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半滑舌鳎TLR5S三种剪切型基因的克隆与表达分析 [中文引用][英文引用]

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 
作者(英文):ZHANG Wenting1 2 3  XIANG Jinsong2 3 4  LI Hailong2 3  ZHANG Ning2 3  DONG Zhongdian2 3  GAO Fengtao2 3  CHEN Songlin2 3 
关键词(英文):Cynoglossus semilaevis  TLR5S  Vibrio anguillarum  gene cloning  expression 
分类号:S917
出版年·卷·期(页码):2016·23·第1期(10-20)
DOI: 10.3724/SP.J.1118.2016.15073
-----摘要:-------------------------------------------------------------------------------------------

Cynoglossus semilaevis)TLR5S全长cDNA序列。TLR5ScDNA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 kD,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。

-----英文摘要:---------------------------------------------------------------------------------------

TLR5S cDNA was cloned using homologous cloning and rapid amplification of cDNA ends techniques to study the regulatory role of TLR5S in the innate immune response of half-smooth tongue sole, Cynoglossus semilaevis, (designated Cs.TLR5S). Three alternative splicing variants (Cs.TLR5S x1, Cs.TLR5S x2, and Cs.TLR5S x3) of the Cs.TLR5S cDNA sequence were found in C. semilaevis. The full-length CsTLR5S 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 sequences from teleosts were clustered into a clade, and Cs.TLR5S was separated from another clade with amphibians, mammals, and other vertebrates. A tissue expression profile analysis using the quantitative real-time polymerase chain reaction (qRT-PCR) showed that Cs.TLR5S 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 x1 was expressed in liver and intestine. In addition, Cs.TLR5S was expressed at different levels in liver, spleen, intestine, and head kidney after a Vibrio anguillarum challenge. These results suggest that expression of the C. semilaevis Cs.TLR5S variants are differentially regulated in different tissues and play important roles in the immune response against bacterial pathogens.

-----参考文献:---------------------------------------------------------------------------------------

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中文著录格式: 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.半滑舌鳎TLR5S三种剪切型基因的克隆与表达分析.中国水产科学.2016;23(1):10-20.
英文著录格式: ZHANG,Wenting1,2,3,,XIANG,Jinsong2,3,4,,LI,Hailong2,3,,ZHANG,Ning2,3,,DONG,Zhongdian2,3,,GAO,Fengtao2,3,,CHEN,Songlin2,3.Molecular cloning, characterization, and expression of three TLR5S splicing variants in half-smooth tongue sole.No Title Settings.2016;23(1):10-20.

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