Cloning of two new CDPK isoforms from Solanum tuberosum

Abstract

Calcium-dependent protein kinases (CDPKs) comprise a multigene family of calcium sensors that play important roles regulating plant growth and development and plant responses to environmental stresses. To the moment six isoforms have been characterized in potato plants. In silico analysis of Solanum phureja genome allowed us to identify other 20 candidate genes encoding potential CDPK isoforms that cluster into four subgroups. Expression of 13 of these isoforms was detected in leaves and stolons from Solanum tuberosum cv Desirée plants. Using primers directed against their 5´and 3´utr sequences we amplified isoforms StCDPK25 and StCDPK17 belonging to subgroup 1 and 3 respectively. These fragments were cloned and sequenced. Isoform StCDPK25 shares 99% identity with the S. phureja coding sequence and presented all the characteristics of a CDPK. Cloning of StCDPK25 coding sequence in pDEST expression vector in order to obtain the recombinant enzyme tagged to 6xHis is currently in progress. Analysis of its sequence indicated that it could be targeted to chloroplasts; the N-terminal sequence will be fused to GFP and transient expression assays will be performed in Nicotiana benthamiana. On the contrary, only a 1 kb fragment containing exons 1, 2 and 7 was amplified for StCDPK17 suggesting that alternative splicing has occurred.