绵羊CCL19基因启动子活性分析及其转录调控元件筛选

doi:10.16431/j.cnki.1671-7236.2022.04.002

Abstract

Abstract: 【Objective】 The objective of this study was to identify the core promoter region and key transcription factors of sheep C-C motif chemokine ligand 19(CCL19) gene,and explore the transcription regulation mechanism of CCL19 gene.【Method】 Using sheep genomic DNA as template,the 5'-flanking sequence 1 000 bp of CCL19 gene was amplified by PCR.Moreover,seven active region sequences of CCL19 gene promoter were amplified and connected to the pGL3-Basic promoter vector.The recombined plasmid and pRL-TK plasmid were co-transfected into 293T cells,and the activity of relative luciferin enzymes was measured through dual-luciferase detection system.Transcription binding factor sites(TFBSs)prediction was made in the CCL19 gene promoter region by bioinformational method.The luciferase reporter vectors with TFBSs deletion were constructed using site mutation technology and co-transfected into 293T cells,the relative fluorescence activity of these deletion plasmids were analyzed.【Result】 The results showed that seven different length of CCL19 gene promoter fragments were successfully amplified and their dual-luciferase reporting vector (pGL3-P,pGL3-P1,pGL3-P2,pGL3-P3,pGL3-P4,pGL3-P5 and pGL3-P6) were constructed.The active detection of different length promoter fragments found that －256/－186 bp was the core promoter region of CCL19 gene,indicating that this region played an important role in the transcription regulation of CCL19 gene.Bio-information analysis of the CCL19 gene promoter region sequence found that there were 5 TFBSs in this region,including POU5F1(-201/-189 bp),ZBTB26(-228/-217 bp),FOXI1(-239/-228 bp),GLI2(-255/-243 bp) and SP2(-219/-211 bp),and the luciferase reporter vectors with TFBSs deletion were constructed.Dual-luciferase report results showed that the deletion of POU5F1 binding site extremely significantly reduced the transcriptional activity of CCL19 gene in sheep (P<0.01),and FOXI1,ZBTB26 and SP2 extremely significantly enhanced the transcriptional activity of CCL19 gene in sheep (P<0.01).【Conclusion】 This study indicated that the luciferase reporter vector of CCL19 gene promoter was successfully constructed and the core region of CCL19 gene promoter was determined.TFBSs of POU5F1 might be the transcriptional regulation site of CCL19 gene.This result could provide the theoretical basis for further exploring the function of CCL19 gene in lymphocyte migration,innate and adaptive immunity of sheep.

Key words: sheep; CCL19 gene; promoter; transcriptional regulation

CLC Number:

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.

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Analysis of Promoter Activity and Screening of Transcription Regulatory Elements of CCL19 Gene in Sheep

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