半滑舌鳎TLR5S三种剪切型基因的克隆与表达分析  

Molecular cloning,characterization,and expression of three TLR5S splicing variants in half-smooth tongue sole

作　　者：张文婷[1,2,3] 向晋松[2,3,4] 李海龙[2,3] 张宁[2,3] 董忠典[2,3] 高峰涛[2,3] 陈松林[2,3] 

机构地区：[1]大连海洋大学水产与生命学院,辽宁大连116023 [2]中国水产科学研究院黄海水产研究所农业部海洋渔业资源重点实验室,山东青岛266071 [3]青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室,山东青岛266071 [4]上海海洋大学水产与生命学院,上海201306

出　　处：《中国水产科学》2016年第1期10-20,共11页Journal of Fishery Sciences of China

基　　金：国家自然科学基金重点项目(31530078);山东省泰山学者攀登计划专项

摘　　要：Toll样受体5(Toll-like receptor 5,TLR5)是TLRs家族成员之一,可分为跨膜型TLR5M和鱼类特有的可溶型TLR5S,它们可以识别致病菌表面的鞭毛蛋白并协同作用激活免疫反应。为了研究半滑舌鳎受到病原感染后TLR5S参与免疫反应的作用,本研究使用RACE技术获得了半滑舌鳎(Cynoglossus semilaevis)TLR5S全长c DNA序列。TLR5S c DNA有3种剪切型:Cs.TLR5S x1,Cs.TLR5S x2和Cs.TLR5S x3。这3种剪切型的相同区域为308 bp 5′非编码区(5′UTR)和1701 bp开放阅读框(ORF),不同的3′UTR分别为138 bp、364 bp和637 bp。Cs.TLR5S共编码567个氨基酸,预测编码蛋白质分子量为64.03 k D,等电点为8.49。氨基酸多重序列比对结果显示,Cs.TLR5S氨基酸序列与其他脊椎动物TLR5S氨基酸序列具有较高的相似性,其中与牙鲆相似度高达61%,表明Cs.TLR5S在进化上的具有一定的保守性。Real-time PCR结果表明该基因在半滑舌鳎的不同组织均有表达,其中在肝的表达量最高,在脾的表达量最低。此外,检测Cs.TLR5S 3′端的不同剪切型在肝、脾、头肾、小肠中的表达,结果显示Cs.TLR5S x3只在肝中高表达,而Cs.TLR5S x1则在肝和小肠中都有中等程度表达。鳗弧菌(Vibrio anguillarum)感染半滑舌鳎实验表明,注射菌液6 h后,Cs.TLR5S基因在肾、小肠、肝和脾4个组织中的表达量都有显著上升;注射鳗弧菌48 h后,以上4种组织中表达量均呈现降低的变化。上述实验结果说明,Cs.TLR5S基因可能参与了半滑舌鳎抗弧菌感染的免疫反应。Toll-like receptor （TLR） 5 plays a vital role in bacterial flagellin recognition and immune response alerts in vertebrates. In the present study, the synergistic role of the TLR5 membrane form and the TLR5 soluble form （TLRSS） are reported in a bony fish. The full-length TLR5S cDNA was cloned using homologous cloning and rapid amplification of cDNA ends techniques to study the regulatory role of TLRSS in the innate immune re- sponse of half-smooth tongue sole, Cynoglossus semilaevis, （designated Cs.TLR5S）. Three alternative splicing variants （Cs.TLRSS xl, Cs.TLRSS x2, and Cs.TLRSS x3） of the Cs.TLRSS cDNA sequence were found in C. semi- laevis. The full-length CsTLRSS cDNA included a 308 bp 5＇-untranslated region （UTR）, a 1701 bp open reading frame, and 138 bp, 364 bp, and 637 bp 3＇-UTRs, respectively. The cDNA encoded a polypeptide of 567 amino acids, with a molecular mass of 64.03 kD and an isoelectric point of 8.49. Multiple sequence alignment revealed that the Cs.TLR5S proteins are well conserved with a typical modular architecture and identical active sites throughout vertebrates, and shared the highest identity with Paralichthys olivaceus TLR5S （61%）, suggesting a conserved function for TLR5S. A phylogenetic analysis indicated that Cs.TLR5S and homologous TLR5S se- quences from teleosts were clustered into a clade, and Cs. TLR5S was separated from another clade with amphibi- ans, mammals, and other vertebrates. A tissue expression profile analysis using the quantitative real-time poly- merase chain reaction （qRT-PCR） showed that Cs. TLRSS mRNA was constitutively expressed in all tested tissues, with predominant expression in liver and the lowest expression in spleen. Alternative splicing of the 3＇-UTR using qRT-PCR showed that Cs. TLR5S x3 was only expressed in liver, whereas Cs. TLR5S xl was expressed in liver and intestine. In addition, Cs. TLRSS was expressed at different levels in liver, spleen, intestine, and head kidney after a Vibrio anguillarum challenge. These resu

关 键 词：半滑舌鳎 TLR5S 鳗弧菌 基因克隆 表达 

分 类 号：S917[农业科学—水产科学]