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本研究利用加州大学圣克鲁兹分校基因组浏览器(University of California Santa Cruz (UCSC) Genome Browse)数据库预测斑马鱼ifn-γ基因的启动子序列并通过PromoterScan和AliBaba软件预测转录因子结合位点,运用Methprimer-Design软件预测CpG岛。之后克隆ifn-γ启动子,并构建成pGL3-ifn-γ-promoter-enhancer表达载体,经双酶切验证和测序鉴定后,通过双荧光素酶报告基因实验检测pGL3-ifn-γ-promoter-enhancer重组载体的活性。结果显示,利用PromoterScan软件预测ifn-γ启动子上存在Promoter区,AliBaba在线分析发现该启动子上存在CRE-BP、CREB、TBP、ICSBP、C/EBP、HNF、c-Fos、TEC、Spl、AP-1、ATF、GATA、RSRFC、Oct、NF-1、c-Jun、TFIID和NF-kappaB等重要的转录因子结合位点,Methprimer-Design在线分析没有预测到CpG岛,但是其5′侧翼序列含有与转录密切相关的TATA Box和CAAT Box转录元件。之后将克隆的ifn-γ启动子片段构建成pGL3-ifn-y-promoter-enhancer表达载体,双酶切后显示目的片段大小和序列正确。最后,在RAW264.7和HEK293T细胞系中证实构建的报告基因载体具有启动子活性。以上结果表明,构建的斑马鱼ifn-γ基因启动子报告基因载体可为详细研究免疫蛋白的功能及其参与的免疫相关信号通路之间的调控作用提供有力的研究工具。
Abstract：In this study,we used University of California Santa Cruz(UCSC) Genome Browse Database to predict and analyze the promoter sequences of ifn-γ gene in zebrafish(Danio rerio),used PromoterScan and Alibaba to predict the transcription factor binding site,used Methprimer-Design to predict the CpG plot.We cloned the ifn-γpromoter and constructed pGL3-ifn-γ-promoter-enhancer expression vector,and confirmed their sequences with the double enzyme digestion and sequencing,used the dual luciferase reporter assay system detecting the activity of pGL3-ifn-γ-promoter-enhancer reorganized vectors.The results showed that there was the presence of promoter region on the ifn-γ gene using PromoterScan software.On-line analysis of the AliBaba revealed the presence of many important transcription factor binding site on the ifn-γ promoter,including CRE-BP,CREB,TBP,ICSBP,C/EBP,HNF,c-Fos,TEC,Spl,AP-1,ATF,GAT A,RSRFC,Oct,NF-1,c-Jun,TFIID and NF-kappa.The CpG island was not found in ifn-γ promoter region according to Methprimer-Design online analyses,but its 5' flanking sequences contain the transcription components including:TATA Box and CAAT Box.Then the ifn-γ promoter fragment was constructed into pGL3-ifn-γ-promoter-enhancer expression vector,fragment sizes were consistent by enzyme digestion,and the sequences were correct.At last,we proved that reorganized vector had promoter activity in RAW264.7 and HEK293 T cell line.To conclude,our results indicate that the construction of zebrafish ifn-γ gene promoter reporter vector could provide a powerful research tool to study of the function of immune-related proteins involved in the regulation of signaling pathways.
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基本信息:
DOI：10.13417/j.gab.038.004928
中图分类号:S917.4
引用信息:
[1]岳倩文,丁安,张秋月,等.斑马鱼ifn-γ基因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,2019,38(11):4928-4937.DOI:10.13417/j.gab.038.004928.
基金信息:
教育部留学回国人员科研启动基金(D-8002-15-0042);; 上海市教委重点创新项目(13ZZ127)、上海市教委水产高峰学科项目共同资助;; 中美海洋研究中心基金(A1-0209-15-0806)
2018-05-20
2018-05-20
2018-05-20