A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.
A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.
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一种新型的FAS配体在碎屑的细胞凋亡中起着重要作用：Chfasl的分子克隆，表达谱和功能鉴定。
DOI：
10.3389/fimmu.2023.1267772
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发表时间：
2023
影响因子：
7.3
通讯作者：
Zhang, Yuehuan
中科院分区：
文献类型：
--
作者：
Qin, Yanping;Wan, Weitao;Li, Jiangwei;Wang, Zhongyu;Yang, Yue;Li, Jun;Ma, Haitao;Yu, Ziniu;Xiang, Zhiming;Zhang, Yuehuan
细胞凋亡通过清除有害或病变细胞来调节正常发育、稳态、免疫耐受和对环境胁迫的反应，在无脊椎动物非特异性免疫中起着关键作用。死亡受体和死亡配体介导的外源性途径是细胞凋亡的重要途径。死亡配体主要是肿瘤坏死因子（TNF）家族的成员，其中FasL是重要成员。FasL在脊椎动物细胞凋亡和免疫中的作用已被多次报道，但贝类中FasL基因的研究较少，其在牡蛎细胞凋亡和免疫中的功能意义尚不清楚。以香港牡蛎（Crassostrea hongkongensis）基因组为模板，克隆了ChFasL基因的全长。采用定量PCR方法检测ChFasL在不同发育阶段和组织中的相对表达量，以及细菌感染后血细胞中相对表达量的变化。亚细胞定位法定位ChFasL在HEK 293 T细胞中的表达位置，双荧光报告基因法研究重组蛋白含量增加对报告基因p53和p21活性的影响。最后，利用RNA干扰技术检测ChFasL基因沉默后血细胞凋亡率的变化。我们从C. hongkongensis，命名为ChFasL。我们发现ChFasL具有潜在的N-糖基化位点、跨膜结构域和TNF区，这是TNF家族的典型特征。ChFasL在各发育阶段的幼虫和牡蛎的所有组织中表达。在溶藻弧菌或S.结果表明，ChFasL在溶血性弧菌血细胞中的相对表达量显著增加，提示ChFasL深入参与了溶血性弧菌的免疫应答过程。hongkongensis对外界微生物刺激的敏感性。亚细胞定位结果显示，ChFasL主要分布于HEK 293 T细胞的胞浆中。重组蛋白pcDNA 31- ChFasL的过表达可显著提高p53和p21的活性，表现出正性调节作用。dsRNA成功下调ChFasL相对表达后，血细胞凋亡率明显低于dsGFP组。这些结果全面证实了ChFasL在C. hongkongensis，为深入了解贝类细胞凋亡的免疫功能提供了基础和前提，也为海洋双壳贝类的致病死亡机制和抗病育种研究做出了贡献。
Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dual-fluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves.
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影响因子：
3.6
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通讯作者：
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