缢蛏2-CRD型半乳糖凝集素(Galectin)基因的克隆和表达分析

利用cDNA末端快速扩增(RACE)技术获得了缢蛏半乳糖凝集素基因(ScGL)。ScGL全长为1 282 bp,5′非编码区35 bp,3′非编码区329 bp,开放阅读框(ORF) 918 bp,编码305个氨基酸。氨基酸序列分析显示,ScGL无跨膜结构域,与已报道的缢蛏半乳糖凝集素含1个糖识别结构域(CRD)不同,ScGL具有2个CRD。相似性分析显示,ScGL与其他软体动物的半乳糖凝集素具有较高的相似性,与菲律宾蛤仔相似性最高,达65%;与已报道的2个缢蛏半乳糖凝集素相似性分别为39.74%和44.76%。系统进化上ScGL与菲律宾蛤仔半乳糖凝集素聚为一支。重组表达发现ScGL在包涵体表达,分子量约34.4 ku。荧光定量PCR分析显示,ScGL在缢蛏鳃、肠、唇瓣、外套膜、出水管、入水管、足和内脏团中均表达;其中肠、内脏团、唇瓣和足中表达量较高,出水管和入水管中表达量最低。消化腺ScGL表达量分别在金黄色葡萄球菌感染后3 h和48 h显著高于对照组;鳃ScGL表达量分别在鳗弧菌和金黄色葡萄球菌感染后6 h和48 h显著高于对照组,说明ScGL参与了病原体诱导的缢蛏免疫应答。本研究为深入探索ScGL在缢蛏免疫中的作用奠定基础。

Cloning and expression analysis of the 2-CRD galectin gene (ScGL) from Sinonovacula constricta

Galectin is a pattern recognition protein, which is mainly involved in immune recognition, cell phagocytosis, and immune elimination in invertebrate. Taking the Sinonovacula constricta that important tidal flat cultured shellfish in China as the research object, the galectin gene from Sinonovacula constricta (named ScGL) was cloned using RACE method. The full length cDNA sequence of ScGL was 1282 bp, which contains a 35 bp 5''UTR, 329 bp 3''UTR and 918 bp open reading frame (ORF) that encoded 305 amino acid residues. Analysis of amino acid sequences showed that ScGL lacks a transmembrane domain and contains 2 CRD unlike the galectin contains one CRD that had been identified in Sinonovacula constricta. Multiple sequence alignment and phylogenetic analysis showed that ScGL shared a hige degree of conservatism with galectin of other species and had the highest identity with Ruditapes philippinesis (65%), 39.74% and 44.76% identical with two galectins reported in the Sinonovacula constricta. ScGL was expressed in inclusion bodies with a calculated molecular mass of 34.4 ku. Quantitative real-time PCR detection results indicated that the ScGL gene were expressed widely in gill, intestine, labial palpus, mantle, exhalent siphon, inhalent siphon, foot and visceral mass, the highest expression level was observed in the intestinal, visceral mass, labial palpus and foot, and the lowest expression in the exhalent siphon, Inhalent siphon. After the Staphylococcus aureus challenge, the expression of ScGL in the digestive gland was significantly up-regulated at 3 hpi and 48 hpi (hours past infection), upon Vibrio anguillarum and Staphylococcus aureus challenge, the expression of ScGL was significantly up-regulated in the gill was respectively up-regulated at 6 hpi and 48 hpi. The result suggests that ScGL might participate in the innate immune response of Sinonovacula constricta triggered by pathogens. The study provides the good foundation for further research of the ScGL in Sinonovacula constricta immunity.