水牛HSD17B1基因启动子荧光素酶报告基因载体构建与分析

doi:10.16431/j.cnki.1671-7236.2021.10.002

Abstract

Abstract: The research was designed to screen the active region and influence factors of buffalo HSD17B1 gene promoter, predict its transcription binding factor, and provide a theoretical basis for exploring the regulatory mechanism of HSD17B1 gene in buffalo reproductive performance. Using buffalo blood genomic DNA as template, PCR was used to amplify three active region sequences of HSD17B1 gene promoter, and then was directed to the pGL3-promoter vector. Recombinant plasmids were transfected into buffalo follicle granulosa cells, and the activity of relative luciferin enzymes was measured through a dual-luciferase detection system and their relationship to luteinizing hormone (LH) and follicle stimulating hormone (FSH) were explored. Transcription binding factor prediction was made in the HSD17B1 gene promoter region by bio-informational method. The results showed that three different length of HSD17B1 gene promoter fragments were successfully cloned and their dual-luciferase reporting vector were successfully constructed. The active detection of different length promoter fragments found that pGL-pro-HSD17B1-1500 was the most active, and -866/-1 500 bp was the core promoter region of HSD17B1 gene, indicating that this region played an important role in the transcription regulation of HSD17B1 gene. The luciferase activity test results showed that the addition of LH could enhance the activity of HSD17B1 gene promoter. Bio-information analysis of the HSD17B1 gene promoter region sequence found that there were 6 transcription factor binding site (Sp1(-2 327/-2 317 bp), HOXA4(-2 162/-2 146 bp), Sp1(-1 409/-1 395 bp), Sp1(-1 391/-1 380 bp), Sp1(-1 345/-1 319 bp) and GATA1(-812/-801 bp)), but no CpG island, and there were one TATA-box and two CAAT-box. The results indicated that the luciferase reporter gene vector of buffalo HSD17B1 gene promoter was successfully constructed, the core region of HSD17B1 gene promoter was determined, and the activity of the promoter was enhanced by LH adding.

Key words: buffalo; HSD17B1 gene; promoter

CLC Number:

LU Xingrong, DUAN Anqin, MA Xiaoya, LIANG Shasha, DENG Tingxian. Construction and Analysis of the Luciferase Reporter Gene Vector of HSD17B1 Gene Promoter in Buffalo[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3512-3521.

