实验海洋生物学重点实验室知识库

血细胞和淋巴器官（Oka）是对虾重要的免疫组织或器官，在对虾先天免疫中发挥着重要作用。尽管已有研究对血细胞在对虾先天免疫中的功能有了一定认识，也探讨了对虾淋巴器官的形态特征和免疫功能。然而，目前对不同类型血细胞在对虾先天免疫中的功能关系尚缺乏系统认识，对对虾淋巴器官的免疫学功能缺乏系统而深入的研究。本论文以凡纳滨对虾为研究对象，建立了不同类型血细胞分离的技术，并通过转录组测序和生物信息学分析，系统探讨了对虾不同类型血细胞之间的功能关系；分析了对虾淋巴器官发挥生物学功能的分子基础，并筛选获得了对虾淋巴器官特异表达的基因，研究了相关基因的功能。研究结果为系统阐释对虾的先天免疫提供了重要基础，也为发展对虾病害防治的有效策略提供了重要参考。论文的主要进展如下：

1. 对虾血细胞和淋巴器官的比较转录组分析：对凡纳滨对虾4个重要免疫组织包括血细胞、淋巴器官（Oka）、肝胰腺和鳃进行了转录组测序和生物信息学分析，通过对在血细胞和淋巴器官中高表达基因（RPKM > 50）进行功能分类，发现在血细胞和Oka中，与内吞作用、酚氧化物酶系统、细胞凋亡等过程相关的基因以及抗菌肽基因呈现高丰度表达；比较上述基因在血细胞和Oka中的表达丰度，发现细胞凋亡、酚氧化物酶系统和部分抗菌肽基因在血细胞中的表达量高于Oka，而内吞作用相关基因和部分抗菌肽如ALF等在Oka中的表达量高于血细胞。进一步比较分析了血细胞和Oka中与其他两种免疫组织鳃和肝胰腺中的差异表达基因，并进行富集分析，发现血细胞与其他三种组织比较，NLR、MAPK、JAK-STAT和Wnt等信号通路相关基因呈现高表达；Oka与其他三种组织比较，在TGF-beta、JAK-STAT、MAPK和Hedgehog等信号通路中的相关基因及胞外基质-受体互作相关基因呈现高表达。以上分析表明，对虾血细胞和Oka在先天免疫中的功能既有相似性，又存在明显差异。
2. 对虾不同类型血细胞的功能关系分析：在建立了利用Percoll密度梯度离心法分离不同类型血细胞的基础上，将凡纳滨对虾的血细胞分为三个类群：颗粒细胞、半颗粒细胞和透明细胞；通过对不同类型血细胞进行转录组测序和生物信息学分析发现，参与吞噬作用，如吞噬受体、细胞骨架重排和溶酶体形成等相关基因在透明细胞中呈现明显的高表达，调节先天免疫信号通路的相关基因在半颗粒细胞呈现高表达，而在颗粒细胞中与酚氧化物酶系统相关基因呈现高表达，提示不同类型的血细胞行使的功能存在差异：透明细胞主要行使细胞吞噬的作用，半颗粒细胞主要参与体液免疫反应，而酚氧化酶系统主要分布在颗粒细胞。利用流式细胞术进一步检测发现，透明细胞摄取微球的能力显著高于半颗粒细胞和颗粒细胞，且通过荧光定量PCR检测发现，吞噬相关受体如甘露糖受体、actin、cathepsin等与吞噬相关基因在透明细胞中的表达水平显著高于其他两种类型的细胞，进一步支持透明细胞是将外源物质吞噬并将其内化的主要细胞类群。利用不同的抑制剂对内吞途径进行抑制时发现，巨胞饮抑制剂IPA-3和NSC23766可以明显抑制透明细胞的吞噬，说明巨胞饮作用途径是透明细胞内化微球的重要途径。

对虾E3泛素连接酶TRIM9基因的鉴定和功能分析：在凡纳滨对虾中鉴定了两个TRIM9基因——LvTRIM9-1和LvTRIM9-2。LvTRIM9-1主要在Oka中表达，而LvTRIM9-2呈现泛表达模式，在肠和神经等组织中表达量较高。在WSSV感染后，LvTRIM9-1和LvTRIM9-2的表达呈现明显不同的表达模式，说明其在WSSV感染中的作用存在差异。利用RNAi技术分别沉默两者的表达，均能抑制WSSV在对虾体内的增殖。酵母双杂交与基因表达调控分析结果显示，LvTRIM9-1能够作用于LvIMD从而正向调控NF-κB途径，而LvTRIM9-2可以作用于Lvβ-TrCP来负向调控NF-κB途径。说明LvTRIM9-1和LvTRIM9-2通过不同的作用方式来调控对虾的NF-κB信号通路，从而在对虾体液免疫中发挥作用。

