[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide-specific phospholipase Cs (PI-PLCs) are important enzymes in eukaryotes, which catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. The Arabidopsis genome contains nine putative PI-PLC genes. AtPLC4, an abiotic stress induced gene, has been reported to encode an active PI-PLC isoform. However, the exact roles of putative AtPLC4 in plant remain to be elicited. The first 108 amino acid residues of the N-terminal of AtPLC4, referred to as AtPLC4 N, was expressed as a recombinant protein in Escherichia coli and used as antigen in generating antibody. Purified recombinant proteins including AtPLC1 to AtPLC5, AtPLC8, AtPLC9 and AtPLC4 N were transferred onto the same blot to test specificity of the prepared antibody. Western blot result shows that only AtPLC4 and AtPLC4 N can be recognized by the antibody. The antibody recognized a protein of approximately 68kDa in the plasma membrane fraction and cytosolic fractions prepared from Arabidopsis thaliana plants. This corresponds very well with the calculated molecular weight of AtPLC4. The results suggest that AtPLC4 may encode a plasma membrane-associated protein.
Full-text · Article · May 2007 · Protein Expression and Purification
[Show abstract][Hide abstract] ABSTRACT: A full-length cDNA clone corresponding to a putative phosphatidylinositol-specific phospholipase C (PI-PLC) was isolated from
Arabidopsis thaliana by screening a cDNA library and using RT-PCR strategy. The cDNA, designated AtPLC6, encodes a putative polypeptide of 578 amino acid residues with a calculated molecular mass of 66251.84 D and a pI of 7.24.
The sequence analysis indicates that the polypeptide contains X, Y, EF-hand and C2 domains. The overall structure of putative
AtPLC6 protein, like other plant PI-PLCs, is most similar to that of mammalian PLCS. The recombinant AtPLC6 protein expressed
in E. coli was able to hydrolyze phosphatidylinositol 4,5-biophosphate (PIP2) to generate inositol 1,4,5-trisphate (TP3) and 1,2-diacylglycerol
(DAG). The protein hydrolyzes PIP2 in a Ca2+-dependent manner and the optimum concentration of Ca2+ is 10 μmol/L. These results suggested that AtPLC6 gene encodes a genuine PI-PLC. Northern blot analysis showed that the AtPLC6 gene is expressed at low level in all examined tissues, such as roots, stems, leaves, flowers, siliques and seedlings under
normal growth conditions. The gene is strongly induced under low temperature and weakly induced under various stresses, such
as ABA, high-salt stress and heat. These results suggested that AtPLC6 might be involved in the signal-trans-duction pathways
of cold responses of the plants.
Preview · Article · Mar 2004 · Chinese Science Bulletin