CAJ | 학술논문

本研究旨在分析猪RELMβ基因启动子结构，初步探索RELMβ基因表达调控机制。通过PCR方法扩增RELMβ基因的系列启动子缺失片段并分别克隆到荧光素酶报告基因表达载体pGL3-Enhancer中，经酶切、测序和生物信息学分析，构建包含RELMβ启动子系列截短的荧光素酶报告基因重组质粒，脂质体转染至HT29和293T细胞，应用双荧光素酶活性检测系统检测启动子活性。试验获得了猪RELMβ基因约1 kb的启动子序列，序列比对发现猪和人物种间相似性仅34.9%，猪和小鼠的同源性是82.4%。生物信息学分析预测猪RELMβ基因转录起始位点在-556 bp处，猪和人RELMβ基因启动子存在系列保守的转录因子结合位点，包括Cdx2、SRY、NFKB、NKX-2、c-Myb、GATA-1、GATA-3、C/EBP、MZF1等。细胞检测结果显示， pGL-RELMβ(-574~+215)活性最强，推测在-574~-182 bp位点之间存在RELMβ基因启动子的关键顺式调控元件，这一区域发现SRY、Cdx2、GATA-1和MZF1等关键转录因子结合位点。
본연구지재분석저RELMβ기인계동자결구，초보탐색RELMβ기인표체조공궤제。통과PCR방법확증RELMβ기인적계렬계동자결실편단병분별극륭도형광소매보고기인표체재체pGL3-Enhancer중，경매절、측서화생물신식학분석，구건포함RELMβ계동자계렬절단적형광소매보고기인중조질립，지질체전염지HT29화293T세포，응용쌍형광소매활성검측계통검측계동자활성。시험획득료저RELMβ기인약1 kb적계동자서렬，서렬비대발현저화인물충간상사성부34.9%，저화소서적동원성시82.4%。생물신식학분석예측저RELMβ기인전록기시위점재-556 bp처，저화인RELMβ기인계동자존재계렬보수적전록인자결합위점，포괄Cdx2、SRY、NFKB、NKX-2、c-Myb、GATA-1、GATA-3、C/EBP、MZF1등。세포검측결과현시， pGL-RELMβ(-574~+215)활성최강，추측재-574~-182 bp위점지간존재RELMβ기인계동자적관건순식조공원건，저일구역발현SRY、Cdx2、GATA-1화MZF1등관건전록인자결합위점。
The aim of this study was to identify the promoter structure of porcine RELMβ gene,and investigate its transcriptional regulation mechanism. The deletion fragments of porcine RELMβ gene promoter region were amplified by PCR method, cloned into pGL3-Enhancer plasmid by digestion, sequencing and bioinformatics analysis, and transfected in-to HT29 and 293T cells. The promoter activities were determined by dual-luciferase assay system. An approximate 1 kb promoter sequence was acquired and the homologies were 34. 9% and 82. 4% to RELMβgene of human and mouse, respec-tively. Bioinformatics analysis found a potential transcription start site located at -556 bp, and the conservative transcrip-tion factor binding sites in pig and human RELMβgene included Cdx2, SRY, NFKB, NKX-2,c-Myb, GATA-1, GATA-3, C/EBP and MZF1. pGL-RELMβ(-574-+215) exhibited the maximal promoter activities, suggesting the region of-574--182 bp contained the key cis-regulatory elements. Further bioinformatics analysis found some key transcrip-tion factor binding sites, including SRY, Cdx2, GATA-1 and MZF1.