实验海洋生物学重点实验室知识库

    对虾是对虾科动物的统称，属于甲壳动物亚门十足目，是水生动物的重要代表类群，具有不可替代的科学价值。同时，对虾又作为世界重要的水产养殖对象，具有重要的经济价值。对虾基因组的破译和大量组学数据的积累，不仅对阐明对虾特殊的生命现象、解析对虾的适应性机制、研究对虾乃至甲壳动物的演化历史和生态地位具有重要意义，而且可以为以对虾代表的经济甲壳动物的遗传育种奠定重要的基础。本论文以凡纳滨对虾（Litopenaeus vannamei）的组学数据为基础，系统研究了对虾基因组中三个基因家族的扩张和基因可变剪切与对虾环境适应的关系。本研究不仅为阐明对虾等甲壳动物对环境适应的分子机制奠定重要基础，而且对对虾养殖环境和生态调控具有一定指导意义。主要研究结果如下：
1. 凡纳滨对虾肌动蛋白（Actin）和肌球蛋白重链（Myosin heavy chain，MYH）基因家族的结构和功能研究
    凡纳滨对虾具有发达的腹部肌肉系统，使其能够迅速躲避敌害袭击，适应底栖生活，而Actin和MYH是两种主要的肌肉组成蛋白。本论文结合基因组和转录组数据，从凡纳滨对虾基因组中鉴定出37个Actin基因和16个MYH基因。综合序列结构、功能域和系统发生分析的结果，将它们分为4类：快肌型、慢肌型、心肌型和细胞质型。相比于其他近缘物种，快肌型Actin、慢肌型Actin和快肌型MYH基因在对虾基因组中均发生了显著扩张。这些基因家族成员之间高度的序列相似性和在基因组上成簇分布的特征表明它们可能是通过串联复制实现扩张的。不同类型的对虾Actin和MYH基因在时空表达上存在显著差异。在不同组织中的表达分析结果表明，快肌型Actin和MYH基因主要在腹部肌肉中高表达，慢肌型Actin和MYH基因主要在包含肌肉成分的组织中表达，心肌型Actin和MYH基因在心脏中高表达，而细胞质型Actin和MYH基因在所有组织中均表达。大多数Actin和MYH基因在胚胎发育的肢芽期至膜内无节幼体期开始转录表达，并随着幼体的发育和运动能力的加强，表达量逐渐提高，显示对虾肌肉系统主要在溞状幼体期和糠虾幼体期开始发育。通过对慢肌型Actin和MYH基因的原位杂交，证实对虾腹部的表层肌肉和游泳足内的肌肉属于慢肌型。以上结果表明，对虾基因组中显著扩张的快肌型Actin和MYH可能是对虾腹部肌肉发达的原因之一，从而使其拥有很强的爆发力；显著扩张的慢肌型Actin使对虾的游泳足可以持久游泳。这两者的配合使对虾既具有快速逃逸躲避敌害的能力，又具有比其他甲壳动物更强的游泳能力。
2. 凡纳滨对虾保幼激素酯酶样羧酸酯酶（CXE）基因家族的结构和功能多样性分析
    对虾一生要经历约50次左右蜕皮，如此频繁的蜕皮为对虾快速生长提供了保障。昆虫的保幼激素酯酶（juvenile hormone esterase，JHE）在早期发育和蜕皮过程中起重要作用，甲壳动物中的保幼激素酯酶样羧酸酯酶（JHE-like carboxylesterase，CXE）与JHE基因同源，但是在对虾复杂的蜕皮通路中，CXE基因的功能尚不清楚。通过对对虾基因组和转录组数据进行分析，本研究鉴定出21条CXE基因（LvCXE），为对虾基因组中发生显著扩张的基因家族之一。所有LvCXE均有保守的催化三联体位点和特征性的GxSxG结构域，其中有14个基因的特征性结构域为GESAG，并且13个含GESAG的LvCXE在系统发育树中聚为一支，表明含GESAG结构域的CXE基因在对虾中发生特异性扩张。大多数LvCXE在对虾的肝胰腺、肠道和卵巢中高表达，其组织分布特征与甲基法尼酯（MF）降解的主要部位相吻合，暗示它们之间可能存在功能关系。从对虾不同的发育时期来看，有10个LvCXE在溞状幼体期至仔虾期高表达，其中包含7个含GESAG结构域的基因。在对虾的不同蜕皮时期，有16个LvCXE（包含11个含GESAG结构域的基因）的表达模式相似：在蜕皮间期和D0期高表达，D3期表达量降到最低，D4期表达量又急剧升高。在对虾蜕皮前期注射昆虫CXE的抑制剂OTFP后，可导致对虾不能完成蜕皮过程而死亡，且OTFP的注射可引起蜕皮激素应答关键转录因子基因如LvE75、LvBr-c、LvHr3、LvFtz-f1等的表达量显著下调，说明LvCXE在对虾蜕皮过程中发挥着重要作用。以上结果表明对虾中特异性扩张的LvCXE具有功能多样性，在幼体发育和生长蜕皮过程中均起重要的调节作用。
3. 凡纳滨对虾全基因组范围可变剪切事件及功能多样性研究
    可变剪切是调节基因表达和产生蛋白质组多样性的主要方式之一，然而对虾中可变剪切相关研究的报道很少。结合对虾基因组和多个转录组数据，本研究在对虾中共鉴定到9209个基因存在38,781个可变剪切事件，其中外显子跳跃（ES）、3’端可变剪切（A3SS）、5’端可变剪切（A5SS）、内含子保留（RI）和外显子互斥（MXE）的比例分别为13.35%、11.43%、10.8%、6.77%、2.36%。所有基因的内含子边界剪切模式以GT-AG模式最多，占92.3%，其次是GC-AG，AT-TC，AT-AC。与非可变剪切基因相比，可变剪切基因的平均长度更长、外显子数目更多、外显子长度更长、内含子更短。非可变剪切基因的功能主要包括：DNA整合、DNA代谢、核酸代谢、神经系统等生物学过程，而多可变剪切基因的功能主要包括响应生物胁迫和响应外界刺激等相关过程。根据剪切形式增加趋势的差异，可变剪切基因可分为type Ⅰ基因和type Ⅱ基因。type Ⅰ基因是指只在胁迫后发生可变剪切的基因，即这类基因在正常状态下只有一种转录本，但是胁迫后转录多种形式的转录本；type Ⅱ基因是指正常状态下已经可以转录多种可变剪切转录本，但是一些特定剪切形式的转录本只在胁迫状态后转录。微生物（白斑综合症病毒WSSV、副溶血弧菌、金黄色葡萄球菌）感染后，对虾全基因组可变剪切事件的总数目显著增多，而且各种类型的可变剪切的数目均大幅度增加。其中type Ⅰ基因的功能相似，主要与物质和能量代谢相关，起到维持基础代谢平衡、确保物质和能量供应的作用；type Ⅱ基因的功能主要和免疫相关，但针对不同病原有所差异：WSSV组主要富集在NLR受体通路；副溶血弧菌组主要富集到细胞外基质受体相互作用和产生IgA的肠道免疫网络通路；金黄色葡萄球菌组显著富集到“志贺氏菌病”、“致病性大肠杆菌感染”、“上皮细胞的细菌感染”等抵御病原的通路。该结果表明对虾在受到微生物感染后，通过改变免疫相关基因的可变剪切模式达到清除不同病原的目的。同时有7个剪切因子（splicing factor）基因的表达量在微生物感染后显著上调，表明可变剪切在微生物感染过程中的调控有一定的相似性。可变剪切在物理因子胁迫过程中也发挥着重要作用。对虾全基因组的可变剪切事件的数量在低盐胁迫后显著增加，type Ⅰ基因主要富集到嘌呤代谢和过氧化物酶体等过程，type Ⅱ基因主要和阳离子结合、离子结合、甜菜碱合成等过程相关，维持机体内外的渗透压平衡。另外，高温胁迫促进了ES、A3SS、A5SS的特异性可变剪切而抑制了RI的特异性可变剪切；其中鳃中与RNA结合、结合、核酸结合等过程相关基因的可变剪切模式的变化可能是下游大规模的可变剪切调节或转录表达调控的基础；肝胰腺中与泛素转移酶活性、核内泛素连接酶复合物过程相关基因的可变剪切模式的变化，可能与机体内错误折叠蛋白的清除过程相关。以上结果表明可变剪切在凡纳滨对虾响应外界环境胁迫过程中起着重要作用。
