Heat shock protein 90 (HSP90) is a highly conserved protein and plays an important role in maintaining the structure of protein, participating in the immunity and regulating the cell cycle. Using the rapid amplification of cDNA ends (RACE) techniques, the cDNA sequence of HSP90 gene (designated Sp-HSP90) was cloned and characterized from the mud crab Scylla paramamosain. The full-length cDNA of Sp-HSP90 is 2677 bp with a complete open reading frame (ORF) of 2166 bp, which encodes a polypeptide of 721 amino acids. Five conserved blocks defining HSP90 protein family were found in the deduced amino acid sequence of Sp-HSP90. It contains an adenosine-5’-triphosphatase (ATPase) domain in the N-terminal and a conserved signature sequence MEEVD in the C-terminal. Quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) analyses revealed the distribution of Sp-HSP90 mRNA in different tissues and its temporal expression in haemocytes of the crabs challenged with Vibrio parahaemolyticus. Different levels of Sp-HSP90 mRNA were detected in heart, hepatopancreas, muscle, haemocytes, testis and ovary except eyestalk. The expression level of Sp-Hsp90 mRNA in hemocytes was found to be obviously up-regulated after live and heat-killed bacterial challenge and significantly higher in live bacteria group than that in heat-killed bacteria group. These results suggest that Sp-HSP90 gene might act on immunity and resistance to infection in S. paramamosain.
Key words: Heat shock protein 90, quantitative real-time polymerase chain reaction (PCR), Scylla paramamosain, Vibrio parahaemolyticus.
HSP90, Heat shock protein 90; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; qRT-PCR, quantitative real-time PCR; ALF, anti-lipopolysaccharide factor;RT-PCR, reverse-transcription polymerase chain reaction; ORF, open reading frame; UTR, untranslated region; Sp-HSP90, the cDNA sequence of HSP90 gene.
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