四角蛤蜊对典型PBDEs胁迫的分子响应机制

Polybrominated diphenyl ethers (PBDEs) are used world-wide as brominated flame retardants, which can be released into the environment through emissions during the production and application processes, and leakage from products during and after use. High concentrations of PBDEs have been detected in sediment and mollusk tissues with BDE-209 and BDE-47 being the most prominent. Especially in Laizhou Bay, where PBDEs manufactories are clustered, the concentrations are among the highest in the world. Toxic effects of PBDEs such as immunotoxicity, developmental toxicity, and endocrine disruption have been identified in many organisms. However, the toxic effect of PBDEs to mollusk is not clearly known. And it is quite required to evaluate ecological risk of PBDEs in marine system. Thus, the molecular response mechanism to BDE-209 and BDE-47 in Mactra veneriformis, a typical type beach bivalve with buried habitat, would be researched in this project. The response of cytochrome P450 (CYP), glutathione S transferases (GST), and p-glycoprotein (P-gp), which play important roles in metabolism and detoxification of xenobiotics would be detected at mRNA and protein level. Moreover, mRNA expression and enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase, which can protect organism from oxidative damage, would be determined. Furthermore, the roles of MAP kinase signaling pathway in mRNA expression regulation of the above genes under PBDEs stress would be discussed. Finally, molecular response mechanism to typical PBDEs stress in Mactra veneriformis would be clarified. It can provide scientific basis to PBDEs ecological assessment as well as the sustainable development of marine environment and mollusk resources.

多溴联苯醚(PBDEs) 对生物体有免疫毒性、发育毒性、内分泌干扰毒性等多种毒性效应，针对我国近岸海域PBDEs污染的特点和PBDEs生态风险评价的迫切需求，选择典型的埋栖型滩涂贝类四角蛤蜊(Mactra veneriformis)为实验生物，通过室内胁迫实验，查明四角蛤蜊体内参与污染物解毒的三相酶/蛋白和抗氧化防御成分在mRNA和酶活/蛋白水平对典型PBDEs胁迫响应的时空特征，探明MAPK信号通路在响应典型PBDEs胁迫和防御成分基因表达调控中的作用，初步阐明四角蛤蜊对典型PBDEs胁迫的分子响应机制，为评估PBDEs生态风险提供科学依据，为贝类资源与环境的可持续利用提供理论参考。

本项目通过室内胁迫实验和野外实验相结合，探讨了四角蛤蜊对典型PBDEs BDE-47和BDE-208胁迫响应的分子机制，主要结果如下：（1）查明了四角蛤蜊转录组对BDE-47胁迫的响应，组装得到溶剂对照组127648个Unigene，胁迫组154,225个Unigene。19625基因表达量出现差异，其中9597个基因表现为上调，10028个基因表现为下调。差异表达的基因涉及255个通路。（2）探明了四角蛤蜊参与污染物代谢和解毒的三相酶系对BDE-47和BDE-209胁迫的响应情况，克隆获得了三相酶系代表蛋白细胞色素P450 4 （MvCYP4）、谷胱甘肽硫转移酶（MvGST）和P-糖蛋白（MvPgp）的cDNA序列，该三种蛋白在mRNA水平对BDE-47和BDE-209胁迫响应敏感。GST的酶活性因BDE-47和BDE-209的胁迫出现波动，但是和GST基因表达量之间没有显著相关关系。（3）阐明了四角蛤蜊抗氧化系统关键酶类超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物（GPx）对BDE-47和BDE-209胁迫的响应，克隆获得了MvCAT、MvGPx3和MvSe-GPxcDNA序列。SOD、CAT、GPx3和Se-GPx在mRNA和酶活水平均可响应BDE-47和BDE-209胁迫。就基因表达量而言，SOD波动范围最大，而就酶活而言，CAT波动范围最大。（4）摸清了MAPK信号通路对BDE-47胁迫的响应，10μg/L BDE-47可以引起四角蛤蜊鳃组织p38、JNK和ERK三个蛋白磷酸化水平的上调，p38和JNK信号通路对BDE-47响应敏感，只在胁迫第3天，检测到ERK蛋白磷酸化水平的升高，其它时间磷酸化水平和对照相比未发生显著变化。（5）评估了防御成分对海域PBDEs污染的指示作用，BDE-47和BDE-209是多溴联苯醚生产厂家集中的莱州湾西岸5个站位中，四角蛤蜊样品富集量最高的PBDEs。PBDEs富集量高的站位，防御成分基因的表达量尤其是CYP4和GST呈现增加的趋势。研究结果可为PBDEs风险评估和贝类与环境的可持续利用提供科学参考。