| 摘要 | 深海热液、冷泉化能环境中黑暗、高压，富含硫化氢、甲烷及重金属等物质，生物对此极端环境的适应机制是国际研究的热点。眼睛是重要的视觉器官，已有研究发现其在深海动物中产生了多种多样的变化。长角阿尔文虾是少数在深海热液、冷泉环境中均有分布的优势甲壳动物之一，仍然保留着眼睛结构，并呈现出白色眼睛和橙色眼睛两种表型，成为研究深海化能极端环境中大型动物视觉器官适应性进化的良好材料。本研究首先对生活于深海化能环境中的长角阿尔文虾（Alvinocaris longirostris）和浅海种脊尾白虾（Paraemon carinicauda）的眼组织进行了转录组测定和比较。在长角阿尔文虾和脊尾白虾中分别组装得到了64,352和46,709个 unigenes，其中 21,922（34.07％）和 16,951（36.29％）经 NR、KOG、GO和 KEGG 等数据库得到注释，GC 含量分别为 39％和 40％。分析 20 种氨基酸所占比例显示两物种中有两个氨基酸之间的差异大于 0.30％，显示深海长角阿尔文虾中带正电荷的赖氨酸较多，非极性氨基酸脯氨酸较少；在密码子使
用偏好性方面，ΔRSCU 值介于 0 到 0.56 之间，有八个密码子显示出强烈的负向或正向密码子使用偏好性，这些特点可能与其对深海环境的适应有关。进一步分析发现两物种中都存在较为完整且保守的视网膜决定基因网络，
均鉴定出眼睛发育过程中必不可少的转录因子：两个 Pax6 同源基因，eyeless 和twin of eyeless，提示两种虾眼睛的早期幼体发育阶段可能是相似的。然而，与浅海脊尾白虾相比，由于适应深海暗环境，长角阿尔文虾的光转导信号通路中相关组分的数量显著减少，且表达水平显著降低。特别在深海长角阿尔文虾眼睛中不存在短波/紫外线敏感的视蛋白（SWS / UVS）的表达，并且从该物种转
录组中仅鉴定出一种中波敏感视蛋白（MWS）。 对关键长波敏感视蛋白（LWS）的比较分析发现，相较浅海类群，深海虾类的氨基酸序列结构中没有发现特异的氨基酸突变位点，提示这些深海甲壳动物中的关键视蛋白仍保留其感光和信号转导功能。系统发育分析还表明节肢动物视蛋白可能发生了基因复制事件。 颜色是重要的表型特，发挥多种适应功能。前期研究在长角阿尔文虾中发现存在白眼（AW）和橙眼（AO）两种表型。通过比较转录组分析，在 AW深海化能极端环境中长角阿尔文虾眼睛适应性的转录组学解析和 AO 之间鉴定到 1491 个差异表达基因（DEG）。其中，许多差异表达基因功能与免疫，抗氧化和解毒有关。色素的生物合成途径中涉及的两个重要的酶编码基因黄嘌呤脱氢酶（xanthine dehydrogenase）和色氨酸氧化酶（tryptophanoxidase）分别在 AW 和 AO 中上调，这可能直接与白眼和橙眼表型的差异有关。此外，单核苷酸多态性（SNP）检测到分布在 14 个 unigenes 中的 28 个 SNP 的基因型在AW 和AO间完全不同。其中免疫相关基因crustin Pm5和antimicrobial peptide 中分别存在三个和两个非同义突变。以上结果提示长角阿尔文虾眼睛颜色的差异可能是由于阿尔文虾在扩散过程中遇到不同的微环境（如微生物，病原体和有毒物质）引起的免疫反应，并在长期的适应过程中固定下来。综上，本研究对深海化能环境中的阿尔文虾转录组的测定、对眼睛形成和光转导分子组分的解析，以及对其不同眼睛表型形成的分子基础的阐释提供了对深海化能极端环境下特殊生命过程的新认知，丰富了深海生物组学数据资源。为进一步阐明深海化能环境中大型甲壳动物眼睛的发育机制及其应对暗光等极端环境的适应性进化机制奠定了基础。
The deep-sea cold seep and hydrothermal vent are unique chemoautotrophic ecosystems with special physical and chemical properties: high static pressure, low oxygen concentration, enrichment of heavy metals, and accumulation of chemical toxins. The adaptative mechanism of organisms to this extreme environment is a hot spot in international research. As an important visual organ, eye has been found to have a variety of changes in deep-sea animals. The shrimp Alvinocaris longirostris is one of the few dominant crustaceans distributed in both deep-sea hydrothermal vent and cold seep environments. The eye structure of this shrimp is still preserved, and it exhibits two phenotypes, white eyes and orange eyes. Thereore, eye of A. longirostris has become a good material for studying the adaptive evolution of visual organs of large animals in the extreme environment of deep-sea chemosynthetic ecosystem. In this study, transcriptome of A. longirostris eyes from deep-sea chemosynthetic environment was sequenced and compared with a newly sequenced transcriptome of the shallow-water shrimp Palaemon carinicauda. In total, 64,352 and 46,709 unigenes were assembled from A. longirostris andP . carinicauda, respectively, of which 21,922 (34.07%) and 16,951 (36.29%) were annotated with databases such as NR, KOG, GO, and KEGG. The GC contents of the transcriptomes were 39% and 40% in A. longirostris and P . carinicauda. Analysis of the proportion of 20 amino acids showed that the difference of two amino acids between the two species was greater than 0.30%, indicating that there were more positively charged lysine and less non-polar amino acid proline in deep-sea A. longirostris. In terms of codon usage preference in A. longirostris, ΔRSCU values ranged from 0 to 0.56, and eight codons showed strong negative or positive codon usage preference, which