| In this sudy, we described cDNA cloing, mRNA tissue distribution and gene expression of heat shock protein 60,70 and 90 (HSP60, HSP70 and HSP90) in T. albonubes. The results as follows:1. HSP60, HSP70 and HSP90 gene cDNA full-length cloning and analysisTanichthys albonubes HSP60, HSP70 and HSP90 gene cDNAs full-length were obtained using homology cloning and RACE technique, respectively. TaHSP60 (Genbank number:HM234132) cDNA sequence full-length was 2486 bp and encoded 575 amino acid residues, which included typical mt-HSP60 features sequence, ATP binding sites and C-terminal conservative repeat regional GGM motif. TaHSP70 (Genbank number: HQ007352) cDNA was 2398 bp in length and encoded 643 amino acid residues, which included three signature sequences (IDLGTTYS, IFDLGGGTFDVSIL and VLVGGSTRI PKIQK) and cytoplasmic HSP70 C-terminal feature motif EEVD. TaHSP90 (Genbank number:HM234131) cDNA was 2686 bp in length and encoded 726 amino acid residues, which included five highly conservative HSP90 signature sequences (NKEIFLRELISNA [S/A][K/DALDKIR, LGTIAIGQFGVGFYSASGTR], [Y/F]LVA[E/D], IKLYVRRVFI and GVVDS[E/D][I/V]DLP LNSRE) and C-terminal cytoplasmic HSP90 peculiar motif MEEVD.2. HSP60, HSP70 and HSP90 tissues expression analysisDetermination of the expression levels of T. albonubes heat shock protein gene in different tissues of the adult fish was made by using fluorescence quantitative PCR. The results showed that, HSP60 mRNA gene expression level was the highest in the liver, followed by in the muscle tissue (about 0.67 times of which in the liver, P<0.05), while HSP60 expression was the lowest in both the gill and the fin (about 0.04 times of which in the liver (P<0.05); HSP60 mRNA expressions in the intestine, eyes, ovaries, muscle and brain were 0.45,0.18,0.10,0.67 and 0.05 times (P<0.05) of which in the liver, respectively. HSP70 mRNA gene expression levels in the liver was the highest, there were significantly differences compared with in the other tissues except muscle (P<0.05); HSP70 expression levels was the lowest in the eyes (about 0.38 times of which in the liver); intestine, gill, fin, ovary and brain expression levels of HSP70 mRNA were 0.60, 0.58,0.49,0.42 and 0.39 times of which in the liver, respectively. HSP90 mRNA gene expression was also the highest in the liver, there were significant difference compared with in the other tissues except muscle and gill (0.98 and 0.85 times of which in the liver, respectively) (P<0.05); HSP90 expression levels was the lowest in the ovary (about 0.40 times of which in the liver); fin, eyes, intestines and brain expression levels of HSP90 mRNA were 0.70,0.49,0.70 and 0.47 times of which in the liver, respectively.3. Effect of heavy metal exposure on the transcription of HSP60, HSP70 and HSP90Determination of the changes of HSP60, HSP70 and HSP90 gene mRNA expression levels after exposed to heavy metals (Cu2+and Cd2+) was made by using the fluorescence quantitative PCR. The results showed that different heat shock proteins had different reactions on the Cu2+ and Cd2+, even the same HSP family in different tissues of the same organism had different patterns. T. albonubes exposed to copper and cadmium could induce significantly increment (P<0.05) of HSP60 mRNA expression in 96 h, and the induction intensity of copper was significantly higher than of cadmium.13.50μg/L of copper exposure, HSP70 mRNA expression did not change significantly in liver and gill; 27.00μg/L of copper exposure, as the exposing time extended, TaHSP70 mRNA expression constantly increased within 96 h in the liver and the gill.1.15 mg/L of cadmium exposure, HSP70 mRNA expression did not change significantly in liver with 96 h, while HSP70 mRNA expression was significantly raised to the peak in gill with 72 h (P<0.05); 2.31 mg/L of cadmium exposure, expression levels of HSP70 mRNA was significantly raised to the peak (P<0.05) in liver and gill, then decreased,96 h expression levels were significantly lower than the control group (P<0.05). HSP90 expression levels in copper and cadmium exposure was also different. In the liver and the gill, HSP90 were significantly increased 24 h after copper exposure (P<0.05), then decreased; and in the late period of treatment, the gene expression level declined and significantly lower than the control group in the gill (P<0.05). After cadmium exposure, the expression levels of HSP90 mRNA was significantly inhibited and the expression was significantly lower than the control group (P<0.05) both in the liver and the gill. |
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