摘要
体色变异大是仿刺参的特征之一,黄褐色是最常见的体色,而通体白色的刺参则十分罕见,被誉为参中“极品”,培育优良的白刺参品系必然有广阔的市场前景。在白刺参人工繁育过程中子代出现体色分离和生长存活差异的现象。刺参基因组序列的缺乏导致关于刺参白化发生机理和白参体色分离子代基因表达差异的研究报道较少。本文借助高通量测序技术,研究刺参白化发生的机制和白参子代的基因表达差异,为白刺参的遗传育种提供科学依据,为培育优良的白刺参品系提供理论支持。主要研究结果如下:
1.在白刺参和青刺参体壁和肠道构建的转录组库中,分别利用454测序平台得到27357个和57119个Unigene,被注释的分别有6539个和5822个,通过GO注释的分别有3983个和2918个。6162个Unigene匹配到15个物种,其中匹配到紫海胆和仿刺参的数量最多。GO分析发现白刺参和青刺参在“色素沉积”、“繁育”、“细胞组分生物合成”方面差异较大。黑色素合成信号通路分析发现在白刺参中参与酪氨酸代谢的HGO基因显著上调,使黑色素合成底物酪氨酸减少,从而使黑色素的合成减少。而与黑色素合成相偶联的MAPK信号通路、Wnt信号通路、Jak-STAT信号通路以及黑色素合成过程中都有显著下调的基因,如Ras基因、PKA基因、PKC基因等,这些基因表达受到抑制,使黑色素合成的信号传递过程在白刺参中传递受阻,从而使黑色素的合成减少。
2.与对照青刺参相比,在白色幼参体壁中有476个上调基因和362个下调基因,其中表达量改变5倍以上有71个的(上调22个,下调49个)。上调5倍以上的注释基因有9个,主要参与免疫防御;下调5倍以上的注释基因有21个,主要参与生长发育和代谢过程。与对照青刺参相比,在体色分离幼参体壁中有362个上调基因和222个下调基因,其中52个基因的表达量改变5倍以上(上调23个,下调29个)。上调5倍以上的注释基因有12个,主要参与免疫防御;下调5倍以上的注释基因有16个,主要参与信号转导和生长发育。RT-PCR验证的差异基因表达趋势与RNA-Seq结果一致。信号通路富集分析发现在白色幼参中显著富集上调基因的信号通路有6条(Q value <0.05),分别参与吞噬作用、免疫应答、病理反应及凋亡;而显著富集下调基因的信号通路有1条,参与细胞因子的相互作用。而在体色分离幼参中显著富集上调基因的信号通路有2条(Q value<0.05),分别参与吞噬作用和抗原感染;显著富集下调基因的信号通路也有2条,分别参与ECM受体相互作用和焦点粘连作用。
3.体色分离幼参的unique reads比白色幼参少30522条,这些缺失的unique reads可能会导致白刺参F1代幼参生理状态的差异。相对于同批次白色幼参,在白刺参F1代体色分离幼参体壁中有161个上调基因和111个下调基因。较ALB库,在SEP库中共有42个基因的表达量改变5倍以上,上调的有31个,下调的有11个。上调5倍以上的注释基因有12个,主要与生长发育和转运相关;下调5倍以上的注释基因有6个,主要是线粒体呼吸链组分。相对于白色幼参,在体色分离幼参中有两条显著富集上调基因的信号通路,它们分别与金黄色葡萄球菌感染和补体和凝结级联反应相关。有一条显著富集下调基因的信号通路,它参与吞噬作用。
4. AjCREB基因cDNA全长为2503bp,其中5’UTR为277bp,3’UTR为1262bp,开放阅读框为963bp,可以编码含320个氨基酸残基的蛋白。在AjCREB cDNA序列推导的氨基酸序列中,Gln220至Ala269的氨基酸序列是非常保守的具有典型的α螺旋结构的BRLZ结构域。AjCREB与其他物种的同源性较低,与其一致性最高是Homo sapiens(I=16.1%),与其进化地位接近的Strongylocentrotuspurpuratus的一致性仅为10.7%。AjCREB单独聚在在构建的进化树的一支中,而在这一支中S. purpuratus与S. kowalevskii聚在一起。
这些研究成果将为深入开展刺参cAMP黑色素合成调控通路、外界环境对白刺参生长发育影响和白刺参免疫能力的研究提供候选基因,为可以稳定遗传的生长良好的白刺参品系的培育打下坚实的理论基础。
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.
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