References

[1] SALOIEMI T, JOKELA H, STRAUSS L, et al.The diversity of sex steroid action:Novel functions of hydroxysteroid (17beta) dehydrogenases as revealed by genetically modified mouse models[J]. Journal of Endocrinology the Journal of the Society for Endocrinology, 2012, 212(1):27-40.
[2] ZHANG C, CHEN J, YIN D, et al.The contribution of 17beta-hydroxysteroid dehydrogenase type 1 to the estradiol-estrone ratio in estrogen-sensitive breast cancer cells[J]. PLoS One, 2012, 7(1):e29835.
[3] HAKKARAINEN J, JOKELA H, PAKARINEN P, et al.Hydroxysteroid (17β)-dehydrogenase 1-deficient female mice present with normal puberty onset but are severely subfertile due to a defect in luteinization and progesterone production[J]. Official Publication of the Federation of American Societies for Experimental Biology, 2015, 29(9):3806-3816.
[4] CHENG C, SHEN F, DING G, et al.Lepidiline a improves the balance of endogenous sex hormones and increases fecundity by targeting HSD17B1[J]. Molecular Nutrition & Food Research, 2020, 64(10):e1900706.
[5] YUAN X, ZHOU X, QIAO X, et al.FoxA2 and p53 regulate the transcription of HSD17B1 in ovarian granulosa cells of pigs[J]. Reproduction In Domestic Animals, 2021, 56(1):74-82.
[6] 朱鹏, 庞春英, 邓廷贤, 等.水牛HSD17B1基因克隆分析及其真核表达载体构建[J]. 中国畜牧兽医, 2016, 43(3):559-567. ZHU P, PANG C Y, DENG T X, et al.Cloning and construction of eukaryotic expression vector of buffalo HSD17B1 gene[J]. China Animal Husbandry & Veterinary Medicine, 2016, 43(3):559-567.(in Chinese)
[7] 徐庚全, 樊江峰, 杨世华, 等.FSH、LH对体外培养的牦牛卵泡颗粒细胞凋亡及E₂、P分泌功能的影响[J]. 畜牧兽医学报, 2015, 46(6):932-932. XU G Q, FAN J F, YANG S H, et al.The effects of FSH, LH on apoptosis and E₂, P secreting of Yak's granulosa cells cultured in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 46(6):932-939.(in Chinese)
[8] 陆杏蓉, 庞春英, 邓廷贤, 等.水牛全血基因组抽提方法优化[J]. 中国牛业科学, 2016, 42(2):28-31. LU X R, PANG C Y, DENG T X, et al.Optimization of methods for genomic extrction of buffalo blood[J]. China Cattle Science, 2016, 42(2):28-31.(in Chinese)
[9] 李丽莎, 彭永东, 郑晓宁, 等.山羊PMEL基因启动子活性及转录调控元件分析[J]. 畜牧兽医学报, 2017, 48(5):826-835. LI L S, PENG Y D, ZHENG X Y, et al.Analysis of the promoter activity and transcriptional regulatory elements of goat PMEL gene[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(5):826-835.(in Chinese)
[10] 张秋月, 丁安, 张哲, 等.斑马鱼tnfb启动子的克隆和报告基因载体的活性分析[J]. 基因组学与应用生物学, 2018, 37(8):3296-3303. ZHANG Q Y, DING A, ZHANG Z, et al.Cloning of tnfb promoter from zebrafish (Danio rerio) and activity analysis of reporter fene vector[J]. Genomics and Applied Biology, 2018, 37(8):3296-3303.(in Chinese)
[11] HE W, MISRA G, LI T, et al.Current knowledge of the multifunctional 17-beta-hydroxysteroid dehydrogenase type 1[J]. Gene, 2016, 588(1):54-61.
[12] LERNER A, OWENS L A, COATES M, et al.Expression of genes controlling steroid metabolism and action in granulosa-lutein cells of women with polycystic ovaries[J]. Molecular Cellular Endocrinology, 2019, 486:47-54.
[13] LIU L Q, LI F E, DENG C Y, et al.Polymorphism of the pig 17 beta-hydroxysteroid dehydrogenase type 1(HSD17B1) gene and its association with reproductive traits[J]. Animal Reproduction Science, 2009, 114(1-3):318-323.
