Mechanism Of Albinism In Sea Cucumber Apostichous Japonicus And Characterization Of Body Color Separation Albino Offspring Based On High Throughput Sequencing

Color mutation is one of the most significant characteristics in sea cucumbersApostichopus japonicus. In general, the body colors of A. japonicus farmed along theChinese coasts are dorsally tawny and ventrally fawn. Sea cucumbers with an entirelywhite body are found rarely and regarded as precious. The successful cultivation ofexcellent albino line must have broad market prospect. The color of offspring fromalbino cross was separated in body color. Those albino juvenile progenies were underpoor growth compared with the corresponding control, and the number was found todecrease. Lack of genomic information in A. japonicus caused few reports onmechanism of albinism and differences of gene expression in this sea cucumber.Hence, this research investigated the mechanism of albinism and differences of geneexpression in the albino offspring with the help of high throughput sequencingtechnologies. The research findings will support scientific basis for genetic breedingof albino sea cucmbers and the theoretical basis for cultivating the excellent albinoline. The main results were as follows:1. Using the454GS-FLX platform, there were27357and57119unigenesassembled in the albino and control transcriptome libraries, of which6539and5822were annotated, including3983and2918GO annotated, respectively. The6162of6539unigenes were matched to the top15species. The two top-ranked matchingspecies were S. purpuratus and A. japonicus. In the GO terms of “Reproduction” and“Cellular component biogenesis” and “Pigmentation”, there were notably different inthe albino library compared with those in the control library. Melanogenesispathway-related analysis showed that the enhancement of HGO transcriptional activity made tyrosine as the substrate of tyrosinase insufficient for synthesizingmelanin in the albino. Significantly downregulated genes in signalling pathwaysassociated with melanin synthesis, i.e., Ras, PKA and PKC, were found in the albinolibrary. Their expression was inhibited so that the signal transmission for synthesizingmelanin was blocked in the albino sea cucumbers. Further, the melanin productionwas decreased.2. Using RNA-Seq, there were837DEGs (475upregulated and362downregulated)in the ALB library compared with the CON library. A total of71DEGs (22upregulated and49downregulated) changed5-fold in expression magnitude. Therewere nine upregulated DEGs annotated, major of which involve in immunologicaldefense. As to the forty-nine downregulated DEGs, there were twenty-one underannotation. Among them, five were thought to involve in growth and development.There were583DEGs (361upregulated and222downregulated) in the SEP librarycompared with the CON library. There were23upregulated genes and29downregulated genes with5-fold differences. The twelve annotated DEGs with5-fold upregulation were mainly related to immunological defense. A total of sixteenof the29downregulated genes with5-fold changes were annotated. These majorannotated DEGs were relevant to signal transduction and growth and development.The expression levels of all DEGs in ALB and SEP libraries were checked byreal-time PCR and the results were in full accord with with the RNA-Seq expressiontrends. There were six significantly enriched pathways in relation to upregulatedDEGs, involving in phagocytosis, immune response, pathological reaction, andapoptosis and cytokines were the only notably enriched pathway for thedownregulated DEGs in the albino juvenile sea cucumbers. There were only twopathways significantly enriched by the upregulated DEGs, involving in phagocytosisand pathogen infection, and ECM-receptor interaction and focal adhesion were theonly significantly affected pathway for the downregulated DEGs in thealbino-separative samples (Q0.05).3. There were30522fewer unique reads in the separation (SEP) library comparedwith the albino (ALB) library, which possibly represented differences of the physiological status of the juvenile progenies of albino sea cucumbers. There were272DEGs (161upregulated and111downregulated) in the SEP library comparedwith the ALB library. A total of31upregulated genes and11downregulated genespresented5-fold differences. The twelve annotated DEGs with5-fold upregulationwere mainly related to growth and development and transport. Six downregulatedgenes with5-fold changes were annotated. These major annotated DEGs werecomponents of mitochondrial respiratory chain. There were two pathwayssignificantly enriched by the upregulated DEGs, which involved in Staphylococcusaureus infection and complement and coagulation cascades. Phagocytosis was theonly significantly affected pathway for the downregulated DEGs (Q0.05).4. The cDNAfull length of AjCREB gene was2503bp, including277bp in5’UTR,1262bp in3’ UTR, and963bp in open reading frame. This gene encoded a proteincontaining320amino acid residues. In the amino acid sequence deduced fromAjCREB cDNA sequence, the sequence fragment from Gln220to Ala269was aconservative BRLZ domain (basic region leucin zipper), which was a typicalalpha-helical structure. The CREB homology between A. japonicus and other specieswas lower. The highest consistency of AjCREB was Homo sapiens’(I=16.1%), whilethe CREB consistency between A. japonicusi and S. purpuratus was10.7%. In aphylogenetic constructed tree, AjCREB was alone together in one branch, and in thisbranch, the CREBs of S. purpuratus and S. kowalevskii were clustered together.These results will provide candidate genes for studying cAMP pathway ofmelanin synthesis in A. japonicus, effect of external environment on growth anddevelopment, and immunocompetence of albino sea cucumbers. Additionally, theywill lay substantial theoretical basis for cultivating stably genetic and well-grownalbino lines of sea cucumbers.