A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning, tissue localization and expression profile after challenge

Transglutaminase can catalyze the cross-linking reaction between soluble clotting protein molecules from the plasma for prevention of excess blood loss from a wound and obstructing micro-organisms from invading the wound in crustaceans. A novel transglutaminase (FcTG) gene was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 2972 bp, encoding 757 amino acids with a calculated molecular mass of 84.96 kDa and a theoretical isoelectric point of 5.61. FcTG contains a typical transglutaminase-like homologue (TGc domain: E-value = 1.94e-38). Three catalytic sites (Cys-324, His-391 and Asp-414) are present in this domain. The deduced amino acid sequence of FcTG showed high identity with black tiger shrimp TG, kuruma shrimp TG and crayfish TG. Transcripts of FcTG mRNA were mainly detected in gill, lymphoid organ and hemocytes by RT-PCR. RNA in situ hybridization further confirmed that FcTG was constitutively expressed in hemocytes both in the circulatory system and lymphoid organ. The variation of mRNA transcription level in hemocytes and lymphoid organ following injection of killed bacteria or infection with white spot syndrome virus (WSSV) was quantified by RT-PCR. The up-regulated expression of FcTG in shrimp lymphoid organ following injection of bacteria indicates that it is inducible and might be associated with bacterial challenge. (c) 2006 Elsevier Ltd. All rights reserved.

Discovery of genes involved in defense/immunity functions in a haemocytes cDNA library from Fenneropenaeus chinensis by ESTs annotation

Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and inter-cellular signal communication. Expressed sequence tags (ESTs) analysis is proved to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. In order to further understand the innate immune system and defense mechanisms of Chinese shrimp at molecular level, complementary DNA library is constructed from the haemocyte tissue of Fenneropenaeus chinensis. A total of 2371 cDNA clones are successfully sequenced and the average sequence length is 460 bp. About 50% are identified as orthologs of known genes from other organisms by BLASTx and BLASTn program. By sequences comparability and analysis, 34 important genes including 177 ESTs are identified that may be involved in defense or immune functions in shrimp based on the known knowledge. These genes are categorized into five categories according to their putative functions in shrimp immune system: 13 genes are different types of antimicrobial peptides (AMP, penaeidin, antilipopolysaccharide factor, etc.), and their proportion is about 3 8%; 11 genes belong to prophenoloxidase system (prophenoloxidase, serine proteinase, serine proteinase inhibitor, etc.), and their proportion is about 32%; five genes have high homology with clotting protein (lectin, transglutaminase, etc), and their proportion is about 15%; three genes may be involved in inter-cell signal communication (peroxinectin, integrin), and their proportion is about 9%; two genes have been identified to be chaperone proteins (Hsc70, thioredoxin peroxidase), and their proportion is about 6%. These EST sequences enrich our understanding of the immune genes of F chinensis and will help farther experimental research into immune factors and improve our knowledge of the immune mechanisms of shrimp. (c) 2007 Elsevier B.V. All rights reserved.

中国明对虾体内凝结作用相关免疫基因的克隆与表达研究

对虾疾病在世界范围内的频频爆发，给地区经济造成了重大损失。然而到目前为止，我们对于对虾免疫系统的分子机制还知之甚少，深入了解其免疫应答过程，包括异物识别，信息传递以及作用方式等是从根本上解决疾病问题的关键之一。本论文从中国明对虾血细胞中分别克隆了一种C型凝集素基因（Fclectin）、参与凝结过程的谷氨酰胺转移酶基因（FcTG）以及参与凝结级联反应和酚氧化酶原激活系统的两种丝氨酸蛋白酶基因（SP-1和SP-2）和两种丝氨酸蛋白酶抑制剂基因（SPI-1和SPI-2），分析了它们的分子结构特征，预测了其可能的作用，并对它们的组织分布及应答不同病原感染的表达变化模式进行了研究。 首次从对虾中克隆了Fclectin基因，比对结果发现该基因属于C型凝集素超家族的成员之一；Northern blot和原位杂交结果显示，Fclectin基因在部分血细胞中呈组成型表达；利用毛细管电泳半定量RT-PCR方法分别研究了细菌和病毒感染后该基因的表达特征，并初步尝试了利用体外培养的原代血细胞系统研究LPS刺激后Fclectin的表达变化。该基因在感染或刺激后表达水平有明显的改变。 利用RACE技术从中国明对虾血细胞中克隆了一个FcTG基因，它与斑节对虾的谷氨酰胺转移酶基因有93%的相似性，可能编码一种具有活性的TG；原位杂交结果FcTG主要在血细胞中表达，在淋巴器官管腔的血细胞中表达尤为丰富，推断该基因可能主要在吞噬细胞中表达；病原的刺激未能使该基因的表达明显改变，但损伤（注射）的刺激会对其表达产生一定影响。 利用本组构建的对虾血液cDNA文库，克隆到了两个不同的丝氨酸蛋白酶基因，命名为SP-1和SP-2。前者为具有假clip结构域的胰蛋白酶样SP类似物（SPH），后者是一个具有完整的clip结构域的SPH，这在对虾中是首次发现。SP-1和SP-2都主要在血细胞中表达，此外SP-1在淋巴器官中的表达水平也很高；细菌的刺激对SP-1的影响不大，但会诱导SP-2表达量的增加，这两个基因的表达模式在病毒刺激后很相似，都出现先上调后下降的过程，可见病毒的感染会导致这两个基因转录的增强。 利用SMART-RACE技术结合对虾血液cDNA文库的利用，从中国明对虾血细胞中克隆到了两种SPI基因，它们分别为kazal-SPI和serpin-SPI，属于两个不同的家族。SPI-1与斑节对虾的SPI有76%的相似性，推断SPI-1可能主要对弹性蛋白酶、枯草杆菌蛋白酶、胰凝乳蛋白酶等有抑制作用；SPI-2为对虾中首次报道的serpin型抑制剂，它与淡水螯虾的serpin有42%的相似性，SPI-2可能主要对胰蛋白酶、酚氧化酶原激活酶、凝血酶和凝结酶有抑制活性。组织分别特征显示这两个抑制剂基因都在鳃、血细胞和淋巴器官中有高水平的表达。细菌的刺激都会导致这两个基因的表达在感染后出现下调，随后又上升至原有水平；病毒感染后，SPI-1和SPI-2的表达变化情况相似，感染后前12h基本没有明显的改变，到了感染的晚期（感染后24h和48h），随着病毒在体内大量复制，这两个基因的表达都急剧下降至接近基因关闭的状态，这可能暗示着与SPI相对应的蛋白酶的活力将很大程度的异常的增加，机体内稳定的免疫系统平衡状态可能已被破坏。