A Calcium-Dependent Protein Kinase In Apple: Molecular Cloning, Expression, Localization And Up-regulation By Abscisic Acid

Abscisic acid is a important regulator in fruit development.It is a complex life course from produce Abscisic acid to physiological response.Protein kinases play important roles in cell signal transduction.lt is very inportent to study protein kinase function in abscisic acid signal transduction.To investigate a function of CDPKs in apple fruits which could be involved in the regulation of fruit development, we isolated a full-length cDNA encoding a calcium-dependent protein kinase(CDPK) designated MdCPKl from apple fruit(Malus Domestica Bork cv.Starkrimson/Malus micromalus.).The main part of the MdCPKl cDNA was amplified in a RT-PCR reaction with two degenerate oligonucleotides that corresponded to the catalytic domain of CDPKs. PCR products were cloned into a pMD18-T (TaKaRa) plasmid and were confirmed by sequence analysis. The full-length cDNA was found to be 1993bp(GenBank accession No.AY395701). It contains 1632bp long from the ATG to the stop codon, encoding a predicated protein of 60kD and is composed of 544 amino acids. In the 5' region of the cDNA a 208bp sequence precedes the ORF (open reading frame). A 157bp untranslated sequence is present in the 3' region of the cDNA.The amino acid sequences of MdCPKl suggest it contains all of the characteristic features of a CDPK,including amino-terminal variable domain of any CDPK characterized to date,conserved serine/threonine kinase domains,a junction domain and a calcium-like domain with four predicated calcium-binding EF hands. The MdCPKl amino acid homologies were analyzed to be 75.09% and 74.55% to tobacco and tomato, respectively. Protein has been predicated two transmembrane domains. Southern blot analysis with the radiolabeled 5'-untranslated region of MdCPKl cDNA as a probe exhibited two hybridizing bands. These results suggest that perhaps two copys of MdCPK1-related genes are present in the apple trees.In order to study the function of MdCPKl, RT-PCR fragments were inserted into the downstream of the glutathione S-transferase (GST) sequence of pGEX-4T-l vector (Amersham Pharmacia Biotech) as GST fusion proteins. These constructs were verified the absence of PCR mistakes by DNA sequencing. Two constructs were expressed as C-terminal glutathione S-transferase (GST) fusion proteins in E.coli and were designated pGEX—MdCPKL and pGEX-MdCPKN, respectively. CPKL codes for the entire open reading frame and CPKN codes for a N-terminal protein. They were transformed into E.coli DH5a. Expressions were induced by the addition of IPTG under the control of (IPTG)-inducible tac promoter. The 86kD GST-MdCPKL fusion proteins and 32kD GST-MdCPKN were expressed and purified from IPTG-induced cultures by affinity chromatography.They were analyzed on 12% SDS-polyacrylamide gels. Proteins detected by immunoblotting. GST-MdCPKL and GST-MdCPKN mouse polyclonal antibody were made. Protein dot blot and ELISA show that two antibodys have good sensitatives.To determine Potential subcellular locations for MdCPKl. MdCPKl open reading frame was amplified by RT-PCR .RT-PCR product was digested and purified, inserted into sites of pEZS-NL undercontrol of the cauliflower mosaic virus 35S promoter that allows the transient expression of protein in C-terminal fusion with enhanced GFP.The construct(35S-MdCPKL-GFP) was sequenced to vertify the absence of PCR mistakes and was delivered into onion(allium cepa) epidermal cells using a particle inflow gun.and also was transformed into protoplast cells of Arabidopsis by PEG4000.Images of MdCPKL-GFP fusion proteins were captured with a confocal laser scanning microscope. Results indicated that MdCPKl was mainly targeted to the cell membrane. Immunoblotting also shows that MdCPK1 only exists in the membrane fraction of apple fruit.The expression of MdCPK1 was examined under ABA treament. Control fruits treated with 0.1%(v/v) ethanol. After each treatment, the fruits were collected.Total RNA and total proteins were isolated. Results show that the expression of MdCPKl was enhanced in apple fruit treated with ABA .To examine tissue-specific expression o...