Biochemical Characterization And Function Analysis Of A Calcium-dependent Protein Kinase From Tomato

With the tobacco calcium-dependent protein kinase gene NtCPK5 cDNA (AY971376) as an information probe to screen tomato dbEST by program blastn (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A pool of ESTs sharing high homology with NtCPK5 were captured and assembled to a contig with the help of software DNAMAN. A couple of specific primers were designed based on 5'-terminal and 3'-terminal nucleotide sequences of the assembled contig and used in a RT-PCR to isolate the cDNA. The obtained fragment sharing high identity with the electronic contig in the nucleotide sequence was 1982 bp long with an opening frame (ORF) of 1698 bp encoding of 565 amino acids and was designated as LeCPK2 (GenBank GQ205414).The deduced amino acid sequence of LeCPK2 shared high identity with those of other plant CDPKs and contained the kinase, autoinhibitory, and calmodulin-like domains typical of CDPKs. The phylogenic tree analysis suggested that LeCPK2 may be an evolutionary link between CDPKs and CRKs (CDPK-related protein kinases).To isolate the promoter sequence of LeCPK2, a part of the LeCPK2 cDNA was used as information probe to search the tomato genome database and a annotated gene was isolated which shared 100% homology with LeCPK2 in amino acid sequence.The annotated gene was then located in a contig which exhibits the nucleotide sequences of LeCPK2 and its upstream region. Based the upstream sequence, a set of specific primers were designed and used in a PCR to isolate the promoter sequence of LeCPK2 from tomato DNAs. A 1700 bp-length fragment was obtained by the PCR,which was identical with the sequence exacted from the tomato genome database and used to perform the promoter sequence analysis of LeCPK2.The motif scan of the regulatory region of LeCPK2 revealed that it was a TATA-less promoter lacking typical TATA-box and contained fews of light responsive elements,10 regulatory elements responsible for pollen specific activation,4 heat stress responsive elements and some hormone-responsive elements and a fungal elicitor responsive element, suggesting that LeCPK2 might play important roles in the heat stress responses, pollen activation and light responses such as photosynthesis, light-regulatory growth and development or light stress.A full-length and a truncated recombinant proteins were expressed in E.coli Rosetta 2 (DE3) and purified by Ni2+ affinity chromatography. The assay for kinase activity revealed that the activity of LeCPK2 was calcium dependent and the remove of C-terminal CaM-like domain inhibited the kinase activity no matter of the presence of Ca2+, which was in accord with the mechanism by which the Ca2+ binds to the CaM-like domain to release the autoinhibitory domain from the active region of the kinase and then elicit the kinase activity. The activity of LeCPK2 was sharply stimulated by Ca2+ with K0.5 (concentration of Ca2+ for half-maximal activity of LeCPK2) of 48.8 nM on substrate histone Ills and 45.5 nM on syntide 2. The optimal concentration of Mg2+ for LeCPK2 activity was 20 mM on substrate Histone Ills and 10 mM on syntide 2. The Km value of LeCPK2 toward HistoneⅢs was 44.9μg/ml and toward syntide 2 was 89.52μM.Expression profiling indicated that LeCPK2 expressed predominantly in flowers and responded divergently to heat and cold stress, in which an obvious mRNA accumulation was detected at 4 h under 42℃stress, but no change of LeCPK2 mRNA levels was observed in 6 h at 4℃. Mechanical wounding and phytohormones such as ethylene, methyl jasmonate and salicylic acid, were also observed to arouse the expression of LeCPK2 with a similar pattern:mRNA accumulation was enhanced at 30 min and reached the maximum at 3 h followed by a decrease until the normal level. In addition, LeCPK2 was not observed to respond in the salt and drought stresses. The expression of LeCPK2 under various environmental stresses were agreement with the analysis of the cis-acting elements of LeCPK2.LeCPK2 was overexpressed in the transgenic tobacco plants to investigate its physiological roles. The control and transgenic plants exhibited no significant change in phenotype after treated at 42℃for 10 h. However, when cultured in strong light for another 1 h, the control plants were wilting and coupled with the increase of malonaldehyde (MDA) content in leaves while the transgenic plants and the control plants no suffering heat treatment were normal in phenotype, suggesting that the light induced but not caused the occurence of heat harm. The harm induced by light might be the result of photooxidation. The normal growth of the transgenic plants after heat and light treatments suggested the roles of LeCPK2 in the protection of plants from heat harms.