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摘要生物监测是环境监测重要部分，当前我国环境监测主要依赖化学分析监测，近年来虽然国际上生物监测技术发展比较迅猛，但基于我国环境监测标准限制，我国生物监测发展一直处于研究阶段，当前生物监测主要依赖于藻、溞和鱼类水生生物物种，对于大型底栖生物目前监测仅依赖于生物多样性调查，因此亟待发展我国水生实验生物，尤其需要加强大型底栖生物生物标志物筛选以及模式化研究。因此本文采用我国广泛分布的软体动物河蚬作为实验生物，为加强其背景生物学研究，系统研究了河蚬生物标志物，最后利用上述标志物探讨了环境污染物对河蚬作用机制。主要研究内容与结果如下：1）利用Illumina技术获得了河蚬miRNA信息转录组信息，获得了条高质量序列，鉴定了45条保守的和39条新的河蚬miRNAs；荧光定量PCR定量结果表明12个miRNA中9个miRNAs在腹足中表达量最高，而miR-10和Novel-2各自在鳃和内脏团中表达最高；预测软件结果发现miRNAs和环境污染相关基因相关，为河蚬作为实验生物提供了分子生物学基础。2）采用RACE技术，成功克隆获得河蚬CCK,Conopression和FFamide的全长cDNA序列，预测了河蚬CCK,Conopression和FFamide蛋白的分子结构特征，并分析了神经肽在河蚬不同组织中的表达分布；有机磷酸酯对神经肽显著调节，表明了CCK,Conopression和Ffamide适合作为河蚬生物标志物，为环境污染物生物监测提供技术手段。3）结合转录组测序技术和荧光定量PCR技术分析了典型有机磷酸酯（TDCPP和TBP）毒理作用机制，多指标结果表明长期低剂量暴露下，主要影响相关通路为凋亡通路、黏着斑通路、小细胞肺癌通路、细胞黏附分子通路，酶活指标和凋亡相关基因指标证实了典型有机磷酸酯显著诱导河蚬细胞凋亡。关键词：河蚬，miRNA，有机磷酸酯，凋亡通路，生物标志物ABSTRACTBiologicalmonitoringisanimportantpartofenvironmentalmonitoring.CurrentlytheenvironmentalmonitoringinChinamainlydependedonthechemicalanalysisandmonitoring.Inrecentyears,althoughtheinternationallybiologicalmonitoringtechnologydevelopmentisrapid,basedonrestrictionsofourstandardofenvironmentalmonitoring,thedevelopmentofbiologicalmonitoringinourcountryhasbeenintheresearchstage.Currently,biologicalmonitoringmanlyreliesonalgae,Daphniaandfishaquaticspecies,forlargebenthiccurrentmonitoringdependsonlyonbiodiversitysurvey.Therefore,itisurgenttodevelopaquaticlaboratoryanimalsinourcountry,particularlytostrengthenthescreeningoflargebenthicbiomarkersandmodelingstudy.Thereforeinthisstudythewidespreadmollusk-Corbiculaflumineainourcountrywastakenasanexperimentalorganism,tostrengthenthebiologicalbackgroundresearch,wesystematicallystudybiomarkersoftheCorbiculaflumineainmonitoringenvironmentalpollutants.Finally,wediscussmechanismofenvironmentalpollutantsonCorbiculaflumineathroughtheabovemarkers.Themainresearchcontentsandresultsareasfollows:1)ThroughIlluminasequencingweobtainedtheCorbiculaflumineamiRNAbiologicalinformation,andobtainedthehighqualitysequences,thenidentified45conservativeand39newCorbiculaflumineamiRNAs;FluorescencequantitativePCRresultsshowedthatninemiRNAsof12wereexpressedhighestinthegastropod,whilemiR-10andNovel-2wasexpressedhighestrespectivelyingillandvisceralmass;SoftwarepredictionresultsshowedthatmiRNAsrelatedtoenvironmentalpollutiongenes,thisprovidesamolecularbiologicalbasisforCorbiculaflumineaasanexperimentalorganism.2)ThroughRACEtechnology,wesuccessfullyclonedtheCorbiculaflumineathefull-lengthcDNAsequenceofCCK,ConopressionandFFamide,predictedthemolecularstructureofCorbiculaflumineaCCK,ConopressionandFFamideprotein,andanalyzedthedistributionofneuropeptideexpressionindifferenttissuesofCorbiculafluminea;theorganicphosphatesignificantlyincreasedtheexpressionofneuropeptide,indicatingthattheCCK,andFfamideConopressioncanbetakenasCorbiculaflumineabiomarkersandthisprovidesatechnicalmeansforthebiologicalmonitoringofenvironmentalpollutants.3)By卡力孜然酊北京白癜风