Functional Identification of Minimal Promoter Region of Elongation of Very Long Chain Fatty Acids 2 (ELOVL2) Gene in Chicken Hepatocytes
Jakkoju Theja Sri *
PVNRTVU, College of Veterinary Science, Animal Genetics and Breeding division, Rajendranagar, Telangana, India.
Durgam Sakaram
PVNRTVU, College of Fishery Science, Pebbair, Telangana, India.
Samala Sai Reddy
PVNRTVU, College of Veterinary Science, Animal Genetics and Breeding division, Rajendranagar, Telangana, India.
Suresh Rathod
PVNRTVU, College of Dairy Technology, Kamareddy, Telangana, India.
Devara Divya
ICAR-CIRC, Meerut, U.P, India.
Tarun kumar Bhattacharya
ICAR-NRCE, Hissar, Haryana, India.
*Author to whom correspondence should be addressed.
Abstract
Background: The enzyme elongation of very long chain fatty acids protein 2 (ELOVL2) plays a crucial role in the long-chain fatty acid elongation cycle. The chicken is known to contain abundant quantities of PUFAs that are suitable for human consumption and is regarded as a perfect model species for studying the synthesis of PUFAs. However, the molecular processes controlling the synthesis of PUFAs in chickens need to be further explored, and a better understanding of ELOVL2 regulation may provide information useful for identifying regulatory genetic variants associated with fatty-acid composition and other economically important traits in chickens. Identification of the minimal promoter region will provide a basis for understanding the transcriptional regulation of ELOVL2 and its potential contribution to fatty-acid metabolism so that the function of the gene may be altered to obtain a desired phenotype.
Aim: For this purpose, an in vitro cell culture experiment was conducted. This investigation was carried out with the aim of identifying the minimal promoter region of ELOVL2 gene.
Place and Duration of Study: The study was carried out at ICAR-Directorate of Poultry Research, Hyderabad, during 2021-2022.
Methodology: 0.5 to 1.0 mL of blood was collected from the wing vein of a control-layer chicken and genomic DNA was isolated using the standard phenol-chloroform method. PCR was performed to amplify two overlapping ELOVL2 promoter fragments of 587 bp and 1558 bp using specific primers, cloned into the reporter vector pAcGFP1-1 (promoterless) with GFP as a marker protein, and then transformed into DH5α competent cells. The pAcGFP1-1 recombinant plasmid was transfected into a primary chicken embryo hepatocyte culture to check functionality, which was reflected by the expression level of GFP protein in hepatocyte cells.
Results: The expression of GFP in chicken embryo hepatocytes was evaluated through real-time PCR. A 1.19-fold higher GFP expression was observed in cells with the 1558 bp fragment as the promoter than in cells with the 587 bp fragment.
Conclusion: Thus, it can be concluded that both the promoter fragments of ELOVL2 are capable of regulating gene expression even in a cell culture system with the larger fragment being more effective. This identified promoter region can be used as a target in further research involving promoter mutations, transcription-factor analysis, gene regulation, and functional characterization of ELOVL2. The observed findings may also support future research in poultry nutrition, lipid metabolism, functional genomics, and molecular breeding.
Keywords: ELOVL2, minimal promoter, chicken embryo hepatocytes, promoter activity, GFP reporter, fatty acid elongation, polyunsaturated fatty acids, transcriptional regulation, primary cell culture, reporter assay