Purification, Sequencing And Subcellular Localization Of An Abscisic Acid-Activated Calcium-Dependent Protein Kinase From Grape Berry

Abscisic acid (ABA) plays essential roles for the fruit growth and development. To understand the mechanisms of fruit growth and development regulated by abscisic acid, it is important to identify its signal components. Protein phosphorylation catalyzed by protein kinases is critical in the signal transduction pathway. Calcium-dependent protein kinase (CDPK) have been found in plant, firstly, but now, its function in the ABA signal transduction pathway are not clear.In the gel autophosphorylation assay of the protein kinase, a 62.5KD CDPK has been identified in the microsome of the grape berries and it was able to be activated by extrinsic ABA. Since this CDPK is involved in the signal pathway, and accordingly is the important role, we focused on the purification of the protein kinase.Microsomal fraction of grape fruit are isolated, separated on a SDS-PAGE, then eluted the target protein from the gel. lyophilizing and gathering, we got the protein of the electrophoretically purity. Two-dimentional gel electrophoesis experiments established that two kinds of proteins, whose molecular weight are close but pI were different, are presented in the purified products. Western blot analyzing the proteins separated from 2D with anti-CDPK full-length sequence from grape berries and N-terminal special antibody, respectively, further confirmes the isolation of the 62.5KD CDPK from the purified proteins. Applying the LS-MS/MS to determine the the CDPK amino acid sequeces hydrolyzed by protease, we got the sequeces of MAPDKPLDSAVLSR; VIAEGLSEEEIGGLR; LTAHEVLSHPWWDDR and NNLNLGDVLGIPDMR, etc. Database searches revealed that purified CDPK was identical to the VCPK1 cloned by our lab by means of screening the grape berries cDNA library using the degenerate primers. So we concluded that they hold the same identity. The accession no. of the gene and the deduced protein is gi 39598579 / gb AAR 28766.1, respectively.To determine where the VCPK1 localizes, our experiments is threefolds: first, the biochemical localization: membrane system isolated through Dextran-PEG two-phase system then immunoblotting with the antibody against the CDPK full-length sequence and the N-terminal special antibody, respectively. Second, the subcellular localization: using the immunogold electron-microscopy technique to determine the subcellular localization of VCPK1 in developing grape berry. Third, the molecular localization: we constructed the vector contains a CaMV-35S promoter, GFP and VCPK1, andtransformed the E.coli with the recombinant vector, and then using transient expression systems in Arabidopsis leaves, the onion lower epidermis and protoplast of Arabidopsis, scrutinazing the results by CLSM (confocal laser scanning microscopy). Based on the analysis of the results above, it suggests that VCPK1 was primarily targeted to the plasma membrane of the flesh parenchyma cells and the chloroplast, the absence of VCPK1 in the subcellular unit else , cytoplasm and vacuole.