【作者】 孟晓林；

【导师】 王雷；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 水产养殖， 2013， 博士

【摘要】 虾夷扇贝（Mizuhopecten yessoensis）是我国北方重要的海水养殖贝类。随着近年来经济的高速发展，逐渐严重的海洋污染给虾夷扇贝养殖业造成了巨大的损失，尤其是生物毒素和持久有机污染物等蓄积引发的食品安全问题，严重制约了产业的健康可持续发展。但是，由于基因组信息资源的缺乏，对于各类海洋污染物，尤其是重金属对虾夷扇贝毒性作用机制的深入研究相对比较匮乏。本研究利用生物化学、分子生物学及生物信息学技术，分别研究了重金属镉（Cd）、铜（Cu）在虾夷扇贝鳃、消化腺组织内的蓄积规律；镉、铜对虾夷扇贝鳃、消化腺组织内SOD、CAT、GPx酶活性的影响；成体虾夷扇贝的全转录组测序及在镉、铜分别胁迫条件下，虾夷扇贝鳃、消化腺组织基因差异表达谱等。主要结果如下：1.研究了镉在虾夷扇贝鳃和消化腺两组织的累积规律，结果表明累积量与胁迫的剂量呈现出线性相关性，同时随着胁迫时间的延长，蓄积量表现出曲线变化规律。两组织中最大Cd蓄积分别出现在10d，0.20mg/L胁迫组（鳃，134.20μg/g）和14d，0.10mg/L胁迫组（消化腺，109.20μg/g）。鳃组织中镉的蓄积量要高于消化腺组织，其相对为虾夷扇贝镉的主要富集组织。2.研究了铜在虾夷扇贝鳃、消化腺两组织中的累积规律，结果表明，0.025mg/L胁迫组在鳃中最高累积出现在14d（347.55μg/g），0.05mg/L、0.10mg/L胁迫组最高累积均出现在6d，分别为340.72μg/g和302.30μg/g。除0.025mg/L胁迫组外，整体随时间呈曲线变化关系。消化腺组织中，0.10mg/L胁迫组铜累积随时间呈线性关系，最高累积出现在14（d60.88μg/g），而0.025mg/L与0.05mg/L组随暴露时间的延长均表现出曲线变化关系。分析表明，鳃是虾夷扇贝铜的主要蓄积部位。消化腺组织对铜的累积不存在浓度依赖性关系，对高铜胁迫累积反应较为敏感。3.研究了镉胁迫对虾夷扇贝两组织的主要抗氧化酶活性的影响。结果表明镉胁迫下鳃、消化腺组织内SOD、CAT、GPx活性基本呈现峰值分布。两组织中，0.10mg/L、0.20mg/L胁迫组SOD活性均在胁迫1d时达到最大值，而0.05mg/L胁迫组峰值均出现在3d。对CAT活性研究表明，鳃组织各胁迫浓度组变化趋势与SOD一致，而消化腺组织则随胁迫浓度增加，最高活性分别出现在10d、6d和1d。对GPx活性研究表明，鳃组织中随胁迫浓度升高，活性最高值分别出现在6d、3d和1d，消化腺组织则分别在3d和1d达到最高。SOD是镉胁迫下虾夷扇贝鳃组织内的主要抗氧化酶，而CAT是消化腺组织在镉胁迫条件下的主要抗氧化酶。4.研究了不同铜胁迫浓度对虾夷扇贝两种组织的主要抗氧化酶SOD、CAT和GPx活性的影响。结果显示，鳃组织各抗氧化酶在胁迫期内均被显著性抑制。消化腺组织中，除0.05mg/L胁迫组对GPx酶活性24h时显著抑制外，0.025mg/L、0.05mg/L胁迫浓度在24h内均可诱导抗氧化酶显著表达，但随胁迫期的延长逐渐降低。0.10mg/L胁迫组各酶活性在胁迫期内显著性抑制。与镉对虾夷扇贝的毒性相比，铜的毒性作用更强，对虾夷扇贝抗氧化防御系统的破坏性更大。5.通过RNA-Seq技术，对成体虾夷扇贝鳃、消化腺、性腺、闭壳肌、外套膜、肾脏及内脏团组织混合样本进行了全转录组分析，共产生194,839条非冗余读段，其中14,240条读段获得了明确功能注释。同时基于GO分析获得了721个与刺激反应（response to stimulus）相关的转录本。通过KEGG分析，获得了251个包含在13个免疫通路中的转录本。6.对虾夷扇贝镉胁迫条件下的鳃、消化腺组织进行差异表达基因研究。鳃组织中共确认了7,556个显著性差异表达基因，其中，3,673个基因上调，3,883个基因下调。消化腺组织中共发现3,002个差异表达基因，其中，上调基因1,627个，下调基因1,375个。通过代谢通路富集性分析确认了大量与环境胁迫相关的差异表达基因。7.对虾夷扇贝铜胁迫条件下的鳃、消化腺组织差异表达基因进行研究。鳃组织中共确认3,459个显著性差异表达基因，其中，1,312个基因上调，2,237个基因下调。消化腺组织中共确认1,718个差异表达基因，其中，上调基因841个，下调基因877个。通过GO富集性分析，发现了大量铜胁迫条件下与化学物质刺激反应相关的差异表达转录本。通过KEGG富集性分析，确认了许多虾夷扇贝在受到铜胁迫时免疫系统相关基因的表达变化情况。本研究初步了解了镉、铜对虾夷扇贝的分子毒性作用机制，将为进一步深入研究海洋污染物对虾夷扇贝的毒性作用机理奠定一定的理论基础。同时，虾夷扇贝的全转录组测序数据极大程度的丰富虾夷扇贝序列数据库，筛选得到的敏感差异表达基因可作为候选基因，构建虾夷扇贝养殖预警预报体系平台，以期为我国虾夷扇贝的健康、安全养殖提供一定的科学依据。更多还原

