| The HSP70 and HSP90 cDNA cloned from fresh water mussle, Cristaria plicata, using rapid amplification of cDNA ends, and the sequences of HSP70 and HSP90 genome were cloned from genomic DNA of C. plicata.The full-length cDNA sequence of HSP70 was 2664bp (GenBank accession number:ADM64336), consisting of a 5'-terminal untranslated region (UTR) of 364 bp, a 3'-terminal UTR of 383 bp with a 26bp poly (A) tail and four ATTTA (RNA instability motifs), and the open reading frame of 1917bp. The HSP70 cDNA encoded a polypeptide of 638 amino acids with a predicted molecular mass of 70.3 kDa, and a theoretical isoelectric point of 5.61. The deduced amino acid sequence of HSP70 cDNA includes three HSP70 family signatures. There were 4 glycosylation sites and a highly conserved, the cytoplasmic HSP70 carboxyl terminal region of EE (V/M) D (residues 635-638). The amino acid sequence of HSP70 was included of an ATPase composed of 382 amino acids, the substrate peptide binding domain composed of 159 amino acids, and the C-terminus domain composed of 82 amino acids. There was no intron in ORF of HSP70 cDNA.The full-length cDNA sequence of HSP90 was 2674bp (GenBank accession number:ADN87332), consisting of a 5'-UTR of 83 bp, a 3'-terminal UTR of 410 bp with a canonical polyadenylation signal sequence AATAAA, a 28bp poly (A) tail and a ATTTA. The open reading frame of HSP90 was 2181 bp, which encoded a polypeptide of 726 amino acids with a predicted molecular mass of 83.47 kDa and a theoretical isoelectric point of 5.05. The deduced amino acid sequence of HSP90 cDNA includes five HSP70 family signatures. There were 3 glycosylation sites and a highly conserved, the cytoplasmic HSP90 carboxyl terminal region of MEEVD. The HSP90 cDNA was deduced that an ATPase domain was composed of 155 amino acids. The full length sequence of HSP90 genome was 9739 bp, with 7 introns and 8 extrons in ORF of HSP90.The temporal expression of HSP70 mRNA and HSP90 mRAN were measured by semi-quantiative RT-PCR and fluorescent real-time quantitative RT-PCR after thermal treatment, bacterial challenge and heavy metal stress.After thermal treatment, the HSP70 mRNA relative express value in five tissues from C. plicata were significantly increased compared with the control group, and the HSP70 levels of blood and gills were the most significant, then were the mantle and hepatopancreas, the least were muscles. The pathogenic bacteria Aeromonas hydrophila challenged, the relative express value were increased obviously in only blood and mantles at 6h, but were not obvious in other tissues. The HSP70 levels reached the peak at 12h, then decreased gradually, and recovered the normal level after 48h.Fluorescent real-time quantitative RT-PCR showed the HSP70 levels in blood were increased significantly at different temperature, the maximum levels at 35℃were the 99042-fold higher than that at 5℃, however, the levels at 15℃and 25℃were 16.1- and 13.4-fold respectively higher than that at 5℃. At heavy metal (Cd2+) stressed, levels of HSP70 mRNA were increased with temperature ascended compared with control group, the expression peak was at 15d, however, the levels were rapidly dropped, were got close to the normal level at 20d. At heavy metal (Cr6+) stressed, HSP70 mRNA levels were not obviously increased at 5d, however, with the stressed time lengthened, the express value rose rapidly. The maximum levels were at 15d, followed the level was descended gradually, the expression of HSP70 recover the control level at 20d.The HSP90 levels in five tissues from C. plicata were significantly increased compared with the control group after thermal treatment. The relatively higher expression was in blood, muscles and hepatopancreas, while mantles and gills were lower level. At A. hydrophila challenged, the relative express value was not significant difference among five tissues. At different time points, the HSP90 level reached the peak at 12 h, followed maintained a relatively high level, then decreased gradually, and recovered the normal level after 48h.HSP90 expression level increased with the temperature lengthened at different temperature treatment, the maximum level were at 35℃,130-fold higher than that at 5℃. HSP90 levels showed different change tendency at different heavy metal Cd2+ concentration stressed, but all showed up-regulated. When the Cd2+ concentrations were 50μg/L and 100μg/L, the peak of HSP90 mRNA expression appeared at 15d and 20d, respectively, followed the level were descended gradually, however, when the Cd2+ concentrations were 200μg/L, the peak appeared at 5d, followed maintained a relatively high level compared with the control group. At heavy metal (Cr6+) stressed, levels of HSP90 mRNA were increased with stress temperature lengthened. When the Cr6+ concentrations were 1μg/L and 50μg/L, the peak of HSP90 mRNA expression appeared at 15d, while the Cr6+ concentration was 100μg/L, the peak appeared at 10d, followed the level were descended gradually, then recovered the normal level at 20d.In order to further inquire into the functions of HSP70 and HSP90, pET30-HSP70 and pET30-HSP90 prokaryotic expression system were constructed, and successfully expressed these proteins. SDS-PAGE analysis showed HSP70 and HSP90 expression levels were increased with induced time to add, by IPTG induction, the maximum level was at 8h.Polyclonal antibodies were prepared with rabbit and mouses using purified recombination proteins. Western-blotting was used to detect the expression level of HSP70 and HSP90 in bloods, gills, muscles, hepatopancreas and mantles from C. plicata. The results showed that expression levels of HSP70 were increased rapidly in five tissues at heat shock temperature (35℃) compared with at room temperature (20℃), relatively higher expression was detected in blood, gills, hepatopancreas and mantles were high, while muscles were relatively low. The expression level of HSP90 were also increased obviously in these tissues at heat shock temperature (35℃) compared with 20℃group, and the maximum was in bloods, then were muscles and hepatopancreas, the least were the mantle andgills.The morphology of C. plicata and Anodonta woodiana was exceedingly similar, which could be divided into four subpopulation, large granulocytes, small granulocytes, hyalinocytes and lymphoid hemocytes. The ratio of different subpopulations in two mussles was obviously difference, small granulocytes were the most subpopulation in two mussles, (56.6±1.41)% and (59.28±0.89)% in the hemocyte population, respectively. Hyalinocytes were also the main types, (37.8±2.11)% and (27.74±0.56)%, respectively. Large granulocytes were the little percentage in hemocyte population, (4.2±0.23)% and (4.28±0.35)%. The ratio of lymphoid hemocytes was(2.4±0.12)% and(8.70±0.23)%,respectively.
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