| 摘要: |
| 作为无颌类脊椎动物的现存代表之一,日本七鳃鳗(Lampetra japonica)是研究免疫系统起源与进化的重要模型。为了探究TGF-β(Transforming growth factor beta)信号通路在日本七鳃鳗免疫调节中的功能,本研究利用PCR技术克隆了日本七鳃鳗TGF-β I型受体基因(L-Tgfbr1)的编码序列,开放阅读框长度为1 335 bp,编码444个氨基酸残基。L-Tgfbr1蛋白含有已知的TGF-β I型受体分子的主要功能结构域,其中位于胞内的丝/苏氨酸激酶催化结构域保守性较高。系统进化树分析表明,L-Tgfbr1处于脊椎动物Tgfbr1蛋白的底端进化枝上,表明其在Tgfbr1进化史中具有原始性地位。实时定量PCR结果发现,L-Tgfbr1在心脏等组织中的转录水平较高。利用脂多糖注入七鳃鳗激活其先天性免疫应答,L-Tgfbr1在肾脏、鳃、髓小体、肝脏、白细胞、口腔腺中的转录水平呈现一过性的迅速上调。利用免疫印迹进一步验证了脂多糖免疫24 h时后L-Tgfbr1在白细胞中的蛋白表达水平显著上调。利用免疫荧光染色发现L-Tgfbr1蛋白主要分布于七鳃鳗白细胞和髓小体细胞的细胞质中。以上结果表明L-Tgfbr1及其介导的TGF-β通路可能在七鳃鳗免疫调控中发挥重要功能。 |
| 关键词: 日本七鳃鳗 转化生长因子βⅠ型受体 基因克隆 基因表达 |
| DOI：10.11759/hykx20201228004 |
| 分类号:Q78;Q17 |
| 基金项目:国家自然科学基金项目(31601150);辽宁省大学生创新创业训练计划项目(201910165242) |
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| Cloning and expression analysis of transforming growth factor β type I receptor gene (L-Tgfbr1) of Lampetra japonica |
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HOU Yun-xuan1, LI Wen-na1,2, YANG Xiao-xuan1, LEI Li-tong1, ZHANG Dai-yun1, MA Chang-wei1, WANG Hao1,2
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1.School of Life Science, Liaoning Normal University, Dalian 116081, China;2.Lamprey Research Center, Liaoning Normal University, Dalian 116081, China
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| Abstract: |
| As one of the extant representatives of jawless vertebrates, the Japanese lamprey (Lampetra japonica) is an essential model for studying the origin and evolution of the immune system. The TGF-β (Transforming growth factor beta) signaling pathway plays an important regulatory role in the immune response. As a key component in the evolution of the TGF-β signaling pathway, the TGF-β pathway in lamprey has rarely been reported. Therefore, it is significant for the systematic characterization and evolutionary analysis of the members of this pathway in lamprey. TGF-β type I receptor is one of the key receptor molecules in the TGF-β signaling pathway. In this study, the coding sequence of the TGF-β type I receptor gene (L-TβR I) of Japanese lamprey was cloned by PCR. The open reading frame was 1335 bp in length and encoded 444 amino acid residues. The L-Tgfbr1 protein contained the main functional domains of known TGF-β type I receptor molecules. Multiple sequence alignment showed that the amino acid sequence of L-Tgfbr1 had high similarity compared to representative species of mammals, birds, bony fishes and cartilaginous fishes. Compared with the extracellular domains, both the GS domain and the serine/threonine kinase catalytic domain of L-Tgfbr1 protein were significantly conserved, which were two key intracellular domains involved in receptor signal transduction. Phylogenetic tree analysis showed that L-Tgfbr1 was located on the bottom clade of all vertebrate Tgfbr1 proteins and was more closely related to the bony fishes. The position of L-Tgfbr1 in the phylogenetic tree was consistent with the evolutionary position of the lamprey as the most primitive vertebrate. Furthermore, the real-time quantitative PCR found that L-Tgfbr1 was widely expressed in various tissues, with higher expression in the heart, gonads, oral glands, leukocytes and liver. After lipopolysaccharide (LPS) was injected into lampreys for activating the innate immune response, the transcription levels of L-Tgfbr1 in the kidney, gills, supraneural myeloid body, liver, leukocytes, and oral glands were transiently and rapidly up-regulated. Western blotting further verified that the protein expression level of L-Tgfbr1 in leukocytes was significantly up-regulated after 24 hours of LPS immunization. Immunofluorescence staining revealed that L-Tgfbr1 protein was mainly distributed in the cytoplasm of lamprey leukocytes and myeloid body cells. Our results would contribute to further revealing the essential roles of L-Tgfbr1 in the immune regulation of lamprey, and provide valuable clues about the origin and evolution of TGF-β receptor in vertebrates. |
| Key words: Japanese lamprey transforming growth factor β type I receptor gene cloning gene expression |
