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| Cloning and Activity Analysis of Promoter of Bovine Stearoyl-CoA Desaturase Gene(SCD) |
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Abstract Stearoyl-CoA desaturase(SCD) is the rate-limiting enzyme of mono-unsaturated fatty acids synthesis. To investigate the transcriptional regulation of bovine SCD gene, 4 fragment (212, 380, 416 and 760 bp) of promoter of bovine SCD were amplified from the Holstein bovine tissue by PCR using primers containing a recognition site. The potential transcriptional biding sites were predicted with bioinformatics method. Promoter fragments were cloned and ligated with a promoter luciferase reporter vector PGL3-basic. Both fragments and vectors were digested with KpnⅠ and XhoⅠ and constructed recombinant vectors pGL3-SCD1, pGL3-SCD2, pGL3-SCD3 and pGL3-SCD4.The promoter activities were determined in transiently transfected HepG2 cell treating with Insulin(10 IU/mL)and linoleic acid(120 μm) by the dual-luciferase assay system. The results indicated that binding sites for transcription factors of bovine sterol-regulated element-binding protein were present in the bovine SCD promoter gene. There was a significantly promoter activity increase with the enlargement of the SCD promoter vector length and pGL3-SCD4 showed the highest level of promoter activity. Compared with the control group, supplementing with insulin significantly increased the promoter activity of pGL3-SCD1~4 vectors in HepG2 cell (P<0.05) and the relative activity of pGL3-SCD4 was the highest of 46.93 in the four vectors. The promoter activities of pGL3-SCD1~3 were not significantly affected by linoleic acid and the activity of pGL3-SCD4 was reduced (P<0.05) with the linoleic acid treatment.These results demonstrated that the region from -398 to -742 bp of promoter pGL3-SCD4 was important for the regulation of SCD gene expression.
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Received: 05 September 2012
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ZHAO Zhi-Dong, TIAN Hong-Shan, JIANG Yan-Yan, SHI Bin-Gang, LIU Xiu, LI Xu-Peng, WANG Deng-Zhe, CHEN Jin-Lin, HU Jiang. Polymorphisms of ACSL1 Gene Promoter and Their Association Analysis with Milk Quality Traits in Yak (Bos grunniens)[J]. 农业生物技术学报, 2019, 27(9): 1596-1603. |
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