【Abstract】 Japanese scallop (Mizuhopecten yessoensis) has been cultured on a large-scale inChina for many years. However, serious marine pollution in recent years has resultedin considerable loss to this industry, especially the safety problems caused by heavymetal and POPs, etc. Moreover, due to the lack of genomic resources, limited relatedresearch about the toxicology mechanism caused by the marine pollutions has beencarried out on this species. In this study, the bioaccumulation in gill or digestive glandfollowing Cd or Cu exposure was investigated. Also, the responses of the antioxidantdefense system including SOD, CAT and GPx were studied. In order to betterannotation the transcripts, the transcriptome of adult M.yessoensis were investigatedbased on RNA-Seq technology. Then the different expressed genes (DEGs) weredetected in gill and digestive gland following Cd or Cu exposure. The detail resultswere as follows:1. The Cd concentration in gill or digestive gland of M.yessoensis displayed alinear relationship with the exposure doses and had a curvilinear change with timeextension. The maximum Cd concentration presented at10d (0.20mg/L,134.20μg/g)in gill and14d (0.1mg/L,109.2μg/g) in digestive gland, respectively. Theconcentration of Cd in gill was larger than that in digestive gland and suggested thatgill might be the main accumulation tissue of Cd in M.yessoensis.2. The maximum accumulation of copper (0.025mg/L,0.05mg/L,0.10mg/L) ingill occurred at14d (347.55μg/g),6d (340.72μg/g) and6d (302.30μg/g) respectively.With the time extension, accumulation of copper has a curvilinear relationship except0.10mg/L group. However, concentration of copper in0.10mg/L group of digestivegland presented liner changes with exposure time and the peak occurred at14d(60.88μg/g). The corresponding changes in0.025mg/L and0.05mg/L were curvilinear and even be decreased. Gill was the main accumulation tissue of Cu in M.yessoensis.3. SOD, CAT and GPx activities in gill or digestive gland respond to cadmiumexposure displayed curvilinear relationship with time extension. The SOD activity in0.10mg/L and0.20mg/L groups of both tissues reached to the peak at24h and that of0.05mg/L group both reached at72h. CAT activity in gill following Cd exposurepresented the same tendency with SOD activity. However, the peak value of CAT indigestive gland occurred at10d,6d and1d with doses increasing. GPx activity in gillfollowing Cd exposure presented the peak at6d,3d and1d with doses increasing and3d and1d in digestive gland. SOD might be the main antioxidant enzyme in gill ofM.yessoensis and CAT was the main antioxidant enzyme in digestive gland.4. SOD, CAT and GPx activities were significantly suppressed in gill followingcopper exposure. However, in digestive gland,0.025mg/L and0.05mg/L groups couldinduce the express of antioxidant enzymes in24h except GPx in0.05mg/L group. Theactivities of all the three enzymes in0.10mg/L group were suppressed during all theexperiment.5. Transcriptome of adult M. yessoensis (adductor muscle, digestive gland, gill,gonad (male and female), kidney, visceral mass and mantle) was analyzed byRNA-Seq. Totally194,839clean reads were produced and14,240clean reads wereclearly annotated in database. Totally721transcripts were founded in the GO term of‘response to stimulus’ and251transcripts were detected involved in13immunesystem pathways.6. Based on RNA-Seq (Quantification), totally7,556different expressed geneswere identified in gill following Cd exposure. Among them,3,673transcripts wereup-regulated and3,883were down-regulated. In digestive gland, a total of3,002DEGs were detected, with1,627up-regulated genes and1,375down-regulated genes.Also, amount of DEGs related with environment stress were founded by KEGGenrichment analysis.7. A total of3,459DEGs were detected in gill following copper exposure, with1,312genes up-regulated and2,237down-regulated. In digestive gland, totally1,718 DEGs were identified. Among them841genes were up-regulated and877genes weredown-regulated. At the same time, amount of DEGs related to ‘response to chemicalstimulus’ was detected by GO enrichment analysis. Also, the expressed level of genesinvolved in immune system was analyzed by KEGG.This thesis investigated the toxic mechanism of M.yessoensis respond tocadmium or copper and would be helpful for researching the toxicology of marinepollutants. Also, transcriptome of M. yessoensis would great enrich the sequencesresources in public database. These sensitive expressed genes could be used aspotential candidate genes for constructing early warning system and platform duringthe healthy aquaculture of Japanese scallop.更多还原