Abstract:

Fifteen Pomacea canaliculata were exposed to 100 μg/L Cu²⁺ for 0, 1, 7, 14 and 21 days. The total RNA was isolated from livers of three P. canaliculate at each time points and the expression level of P. canaliculate metallothionein (PcMT) mRNA was detected by real-time fluorescence quantitative PCR. The DNA coding for the full-length PcMT was amplified from P. canaliculata total cDNA, and then cloned into the bacterial expression vector pET28a. The recombinant plasmid DNA was sequenced to confirm the correct insert, then transformed into E. coil BL21 (DE3). The recombinant PcMT protein was expressed in BL21 under induction of IPTG and analyzed by SDS-PAGE. The results showed that the expression of PcMT mRNA in the liver of P. canaliculate was continuously upregulated upto 14 days under stress of 100 μg/L Cu²⁺ and reached to a peak value of 2.1 ± 0.2, then decreased to 1.1 ± 0.1 on Day 21. The full length DNA of PcMT gene was about 300 bp. The correct insert in the constructed recombinant pET28a-PcMT plasmid was confirmed by PCR, double digestion and DNA sequencing. The recombinant PcMT protein was successfully expressed in BL21 under induction of IPTG and conformed by SDS-PAGE with relative molecular weight (M_r) of 17 000. These results provide information for further study on the dynamic expression of PcMT in P. canaliculata under stress of heavy metals and its role in the viability of the snail.

Key words: Pomacea canaliculata, Cu²⁺ stress, Metallothionein, Gene expression analysis