Salinity Adaptation Studies On Heat Shock Protein HSP60and HSP90α Of The Swimming Crab Portunus Trituberculatus

The swimming crab, Portunus trituberculatus, is an important marine fishery andaquaculture species in China. P.trituberculatus is a euryhaline, surviving in wide-rangesalinity conditions, but different water salinity condition might influence its entire lifehistory. To investigate the molecular mechanism of osmotic regulation has greatsignificance on the improvement of germplasm resources and artificial propagation ofP.trituberculatus. Heat shock protein (HSPs) is a group of highly conserved proteins inthe process of biological evolution. HSP can recognize hydrophobic regions ofintracellular denatured proteins to restore the native conformation, and to improve thecellular tolerance against adverse stress. Among the heat shock proteins, HSP60andHSP90a were two main HSP molecular. HSP60take priority over other HSPs onbinding to denatured protein and protecting cells against adverse stress. One of the mainfunctions of HSP90is to protect cells from damage. In addition, HSP90can also bindwith their―client proteins‖to alter these protein functional status.We have successfully cloned PtHSP60with an open read frame (ORF) sequence of1734bp. The putative ORF encoded a polypeptide of577amino acids with an estimatedmolecular mass of61.25kDa and a predicted isoelectric point (pI) of5.23. The aminoacids sequence contained PtHSP60family signatures and characteristic motifs.Homology and phylogenetic analysis showed that the deduced amino acid sequence ofHSP60shared96%similarity with previously described PtHSP60sequence fromL.vannamei.Semi-quantitative RT-PCR analysis revealed that mRNA expressions of PtHSP60could be detected in all tested tissues (liver, heart, ovary, abdominal muscle, antennal,galand, dermis, intestine, appendage muscle, gill), suggesting it was widely expressedgene. Before salinity challenges, mRNA expression level of PtHSP60in tissues weresimilar, except for antennal gland and intestine. During low salinity stress (10‰), thePtHSP60mRNA expressions in ovary increased5.52-fold significantly (p<0.05), followed by the gills, heart and appendage muscle increased to2.14-fold,2.0-fold and1.96-fold respectively, while the antennal gland, intestinal expressions decreasedsignificantly to0.44-fold and0.36-fold respectively (p<0.05). During high salinitystress (40‰), the PtHSP60mRNA expressions in ovary increased significantly to5.57-flod (p<0.001), followed by the dermis, gills increased to3.66-fold and2.4-foldrespectively. Salinity stress didn’t influence the PtHSP60mRNA expression of liver andabdominal muscle.We used semi-quantitative RT-PCR and western blotting to detect gill HSP60mRNA and protein expressions of P. trituberculatus. The result showed that under lowsalinity stress, HSP60mRNA expressions increased gradually to2.5-fold within24hr.However, when under high salinity stress, HSP60mRNA expressions increased rapidlyto the maximum, approximately4.26-flod, and then gradually decreased. Salinity couldinduce and remain HSP60proteins expression after48hr.Semi-quantitative RT-PCR analysis of gill HSP90α mRNA expressions revealedthat HSP90α mRNA expression gradually decreased to0.12-fold (p<0.05) under10‰low salinity. While under40‰high salinity, HSP90α mRNA expression increasedwithin24hr,followed by decreasing to0.22-fold after120hr.In order to prove the correlations between HSP90a protein and salinity stress, weinvestigated the prokaryotic expression of P. trituberculatus HSP90α recombinantprotein under a series of salinity stresses. Based on the coding sequences of P.trituberculatus HSP90α protein in GenBank, we cloned the full length of HSP90a gene.Recombinant pET28-HSP90α prokaryotic expression recombinant plasmid wasconstructed and expressed in Escherichia coli DE3(BL21) under a series of salinitystresses. Results showed that the survival rate of recombinant plasmid transferredEscherichia coli was higher than that of empty vector transferred cells. When thesalinity challenge was closed to the salinity tolerance maximum value of E. coli, thesurvival rate between those two kinds of cells became more significantly. For example,at the highest salinity challenge condition (1050mmol/L), the survival rate ofrecombinant plasmid transferred E. coli was10.7times higher than that of empty vectortransferred cells. Therefore, our results indicated that P. trituberculatus HSP90α proteinpossessed protective effect against salinity stress and HSP90α protein might be involvedin salinity adaptation physiological process in P. trituberculatus.In general,under salinity stress, PtHSP60mRNA and protein expressions were increased,and the result of gill PtHSP90α mRNA expression in vivo and prokaryoticexpression in vitro,both implied that PtHSP60and PtHSP90α may be involved insalinity adaptation physiological process in P. trituberculatus. This study enriched theHSP molecular function,and provided a theoretical basis for the development offreshwater P.trituberculatus.