may be related to their adaptation to deep-sea environment. Further analysis revealed that a relatively complete and conserved retinal determination gene network existed in A. longirostris and P . carinicauda. The essential transcription factors during eye development were identified in both species, including two Pax6 homologs genes, eyeless and twin of eyeless, which suggests that the development of the two shrimp eyes at the early embryo-larvae stages might be similar. However, the number of phototransduction components was significantly reduced in A. longirostris in comparison with the shallow water shrimp, as well as the expression level due to adaptation to the dark environment of deep sea. Particularly, photoreceptor hortwavelength/UV-sensitive (SWS/UVS) opsin was absent in the deep-sea A. longirostris, and only one middle-wavelength-sensitive (MWS) opsin was identified in this species. A comparision of key long-wavelength-sensitive (LWS) opsins between deep-sea and shallow-water shrimp groups showed that no specific amino acid mutations were detected in the sequence of deep-sea shrimps, suggesting that key opsins in these deep-sea crustaceans still conserve the light sensing and signal transduction functions. Phylogenetic analyses also revealed that gene replication events may occur in the evolution of of arthropoda opsins. Coloration is an important phenotypic trait which exerts multiple adaptive functions. Earlier studies found that there are white-eye (AW) and orange-eye (AO) phenotypes in the shrimp A. longirostris. By comparative transcriptome analyses, 1491 differentially expressed genes (DEGs) were identified between AW and AO. Among them, many DEGs were associated with immunity, antioxidation, and detoxification. Two significant enzyme encoding genes, xanthine dehydrogenase and tryptophan oxidase involved in pigment biosynthesis pathways were up-regulated in AW and AO, respectively, which might be related to the differences of white- and orange- eye phenotypes. Moreover, single nucleotide polymorphism (SNP) calling detected that genotypes of 28 SNP distributing in 14 unigenes were completely different between AW and AO. There were three and two non-synonymous mutations in immune genes crustin Pm5 and antimicrobial peptide, respectively. The above results indicate that the difference of the eye color of Alvinocaridid shrimps is probably resulted from immune response caused by different microenvironments (such as microbes, pathogens and toxic substances) during the diffusion process, and finally fixed during long-term daptation. To sum up, this study provided the eye transcriptome of Alvinocaridid shrimps in deep-sea chemosynthetic environments, identified key component involved in eye development and phototransduction, and primarily explained the molecular basis for the formation of different eye phenotypes in A. longirostris. This study provides new insights into the special life processes in the extreme environment of deep-sea chemosynthetic ecosystem and greatly enriches the gene resources of deep-sea animals. Specifically, the study lays the foundation for further elucidating the developmental mechanism of the eyes of large crustaceans in deep-sea chemosynthetic environments and the adaptive evolutionary mechanism during their coping with extreme environments such as dark light. |
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