Heat Shock Protein Gene Cloning And Responses Of Sea Cucumber (Apostichopus Japonicus Selenka) To Temperature And Salinity Stress

1. Molecular cloning and sequence analysis of hsp70and hsp90in sea cucumberApostichopus japonicusTotal RNA was extracted from sea cucumber (Apostichopus japonicus Selenka),Hsp70gene with GenBank number of EU930813and Hsp90gene with GenBank number ofJF907619were obtained by using RT-PCR and RACE. The sea cucumber Hsp70genecDNA sequence was2176bp and the length open reading frame was1950bp that encodinga649amino-acid polypeptide. The sea cucumber Hsp90gene cDNA sequence was2293bpand the length open reading frame was2184bp that encoding a727amino-acid polypeptide.The deduced amino-acid polypeptide of Hsp70and Hsp90possessed Hsp70and Hsp90protein family signature respectively, and have highly homology contrasting to otherspecies Hsp70and Hsp90(Hsp70:72-76%; Hsp90:76-81%). Molecular cloning andsequence analysis of hsp70and hsp90will be helpful to clarify the physiological adaptionof sea cucumber Apostichopus japonicus to the environment.2. Effects of Salinity Fluctuation on the Expression of hsp70and hsp90s genes in seacucumber, Apostichopus japonicus SelenkaThe expression of hsp70, hsp90a and hsp90b genes in sea cucumber (Apostichopusjaponicus Selenka) was studied in a simulated field salinity decrease after a heavy rainfall insummer. Salinity firstly decreased gradually from30to20at a rate of2.5every6h, and thenwas maintained at20. After96h at the low salinity (20), salinity was increased gradually to30at a rate of2.5every6h, and then was maintained at30for96h. Results showed thatexpressions of hsp70, hsp90a and hsp90b genes all increased after salinity decrease, led to the maximum levels at the lowest salinity, and then dropped to the original level after72h atlow-salinity exposure. Similarly, the expression of these three genes in sea cucumberincreased with the salinity increase, and decreased to the original level after72h atlow-salinity exposure. Compared to the second salinity fluctuation, the gene expressions werelower than those of the first salinity fluctuation, which might be caused by energy budget. Inthis study the expression of hsp90a and hsp90b genes was similar to that of hsp70, andhsp90s also could be regarded as biomarkers to environmental stress.3. Changes in Oxygen Consumption Rate, hsp70and hsp90gene expression of seacucumber, Apostichopus japonicus in fluctuating temperature modePhysiological responses to temperature reflect the evolutionary adaptations oforganisms to their thermal environment and the capability of animals to tolerate thermalstress. To study the physiological adaptations of an intertidal-subtidal species to the extremeand unpredictable heat stress of the intertidal zone, oxygen consumption rate and heat shockprotein expression were quantified in the sea cucumber Apostichopus japonicus. Usingsimulate natural temperatures, the relationship between temperature, physiologicalperformance (oxygen consumption and heat shock proteins) and thermotolerance wereassessed. Depression of oxygen consumption rate and upregulation of heat shock proteingenes (hsps) occurred in sequence when ambient temperature was increased from24to30C. Large-scale mortality of the sea cucumber occurred when temperatures rose beyond30C, suggesting that the upregulation of heat shock proteins and mortality are closelyrelated to the depression of aerobic metabolism, a phenomenon that is in line with theconcept of oxygen-and capacity-limited thermal tolerance (OCLTT). Thephysiologically-related thermotolerance of this sea cucumber should be an adaptation to itslocal environment.