Gene Expression And Activity Analysis Of Pocillopora Damicornis HSP70 Under Heat Stress

Coral reef ecosystem is one of the most important marine ecosystems.Coral reef ecosystem is being threat seriously by the elevated sea surface temperatures,which causes coral bleaching and mortality.Hermatypic coral Pocillopora damicornis is not only listed as a biology model in the simulation study of global climate change,but also servered as a model organism in coral ecological research.Therefore,the study on the molecular mechanism of coral thermal stress is imperative and significant.It is well-known that heat shock protein plays important role through recovering proteins from misfolded conformation and maintaining cells homeostasis in the heat resistance process.P.damicornis HSP70(PdHSP70)was studied in this research to explore its expression pattern and biological activities under thermal stress.The whole cDNA sequence of PdHSP70 was obtained through RACE PCR method.The whole length of PdHSP70 was of 2346 bp,including a 5'UTR(Untranslated region)of 103 bp,a 3' UTR of 290 bp and an ORF(Open reading frame)of 1953 bp.The ORF encoded a polypeptide containing 650 amino acids with a molecular weight of 71.93 kDa.A HSP70 domain(8-616 amino acids)was predicted in the peptide using SMART method,while three domains predicted through InterPro method contained an actin-like ATPase domain(8-385 amino acids),a peptide-binding domain(390-547 amino acid)and a C-terminal domain(523-623 amino acid).The expression level of PdHSP70 mRNA was determined by real-time RT-PCR after heat stress(32?),and it increased significantly at 12 h and return to the normal level in 24 h.Furthermore,there was not significant change in the expression level of PdHSP70 mRNA in the control group during whole experiment process.Recombinant PdHSP70 protein was expressed in Escherichia coli and puried using nickel affinity chromatography.The ATPase activity of recombinant PdHSP70 under 25,30,35 and 40 ? were 21.76,21.88,13.60 and 16.20 nmol mg-1 min-1,respectively.No significant diffierences were observed among these activities.The present study suggested that PdHSP70 mRNA expresion could be induced after heat stress to clear up the misfolded protein and maintain the normal physiological functions..When the coral had adapted the thermal stress,the expression level of PdHSP70 mRNA would return to the normal level to restore the cell homeostasis.As a kind of heat-induced protein,PdHSP70 played a significant role in heat acclimation and homeostasis maintaining of coral P.damicornis.The molecular mechanism of heat stress and acclimation in hermatypic coral had been preliminarily revealed according to this study related to PdHSP70.It would not only provide theoretical basis on coral ecosystem protection but also enriched and explored the content of tropical marine biology.