A MAPKK kinase gene was firstly cloned from Dunaliela salina by RT-PCR and RACE technology.The full length of cDNA for MAPKKK gene was 1 460 bp with a 27bp 5UTR,a 554bp 3UTR and a 879bp open reading frame coding 292 amino acids.MAPKKK was anlysed by several bioinformatics softwares and the result were as follows: This protein was unstable,hydrophilic and located in cytoplasmic matrix without signal peptide and transmembrane region.The amino acids sequence contained a protein kinase ATP-binding region spanning from 24rd to 45rd amino acid and a Serine/Threonine protein kinase region spanning from 137rd to 149 rd amino acids.Several potential phosphorylation sites and an important catalytic active site Asp141 were found in this protein.The main components of protein secondary structure were α-helix and random coil.A 3D model of protein tertiary structure was built successfully and shown in ribbons stereogram and hydrophobic surface graphic model.Both the multiple sequence alignment and phylogenic analysis showed this protein had the closest evolutionary relationship with the corresponding proteins of Volvox carteri f.Nagariensis and Chlamydomonas reinhardtii.The prokaryotic expression vector pGS21a-MAPKKK was constructed by using T4 ligase to link the ORF of MAPKKK and the vector pGS21a.The recombinant plasmid was conducted into E.coli BL21(DE3)and induced by IPTG.The fusion protein was expressed successfully in E.coli BL21.Detected by SDS-PAGE,the fusion protein had existed in both soluble and inclusion body.The soluble protein was purified after optimizing induced conditions,then the highly purified soluble fusion protein was obtained.The result of western blotting demonstrated this fusion protein was MAPKKK protein with a GST label.