Cloning, Expression and Promoter Analysis of Adipokine NRG4 Gene in Chicken (Gallus domesticus)
GUO Ya-Qi1,2,3, WANG Wei-Jia1,2,3, GAO Zhi-Hui1,2,3, MU Fang1,2,3, XU Hai-Dong1,2,3, LI Hui1,2,3, WANG Ning1,2,3,*
1 Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Harbin 150030, China; 2 Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China; 3 College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China
Abstract：Neuregulin-4 (NRG4) is a cytokine highly expressed in mammalian brown adipose tissue. NRG4 is involved in the regulation of energy balance and glucolipid metabolism through the activation of ErbB4 signaling pathway. To date, due to the lack of brown adipose tissue in chicken (Gallus domesticus), the tissue expression and function of NRG4 gene in chickens have not been investigated. In this study, the full-length coding sequence of chicken NRG4 gene was amplified by PCR from the cDNA of chicken adipose tissue, and cloned to yield the NRG4 expression vector. Expression profiling of NRG4 gene was performed using qPCR on Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). Four promoter fragments of NRG4 gene were amplified by PCR from the chicken genomic DNA, cloned, sequenced and analyzed by bioinformatics. A series of NRG4 promoter reporters were constructed and their promoter reporter activities were determined and compared. The results showed that chicken NRG4 gene encoded a protein of 112 amino acids and shared low amino acid identity with human (Homo sapiens) and mouse (Mus musculus) NRG4, 57.76% and 52.59%, respectively. Tissue expression profiling analysis showed that NRG4 gene was widely expressed in various chicken tissues, with the highest expression in the kidney and the lowest expression in the spleen. Expression analysis of NRG4 gene in NEAUHLF showed that NRG4 gene expression in crop fat, abdominal fat and gizzard fat was significantly higher in the fat line than that in the lean line (P<0.01 or P<0.05). Reporter gene assays showed that compared with the empty vector pGL3-basic, pGL3-NRG4 (-2854/-2076) and pGL3-NRG4 (-1451/+117) had high promoter activity (P<0.01 or P<0.05). This study paves the way for further studies on the role and mechanism of chicken NRG4 in adipogenesis and adipose tissue development.
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