    对虾基因组中基因家族的扩张和可变剪切形式的增加丰富了基因组成和表达形式的多样性，进而促进了基因功能多样性的发挥。Actin和MYH基因家族的显著扩张促进了对虾快肌和慢肌的进化，提升了对虾迅速躲避敌害袭击的能力；CXE基因家族的扩张则为对虾的频繁蜕皮提供精确的调控保障；对虾中丰富的可变剪切事件在增加了转录本和蛋白质的结构与功能多样性的同时，也使得对虾获得较强的环境适应能力，在应对生物因子和物理因子刺激时，体现出了独特的响应和适应机制。以上结果表明，基因扩张和可变剪切均是对虾适应环境的重要途径。

    Penaeid shrimp, belonging to Decapoda, Crustacea. They are representative species of aquatic animals with irreplaceable scientific value. Furthermore, as important aquaculture species in the world, shrimp presents great economic value. Deciphering the shrimp genome and accumulation of a large number of “omics” data are of great significance to clarify the special life phenomenon, illustrate the mechanism of evolutionary adaptation, analyze the evolutionary history and ecological position of shrimp, even to crustacean. What’s more, it plays an important foundation for the genetic breeding of economic crustacean represented by shrimp. In this paper, the relationships between the expansion of three gene families and alternative splicing events of the whole genome and the environmental adaptation were systematically analyzed based on the genomic data and transcriptomic data of L. vannamei. The researches will not only lay an important foundation for clarifying the molecular mechanism of environmental adaptation of shrimp and other crustaceans, but also have some guiding significance for the regulation of culture environment and ecology of shrimp. The major results are as follows:
1.    Studies on the structure and function of Actin and Myosin heavy chain (MYH) gene families in L. vannamei.
    In order to adapt to benthic life, the well-developed abdominal muscle system enables shrimp to escape from the predators quickly. Actin and MYH are both major muscle constituent proteins. Combined with genomic and transcriptomic data, 37 Actin and 16 MYH genes were characterized in shrimp. Based on the sequence structure, functional domain and phylogenetic tree, they could be classified into four types: fast muscle type, slow muscle type, heart type and cytoplasmic type. Compared with other related species, fast type Actin, slow type Actin and fast type MYH were significantly expanded in shrimp genome. The feature of high sequence similarity and clustered distribution in the genome indicated that they might expand through tandem replication. The temporal and spatial expressions of different types of Actins and MYHs were significantly different in shrimp. The fast type Actin and MYH were highly expressed in the abdominal muscles, the slow type Actin and MYH were highly expressed in muscle-containing tissues, the heart type Actin and MYH were highly expressed