山羊DCT基因启动子活性区及其转录因子调控探究

doi:10.11843/j.issn.0366-6964.2018.01.007

摘要/Abstract

摘要：

旨在探究山羊DCT基因启动子活性区及相关转录因子对该基因的调控作用，为山羊DCT基因的表达调控提供理论依据。通过对山羊DCT基因5'侧翼区序列及第一外显子区序列进行生物信息学分析，并与人和小鼠DCT基因启动子序列进行比对，同时结合在线启动子预测结果，采用快速克隆的方法构建5个5'系列缺失序列的启动子报告基因载体，以此为基础构建3'缺失序列的6个报告基因载体，并构建SOX10、MITF和OTX2转录因子结合位点点突变的6个报告基因载体，以瞬时转染的方法转染A375细胞，双荧光素酶检测试剂盒检测缺失片段和点突变片段的启动子活性。结果表明，成功构建了山羊DCT基因11个不同长度的启动子报告基因载体，-990~+232 bp的P3片段荧光素酶活性极显著高于其他片段（P<0.01），基于P3构建的3'系列缺失片段中-881~-154 bp的P8片段荧光素酶活性极显著高于其他片段（P<0.01）。转录因子SOX10结合位点突变的载体荧光素酶活性极显著降低（P<0.01），MITF和OTX2结合位点突变的载体荧光素酶活性极显著增强（P<0.01）。山羊DCT基因启动子核心调控区位于-881~-154 bp区域，转录因子SOX10对山羊DCT基因发挥正调控作用，而转录因子MITF和OTX2对山羊DCT基因的调控作用尚需深入研究。

Abstract:

The research aimed to study the promoter activity area and to explore the regulation mechanism of some related transcription factors of goat DCT gene, which would help to find the theoretical reference for expression regulation of goat DCT. 5' flanking region and the first exon sequences were analyzed by bioinformatics and blasted with mouse and human DCT gene promoter sequences. Based on the results of bioinformatics analysis and online promoter prediction, five 5'-terminal and six 3'-terminal deleted fragment promoter reporter gene vectors were constructed with the rapid amplification kit. Six mutation reporter gene vectors for SOX10, MITF and OTX2 transcription factor binding sites were built using P8 fragment sequences as the template. These vectors were transfected into A375 cells by transient transfection and their promoter activities were detected with the dual-luciferase detection reagent. The results showed that 11 deleted fragment promoter reporter gene vectors with different length were successfully constructed. The luciferase activity of P3 vector for -990-+232 bp was significantly higher than the others (P<0.01). And P8 vector for -881——154 bp based on P3 fragment had the highest luciferase activity among the serial 3'-terminal deletion vectors (P<0.01). The mutation vectors of SOX10 binding site showed extremely significantly lower luciferase activity compared with the original vector (P<0.01), while the MITF and OTX2 binding sites mutation vectors both showed extremely significantly higher luciferase activity compared with the original vectors (P<0.01). The core promoter region of goat DCT gene is located at -881——154 bp. Transcription factor SOX10 up-regulate the expression of DCT gene, while the regulating role of transcription factor MITF and OTX2 should be thoroughly studied.

中图分类号:

S827.2

刘春杨, 张乐超, 王麒, 周荣艳, 李兰会, 李祥龙. 山羊DCT基因启动子活性区及其转录因子调控探究[J]. 畜牧兽医学报, 2018, 49(1): 55-64.

LIU Chun-yang, ZHANG Le-chao, WANG Qi, ZHOU Rong-yan, LI Lan-hui, LI Xiang-long. The Exploration of the Promoter Activity Area and Regulation by Transcription Factors of Goat DCT Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 55-64.

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