鸭TLR4基因可变剪接体的克隆、鉴定及组织表达分析

摘要

This experiment was conducted to clone and identify the spliced variants of the TLR4 gene,and analyze the regulation of their gene expression in duck.Based on the sequence of TLR4 gene in GenBank(accession number:JN048668),the primers were designed,and the TLR4 gene was obtained by the RT-PCR.The temporal and spatial expression pattern was detected by qRTPCR to analysis the levels of transcript variants in different tissues by constructing the polyI:C infection duck model.One new transcript variants(TLR4-b) of duck TLR4 gene was found,and sequences analysis showed that it was an alternative exon between the first and the second exon.The tissue expression analysis showed that the expression of TLR4 was mainly wild type(TLR4a).And the expression of TLR4-a mRNA always increased firstly,then decreased in lung and spleen after the stimulation of ployI:C,but the expression of TLR4-b had little change.The new splicing isoforms(TLR4-b) of TLR4 gene was cloned and identified sucessfully in duck.The expression of TLR4-a was higher than that of TLR4-b under normal conditions,but the expression level of TLR4-a gene firstly increased,then decreased after ployI:C stimulation.The results preliminary reveal the structure of TLR4 gene transcript variant in duck,and its function mainly depends on TLR4-a gene.%本研究旨在克隆、鉴定鸭TLR4基因的可变剪接体,并对TLR4基因的可变剪接体表达规律进行分析.根据GenBank中鸭TLR4基因的mRNA序列(登录号:JN048668)设计引物,利用RT-PCR扩增鸭CDS区,以获取TLR4可变剪接体;并通过构建雏鸭PolyI:C感染模型,利用qRT-PCR检测TLR4可变剪接体的时空表达变化.结果,获得一种鸭TLR4基因新的可变剪接体(TLR4-b),并鉴定其剪接形式为跨内含子剪接.组织表达分析表明,TLR4基因的表达主要以野生型(TLR4-a)为主,且在PolyI:C等抗原刺激后,肺、脾脏等组织中TLR4-a mRNA表达量总体表现为先上升,后下降,而TLR4-b表达变化不大.本研究成功克隆并鉴定了鸭TLR4基因新的可变剪接体,在正常情况下TLR4-a表达量显著高于TLR4-b,在PolyI:C感染下,TLR4-a表现为先上升,后下降.研究结果初步揭示了鸭TLR4基因可变剪接体的结构,且其功能主要取决于TLR4-a.