乌鳢半胱氨酸蛋白酶3基因克隆及表达分析  

Cloning and expression analysis of cysteinyl aspartate specific proteinase 3 gene(caspase-3)in Channa argus

作　　者：赵浩航 丘金珠 周秀莹 唐怀庆 赵天珍 梁富 宋海霞 喻大鹏 宋长江 ZHAO Hao-hang;QIU Jin-zhu;ZHOU Xiu-ying;TANG Huai-qing;ZHAO Tian-zhen;LIANG Fu;SONG Hai-xia;YU Da-peng;SONG Chang-jiang(Shenzhen Institute,Guangdong Ocean University,Shenzhen,Guangdong 510000,China;Guangdong OceanUniversity,Zhanjiang,Guangdong 524088,China;Zhongshan Institute for Quality and SafetyInspection of Agricultural Products,Zhongshan,Guangdong 528400,China)

机构地区：[1]广东海洋大学深圳研究院,广东深圳510000 [2]广东海洋大学,广东湛江524088 [3]中山市农产品质量安全检验所,广东中山528400

出　　处：《南方农业学报》2023年第11期3405-3415,共11页Journal of Southern Agriculture

基　　金：国家重点研发计划项目(2020YFD0900201);中山市第二批社会公益与基础研究项目(2021B2058)。

摘　　要：【目的】克隆和表达分析乌鳢(Channa argus)的半胱氨酸蛋白酶(Cysteinyl aspartate specific proteinase)3基因(caspase-3),为探究该基因在乌鳢机体中的免疫应答作用及在其他鱼类免疫上的作用提供理论依据。【方法】根据NCBI公布的乌鳢基因组信息,对乌鳢的caspase-3基因序列进行克隆,对推导的caspase-3氨基酸序列进行生物信息学分析,采用实时荧光定量PCR检测caspase-3基因在健康乌鳢各组织中的表达分布及在不同刺激物[鰤鱼(Seriola quinqueradiata)诺卡氏菌(Nocardia seriolae)、脂多糖(LPS)和聚肌胞苷酸(PolyI:C)]刺激下的时序表达情况。【结果】caspase-3基因序列全长855 bp,编码284个氨基酸残基,蛋白分子量为31.069 kD,理论等电点(p I)为6.17。caspase-3蛋白含有1个CASc结构域。多序列比对结果显示,乌鳢的caspase-3氨基酸序列与秘鲁笛鲷(Lutjanus peru)的caspase-3氨基酸序列相似度最高,达81.7%;系统发育进化树分析结果表明,乌鳢的caspase-3氨基酸序列与秘鲁笛鲷的caspase-3氨基酸序列聚为一支。实时荧光定量PCR检测结果表明,乌鳢caspase-3基因在肌肉、皮肤、鳃、心脏、胸腺、脑、头肾、脾脏、肝脏和血液等组织中均有表达,其中在脾脏和头肾的表达量最高;在不同刺激物刺激下,caspase-3基因在乌鳢脾脏、肾脏、血液和肝脏组织中的表达量均发生显著变化(P<0.05);用灭活鰤鱼诺卡氏菌、LPS和PolyI:C孵育乌鳢头肾白细胞后,caspase-3基因表达量均显著升高,且具有明显的时间依赖性。【结论】乌鳢caspase-3基因可能在乌鳢抵御细菌和病毒入侵的免疫反应中发挥着重要作用。【Objective】Cloning and expression analysis of cysteinyl aspartate specific proteinase-3 gene(caspase-3)of Channa argus were conducted,which provided a theoretical basis for exploring the role of this gene in the immune response of C.argus and its role in the immunity of other fishes.【Method】According to the genome information of C.ar‐gus published by NCBI,the caspase-3 gene sequence was cloned and the deduced caspase-3 amino acid sequence bioin‐formatics analysis was performed by real-time fluorescence quantitative PCR to detect the expression distribution of cas‐pase-3 gene in various tissues of healthy C.argus and the temporal expression under different stimuli[(Seriola quinquera‐diata Nocardia seriolae,lipopolysaccharide(LPS)and polymyxcytidylic acid(PolyI:C)].【Result】The caspase-3 gene sequence was 855 bp and encoded 284 amino acid residues,with a protein molecular mass of 31.069 kD and a theoretical isoelectric point(pI)of 6.17.The caspase-3 protein contained 1 CASc domain.Multiple sequence alignment showed that the amino acid sequence of caspase-3 of C.argus and the caspase-3 of Lutjanus peru,reaching 81.7%.Phylogenetic analy‐sis showed that caspase-3 of C.argus and caspase-3 of L.peru clustered into a single lineage.The results of real-time fluo‐rescence quantitative PCR showed that,the caspase-3 gene of C.argus in the tested tissue(muscle,skin,gills,heart,thymus,brain,head kidney,spleen,liver and blood)were expressed.The spleen and head kidney had the highest expres‐sion.Upon stimulation with the different stimuli,the expression level of caspase-3 gene in the spleen,head kidney,blood and liver tissues of C.argus all changed significantly(P<0.05);After incubation of head kidney leukocyte with S.quinqueradiata N.seriolae,LPS and PolyI:C,the caspase-3 gene expression was all significantly increased,and it had an obvious time-dependence.【Conclusion】The caspase-3 gene of C.argus may play an important role in the immune re‐sponse against bacterial and viral invasion.

关 键 词：乌鳢 半胱氨酸蛋白酶3基因(caspase-3) 组织分布 免疫应答 

分 类 号：S965.125[农业科学—水产养殖]