Ainley W M, Key J L
Botany Department, University of Georgia, Athens.
Plant Mol Biol. 1990 Jun;14(6):949-67. doi: 10.1007/BF00019392.
A heat-inducible expression cassette has been constructed to study the conditional expression of sense or antisense orientations of any sequence of interest in transgenic plants or plant tissues. The construct includes the promoter and all but 5 bases of the mRNA leader from the soybean Gmhsp17.5-E gene, the polylinker from pUC18 (modified to remove the ATG), and a fragment that contains the polyadenylation signal and site from the nopaline synthase gene. Analysis of transient expression of a construct containing the beta-glucuronidase (GUS) coding sequence cloned in the cassette and introduced into Nicotiana plumbaginifolia protoplasts by electroporation shows that the promoter has high expression at heat shock temperatures. This construct is expressed at a roughly 80-fold higher level per unit time than a cauliflower mosaic virus 35S gene promoter-GUS construction. The heat shock promoter is regulated positively by supercoiling in this transient assay system. The level of expression of HS-GUS constructions with the polyadenylation sites from either the nopaline synthase gene or the Gmhsp17.5-E gene was similar. Constructs with a perfect fusion at the 5' end had higher levels of expression than those with the corresponding nonperfect transcriptional fusion.
已构建了一个热诱导表达盒，用于研究转基因植物或植物组织中任何感兴趣序列的正义或反义方向的条件性表达。该构建体包括来自大豆Gmhsp17.5 - E基因的启动子和mRNA前导序列除5个碱基外的所有部分、来自pUC18的多克隆位点（经修饰去除了ATG）以及一个包含胭脂碱合酶基因的聚腺苷酸化信号和位点的片段。对通过电穿孔导入烟草叶肉原生质体的、含有克隆在该盒中的β - 葡萄糖醛酸酶（GUS）编码序列的构建体进行瞬时表达分析表明，该启动子在热激温度下具有高表达。与花椰菜花叶病毒35S基因启动子 - GUS构建体相比，该构建体每单位时间的表达水平大约高80倍。在这个瞬时分析系统中，热激启动子受超螺旋正向调控。具有来自胭脂碱合酶基因或Gmhsp17.5 - E基因的聚腺苷酸化位点的HS - GUS构建体的表达水平相似。在5'端具有完美融合的构建体比具有相应非完美转录融合的构建体具有更高的表达水平。
