一种适于家蚕细胞激素研究的双荧光素酶报告基因系统的内参质粒的构建(英文)  

Establishment of a suitable control reporter plasmid of a dual luciferase reporter gene system for hormone research in silkworm cell lines

作　　者：刘红玲[1] 林英[1,2] 沈关望 顾健健[1] 张海燕 吴金鑫 徐银莹[1] 龙威[1] 夏庆友 Hongling Liu1, Ying Lin1,2, Guanwang Shen1,2, Jianjian Gu1, Haiyan Zhang1, Jinxin Wu1, Yinying Xu1, Wei Long1, and Qingyou Xia1,2(1 State Key Laboratory ofSillcworm Genome Biology, Southwest University, Chongqing 400716, China ;2 Chongqing Engineering and Technology Research Center for Novel Silk Materials, Chongqing 400716, China)

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400716 [2]重庆市蚕丝纤维新材料工程技术研究中心,重庆400716

出　　处：《生物工程学报》2018年第10期1631-1641,共11页Chinese Journal of Biotechnology

基　　金：State Key Program of National Natural Science of China(No.31530071),National Natural Science Foundation of China(No.3147254); Fundamental Research Funds for the Central Universities(No.XDJK2017B006)

摘　　要：双荧光素酶报告基因系统能够提供灵敏的读数,但该系统需要依赖组成型表达的内参对读数进行归一化。然而,大多数内参并不是在所有条件下都组成型表达。为此,文中建立了一个有效的方法制备适于家蚕细胞双荧光素酶报告基因系统的内参质粒。首先,突变BmV gP78启动子上的激素应答相关元件,获得了在家蚕细胞中稳定表达的组成型启动子BmV gP78M;然后,用BmV gP78M替换pRL-SV40质粒上的SV40启动子和嵌合内含子序列,成功构建了pRL-V gP78M内参质粒;最后,通过细胞转染实验证实pRL-V gP78M内参在家蚕细胞系中稳定表达,并且pRL-V gP78M内参的表达活性不受蜕皮激素、保幼激素及激素相关转录因子的影响。最终,获得了在家蚕细胞中稳定表达且表达量适中的内参质粒pRL-V gP78M。该内参可以有效地作为双荧光素酶报告基因系统的内参质粒用于家蚕细胞系中激素的研究。同时,该内参质粒的构建方法也为构建适于其他物种细胞系的双荧光素酶报告基因系统的内参质粒提供了参考。The dual luciferase reporter gene system provides sensitive readout, while it relies on a constitutively-expressed control gene for readout normalization. However, most standard control reporter genes are not constitutively expressed under all conditions. Here, we report an effective method to construct a control reporter plasmid for the dual luciferase reporter gene system that would be suitable for hormone research in silkworm cell lines. First, we modified BmVgP78M, a stably-expressed constitutive promoter in silkworm cells by mutating its hormone-related element. Then, we constructed the pRL-VgP78M control reporter plasmid by replacing the SV40 promoter and chimeric intron sequences in pRL-SV40 with the BmVgP78M sequence. Finally, we confirmed that the pRL-VgP78M control reporter plasmid could be stably expressed in silkworm cell lines via cell transfection experiments, and it was unresponsive to the induction of ecdysone, juvenile hormone, or their transcription factors. We thus obtained a control reporter plasmid pRL-VgP78M that could be expressed stably and moderately in silkworm cells. It can be readily used as the control reporter plasmid of the dual luciferase reporter gene system for hormone research in silkworm cell lines. It will also provide a reference for construction of control reporter plasmids of dual luciferase reporter gene systems that are adaptable to cell lines isolated from other species.

关 键 词：家蚕 双荧光素酶报告基因系统 内参质粒 激素 方法 

分 类 号：Q78[生物学—分子生物学]