Cu2+胁迫对福寿螺金属硫蛋白基因mRNA表达的影响

Effects of Cu2+ stress on the expression of metallothionein mRNA in Pomacea canaliculata

探讨不同浓度Cu2+胁迫对福寿螺肝胰腺组织金属硫蛋白(metallothionein, MT)基因表达的影响,取15只福寿螺,在100μg/L Cu^2+离子胁迫后的0、 1、 7、 14、 21 d,各取3只福寿螺,分离肝脏,提取RNA,反转录为c DNA,实时荧光定量PCR检测福寿螺金属硫蛋白基因(Pc MT) mRNA的相对表达水平。以cDNA为模板, PCR扩增Pc MT基因完整编码序列,连接至pET28a质粒,转入大肠埃希菌BL21 (DE3)感受态细胞中,挑取阳性克隆进行鉴定与测序。用异丙基-β-D-硫代半乳糖苷诱导重组蛋白表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。结果显示, Cu2+胁迫0~14 d,福寿螺肝脏中Pc MT基因mRNA的表达量持续上升,峰值为2.1±0.2, 21 d时下降为1.1±0.1。PCR扩增Pc MT基因,获得长度约300 bp的片段,构建的pET28a-Pc MT质粒经PCR、双酶切和测序鉴定,均与预期大小一致。SDS-PAGE结果显示, Pc MT蛋白的相对分子质量(Mr)约为17 000。

Fifteen Pomacea canaliculata were exposed to 100 μg/L Cu^2+ 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 100μg/L Cu^2+ 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 (Mr) 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.