School of Environmental Engineering, Graduate School of Energy and Environmental System Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743, Republic of Korea.
Comp Biochem Physiol C Toxicol Pharmacol. 2011 Nov;154(4):399-408. doi: 10.1016/j.cbpc.2011.07.008. Epub 2011 Jul 22.
Antioxidant enzymes play important roles in the protection against oxidative damage caused by environmental pollutants by scavenging high levels of reactive oxygen species and have been quantified as oxidative stress markers. However, combining mRNA expressions of genes coding for detoxification enzymes along with enzyme activities will be more useful biomarkers of stress. Therefore, in this study the cDNA of the catalase gene from the aquatic midge, Chironomus riparius (CrCAT) was sequenced using 454 pyrosequencing. The 2139 bp CrCAT cDNA included an open reading frame of 1503 bp encoding a putative protein of 500 amino acids with a predicted molecular mass of 56.72 kDa. There was an 18 bp 5' and a long 618 bp 3' untranslated region with a polyadenylation signal site (AATAAA). The deduced amino acid sequence of CrCAT contained several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTX and the proximal active site signature FXRERIPERVVHAKGXGA. A comparative analysis showed the presence of conserved amino acid residues and all of the catalytic amino acids (His(70), Asn(143), and Tyr(353)) were conserved in all species. The CrCAT contained three potential glycosylation sites and a peroxisome targeting signal of 'AKM'. The mRNA was detected using RT-PCR at all developmental stages. The time-course expression of CrCAT was measured using quantitative real-time PCR after exposure to different concentration and durations of Paraquat (PQ), cadmium chloride (Cd) and nonylphenol (NP). The expression of CrCAT was significantly up regulated on exposure to 50 and 100mg/L PQ for 12 and 24h. Among the different concentrations and durations of Cd tested, significantly highest level of expression for CrCAT mRNA and catalase enzyme activity was observed on exposure to 10mg/L for 24h. In the case of NP, the highest level of CrCAT expression was observed after exposure to 100 μg/L for 24h. The expression profiles of three selected C. riparius glutathione S-transferase genes (CrGSTs) viz. CrGSTdelta3, CrGSTsigma4 and CrGSTepsilon1 was also studied on exposure to NP and were up or down regulated at different time points and concentrations. Significantly highest level of expression for CrGSTdelta3 was observed after 48 h and for CrGSTsigma4 and CrGSTepsilon1 after 24h exposure to 100 μg/L of NP. The results show that CrGSTs and CrCAT could be used as potential biomarkers in C. riparius for aquatic ecotoxicological studies.
抗氧化酶通过清除高水平的活性氧物种，在防止环境污染物引起的氧化损伤方面发挥着重要作用，并且已被量化为氧化应激标志物。然而，结合编码解毒酶的基因的 mRNA 表达和酶活性将是更有用的应激生物标志物。因此，在这项研究中，使用 454 焦磷酸测序对水生摇蚊 Chironomus riparius（CrCAT）的过氧化氢酶基因的 cDNA 进行了测序。2139bp 的 CrCAT cDNA 包括一个 1503bp 的开放阅读框，编码一个由 500 个氨基酸组成的假定蛋白质，预测分子量为 56.72kDa。有一个 18bp 的 5'非翻译区和一个长 618bp 的 3'非翻译区，其中包含一个 polyadenylation 信号位点（AATAAA）。CrCAT 的推导氨基酸序列包含几个高度保守的基序，包括近端血红素配体特征序列 RLFSYNDTX 和近端活性位点特征序列 FXRERIPERVVHAKGXGA。比较分析表明，所有物种都存在保守的氨基酸残基和所有的催化氨基酸（His(70)、Asn(143)和 Tyr(353)）。CrCAT 含有三个潜在的糖基化位点和一个过氧化物酶体靶向信号 'AKM'。使用 RT-PCR 在所有发育阶段检测到 mRNA。使用定量实时 PCR 测量暴露于不同浓度和持续时间的百草枯（PQ）、氯化镉（Cd）和壬基酚（NP）后 CrCAT 的时间过程表达。在暴露于 50 和 100mg/L PQ 12 和 24h 后，CrCAT 的表达明显上调。在所测试的 Cd 的不同浓度和持续时间中，在暴露于 10mg/L 24h 时观察到 CrCAT mRNA 和过氧化氢酶活性的最高表达水平。在 NP 的情况下，在暴露于 100μg/L 24h 后观察到 CrCAT 表达水平最高。还研究了三种选定的 Chironomus riparius 谷胱甘肽 S-转移酶基因（CrGSTs）即 CrGSTdelta3、CrGSTsigma4 和 CrGSTepsilon1 的表达谱在暴露于 NP 时的不同时间点和浓度下上调或下调。CrGSTdelta3 的表达水平在 48h 时最高，CrGSTsigma4 和 CrGSTepsilon1 在暴露于 100μg/L NP 24h 时最高。结果表明，CrGSTs 和 CrCAT 可作为水生毒理学研究中 Chironomus riparius 的潜在生物标志物。