in heart, and the cytoplasmic type Actin and MYH were highly expressed in all detected tissues. Most Actins and MYHs began to be expressed from limb bud stage to nauplii in membrane stage, and the expression increased gradually with the development of larvae and the enhancement of motor ability. These results suggested that shrimp muscle system mainly developed at zoea and mysis stage. Through in situ hybridization analysis, we found that slow type LvActinSSK5 and LvMYH5 genes were both mainly expressed in the superficial ventral muscle and pleopod muscles, which are regarded as slow muscle. Therefore, the expanded fast-type Actin and MYH may explain the well-developed muscle system of shrimp, which makes them have excellent explosive power. The expanded slow-type Actin make them have long-term swimming ability. The cooperation of explosive power and swimming ability enable the shrimp to escape from predators quickly.
2.    Analysis on the structure and function diversity of juvenile hormone esterase-like carboxylesterase (CXE) gene family in L. vannamei.
    Shrimp usually experiences about 50 molts during their lifetime. Such frequent molting provides a guarantee for rapid growth of shrimp. Juvenile hormone esterase (JHE) plays an important role in the early development and molting process in insects. CXE gene in crustacean is regarded as a homologue with JHE gene, but its function in the complex molting pathway of shrimp is unclear. Based on genomic and transcriptomic data, 21 LvCXEs were characterized in L. vannamei, which was one of significantly expanded gene families in shrimp. All LvCXEs have a conserved triplet catalytic site and a characteristic GxSxG motif. Among them, 13 of 14 LvCXEs containing a GESAG motif were clustered in the phylogenetic tree, indicating that they were specifically expanded in shrimp. Most of LvCXEs were highly expressed in hepatopancreas, intestine and ovary, which were the major sites for methyl farnesyl (MF) degradation. It suggested the potential functional relationships between LvCXE and MF. Among them, 10 LvCXEs, including 7 genes containing GESAG motif, were highly expressed from zoeae stage to post-larval stage. Totally 16 LvCXEs, including 11 genes containing GESAG motif, presented molt-dependent expressions, with the lowest expression at pre-molt D3 stage during the molt cycle. When injecting OTFP, a specific inhibitor of JHE in insects, at the pre-molt stage of shrimp, they failed to molt and died finally. Furthermore, the expression levels of four key ecdysone responsive transcription factors, including LvE75, LvBr-c, LvHr3 and LvFtz-f1, were down-regulated after OTFP injection. These results suggested that the important role of LvCXEs in shrimp molting process. In conclusion, the functional diversity of expanded LvCXEs plays vital roles in the embryo development and molting process of shrimp.
