Cu^(2+)胁迫下福寿螺谷胱甘肽S转移酶基因表达分析

Analysis of glutathione S-transferase gene expression in Pomacea canaliculata under Cu^(2+) stress

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 Cu^2+ 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 Cu^2+ 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 Cu^2+ 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 Cu^2+ 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 Cu^2+ stress was compared by t-test.Results The relative expression of PcGST mRNA in P.canaliculata tissue without Cu^2+ stress was set as 1.0 ± 0.0.In the liver tissue,under the stress of 50 μg/L Cu^2+,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 Cu^2+ 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 Cu^2+ 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 Cu^2+ 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 of 100 μg/L Cu^2+,the relative expression of PcGST mRNA reached the peak of 18.0 ± 0.8 on 14 d under the stress of 150 μg/L Cu^2+.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 Cu^2+.Conclusion PcGST gene was upregulated in P.canaliculate tissue under Cu^2+ stresss.