| Polycyclic aromatic hydrocarbons (PAHs) are one group of persistent organic pollutants (POPs), widely distributed in the environment. With the rapid development of industries and maritime oil transportion and exploitation, the PAHs pollution in the ocean is more and more serious and has induced the damage to marine life in direct or indirect way. As one of the organisms which have high species diversity, abundant biomass and play an important role in the marine ecosystem, copepods are also threated by PAHs. They have small body size, short life-cycle and are sensitive to pollutants, which make them the excellent candidates to study the effects of anthropogenic pollutants. So the study on responses of copepods to PAHs is of great importance to marine environmental monitoring and healthy assessment. With the rapid progress of molecular technique, the study on molecular mechanism of copepods exposed to PAHs at the transcriptomic level could assess the potential threat more quickly and precisely, which has important theoretical and practical significance. However, copepods are of small body size and it is difficult to obtain sufficient RNA for transcriptomic research, which limits the application of molecular technique in copepods. In this study, full-length cDNA library of Acartia pcifica with GeneRacer kit was constructed, and common sequence was found at the 5’end of their mRNAs. With this common sequence, subtractive hybridization libraries exposure to two PAHs (1,2-dimethylnaphthalene and pyrene) of Pseudodiaptomus poplesia were constructed and the full-length cDNA sequences of functional genes responded to PAHs were obtained. With the RT-qPCR technique, expression profiles of these functional genes in P. poplesia exposed to PAHs were studied. This study provides theoretical basis and methodological reference for exploring the molecular mechanism of copepods responded to pollutants and molecular biomarkers for PAHs monitoring in the marine environment. The main results are as follows:(1) Full-length cDNA libraries of A. pcifica were constructed and the common sequence (CS) was found to exist at the 5’end of mRNA in Calanoida copepods by bioinformatics analysis.(2) Full-length cDNA libraries of different copepods were constructed with 5’-CS. Lots of full-length genes related to signal transduction, gene regulation, metabolism process and transport were acquired.(3) Cytoplasmic ribosomal protein (cRP) genes were analysed. A complete set of 79 cRPs from P. poplesia were obtained, including 32 40S subunit RPs and 47 60S subunit RPs. The characteristics of RPs from this copepod include:higher GC content in the coding regions (CDs) than that in the untranslated regions (UTRs), and significantly longer 3’-UTRs than 5’-UTRs; significantly species codon usage bias; highly proportion of the positive residues (arginine and lysine) in the deduced RP amino acid sequences; high pI values, concentrating at 10-12.(4) The acute toxicity test indicated that P. poplesia was relatively sensitive to PAHs compared to other copepods. The 96h-LC5o of P. poplesia responded to 1, 2-dimethylnaphthalene and pyrene were 788.98 (681.93-912.82) μg/L and 54.68 (47.88-62.45) μg/L, respectively.(5) Suppression subtractive hybridization (SSH) libraries of P. poplesia under the PAHs stress were constructed by "biotin-labeled with magnetic beads" method based on the 5’-CS. Full-length cDNA sequences of genes related to PAHs exposure were recognized firstly from P. poplesia. A serial of interesting genes including:glutathione S-transferase delta-epsilon(GST (δ-ε)), glutathione S-transferase theta (GST (θ)), phospholipid-hydroperoxide glutathione pcroxidase (PHGPx, GPx4, Glutathione peroxidase 2 (GPx2), peroxiredoxin 6 (Prdx6) and ras-related C3 botulinum toxin substrate 1 (Rac1) were further studied with RT-qPCR technique.(6) The expression profiles of important functional genes of P. poplesia exposure to sublethal PAHs were analysed. The main results were summarized as follows:① RPL15 and RPS20 were proved to be suitable as the reference genes for P. poplesia exprosure to 1,2-dimethylnaphthalene; otherwise, RPL15 and EF-1-delta were appropriate to be the reference genes for P. poplesia exprosure to pyrene.②The gene expression study of P. poplesia in this thesis indicated that different genes, even different isoenzymes of the same superfamily had different profiles under different conditions. Compared to 1,2-dimethylnaphthalene, the reponse of seven genes studied was more sensitive to pyrene. According to the reponse to PAHs, GST (δ-ε)、Prdx6 could be the potential biomarker for environmental bio-monitoring of pyrene.③P. poplesia was sensitive to pyrene. Short term (24h) or low concentration (0.5% of LC50) of pyrene could induce or repress the expression of most genes in our study.④The expression of GST (δ-ε) was up-regulated significantly after 24h exposed to pyrene. With the increase of the time, its expression returned to the control level. This result indicated that GST (δ-ε) played an important role in the detoxification process of P. poplesia under pyrene stress and was very sensentive.⑤Prdx6 gene was considerably induced after 24h exposed to pyrene, and showed positive correlationship with concentration. Howerer, with the extenstion of exposure time, the expression level of Prdx6 was downregulated. The results showed that short term exposure to pyrene would up-regulated Prdx6 expression to protect P. poplesia against oxidative stress damage; with the increasing of concentration and time, the organisms would be damaged irreversibly by excess reactive oxygen species (ROS) and inhibit the expression of Prdx6.⑥The expression of Rac1 was induced significantly after 24h, above 4 folds of control at the low concentration. Our results showed that pyrene could induce Racl expression under short-time exposure to enhance the physiological and biochemical signal transduction. Rac1 may play vital role in the response to PAHs. Since there is not much information about this gene in copepods, more studies need to be done to explore its function and molecular mechanism. |
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