Proteins in the tumor necrosis factor receptor (TNFR) superfamily play significant roles in many physiological and pathological events, such as inflammation, apoptosis, autoimmunity, and organogenesis. Here, two TNFR gene homologs (PmTNFR1 and PmTNFR5) were identified in the pearl oyster Pinctada fucata martensii. The predicted PmTNFR1 and PmTNFR5 protein sequences were 406 and 533 amino acids long, respectively, and both possessed motifs characteristic of the TNFR family, including a TNFR homology domain (CRD), a transmembrane domain (TM), and death domains. However, the predicted amino acid sequences of PmTNFR1 and PmTNFR5 had low identity (~16-23%) with sequences of vertebrate TNFR family proteins. Furthermore, PmTNFR5 had a death domain at the C-terminal, indicating that this protein may be a novel member of the TNFR superfamily. Constitutive PmTNFR1 and PmTNFR5 mRNA expression was detected in all six pearl oyster tissues tested, with comparatively greater transcript abundance in the hepatopancreas and gill. The gene expression levels of PmTNFR1 and PmTNFR5, as well as those of downstream signaling molecules related to the NF-κB pathway (RIP, TRAF2, TRAF3, IKK, and NF-κB), were quantified in the gill after LPS challenge and in the hemocytes after nucleus insertion surgery using real-time PCR (qRT-PCR). We found that all genes were significantly upregulated at 6 h and 12 h post-injection, as well as at 15 d post-insertion. We used RNAi to inhibit the expression of the PmTNFR1 and PmTNFR5 genes. We then quantified the expression levels of PmTNFR1 and PmTNFR5, as well as downstream genes, using qRT-PCR. We found that RNAi inhibition of PmTNFR1 and PmTNFR5 downregulated the downstream genes (RIP, TRAF2, TRAF3, IKK, and NF-κB). Therefore, our results suggested that PmTNFR1 and PmTNFR5 mediate the NF-κB signaling pathway, and are closely related to immune defense, particularly allograft immunity, in the pearl oyster P. fucata martensii.

中文翻译：

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珍珠牡蛎Pinctada fucata martensii的两种TNFR的分子克隆，表征和表达。

肿瘤坏死因子受体（TNFR）超家族中的蛋白质在许多生理和病理事件中起重要作用，例如炎症，凋亡，自身免疫和器官发生。在这里，在珍珠贝牡蛎Pinctada fucata martensii中鉴定出两个TNFR基因同源物（PmTNFR1和PmTNFR5）。预测的PmTNFR1和PmTNFR5蛋白序列分别长406和533个氨基酸，并且都具有TNFR家族的特征基序，包括TNFR同源结构域（CRD），跨膜结构域（TM）和死亡结构域。但是，PmTNFR1和PmTNFR5的预测氨基酸序列与脊椎动物TNFR家族蛋白的序列具有较低的同一性（〜16-23％）。此外，PmTNFR5在C末端有一个死亡域，表明该蛋白可能是TNFR超家族的新成员。在所有测试的六个牡蛎组织中均检测到组成型PmTNFR1和PmTNFR5 mRNA表达，肝胰腺和腮中的转录本丰度相对较高。在LPS攻击后的腮中和在细胞中，定量了PmTNFR1和PmTNFR5以及与NF-κB通路相关的下游信号分子（RIP，TRAF2，TRAF3，IKK和NF-κB）的基因表达水平。使用实时PCR（qRT-PCR）进行核插入手术后的血细胞。我们发现，所有基因在注射后6 h和12 h以及插入后15 d均显着上调。我们使用RNAi来抑制PmTNFR1和PmTNFR5基因的表达。然后，我们使用qRT-PCR量化了PmTNFR1和PmTNFR5以及下游基因的表达水平。我们发现RNAi对PmTNFR1和PmTNFR5的抑制下调了下游基因（RIP，TRAF2，TRAF3，IKK和NF-κB）。因此，我们的结果表明，PmTNFR1和PmTNFR5介导了珍珠牡蛎P. fucata martensii的免疫防御，尤其是同种异体移植免疫，与NF-κB信号通路密切相关。