›› 2010, Vol. 53 ›› Issue (3): 279-285.doi:

• 研究论文 • 上一篇    下一篇



  • 出版日期:2010-05-07 发布日期:2010-03-20
  • 通讯作者: 蒋明星

Comparison of transcriptional activities of eight promoters and application of hr5-IE1 promoter to realize the expression of EGFP in Bombyx mori BmN cells

WANG Na, DU Xi-Kuan, JIANG Ming-Xing, ZHANG Chuan-Xi, CHENG Jia-An   

  • Online:2010-05-07 Published:2010-03-20

摘要: 为筛选出一个较强的启动子用于提高转座子piggyBac在家蚕Bombyx mori细胞中的转化效率,采用双荧光素酶报告基因检测(dual-luciferase reporter assay)技术比较了热激蛋白启动子(hsp70和hsp82)、家蚕肌动蛋白启动子(A3)、多聚泛素(polyubiquitin)启动子(PUB)、α微管蛋白启动子(α-tub)、丝素轻链启动子(Fib-L)、人工合成启动子3×P3及苜蓿丫纹夜蛾多角体病毒(AcNPV)增强子-启动子组合(hr5-IE1)8种启动子在家蚕细胞株BmN内的活性。结果显示hr5-IE1活性最强,A3次之,其余启动子活性均较弱。构建含有hr5-IE1启动子和piggyBac的转座酶编码区的质粒作为辅助质粒,与EGFP载体质粒一起转染家蚕细胞后,实现了EGFP基因整合到细胞基因组中。因此,今后可考虑将hr5-IE1用于家蚕细胞遗传转化的研究中,以提高细胞转化的效率。

关键词: 家蚕, hr5-IE1, 荧光素酶分析, piggyBac, 启动子, 遗传转化育

Abstract: In order to find a strong promoter that can be used to promote expression of piggyBac transposase and thereby increase genetic transformation efficiency in cells of the silkworm, Bombyx mori, the transcriptional activities of eight promoters were compared in the BmN cells of B. mori, including hsp70, hsp82, actin3 (A3), polyubiquitin (PUB), α-tubulin, fibroin-L, artificial promoter 3×P3 and immediately early 1 gene promoter flanked by the hr5 enhancer element (hr5-IE1). The results showed that hr5-IE1 displayed the highest transcriptional activity, followed by A3, while the activities of the other six promoters were relatively low. When transfected with an EGFP vector and a piggyBac helper plasmid, in which the expression of piggyBac transposase was driven by hr5-IE1, the EGFP cassette was successfully integrated into the genome of BmN cells. Therefore, hr5-IE1 has the potential of serving as a sound element in future piggyBac-based transgenic research of B. mori with the capability of increasing transformation efficiency.

Key words: Bombyx mori, hr5-IE1, luciferase assay, piggyBac, promoter, genetic transformation