| at shock proteins 70kDa (HSP70s) were the most important and highly con(?)d proteins among members of the Heat shock protein family, and mainly function;as molecular chaperone which play an important role in protein folding.sent researches showed that marine bivalves HSP70s could be induced by a wid(?) ange of marine environmental pollutants. The bivalve HSP70s are ideal candi(?)es for comprehensive biomarkers of early marine environmental pollution. However, the sensitivity of HSP70s expression response to pollutants and the times of sig(?)icantly higher expression duration are unknown, which are important for correcdy evaluate feasibility and application range of HSP70s as early comp(?)ensive biomarkers of marine environmental pollution. In order to solve the problems three HSP70s (HSC70, HSP69 and HSP68) of Crassostrea hong(?) gensis (Lam and Morton,2003) were cloned with RT-PCR, RACE and geno(?) walker methods. Then, the 5'-flanking regions of them were also cloned by mean(?) of genome walker method. At the same time, we get one isogene of HSP69 and (?) HSC70, respectively. On this basis, the sensitivity of HSC70 gene expression and t naintenance times of significantly higher expression duration response to pollu(?)s are studied.Through PCR amplification in C. hongkongensis genome DNA, six introns were found in the HSC70 gene. And the longest intron (322bp) was located in the 5'unt islated region (UTR) and the others (length:119bp,203bp,104bp,110bp and 21(?)bp) in the open reading frame (ORF) region. A 1231 bp 5'-flanking region of HSC70 gene was obtained using genome walker method, including a putative core moter region and transcription elements including 6 heat shock elements (HSF(?)one CAAT box and a few poly C region. At the same time, one isogene of HSC fragment was cloned. There are 2789bp sequences including 6 putative intro with differences only in the first intron of both the gene and isogene of HSC70 Sequence analysis revealed that the identity of the exon nucleotide seque(?)between the gene and its isogene we obtained in the HSC70 is 99.48%. and th(?) of the partially deduced amino acid sequence is 100%.terms of the features of the HSP69 in C. hongkongensis, it was found that the f(?)ength HSP69 cDNA of 2251 bp contained an open reading frame (ORF) of 1911bp with the 5'UTR of 130 bp and the 3'UTR of 218 bp. The deduced 633 amino acid sequence showed the highest identity with HSP69 of other mollusks. No intron was found in the HSP69 gene. The 5'-flanking region sequence of the HSP69 gene contained a putative core promoter region and transcription elements including 6 heat shock element (HSE) and GC box. At the same time, one isogene of HSP69 fragment was cloned, obtaining 1933bp sequence including one ORF of 1911bp that encoded 633 amino acids. Sequence analysis revealed the identities of ORF nucleotide sequence and amino acid sequence between the gene and isogene of HSP69 were 98.42% and 96.48%, respectively. A 506 bp 5'upstream sequence of the HSP69 isogene contained a putative core promoter region and 3 heat shock elements (HSE). The results indicated that HSP69 isogene expression could be induced by heat.A 2794bp sequence of C. hongkongensis HSP68 gene was obtained using genome walker method, including a ORF of 1848bp that encoded 615 amino acids. Three classical HSP70s signature motifs were detected in the deduced amino acid sequence. A ployadenylation signal was found on the 744bp of 3'end, and a putative core promoter region and a heat shock element (HSE) in 126bp 5' upstream sequences.The investigation about tissues distribution of HSC70, HSP69 and HSP68 with real-time fluorescence quantitative RT-PCR indicated that HSC70, HSP69 and HSP68 transcriptional expression levels were detectible in digestive gland, muscle, mantle, gill and heart from un-stressed C. hongkongensis. Tissue-specific variation was observed in all three HSP70s:relatively high expression in muscle and lowest in mantle. A similar trend occurred that significantly increased at 3-6 hours and then dropped and returned to the control level after heat shock(24℃to 37℃, 1h). Significantly higher expression of the genes could maintain about 24 hours for HSC70, about 12 hours for HSP69 and just at 3 hours for HSP68. The highest gene expression levels of three HSP70s varied in different organs.Our results showed that expression levels were different among various tissues or organs of small and large individuals after malachite green, formaldehyde and Cu2+challenge.The mRNA transcription levels of HSC70 were analyzed with real-time fluorescence quantitative RT-PCR technique in different organs of C. hongkongensis responding to a series of concentrations for Cu2+, Zn2+, malachite green, formaldehyde and deltamethrin. The results showed that the threshold induction concentration of HSC70 expression was 0.1-1μg/l in the digestive gland for Cu2+,0.1-1μg/l in the gill for Zn2+,0.001-0.01μg/1 in the digestive gland for malachite green,0.0001-0.001μg/l in the gill for formaldehyde and 0.05-0.1μg/l in the gill and digestive gland for deltamethrin, respectively. All these proved that the HSC70 gene was sensitive to pollutants.HSC70 expressions were significantly induced at different time after pollutants exposure when treated concentration was greater than or equal to the upper limit of the threshold induction concentration range. The overall response rule was as follows:with the treatment time elongation, the expression level of HSC70 gene increased at first, end then decreased, gradually returned to control levels, even became significantly lower than the control; with increastment of pollutant concentration, HSC70 expression levels also raised gradually to a maximum value, and then the expression level progressively decreased, meanwhile, the peak expression time point gradually went ahead. The rules described above always occurred prominently when the less toxics, malachite green and two heavy metals (Cu2+and Zn2+), treated C. hongkongensis. However, HSC70 expression levels were significantly higher than control group only in a narrow range of concentrations of formaldehyde and deltamethrin, and the HSC70 expression will be inhibited when the treated concentration exceeds the scope. At the same time, the higher the concentration treated, the earlier inhibition occurred.The expression time of the oyster HSC70 at a significantly higher level than the control was different after treatment with different concentrations of various pollutants. The higher the treated concentration in a range of pollutants, the longer the HSC70 expression time, but when the concentration was over some upper limit of range, the expression time became shorter. In general, the significantly higher expression time maintained for several days, up to more than ten days. HSC70 expression time in gill was longer than that in the mantle and digestive gland.When the oysters were exposed to either the mixture of malachite green and trichlorfon (no matter the two pollutants was at over or blow the threshold induction concentrations) for 35 days or trichlorfon for 10 days at first, then malachite green for another 25 days, the two pollutants induced HSC70 mRNA expression in a synergistic effect in the oyster gill. On the basis of the above-mentioned results, the HSC70 gene of C. hongkongensis was sensitive to pollutants, and the significantly higher expression time maintained for several days, up to more than ten days. Therefore, it is feasible that the HSC70 gene of C. hongkongensis was used as early comprehensive warning biomarker of marine pollution. Due to the significant difference of HSC70 expression between small and large individuals, the same or similar size oysters should be used for research and practical application on early warning of marine environment pollution. At the same time, the gill of C. hongkongensis was the most appropriate organs for indicator, and digestive gland could also be used. |
