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| Eukaryotic Expression Vector Construction and Promoter Transcription Efficiency Analysis of Chicken (Gallus gallus) Chemerin Gene |
| WANG Peng, YIN Xiao-Hang, XIE Yao-Yao, CHEN Man-Yu, ZHANG Jing, LU Qing-Shuang, QIAN Dan-Feng, XU Lu, YU Jian-Feng, GU Zhi-Liang* |
| School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, China |
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Abstract Chemerin is an adipocytokine secreted by adipose tissue, which can regulate biological processes such as adipocyte generation and metabolism. In this study, the total RNA of chicken (Gallus gallus) liver tissue was used as the template, and the full-length Chemerin cDNA sequence of chicken was amplified by reverse transcription PCR (RT-PCR) and rapid amplified cDNA ends (RACE). qPCR was used to detect the expression of Chemerin gene in different tissues. The eukaryotic expression vector of Chemerin-Myc fusion protein was constructed. The transcription efficiency of chicken Chemerin gene promoter was assessed by dual luciferase reporter vectors with various promoter fragments. The results showed that the full length of the cDNA was 733 bp, encoding 162 amino acids, including a 55 bp 5'UTR and a 189 bp 3'UTR. Chemerin gene was mainly expressed in liver, kidney and adipose tissue. The eukaryotic expression vector pCMV-3Tag-9-gga-chemerin-Myc was successfully constructed. After transfection into chicken Leghorn male hepatoma (LMH) cells, fusion protein of Myc and Chemerin was detected by Western blot. The 4 constructed pGL3b-chemerin-F1/F2/F3/F4 vectors all showed that the luciferase activity was very significantly increased compared with the control group (P<0.01), and the promoter transcription efficiency of F1 and F3 was the highest. The luciferase activity of pGL3b-chemerin-mutF1 was very extremely significantly decreased after the site-directed mutation of Kruppel-like factor 5 (KLF5) binding site in pGL3b-chemerin-F1 (P<0.001). After the interference of KLF5 gene expression in LMH cells, the mRNA expression of Chemerin gene significantly decreased (P<0.01), which implied that there was a cis-element with positive regulator KLF5 binding in F1 fragment. These results provide basic data for further study on the function of Chemerin gene in chicken.
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Received: 07 January 2021
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Corresponding Authors:
* zhilianggu88@hotmail.com
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