猪DKK1基因启动子区的克隆及其活性分析

doi:10.11843/j.issn.0366-6964.2017.06.020

Abstract

Abstract:

To further investigate the transcriptional regulatory mechanism of DKK1(Dickkopf1) gene, the sequence features were analyzed by promoter online prediction tools, which were based on the 5'-flanking sequence of swine DKK1 gene published by Ensembl database. Specific primers were designed by Primer Premier 5.0 software to amplify DKK1 gene. To analyze its transcriptional activity, pGL3-DKK1 promoter luciferase reporter gene vectors were constructed and transfected into 293T cells and Hela cells, respectively. The results showed that the promoter region of DKK1 gene contained a TATA-box, a variety of transcription factors and a CpG island. Meanwhile, the promoter of DKK1 gene had a preference for 239T cells, and the sequence of -1 679/+292 bp had the highest promoter activity, which was obviously higher than that of other fragments (P<0.01). Further analysis revealed that there were core promoter region (-953/-1 679), negative (-586/-953) and positive (-953/-1 679) regulatory regions, respectively. The 5'-flanking sequence of swine DKK1 was analyzed by bioinformatics combined with the reporter gene activity detection of promoter fragments with different length, this result demonstrated that it had transcriptional activity of promoter, and its promoter region was preliminarily determined, and the core promoter region and the main regulatory region were successfully identified, which laid a foundation for further studying on the transcriptional regulatory mechanism of the DKK1.

CLC Number:

S828
Q785

HU Hui-yan, JIA Qing, HOU Sheng-kui, LIU Jin, ZHAO Si-si, ZHANG Wei-feng, ZHANG Jun, ZHANG Jian-ting, BIAN Hui-min. Cloning and Activity Analysis of the Promoter Region of Swine DKK1 Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1150-1157.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2017.06.020

