| Calcium dependent protein kinases (CDPKs) are a kind of serine/threonine protein kinases that are only present in plants and some protests. CDPKs were firstly reported in pea by Hetherington and Trewavas(1982) and were initially purified and identified from soybean by Harmon et al (1987). Since the first gene encoding CDPK was cloned from soybean by Harper et al (1991), 95 genes encoding CDPKs have been so far cloned from 16 higher plants, 6 lower plants and 4 protests. CDPKs are a multigene family and there are 34 CDPKs in Arabidopsis thaliana. CDPKs play important roles in plant calcium signal transduction. There is increasing evidence that CDPKs participate in C/N metabolism, transmembrane transport of ion and water, cytoskeletal regulation, stomatal movement regulation, growth and development regulation and biotic and abiotic stress responses in plants.Using RT-PCR and RACE techniques, a full-length cDNA VfCPKl and a partial cDNA VJCPK2 were firstly successfully cloned from broad bean epidermal peels and leaves respectively, and a preliminary study was conducted on the expression pattern of VfCPKl. The results indicated that using RT-PCR with two degenerate primes designed from conserved amino acids of CDPK kinase domains, three 141 bp cDNA fragments (141-1, 141-2 and 141-3) were cloned from broad bean epidermal peels, and by RACE techniques, a full-length cDNA VfCPKl was cloned from broad bean epidermal peels with two gene-specific primes designed according to the 141-1 sequence. Using RT-PCR with two degenerate primes designed from conserved amino acids of CDPK kinase and autoinhibitory domains, three 618 bp cDNA fragments (618-1. 618-2 and 618-3) were also isolated from broad bean leaves, and a partial cDNA VJCPK2 lacking 5' end was isolated from broad bean leaves with two gene-specific primers designed according to the 618-1 sequence.The VfCPKl cDNA is 1785 bp long with an open reading frame of 1479 bp encoding for a protein of 493 amino acids, a 5'-untranslated region of 127 bp and a 3'-untranslated region of 179 bp containing AATAAA and poly (A) tail. The predicted VfCPKl protein with 493 amino acids contains all the structural characteristics of reported CDPKs and there are four domains of a variable domain, a kinase domain, an autoinhibitory domain and a calmodulin-like domain from N- to C-terminal end. At the N-terminal end is the variable domain of 26 amino acids in length, which has no conserved MGXXC(S/Q)XXT sequence essential for N-myristoylation. So it is conferred that the VfCPKl is a soluble protein. The VfCPKl has an obvious kinase catalytic domain for Ca2+/calmodulin dependent protein kinases and serine/ threonine protein kinases, and a calcium-binding domain like calmodulin. The kinase catalytic domain is 264 amino acids in length and contains all of 11 conserved subdomains and 15 invariant amino acid residues essential for eukaryotic serine/threonine protein kinases. The autoinhibitory domain is 31 amino acids in length. At the C-terminal end is a calmodulin-like domain of 162 amino acids in length, which has four highly conserved Ca2+-binding EF hands.The putative protein sequence of VfCPKl shares a remarkably high degree of amino acid identity with other CDPKs in plants, including seed CDPKa in soybean (82% identity; GenBank 29892113), SK5 (CDPKa) in soybean (82% identity; GenBank 116054), seed CDPKb in soybean(80% identity; GenBank 29892204), RiCDPK2 in potato (79% identity; GenBank 15289760), seed CDPKP in soybean (79% identity; GenBank 2501764), a CDPK in rice (78% identity; Genbank 34147319), ZmCPK11 in maize (78% identity; GenBank 31747505), and so on. When compared to 34 Arabidopsis CDPKs, VfCPKl shows higher homology to AtCPK12, AtCPK4 and AtCPK11 with amino acid identity 77%, 75% and 74% respectively.By Northern and Western blot analysis, we examined expression pattern of VfCPK1 in different parts including roots, stems, leaves, mesophylls and epidermal peels in broad bean at mRNA or protein levels, and effects of different external stress treatments on VfCPKl...
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