Cu~(2+)胁迫下福寿螺谷胱甘肽S转移酶基因表达分析
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摘要
目的分析Cu~(2+)胁迫前后福寿螺谷胱甘肽S转移酶(PcGST)基因的表达规律,探讨福寿螺适应Cu~(2+)的相关机制。方法 80只福寿螺随机分为4组,每组20只福寿螺, Cu~(2+)胁迫浓度分别为0、 50、 100、 150μg/L。分别于Cu~(2+)胁迫后的0、 1、 7、 14 d,各组随机取3只福寿螺,分离其肝脏、鳃、肾脏组织,分别提取组织中的RNA,逆转录为cDNA,实时荧光定量PCR检测Cu~(2+)胁迫前后PcGST m RNA的相对表达量。基于Cu~(2+)胁迫下的福寿螺基因转录组筛选获得PcGST基因,构建pET-28a-PcGST重组质粒,转化至大肠埃希菌(E. coil) BL21 (DE3)感受态细胞中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)分析重组蛋白表达情况。将转化菌株(含PcGST基因的E. coil)和非转化菌株(未转化的E. coil)等量分成6份,随机取3份作为Cu~(2+)胁迫组(含0.2 mmol/L的CuSO4),另3份作为对照组(不含CuSO4), 20℃、 150 r/min摇床培养。每隔1 h测定一次菌液吸光度(A600)值,连续测定6 h。采用t检验比较转化菌株和非转化菌株对Cu~(2+)的耐受能力。结果设定无Cu~(2+)胁迫下的福寿螺组织中PcGST mRNA的相对表达量为1.0±0.0。肝脏组织中, 50μg/L Cu~(2+)胁迫下, PcGST mRNA的相对表达量于0～14 d呈持续上升趋势,峰值为4.9±0.5; 100μg/L、 150μg/L Cu~(2+)胁迫下, PcGST mRNA的相对表达量先上升后下降,峰值均于7 d出现,分别为13.0±3.0和12.2±2.2。鳃组织中,50μg/L、 100μg/L Cu~(2+)胁迫下, PcGST mRNA的相对表达量先上升后下降,峰值均于7 d出现,分别为5.3±0.7和23.3±16.5; 150μg/L Cu~(2+)胁迫下, PcGST mRNA的相对表达量也呈现先上升后下降的趋势,峰值于1 d出现,为9.8±3.3。肾脏组织中, 50μg/L Cu~(2+)胁迫下, PcGST mRNA的相对表达量随时间变化不显著; 100μg/L Cu~(2+)胁迫下, PcGST m RNA的相对表达量前期随时间变化不显著,于14 d出现显著增加,为3.9±1.0; 150μg/L Cu~(2+)胁迫下, PcGST mRNA的相对表达量于0～14 d呈持续上升趋势,峰值为18.0±0.8。PcGST基因PCR扩增产物约为600 bp。SDS-PAGE结果显示, PcGST蛋白的相对分子质量(Mr)约为26 370。LB液体培养基中,转化菌株与非转化菌株的生长曲线接近,最大A600值分别为1.5±0.0和1.4±0.0,差异无统计学意义(P> 0.05);含0.2 mmol/L CuSO4的LB液体培养基中,转化菌株的生长曲线优于非转化菌株,最大A600值分别为1.5±0.1和1.0±0.0,差异有统计学意义(P <0.05)。结论Cu~(2+)胁迫能促进福寿螺体内PcGST基因的表达。
Objective The expression of glutathione S-transferase gene in Pomacea canaliculata(PcGST) was analyzed under Cu~(2+)stress which is toxic to snail, and the related mechanism of P. canaliculata adaptation to Cu~(2+)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-28 a expression plasmid. The recombinant plasmid pET-28 a-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 m RNA 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 under150 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 Cu~(2+)till 7 d and reached the peak of 3.9 ± 1.0 under the stress of100 μg/L Cu~(2+), the relative expression of PcGST mRNA reached the peak of 18.0 ± 0.8 on 14 d under the stress of150 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-28 a. 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 Cu~(2+).Conclusion PcGST gene was upregulated in P. canaliculate tissue under Cu~(2+)stresss.
Objective The expression of glutathione S-transferase gene in Pomacea canaliculata(PcGST) was analyzed under Cu~(2+)stress which is toxic to snail, and the related mechanism of P. canaliculata adaptation to Cu~(2+)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-28 a expression plasmid. The recombinant plasmid pET-28 a-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 m RNA 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 under150 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 Cu~(2+)till 7 d and reached the peak of 3.9 ± 1.0 under the stress of100 μg/L Cu~(2+), the relative expression of PcGST mRNA reached the peak of 18.0 ± 0.8 on 14 d under the stress of150 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-28 a. 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 Cu~(2+).Conclusion PcGST gene was upregulated in P. canaliculate tissue under Cu~(2+)stresss.
引文
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[16]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.
