香鱼白细胞介素17C基因克隆及鳗利斯顿氏菌侵染后的时空表达

为了解香鱼白细胞介素17(interleukin 17,IL-17)的序列特征、系统发育及其与鳗利斯顿氏菌侵染的相关性,实验采用RACE-PCR方法扩增出香鱼IL-17C基因c DNA序列,并通过实时荧光定量PCR(q RT-PCR)技术检测了鳗利斯顿氏菌侵染后香鱼各组织中IL-17C基因的表达变化。结果显示,香鱼IL-17C基因c DNA序列全长1 087 bp,含534 bp的开放阅读框,在3′端非编码区存在7个m RNA不稳定信号(ATTTA)。预测该基因编码177个氨基酸,N端19个氨基酸为信号肽序列,成熟蛋白中含有8个半胱氨酸残基。系统进化树分析显示不同物种的相同IL-17亚型聚为一支,香鱼IL-17C与其他脊椎动物IL-17C聚为一支;同源比对结果显示香鱼IL-17C与虹鳟的IL-17C1和IL-17C2亲缘关系最近,相似性分别为40.72%和33.51%。q RT-PCR检测显示IL-17C基因在健康香鱼的心、肝、脾、头肾、鳃、脑、肌肉和肠等组织中均有表达,鳃和头肾中表达量较高。经鳗利斯顿氏菌侵染后,香鱼肝脏中IL-17C基因表达变化最大,菌侵染8 h时的表达量为对照组的9.17倍;其次为脾,菌侵染4 h时的表达量为对照组的6.69倍。研究表明,香鱼IL-17C基因的表达与病原菌侵染密切相关,可能在免疫功能中具有重要作用。

Cloning of IL-17C gene from Plecoglossus altivelis and the expression pattern analysis after Listonella anguillarum infection

To understand the sequence features and phylogeny of interleukin 17 (IL-17) in sweet fish (Plecoglossus altivelis), and its correlation to Listonella anguillarum infection, the cDNA sequence of IL-17C was cloned from P. altivelis by RACE-PCR, and the expression changes of different tissues were detected after L. anguillarum infection by real-time quantitative PCR (qRT-PCR). The full length of P. altivelis IL-17C cDNA was 1 087 bp including a 534 bp ORF and seven 3''UTR instability motifs (ATTTA), and it encoded 177 amino acids with eight conservation cysteine (Cys) and a 19 amino acids N-terminal signal peptide. Phylogenetic tree analysis showed that the same IL-17 subtype homologue from different species gathered together, and the P. altivelis IL-17C was clustered in one group with other vertebrate IL-17C. Multiple sequence alignment and phylogenetic analysis showed that P. altivelis IL-17C has higher similarity to Oncorhynchus mykiss IL-17C1 (40.72%) and O. mykiss IL-17C2 (34.04%). In healthy P. altivelis, IL-17C mRNA expression was detected in heart, liver, spleen, head kidney, gill, brain, muscle and intestine, high expression was shown in gill and head kidney. After L. anguillarum infection, the expression of IL-17C increased 9.17 folds in liver at 8 hpi (hours post infection) and 6.69 folds in spleen at 4 hpi compared to the control group. IL-17C gene is closely related to the pathogenic bacteria infection of P. altivelis, and it may play an important role in inflammatory immune response.