李辉, 冯伟, 唐永凯, 苏胜彦, 王美垚, 李建林. 2021: 中华绒螯蟹过氧化物还原酶基因克隆及其在鳗弧菌感染下的表达分析. 南方农业学报, 52(8): 2267-2275. DOI: 10.3969/j.issn.2095-1191.2021.08.027
引用本文: 李辉, 冯伟, 唐永凯, 苏胜彦, 王美垚, 李建林. 2021: 中华绒螯蟹过氧化物还原酶基因克隆及其在鳗弧菌感染下的表达分析. 南方农业学报, 52(8): 2267-2275. DOI: 10.3969/j.issn.2095-1191.2021.08.027
LI Hui, FENG Wei, TANG Yong-kai, SU Sheng-yan, WANG Mei-yao, LI Jian-lin. 2021: Cloning of peroxiredoxin gene (EsPrx) in Eriocheir sinensis and its expression analysis under Vibrio anguillarum infection. Journal of Southern Agriculture, 52(8): 2267-2275. DOI: 10.3969/j.issn.2095-1191.2021.08.027
Citation: LI Hui, FENG Wei, TANG Yong-kai, SU Sheng-yan, WANG Mei-yao, LI Jian-lin. 2021: Cloning of peroxiredoxin gene (EsPrx) in Eriocheir sinensis and its expression analysis under Vibrio anguillarum infection. Journal of Southern Agriculture, 52(8): 2267-2275. DOI: 10.3969/j.issn.2095-1191.2021.08.027

中华绒螯蟹过氧化物还原酶基因克隆及其在鳗弧菌感染下的表达分析

Cloning of peroxiredoxin gene (EsPrx) in Eriocheir sinensis and its expression analysis under Vibrio anguillarum infection

  • 摘要: 【目的】克隆中华绒螯蟹(Eriocheir sinensis)过氧化物还原酶基因(EsPrx)的开放阅读框(ORF),并分析EsPrx基因的组织表达特征及细菌感染下的表达情况,为中华绒螯蟹的先天免疫和抗病机制研究提供参考依据。【方法】通过RT-PCR结合转录组序列比对克隆出EsPrx基因,利用DNAStar、ClustalW、ExPASy、TMHMM Server v.2.0、SignalP 5.0及NetPhos 3.1 Server等在线软件进行生物信息学分析,并以实时荧光定量PCR检测EsPrx基因的组织表达特征及鳗弧菌(Vibrio anguillarum)感染下的表达情况。【结果】EsPrx基因ORF为597 bp,共编码198个氨基酸残基,含有Prx特有的2个结构域(FYPLDFTFVCPTEI和GEVCPA)。EsPrx蛋白分子式为C994H1544N258O293S8,分子量为22.05 kD,理论等电点(pI)为5.67,无跨膜域和信号肽,属于非分泌性蛋白。EsPrx氨基酸序列与扁额青蟹(Eurypanopeus depressus)、拟穴青蟹(Scylla paramamosain)和三疣梭子蟹(Portunus trituberculatus)的Prx氨基酸序列高度同源,对应的相似性分别89%、89%和88%;基于Prx氨基酸序列相似性构建的系统发育进化树也显示,EsPrx氨基酸序列先与拟穴青蟹、扁额青蟹及南美白对虾(Penaeus vannamei)的Prx氨基酸序列聚在一起,再与其他动物的2-Cys Prx氨基酸序列聚类,证实EsPrx基因属于未分化的Prx基因,即Prx1/2基因。EsPrx基因在中华绒螯蟹各组织中广泛表达,以肝胰腺的相对表达量最高,极显著高于在其他组织中的相对表达量(P<0.01);EsPrx基因表达在鳗弧菌感染不同时间段存在明显差异,感染6~24 h其相对表达量显著升高(P<0.05),而后迅速降至正常水平。【结论】EsPrx基因为甲壳动物未分化的Prx1/2基因,在中华绒螯蟹肝胰腺中高表达,并参与鳗弧菌感染的抗氧化应激反应,即在抗逆过程中发挥重要作用。

     

    Abstract: 【Objective】 Cloned the open reading frame(ORF) of peroxiredoxin gene(EsPrx) of Eriocheir sinensis, and analyzed the tissue expression characteristics of EsPrx gene and its expression under bacterial infection, which provided reference for the study of innate immunity and disease resistance mechanism of E. sinensis.【Method】 The EsPrx gene was cloned by combining RT-PCR with transcriptome sequence alignment analysis. Bioinformatics analysis was performed using online software such as DNAStar, ClustalW, ExPASy, TMHMM Server v.2.0, SignalP 5.0, and NetPhos 3.1 Server. The tissue expression characteristics of the EsPrx gene and its expression in the presence of Vibrio anguillarum infection were detected by real-time fluorescence quantitative PCR.【Result】 The ORF of EsPrx gene was 597 bp, encoding a total of 198 amino acid residues, and it contained two domains specific to Prx(FYPLDFTFVCPTEI and GEVCPA). The molecular formula of EsPrx protein was C994H1544N258O293S8, with a molecular weight of 22.05 kDa and a theoretical isoelectric point(pI) of 5.67. Without transmembrane domain and signal peptide, it was a non-secretory protein. The amino acid sequence of EsPrx was highly homologous with the Prx amino acid sequence of Eurypanopeus depressus, Scylla paramamosain and Portunustri tuberculatus, with the corresponding similarities of 89%, 89% and 88%, respectively. The phylogenetic tree constructed based on the similarity of Prx amino acid sequence showed that the amino acid sequence of EsPrx was first clustered with the Prx amino acid sequence of S. paramamosain, E. depressus and Penaeus vannamei, and then clustered with the 2-Cys Prx amino acid sequence of other animals, confirming that the EsPrx gene belonged to the undifferentiated Prx gene, namely, Prx1/2 gene. The EsPrx gene was widely expressed in various tissues of E. sinensis, and the relative expression level of EsPrx gene in hepatopancreas was the highest, which was extremely significantly higher than that in other tissues(P<0.01). The expression of EsPrx gene was greatly different at different time points of Vibrio anguillarum infection, and its relative expression was significantly increased 6-24 h after infection(P<0.05), and then rapidly decreased to normal level.【Conclusion】 EsPrx gene is an undifferentiated Prx1/2 gene of crustacean, which is highly expressed in hepatopancreas of E. sinensis, and participates in the anti-oxidative stress response of V.anguillarum infection, that is, plays an important role in the resistance process.

     

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