Loxp序列位置对基因表达的影响

doi:10.3864/j.issn.0578-1752.2018.13.013

摘要/Abstract

摘要： 【目的】先选用绿色荧光蛋白基因（egfp）作为试验基因构建loxp位点位于egfp基因上游或下游不同位置的表达结构，对loxp位点的位置对基因表达的影响进行初步分析。然后以植酸酶（phytase）基因为另一个试验基因对通过egfp基因得出的结果进行进一步验证。研究结果为开展通过基因打靶技术将外源基因与内源基因通过2A序列连接共表达的转基因动物制作中，打靶位点的选择提供可靠依据。【方法】先选择增强绿色荧光蛋白基因（egfp）为试验基因,红色荧光蛋白基因（dsred2）为内参基因。以pEGFP-N2、pGEM-5zf-loxp质粒为基础，构建了ploxp-EGFP（loxp序列位于egfp基因开放阅读框的Kozak序列上游的5′非翻译区）、pEGFP-loxp（loxp序列位于egfp基因开放阅读框的终止密码子下游的3′非翻译区）、ploxp-EGFP-loxp（egfp基因开放阅读框的Kozak序列上游5′非翻译区和终止密码子下游3′非翻译区各有一个loxp序列）。以pEGFP-N2质粒为对照质粒，以pDsRed2-N1为内参质粒，与所构建的egfp基因各表达质粒共转染PK15细胞，转染24h后在荧光显微镜下观察荧光表达情况，用荧光分析软件Image J进行荧光强度分析并记录荧光强度，进而应用SPSS19.0软件对各转染荧光表达情况进行分析，确定loxp序列的位置对基因表达的影响。为了对以egfp基因为试验基因所得到的结果进行进一步验证，本试验选择植酸酶（phytase基因）基因为试验基因，egfp基因为转染内参基因萤火虫荧光素酶基因（luciferase基因）为表达内参基因。以pIREsNEo、pGEM-5zf-loxp和pT-phytase、pGL4.13[luc2/SV40]质粒为基础。构建了p-SV40-luciferase-CMV-loxp-phytase（loxp序列位于phytase基因开放阅读框的Kozak序列上游的5′非翻译区）、p-SV40-luciferase-CMV-loxp-phytase-loxp（phytase基因开放阅读框的Kozak序列上游5′非翻译区和终止密码子下游3′非翻译区各有一个loxp序列）、p-SV40-luciferase-CMV-phytase-loxp（loxp序列位于phytase基因开放阅读框的终止密码子下游的3′非翻译区）和p-SV40-luciferase-CMV-phytase（phytase基因开放阅读框的上下游均无loxp序列）等试验质粒,以egfp基因(pEGFP-N2)为转染内参基因与所构建的各种phytase基因表达结构共转染PK15细胞，同时设置一个转pEGFP-N2+ pDsRed2-N1的空白对照。转染48h后在荧光显微镜下观察绿色荧光蛋白基因表达情况，用荧光分析软件Image J进行荧光强度分析并记录荧光强度，应用SPSS19.0软件对各转染绿色荧光表达情况进行分析。同时应用钼蓝法测定各转染的植酸酶活性。应用萤火虫萤光素酶报告基因检测试剂盒检测各转染萤火虫荧光素酶活性。用SPSS19.0软件对各转染的植酸酶和萤火虫荧光素酶表达水平（活性）进行统计分析。【结果】试验中以dsred2为内参基因，对转染后每个转染的不同区域红色荧光强度平均值(代表各转染红色荧光蛋白的表达水平)进行统计分析表明egfp基因各结构每个转染的间红色荧光蛋白表达水平差异不显著，表明不同转染间排除了转染操作、质粒纯度、细胞活性等的差异对分析结果的影响，转染前质粒的电泳结果表明，各质粒间不同构象的质粒比例基本一致，这也基本排除了转染用质粒质量的差异对转染效果的影响，对各转染不同区域绿色荧光强度的平均值（代表各转染的egfp基因表达水平）进行的统计分析表明，同一结构不同转染间egfp基因表达水平差异不显著，不同结构的转染间存在差异，其中ploxp-EGFP和ploxp-EGFP-loxp结构转染中绿色荧光蛋白的表达水平显著低于pEGFP-loxp和pEGFP-N2，而pEGFP-loxp和pEGFP-N2转染间egfp基因间的表达差异不显著。这一结果初步说明loxp序列在外源基因开放阅读框下游时对基因表达水平没有影响，在外源基因开放阅读框上游时对基因表达呈负影响，为了进一步验证上述结果的可靠性，本研究以植酸酶基因作为另一试验基因，以萤火虫荧光素酶基因为表达内参基因，以egfp基因为转染内参基因再次进行了验证，验证试验得到了相似的结果。【结论】开展通过基因打靶技术将外源基因与内源基因通过2A序列连接，实现外源基因和内源基因共表达的转基因动物制作研究中，以Cre/loxp系统作为报告基因删除工具时，则内源基因的下游为最佳的打靶位点。

