中华绒螯蟹EsPrx4基因的克隆及抗菌功能分析  

Cloning and Anti-bacterial Functional Analysis of EsPrx4 in Eriocheir sinensis

作　　者：梁猛 王美垚[1,2] 李建林[1,2] 颜逢缘 周军[4] 唐永凯[1,2,3] LIANG Meng;WANG Mei-Yao;LI Jian-Lin;YAN Feng-Yuan;ZHOU Jun;TANG Yong-Kai(Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214128,China;Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,Ministry of Agriculture and Rural Affairs,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Freshwater Fisheries Research Institute of Jiangsu Province,Nanjing 210017,China)

机构地区：[1]南京农业大学无锡渔业学院,无锡214128 [2]中国水产科学研究院淡水渔业研究中心/农业农村部淡水渔业和种质资源利用重点实验室,无锡214081 [3]上海海洋大学水产与生命学院,上海201306 [4]江苏省淡水水产研究所,南京210017

出　　处：《农业生物技术学报》2023年第4期824-832,共9页Journal of Agricultural Biotechnology

基　　金：江苏省农业重大新品种创制(PZCZ201749);中央级基本科研业务费(2020TD36);江苏省种业振兴项目(JBGS〔2021〕031);江苏省青年科学基金(SBK2020044520)。

摘　　要：近年来,养殖水环境恶化和养殖密度的提高,病害的发生和非生物的胁迫应激,严重影响着中华绒螯蟹(Eriocheir sinensis)的生长和发育。过氧化物还原酶(peroxiredoxin,Prx)在氧化应激应答过程中发挥重要作用。Prx4为Prx家族的重要成员,为了探讨Prx4在中华绒螯蟹抗菌免疫应答中的调控功能,本研究应用同源克隆技术从中华绒螯蟹的肝胰腺中克隆出EsPrx4基因(GenBank No.ON685195)。序列分析表明,EsPrx4的编码区为738 bp,共编码245个氨基酸。理化性质分析表明,EsPrx4蛋白的分子量为27.31 kD,理论等电点为5.55,总平均亲水性为-0.181,表明EsPrx4蛋白为亲水性蛋白。系统进化分析表明,中华绒螯蟹EsPrx4与拟穴青蟹(Scylla paramamosain)、斑节对虾(Penaeus monodon)和南美白对虾(Litopenaeus vannamei)等虾蟹类的Prx4基因具有高度同源性,表明虾蟹中Prx4具有较高的保守性。qPCR结果显示,EsPrx4在中华绒螯蟹体内广泛表达,在肝胰腺中表达量最高,在肠中表达量最低。在鳗弧菌(Listonella anguillarum)刺激下,EsPrx4迅速应答,在24 h表达量达到最高,72 h时恢复至正常水平,表明EsPrx4参与了鳗弧菌刺激引起的反应,在中华绒螯蟹免疫应答中发挥重要作用。本研究为中华绒螯蟹先天免疫和Prx基因家族的深入研究提供理论支持。In recent years,deterioration of water environment,elevation of stocking density,occurrence of disease and abiotic stress have seriously affected growth and development of Eriocheir sinensis.Peroxiredoxin(Prx)plays an important role in oxidative stress response.Prx4 is an important member of Prx family.To discuss the role of Prx4 in the anti-bacterial response of E.sinensis,EsPrx4(GenBank No.ON685195)was cloned from hepatopancreas with homologous cloning method.Sequence analysis showed that the coding region of EsPrx4 was 738 bp,encoding 245 amino acids.The physical and chemical propertiese analysis showed that the molecular weight of EsPrx4 protein was 27.31 kD,the theoretical isoelectric point was 5.55,and the total average hydrophilicity was-0.181,indicating that EsPrx4 protein was hydrophilic.Phylogenetic analysis showed that the EsPrx4 has high homology to that of Scylla paramamosain,Penaeus monodon and Litopenaeus vannamei,which indicated high conservation in Prx4 of shrimp and crab.Results of q PCR indicated that EsPrx4 was widely expressed in E.sinensis,it had the highest expression in hepatopancreas and the lowest expression in intestine.Under the stimulation of Listonella anguillarum,the expression of EsPrx4 reached the highest level at 24 h and recovered to the normal level at 72 h,indicating that EsPrx4 plays an important role in the immune response of E.sinensis.This study provides theoretical support for further research on innate immunity and Prx gene family of E.sinensis.

关 键 词：中华绒螯蟹 过氧化物还原酶(Prx) 克隆 抗菌 基因表达 

分 类 号：S917[农业科学—水产科学]