【作者】 张琳琳；

【导师】 张国范；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 水产养殖学， 2012， 博士

【摘要】 潮间带是介于海洋和大陆之间的过渡地带，是潮区最高高潮线至最低低潮线之间的海岸带，包含了沙滩、岩礁、盐沼、河口等多种多样的地理环境，是重要的海洋极端环境。长牡蛎（Crassostrea gigas）是潮间带的典型物种，也是研究海洋生物对潮间带环境适应的极佳模式生物。借鉴海胆和果蝇等模式生物防御相关基因的研究成果，利用长牡蛎基因组序列图谱，EST文库，不同胁迫状态下的转录组等多种功能基因组数据，借助生物信息学的研究方法，我们分析了长牡蛎的防御系统，鉴定了为数众多的防御系统相关基因；并从比较基因组学的角度探讨了该系统关键基因的进化历程；对牡蛎胁迫状态下的防御相关基因表达模式进行了系统的分析。另方面，本文克隆了防御系统的些关键基因，并从基因家族分析的角度对它们进行了系统进化研究和表达模式探讨。总之，本研究以高度适应潮间带广温、广盐、缺氧及不稳定环境，且生理生态特殊的长牡蛎为研究对象，从基因组水平和转录组水平上研究潮间带环境适应中关键基因的适应性进化，及其胁迫状态下响应模式。获得的主要结果如下：1.长牡蛎基因组进化与防御相关基因利用长牡蛎的基因组序列图谱与物种进化树上的代表种（人，海胆，果蝇，蜜蜂，牡蛎，水蛭，帽贝，小头虫）以及冠轮动物超门的其他测序物种（水蛭，帽贝，小头虫）的基因组序列进行基因家族收缩和扩张的比较分析，发现长牡蛎中扩张的基因家族其功能富集结果偏向于防御相关，富集到的条目包括“凋亡”、“免疫反应”、“防御反应”和“蛋白泛素化”等，提示长牡蛎基因组在进化中积累了相对较多的防御系统基因资源用于适应复杂多变的潮间带环境。另方面，对不同胁迫状态下的转录组数据分析表明，应激调控基因倾向于为倍增基因，暗示长牡蛎防御相关基因的扩张可能是复制而来。进步对基因倍增的机制进行深入发掘，发现串联复制可能是长牡蛎基因倍增的重要来源，并且在倍增基因形成的早期即发生表达分化。2.长牡蛎防御相关基因本研究利用模式生物相关领域的研究基础和长牡蛎的基因组序列图谱，对长牡蛎的防御系统首次进行了基因组水平上的全面分析，从整体上刻画了长牡蛎的防御系统特征。我们发现长牡蛎的应激防御系统复杂完善，其胞内反应的核心成员热激蛋白HSP70和凋亡抑制蛋白IAP家族发生明显扩张，分别在长牡蛎基因组中有88和48个拷贝。长牡蛎基因组还编码了136个细胞色素氧化酶45（0CYP450）基因，在CYPⅡ亚家族存在显著扩张，推测它们可能在抵抗异生物质过程中具有重要作用。此外，典型免疫识别受体（Toll样受体，含C1q结构域蛋白,纤维蛋白原相关蛋白和C型凝集素）也经历了大规模的基因扩增，结合海胆和文昌鱼基因组中发生的类似情况，我们猜测胚系编码的大规模、多类型的免疫识别受体是不具备获得性免疫的无脊椎动物的主要和特征性免疫识别机制。3.基于RNA-seq的不同胁迫状态转录组分析本研究利用RNA-seq测序技术，对长牡蛎的69个胁迫转录组（梯度温度，盐度，不同重金属、不同诱导时间的不同病原，不同时间干露条件）进行比较分析，筛选得到7,440个应激调控基因。相关基因的功能分类显示，胁迫中上调的基因主要有电子传递链基因，核糖体相关蛋白，物质能量代谢相关基因以及稳定蛋白和DNA的相关基因。此外，本文还从差异基因和模块（功能网络，候选相互作用的基因等）的角度进行了不同应激原诱导的应激调控基因的关联性分析，结果显示同个差异基因或者模块参与不同胁迫条件的调控。胞内应激系统的关键基因如分子伴侣等均在应激状态下大量表达，说明胁迫状态下，胞内应激系统功能全面激活，维持蛋白代谢乃至细胞到生物体的稳态平衡。温度胁迫条件下筛选到表达量下调基因455个，上调基因320个，其中高度诱导的HSP70在35°C高温胁迫12h时上调高达2,000倍，HSP70的扩张和高温下的高度诱导表达可能与长牡蛎适应潮间带正午曝晒时接近49°C的高温的能力有关，这些高度诱导表达的HSP70在系统发育树中聚类为枝，暗示其扩张发生在近期。盐度胁迫筛选到下调基因531个，上调基因492个。渗透物质自由氨基酸（FAA）代谢相关的基因在低盐转录组中显著差异表达，暗示FAA在渗透调控以及低盐胁迫中的重要作用，牡蛎具有高含量的FAA，可能是其适应潮间带降雨或河口所致低盐的重要原因；重金属胁迫筛选到下调基因1,095个，上调基因605个，金属硫蛋白（MT）在镉和汞胁迫下高表达，暗示MT可能是长牡蛎能够耐受高负荷重金属的重要原因；此外，本文还筛选到弧菌诱导差异基因829个，帕金虫诱导差异基因494个，这些基因为研究长牡蛎能够耐受高病原负荷的机制提供了基础资源；最后，干露胁迫条件下筛选到下调基因1,974个，上调基因2,445个。二维聚类结果暗示，闭壳肌和鳃在干露胁迫的应对中有不同的分工，闭壳肌作为糖原的储存场所，在干露的基因应激表达以及调控中发挥更重要作用。IAP在干露条件下高表达，其中高度诱导的5个IAP基因在干露状态闭壳肌中上调66倍，结合IAP抑制凋亡酶的功能机制，我们猜测IAP的扩张及其高度诱导表达可能是长牡蛎适应潮间带长时间露空的重要组学基础。4.长牡蛎TLR信号传导通路TLR信号传导通路是免疫反应中的古老通路和基本成分。本文从cDNA和gDNA水平克隆了CgToll-1基因，并调查了CgToll-1在不同器官和弧菌诱导下的表达模式，为长牡蛎免疫反应研究提供了基础资料。在此基础上，本文寻找了贝类中TLR通路的关键基因，并将之与海葵，线虫，果蝇和海胆在系统发育树，蛋白比对，密码子使用偏向性方面进行了比较分析，结果表明TLR信号通路下游的基因比上游的基因更具有保守性。5.长牡蛎凋亡调控网络凋亡系统在防御系统和生长发育中起到重要作用，但在贝类中研究较少，本文利用长牡蛎EST库和基因组序列对凋亡系统关键基因进行鉴定，发现凋亡系统的主要基因在牡蛎中是保守的，IAP和凋亡酶在长牡蛎基因组中发生了扩张。另外，本研究还克隆了四个凋亡关键基因CgFADD, CgIAP, CgCaspase-1和CgCaspase-2，对上述基因进行了系统进化和诱导表达分析。结果表明，凋亡系统在生物应对胁迫时基因表达模式差异明显，在应激反应中有不可替代的重要作用。更多还原

【Abstract】 The intertidal zone of the sea is the littoral area between the high tide mark andthe low tide mark, which includes many types of inhabits, such as sand beach, rock,salt marsh and estuary. The intertidal zone is important marginal environment of thesea. The Pacific oyster (Crassostrea gigas) is typical species of the intertidal zone,and also important species in studying organism response to environment. Comparedwith the defensome study in the sea urchin and the fly, and based on the C. gigasdatabase including genome sequence, EST database, transcriptomes under variouschallenges, we identified lots of defense-related genes and gave a detail study of theirevolution using bioinformatics method. Their expression patterns were alsoinvestigated. On the other hand, we cloned