https://www.xmsyxb.com/EN/Y2017/V48/I6/1150

References

[1] BROTT B K, SOKOL S Y. Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins[J]. Mol Cell Biol, 2002, 22(17):6100-6110.
[2] CAI T, SUN D Q, DUAN Y, et al. WNT/β-catenin signaling promotes VSMCs to osteogenic transdifferentiation and calcification through directly modulating Runx2 gene expression[J]. Exp Cell Res, 2016, 345(2):206-217.
[3] MAO B Y, WU W, DAVIDSON G, et al. Kremen proteins are Dickkopf receptors that regulate Wnt/β-catenin signalling[J]. Nature, 2002, 417(6889):664-667.
[4] PHILLIPS M D, MUKHOPADHYAY M, POSCABLO C, et al. Dkk1 and Dkk2 regulate epicardial specification during mouse heart development[J]. Int J Cardiol, 2011, 150(2):186-192.
[5] NALESSO G, SHERWOOD J, BERTRAND J, et al. WNT-3A modulates articular chondrocyte phenotype by activating both canonical and noncanonical pathways[J]. J Cell Biol, 2011, 193(3):551-564.
[6] DESERT R, MEBARKI S, DESILLE M, et al. "Fibrous nests" in human hepatocellular carcinoma express a Wnt-induced gene signature associated with poor clinical outcome[J]. Int J Biochem Cell Biol, 2016, 81:195-207.
[7] LI J, SAROSI I, CATTLEY R C, et al. Dkk1-mediated inhibition of Wnt signaling in bone results in osteopenia[J]. Bone, 2006, 39(4):754-766.
[8] JIN H T, WANG B L, LI J, et al. Anti-DKK1 antibody promotes bone fracture healing through activation of β-catenin signaling[J]. Bone, 2015, 71:63-75.
[9] 刘亚龙, 武宇赤, 刘国栋, 等. DKK1在大鼠骨髓间充质干细胞成骨、成脂分化早期的差异性表达[J]. 中国骨质疏松杂志, 2014, 20(11):1290-1297.
LIU Y L, WU Y C, LIU G D, et al. Differential expression of DKK1 during early osteogenic and adipogenic differentiation in rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Osteoporosis, 2014, 20(11):1290-1297. (in Chinese)
[10] 李哲海. Dkk1在骨髓间充质干细胞成骨、成脂分化的分子调节机制的实验研究[D]. 长沙:中南大学, 2014.
LI Z H. Regulate of the adipogenic and osteogenic differentiation of BMSCs by Sh-RNA-DKKl[D]. Changsha:Central South University, 2014. (in Chinese)
[11] KATOH M, KATOH M. WNT signaling pathway and stem cell signaling network[J]. Clin Cancer Res, 2007, 13(14):4042-4045.
[12] GARCIA-MARTÍN A, REYES-GARCIA R, GARCÍA-FONTANA B, et al. Relationship of Dickkopf1 (DKK1) with cardiovascular disease and bone metabolism in Caucasian type 2 diabetes mellitus[J]. PLoS One, 2015, 10(1):e0117687.
[13] NIEHRS C. Function and biological roles of the Dickkopf family of Wnt modulators[J]. Oncogene, 2006, 25(57):7469-7481.
[14] LIU C X, GAO H, ZHAI S L, et al. Molecular characterization, chromosomal localization, expression profile and association analysis with carcass traits of the porcine dickkopf homolog1 gene[J]. Mol Biol Rep, 2011, 38(3):1929-1934.
[15] KIM M B, SONG Y, KIM C, et al. Kirenol inhibits adipogenesis through activation of the Wnt/β-catenin signaling pathway in 3T3-L1 adipocytes[J]. Biochem Biophys Res Commun, 2014, 445(2):433-438.
[16] ANDI T, REDDY S T, GADDAPARA T, et al. WNT signals are required for the initiation of hair follicle development[J]. Dev Cell, 2002, 2(5):643-653.
[17] SICK S, REINKER S, TIMMER J, et al. WNT and DKK determine hair follicle spacing through a reaction-diffusion mechanism[J]. Science, 2006, 314(5804):1447-1450.
[18] 刘薇. DKK1转基因体细胞克隆西藏小型猪的制备[D]. 广州:南方医科大学, 2013.
LIU W. Generation of DKK1 transgenic Tibet minipigs by somatic cell nuclear transfer (SCNT)[D]. Guangzhou:Southern Medical University, 2013. (in Chinese)
[19] 周纬男, 史铭欣, 乔书培, 等. 鸡Perilipin1基因启动子的克隆及分析[J]. 畜牧兽医学报,2016, 47(2):249-259.
ZHOU W N, SHI M X, QIAO S P, et al. Promoter cloning and analysis of chicken Perilipin1 gene[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(2):249-259. (in Chinese)
[20] 万雷, 黄宏兴, 黄红, 等. 过表达DKK1、Sost重组腺病毒载体的构建及其对MG63细胞和相关蛋白的影响[J]. 广州中医药大学学报, 2016, 33(4):578-584.
WAN L, HUANG H X, HUANG H, et al. Construction of DKK1, Sost overexpressing recombinant adenovirus vector and effect of overexpression on MG63 cells and related proteins[J]. Journal of Guangzhou University of Traditional Chinese Medicine, 2016, 33(4):578-584. (in Chinese)
[21] ANNICOTTE J S, SCHOONJANS K, HABY C, et al. An E-box in pGL3 reporter vectors precludes their use for the study of sterol regulatory element-binding proteins[J]. Biotechniques, 2001, 31(5):993-994, 996.
[22] 曹贵玲, 张渝洁, 李标, 等. 山羊Dkk1基因的结构、启动子区的多态性及其与生产性状的关联分析[J]. 畜牧兽医学报, 2010, 41(11):1394-1400.
CAO G L, ZHANG Y J, LI B, et al. Genomic structure, polymorphism in promoter region and their associations with production traits of Dkk1 gene in goats[J]. Acta Veterinaria et Zootechnica Sinica, 2010, 41(11):1394-1400. (in Chinese)
[23] JOKELA T A, MAKKONEN K M, OIKARI S, et al. Cellular content of UDP-N-acetylhexosamines controls hyaluronan synthase 2 expression and correlates with O-linked N-acetylglucosamine modification of transcription factors YY1 and SP1[J]. J Biol Chem, 2011, 286(38):33632-33640.
[24] LUI W Y, SZE K L, LEE W M. Nectin-2 expression in testicular cells is controlled via the functional cooperation between transcription factors of the Sp1, CREB, and AP-1 families[J]. J Cell Physiol, 2006, 207(1):144-157.
[25] EMILI A, GREENBLATT J, INGLES C J. Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein[J]. Mol Cell Biol, 1994, 14(3):1582-1593.
[26] CHOI M, WANG S E, KO S Y, et al. Overexpression of human GATA-1 and GATA-2 interferes with spine formation and produces depressive behavior in rats[J]. PLoS One, 2014, 9(10):e109253.
[27] PACKER A I, JANE-WIT D, MCLEAN L, et al. Hoxa4 expression in developing mouse hair follicles and skin[J]. Mech Dev, 2000, 99(1-2):153-157.

