Osuna-Jiménez Inmaculada, Abril Nieves, Vioque-Fernández Amalia, Gómez-Ariza José Luis, Prieto-Álamo María-José, Pueyo Carmen
Department of Biochemistry and Molecular Biology, Agrifood Campus of International Excellence (ceiA3), University of Córdoba, Severo Ochoa Building, Rabanales Campus, 14071 Córdoba, Spain.
Department of Chemistry and Materials Science, Faculty of Experimental Science, University of Huelva, El Carmen Campus, 21007 Huelva, Spain.
Fish Shellfish Immunol. 2014 Sep;40(1):136-45. doi: 10.1016/j.fsi.2014.06.031. Epub 2014 Jul 5.
This study aimed to identify differentially expressed genes in Procambarus clarkii crayfish collected from locations of different environmental qualities in the Doñana National Park surrounding areas. The pollution sustained by the crayfish was confirmed by their hepatopancreatic metal concentration. We generated forward and reverse libraries by suppression subtractive hybridization (SSH) to analyze the transcriptional profiles of crayfish from moderately and highly polluted zones in comparison with the control site within the Doñana Biological Reserve. Forty-three differentially expressed genes were detected, and most of them were identified as genes involved in a variety of biological functions, particularly in the innate immune response. To verify the SSH results and assess interindividual variability nine transcripts (ALP, AST, BTF3, CHIT, CTS, ferritin, HC, HC2, and SPINK4) were selected for absolute quantification by real-time qRT-PCR. The qRT-PCR data revealed substantial differences in the absolute amounts of the nine transcripts and confirmed their up- or down-regulation in the polluted sites. Additionally, a positive and significant linear correlation was found between the hepatopancreatic copper concentration and the levels of the transcripts encoding hemocyanins. Finally, the transcriptomic study was complemented with a detailed analysis of SNP profiles of the selected transcripts that revealed point mutations that might underlie adaptive response to environmental stress in P. clarkii. Overall, this work provides novel insights into the molecular pathways that could mediate the response to environmental pollutants in P. clarkii emphasizing the central role of the immune function and thus, should clearly benefit further immunotoxicological research in this organism.
本研究旨在鉴定从多尼亚纳国家公园周边不同环境质量地点采集的克氏原螯虾中差异表达的基因。螯虾所遭受的污染通过其肝胰腺金属浓度得以证实。我们通过抑制性消减杂交（SSH）构建了正向和反向文库，以分析来自多尼亚纳生物保护区内中度和高度污染区域的螯虾与对照位点相比的转录谱。检测到43个差异表达基因，其中大多数被鉴定为参与多种生物学功能的基因，特别是先天免疫反应。为了验证SSH结果并评估个体间的变异性，选择了9个转录本（碱性磷酸酶、天冬氨酸转氨酶、BTF3、几丁质、组织蛋白酶、铁蛋白、血蓝蛋白、血蓝蛋白2和丝氨酸蛋白酶抑制剂Kazal型4）通过实时定量逆转录聚合酶链反应（qRT-PCR）进行绝对定量。qRT-PCR数据显示这9个转录本的绝对量存在显著差异，并证实了它们在污染位点的上调或下调。此外，还发现肝胰腺铜浓度与编码血蓝蛋白的转录本水平之间存在正相关且显著的线性关系。最后，转录组学研究辅以对所选转录本的单核苷酸多态性（SNP）谱的详细分析，揭示了可能是克氏原螯虾对环境应激适应性反应基础的点突变。总体而言，这项工作为介导克氏原螯虾对环境污染物反应的分子途径提供了新的见解，强调了免疫功能的核心作用，因此，显然将有利于该生物体进一步的免疫毒理学研究。
