Cloning HSP70 And Hsp90 Genes Of Huso Dauricus And The Effects Of Temperature And Salinity Stress On Their Gene Expression

Huso dauricus is a cold freshwater fish belonging to the Acipenseri form family that inhabits the main stream and tributaries of the Heilongjiang River in China. H. dauricus show the better characters such as larger body size, higher disease resistance and later sexual maturity than other fish. Harvesting caviar is the farmed main purpose of H. dauricus. At present, the cultured populations of H. dauricus have been moving south from frigid Heilongjiang to Beijing, Hebei,Hubei, Sichuan, Chongqing, Yunnan and other places. H. dauricus facing increasingly severe surroundings of survival for the increasing in temperature and in transport frequency. Heat shock proteins(HSP) are important anti-stress proteins in organisms, with an important role in maintaining homeostasis of the body. The main purpose of this paper is the study of the mechanisms of stress response when addressing serious environmental challenges.This study provides a theoretical basis for farming, transport and protecting artificial population of H. dauricus.The genes encoding HSP70 and HSP90 proteins were isolated from H. dauricus by homologous cloning and rapid amplification of c DNA ends(RACE). We also analyzed patterns of HSP70 and HSP90 expression in the muscle, gill, and liver of H. dauricus under different combinations of temperature and salinity stress, including temperatures of 4, 10, 25, 16 and 28°C,and salinities of 0, 10, 20, 30, and 40 ppt, where 16°C at 0 ppt served as the control. In addition,11 different tissues of HSP70 and HSP90 were detected by fluorescent real-time RT-PCR under unstressed conditions. The test results show that the full-length HSP70 c DNA from H. dauricus(Gen Bank accession no. KP050541) was 2275 bp and contained an 85-bp5′-terminaluntranslated region(UTR) and a 237-bp3′-terminal UTR. The open reading frame(ORF) was 1953 bp and encoded a protein of 650 amino acids. The theoretical isoelectric point and molecular weight(p I/Mw) of the HSP70 gene was 5.215/71131.33. Three highly conserved signatures, including9–16(IDLGTTYS), 197–210(IFDLGGGTFDVSIL), and 334–348(PTIEEVD). The full-length HSP90 c DNA from H. dauricus(Gen Bank accession no.KP050542) was 2718 bp and contained a 44-bp5′-terminal UTR and a 497-bp3′-terminal UTR. The ORF was 2178 bp, encoding a protein of 725 amino acids. The theoretical isoelectric point and molecular weight(p I/Mw) of the HSP90 gene was 4.846/83623.73. Only one highly conserved signature(32–41:YSNKEIFLRE). Fluorescence quantitative PCR results showed that HSP70 and HSP90 m RNA were widely expressed in 11 tissues of H. dauricus, including muscle, gill, brain, beard, heart,liver, spleen, stomach, intestine, swim bladder, and gonads. m RNA expression of the HSP70 and HSP90 genes in H. dauricus was highest in the intestine and lowest in muscle tissue. In addition,trends in the levels of expression were consistent between the HSP70 and HSP90 genes. In the experiment of temperature stress, m RNA expression of HSP70 and HSP90 was highest in all tissues(muscle, gill, and liver) in the 4°C cold stress treatment. The levels of HSP70 and HSP90 expression were more sensitive under cold stress(4 and 10°C) than heat stress(25 and 28°C).HSP70 and HSP90 expression changed little during salinity stress.In this study, levels of HSP70 and HSP90 expression were highest in the 4°C treatment,indicating that they play a very important role during cold stress. Levels of HSP70 and HSP90 expression were higher than the control group under heat stress, indicating that the heat shock proteins play an important role in the situation. In addition, HSP70 and HSP90 expression changed little during salinity stress, showing that H. dauricus may be better able to respond tochanges in osmotic stress. In practice, H. dauricus commonly encountered temperature stress.There is a question hanging unresolved that is whether H. dauricus grow health under temperature stress. In this study, these data can be well supported on the subject. In aquaculture,fish are usually disinfected at salinities of 30–50ppt, and fish are generally transported in low-salinity waters. The present study revealed the molecular adaptation of H. dauricus to saline water. Thus, these data are useful for further study of HSP expression under environmental stress,and they further our understanding of the mechanisms of HSP70 and HSP90 stress response in cold freshwater fish.