Spliced leader-based analyses reveal the effects of polycyclic aromatic hydrocarbons on gene expression in the copepod Pseudodiaptomus poplesia
Spliced leader-based analyses reveal the effects of polycyclic aromatic hydrocarbons on gene expression in the copepod Pseudodiaptomus poplesia
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基于剪接前导序列的分析揭示了多环芳烃对桡足类 Pseudodiaptomus poplesia 基因表达的影响
DOI：
10.1016/j.aquatox.2016.12.014
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发表时间：
2017
影响因子：
4.5
通讯作者：
Guangxing Liu
中科院分区：
文献类型：
--
作者：
Yunyun Zhuang;Feifei Yang;Donghui Xu;Hongju Chen;Huan Zhang;Guangxing Liu
多环芳烃（PAHs）是一组有毒和致癌的污染物，可对包括桡足类在内的海洋生物的发育、生长和繁殖产生不利影响。然而，在海洋浮游桡足类调节多环芳烃暴露的分子机制的知识是有限的。本文研究了1，2-二甲基萘（1，2-NAPH）和芘对羊伪镖水蚤（Pseudodiaptomus poplesiau）存活和基因表达的影响。急性毒性反应导致1，2-NAPH和芘的96小时LC 50分别为788.98 μg L− 1和54.68 μg L− 1。使用最近发现的桡足类剪接的领导人作为引物，我们构建了全长cDNA文库暴露于亚致死浓度的桡足类，并揭示了289个独特的基因的不同功能，包括应激反应基因和新的基因以前没有记录的这个物种。83个基因家族在PAH暴露文库中特异表达。我们进一步分析了七个目标基因的表达，通过逆转录-定量PCR在一个时间过程中的测试与三个亚致死浓度。这些靶基因在解毒、氧化防御和信号转导中具有主要作用，并且包括不同形式的谷胱甘肽S-转移酶（GST）、谷胱甘肽过氧化物酶（GPX）、过氧化物氧还蛋白（PRDX）、甲基丙二酸半醛脱氢酶（MSDH）和ras相关的C3肉毒杆菌毒素底物（RAC 1）。对7个候选参考基因的表达稳定性进行了评价，并将其中两个最稳定的参考基因（RPL 15和RPS 20（1，2-NAPH暴露），RPL 15和EF 1D（芘暴露））用于标准化目的基因的表达水平。1，2-NAPH暴露后，GST-T、GST-DE、GPX 4、PRDX 6和RAC 1表达显著上调，芘暴露后，GST-DE和MSDH表达显著上调。这些结果表明，多环芳烃暴露在磷诱导氧化应激，信号转导可能受到影响。天花。然而，基因上调后，表达水平下降到96小时，表明阈值的曝光时间，导致抑郁的基因表达。长期接触可引起解毒和抗氧化机制的功能障碍。天花。GST-T、GPX 2和GPX 4对芘的转录响应很小。我们的结果揭示了不同的敏感性P。在个体水平和转录水平上，poplesiato两种多环芳烃。作为首次尝试，本研究证明了桡足类剪接前导序列是获得全长cDNA的有效途径。poplexia暴露于多环芳烃，并提供了宝贵的基因资源，这一非模式物种。这种方法可以适用于其他哲水蚤桡足类暴露于各种压力，特别是在野外条件下。
Polycyclic aromatic hydrocarbons (PAHs) are a group of toxic and carcinogenic pollutants that can adversely affect the development, growth and reproduction of marine organisms including copepods. However, knowledge on the molecular mechanisms regulating the response to PAH exposure in marine planktonic copepods is limited. In this study, we investigated the survival and gene expression of the calanoid copepodPseudodiaptomus poplesiaupon exposure to two PAHs, 1, 2-dimethylnaphthalene (1, 2-NAPH) and pyrene. Acute toxicity responses resulted in 96-h LC50of 788.98 μg L−1and 54.68 μg L−1for 1, 2-NAPH and pyrene, respectively. Using the recently discovered copepod spliced leader as a primer, we constructed full-length cDNA libraries from copepods exposed to sublethal concentrations and revealed 289 unique genes of diverse functions, including stress response genes and novel genes previously undocumented for this species. Eighty-three gene families were specifically expressed in PAH exposure libraries. We further analyzed the expression of seven target genes by reverse transcription-quantitative PCR in a time-course test with three sublethal concentrations. These target genes have primary roles in detoxification, oxidative defense, and signal transduction, and include different forms of glutathioneS-transferase (GST), glutathione peroxidases (GPX), peroxiredoxin (PRDX), methylmalonate-semialdehyde dehydrogenase (MSDH) and ras-related C3 botulinum toxin substrate (RAC1). Expression stability of seven candidate reference genes were evaluated and the two most stable ones (RPL15andRPS20for 1, 2-NAPH exposure,RPL15andEF1Dfor pyrene exposure) were used to normalize the expression levels of the target genes. Significant upregulation was detected inGST-T,GST-DE,GPX4,PRDX6andRAC1upon 1, 2-NAPH exposure, andGST-DEandMSDHupon pyrene exposure. These results indicated that the oxidative stress was induced and that signal transduction might be affected by PAH exposure inP. poplesia. However, gene upregulation was followed by a reduction in expression level towards 96 h, indicating a threshold value of exposure time that leads to depressed gene expression. Prolonged exposure may cause dysfunction of detoxification and antioxidant machinery inP. poplesia. The transcriptional responses ofGST-T,GPX2andGPX4upon pyrene exposure were minimal. Our results reveal the different sensitivity ofP. poplesiato two PAHs at both the individual and transcriptional levels. As the first attempt, this study proved that copepod spliced leader is useful for obtaining full-length cDNA inP. poplesiaexposed to PAHs and provided a valuable gene resource for this non-model species. This approach can be applied to other calanoid copepods exposed to various stressors, particularly under field conditions.