Hemocytes and lymphoid organ (named as Oka) are key immune tissues or organs in shrimp, and they play essential roles in innate imunity of shrimp. Till now, previous studies focused on function of all hemocytes in innate immunity and the morphological observation of Oka, but systematic knowledge of the immune relationship among different types of hemocytes and immunological function of Oka is very limited. Therefore, based on the separation technique of different types of hemocytes, we conducted transcriptome sequencing and bioinformatics analysis to systematically investigate the immunological function of different types of hemocytes in Pacific white shrimp Litopenaeus vannamei. Moreover, we filtered the specifically expressed genes of Oka using the differential expressed genes (DEGs) based on the transcriptome of different tissues, and further analyzed the immunological functions of these selected genes to systematically uncover the immune roles of Oka in the dissertation. This study provided important basis for systematic interpretation of innate immunity, and essential reference for effective stragy of disease prevention in shrimp.

1. Comparative transcriptome analysis of hemocytes and lymphoid organ in shrimp. Four essential immune tissues of shrimp including hemocytes, lymphoid organ, hepatopancreas and gills were selected to conduct the transcriptome sequence and bioinformatics analysis. We analysed the annotations of genes highly expressed in hemocyte or Oka (RPKM > 50), and found that several immune genes including endocytosis, antibacterial peptides (AMP), phenoloxidase (PO) system and apoptosis were both highly expressed in hemocytes and Oka. Genes related to apoptosis, PO system and some AMP genes in hemocytes showed higher expression levels than those in Oka, while genes related to endocytosis and some ALF genes showed higher expression levels in Oka. Compared with the genes expressed in other immunological tissues like gill, hepatopancreas and Oka, several signaling pathways like NOD-like receptor, MAPK, JAK-STAT and Wnt signaling pathway were found to highly expressed in hemocytes. And several signaling pathways like TGF-beta, JAK-STAT, MAPK, Hedgehog and ECM-receptor interaction pathway were found to highly express in Oka compared to ther immunological tissues like gill, hepatopancreas and hemocytes. Oka and hemocytes showed great similarity and difference in the functions of shrimp innate immunity.
2. Analysis of functional relationship among different types of hemocytes in shrimp. Total hemocytes were successfully separated into three types including hyalinocytes, semi-granulocytes and granulocytes by Percoll gradient centrifugation, and then were conducted transcriptome sequence and bioinformatics analysis. The data showed that different types of hemocytes showed significantly differential expression patterns at transcriptional level. The genes involved in phagocytosis like phagocytic receptors, lectins and actin cytoskeleton were highly expressed in hyalinocytes, while genes involved in the humoral immunity signaling pathways were highly expressed in semi-granulocytes, and genes involved in prophenoloxidase (proPO) system were highly expressed granulocytes. Flow cytometry analysis showed that hyalinocytes possessed higher capacity in phagocytizing the fluorescent beads than semi-granulocytes and granulocytes. Several genes related to phagocytosis including actin, cathepsin and phagocytic receptor like mannose receptorshowed higher expression levels in hyalinocytes via qPCR detection. It segguested that hyalinocytes were the main type of hemocytes responsive for ingesting fluorescent beads. Moreover, after treatment with different endocytic inhibitors, the uptake capability of fluorescent beads by hyalinocytes were significantly suppressed by macropinocytosis inhibitor like IPA-3 and NSC23766, suggesting that their ingestion process mainly depends on the endocytic way of macropinocytosis.

Identification and functional study of E3 ubiquitin ligase TRIM9 genes in shrimp. Two TRIM9 genes (designated as LvTRIM9-1 and LvTRIM9-2) were identified from the transcriptome of the Pacific whiteleg shrimp. The transcript of LvTRIM9-1 was specially detected in Oka, while the transcript of LvTRIM9-2 was detected in most tissues of the shrimp, with relatively higher levels in intestines and nerves. The expression levels of LvTRIM9-1 and LvTRIM9-2 were different post white spot syndrome virus (WSSV) infection, indicating that they played distinct roles in WSSV infection. Knockdown of two LvTRIM9 genes’ expressions by double-strand RNA mediated interference could both lead to a decrease of virus copy number in WSSV-infected shrimp. Yeast two-hybrid analysis and gene regulation analysis showed that LvTRIM9-1 could positively regulate NF-κB pathway via directly interact with immune deficiency (LvIMD), and LvTRIM9-2 could negatively regulate NF-κB pathway via directly interact with beta-transducin repeat-containing protein of shrimp (Lvβ-TrCP). These data indicated that LvTRIM9-1 and LvTRIM9-2 affected humoral immunity in different ways via distinct regulation on NF-κB pathway in shrimp.