MOLECULAR CLONING, TISSUE-SPECIFIC EXPRESSION AND RELEVANCE TO DIETARY COPPER AND ZINC OF GENE METALLOTHIONEIN IN MACROBRACHIUM NIPPONENSE

Metallothionein(MT) is a metal binding protein with low molecular weight. The expression of MT can be induced by minerals such as copper or zinc, and it is involved in the metabolism of trace elements. To better understand the physiological and nutritional effects of copper and zinc on Macrobrachium nipponense, we cloned mn-MT from the oriental river prawn using rapid amplification of the cDNA ends(RACE), and evaluated the effects of dietary copper and zinc on the expression of MT. The full length of mn-MT cDNA was 665 bp, and it had a 180 bp open reading frame(ORF) encoding a 59aa peptide. The calculated molecular weight of the peptide was 6.085 kD and the predicted isoe-lectric point was 7.73. The most abundant amino acid in this protein was cysteine residues(30.5%), followed by lysine(16.95%) and serine(10.17%). The similarity analysis showed that mn-MT shared the similarities of 78%, 75% and 75% with the counterparts in the Homarus americanus, Penaeus monodon and Eriocheir sinensis respectively. Quantitative real-time RT-PCR(qRT-PCR) analysis showed that the mn-MT mRNA was expressed in the hepatopancreas, gill, hemocytes, intestine, ovary, muscles and stomach, with the highest level in the hepatopancreas. M. nipponense were fed for 56 days with seven different diets supplemented with Cu at 0, 20, 40 and 160 mg/kg diet and Zn at 0, 35, 210 mg/kg diet, and then we analyzed the expression of mn-MT mRNA in the hepatopancreas. The expression of mn-MT mRNA was first elevated along with the increase in dietary Cu level, reached to the maximum at 40 mg/kg diet, and then dropped as the Cu concentration was further increased. The expression of mn-MT mRNA was higher in prawns fed with high Zn(210 mg/kg diet) than that in prawns fed with 35 mg Zn/kg diet or 0 mg Zn/kg diet(P0.05). There was no significant difference between the 0 mg Zn/kg group and the 35 mg Zn/kg group(P0.05). These results suggested that the expression of mn-MT mRNA could be affected by dietary copper and zinc with different dose-dependent response curves.