摘要
【目的】克隆尼罗罗非鱼白细胞介素-34(IL-34)基因,开展生物信息学分析,研究其在罗非鱼不同组织的表达模式,并探究其在不同刺激下头肾、脾脏和外周血的表达差异,为IL-34在罗非鱼细菌感染中的免疫调控作用提供参考。【方法】根据尼罗罗非鱼OnIL-34基因序列设计引物,鉴定出OnIL-34同源物,克隆OnIL-34编码区序列(CDS),对其氨基酸序列、蛋白结构域组成及系统发育进行分析,并通过实时荧光定量PCR技术(qRT-PCR)检测该基因在不同健康组织中的分布,以及在无乳链球菌、嗜水气单胞菌和脂多糖(LPS)刺激后的表达变化。【结果】OnIL-34开放阅读框(ORF)长648 bp,编码215个氨基酸,预测蛋白分子量为25.26 kDa,理论等电点为8.52。蛋白序列包含1个含20个氨基酸的跨膜结构域,N端有含189个氨基酸的胞外结构域。系统进化树分析表明,尼罗罗非鱼IL-34氨基酸序列与图形斑马鼻蝶(Simochromis diagramma)相似度最高、为98.6%,且系统进化树结果显示其与图形斑马鼻蝶IL-34聚为一支。qRT-PCR结果显示,OnIL-34在健康尼罗罗非鱼各组织中均有表达,其中在胃中表达量最高,是肠道组织的26.26倍;其次为肝脏、心脏、和鳃,分别为肠道组织的26.09、17.14、15.85倍。罗非鱼感染无乳链球菌和嗜水气单胞菌后,IL-34在头肾中的表达显著上调(P<0.05);而在LPS刺激6 h后IL-34在外周血中表达显著上调,24 h后在头肾中表达显著上调;在无乳链球菌、嗜水气单胞菌和LPS体外刺激后,头肾、脾脏和外周血白细胞中IL-34表达量均显著上调。【结论】在病原菌感染和LPS刺激后,尼罗罗非鱼IL-34在免疫组织中表达上调,初步证实其在病原体感染、宿主免疫防御和炎症反应具有重要作用,但其具体的免疫调控途径还需进一步探究。
【Objective】To clone the interleukin-34(IL-34)gene in Nile tilapia,conducting bioinformatics analysis,and investigating its expression pattern in different tissues of tilapia.Further more,the expression differences of IL-34 in the head kidney,spleen and peripheral blood under different stimuli were explored to provide a reference for the immune regulatory role of IL-34 in bacterial infection of tilapia.【Method】Primers were designed based on the OnIL-34 gene sequence of Nile tilapia,homologous genes were identified,and the coding region(CDS)of OnIL-34 was cloned.The amino acid sequence,protein domain composition,and phylogenetic analysis were conducted,followed by real-time quantitative PCR(qRT-PCR)to detect gene distribution in healthy tissues and expression changes induced by Streptococcus agalactiae,Aeromonas hydrophila,and lipopolysaccharide(LPS).【Result】The open reading frame(ORF)of OnIL-34 is 648 bp long,encoding 215 amino acids with a predicted molecular weight of 25.26 kDa and theoretical isoelectric point of 8.52.The protein sequence contains a 20-aminoacid transmembrane domain and an 189-amino-acid extracellular domain at the N-terminus.Phylogenetic analysis showed that the Nile tilapia IL-34 amino acid sequence exhibited the highest similarity(98.6%)to Simochromis diagramma,with phylogenetic tree results indicating coalescence with the S.diagramma IL-34 lineage.qRT-PCR results demonstrated that OnIL-34 is expressed in all tissues of healthy Nile tilapia,with the highest expression detected in the stomach(26.26-fold higher than intestinal tissue).The liver,heart,and gill tissues showed significantly lower levels at 26.09,17.14,and 15.85 times the intestinal level,respectively.After infection by S.agalactiae and A.hydrophila,IL-34 expression in the head kidney was significantly elevated(P<0.05).Following LPS stimulation for 6 hours,peripheral blood IL-34 levels increased markedly,with a significant rise in the head kidney at 24 hours.All tissues including head kidney,spleen,and peripheral blood leukocytes exhibited elevated IL-34 expression following pathogen exposure or LPS stimulation.【Conclusion】The upregulated IL-34 in immune tissues following pathogen infection and LPS stimulation preliminarily confirms its critical role in host immunity and inflammatory responses.However,the specific immunomodulatory mechanisms require further investigation.
作者
梁富
张璐
李晓暖
唐怀庆
谢理滔
梁莹
黄振檀
喻大鹏
宋海霞
LIANG Fu;ZHANG Lu;LI Xiaonuan;TANG Huaiqing;XIE Litao;LIANG Ying;HUANG Zhentan;YU Dapeng;SONG Haixia(Agricultural Products Quality&Safety Testing Institute of Zhongshan,Zhongshan 528437,China;School of Life Sciences,South China Normal University,Guangzhou 510631,China;Agricultural Technology Promotion Center of Zhongshan,Zhongshan 528437,China;Shenzhen Research Institute,Guangdong Ocean University,Shenzhen 518108,China)
出处
《广东农业科学》
2025年第6期130-142,共13页
Guangdong Agricultural Sciences
基金
中山市第二批社会公益与基础研究项目(2023B2053)。
