Cloning, Expression And Applied Of HSP70 Gene Form Pacific Abalone (Haliotis Discus Hannai Ino)

Heat shock protein 70kDa (HSP70s) is the most important proteins amongmembers of heat shock proteins, and functions mainly as molecular chaperones.Moreover, they play important roles in cellular protection, anti-apoptotic effects, andimmune therapy of tumor. Therefore, researches about HSP70s have been one ofimportant and prospective focuses in fields of life sciences. The HSP70 cDNA clonedfrom Pacific abalone(Haliotis discus hannai Ino.)using rapid amplification of cDNAends (RACE), was highly homologous to other HSP70 genes. The full-length cDNAof the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslatedregion (UTR) of 90bp, a 3'-terminal UTR of 573 bp with a canonical polyadenylationsingnal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp.The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domainof 382 amino acids, the substrate peptide binding domain of 161 amino acids and aC-terminus domain of 112 amino acids. In order to acquire the recombined HSP70expressed in vitro, we successfully constructed pProEXTMHT-HSP70 prokaryoticexpression system, which was essential to the preparation of antibody and the measureof HSP70 expression of abalone by Western blot blotting or ELISAThe temporal expression of HSP70 was measured by semi-quantitative RT-PCRafter heat shock and bacterial challenge. Challenge of Pacific abalone with heat shockor the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in theexpression of HSP70 mRNA level in muscle, followed by a recovery to normal levelafter 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the topat 12 h and maintained a relatively high level compared with the control after thermaland bacterial challenge. The upregulated mRNA expression of HSP70 in the abalonefollowing heat shock and infection response indicates that the HSP70 gene isinducible and involved in immune response.Here we report the growth rates and Hsp70 expressions in inbred and outbredpopulations of abalone. In abalone, inbred populations expressed more Hsp70 thanoutbred populations at all temperatures except at very high temperatures close to thephysiological limit. At benign temperatures, there was a clear trend towards higherHsp70 expression in inbred than outbred populations, whereas at higher temperatures,a trend in the opposite direction was observed. The temperature of maximal Hsp70expression (Tpeak) varied with the population type. The Tpeak of inbred populations(26°C) was lower than that of the outbred populations (28°C). The maximum ofHsp70 of inbred populations was higher than that of outbred populations. The resultsshowed a trend towards higher expression in inbred at lower temperature. Theseresults provide the first direct experimental evidence that hybrid can cope with theintrinsic stress even at non-stressful temperatures. The constitutive Hsp70 maytherefore be used as MAS in breeding.Real-Time PCR measured the temporal expression of HSP70 of abalones afterheat shock for different time of recovery. The results were similar with that ofmeasured by RT-PCR, which showed that the sensitivity of RT-PCR and Real timePCR is different. Real-Time PCR measured the temporal expression of HSP70 ofabalone living through the winter in the north and the south respectively with red andgreen shell color. The results showed because the temperature of habitat is different,the heat shock response were different for the abalones living through the winter inthe north and south respectively. Three salient findings in this study address theeffects of population and temperature on the heat-shock response and its regulation inthe abalone. First, at began temperature, the constitutive HSP70 in population livingthrough the winter in the south is excess that in population living through the winterin the north. Second, with the increase of temperature, the HSP70 expressionincreased. The amplitude of north population is faster than that of south population.At last, the degree of decrease is different for the two populations..