Loxp序列位置对基因表达的影响  

Researching on the Effect of the Location of Loxp Sequence on the Gene

作　　者：许盈盈[1] 靳伟 代敏敏 樊宝良[1] XU YingYing;JIN Wei;DAI MinMin;FAN BaoLiang(College of Animal Sciences,Hebei Agricultural University,Baoding 071001,Hebei)

机构地区：[1]河北农业大学动物科技学院,河北保定071001

出　　处：《中国农业科学》2018年第13期2579-2591,共13页Scientia Agricultura Sinica

基　　金：转基因重大专项(2014ZX08006-005);国家自然科学基金(30972081);河北省现代农业产业技术体系蛋肉鸡产业创新团队遗传资源开发与利用岗位项目(HBCT2018150201);河北省科技支撑计划项目(14236602D-4(2014))

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p EGFP-N2、p GEM-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)、p EGFP-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p EGFP-N2质粒为对照质粒,以p Ds Red2-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p IREs NEo、p GEM-5zf-loxp和p T-phytase、p GL4.13[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-lo【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 2 A 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 p EGFP-N2, p GEM-5 zf-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）, p EGFP-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 p Ds Red2-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 p IREs Neo, p GEM-5 zf-loxp, p T-phytase, p GL4.13[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-luci

关 键 词：loxp序列 基因表达 2A序列 基因打靶 

分 类 号：Q78[生物学—分子生物学]