3.    Studies on alternative splicing events and functional diversity in the whole genome of L. vannamei.
    Alternative splicing is one of the main ways to regulate gene expression and generate proteome diversity. However, research on alternative splicing of shrimp is very limited. Combined with genome and multiple transcriptome libraries, 38,781 alternative splicing events of 9209 genes were identified in shrimp. The ratio of exon skipping (ES), alternative 3’ splice sites (A3SS), alternative 5’ splice site (A5SS), intron retention (RI) and mutually exclusive exon (MXE) was 13.35%, 11.43%, 10.8%, 6.77%, 2.36%, respectively. Among all transcripts, 92.93% used the canonical GT-AG dinucleotides at the 5’ and 3’ sites, followed by GC-AG and AT-TC. Compared with non-AS genes, AS genes were longer and contained more and shorter exons and longer introns. The functions of non-AS genes were mainly related to DNA integration, DNA metabolic process, nucleic acid metabolic process and neurological system process, while multi-AS genes were mainly responsive to biotic and environmental stress. According to the difference in the increasing trend of the splicing form, AS genes can be divided into type Ⅰ genes and type Ⅱ genes. Type Ⅰ genes possess only one transcript in the control group but they are alternatively spliced in the experimental group of stressed libraries. Type Ⅱ genes possess specific AS events in the experimental group under stressed conditions compared with the control group. After injection of white spot syndrome virus (WSSV), Vibrio parahaemolyticus and Staphylococcus aureus, the total number of AS events in the whole genome increased significantly, and the number of various types of AS increased overall. The function of type Ⅰ genes was similar and mainly related to substance metabolism or energy metabolism, which plays a role in maintaining the basic metabolic balance and ensuring the material and energy supply. The function of type Ⅱ genes was mainly related to immunology, but it was different for different pathogens. In detail, the function of type Ⅱ genes was related to NOD-like receptor signaling pathway in WSSV group, ECM receptor interaction and intestinal immune network for IgA production pathway in V. parahaemolyticus group, ECM receptor interaction, shigellosis, pathogenic Escherichia coli infection, bacterial invasion of epithelial cells pathway in S. aureus group. These results showed that the pathogen could be eliminated by changing the AS pattern of immune related genes when shrimp was infected by microorganisms. What’s more, the expression levels of seven splicing factors were up-regulated in above three groups, suggesting some similarity of AS regulation during microbial challenge. Additionally, AS also plays an important role under the stress of physical environmental factors in shrimp. The total number of AS events also increased significantly under low salinity stress. Type Ⅰ genes were mainly enriched in peroxisome and purine metabolism process, and type Ⅱ genes were mainly related to cation binding, ion binding and betalain biosynthesis process. These processes could maintain the balance of osmotic pressure in shrimp. What’s more, heat stress induced the specific AS events of ES, A3SS and A5SS, but repressed RI. The AS patterns of genes related to RNA binding, binding and nucleic acid binding were changed to amplify the downstream regulation of AS and transcription in gill under heat stress. The AS patterns of genes related to ubiquitin-protein transferase activity and nuclear ubiquitin ligase complex process were changed to remove misfolded proteins in hepatopancreas. The above results indicated that AS plays a crucial role in response to external environmental stress in shrimp.
In shrimp genome, the expansion of gene families and the increase of AS events enrich the diversity of gene composition and expression, and then promote the functional diversity of genes. The significant expansion of Actin and MYH gene families promoted the evolution of fast type and slow type muscles, and enhanced the ability of shrimp to escape from predators quickly. The significant expansion of CXE gene family provided precise regulations for frequent molting in shrimp. The abundant AS events not only increase the structural and functional diversity of transcripts and proteins, but also enable shrimp to acquire strong environmental adaptability. It reflects a unique response and adaptation mechanism in shrimp under the stimulation of biological factors or physical environmental factors. In conclusion, gene expansion and AS are both important ways for shrimp to adapt to the environment.