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Molecular Cloning and Functional Analysis of the Duck TLR4 Gene
Jiangsu Key Laboratory for Animal Genetic, Breeding and Molecular Design, Yangzhou University, Yangzhou 225009, Jiangsu, China
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed.
Received: 10 August 2013; in revised form: 26 August 2013 / Accepted: 29 August 2013 / Published: 10 September 2013
Abstract: Toll-like receptor 4 (TLR4) recognizes pathogen-associated molecular patterns in some animals and has been shown to be closely associated with several diseases such as tumors, atherosclerosis, and asthma. However, its function in ducks is not clear. Alternative splicing of the TLR4 gene has been identified in pigs, sheep, mice, and other species, but has not yet been reported in the duck. In this study, alternative splicing of the duck TLR4 gene was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Duck TLR4 gene (duTLR4, accession number: KF278109) was found to consist of 3367 nucleotides of coding sequence. An alternative splice form, TLR4-b, was identified and shown by alignment to retain the intron between exons 1 and 2. Real-time quantitative polymerase chain reaction (qPCR) analyses suggested that duTLR4-a (wild-type) mRNA is widely expressed in various healthy tissues, whereas TLR4-b is expressed at only low levels. Following stimulation of normal duck embryo fibroblasts with lipopolysaccharide, the expression of both isoforms initially increased and then decreased. Expression of the wild-type isoform subsequently increased again, while that of the variant remained low. The expression levels of wild-type TLR4 were further analyzed by transient transfection of a pcDNA3.1(+)-TLR4-a overexpression vector into duck embryo fibroblasts. qRT-PCR analyses showed that after stimulation with LPS and poly(I:C) the expression levels of IL-1β, IL6, and MHC II increased with a response-efficacy relationship. Our experimental results indicate that TLR4 plays an important role in resistance to both bacterial and viral infections in the duck.
Keywords: duck; TLR4; alternative splicing; expression analysis
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MDPI and ACS Style
Zhao, W.; Huang, Z.; Chen, Y.; Zhang, Y.; Rong, G.; Mu, C.; Xu, Q.; Chen, G. Molecular Cloning and Functional Analysis of the Duck TLR4 Gene. Int. J. Mol. Sci. 2013, 14, 18615-18628.
Zhao W, Huang Z, Chen Y, Zhang Y, Rong G, Mu C, Xu Q, Chen G. Molecular Cloning and Functional Analysis of the Duck TLR4 Gene. International Journal of Molecular Sciences. 2013; 14(9):18615-18628.
Zhao, Wenming; Huang, Zhengyang; Chen, Yang; Zhang, Yang; Rong, Guanghui; Mu, Chunyu; Xu, Qi; Chen, Guohong. 2013. "Molecular Cloning and Functional Analysis of the Duck TLR4 Gene." Int. J. Mol. Sci. 14, no. 9: 18615-18628.