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邻苯二甲酸二丁酯暴露下大型溞miR-34和Bcl-2基因的表达和功能分析
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Abstract:
本文旨在探究miR-34靶基因的功能及邻苯二甲酸二丁酯(Dinbutyl phthalate, DBP)暴露下大型溞miR-34与其靶基因Bcl-2的表达调控关系。首先利用miRbase数据库、Clustal W和MEGA7.0软件分析各物种miR-34序列的保守性；其次通过Targetscan和miRanda分析miR-34的靶基因，并运用GO和KEGG进行miR-34靶基因的功能富集分析；最后采用实时荧光定量PCR方法检测大型溞在环境浓度6 mg/L DBP暴露24 h和48 h后，大型溞miR-34及其靶基因Bcl-2的表达变化。结果显示，miR-34序列在物种进化过程中具有高度保守性，13个物种的序列有17个碱基完全一致。miR-34靶基因的GO和KEGG的功能聚类分析发现，这些靶基因参与多个信号通路，主要富集在细胞内转录调控、信号转导和胞吞作用等生物学过程中。DBP暴露48 h大型溞miR-34的相对表达水平明显上调，靶基因Bcl-2的mRNA相对表达则相反，表明大型溞miR-34与Bcl-2的负调控关系。结果提示miR-34可能在大型溞对DBP毒性作用的响应过程中发挥作用，其中miR-34可通过调控Bcl-2的表达水平来影响大型溞细胞的生命活动。这对于分析水生无脊椎动物在毒性胁迫时的调节应对机制有一定的借鉴作用。
The purpose of this study was to investigate the function of miR-34 targeted genes and the expression and regulation relationship between miR-34 and its target gene Bcl-2 in Daphnia magna exposed to Dibutyl phthalate (DBP). Firstly, miRbase database, Clustal W and MEGA7.0 software were used to analyze the sequence conservation of miR-34 in various species. The target genes of miR-34 were analyzed by Targetscan and miRanda. The functional enrichment analysis of miR-34 target genes was performed using GO and KEGG. Finally, real-time quantitative PCR method was used to detect the expression changes of miR-34 and its target gene Bcl-2 in D. magna exposed to environmental concentration of 6 mg/L DBP for 24 h and 48 h. The results showed that the miR-34 sequence was highly conserved during species evolution. The sequences of 13 species have 17 bases identical. GO and KEGG functional clustering analysis of miR-34 target genes found that these target genes were involved in multiple signaling pathways. The main biological processes analyzed for GO enrichment are intracellular transcriptional regulation, signal transduction and endocytosis. The relative ex-pression level of miR-34 was significantly up-regulated in Daphnia magna after DBP exposure for 48 h. The relative mRNA expression of the target gene Bcl-2 was opposite. It indicated the negative regulation relationship between miR-34 and Bcl-2 in D. magna. The results suggest that miR-34 may play a role in the response of D. magna to DBP toxicity. Among them, miR-34 affects the life ac-tivities of cells by regulating the expression level of Bcl-2. This has a certain reference for studying the regulatory response mechanism of aquatic invertebrates under toxic stress.
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