several key defense-related genes andconducted phylogeny and gene expression study. In all, we conducted genome-leveland trancriptome-level gene-environment study based on the organism C. gigas,which is well represented for their special physiological mechanims in intertidal zoneadaption. The main results are as follows:1. Genome evolution and defense related genes in C. gigasGene family construction and expansion study was carried out with the modelspecies (human, sea urchin, fly, honeybee, oyster, leech, limpet and capitella) andsequenced Lophotrochozoa (oyster, leech, limpet and capitella). Among5,942oyster-specific genes identified by comparing four sequenced Lophotrochozoangenomes, GO terms related to defense, such as ‘metal binding’,‘protein binding andubiquitination’,‘apoptosis and apoptosis regulation’,‘immune response’,‘response tobiotic stimulus’and ‘defense response’, were highly enriched. The enrichment ofthese defense related genes in C. gigas reflects striking adaptation of the oystergenome for survival in a highly stressful environment. On the other hand, duplicationand function divergence after duplication are major reason of genome evolution.Genes differentially expressed in response to stress are more likely to have paralogousduplications, providing evidence for selective retention of duplicated defense-related genes in C. gigas. Our result further suggests tandem duplication may be the majorreason of duplication and the duplication diverged even when the duplicated.2. Defensome of C. gigasWe report the first comprehensive analysis of the oyster defense system based onthe genome sequence of the C. gigas. The annotated oyster gene-set includes a richrepertoire of genes related to defense against biotic and abiotic stress. The oystergenome contains88HSP70genes, which play crucial roles in protecting cells againstheat and other stresses, compared with~14in human and40in sea urchin. Inhibitor ofapoptosis proteins (IAPs), were also expanded in C. gigas (48), indicating a powerfulapoptosis system which may be important in stress response. Expanded familiesinclude multicopper oxidase important in defense against oxidative stress, andcytochrome P450(CYP450) families important in biotransformation of endobiotic andxenobiotic chemicals. Genes encoding lectin-like proteins including C-type lectin(C-lectin), fibrinogen-related proteins (FREP), and globular head C1qdomain-containing proteins (ghC1q) are highly overrepresented in oyster genome;these genes play important roles in innate immune response in invertebrates. Thosedata suggest the vast expansion of recognition receptor may represent a commonpathogenic strategy in invertebrates without the adaptive immune system.3. Transcriptomes under challengesTo investigate genome-wide response to stress, we sequenced69transcriptomesfrom C. gigas subjected to nine stressors including temperature, salinity, six heavymetals, vibrio, perkinsus and air exposure. We found7,440genes differentiallyexpressed under at least one stressor, and genes responding to different stressorsshowed significant overlap. Under most stressors, electron transfer chain, ribosomeproteins, substance&energy metabolism and DNA&protein stabilization wereup-regulated, and those for protein degradation down-regulated, pointing to concertedresponses to maintain cellular homeostasis. Up-regulation of transcripts for proteins inendoplasmic reticulum (e.g. Calreticulin,94kDa