Effect Of Temperature And Salinity On The Manila Clam(Ruditapes Philippinarum) Heat Shock Protein Family Gene Expression

The HSP60 gene of Ruditapes philippinarum was cloned by using the rapid amplification of c DNA ends(RACE) method. The full-length c DNA of HSP60(including poly-A sequences) was 1777 bp containing a 21 bp 5’UTR and a 49 bp 3’UTR respectively, the open reading frame was 1728 bp encoding 575 amino acids with calculated molecular mass of 61.25 k Da and an isoelectric point of 5.04. No signal peptide structure was found in the N terminal of the deduced protein. By multiple sequence alignment, the deduced amino acid sequence of Rp HSP60 exhibited a 80% identity with the HSP60 proteins from other species.The expression of HSP40, HSP60 and HSP90 genes in different tissues(including blood cells, adductor muscle, mantle, foot, gills and digestive gland) of wild and three varieties of shell colour strains(orange clam, zebra clam, white clam) was examined by quantitative real-time PCR technology. It showed that the HSP40, HSP60 and HSP90 were expressed in all the detected tissues. By comparison with the expression level in the adductor muscle, the highest level of HSP40 was detected in the gill tissues(p<0.01), and the lowest was found in the adductor muscle of the wild and three kinds of shell colour strains; the highest level of HSP60 was detected in hemocytes of wild clam, orange calm and zebra clam strains(p<0.01), and in the gill tissues of the white clam strains(p<0.01), and the lowest level were found in the adductor muscle in the wild and three kinds of shell colour clam strains(p<0.01); the highest level of HSP90 was detected in hemocytes of the wild and zebra clam strains(p<0.01), and in the gills of the ocean orange and white clam strains(p<0.01), and the lowest level of HSP90 was found in adductor of the wild and three kinds of shell colour strains(p<0.01). These results indicated that HSPs gene family in clams may exhibit a tissue specific expression.Under the low salinity(15) stress, the expression level of HSP40 in the gill of the three shell colour strain exhibited a process which rised first then fall, and went up to the higher level, while in white clam strain, the expression was decreased at beginning then increased to the highest level. In addition, the relative expression level of HSP40 in gill in orange clam strain was higher than other three shell color strains at all time points. The expression level of HSP60 in the gill of wild clam showed a process that rised first then fall, and rised the higher level, and the expression of HSP60 gradually increased and reached the highest level at 96 h, 24 h and 72 h in gill of orange clams, zebra and white clam strain(p<0.01)respectively. The highest level of HSP90 was detected at 72 h, 12 h, 72 h in the gill of wild, zebra clam and white clam strains respectively, and there was no significant variation of HSP90 gene expression in the gill of orange clam strain. It indicated that the expression of HSP40, HSP60 and HSP90 gene in R philippinarum could be induced by low salinity stress.Under the higher(30℃)temperature stress, the expression of HSP40 increased gradually first then restored in the gill tissues of the three shell color strains, and the relative expression level of HSP40 was higher in the orange clam gill than the other three shell color strains at all the time points; the highst expression of HSP60 was detected at 6h in gill of the wild populations, at 96 h in the gill of orange clam and white clam strains(orange clam p<0.05, white clam p<0.01), at 48 h and 96 h in Zebra clams(p<0.05); the expression HSP90 in the gill tissues of the four kinds of shell color clams increased in first 6h and reach the peak then decreased to normal levels.Under the lower(-1℃) temperature stress, the expression of HSP40 increased and reached the highest level in gill in gill tissues of wild populations at 6 h, in the gill of orange clam and zebra clams strains at 24 h,and in the gill of white clam strain at 48 h(p<0.05); the HSP60 gene expressed stably in the gill tissuesof wild population, and, the expression level of HSP60 in the gill of orange clam and white clam strains increased and arrived at the highest value at 48 h, while the expression of HSP60 gene in the gill of zebra clam strain decrease slightly in first 48 h then restored at 72h; the expression of HSP90 gene in the gill tissue of wild population decrease in first 48 h and the minimum level was detected at 6 h, 12 h, then restored at 96h; while the HSP90 expression level increased in the gill tissue of the three shell colour strains after the lower temperature stimulation, and the highest level was found at 48 h in the gill of orange calm strain, and at 72 h in the gill of zebra clam strain, and at 48 h in the gill of white clam strain.These results demonstrated that the HSP40, HSP60 and HSP90 genes were susceptible to temperature and low salinity stress and could be served as marker gene to temperature and salinity stress.