Abstract:

Objective The expression of glutathione S-transferase gene in Pomacea canaliculata (PcGST) was analyzed under Cu²⁺ stress which is toxic to snail, and the related mechanism of P. canaliculata adaptation to Cu²⁺ stress was discussed. Methods Eighty snails were randomly divided into four groups. Four Cu²⁺ stress concentrations (0, 50, 100, 150 μg/L) were set to each group of 20 snails. Three snails from each group were randomly selected on 0, 1, 7, and 14 d after Cu²⁺ stress, and the liver, gill and kidney tissues were isolated. Total RNA were extracted from these tissues of P. canaliculata and reverse transcribed into cDNA. Real-time quantitative PCR was used to detect the relative expression of PcGST mRNA under Cu²⁺ stress. The PcGST gene was amplified from P. canaliculata tissue and then cloned into pET-28a expression plasmid. The recombinant plasmid pET-28a-PcGST was transformed into Escherichia coli BL21(DE3) bacteria and the recombinant PcGST was expressed under induction of isopropyl-β-D-thiogalactoside(IPTG). The expressed recombinant PcGST was analyzed by SDS-PAGE. The PcGST transformed E. coli BL21 and untransformed plain BL21 were equally divided into 6 aliquots. Three aliquots were randomly selected as Cu²⁺ stress group (with 0.2 mmol/L CuSO₄) and the other three as control group (without CuSO₄). All bacterial aliquots were cultured in a shaking incubator at 20 ℃ 150 r/min. A₆₀₀ of bacterial culture was measured every 1 hour until 6 hours. The tolerance of PcGST transformed and non-transformed bacteria to Cu²⁺ stress was compared by t-test. Results The relative expression of PcGST mRNA in P. canaliculata tissue without Cu²⁺ stress was set as 1.0 ± 0.0. In the liver tissue, under the stress of 50 μg/L Cu²⁺, the relative expression of PcGST mRNA increased continuously from 0 to 14 days, with a peak value of 4.9 ± 0.5, the relative expression of PcGST mRNA have gone up and then down, with a peak value of 13.0 ± 3.0 and 12.2 ± 2.2 on 7 d under 100 μg/L and 150 μg/L Cu²⁺ stress, respectively. In the gill tissue, the relative expression of PcGST mRNA have gone up and then down, with a peak value of 5.3 ± 0.7 and 23.3 ± 16.5 on 7 d under 50 μg/L and 100 μg/L Cu²⁺ stress, respectively, the relative expression of PcGST mRNA reached the peak of 9.8 ± 3.3 on 1 d then decreased under 150 μg/L Cu²⁺ stress. In the kidney tissue, the relative expression of PcGST mRNA did not increase at the early incubation under the stress of 50 and 100 μg/L Cu²⁺ till 7 d and reached the peak of 3.9 ± 1.0 under the stress of 100 μg/L Cu²⁺, the relative expression of PcGST mRNA reached the peak of 18.0 ± 0.8 on 14 d under the stress of 150 μg/L Cu²⁺. The The PcGST DNA was successfully amplified from P. canaliculate tissue with a size of approximate 600 bp and cloned into expression vector pET-28a. After being expressed in E. coli BL21, the expressed recombinant PcGST was appeared as 26 370 on SDS-PAGE. The PcGST transformed E. coli BL21 were grown better than the E. coli BL21 without PcGST transformed in LB liquid medium containing 0.2 mmol/L, with A₆₀₀ of 1.5 ± 0.1 and 1.0 ± 0.0, respectively, after 6 h culture, with statistical difference (P < 0.05), while the PcGST transformed and untransformed bacteria grew at the similar rate in LB liquid medium without Cu²⁺.Conclusion PcGST gene was upregulated in P. canaliculate tissue under Cu²⁺ stresss.

Key words: Pomacea canaliculata, Cu²⁺ stress, Glutathione S-transferase, Analysis of gene expression