[17]Liu HH,He JY,Zhao RT,et al.A novel biomarker for marine environmental pollution of pi-class glutathione S-transferase from Mytilus coruscus[J].Ecotox Environ Safe,2015,118:47-54.
[18]Wan Q,Whang I,Lee JS,et al.Novel omega glutathione S-transferases in disk abalone:characterization and protective roles against environmental stress[J].Comp Biochem Physiol C Toxicol Pharmacol,2009,150(4):558-568.
[19]张林宝.菲律宾蛤仔对典型污染物胁迫响应的分子生物标志物研究[D].北京:中国科学院,2012.
[20]朱丹丹.湖北钉螺肝脏cDNA文库的构建和序列分析[D].北京:中国科学院研究生院,2010.
[21]罗鸣.近江牡蛎一种四跨膜素鉴定及功能研究[D].杭州:浙江大学,2012.
[22]Chan PC,Shiu CK,Wong FW,et al.Common carp metallothionein-1 gene:cDNA cloning,gene structure and expression studies[J].Biochim Biophys Acta,2004,1676(2):162-171.
[23]李姣,夏媛媛,程天印.福寿螺肾脏组织学观察[J].畜牧兽医科技信息,2011(4):30-31.
[2]Carlsson NOL,Br觟nmark C,Hansson LA.Invading herbivory:the golden apple snail alters ecosystem functioning in Asian wetlands[J].Ecology,2004,85(6):1575-1580.
[3]刘义,高世同,耿艺介,等.广州管圆线虫对淡水螺感染性的实验研究[J].热带医学杂志,2007,7(8):739-741,734.
[4]Qiu JW,Kwong KL.Effects of macrophytes on feeding and life-history traits of the invasive apple snail Pomacea canaliculata[J].Freshwater Biology,2009,54(8):1720-1730.
[5]周天泽.某些无机成分在环境水体中的存在形式[J].环境科学,1979(3):57-63.
[6]Dummee V,Tanhan P,Kruatrachue M,et al.Histopathological changes in snail,Pomacea canaliculata,exposed to sub-lethal copper sulfate concentrations[J].Ecotoxicol Environ Safe,2015,122:290-295.
[7]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.
[8]Londhe S,Kamble N.Histopathology of cerebro neuronal cells in freshwater snail Bellamya bengalensis:impact on respiratory physiology,by acute poisoning of mercuric and zinc chloride[J].Toxico Environ Chem,2013,95(2):304-317.
[9]吕耀平,黄佩佩,刘子明,等.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):33-38.
[10]包坚敏,王志铮,陈启恒,等.4种重金属对泥螺的急性毒性和联合毒性研究[J].浙江海洋学院学报(自然科学版),2007,26(3):252-256.
[11]Zhang YM,Wang YJ,Yu RL,et al.Effects of heavy metals Cd2+,Pb2+and Zn2+on DNA damage of loach Misgurnus anguillicaudatus[J].中国高等学校学术文摘·生物学,2008,3(1):50-54.
[12]Bartling D,Radzio R,Steiner U,et al.A glutathione S-transferase with glutathione-peroxidase activity from Arabidopsis thaliana.Molecular cloning and functional characterization[J].Eur J Biochem,1993,216(2):579-586.
[13]陈荣,刘辉,李东晓,等.水生动物谷胱甘肽硫转移酶研究进展[J].厦门大学学报(自然科学版),2006,45(S2):176-184.
[14]Morris MJ,Liu D,Weaver LM,et al.A structural basis for cellular uptake of GST-fold proteins[J].PLoS One,2011,6(3):e17864.
[15]Dummee V,Tanhan P,Kruatrachue M,et al.Histopathological changes in snail,Pomacea canaliculata,exposed to sub-lethal copper sulfate concentrations[J].Ecoto Environ Safe,2015,122(4):290-295.
[16]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.
[17]Liu HH,He JY,Zhao RT,et al.A novel biomarker for marine environmental pollution of pi-class glutathione S-transferase from Mytilus coruscus[J].Ecotox Environ Safe,2015,118:47-54.
[18]Wan Q,Whang I,Lee JS,et al.Novel omega glutathione S-transferases in disk abalone:characterization and protective roles against environmental stress[J].Comp Biochem Physiol C Toxicol Pharmacol,2009,150(4):558-568.
[19]张林宝.菲律宾蛤仔对典型污染物胁迫响应的分子生物标志物研究[D].北京:中国科学院,2012.
[20]朱丹丹.湖北钉螺肝脏cDNA文库的构建和序列分析[D].北京:中国科学院研究生院,2010.
[21]罗鸣.近江牡蛎一种四跨膜素鉴定及功能研究[D].杭州:浙江大学,2012.
[22]Chan PC,Shiu CK,Wong FW,et al.Common carp metallothionein-1 gene:cDNA cloning,gene structure and expression studies[J].Biochim Biophys Acta,2004,1676(2):162-171.
[23]李姣,夏媛媛,程天印.福寿螺肾脏组织学观察[J].畜牧兽医科技信息,2011(4):30-31.