Cu~(2+)胁迫对福寿螺金属硫蛋白基因mRNA表达的影响
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摘要
探讨不同浓度Cu~(2+)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, Cu~(2+)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(Pc MT) mRNA was detected by real-time fluorescence quantitative PCR. The DNA coding for the full-length Pc MT was amplified from P. canaliculata total cDNA, and then cloned into the bacterial expression vector pET28 a. The recombinant plasmid DNA was sequenced to confirm the correct insert, then transformed into E. coil BL21(DE3). The recombinant Pc MT protein was expressed in BL21 under induction of IPTG and analyzed by SDS-PAGE. The results showed that the expression of Pc MT m RNA in the liver of P. canaliculate was continuously upregulated upto 14 days under stress of 100 μg/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 Pc MT gene was about 300 bp. The correct insert in the constructed recombinant pET28 a-Pc MT plasmid was confirmed by PCR, double digestion and DNA sequencing.The recombinant Pc MT 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 Pc MT in P. canaliculata under stress of heavy metals and its role in the viability of the snail.
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(Pc MT) mRNA was detected by real-time fluorescence quantitative PCR. The DNA coding for the full-length Pc MT was amplified from P. canaliculata total cDNA, and then cloned into the bacterial expression vector pET28 a. The recombinant plasmid DNA was sequenced to confirm the correct insert, then transformed into E. coil BL21(DE3). The recombinant Pc MT protein was expressed in BL21 under induction of IPTG and analyzed by SDS-PAGE. The results showed that the expression of Pc MT m RNA in the liver of P. canaliculate was continuously upregulated upto 14 days under stress of 100 μg/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 Pc MT gene was about 300 bp. The correct insert in the constructed recombinant pET28 a-Pc MT plasmid was confirmed by PCR, double digestion and DNA sequencing.The recombinant Pc MT 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 Pc MT in P. canaliculata under stress of heavy metals and its role in the viability of the snail.
引文
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[19]阎光宇,孙继鹏,易瑞灶,等.褶牡蛎金属硫蛋白基因的克隆及组织表达分析[J].水产科学,2017,36(5):634-641.
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[21]武阳.重组华溪蟹金属硫蛋白在SUMO融合系统中的表达与纯化及金属螯合能力的研究[D].太原:山西大学,2012:20-43.
[22] Malakhov MP,Mattern MR,Malakhova OA,et al. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins[J]. J Struct Funct Genomics,2004,5(1-2):75-86.
[23] Zahid MT,Shakoori FR,Zulifqar S,et al. Molecular characterization of a copper metallothionein gene from a ciliate tetrahymena farahensis[J]. J Cell Biochem,2016,117(8):1843-1854.
[2]胡求安,张仪,郭云海,等.广东省南澳岛福寿螺和鼠类密度及其广州管圆线虫感染现状调查[J].中国寄生虫学与寄生虫病杂志,2017,35(3):239-245.
[3]薛明,柯才焕,周时强,等.重金属对波部东风螺早期发育的毒性及EDTA的解毒效果[J].热带海洋学报,2004,23(1):44-50.
[4] Gupta PK,Khangarot BS,Durve VS. The temperature dependence of the acute toxicity of copper to a freshwater pond snail,Viviparus bengalensis L[J]. Hydrobiologia,1981,83(3):461-464.
[5] Dummee V,Tanhan P,Kruatrachue M,et al. Histopathological changes in snail,Pomacea canaliculata,exposed to sublethal copper sulfate concentrations[J]. Ecotoxicol Environ Saf,2015,122:290-295.
[6] Khangarot BS,Mathur S,Durve VS. Comparative toxicity of heavy metals and interaction of metals on a freshwater pulmonate snail Lymnaea acuminata(lamarck)[J]. Acta Hydrochimica et Hydrobiologica,1982,10(4):367-375.
[7]吕耀平,黄佩佩,刘子明.铜锌汞铬4种重金属离子对方形环棱螺的急性毒性效应[J].丽水学院学报,2014,36(2):2095-3801.
[8] Nam YK,Kim EJ. Diversification and domain evolution of molluskan metallothioneins:a mini review[J]. Can J Fish Aquat Sci,2017,20(1):8-20.
[9]张清顺,侯建军,刘香江,等.铜对梨形环棱螺抗氧化酶活性和金属硫蛋白含量的影响[J].水生生物学报,2009,33(4):717-725.
[10]王昊盛,宋鑫金,董文强,等.厚壳贻贝(Mytilus coruscus)金属硫蛋白MT-10:cDNA克隆、结构分析及铜离子胁迫下的表达[J].海洋与湖沼,2017,48(4):864-869.
[11]张文领,牟希东,胡隐昌,等.福寿螺细胞色素P450基因CYP3192A1的克隆与表达分析[J].南方水产科学,2017,13(1):66-75.
[12]任丹丹.南极红酵母重金属适应性相关基因的克隆与表达分析[D].哈尔滨:哈尔滨工业大学,2016.
[13] Schicht O,Freisinger E. Spectroscopic characterization of cicer arietinum metallothionein 1[J]. Inorganica Chimica Acta,2009,362(3):714-724.
[14]王清路.嗜热四膜虫金属硫蛋白MTT1和MTT2的结构与功能分析[D].太原:山西大学,2012.
[15]谢红.重金属在金属硫蛋白基因表达中的调控作用[J].国外医学:卫生学分册,1999,26(3):151-155.
[16] Vasak M. Metal removal and substitution in vertebrate and invertebrate metallothioneins[J]. Meth Enzymol,1991,205:452-458.
[17] Mao H,Wang DH,Yang WX. The involvement of metallothionein in the development of aquatic invertebrate[J].Aquat Toxicol,2012,110:208-213.
[18]魏华,佟金凤,赵莹莹,等.镉和锌对中华小长臂虾(Palaemonetes sinensis)金属硫蛋白表达的影响[J].沈阳农业大学学报,2018,49(3):302-308.
[19]阎光宇,孙继鹏,易瑞灶,等.褶牡蛎金属硫蛋白基因的克隆及组织表达分析[J].水产科学,2017,36(5):634-641.
[20]刘兰芳.蚯蚓MT基因克隆和原核表达及转拟南芥研究[D].保定:河北大学,2010:20-40.
[21]武阳.重组华溪蟹金属硫蛋白在SUMO融合系统中的表达与纯化及金属螯合能力的研究[D].太原:山西大学,2012:20-43.
[22] Malakhov MP,Mattern MR,Malakhova OA,et al. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins[J]. J Struct Funct Genomics,2004,5(1-2):75-86.
[23] Zahid MT,Shakoori FR,Zulifqar S,et al. Molecular characterization of a copper metallothionein gene from a ciliate tetrahymena farahensis[J]. J Cell Biochem,2016,117(8):1843-1854.