[14] GROSKREUTZ D, SHERF B, WOOD K, et al.Increased expression and convenience with the new pGL3 luciferase reporter vectors[J]. Promega Notes Magazine, 1995, 50(2):1-5.
[15] ALAM J, COOK J L.Reporter genes:Application to the study of mammalian gene transcription[J]. Analytical Biochemistry, 1990, 188(2):245-254.
[16] DANINO Y M, EVEN D, IDESES D, et al.The core promoter:At the heart of gene expression[J]. Biochimica et Biophysica Acta-Gene Regulatory Mechanisms, 2015, 1849(8):1116-1131.
[17] 吴琦.猪HSD17B1基因启动子活性分析及其表达调控的相关研究[D].广州:华南农业大学, 2017. WU Q.Expression and regulation mechanism of porcine genes HSD17B1[D].Guangzhou:South China Agricultural University, 2017.(in Chinese)
[18] 马冬梅, 韩林强, 白俊杰.大口黑鲈GHRH基因启动子区域序列分析及其活性检测[J]. 海洋渔业, 2016, 38(4):383-390. MA D M, HAN L Q, BAI J J.Sequence and activity analysis of GHRH promoter region from Micropterus salmoides[J]. Marine Fisheries, 2016, 38(4):383-390.(in Chinese)
[19] 周琳, 高春芳, 王皓, 等.人B细胞活化因子基因启动子活性研究[J]. 现代免疫学, 2006, 26(5):367-372. ZHOU L, GAO C F, WANG H, et al.Analysis on the activity of promoter from human B cell activating factor gene[J]. Current Immunololgy, 2006, 26(5):367-372.(in Chinese)
[20] 胡杨, 吴雄志.特异性蛋白转录因子1在乳腺癌中的研究进展[J]. 天津医科大学学报, 2019, 25(2):184-188. HU Y, WU X Z.Research progress of specific protein transcription factor 1 in breast cancer[J]. Journal of Tianjin Medical University, 2019, 25(2):184-188.(in Chinese)
[21] SAFE S, ABDELRAHIM M.Sp transcription factor family and its role in cancer[J]. European Journal of Cancer, 2005, 41(16):2438-2448.
[22] SAMSON S.Role of Sp1 in insulin regulation of gene expression[J]. Journal of Molecular Endocrinology, 2002, 29(3):265-279.
[23] MARIN M, KARIS A, VISSER P, et al.Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation[J]. Cell, 1997, 89:619-628.
[24] ZHAO C, MENG A.Sp1-like transcription factors are regulators of embryonic development in vertebrates[J]. Development, Growth Differentiation, 2005, 47(4):201-211.
[25] THOMAS K, WU J, SUNG D Y, et al.SP1 transcription factors in male germ cell development and differentiation[J]. Molecular Cellular Endocrinology, 2007, 270(1-2):1-7.
[26] NAKAMURA H, EDWARD D P, SUGAR J, et al.Expression of Sp1 and KLF6 in the developing human cornea[J]. Molecular Vision, 2007, 13:1451-1457.
[27] XIAO C L, MAI Z B, LIAN X L, et al.FANSe2:A robust and cost-efficient alignment tool for quantitative next-generation sequencing applications[J]. PLoS One, 2014, 9(4):e94250.
[28] GRAY S, PANDHA H S, MICHAEL A, et al.HOX genes in pancreatic development and cancer[J]. Journal of the Pancreas, 2011, 12(3):216-219.
[29] 杨帆.Cul4a通过上调scl和gata1表达促进斑马鱼初级造血红系发育[D].济南:山东大学, 2020. YANG F.Cul4a promotes zebrafish primitive erythropoiesis via upregulating scl and gata1 expression[D].Jinan:Shandong University, 2020.(in Chinese)
[30] SHIN E, JEONG J G, CHUNG H, et al.The Gata1 low murine megakaryocyte-erythroid progenitor cells expand robustly and alter differentiation potential[J]. Biochemical Biophysical Research Communications, 2020, 528(1):46-53.
[31] 薛梅, 李旭, 陈葳.人UCA1基因转录调控的生物信息学分析与鉴定[J]. 南方医科大学学报, 2013, 33(11):1596-1599. XUE M, LI X, CHEN W.Bioinformatics analysis and identification of transcriptional regulation of human UCA1 gene[J]. Journal of Southern Medical University, 2013, 33(11):1596-1599.(in Chinese)

