Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate
70 (HSC70) and inducible heat shock protein 70 (HSP70) were isolated from the liver
of Wuchang bream (Megalobrama amblycephala Y.) using RT-PCR and rapid amplification
of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70, respectively. The cDNAs
were 2336 and 2224 bp in length [not including poly (A)] and contained 1950 and 1932
bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and
643 amino acids with predicted molecular weights of 71.24 and 70.52 kDa, and theoretical
isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis
revealed that Ma-HSC70 gene contained seven introns with all introns conforming to
the GT/AG rule whereas Ma-HSP70 gene did not contain any intron in the coding region.
Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three
signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization
signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis
revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other
vertebrates, while Ma-HSP70 shared more than 85.0% identity with the known HSP70s
of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics
analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic,
rich in B cells antigenic sites, without any signal peptide or transmembrane region.
The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation
sites, casein kinase II phosphorylation sites, and N-glycosylation sites, predicting
that they could play essential roles in protein folding, translocation, intracellular
localization, signal transduction and regulation. The predominant secondary structures
of the two proteins were alpha-helix and random coil. Fluorescent real-time quantitative
RT-PCR was used to study the effects of heat shock (34 degrees C), crowding stress
(100g L(-1)) and challenge with bacteria Aeromonas hydrophila on the mRNA expression
of the two HSP70s in Wuchang bream liver. The results indicated that, during 24 h
stress, Ma-HSC70 mRNA expression decreased at first and then rose to the level before
stress under heat shock and crowding stress, but Ma-HSP70 mRNA expression increased
at first and then decreased under heat stress, and appeared to increase continuously
under crowding stress. After bacterial challenge, the mRNA levels of both Ma-HSC70
and Ma-HSP70 increased at first and then decreased. The cloning and expression analysis
of the two HSP70s provide theoretical basis to further study the mechanism of anti-adverseness
and expression characteristics under stress conditions of Wuchang bream.