拟穴青蟹两种新C-型凝集素基因的克隆与表达分析

摘要

C-type lectins are a group of carbohydrate binding proteins which contain one or more Ca2+ dependent car-bohydrate recognition domains (CRDs). They have been regarded as one of the most important pattern recognition re-ceptors in invertebrates. In this study, we cloned two novel C-type lectins, namely Sp-lectin3 and Sp-lectin4, from the hepatopancreas of Scylla paramamosain. The predicted amino acid sequences of Sp-lectin3 and Sp-lectin4 possessed several conserved properties of C-type lectins, such as a signal peptide, a single CRD, and a QPD motif (only in Sp-lectin4). The open reading frame of Sp-lectin3 consisted of 5 exons and 4 introns, while that of Sp-lectin4 was composed of 4 exons and 3 introns. In normal cultivated crabs expression of both genes could be detected in almost all the eleven tested tissues and organs, such as hepatopancreas, intestine, gill, haemocytes, and different parts of the re-productive duct. The expression of Sp-lectin3 was highest in hepatopancreas and second highest in ejaculatory ducts;for Sp-lectin4 the highest expression was observed also in hepatopancreas and then in the intestine. We further investigated the expression of Sp-lectin3 and Sp-lectin4 at different developmental stages. The results showed that the expression of both genes increased gradually and reached a peak at the embryonic stage of zoea, then declined sharply at the stage of megalopa, and finally recovered at the stage of crablet. When artificially infected with Vibrio parahaemolyticus, the expression of Sp-lectin3 in ejaculatory ducts was significantly up-regulated at 6h after the infection, and the expression in seminal vesicles was boosted at 12h post infection (P<0.05). For Sp-lectin4, the expression was significantly elevated only in hepatopancreas both at 12h and 18h post infection (P<0.05). Our results suggested that the two C-type lectins might be involved in the immune response to bacterial infection in Scylla paramamosain. Sp-lectin3 could mainly func-tion in the reproductive system such as the ejaculatory duct and seminal vesicles, while Sp-lectin4 may mainly function in hepatopancreas.%为研究拟穴青蟹(Scylla paramamosain)中 C-型凝集素(C-lectin)的功能,从其肝胰腺中克隆获得全长858 bp和598 bp的两个C-型凝集素分子,分别命名为Sp-lectin3和Sp-lectin4,其推导的氨基酸序列含有信号肽和一个CRD,其中Sp-lectin4还具有糖类结合的特征性基序“QPD”。Sp-lectin3和Sp-lectin4的开放阅读框分别由5个和4个外显子编码。在正常养殖青蟹的肝胰腺中该两个基因表达量最高,其次是射精管(Sp-lectin3)或肠(Sp-lectin4)；除脑和储精囊外的所检测组织/器官中, Sp-lectin4表达量均高于 Sp-lectin3。随着胚胎的发育,该两个基因表达量逐渐升高,峰期为溞状幼体,而大眼幼体期的表达量急剧下降,仔蟹期再回升；在胚胎发育阶段除囊胚期外, Sp-lectin4表达量极显著高于Sp-lectin3(P<0.01),相反,在胚后发育阶段除大眼幼体II期外, Sp-lectin3表达量却极显著高于Sp-lectin4(P<0.01)。副溶血弧菌(Vibrio parahemolyticus)人工感染,可诱导Sp-lectin3在储精囊和射精管中的表达,分别在感染后12h和6h显著上调(P<0.05)；同时,可诱导 Sp-lectin4在肝胰腺中的表达,并在感染后12h 和18h 显著上调(P<0.05)。结果表明, Sp-lectin3和Sp-lectin4可能参与拟穴青蟹的抗细菌感染免疫反应, Sp-lectin3侧重于生殖系统如射精管和储精囊中发挥作用,而Sp-lectin4侧重于在肝胰腺。