hNaDC1基因5''''侧翼区转录调控序列系列载体构建与鉴定

目的构建hNaDC1基因5’侧翼区转录调控序列系列萤火虫荧光素酶报告基因表达载体。方法PCR扩增获得hNaDC1基因5’侧翼转录调控区不同长度片段:hNaDC1A(-2232/ 136,2368bp)、hNaDC1B(-1640/ 136,1776bp)、hNaDC1C(-1084/ 136,1221bp)、hNaDC1D(-253/ 136,389bp)、hNaDC1E(-2232/-12,2244bp),以pGL3-Basic为载体构建hNaDC1基因5’侧翼序列系列缺失质粒。重组体通过特异限制性内切酶酶切鉴定,并送样测序鉴定。结果成功构建hNaDC1基因5’侧翼区转录调控元件萤火虫荧光素酶报告基因表达载体5个:pGL3-hNaDC1A～E。结论为进一步研究NaDC1基因5’侧翼区转录调控元件的分布特点及转录调控元件与转录因子间的相互作用提供了基本实验条件。

Construction and indentification of the vectors for 5'''' flanking regulated elements of hNaDC1 gene

Objective To construct the firefly luciferase report gene vectors for 5' flanking regulated elements of hNaDC1 gene.Methods The DNA fragments hNaDC1A~E(-2 232/ 136) in 5'flanking region of hNaDC1 gene were amplified from the nephridial tissue by using PCR.The PCR products were directionally subcloned into pGL3-Basic vector.The recombined clones were identified by agarose gel electrophoresis after restriction endonuclease digesting and DNA sequencing.Results 5 expression vectors(pGL3-NaDC1A~E)for 5' flanking regulated elements of hNaDC1 gene had been constructed.Conclusion These expression vectors offer the basic experimental conditions for studying the distribution characters of hNaDC1 gene 5' flanking regulations elements and the properties of interactions between the elements and regulation proteins with the help of dual luciferase report gene assay system.