Wandong Fu

Molecular cloning and expression analysis of a cytosolic heat shock protein 70 gene from mud crab Scylla serrata

Heat shock protein 70s (Hsp70s) play important roles in resisting environmental stresses and stimulating innate immune system. To understand the immune defense mechanisms of Scylla serrata, a full-length cytosolic Hsp70 cDNA of S. serrata (designated as SSHsp70) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length of SSHsp70 cDNA was 2235 bp, with a 5 untranslated region of 105 bp, a 3 untranslated region of 174 bp, and an open reading frame of 1956 bp encoding a polypeptide of 651 amino acids with an estimated molecular mass of 71.3 kDa and an estimated isoelectric point of 5.55. The cloned SSHsp70 belonged to a cytosolic Hsp70 family. Three typical Hsp70 signature motifs were detected in SSHsp70 by InterPro analysis. Quantitative PCR (qPCR) was used to detect tissue distribution and mRNA expression levels of SSHsp70 under different stress conditions. The obviously high levels of SSHsp70 transcript were in hemocyte, heart, hepatopancreas and gill, whereas low levels were detected in muscle, eyestalk, stomach, and gut. In different temperature treatments, the expression levels of SSHsp70 in low or high temperatures were higher than those in temperate temperature. In pathogen challenge treatments, the mRNA expression level of SSHsp70 reached a maximum level after 18 h and then dropped progressively. In different salt concentration treatments, the mRNA expression level of SSHsp70 had a minimum level at 25‰ salt concentration and high expression levels at high or low salt concentration. In different nitrite concentration treatments, the mRNA expression level of SSHsp70 increased progressively with the increase of nitrite concentration. The results confirmed Hsp70 could be used as a tool for evolution and phylogenetic analysis, a kind of potential biomarker, and a disease resistance factor used in application.