Construction and Analysis of the Luciferase Reporter Gene Vector of HSD17B1 Gene Promoter in Buffalo

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	XU Yuanyuan, ZHENG Haiying, YANG Chunyan, DENG Tingxian, HUANG Chenqian, FENG Chao, LU Xingrong, DUAN Anqin, MO Xia, MA Xiaoya, SHANG Jianghua. Effects of Inhibition of BMP/Smad Signaling Pathway on the Growth and Steroid Hormone Synthesis of Buffalo Ovarian Granulosa Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2354-2362.
[2]	XIAO Peng, SHANG Jianghua, YANG Chunyan, LI Mengqi, DUAN Anqin, MA Xiaoya, FENG Chao, HUANG Chenqian, ZHANG Bo, ZHOU Jinchen, WEI Kelong, ZHENG Wei, ZHENG Haiying. Effects of Alpha-linolenic Acid on Buffalo Ovarian Granulosa Cells Cultured in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2380-2387.
[3]	SONG Xingchao, MENG Jinzhu, ZHAO Yuanyuan, WU Zhenyang, AN Qingming. Cloning and Bioinformatics Analysis of MyoG Gene Promoter Region Sequence in Guizhou White Goat [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 893-903.
[4]	GAO Xiaomin, ZHOU Shujian, CHEN Chen, JIN Jing, HU Cai, ZHANG Chen, ZUO Qisheng, ZHANG Yani, CHEN Guohong, LI Bichun. Regulation of STAT1 and Histone Acetylation Modification on lncRNA-BMP4 Transcription in Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3321-3332.
[5]	LIU Huayun, ZHANG Lei, XU Guili, ZHANG Tianhao, XIE Yuchun, DUAN Lingxin, LIU Zhengzhu, GONG Yuanfang. Identification of the Core Promoter Region of MC1R Gene in Silver Fox [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2442-2450.
[6]	YANG Yuying, WANG Shaolin. Establishment of the Epidemiological Cut-off Values of Enterococcus Isolated from Animals for Antimicrobial Growth Promoters [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2788-2795.
[7]	ZHAI Zhe, CHEN Si, WU Yanru, WANG Xuemei, LI Chongrui, LIU Zhiyong, CHEN Qiaoling, WANG Fengyang, DU Li, LI Chang, JIN Ningyi. Analysis of Promoter Activity and Screening of Transcription Regulatory Elements of CCL19 Gene in Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1213-1222.
[8]	CHEN Zhe, YANG Pengxia, LEI Mingming, CHEN Jiabin, YAN Leyan. Cloning, Promoter Analysis and Transcriptional Regulation of p21 Gene in Goose [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 405-415.
[9]	LIU Xinyong, TAN Zhengzhun, LI Hui, HUANG Jian, LUO Hua, ZENG Linghu, CHENG Juanru, ZHONG Huapei, YANG Chunyan, WANG Xiaoli, QIN Guangsheng. Effects of Heat Stress on Blood Biochemical Indexes of Murrah and Nili-Ravi Buffaloes [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 521-528.
[10]	XU Yuanyuan, MO Xia, HUANG Chenqian, FENG Chao, LU Xingrong, MA Xiaoya, YANG Chunyan, ZHENG Haiying, SHANG Jianghua. Cloning and Bioinformatics Analysis of TRAIL Gene in Buffalo [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3693-3703.
[11]	LIANG Shasha, PANG Chunying, DENG Tingxian, LU Xingrong, DUAN Anqin, MA Xiaoya, FANG Yanyan, LIANG Xianwei. Polymorphism of MFSD2A Gene and Its Association with Milk Production Traits in Buffalo [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3856-3865.
[12]	ZHENG Haiying, YANG Chunyan, LI Lingyu, TANG Liping, ZHENG Wei, SHANG Jianghua. Effects of Resveratrol on Proliferation of Cumulus Cells Cultured in vitro and mRNA Expression Levels of Related Genes in Buffalo [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 208-215.
[13]	LI Lingyu, ZHENG Haiying, CHEN Mingtang, TANG Liping, SHANG Jianghua, YANG Chunyan. Effects of Oocyte Origin on the Developmental Potential of Buffalo Somatic Cell Nuclear Transfer Embryos [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 241-247.
[14]	DUAN Anqin, LU Xingrong, MA Xiaoya, LIANG Shasha, DENG Tingxian. Effect of HSD17B4 Gene Interference on Milk Fat and Casein in Buffalo Mammary Epithelial Cell [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3128-3137.
[15]	MA Xiaoya, LIANG Yanyin, LU Xingrong, DUAN Anqin, LIANG Shasha, DENG Tingxian. Bioinformatics Analysis of ABCD Gene Family in Buffalo and Expression Analysis of ABCD1 Gene [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3171-3182.