Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (24): 4797-4805.doi: 10.3864/j.issn.0578-1752.2016.24.011


Expression and Localization of Bovine SREBP1 Protein and Regulation of the Transcription of SCD1 Promoter in Bovine Mammary Epithelial Cell

HAN Li-qiang, WANG Yue-ying, WANG Lin-feng, ZHU He-shui, ZHONG Kai, CHU Bei-bei, YANG Guo-yu   

  1. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Key Laboratory of Animal Biochemistry and Nutrition of Ministry of Agriculture, Zhengzhou 450002
  • Received:2015-07-29 Online:2016-12-16 Published:2016-12-16

Abstract: 【Objective】As a member of nuclear transcription factor, sterol regulatory element binding protein1(SREBP1)plays a significant role in the expression of the lipogenic gene . The objective of this study is to investigate the effect of SREBP1 on the regulation of transcription of SCD1 gene in the mammary epithelial cells, which will provide a fundamental basis for the transcription regulation mechanism of SREBP1 to target gene. 【Method】 The coding sequence (CDS) of SREBP1 gene was cloned using the method of subsection cloning with the cDNA of Holstein mammary tissues as the template. The expression vector pcDNA3.1-SREBP1 was recombined and constructed by recombining vector pcDNA3.1. Plasmids were extracted from the constructed vector after being verified by sequencing, and transfected to mammary epithelial cells. With gene EIF3K as the reference gene, the expressional difference of mRNA in gene SREBP1 was detected with Real-time PCR. SREBP1 was marked with the immunofluorescence method. The cell nucleus was restained with DAPI, and the subcellular localization of protein SREBP1 was observed with laser con-focal microscopy. The SCD1 gene promoters with different regulatory elements and 1.0 µg pcDNA3.1- SREBP1 were restained as treatments, and the promoter activity was analyzed systematically with luciferase reporter gene. Vectors pcDNA3.1-SREBP1 of 0.25µg, 0.5µg and 1µg were transfected respectively. The dose-effect relationship between the promoter activity of pGL3-SCD2, pGL3-SCD3 and protein SREBP1 was analyzed. 【Result】 The PCR products cloned in subsection were fragments 1170bp, 1116bp, 363bp and 900 bp respectively. The expression vector pcDNA3.1-SREBP1 was obtained by reconstructing the fragments and vector pcDNA3.1. Confirmed by digestion and sequence, the expression vector, except one nonsense mutation, was exactly the same with the standard sequence. The whole sequence length reached 3510 bp. After vector pcDNA3.1-SREBP1 was transfected into mammary epithelial cells, compared with the control group of the empty transfected vector, the expression mRNA of gene SREBP1 increased by 130.4 times (P<0.001)by the real-time PCR detection. As shown in the observation of the laser con-focal microscopy, the cell nucleus dyed with DAPI was blue, and SREBP1 with immunofluorescent labeling was green, both of which were cyan after being merged and were localized in the mammary epithelial cell nucleus. The results of the detection of the promoter activity showed that, compared with those of pGL3-SCD1 and pGL3SCD 2, the treatment with SREBP1 increased the promoter activity of pGL3-SCD3 and pGL3-CD4 significantly (P<0.001), and their promoter activity increased by 1.0 and 0.7 times. The further study showed that, after treatment with 0.25-1µg pcDNA3.1-SREBP1, compared with the continuous decrease of the promoter activity of pGL3-SCD2, the promoter activity of pGL3-SCD3 increased from 59.81 to 108.43(P<0.001). There is a dose-effect relationship between them. Combined with the element SRE (5′-AGCAGATTGCG-3′), the architectural difference between promoter SCD2 and promoter SCD 3, it could be speculated that the sequence was the incorporating sequence in which SREBP1 regulated the transcription of gene promoter SCD1. 【Conclusion】 It was verified that the gene expression vector SREBP1 was constructed by cloning, the subcellular localization of protein SREBP1 was in the mammary epithelial cell nucleus, and SREBP1 promoted the regulation of the transcription of SCD1 promoter together with regulatory element SRE. 

Key words: bovine, SREBP1, mammary epithelial cell, subcellular localization, gene regulation

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