Cloning and Activity Analysis of the Promoter Region of Swine DKK1 Gene

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	NIU Naiqi, ZHAO Runze, ZONG Wencheng, LIU Xiance, LIU Hai, SHI Guohua, JING Xitao, ZHANG Longchao. Association of Polymorphisms of GREB1L and MIB1 Genes with Rib Number and Carcass Traits in Beijing Black Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 79-86.
[2]	ZHU Xueli, ZHANG Longchao, WANG Lixian, PU Lei, LIU Xin. Association Analysis of AQP9 and RPS10 Gene Polymorphisms with Backfat Thickness in Beijing Black Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 87-98.
[3]	SHI Shengjie, WANG Liguang, GAO Lei, CAI Chuanjiang, HE Weixian, CHU Guiyan. Effect of miR-24-3p on Estradiol Synthesis in Porcine Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 169-178.
[4]	YANG Kai, LU Zhuoda, HE Jian, ZHANG Ruiqi, WANG Suqing, LI Kebiao, ZHAO Yunxiang, ZHU Xiaoping, GUO Jinbiao. The Population Effect of Commercial Pigs on the Accuracy of Genomic Selection for Carcass Traits in Duroc Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 4943-4951.
[5]	LI Chao, ZHAO Xueyan, WANG Yongjun, WANG Yanping, REN Yifan, LI Jingxuan, WANG Huaizhong, WANG Jiying, SONG Qinye. Analysis on the Structural Composition and Function of Bacterial Microbiota of Caecum and Colon in Laiwu and DLY pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5033-5045.
[6]	XU Tingting, QI Fenfang, HUANG Shihui, NIU Xi, LI Sheng, RAN Xueqin, WANG Jiafu, XIE Jian. Study of the Polymorphisms of Structural Variation and the Expression of MAP3K4 gene in Xiang Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5046-5055.
[7]	XUE Hongyan, YANG Mengyu, YANG Huan, DONG Lijun, CAI Xiaqing, ZHAO Zemin, WANG Xianzhong. The Role of ALOX15B-JNK in Heat Stress-induced Oxidative Stress and Apoptosis of Sertoli Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5056-5065.
[8]	HU Ziping, WANG Ligang, ZONG Wencheng, HOU Renda, SU Yanfang, NIU Naiqi, WANG Lixian, WANG Yuan, ZHNAG Longchao. Genetic Structure Analysis of Jianbai Xiang Pig Population Based on Genomic SNP and ROH [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4117-4125.
[9]	JI Zhengyu, NI Mengru, ZHANG Zhaobo, ZHAO Gan, HUANG Zan, LI Pinghua, HUANG Ruihua, HOU Liming. Research on Adipogenic Capacity and Gene Expression Pattern of in Vitro FAPs Cells Derived from Longissimus Dorsi Muscle of Suhuai Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4126-4142.
[10]	BI Huan, QIN Hai, YUAN Wei, ZHANG Yudan, ZHANG Yiyu, CHEN Wei. Effect of Knocking down TYRP1 Gene on Melanin Production in the Xiang Pig Epidermal Melanocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4143-4153.
[11]	LIU Pan, LI Ruiqi, TAN Zhankun, WANG Yifei, CHEN Xiaochen, HE Weixian, DU Renrang, MA Jian, CHU Guiyan, CAI Chuanjiang. Effects of High Fiber Diet on Growth Performance, Meat Quality and Intestinal Microbiome of Growing-finishing Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4247-4259.
[12]	LI Pinghui, PU Guang, WANG Zhongyu, ZHOU Wuduo, NIU Peipei, WU Chengwu, HOU Liming, HUANG Ruihua, LI Pinghua. Effect of Dietary Fiber Level on Blood and Intestinal Immune Indexes of Meishan Pigs and Preliminary Analysis of Its Mechanism [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4260-4277.
[13]	WANG Hui, FENG Baoliang, WU Dan, XIANG Guangming, WANG Nan, MU Yulian, LI Kui, LIU Zhiguo. Research Progress of CD163 Gene and Disease-Resistant Breeding on Porcine Reproductive and Respiratory Syndrome [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3127-3138.
[14]	ZHANG Wanfeng, ZHAO Tianzhi, LI Jiao, YOU Ziwei, YANG Yang, CAI Chunbo, GAO Pengfei, CAO Guoqing, GUO Xiaohong, LI Bugao. Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3242-3251.
[15]	LIAN Yuju, ZHANG Zhiyuan, LIAO Xiaobo, WEI Hongjiang, YIN Yulong, LIU Mei. Breeding Methods and Application Progress of Medical Miniature Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2667-2682.