摘要
目的:探讨杜氏盐藻硝酸盐还原酶(NR)基因5′上游序列(Pnr)是否可以驱动外源基因在杜氏盐藻中的表达。方法:利用改进的重叠区扩增基因拼接法(SOEing),将杜氏盐藻NR基因5′上游序列(Pnr)与除草剂抗性基因bar融合。同时将NR基因3′端序列(Tnr)与克隆载体pEGM-7zf连接,构成中间载体pEGM-7zf-Tnr。最后将融合片段Pnr-Tnr与中间载体连接,构建含Pnr-bar-Tnr表达盒的盐藻真核表达载体p7NBT。电击法转化杜氏盐藻,通过草丁膦培养筛选转化藻株后对其进行分析。结果:转化藻株总RNA的RT-PCR结果扩增出与bar基因序列完全一致的目的片段。结论:杜氏盐藻NR基因Pnr能够驱动外源基因bar的转录。
Aim： To study the role of 5′-flanking region（Pnr） of nitrate reductase（NR） from Dunaliella salina（D.salina） in driving the expression of the heterologous genes.Methods：After an intermediate plasmid pGEM-7zf-Tnr was constructed by ligating Tnr（3′-flanking region of NR of D.salina） into the pEGM-7zf,a fusion gene Pnr-bar produced by gene splicing and overlap extension（SOEing） was inserted into the intermediate plasmid pGEM-7zf-Tnr to create a eukaryotic expression plasmid p7NBT harboring an expression cassette Pnr-bar-Tnr.Subsequently,the plasmid p7NBT was transformed into cells of D.salina and the transformed cells were screened with UTEX liquid medium containing 3 mg/L PPT.RT-PCR was performed to determine the transcription of the bar gene in D.salina.Results： Sequence of the bar gene was amplified in D.salina cells transformed with plasmid p7NBT,but not in cells of control group.Conclusion： Pnr from D.salina could drive the transcription of the bar gene.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2008年第6期1129-1133,共5页
Journal of Zhengzhou University(Medical Sciences)
基金
国家自然科学基金资助项目30270031
