Dey N, Maiti I B
Molecular Plant Virology and Plant Genetic Engineering Laboratory, Tobacco and Health Research Institute, University of Kentucky, Lexington 40546-0236, USA.
Plant Mol Biol. 1999 Jul;40(5):771-82. doi: 10.1023/a:1006285426523.
A full-length transcript (FLt) promoter fragment was isolated from a genomic clone of mirabilis mosaic virus (MMV), a double-stranded DNA plant pararetrovirus belonging to the caulimovirus family. The boundaries required for maximal promoter expression were defined by 5' and 3' deletion analysis of the MMV promoter fragments coupled to a GUS reporter gene. The expression patterns of these chimeric gene constructs were evaluated both in transgenic Nicotiana tabacum cv. Samsun NN plants and in protoplast transient expression experiments. A 360 bp FLt promoter fragment (sequence -297 to +63 from the transcription start site) was found sufficient for strong promoter activity. The transcription start site (TSS) of the MMV FLt promoter was determined by primer extension analysis using total RNA isolated from transgenic plants containing a MMV promoter:uidA fusion gene. Analysis of the 5' and 3' deletion constructs showed that an upstream region (sequence -248 to -193 from the transcription start site) is required for the MMV FLt promoter activity along with the as-1, TATA box regions. In addition, a 31 bp sequence (+33 to +63 from the transcription start site) located downstream of a TATA box is also essential for the maximum expression of the MMV FLt promoter. Analysis of transcripts (mRNA) from these chimeric constructs also indicated that the MMV FLt promoter fragment (-297 to +63 from the transcription start site) has the highest promoter activity. In a comparative analysis the MMV FLt promoter showed much greater activity than the CaMV 35S promoter.
从烟草花叶病毒（MMV）的基因组克隆中分离出一个全长转录本（FLt）启动子片段，MMV是一种双链DNA植物准逆转录病毒，属于花椰菜花叶病毒科。通过将MMV启动子片段与GUS报告基因偶联进行5'和3'缺失分析，确定了最大启动子表达所需的边界。在转基因烟草品种Samsun NN植株和原生质体瞬时表达实验中评估了这些嵌合基因构建体的表达模式。发现一个360 bp的FLt启动子片段（转录起始位点上游-297至+63的序列）足以产生强启动子活性。使用从含有MMV启动子：uidA融合基因的转基因植物中分离的总RNA，通过引物延伸分析确定了MMV FLt启动子的转录起始位点（TSS）。对5'和3'缺失构建体的分析表明，MMV FLt启动子活性需要一个上游区域（转录起始位点上游-248至-193的序列）以及as-1、TATA框区域。此外，位于TATA框下游的一个31 bp序列（转录起始位点上游+33至+63）对于MMV FLt启动子的最大表达也是必不可少的。对这些嵌合构建体的转录本（mRNA）分析还表明，MMV FLt启动子片段（转录起始位点上游-297至+63）具有最高的启动子活性。在比较分析中，MMV FLt启动子显示出比CaMV 35S启动子高得多的活性。
