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LATS1基因启动子转录调控分析
魏大为1, 张久盘2, 杨智燕,等1
1.宁夏大学 农学院,;2.宁夏农林科学院固原分院
摘要:
【目的】探究牛大肿瘤抑制基因1(Large tumor suppressor gene1,LATS1)的组织表达规律,并初步鉴定其启动子区关键转录因子,以期阐明其转录调控机制。【方法】利用荧光定量PCR检测LATS1基因在牛心脏、肝脏、脾脏、肺脏、肾脏、皮下脂肪、背最长肌、大肠、小肠、大脑、皱胃及睾丸组织中的相对表达量。克隆牛LATS1基因启动子的全长序列,通过逐段缺失PCR技术扩增7个缺失不同片段的启动子序列,并构建其双荧光素酶报告载体,分别转染小鼠C2C12和3T3-L1细胞系,通过检测不同缺失片段荧光素酶报告载体的活性,确定LATS1启动子核心区域。使用在线软件预测牛LATS1基因启动子核心区域的关键转录因子。【结果】LATS1基因在大脑、背最长肌、大肠、皱胃、肾脏中高表达。克隆了1 950 bp的LATS1基因启动子序列及其7个逐段缺失片段序列,并成功构建了pLATS1-1783/+167、pLATS1-1449/+167、pLATS1-1149/+167、pLATS1-837/+167、pLATS1-555/+167、pLATS1-298/+167和pLATS1-123/+167双荧光素报告载体。检测到牛LATS1基因启动子核心区域位于-298/-123区域。在线软件预测到牛LATS1基因启动子核心区存在肌细胞增强因子2(MEF2A)、转录激活因子5(STAT5)、同源异型盒基因5(HOXA5)、肌细胞决定基因1(Myod1)和靶向叉头框转录因子1(FoxO1)结合位点。【结论】克隆了牛LATS1基因启动子,明确了其核心区位于-298/-123区域;MEF2A、STAT5、HOXA5、Myod1和FoxO1可能对牛LATS1基因转录活性具有重要的调控作用。
关键词:    LATS1基因  启动子  转录调控
DOI:
分类号:
基金项目:宁夏重点研发项目(引才专项)(2020BEB04011);宁夏托举人才工程项目(TJGC2019076);宁夏大学科研启动金项目(030900002032);宁夏科技重大专项(2019BEF02004)
Transcription regulation analysis of bovine LATS1 gene
WEI Dawei,ZHANG Jiupan,YANG Zhiyan,et al
Abstract:
【Objective】Aiming to elucidate the transcriptional regulation mechanism of bovine large tumor suppressor 1 gene (LATS1),this study explored the expression level of LATS1 gene in different tissues of cattle and identified the key transcription factors in the promoter region.【Method】Real time PCR was used to detect the relative expression of bovine LATS1 gene in heart,liver,spleen,lung,kidney,subcutaneous fat,longissimus dorsi,large intestine,small intestine,brain,abomasum and testicle tissues.The full-length sequence of bovine LATS1 gene promoter was cloned and the luciferase reporter constructs with seven different deletion fragments of LATS1 gene promoter were constructed and transfected into C2C12 and 3T3 L1 cell lines,respectively.The key transcription factors in core promoter regions of bovine LATS1 were also predicted using on-line software.【Result】The bovine LATS1 gene was highly expressed in brain,longissimus dorsi,large intestine,abomasum and kidney.A 1 950 bp promoter region sequence of bovine LATS1 gene and seven deletion fragments were cloned.The luciferase reporter vectors of pLATS1-1783/+167,pLATS1-1449/+167,pLATS1-1149/+167,pLATS1-837/+167,pLATS1-555/+167,pLATS1-298/+167 and pLATS1-123/+167 were successfully constructed.The core promoter region of bovine LATS1 gene was located at -298/-123 bp.The online software predicted myocyte enhancer factor 2A (MEF2A),activator of transcription 5 (STAT5),homeobox A5 (HOXA5),myogenic differentiation1 (Myod1) and forkhead box O1 (FoxO1) transcription factors binding sites on the bovine LATS1 gene.【Conclusion】The bovine LATS1 gene promoter was cloned and its core promoter was located at -298/-123 bp.MEF2A,STAT5,HOXA5,Myod1 and FoxO1 may play important roles in the regulation of bovine LATS1 gene transcription activity.
Key words:  bovine  LATS1 gene  promoter  transcriptional regulation