Molecular Characterization And Function Of Two Different HSP70s In The Prawn, Macrobrachium Rosenbergii

Heat shock protein 70kDa (HSP70s) is the most important proteins among members of heat shock proteins, and functions mainly as molecular chaperones. Moreover, they play important roles in cellular protection, anti-apoptotic effects, and immune therapy of tumor. Therefore, researches about HSP70s have been one of important and prospective focuses in fields of life sciences.Up to now, the researches about HSP70s mainly focused upon vertebrate, and paid little attention to the invertebrates, especially the aquatic invertebrates. It is well known that the aquatics broadly live in the water, and have much closer relations with aquatic environment, relations which to a certain extent depends on HSP70s to establish. So it is of the great significance that researches about relations between HSP70s and aquatics acclimation and tolerance should be carried out.In our present study, we selected Macrobrachium rosenbergii, which is the commercially important organism, as the experimental materials. Two genes encoding heat shock protein 70kDa were cloned from thoracic ganglia and hepatopancreas of 35 ℃ heat-stressed Macrobrachium rosenbergii. Their cDNAs were 2448 and 2173bp in length and contained 1950 and 1734bp open reading frames (ORFs), respectively. The ORFs encoded 649 (Mr 71kDa) and 577 (65kDa) amino acid polypeptides, which were named Mar-HSC70 and Mar-HSP70, respectively. High identities of Mar-HSC70 and Mar-HSP70 amino acids with other known HSP70s indicated that they belong to the two members of HSP70 family. It was found that the major differences between the two proteins were 60 and 14 amino acids absent in the Mar-HSP70, and the deletions have been further identified by sequencing. Genomic DNA sequence analysis revealed that either of the genes has no introns.Using Northern blotting, western blotting and semi-quantitative RT-PCR,expression of Mar-HSC70 and Mar-HSP70 were detected in response to heat, cold shock, heavy metal and bacteria at cellular and protein levels. The results indicated that Mar-HSC70 mRNA nearly have the same levels under stressed conditions, and mainly enriched in ovary; in contrast, Mar-HSP70 was only sensitive to heat shock, and much expressed in the thoracic ganglia, hepatopancreas and muscle at 35 °C. Semi-quantitative RT-PCR confirmed that expression levels of Mar-HSP70 were much lower at 15, 20, 25 and 30'C. Their expression was further confirmed using western blotting, and the bright band (Mw 65kDa) were detected at 35 °C. In general, the results suggested that Mar-HSC70 and Mar-HSP70 belongs to members of constitutive and inducible HSP70 families, respectively. Moreover, It is confirmed that Mar-HSP70 plays crucial roles in cellular protection against heat, roles which may be related to the deletions of N- and C-terminal of Mar-HSP70.Their expression were further localized at cellular levels using in situ hybridization and immunohistochemistry. The results indicated that Mar-HSC70 (25 and 35°C) and Mar-HSP70 (35°C) were expressed in most cells, especially in neurosecretory cells, R- and F-cells, and expression of Mar-HSP70 (35 °C) were significantly higher than that of Mar-HSC70. This suggested that Mar-HSP70 can be expressed rapidly in most cells to protect against the heat stress.To further inquire into the functions of Mar-HSP70, we constructed Mar-HSP70-pET-28a (+) prokaryotic expression system, and successfully expressed the recombinant Mar-HSP70 in E.coli BL21 (DE3). It was found that the expression level was over 50% of total protein in the engineered bacteria after IPTG induction for 5 hr. Immunoblotting proved the immunogenicity of the Mar-HSP70. And the soluble Mar-HSP70 was produced from the bacteria using lower temperature induction (30°C). Mostly important, we established detection system of Mar-HSP70 activity, and found that bacteria expressing Mar-HSP70 can confer thermotolerance to survive. The results indicated that Mar-HSP70 protected bacteria from heat damage to a certain degree, which will provide good model and thoughts for functional studies of inducible HSP70s.In general, this is the first time to clone the two genes encoding heat shock protein70kDa from prawns, and to analyze their expression and functions. The two HSP70s have common characteristics of most HSP70s, in the meanwhile, they have specificity of their own, specificity which will provide theoretical basis to have a better understanding of aquatic invertebrates HSP70.