural fiber used as the raw materials in textile production. Because of their rapid elongation rate, high cellulose content, and lack of cell division, cotton fibers are a good experimental model for studying the molecular mechanism of plant cell elongation, cell wall biogenesis. Cloning the developmentally regulated genes from cotton fibers may play important roles not only in improving the quality of cotton fiber, but also in studying plant cell development and cellulose biosynthesis.The special primer pair was designed based on a cotton fiber EST which is highly homologous to KOR gene of Arabidopsis thaliana, and A full-length cDNA was isolated (which is designated GhCEL) from a Gossypium hirsutum L ZAP II cDNA library by the PCR screening. The clone was submitted to GenBank, with an accession number: AY574906. The cDNA contains 2293bp terminating in a poly (A) ?tail, and an open reading frame encoding a polypeptide of 619-amino acid residues with a theoretical molecular weight of 68.6kDa and a pI of 9.0. The BLAST analysis indicated that GhCEL, the protein of GhCEL had 79% identity to Lycopersicon Cel3, and 74% to Arabidopsis KOR, respectively. Hydropathy analysis of the amino acid sequence showed that the GhCEL was a potential membrane protein which had a trasmembrane hydrophobic region between residues 72-94. Bioinformatics software analysis illuminated that GhCEL had a Glycosyl hydrolases family 9 activity catalyzing domain between residues 109-586, a Proline-rich region between residues 604-619, a possible cAMP- and cGMP-dependent protein kinase phosphorylation site and 9 possibleN-glycosylation site. The complete coding sequence of GhCEL was cloned into the prokaryotic expression vector pGEX-KG plasmid, after induced by IPTG at 30C or 37C for 4h, a fusion protein with a molecular weight of 95kDa was produced in E. coli DH5 a... |
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