Molecular cloning, characterization, and expression of hsp60 in caudal fin regeneration of Misgurnus anguillicaudatus

Abstract

Urodele amphibians and teleost fish are capable of nearly perfect regeneration of lost appendages. The fin constitutes an important model for studying the molecular basis of tissue regeneration. It has been known that heat shock protein 60 (Hsp60) is a multifunctional protein of the heat shock protein family. The purpose of this study is to investigate the role of hsp60 as a part of a stress response system after fin injury or in fin regeneration. We firstly cloned full-length cDNA of hsp60 from Misgurnus anguillicaudatus (designated as MaHsp60) by RACE method. The cDNA contains a 83-bp 5'UTR, a 1,728-bp open reading frame encoding 492 amino acids and a 542-bp 3'UTR (Accession No.: KF537340). The phylogenetic tree shows that the MaHsp60 fits within the hsp60 clade. Then quantitative RT-PCR detected that MaHsp60 began to increase rapidly its expression at 1 dpa and reached its peak at 2 dpa. Next, spatial distribution analysis of MaHsp60 in fins showed that MaHsp60 located mainly in the deeper layer of regenerated epidermis when MaHsp60 expressed most. After the MaHsp60 had been cloned into the pET-32a vector, SDS-PAGE analysis confirmed that the MaHsp60 protein was efficiently expressed in Escherichia coli BL21 and adjustable with the temperature. These findings have revealed that MaHsp60, a highly conserved gene during vertebrate evolution as well as related to stress response, is involved in the formation of wound epidermis which occurs as the first phase of fin regeneration after fin amputation in caudal fin regeneration.