Cloning and prokaryotic expression of the full-length cDNA of CDPK gene from Leymus chinensis

Objective Cloning and prokaryotic expression of CDPK gene from Leymus chinensis would help to further study the function of it, which could probe into its molecule mechanism how L.chinensis adapts itself to the saline-alkali habitat and provide a theoretical basis.

Method CDPK gene of L.chinensis was cloned by RT-PCR and RACE technology, which was named Lc-CDPK.By constructing prokaryotic expression vector of CDPK gene and transforming it into Escherichia coli BL21 (DE3) with IPTG induction, the expression of CDPK protein was analyzed by SDS-PAGE.

Result and conclusion The full-length cDNA sequence of CDPK gene was 1 704 bp with a 1 647 bp ORF (open reading frame) encoding 548 amino acids, and the amino acids from 81 to 339 formed the S-TKc structural domains possessing catalytic activity of serine/threonine protein kinase and having four conservative EF hand structural domains.A comparison with the nucleotide sequence of coding region of L.chinensis and those of other graminaceous crops showed that it has a close similarity with CDPK gene of wheat, sharing at identity of 96%. The relative molecular mass of prokaryotic expressed fusion protein was 61 350 by IPTG induction, which matched up with the anticipated theoretical value.The induced protein reached the highest level at 7 hours of induction, accounting for 49.1% in the total bacterial proteins.