glucose-regulated protein, Calnexinand78kDa glucose-regulated protein) suggests that protein quality control is criticalin cellular homeostasis under stress.Heat stress induced455down-regulated genes and320up-regulated genes,importantly,~2000-fold increase in expression of five highly inducible HSP70s or a13.9-fold increase in average expression of all HSP70s, amounting to4.2%of alltranscripts. The genomic expansion and massive up-regulation of HSPs helps explain why C. gigas can tolerate temperatures as high as49oC when exposed to the summersun at the low tide. Phylogenetic analysis showed that inducible HSP70s were close inevolution, which suggesting that they branched relatively recently. Asosmoconformers living in estuarine environments, oysters must maintain cellularhomeostasis under osmotic stress. Low but not high salinity induced a significanttranscriptomic response, which induced531down-regulated genes and492up-regulated genes. The most obvious change is the up-regulation of genesparticipating in free amino acid (FAA) metabolism, suggesting FAA plays a role inosmotic homeostasis. Metal stresses induced1,095down-regulated genes and605up-regulated genes. Metallothioneins, which are important in metal chelation, werehighly up-regulated under challenges by most but not all metals. Besides, weidentified829vibrio induced genes and494perkinsus induced genes, which providedrich information for further studies. Air exposure induced1,974down-regulated genesand2,445up-regulated genes. Clustering showed the function of adductor muscle andgill under challenge was different. Air exposure induced up to67-fold up-regulationof five highly expressed IAPs. This finding along with the expansion of IAPs suggeststhat powerful inhibition of apoptosis is important for oysters’ amazing endurance toair exposure.4. TLR pathway of the Crassostrea gigasToll-like receptor (TLR) signaling pathway was an important and evolutionarilyconserved innate immune pathway. In this study, we cloned a novel TLR, a keycomponent of TLR pathway in cDNA and gDNA level, from Crassostrea gigas, andnamed it CgToll-1. Real-time reverse transcription polymerase chain reaction analysisrevealed that the highest CgToll-1expression level was in hemolymph, and theexpression pattern in hemolymph dramatically increased in the presence of bacteriaVibrio anguillarum. Furthermore, TLR pathway core genes of mollusks were searchedand compared with model invertebrates with the methods phylogenetic trees,alignment identities and codon usage biases. The results suggest downstream genesare more conserved than upstream genes.5. Apoptosis network in the Crassostrea gigasApoptosis system was reported to play important role in organism immunity, but itwas currently understudied in molluscan immunity researches. Base on the recentgeneration of ESTs and genome sequence of the C. gigas, a survey ofapoptosis-related molecules was conducted, we found that the basic genes and domains in apoptosis-associated proteins were conserved, the overall apoptoticmachinery was complex in C. gigas and that the organism had an expanded number ofputative IAPs and caspases. Moreover, four typical apoptosis-related genes werecloned in C. gigas and compared with the sequences of these genes in Drosophilamelanogaster and Homo sapiens. The expression level of major apoptosis-relatedgenes under challenge increased dramatically indicating their role in oyster defense.更多还原