仿刺参干扰素调节因子3基因的克隆及其功能分析  

Cloning and functional study of interferon regulatory factor 3 of Apostichopus japonicus

作　　者：姜建洋 李宏阳 王称杨 李成华[1] 邵铱娜 JIANG Jianyang;LI Hongyang;WANG Chengyang;LI Chenghua;SHAO Yina(School of Marine Sciences,Ningbo University,Ningbo 315211,China)

机构地区：[1]宁波大学海洋学院,浙江宁波315211

出　　处：《水产学报》2025年第1期89-102,共14页Journal of Fisheries of China

基　　金：国家自然科学基金(42176102)。

摘　　要：【目的】研究仿刺参干扰素调节因子3(interferon regulatory factor 3,IRF3)基因功能。【方法】本研究首先采用RACE技术,通过克隆和序列拼接获得了仿刺参IRF3基因的全长cDNA序列,并命名为AjIRF3。AjIRF3基因全长2023 bp,编码441个氨基酸。【结果】序列分析显示,AjIRF3基因C末端具有1个IAD结构域;系统进化树分析结果显示,AjIRF3能与IRF3家族成员聚合且与软体动物欧洲玉黍螺的亲缘关系最近。组织分布显示,AjIRF3在所有检测的组织中均能表达,其中在肌肉中表达量最高,肠次之,体腔细胞中的表达量最低。灿烂弧菌胁迫下,AjIRF3在仿刺参体腔细胞中的表达量明显上调,且在6 h达到最高值,为对照组的3.64倍;进一步在LPS和poly(I:C)胁迫下,AjIRF3均在48 h达到最高值,分别为对照组的3.20倍和8.57倍。亚细胞定位结果显示,AjIRF3主要分布于细胞质中,但经灿烂弧菌胁迫后AjIRF3主要从细胞质易位到细胞核中。此外,干扰AjIRF3后,仿刺参体腔细胞凋亡率与NC组相比显著下降16%;进一步用灿烂弧菌胁迫后发现,体腔细胞中的胞内菌数量与NC组相比显著上调19.83倍,表明干扰仿刺参IRF3后促进了病原感染。【结论】仿刺参IRF3能够响应病原感染,并在抵御病原感染过程中发挥重要作用。研究结果为防控仿刺参腐皮综合征的发生提供了理论基础。Interferon regulatory factor 3(IRF3)is an important member of the interferon regulatory factor family and plays an important role in defensing against pathogen infection.To investigate the function of the IRF3 gene in Apostichopus japonicus,we obtained the full-length cDNA sequence of the IRF3 gene in A.japonicus by cloning and sequence splicing using RACE technology,and named it AjIRF3.The full-length sequence of AjIRF3 gene is 2023 bp and encodes 441 amino acids.Sequence analysis revealed that the C terminus of AjIRF3 contains an IAD domain.Phylogenetic tree analysis indicated that AjIRF3 is a new member of the IRF3 family and is more closely related to the invertebrate mollusc Littorina littorea.Tissue distribution analysis showed that AjIRF3 is expressed in all tissues,with the highest expression in muscle,followed by intestine,and the lowest expression in coelomocyte.Furthermore,the expression of AjIRF3 in coelomocyte was significantly up-regulated under Vibrio splendidus infection and reached its highest level at 6 h,which was 3.64-fold higher(P<0.05)than that of the control.Moreover,AjIRF3 mRNA expression levels peaked at 48 h after lipopolysaccharide(LPS)and poly(I:C)infection,increasing by 3.20-fold(P<0.01)and 8.57-fold(P<0.01),respectively,compared with the control.Subcellular localization studies showed that AjIRF3 is distributed in the cytoplasm,but it is mainly translocated from the cytoplasm to nucleus after V.splendidus infection.Additionally,we found that the apoptosis rate of coelomocyte was significantly decreased by 16%(P<0.01)following AjIRF3 interference.More importantly,our result showed that the number of intracellular bacteria in the si-AjIRF3 group was significantly increased by 19.83-fold(P<0.05)compared with NC group after V.splendidus infection.Collectively,these findings suggest that AjIRF3 plays important roles in innate immunity and regulates apoptosis to resist pathogen infection.

关 键 词：仿刺参 干扰素调节因子3 表达分析 细胞凋亡 

分 类 号：Q785[生物学—分子生物学] S947.9[农业科学—水产养殖]