IFAPA centro El Toruño, Junta de Andalucía, Camino Tiro de pichón s/n, 11500 El Puerto de Santa María, Cádiz, Spain.
Fish Shellfish Immunol. 2011 Dec;31(6):925-37. doi: 10.1016/j.fsi.2011.08.010. Epub 2011 Aug 31.
The g-type lysozyme is a key protein of the innate immune system to fight bacterial infections. In this study we cloned and characterized the gene encoding for g-type lysozyme in Senegalese sole (Solea senegalensis). The deduced amino acid sequence comprised 195 residues containing the three conserved catalytic residues and two cysteines. A BAC analysis revealed that the gene is structured in 5 exons and 4 introns. Also, two polyadenylation signals that generate two cDNAs differing in 3'-UTR length were detected. Promoter analysis showed the presence of the main cis-acting elements involved in the transcriptional regulation of the gene. At genomic level, the g-type lysozyme was associated with mucolipin 1 and the peptidoglycan recognition protein 2 conforming a cluster of antidefensive genes with a well-conserved synteny across Percomorpha. FISH analysis using the BAC clone revealed a single hybridization signal located in an acrocentric chromosome pair. The phylogenetic analysis confirmed that the g-type lysozyme represents a complex group in fish that has been shaped by gene duplications and diversification with several positions under Darwinian selection. Expression analysis in juvenile tissues indicated that transcript levels were higher in gills, spleen and heart. During development, gene expression activated just at the beginning of metamorphosis, increasing progressively until climax. Hormonal treatments demonstrated that this gene was regulated positively by thyroid hormones during development and negatively by dexamethasone. In contrast, no response was observed after all-trans retinoic acid or 4-diethylaminobenzaldehyde treatments. Finally, treatments using lipopolysaccharide, lipoteichoic acid, peptidoglycan, zymosan and poly(I:C) activated gene expression in a time- and tissue-specific manner. Taken together, data indicate that g-type lysozyme is a high evolutionary conserved gene that diversified to adapt to changing environment and pathogen conditions. Gene expression can be activated by diverse pathogen stimuli and modulated by physiological factors with important consequences for the aquaculture of this species.
g 型溶菌酶是鱼类先天免疫系统抵抗细菌感染的关键蛋白。本研究克隆并鉴定了塞内加尔比目鱼（Solea senegalensis）g 型溶菌酶基因。推导出的氨基酸序列包含 195 个残基，其中包含三个保守的催化残基和两个半胱氨酸。BAC 分析表明，该基因由 5 个外显子和 4 个内含子组成。还检测到两个多聚腺苷酸化信号，它们生成了两种在 3'-UTR 长度上不同的 cDNA。启动子分析表明，该基因存在参与转录调控的主要顺式作用元件。在基因组水平上，g 型溶菌酶与粘蛋白 1和肽聚糖识别蛋白 2相关，形成一个具有良好保守性的防御基因簇，在鲈形目鱼类中具有保守的同线性。使用 BAC 克隆进行的 FISH 分析显示，在一个近端着丝粒染色体对中存在一个单一的杂交信号。系统发育分析证实，g 型溶菌酶在鱼类中代表一个复杂的群体，该群体通过基因复制和多样化形成，其中有几个位置受到达尔文选择的影响。在幼鱼组织中的表达分析表明，在鳃、脾脏和心脏中，转录水平较高。在发育过程中，该基因仅在变态开始时表达激活，逐渐增加直到达到高峰。激素处理表明，该基因在发育过程中受甲状腺激素的正向调节，受地塞米松的负向调节。相比之下，在用全反式视黄酸或 4-二乙氨基苯甲醛处理后，没有观察到反应。最后，用脂多糖、脂磷壁酸、肽聚糖、酵母聚糖和聚（I：C）处理以时间和组织特异性的方式激活基因表达。综上所述，数据表明 g 型溶菌酶是一个高度进化保守的基因，其多样化是为了适应不断变化的环境和病原体条件。基因表达可以被各种病原体刺激激活，并被生理因素调节，这对该物种的水产养殖具有重要意义。
