日本七鳃鳗转化生长因子βⅠ型受体基因(L-Tgfbr1)的克隆与表达分析  

Cloning and expression analysis of transforming growth factor β type Ⅰ receptor gene(L-Tgfbr1)of Lampetra japonica

作　　者：侯蕴轩 李文娜 杨晓萱 雷丽桐 张代云 马畅蔚 王浩[1,2] 无 HOU Yun-xuan;LI Wen-na;YANG Xiao-xuan;LEI Li-tong;ZHANG Dai-yun;MA Chang-wei;WANG Hao;无(School of Life Science,Liaoning Normal University,Dalian 116081,China;Lamprey Research Center,Liaoning Normal University,Dalian 116081,China;Collaborative Innovation Center of Seafood Deep Processing,Dalian Polytechnic University,Dalian 116081,China)

机构地区：[1]辽宁师范大学生命科学学院,辽宁大连116081 [2]辽宁师范大学七鳃鳗研究中心,辽宁大连116081 [3]大连工业大学海洋食品精深加工关键技术省部共建协同创新中心,辽宁大连116081

出　　处：《海洋科学》2023年第1期88-98,共11页Marine Sciences

基　　金：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)。

摘　　要：作为无颌类脊椎动物的现存代表之一,日本七鳃鳗(Lampetrajaponica)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-βⅠ型受体基因(L-Tgfbr1)的编码序列,开放阅读框长度为1335 bp,编码444个氨基酸残基。L-Tgfbr1蛋白含有已知的TGF-βⅠ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。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-βsignaling pathway,the TGF-βpathway 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-βtype I receptor is one of the key receptor molecules in the TGF-βsignaling pathway.In this study,the coding sequence of the TGF-βtype 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-βtype 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

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 

分 类 号：Q78[生物学—分子生物学] Q17