| 其他摘要 | Heavy metal contamination has become a serious problem in coastal area of China at present. It is essential to choose appropriate bionidicators and biomarkers to monitor marine environment. In this study, the bioaccumulation and subcellular distribution of cadmium in digestive gland and gills from manila clams Ruditapes philippinarum with different shell colors (White clam and Zebra clam) were compared. Meanwhile, the full-length cDNAs of metallothionein (RpMT) was cloned from R. philippinarum with RACE approaches, and the expression profile of RpMT transcript was determined in White clam and Zebra clam under Cd2+ exposure. The results are as followed: Laboratory static bioassays were conducted to determine the acute toxicity of Cd2+ to R. philippinarum. The median lethal concentration (LC50) of White clam for 24, 48 and 96 h of exposure are 41.025, 11.854 and 1.362 mg/L, respectively. As concerned to Zebra clam, the LC50 values for 24, 48 and 96 h of exposure are 36.559, 10.597 and 0.633 mg/L, respectively. Accroding to the empirical formula 96 h LC50×f, the safe concentration of Cd2+ for White clam and Zebra clam was estimated to be 1.36×10-2 and 6.33×10-3 mg/L, respectively. R. philippinarum showed significant high Cd2+ bioaccumulation ability. After exposure, the amount of Cd in the tissues of digestive gland and gills increased by 10.22 and 13.78 folds in White clam, and 11.75 and 21.59 folds in Zebra clam, respectively. Additionally, the amounts of Cd in different subcellular fractions were measured. The subcellular distribution of Cd in manila clam followed the general pattern (mean%): proteins > cellular debris > organelles > metal-rich granules. After Cd2+ exposure, the relative distribution of Cd in cellular debris increased significantly except for digestive gland in White clam, and so did the percentage of Cd bound to proteins in gill tissues, compared with the control group. Subcellular distribution of Cd was found to be tissue-dependent. The proportional subcellular distribution of Cd in proteins of digestive gland was higher than that of gills, while the percentage in cellular debris and organelles of gills was higher than that in digestive gland. Moreover, the relative distributions of Cd among subcellular fractions showed differences between White clam and Zebra clam. In the control group, no significant difference was found in digestive gland between White clam and Zebra clam. However, Cd bound to metal-rich granules and cellular debris in gills of White clam was more than that of Zebra clam. After exposure, the relative distribution of Cd in organelles in digestive gland of White clam was higher than that of Zebra clam, but showed no difference in gills. The full-length cDNA of metallothionein was identified from R. philippinarum (named as RpMT) by RACE approaches. The full-length cDNA of RpMT is of 570 bp. The open reading frame (ORF) of RpMT encoded 75 amino acids with 21 cysteine residues and 15 characteristic repeating cysteine motifs Cys-Xn-Cys. The theoretical molecular weight of RpMT is 7.35 kDa and theoretical isoelectric point is 7.75. By quantitative Real-time PCR, the expression profile of RpMT was detected in digestive gland and gills from two shell colors of clam (White clam and Zebra clam) post cadmium exposure. The expression of RpMT gene under Cd2+ stress increased significantly in both shell colors of clam. After Cd2+ exposure, the variation of RpMT mRNA expression in gill tissues was more significant than that in digestive gland in both White and Zebra clams. In addition, the up-regulation of RpMT transcript in gill tissues of White clam was more sensitive to Cd2+ exposure than that in the Zebra clams. |
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