STGC3基因启动子的初步分析及鉴定

发布时间：2018-04-29 13:45

本文选题：鼻咽癌 + STGC3基因　；参考：《南华大学》2012年硕士论文

【摘要】：目的：利用生物信息学及报告基因载体分析方法对STGC3基因启动子区进行预测及活性检测，初步鉴定STGC3基因的启动子区域，为阐明STGC3基因的表达调控机制提供重要的依据。方法：根据STGC3基因的前期研究结果，利用多种生物信息学软件对STGC3基因5’端上游调控区－3046bp至－46bp区域的3000bp序列进行启动子预测，把预测和分析得到的STGC3基因可能的启动子片段构建至萤火虫荧光素酶报告基因pGL3-enhance载体上，然后将重组体转染到人胚肾上皮细胞系293T、人鼻咽癌细胞系CNE2及永生化人鼻咽上皮细胞系NP69，并利用双荧光素酶检测试剂盒检测重组质粒的活性以初步鉴定STGC3基因的启动子区域。结果：生物信息学分析结果显示－3046bp至－46bp区域的转录起始位点有两个，一个在－2348bp处，另一个在－948bp处，而且此区域存在两个CpG岛，CpG岛1位于－1805bp至－1705bp处，长度为101bp; CpG岛2位于－900bp至－684bp处，，长度为217bp。该区域未检测到经典的TATA盒序列，－1140bp和－774bp处有GC盒信号，－441bp处有CAAT盒信号。STGC3基因启动子区分值0.8以上的区域位于－2992bp至－69bp，且－845bp至－795bp区域启动子分值最高达到1.0。实验结果显示pGL3-en283、pGL3-en281、 pGL3-en571质粒在NP69、293T和CNE2三种细胞中相对于阴性对照pGL3-enhance质粒均有较强的启动子活性，其中pGL3-en281质粒在三种细胞中的启动子活性最强，故－934bp至－653bp区域有可能是其核心启动子区，而且可能在－653bp至＋72bp的DNA序列中含有负性调控元件，而三种质粒在293T细胞中比其在CNE2细胞中的启动子活性强，可能在CNE2细胞中存在影响启动子活性的因素。结论：（1） STGC3基因启动子区位于－2992bp至－69bp区域，而－934bp至－653bp区域有可能是其核心启动子区。（2）在－653bp至＋72bp的DNA序列中可能存在负性调控元件，在CNE2细胞中可能存在影响启动子活性的因素。
[Abstract]:Objective: to predict and detect the promoter region of the STGC3 gene by bioinformatics and reporter gene vector analysis, and to identify the promoter region of the STGC3 gene and provide an important basis for clarifying the regulation mechanism of the expression of STGC3 gene.
Methods: Based on the preliminary study results of the STGC3 gene, a variety of bioinformatics software was used to predict the 3000bp sequence of the 3000bp sequence of the STGC3 gene, the upstream region of the upstream and control region of the 5 'end. The promoter fragment of the predicted and analyzed STGC3 gene was constructed to the luciferase luciferase reporter gene pGL3-enhance In vivo, the recombinant was transfected into the human embryonic renal epithelial cell line 293T, human nasopharyngeal carcinoma cell line CNE2 and the immortalized human nasopharyngeal epithelial cell line NP69, and the activity of the recombinant plasmid was detected by double luciferase detection kit to identify the promoter region of the STGC3 gene.
Results: the results of bioinformatics analysis showed that there were two transcriptional starting sites in the 3046bp to 46bp region, one at 2348bp, another at - 948bp, and two CpG islands in this region, and CpG island 1 at - 1805bp to 1705bp, with the length of 101bp; CpG Island 2 in 900bp to - 684bp. The length was unchecked in the region. The classical TATA box sequence, - 1140bp and - 774bp, has a GC box signal, - the region of the CAAT box signal.STGC3 gene promoter region of more than 0.8 is located at - 2992bp to - 69bp, and the - 845bp to 795bp regional promoter is up to the maximum 1.0.
The experimental results show that pGL3-en283, pGL3-en281 and pGL3-en571 plasmids have strong promoter activity compared with negative control pGL3-enhance plasmids in three kinds of NP69293T and CNE2 cells, and the promoter activity of pGL3-en281 plasmid is the strongest in the three cells. Negative regulatory elements can be contained in the DNA sequence of 653bp to + 72bp, and the three plasmids in 293T cells are more active than their promoter in CNE2 cells, and may have factors that affect promoter activity in CNE2 cells.
Conclusion:
(1) the promoter region of STGC3 gene is located in the region of - 29 - 9 - 2 - to - 6 - 9, and the region of - 9 - 3 - 4 - to - 6 - 5 - 3 - 3 may be the core promoter region.
(2) there may be negative regulatory elements in DNA sequences from 653bp to + 72bp, and there may be some factors affecting promoter activity in CNE2 cells.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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