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杨晶, 陆新江, 柴方超, 陈炯. 2016: 一个大黄鱼piscidin基因的分子鉴定和功能分析. 动物学研究, 37(6): 347-355. DOI: 10.13918/j.issn.2095-8137.2016.6.347
引用本文: 杨晶, 陆新江, 柴方超, 陈炯. 2016: 一个大黄鱼piscidin基因的分子鉴定和功能分析. 动物学研究, 37(6): 347-355. DOI: 10.13918/j.issn.2095-8137.2016.6.347
Jing YANG, Xin-Jiang LU, Fang-Chao CHAI, Jiong CHEN. 2016: Molecular characterization and functional analysis of a piscidin gene in large yellow croaker (Larimichthys crocea). Zoological Research, 37(6): 347-355. DOI: 10.13918/j.issn.2095-8137.2016.6.347
Citation: Jing YANG, Xin-Jiang LU, Fang-Chao CHAI, Jiong CHEN. 2016: Molecular characterization and functional analysis of a piscidin gene in large yellow croaker (Larimichthys crocea). Zoological Research, 37(6): 347-355. DOI: 10.13918/j.issn.2095-8137.2016.6.347

一个大黄鱼piscidin基因的分子鉴定和功能分析

Molecular characterization and functional analysis of a piscidin gene in large yellow croaker (Larimichthys crocea)

  • 摘要: Piscidin抗菌肽家族是鱼类抗菌肽之一,具有广谱抗菌活性,在先天性免疫系统中起着重要作用。本研究鉴定了一种大黄鱼piscidin抗菌肽piscidin-5-like type 3(Lcpis5lt3)基因。多重序列比对揭示Lcpis5lt3与其它鱼类piscidin氨基酸序列在信号肽端高度保守。系统进化树显示Lcpis5lt3与其他大黄鱼piscidin-5-like紧密成簇。实时荧光定量PCR(Quantitative PCR)结果显示,Lcpis5lt3在所检测的组织,包括脑、肌肉、鳃、头肾、肠、肾、肝和脾中均有表达,其中在脾和头肾中表达量较高。大黄鱼感染溶藻弧菌后,Lcpis5lt3 mRNA表达量在鳃、头肾、肠、肾和肝组织中的4 h、8 h、12 h和24 h均上调,而在脾组织中表达量无明显变化。用合成的Lcpis5lt3成熟肽进行体外抑菌实验,发现Lcpis5lt3成熟肽对多种细菌有不同的抗菌能力,如嗜水气单胞菌、鳗弧菌、溶藻弧菌、副溶血弧菌、金黄色葡萄球菌和单核细胞增生李斯特菌。感染溶藻弧菌的大黄鱼注射Lcpis5lt3成熟肽后,存活率增加,组织及血液载菌量降低,组织肿瘤坏死因子-α、白细胞介素-1β和白细胞介素-10三种细胞因子的表达量下调。综上,Lcpis5lt3在大黄鱼先天性免疫中起重要作用,并有可能会作为一种新型药物抵抗病原菌的感染。

     

    Abstract: The piscidin family, which includes potent antimicrobial peptides with broad-spectrum activity, plays an important role in the innate immune system of fish. In this study, we cloned piscidin-5-like type 3 (Lcpis5lt3) in large yellow croaker (Larimichthys crocea). Multiple alignments with other known piscidins revealed amino acid conservation throughout the fish, especially at the signal peptide (22 amino acids). The phylogenetic tree confirmed that Lcpis5lt3 and large yellow croaker piscidin-5-like proteins were grouped together to form a branch. Quantitative real-time PCR revealed that Lcpis5lt3 was expressed in a wide range of tissues, including the brain, muscle, gill, head kidney, intestine, kidney, liver, and spleen. The highest mRNA expression level of Lcpis5lt3 was found in the spleen. After Vibrio alginolyticus infection, mRNA expression was rapidly upregulated in the liver, head kidney, gill, kidney, and intestine at 4, 8, 12, and 24 h post infection (hpi), whereas there were no significant changes in the spleen. The antimicrobial spectrum showed that the synthetic mature peptide of Lcpis5lt3 exhibited different activity in vitro against various bacteria, such as Aeromonas hydrophila, V. anguillarum, V. alginolyticus, V. parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes. In addition, survival rates from the in vivo assay indicated that the synthetic peptide of Lcpis5lt3 increased the survival rate of large yellow croaker after V. alginolyticus challenge, resulting in a decline in bacterial burden and mRNA expression levels of interleukin-1β, interleukin-10, and tumor necrosis factor-α. These data suggest that Lcpis5lt3 plays an important role in innate immunity in large yellow croaker and might represent a potential therapeutic agent against pathogen invasion.

     

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