Genes, Their Evolution And Expression Regulation Of The Defense System In The Crassostrea Gigas

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.