关键词: loxp序列, 基因表达, 2A序列, 基因打靶

Abstract: 【Objective】 In order to get a solid foundation for the site selection of a gene targeting experiment which aimed to realize the co-expression of the foreign gene and the endogenous gene linked through 2A sequence, the effect on the gene expression of different location of the loxp sequence at the open reading frame of a gene need to be clarified when the Cre/loxp system was selected as a tool for reporter gene deletion. 【Method】The enhanced green fluorescent protein (egfp) gene was selected as experimental gene, and red fluorescent protein (dsred2) gene as internal reference gene. Based on pEGFP-N2, pGEM-5zf-loxp plasmid, three plasmids with different location of loxp sequence at the open reading frame of egfp gene named ploxp-EGFP (The loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region of egfp gene), pEGFP-loxp (The loxp sequence located at the downstream of the termination codon in the 3′un-translation region of egfp gene) and ploxp-EGFP-loxp (There is one loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of egfp gene) were constructed. As the internal reference, Plasmid pDsRed2-N1 was co-transfected with constructed plasmids into PK15 cells. Twenty-four hours later, the fluorescence intensity of every transfection was observed and analyzed using fluorescence microscope and software Image J. In order to verify the result obtained using egfp gene as experimental gene, phytase gene was selected as another experimental gene. Based on pIREsNeo, pGEM-5zf-loxp, pT-phytase, pGL4.13[luc2/SV40] plasmid and luciferase gene as expression internal reference gene four plasmids with different location of loxp sequence at the open reading frame of phytase gene named p-SV40-luciferase-CMV-loxp-phytase (The loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region of phytase gene, p-SV40-luciferase- CMV-loxp-phytase-loxp (There is one loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of phytase gene), p-SV40-luciferase-CMV- phytase-loxp (The loxp sequence located at the downstream of the termination codon in the 3′un-translation region of phytase gene), p-SV40-luciferase-CMV-phytase (There is no loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of phytase gene)were constructed. As transfection internal reference, pEGFP-N2 was co-transfection with the differenct plasmid constructed above into PK15 cells, pEGFP-N2 and pDsRed2-N1 were co-transfected into PK15 cells to work as blank control for enzyme activity of phytase and luciferase. Forty-eight hours later, the fluorescence intensity of every transfection was observed and analyzed using fluorescence microscope and software Image J. These data were analyzed using statistics software SPSS19.0. At the same time, the activity of phytase and luciferase of every transfection were measured by Molybdenum Blue Method or using Firefly Luciferase Assay Kit, and these data were analyzed using statistics software SPSS19.0.【Result】In the experiment, the dsred2 gene worked as internal reference gene. Statistics analysis using the average value of red fluorescence of different area of every transfection as basic data shows there is no significant difference between every different transfection. This result shows there is no significant difference in purity of plasmid, handling and cell activity between every transfection. Plasmid electrophoresis result shows there is no significant difference in quality between every plasmid used in transfections, this means the difference in quality between every plasmid used in every transfection is not enough to influence the accuracy for analysis. Analysis on the green fluorescence in the same rule shows there is no significant difference between different transfection of the same plasmid and between pEGFP-N2 and pEGFP-loxp, ploxp-EGFP and ploxp-EGFP-loxp, but there is significant difference between pEGFP-N2, pEGFP-loxp and ploxp-EGFP ploxp-EGFP-loxp. These result shows loxp sequence had no effect on egfp gene expression when it is located downstream of the open reading frame of the gene, but had a negative effect on egfp gene expression when it located upstream of the open reading frame of the gene. Replacing the egfp gene by phytase gene and using egfp gene as transfection internal reference gene, luciferase gene as expression internal reference gene, it shows a same result.【Conclusion】These results indicate that if we try to prepare a transgenic animals in which the foreign gene and the endogenous gene is linked by 2A sequence and co-express under the regulation of the full regulation element of the endogenous gene in one copy manner and the Cre/loxp system is selected as a tool for reporter gene deletion, the downstream of the open reading frame of the endogenous gene is the suitable targeting site.

Key words: loxp sequence, gene expression, 2A sequence, gene targeting

许盈盈，靳伟，代敏敏，樊宝良. Loxp序列位置对基因表达的影响[J]. 中国农业科学, 2018, 51(13): 2579-2591.

XU YingYing, JIN Wei, DAI MinMin, FAN BaoLiang. Researching on the Effect of the Location of Loxp Sequence on the Gene[J]. Scientia Agricultura Sinica, 2018, 51(13): 2579-2591.

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