摘要
为初步探索鱼类PKCθ基因在抵御病原菌感染过程中的作用,本研究进行了杂交鳢(Channa maculata♀×C.argus♂)PKCθ(CmaPKCθ)基因克隆及其在舒伯特气单胞菌(Aeromonas schubertii)、鰤鱼诺卡氏菌(Nocardia seriolae)感染后的表达分析。克隆获得CmaPKCθ的完整开放阅读框(ORF)长度为2151 bp,预测编码716个氨基酸,具有保守的C1、C2样、C3和C4结构域。Blast分析表明CmaPKCθ氨基酸序列与尖吻鲈(Lates calcarifer)PKCθ的同源性最高,达92%。氨基酸多重序列比对显示CmaPKCθ具有保守的磷酸化位点和泛素化位点。系统进化树结果显示杂交鳢与其他鱼类的PKCθ聚为一个分支,其中与尖吻鲈PKCθ亲缘关系最近。实时荧光定量PCR结果表明,CmaPKCθ在健康杂交鳢的所有被检组织中广泛表达,其中在肝脏中表达水平最高,其次为脾脏、胸腺和头肾,在肌肉中的表达量最低。舒伯特气单胞菌和鰤鱼诺卡氏菌分别感染杂交鳢后,其肝脏、脾脏和头肾分别在第5天、第5天、第12小时和第10天、第2天、第3天时达到表达峰值(p<0.05),CmaPKCθ的表达变化相似,在不同时间点的肝脏、脾脏和头肾中,均主要表现为上调。上述结果表明,CmaPKCθ可能在杂交鳢激活T细胞增殖活化和抵御病原菌感染过程中发挥重要作用,本研究结果可为病原菌和鱼体的免疫互作机制研究及其病害防控提供参考。
In order to initially explore the role of fish PKCθagainst pathogenic bacteria,PKCθ(CmaPKCθ)from hybrid snakehead(Channa maculata♀×C.argus♂)was cloned and characterized,and the expression profiles were analyzed after infection with two pathogenic bacteria including Aeromonas schubertii and Nocardia seriolae in this study.The open reading frame(ORF)of CmaPKCθwas 2151 bp,which encoded a putative protein of 716 amino acid residues.Analysis of the protein features showed that CmaPKCθcontained four conservative domains of C1,C2 like,C3,and C4.Blast analysis showed that CmaPKCθshared the highest amino acid homology with PKCθof Lates calcarifer(92%).Multiple sequence alignment revealed that CmaPKCθhad several conserved phosphorylation sites and ubiquitination sites.Phylogenetic tree analysis illustrated that the CmaPKCθwas clustered with PKCθof other fish species,and was most closely related to L.calcarifer.Moreover,the m RNA expression levels in different tissues were analyzed by real time quantitative PCR,we found that CmaPKCθcould be detected in all examined tissues but with varied expression levels.CmaPKCθwas expressed strongly in the liver and relatively highly in the spleen,thymus,and head kidney,whereas it was expressed at lowest levels in the muscle.The expression changes of CmaPKCθwere similar and mainly up-regulated in the liver,spleen,and head kidney at different time points after infection with the pathogens A.schubertii and N.seriolae,respectively.CmaPKCθshowed the highest expression in the liver,spleen,and head kidney at 5 d,5 d,and 12 h after infection with A.schubertii,respectively(p<0.05).However,CmaPKCθdisplayed the highest expression in the liver,spleen,and head kidney at 10 d,2 d,and 3 d after infection with N.seriolae,respectively(p<0.05).Together,these results suggested that CmaPKCθmight play an important role in the proliferation and differentiation of T cells and fish defense against pathogen infection,which would provide reference for the research of the immune interaction b
作者
刘付翠
赵飞
何山
谭爱萍
张瑞泉
邓玉婷
姜兰
Liu Fucui;Zhao Fei;He Shan;Tan Aiping;Zhang Ruiquan;Deng Yuting;Jiang Lan(Key Laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou,510380;College of Fishery and Life Science,Shanghai Ocean University,Shanghai,201306)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2020年第5期1990-1998,共9页
Genomics and Applied Biology
基金
广东省渔港建设和渔业产业发展专项(A201601B07)
现代农业产业技术体系专项(CARS-46)
广东省级鱼病防治专项(粤财农[2014]585号)
广东省自然科学基金(2015A030310137)
国家自然科学基金(31402338)共同资助。
关键词
杂交鳢
PKCΘ
基因克隆
病原菌
表达分析
Channa maculata♀×C.argus♂
PKCθ
Gene cloning
Pathogenic bacteria
Expression analysis
