Molecular cloning, sequence analysis and tissue expression of serine/threonine kinases Akt from Eriocheir sinensis

In the present study, full length cDNA encoding the serine/threonine kinases Akt from Eriocheir sinensis (EsAkt) was cloned by using 3′RACE and 5′RACE techniques, and the sequence and structural analysis of the EsAkt was conducted with bioinformatics methods. The results showed that the full length cDNA encoding EsAkt consisted of 2 200 bp nucleic acids in length, including a 5′-UTR of 159 bp, a 3′-UTR of 496 bp and an open reading frame (ORF) of 1 545 bp encoding 514 amino acids. Analysis of the protein domain features showed that the deduced polypeptides contained three conservative domains characteristic of Serine/Threonine protein kinases family. Multiple sequence alignment revealed that the amnio acids sequences of EsAkt have the 0.889 identity with Fenneropenaeus chinensis and Litopenaeus vannamei. The phylogenetic analysis showed that the EsAkt was arranged in the same clade with Akts from other arthropods. The tissue distribution of EsAkt mRNA in sexual maturity individuals and different muscle groups during molt cycle in juvenile crabs were analyzed by quantitative real-time PCR (qRT-PCR). In sexual maturity crabs, the EsAkt transcript was detected in eyestalk, claw muscle, ovary, heart, hepatopancreas, epidermis, testis, gill and triangular membrane, and the expression level was relatively high in ovary, eyestalk and testis, and was low in hepatopancreas. In juvenile crabs, the EsAkt transcript in different muscle groups was different depending on the molt stages. In walking leg muscles, the EsAkt expression level has no obvious change. In abdominal muscles, the EsAkt expression level was much higher in later pre-molt D₃−D₄ stage than post-molt A-B stage and inter-molt C stage. In claw muscles, the EsAkt expression level was decreased rapidly in pre-molt D₃−D₄ stage and increased in post-molt A-B stage, and lasted to inter-molt C stage. These results suggested that the expression of EsAkt transcript was related with the molt stage of E. sinensis, and it is possible that the EsAkt is involved in the muscle atrophy, growth and rebuilding during the molt cycle of E. sinensis.